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Efficacy of the intralesional treatment with Chenopodium ambrosioides in the murine infection by Leishmania amazonensis
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Journal of Ethnopharmacology 115 (2008) 313–319
Efficacy of the intralesional treatment with Chenopodium ambrosioides in the murine infection by Leishmania amazonensis Fernando J. Patr´ıcio a , Graciomar C. Costa a , Paulo V.S. Pereira a , Walmir C. Arag˜ao-Filho a , Sanara M. Sousa a , Josias B. Fraz˜ao a , Wanderson S. Pereira a , M´arcia C.G. Maciel a , Lucilene A. Silva a , Fl´avia M.M. Amaral a , Jos´e M.M. Rebˆelo b , Rosane N.M. Guerra a , Maria Nilce S. Ribeiro c , Fl´avia R.F. Nascimento a,∗ a
Laborat´orio de Imunofisiologia, Departamento de Patologia, Universidade Federal do Maranh˜ao (UFMA), Centro de Ciˆencias Biol´ogicas e da Sa´ude (CCBS), Campus do Bacanga, Av. dos Portugueses s/n, S˜ao Lu´ıs, MA, Brazil, CEP:65085-580 b Laborat´ orio de Entomologia e Vetores, Departamento de Biologia, Universidade Federal do Maranh˜ao (UFMA), Centro de Ciˆencias Biol´ogicas e da Sa´ude (CCBS), Campus do Bacanga, Av. dos Portugueses s/n, S˜ao Lu´ıs, MA, Brazil, CEP:65085-580 c Laborat´ orio de Farmacognosia, Departamento de Farm´acia, Universidade Federal do Maranh˜ao (UFMA), Centro de Ciˆencias Biol´ogicas e da Sa´ude (CCBS), Campus do Bacanga, Av. dos Portugueses s/n, S˜ao Lu´ıs, MA, Brazil, CEP:65085-580 Received 17 February 2007; received in revised form 6 October 2007; accepted 9 October 2007 Available online 16 October 2007
Abstract Aim of the study: Leishmaniasis, caused by protozoan from Leishmania genus, is an endemic disease in the tropical and subtropical regions of the world. The chemotherapy to this disease is not always effective and can cause several side effects. Chenopodium ambrosioides L. (Chenopodiaceae) is used by the native people in the treatment of cutaneous ulcers caused by different species of Leishmania. The aim of this study was to investigate the effect of the treatment with a hydroalcoholic crude extract (HCE) from the leaves of Chenopodium ambrosioides on the murine infection with Leishmania amazonensis. Material and methods: The mice were treated for 4–6 weeks post-infection (p.i.) with HCE (5 mg/kg) or meglumine antimoniate (Sbv ) (28 mg/kg) either by the oral route, once a day, for 15 days or by five intralesional (IL) injections at intervals of 4 days. The thickness of the infected paws was determined weekly and the parasite load evaluated in the draining lymph nodes (LN), the spleen and in the footpad after 7 weeks of infection. The nitric oxide (NO) production was evaluated in cultures with cells from peritoneum or LN. Results: The IL treatment increased the NO production in the LN and peritoneum cultures and reduced the parasite load from the footpad, spleen and LN. On the other hand, the oral treatment decreased did alter neither the NO production nor the parasite load. Conclusions: IL HCE treatment was more efficient than the oral HCE treatment since the former was able to control the dissemination of infection. This effect can be due to either a direct leishmanicidal effect of HCE or the improvement in the NO production by HCE-stimulated macrophages. The results could justify the topical use of the Chenopodium ambrosioides’ leaves in the treatment of the ulcers caused by Leishmania. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Chenopodium ambrosioides; anti-leishmanial activity; nitric oxide; Mastruz; Leishmania amazonensis; Chenopodiaceae; in vivo infection
1. Introduction Species of the Leishmania genus, a protozoan from Trypanosomatidae family, are the causative agents of human
Abbreviations: HCE, hydroalcoholic crude extract; NO, nitric oxide; PBS, phosphate buffered solution; IL, intralesional; p.i., post infection; Sb, meglumine antimoniate (Glucantime® ); wk, weeks; LN, lymph node; FL, footpad lesion. ∗ Corresponding author. Tel.: +55 98 21098548; fax: +55 98 32316844. E-mail address: [email protected] (F.R.F. Nascimento). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.10.009
leishmaniasis. These parasites have a digenetic life cycle that includes an extracellular promastigote form in the sand fly vector and a nonflagellated intracellular amastigote stage within the mononuclear phagocytes of vertebrate hosts (Almeida et al., 2003). Leishmaniasis affects about 2 million people per year and presents a broad clinical spectrum ranging from asymptomatic and self-healing forms to cutaneous and/or visceral forms, causing significant morbidity and mortality. In Brazil, this disease constitutes a serious health problem, which has some endemic
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regions that present both cutaneous and visceral forms of leishmaniasis (Costa et al., 1995; Costa, 1998; Costa et al., 1998). Pentavalent antimonials, that were developed more than 50 years ago, are still the first choice among the drugs used in the treatment of leishmaniasis, despite their cardiac and renal toxicity (Paula et al., 2003; Rath et al., 2003). Although there are several drugs on trial for the human leishmaniasis chemotherapy, most of them are new formulations of old drugs. Considering the present clinical scenario, the development of new drugs is necessary. The lack of an effective anti-leishmanial drug has caused a renewed interest in the study of medicinal plants as possible sources of new chemotherapeutic compounds with better antileishmanial activities and fewer side effects (Iwu et al., 1994; Da Silva et al., 1995). The use of plants in the treatment of leishmaniasis is an old popular practice of the native people from endemic areas (Fournet et al., 1992; Moreira et al., 1998, 2002). In most cases, the therapy consists of either oral or topical administration of plant preparations (Franc¸a et al., 1993; Iwu et al., 1994; Akendengue et al., 1999). In an inquiry conducted among a hundred patients from an endemic area of cutaneous leishmaniasis in the northeastern region of Brazil, forty nine plant species used to treat skin ulceration caused by Leishmania species were identified. One of the most cited plant in this inquiry was Chenopodium ambrosioides L. (Chenopodiaceae) (Franc¸a et al., 1996). Chenopodium ambrosioides is an herbaceous shrub commonly known in Brazil as ‘mastruz’ or ‘erva-de-Santa-Maria’ and as American wormseed, goosefoot, ‘epazote’ and ‘paico’ in other American countries. Infusions and decoctions prepared from the leaves, roots and inflorescences of the Chenopodium ambrosioides have been used for centuries by native people as dietary condiments and as traditional medicine. Chenopodium ambrosioides has been used as anthelmintic, as anti-inflammatory, as anti-tumoral, as healer and as antileishmanial (Conway and Slocumb, 1979; Klicks, 1985; Franc¸a et al., 1996; Moreira et al., 2002). Chenopodium ambrosioides is rich in flavonoids and terpenoids compounds that have diverse pharmacological properties such as antioxidant and cancer chemopreventive effects (Di Carlo et al., 1999; Liu, 2004). It was previously shown that the crude extract from Chenopodium ambrosioides is a strong stimulator of the murine lymphocytes (Rossi-Bergmann et al., 1997). In fact, our group has demonstrated that Chenopodium ambrosioides has a significant anti-tumor activity (Nascimento et al., 2006) and also has an effect on macrophage activation, inducing both nitric oxide (NO) and hydrogen peroxide production and increasing the macrophage spreading ability (Cruz et al., 2007). The in vitro leishmanicidal effects of Chenopodium ambrosioides against Leishmania amazonensis promastigotes was recently shown by our group (Bezerra et al., 2006). In the same way, Monzote et al. (2006) showed that an essential oil from Chenopodium ambrosioides inhibits the progression of leishmanial infection both in vitro and in vivo. Based on these data, an evaluation was made on the effects of the intralesional and oral treatments with hydroalcoholic extract
prepared with the leaves of Chenopodium ambrosioides in the progression of the experimental infection with Leishmania amazonensis in mice. 2. Material and methods 2.1. Mice Male C3H/HePas mice (8/group), 8–12-weeks-old, weighing 20–25 g have been maintained for many generations in the Animal Breeding Unit (Biot´erio Central da Universidade Federal do Maranh˜ao, S˜ao Lu´ıs, MA, Brazil) under standard conditions. The animals were kept in well cross ventilated room at 26 ± 2 ◦ C, relative humidity 44–56%, light and dark cycles of 12 h. The animals had free access to sterilized food and acidified water. All procedures described were reviewed and approved by the Animal Ethics Committee in accordance with COBEA (Brazilian College of Animal Experimentation). 2.2. Plant material Leaves of Chenopodium ambrosioides L. (Chenopodiaceae) ´ were collected and identified at the Atico Seabra Herbarium of the Universidade Federal do Maranh˜ao (S˜ao Lu´ıs, MA, Brazil) (voucher specimen no. 0998). They were dried at 37 ◦ C and later powdered. The dry powdered leaves (200 g) were then extracted with 1 L of ethanol (70%) and mixed each 8 h for 24 h. After this period the hydroalcoholic extract was filtered using a cotton funnel and the same procedure was repeated four times. After this process the hydroalcoholic crude extract (HCE) was concentrated under low pressure. The yield obtained was 10.4% (w/w). Finally, the extract was dried and the remainder was later lyophilized. 2.3. Parasites MHOM/Br/90/BA125 Leishmania amazonensis was kindly provided by Dr. Aldina Barral from Centro de Pesquisas Gonc¸alo Moniz CPQGM/FIOCRUZ–BA. The promastigotes were serially cultured at 26 ◦ C in RPMI 1640 (Sigma, St. Louis, MO, USA) medium supplemented with 10% heat-inactivated fetal calf serum (Sigma), 2 mM l-glutamine (Gibco BRL, Grand Island, NY), penicillin (100 U/mL) and streptomycin (100 g/mL) (Sigma). 2.4. Infection and treatment of infected mice C3H/HePas mice (n = 48) were infected in the right hind footpad with 5 × 105 stationary phase Leishmania amazonensis promastigotes. Three weeks after the infection, the paws were measured and the animals distributed in order to assure similar lesion size average among groups. The animals were then divided in two groups according to the route of treatment: oral and intralesional. Each group was subdivided in three subgroups: Control (treated with phosphate buffered solution-PBS), HCE and Sb (treated with meglumine antimoniate-Sbv , Rhodia).
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The protocol used to treat the animals was adapted from Fournet et al. (1996). The intralesional treatment was initiated on the 23rd day post-infection (p.i.). The infected paws received five injections of 0.025 mL of PBS or HCE (5 mg/kg) or Sbv (28 mg/kg) with an interval of 4 days between the injections. The oral treatment was initiated on the 28th day post infection (4th week p.i.) and was maintained for 15 days. The mice received daily, by gavage, 0.2 mL of PBS or HCE (5 mg/kg/day) or Sbv (28 mg/kg/day). 2.5. Treatment efficacy evaluation During the treatment, the footpad thickness was determined weekly using a digital caliper. Lesion development was expressed as the difference, in size, between the infected footpad and the contralateral uninfected one. At the end of the 7th week post infection, 1 week after the last dose of treatments, the animals were sacrificed. The parasite load was evaluated in the draining lymph nodes, the spleen and in the footpad lesion. The organs and a part of the footpad lesion were removed, weighed, and then homogenized, with a potter glass homogenizer, in RPMI 1640 medium (Sigma) supplemented as described above. The limiting dilution assay was performed as previously described by Buffet et al. (1995). Briefly, under sterile conditions, serial fourfold dilution were prepared and distributed in 96-well microtiter plates (Costar, New York, NY, USA) in duplicates. After 10 days of incubation at 26 ◦ C, the wells were examined in an inverted microscope (Nikon, Inc.), at a magnification of 320×, for the presence or the absence of promastigotes. The final titer was the last dilution for which the well contained at least one parasite. The parasite load (number of parasites/gram of tissue) was calculated as follows: the geometric mean of the reciprocal of the positive titers from each duplicate was divided by the weight of the lymph node or spleen or footpad lesion. The value obtained was multiplied by the reciprocal fraction of the homogenized organ inoculated into the first well.
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2.7. Air pouch model The back of the mice was shaved and 3 mL of sterile air were injected subcutaneously. Immediately after, the pouches were injected with HCE at doses of 5 mg/kg. Control animals received 0.2 mL of sterile phosphate buffered saline (PBS) alone. After 24 h the mice were killed. The cells were aseptically collected by washing the pouches cavity with 3 mL sterile ice-cold PBS devoid of calcium and magnesium ions. For total cell determination, nine volumes of the cellular suspension were added to 1 volume of 0.05% crystal violet dissolved in 30% acetic acid and the cells were counted using a bright-line Neubauer chamber (Sigma). Differential cell counts were determined by cytospin preparations stained with Instant-Prov (Newprov, Pinhais, Brazil). 2.8. Statistical analysis The results are expressed as the mean ± standard deviation from 8 animals per group. The statistical evaluation was done by ANOVA followed by Tukey–Kramer test. Differences were considered significant at p ≤ 0.05 and are represented by an asterisk. All experiments were repeated for at least two times. 3. Results 3.1. Effect of HCE treatment on the footpad lesion progression The infection with Leishmania amazonensis promastigotes in the footpad of C3H/HePas induced a progressive increase in the footpad lesion size in all the mice. However, the intralesional treatment with HCE increased the paw thickness when compared to both the control and the Sb group. (Fig. 1A). On the other hand, the oral treatment with HCE or Sbv decreased the paw thickness when compared to the control group (Fig. 1B).
2.6. Nitric oxide evaluation 3.2. Effect of HCE on parasite load Cells obtained from the peritoneum or from the draining popliteum lymph node of the infected mice were collected, quantified and resuspended in RPMI 1640 medium (Sigma) supplemented as described above at a concentration of 2 × 106 mL−1 or 5 × 106 mL−1 , respectively. One hundred microliters of those cell suspensions were added to each well of a 96 wells flat bottom plate. The plate was cultured for 48 h at 37 ◦ C in a humid atmosphere containing 5% CO2 and 95% air. After the incubation, 50 L of supernatants were collected and incubated with an equal volume of Griess reagent (1% sulfanilamide/0.1% naphthalene diamine dihydrochloride/2.5% H3 PO4 ) for 10 min at room temperature, to quantify the accumulation of nitrite (Ding et al., 1988). The absorbance was determined at 540 nm. Conversion of absorbance to M of NO2 − was done by comparing the samples to a standard curve obtained with known concentrations (5–60 M) of sodium nitrite diluted in RPMI medium.
The limiting dilution assay showed that the HCE treatment significantly reduced the parasite load from the footpad lesion when compared to both the control and Sb groups. It was also observed that the parasite load in the spleen and lymph node from the group treated with HCE was significantly smaller than in the control group (Fig. 2A). Despite the decrease in the paw thickness after the oral treatment with both HCE and Sb, these treatments did not present the same efficacy in reducing the parasite load as observed after the intralesional treatment. In this case there was no significant difference in the parasite load in the three tissues evaluated (Fig. 2B). The lesion imprinting was used to confirm the results of limiting dilution. The animals that did not present any promastigotes in the first dilution did not also have any amastigotes in the imprints (data not shown).
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Fig. 1. Effect of HCE from Chenopodium ambrosioides on lesion growth using different routes of administration. C3H/HePas were infected in the footpad. After 4 weeks, they were treated with PBS or with Sb (28 mg/kg) or with HCE (5 mg/kg) by intralesional (A) or oral (B) routes. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group.
3.3. Effect of HCE treatment on nitric oxide production The NO production by cells from peritoneum or LN from the infected mice was evaluated in the cultures’ supernatants. There were different results according to the treatment route and the kind of cultured cell. The infection per se induced NO production in the culture of the lymph node cells from the control animals. This production was not altered after the oral treatment with HCE. However, the intralesional treatment with HCE induced a significant increase in the NO production (Fig. 3). The infection did not induce per se the NO production in the peritoneal cells cultures. There was NO production only after the intralesional treatment with HCE (Fig. 3).
Fig. 2. Inhibition of parasite growth by the treatment with HCE from Chenopodium ambrosioides. One week after the end of the treatments by intralesional (A) or oral (B) routes, the spleen, lymph node and footpad lesion were removed for parasites burden analysis. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group. # p < 0.05 when compared to Sb group.
to treat these ulcerations. This treatment is made by topical application of the leaves or, sometimes, by the ingestion of an infusion prepared with them. However, the real efficacy of Chenopodium ambrosioides treatment in the infection by Leishmania is not clear since the cutaneous leishmaniasis can have spontaneous cure of skin lesions (Marzochi and Marzochi, 1994). The maintenance and continuity of a Leishmania infection is related to the presence of the parasites in the lesion, and also in its ability to invade new host cells and multiply. The macrophage
3.4. Mononuclear cell migration induced by HCE The injection of HCE (5 mg/kg) into the air pouch caused a marked cell migration which was constituted basically of macrophage (Fig. 4). 4. Discussion In some endemic regions in the northeastern region of Brazil, alternative medicine has been used to treat the ulcerations caused by Leishmania species. Franc¸a et al. (1996) showed that Chenopodium ambrosioides is one of the main plant specie used
Fig. 3. Effect of intralesional and oral treatments with HCE from Chenopodium ambrosioides on the NO production by cells from peritoneum or draining lymph node. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group. # p < 0.05 when compared to IL group.
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Fig. 4. Effect of HCE from Chenopodium ambrosioides on neutrophil and mononuclear cells migration into air pouch. The HCE at the doses of 5 mg/kg was injected in the air pouch. After 24 h the pouches were washed and the cell counting was performed. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group.
constitutes both the host and the effector cell against infection by Leishmania parasites and by other intracellular microorganisms. Thus, macrophage activation is fundamental for the infection control (Liew et al., 1990; MacMicking et al., 1997). We have previously shown that the HCE from the leaves of Chenopodium ambrosioides activates macrophages (Cruz et al., 2007) and also presents a moderate specific leishmanicidal effect in vitro against the promastigotes from Leishmania amazonensis (Bezerra et al., 2006). Therefore, it was reasonable to suppose that the use of Chenopodium ambrosioides in the treatment of Leishmania ulcerations could be really effective in the control of the infection. To test this hypothesis we infected the mice with Leishmania amazonensis, a New World Leishmania specie, which is one of the major agents of diffuse cutaneous leishmaniasis, and usually unresponsive to treatment (Fournet et al., 1994). The C3H/HePas mice were chosen because they are susceptible to the infection by the Leishmania amazonensis promastigotes and develop chronic cutaneous lesions when infected in the footpad (Vanloubbeeck and Jones, 2004). The protocol of treatment was similar to that described by Fournet et al. (1996) which used the oral and intralesional treatments after the 4th week of infection, when the lesion is already established. It is important to emphasize that this protocol permits the reproduction of the methods of treatment used by the population in the endemic areas (Franc¸a et al., 1996). We showed here that the intralesional and oral treatments with HCE induced disparity effects in almost all parameters. The intralesional treatment decreased the parasite load in the footpad lesion, in the spleen and in the lymph node, however, it induced an increase in the infected paw thickness when compared to both the control and the Sb groups (Fig. 1A). On the other hand, the oral treatment decreased the thickness of footpad but had no effect in the parasite load (Fig. 1B). These results seem controversial but are in according to Oliveira et al. (2004) and Teixeira et al. (2006). These authors showed that the reduction of the parasite load does not necessarily reduce the footpad thickness that can be influenced by leukocyte infiltration in the infection site. The increase in the paw thickness induced by HCE was probably a result of the association between the infection
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and a pro-inflammatory effect induced by the HCE treatment. It is likely that the HCE injected in the lesion induce a cellular influx. This hypothesis was reinforced by the fact that the injection of the HCE in the air pouch induced an intense cellular infiltration, which was constituted basically of macrophages. The ability of the HCE to recruit macrophages, a fundamental cell in the control of Leishmania, is important to explain the increase of the footpad size and the concomitant decrease of the parasite load observed after the intralesional treatment. We have previously shown that the same pattern of inflammation was observed when the site of injection was the peritoneal cavity. Beside this, in vivo and in vitro treatments with Chenopodium ambrosioides induced NO production (Cruz et al., 2007). Since the macrophage activation is important in the control of Leishmania infection, mainly due to the NO production by these cells, we investigated here if the Chenopodium ambrosioides treatment by intralesional or oral routes could, in association to the infection, induce the production of this metabolite. It was noted that the cells from the draining lymph nodes, but not from the peritoneum, spontaneously produced NO. This production was likely due the in vivo pre-stimulation by the Leishmania amastigotes present in the draining lymph node. On the other hand, the NO production in the lymph node cells cultures was not altered after the oral treatment with HCE; nevertheless it was significantly increased when the mice were treated with HCE by the intralesional route. This result confirm that the intralesional treatment induce a pro-inflammatory stimulus which induce the migration and activation of NO-producers macrophages. Interestingly, the intralesional treatment also induced a weak NO production by the peritoneal cells, what was not observed after the oral treatment. The NO production by macrophages is important to control the parasite growing (Liew et al., 1990). Despite the NO production induced by HCE not being so intense, it seems to be enough to control the infection. Recently, we demonstrated that NO production is associated to the resistance to some infections in mice, but the overproduction of this molecule can be associated to the susceptibility (Nascimento et al., 2002). Besides, we have demonstrated in another study with the canine leishmaniasis that small levels of NO are related to the asymptomatic disease, while high levels of this molecule are associated to the progression of infection, a result probably related to an immunosuppressive effect of NO (Silva et al., unpublished data). These results are encouraging especially because they were obtained with the crude extract in a small dose (5 mg/kg). Gadano et al. (2002) described that Chenopodium ambrosioides can be genotoxic in vitro. However, we have previously shown that this dose has no toxic effects in mice when used by oral, intraperitoneal or topical route (unpublished data). Beside this we have shown that this dose was an adequate amount to activate macrophages in vivo (Cruz et al., 2007), a crucial cell in the control of Leishmania. In fact, we observed here that the treatment of infected mice with this dose did not alter the weight of liver and kidney and had no pathological alterations in these organs (data not shown).
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Our results corroborate with the results obtained by Monzote et al. (2006) which showed that an essential oil from Chenopodium ambrosioides has a significant effect on macrophages infected in vitro with Leishmania amazonensis and that the in vivo treatment with this oil, by the intraperitoneal route, inhibits the progression of infection in experimentally infected mice. Summed up, the results obtained here suggest that the treatment with HCE by intralesional route affects not only the regulatory mechanisms that control Leishmania amazonensis dissemination but also seems to have a direct leishmanicidal effect. Nevertheless, more studies are necessary to totally exclude the toxic effects of Chenopodium ambrosioides and to validate its use as an accessible alternative topical treatment for Leishmania infection for the population from endemic areas. Acknowledgments We thank Mr. Wickliff Eric for revising the manuscript. We are grateful to Dr. Aldina Barral from FIOCRUZ/BA by the donation of Leishmania amazonensis promastigotes and also to Dr. Ana L´ucia Abreu Silva for helpful suggestions. This work was supported by CNPq (Proc. PNOPG 550433/20015 and 620081/2004-0 ACT). F.J.P., G.C.C., S.M.S., L.A.S., R.N.M.G. and F.R.F.N. were supported by fellowship from CNPq. P.V.S.P., J.B.F and M.C.G.M. were supported by fellowship from FAPEMA. W.C.A.F. was supported by fellowship from PET/SESu/MEC. References Akendengue, B., Ngou-milama, E., Laurens, A., Hocquemiller, R., 1999. Recent advances in the fight against leishmaniasis with natural products. Parasite 6, 3–8. Almeida, M.C., Vilhena, V., Barral, A., Barral-Netto, M., 2003. Leishmanial infection: analysis of its first steps. A review. Mem´orias do Instituto Oswaldo Cruz 98, 861–870. Bezerra, J.L., Costa, G.C., Lopes, T.C., Carvalho, I.C.D.S., Patr´ıcio, F.J., Sousa, S.M., Amaral, F.M.M., Rebelo, J.M.M., Guerra, R.N.M., Ribeiro, M.N.S., Nascimento, F.R.F., 2006. Avaliac¸a˜ o da atividade leishmanicida in vitro de plantas medicinais. Revista Brasileira de Farmacognosia 16, 631–637. Buffet, P.A., Sulahian, A., Garcia, Y.J.F., Nassar, N., Derouin, F., 1995. Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrobial Agents and Chemotherapy 39, 2167–2168. Conway, G.A., Slocumb, J.C., 1979. Plants used as abortifacients and emmenagogues by Espanish New Mexicans. Journal of Ethnopharmacology 1, 241–261. Costa, J.M.L., Viana, G.M.C., Saldanha, A.C.R., Nascimento, M.D.S.B., Alvim, A.C., Burattini, M.N., Silva, A.R., 1995. Visceral leishmaniasis in the state of Maranh˜ao, Brazil: evolution of an epidemic. Cadernos de Sa´ude P´ublica 11, 321–324. Costa, J.M.L., 1998. Estudo da leishmaniose cutˆanea difusa no Estado do Maranh˜ao, Brasil: avaliac¸a˜ o terapˆeutica e correlac¸a˜ o do perfil imunol´ogico entre pacientes e seus familiars. Revista da Sociedade Brasileira de Medicina Tropical 31, 401–403. Costa, J.M.L., Balby, I.T.A., Rocha, E.J.S., Silva, A.R., Rebˆelo, J.M.M., Ferreira, L.A., Gama, M.E.A., Branco, M.R.F.C., Burattini, M.N., Soares, N.J.S., 1998. Estudo comparativo da leishmaniose tegumentar Americana em crianc¸as e adolescentes procedentes das a´ reas endˆemicas de Buriticupu (Maranh˜ao) e Corte da Pedra (Bahia), Brasil. Revista da Sociedade Brasileira de Medicina Tropical 31, 279–288.
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F.J. Patr´ıcio et al. / Journal of Ethnopharmacology 115 (2008) 313–319 Nascimento, F.R.F., Calich, V.L., Rodriguez, D., Russo, M., 2002. Dual role for nitric oxide in paracoccidioidomycosis: essential for resistance, but overproduction associated with susceptibility. Journal of Immunology 168, 4593–4600. Nascimento, F.R.F., Cruz, G.V.B., Pereira, P.V.S., Maciel, M.C.G., Silva, L.A., Azevedo, A.P.S., Barroqueiro, E.S.B., Guerra, R.N.M., 2006. Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment. Life Sciences 78, 2650–2653. Oliveira, C.I., Teixeira, M.J., Teixeira, C.R., Jesus, J.R., Rosato, A.B., Silva, J.S., Brodskyn, C., Barral-Netto, M., Barral, A., 2004. Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice. Microbes and Infection 6, 977–984. Paula, C.D.R., Sampaio, J.H.D., Cardoso, D.R.C., Sampaio, R.N.R., 2003. Estudo comparativo da efic´acia de isotionato de pentamidina administrada em trˆes doses durante uma semana e de N-metil-glucamina 20 mgSbV/kg/dia durante 20 dias para o tratamento da forma cutˆanea da leishmaniose tegu-
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Efficacy of the intralesional treatment with Chenopodium ambrosioides in the murine infection by Leishmania amazonensis Fernando J. Patr´ıcio a , Graciomar C. Costa a , Paulo V.S. Pereira a , Walmir C. Arag˜ao-Filho a , Sanara M. Sousa a , Josias B. Fraz˜ao a , Wanderson S. Pereira a , M´arcia C.G. Maciel a , Lucilene A. Silva a , Fl´avia M.M. Amaral a , Jos´e M.M. Rebˆelo b , Rosane N.M. Guerra a , Maria Nilce S. Ribeiro c , Fl´avia R.F. Nascimento a,∗ a
Laborat´orio de Imunofisiologia, Departamento de Patologia, Universidade Federal do Maranh˜ao (UFMA), Centro de Ciˆencias Biol´ogicas e da Sa´ude (CCBS), Campus do Bacanga, Av. dos Portugueses s/n, S˜ao Lu´ıs, MA, Brazil, CEP:65085-580 b Laborat´ orio de Entomologia e Vetores, Departamento de Biologia, Universidade Federal do Maranh˜ao (UFMA), Centro de Ciˆencias Biol´ogicas e da Sa´ude (CCBS), Campus do Bacanga, Av. dos Portugueses s/n, S˜ao Lu´ıs, MA, Brazil, CEP:65085-580 c Laborat´ orio de Farmacognosia, Departamento de Farm´acia, Universidade Federal do Maranh˜ao (UFMA), Centro de Ciˆencias Biol´ogicas e da Sa´ude (CCBS), Campus do Bacanga, Av. dos Portugueses s/n, S˜ao Lu´ıs, MA, Brazil, CEP:65085-580 Received 17 February 2007; received in revised form 6 October 2007; accepted 9 October 2007 Available online 16 October 2007
Abstract Aim of the study: Leishmaniasis, caused by protozoan from Leishmania genus, is an endemic disease in the tropical and subtropical regions of the world. The chemotherapy to this disease is not always effective and can cause several side effects. Chenopodium ambrosioides L. (Chenopodiaceae) is used by the native people in the treatment of cutaneous ulcers caused by different species of Leishmania. The aim of this study was to investigate the effect of the treatment with a hydroalcoholic crude extract (HCE) from the leaves of Chenopodium ambrosioides on the murine infection with Leishmania amazonensis. Material and methods: The mice were treated for 4–6 weeks post-infection (p.i.) with HCE (5 mg/kg) or meglumine antimoniate (Sbv ) (28 mg/kg) either by the oral route, once a day, for 15 days or by five intralesional (IL) injections at intervals of 4 days. The thickness of the infected paws was determined weekly and the parasite load evaluated in the draining lymph nodes (LN), the spleen and in the footpad after 7 weeks of infection. The nitric oxide (NO) production was evaluated in cultures with cells from peritoneum or LN. Results: The IL treatment increased the NO production in the LN and peritoneum cultures and reduced the parasite load from the footpad, spleen and LN. On the other hand, the oral treatment decreased did alter neither the NO production nor the parasite load. Conclusions: IL HCE treatment was more efficient than the oral HCE treatment since the former was able to control the dissemination of infection. This effect can be due to either a direct leishmanicidal effect of HCE or the improvement in the NO production by HCE-stimulated macrophages. The results could justify the topical use of the Chenopodium ambrosioides’ leaves in the treatment of the ulcers caused by Leishmania. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Chenopodium ambrosioides; anti-leishmanial activity; nitric oxide; Mastruz; Leishmania amazonensis; Chenopodiaceae; in vivo infection
1. Introduction Species of the Leishmania genus, a protozoan from Trypanosomatidae family, are the causative agents of human
Abbreviations: HCE, hydroalcoholic crude extract; NO, nitric oxide; PBS, phosphate buffered solution; IL, intralesional; p.i., post infection; Sb, meglumine antimoniate (Glucantime® ); wk, weeks; LN, lymph node; FL, footpad lesion. ∗ Corresponding author. Tel.: +55 98 21098548; fax: +55 98 32316844. E-mail address: [email protected] (F.R.F. Nascimento). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.10.009
leishmaniasis. These parasites have a digenetic life cycle that includes an extracellular promastigote form in the sand fly vector and a nonflagellated intracellular amastigote stage within the mononuclear phagocytes of vertebrate hosts (Almeida et al., 2003). Leishmaniasis affects about 2 million people per year and presents a broad clinical spectrum ranging from asymptomatic and self-healing forms to cutaneous and/or visceral forms, causing significant morbidity and mortality. In Brazil, this disease constitutes a serious health problem, which has some endemic
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regions that present both cutaneous and visceral forms of leishmaniasis (Costa et al., 1995; Costa, 1998; Costa et al., 1998). Pentavalent antimonials, that were developed more than 50 years ago, are still the first choice among the drugs used in the treatment of leishmaniasis, despite their cardiac and renal toxicity (Paula et al., 2003; Rath et al., 2003). Although there are several drugs on trial for the human leishmaniasis chemotherapy, most of them are new formulations of old drugs. Considering the present clinical scenario, the development of new drugs is necessary. The lack of an effective anti-leishmanial drug has caused a renewed interest in the study of medicinal plants as possible sources of new chemotherapeutic compounds with better antileishmanial activities and fewer side effects (Iwu et al., 1994; Da Silva et al., 1995). The use of plants in the treatment of leishmaniasis is an old popular practice of the native people from endemic areas (Fournet et al., 1992; Moreira et al., 1998, 2002). In most cases, the therapy consists of either oral or topical administration of plant preparations (Franc¸a et al., 1993; Iwu et al., 1994; Akendengue et al., 1999). In an inquiry conducted among a hundred patients from an endemic area of cutaneous leishmaniasis in the northeastern region of Brazil, forty nine plant species used to treat skin ulceration caused by Leishmania species were identified. One of the most cited plant in this inquiry was Chenopodium ambrosioides L. (Chenopodiaceae) (Franc¸a et al., 1996). Chenopodium ambrosioides is an herbaceous shrub commonly known in Brazil as ‘mastruz’ or ‘erva-de-Santa-Maria’ and as American wormseed, goosefoot, ‘epazote’ and ‘paico’ in other American countries. Infusions and decoctions prepared from the leaves, roots and inflorescences of the Chenopodium ambrosioides have been used for centuries by native people as dietary condiments and as traditional medicine. Chenopodium ambrosioides has been used as anthelmintic, as anti-inflammatory, as anti-tumoral, as healer and as antileishmanial (Conway and Slocumb, 1979; Klicks, 1985; Franc¸a et al., 1996; Moreira et al., 2002). Chenopodium ambrosioides is rich in flavonoids and terpenoids compounds that have diverse pharmacological properties such as antioxidant and cancer chemopreventive effects (Di Carlo et al., 1999; Liu, 2004). It was previously shown that the crude extract from Chenopodium ambrosioides is a strong stimulator of the murine lymphocytes (Rossi-Bergmann et al., 1997). In fact, our group has demonstrated that Chenopodium ambrosioides has a significant anti-tumor activity (Nascimento et al., 2006) and also has an effect on macrophage activation, inducing both nitric oxide (NO) and hydrogen peroxide production and increasing the macrophage spreading ability (Cruz et al., 2007). The in vitro leishmanicidal effects of Chenopodium ambrosioides against Leishmania amazonensis promastigotes was recently shown by our group (Bezerra et al., 2006). In the same way, Monzote et al. (2006) showed that an essential oil from Chenopodium ambrosioides inhibits the progression of leishmanial infection both in vitro and in vivo. Based on these data, an evaluation was made on the effects of the intralesional and oral treatments with hydroalcoholic extract
prepared with the leaves of Chenopodium ambrosioides in the progression of the experimental infection with Leishmania amazonensis in mice. 2. Material and methods 2.1. Mice Male C3H/HePas mice (8/group), 8–12-weeks-old, weighing 20–25 g have been maintained for many generations in the Animal Breeding Unit (Biot´erio Central da Universidade Federal do Maranh˜ao, S˜ao Lu´ıs, MA, Brazil) under standard conditions. The animals were kept in well cross ventilated room at 26 ± 2 ◦ C, relative humidity 44–56%, light and dark cycles of 12 h. The animals had free access to sterilized food and acidified water. All procedures described were reviewed and approved by the Animal Ethics Committee in accordance with COBEA (Brazilian College of Animal Experimentation). 2.2. Plant material Leaves of Chenopodium ambrosioides L. (Chenopodiaceae) ´ were collected and identified at the Atico Seabra Herbarium of the Universidade Federal do Maranh˜ao (S˜ao Lu´ıs, MA, Brazil) (voucher specimen no. 0998). They were dried at 37 ◦ C and later powdered. The dry powdered leaves (200 g) were then extracted with 1 L of ethanol (70%) and mixed each 8 h for 24 h. After this period the hydroalcoholic extract was filtered using a cotton funnel and the same procedure was repeated four times. After this process the hydroalcoholic crude extract (HCE) was concentrated under low pressure. The yield obtained was 10.4% (w/w). Finally, the extract was dried and the remainder was later lyophilized. 2.3. Parasites MHOM/Br/90/BA125 Leishmania amazonensis was kindly provided by Dr. Aldina Barral from Centro de Pesquisas Gonc¸alo Moniz CPQGM/FIOCRUZ–BA. The promastigotes were serially cultured at 26 ◦ C in RPMI 1640 (Sigma, St. Louis, MO, USA) medium supplemented with 10% heat-inactivated fetal calf serum (Sigma), 2 mM l-glutamine (Gibco BRL, Grand Island, NY), penicillin (100 U/mL) and streptomycin (100 g/mL) (Sigma). 2.4. Infection and treatment of infected mice C3H/HePas mice (n = 48) were infected in the right hind footpad with 5 × 105 stationary phase Leishmania amazonensis promastigotes. Three weeks after the infection, the paws were measured and the animals distributed in order to assure similar lesion size average among groups. The animals were then divided in two groups according to the route of treatment: oral and intralesional. Each group was subdivided in three subgroups: Control (treated with phosphate buffered solution-PBS), HCE and Sb (treated with meglumine antimoniate-Sbv , Rhodia).
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The protocol used to treat the animals was adapted from Fournet et al. (1996). The intralesional treatment was initiated on the 23rd day post-infection (p.i.). The infected paws received five injections of 0.025 mL of PBS or HCE (5 mg/kg) or Sbv (28 mg/kg) with an interval of 4 days between the injections. The oral treatment was initiated on the 28th day post infection (4th week p.i.) and was maintained for 15 days. The mice received daily, by gavage, 0.2 mL of PBS or HCE (5 mg/kg/day) or Sbv (28 mg/kg/day). 2.5. Treatment efficacy evaluation During the treatment, the footpad thickness was determined weekly using a digital caliper. Lesion development was expressed as the difference, in size, between the infected footpad and the contralateral uninfected one. At the end of the 7th week post infection, 1 week after the last dose of treatments, the animals were sacrificed. The parasite load was evaluated in the draining lymph nodes, the spleen and in the footpad lesion. The organs and a part of the footpad lesion were removed, weighed, and then homogenized, with a potter glass homogenizer, in RPMI 1640 medium (Sigma) supplemented as described above. The limiting dilution assay was performed as previously described by Buffet et al. (1995). Briefly, under sterile conditions, serial fourfold dilution were prepared and distributed in 96-well microtiter plates (Costar, New York, NY, USA) in duplicates. After 10 days of incubation at 26 ◦ C, the wells were examined in an inverted microscope (Nikon, Inc.), at a magnification of 320×, for the presence or the absence of promastigotes. The final titer was the last dilution for which the well contained at least one parasite. The parasite load (number of parasites/gram of tissue) was calculated as follows: the geometric mean of the reciprocal of the positive titers from each duplicate was divided by the weight of the lymph node or spleen or footpad lesion. The value obtained was multiplied by the reciprocal fraction of the homogenized organ inoculated into the first well.
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2.7. Air pouch model The back of the mice was shaved and 3 mL of sterile air were injected subcutaneously. Immediately after, the pouches were injected with HCE at doses of 5 mg/kg. Control animals received 0.2 mL of sterile phosphate buffered saline (PBS) alone. After 24 h the mice were killed. The cells were aseptically collected by washing the pouches cavity with 3 mL sterile ice-cold PBS devoid of calcium and magnesium ions. For total cell determination, nine volumes of the cellular suspension were added to 1 volume of 0.05% crystal violet dissolved in 30% acetic acid and the cells were counted using a bright-line Neubauer chamber (Sigma). Differential cell counts were determined by cytospin preparations stained with Instant-Prov (Newprov, Pinhais, Brazil). 2.8. Statistical analysis The results are expressed as the mean ± standard deviation from 8 animals per group. The statistical evaluation was done by ANOVA followed by Tukey–Kramer test. Differences were considered significant at p ≤ 0.05 and are represented by an asterisk. All experiments were repeated for at least two times. 3. Results 3.1. Effect of HCE treatment on the footpad lesion progression The infection with Leishmania amazonensis promastigotes in the footpad of C3H/HePas induced a progressive increase in the footpad lesion size in all the mice. However, the intralesional treatment with HCE increased the paw thickness when compared to both the control and the Sb group. (Fig. 1A). On the other hand, the oral treatment with HCE or Sbv decreased the paw thickness when compared to the control group (Fig. 1B).
2.6. Nitric oxide evaluation 3.2. Effect of HCE on parasite load Cells obtained from the peritoneum or from the draining popliteum lymph node of the infected mice were collected, quantified and resuspended in RPMI 1640 medium (Sigma) supplemented as described above at a concentration of 2 × 106 mL−1 or 5 × 106 mL−1 , respectively. One hundred microliters of those cell suspensions were added to each well of a 96 wells flat bottom plate. The plate was cultured for 48 h at 37 ◦ C in a humid atmosphere containing 5% CO2 and 95% air. After the incubation, 50 L of supernatants were collected and incubated with an equal volume of Griess reagent (1% sulfanilamide/0.1% naphthalene diamine dihydrochloride/2.5% H3 PO4 ) for 10 min at room temperature, to quantify the accumulation of nitrite (Ding et al., 1988). The absorbance was determined at 540 nm. Conversion of absorbance to M of NO2 − was done by comparing the samples to a standard curve obtained with known concentrations (5–60 M) of sodium nitrite diluted in RPMI medium.
The limiting dilution assay showed that the HCE treatment significantly reduced the parasite load from the footpad lesion when compared to both the control and Sb groups. It was also observed that the parasite load in the spleen and lymph node from the group treated with HCE was significantly smaller than in the control group (Fig. 2A). Despite the decrease in the paw thickness after the oral treatment with both HCE and Sb, these treatments did not present the same efficacy in reducing the parasite load as observed after the intralesional treatment. In this case there was no significant difference in the parasite load in the three tissues evaluated (Fig. 2B). The lesion imprinting was used to confirm the results of limiting dilution. The animals that did not present any promastigotes in the first dilution did not also have any amastigotes in the imprints (data not shown).
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Fig. 1. Effect of HCE from Chenopodium ambrosioides on lesion growth using different routes of administration. C3H/HePas were infected in the footpad. After 4 weeks, they were treated with PBS or with Sb (28 mg/kg) or with HCE (5 mg/kg) by intralesional (A) or oral (B) routes. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group.
3.3. Effect of HCE treatment on nitric oxide production The NO production by cells from peritoneum or LN from the infected mice was evaluated in the cultures’ supernatants. There were different results according to the treatment route and the kind of cultured cell. The infection per se induced NO production in the culture of the lymph node cells from the control animals. This production was not altered after the oral treatment with HCE. However, the intralesional treatment with HCE induced a significant increase in the NO production (Fig. 3). The infection did not induce per se the NO production in the peritoneal cells cultures. There was NO production only after the intralesional treatment with HCE (Fig. 3).
Fig. 2. Inhibition of parasite growth by the treatment with HCE from Chenopodium ambrosioides. One week after the end of the treatments by intralesional (A) or oral (B) routes, the spleen, lymph node and footpad lesion were removed for parasites burden analysis. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group. # p < 0.05 when compared to Sb group.
to treat these ulcerations. This treatment is made by topical application of the leaves or, sometimes, by the ingestion of an infusion prepared with them. However, the real efficacy of Chenopodium ambrosioides treatment in the infection by Leishmania is not clear since the cutaneous leishmaniasis can have spontaneous cure of skin lesions (Marzochi and Marzochi, 1994). The maintenance and continuity of a Leishmania infection is related to the presence of the parasites in the lesion, and also in its ability to invade new host cells and multiply. The macrophage
3.4. Mononuclear cell migration induced by HCE The injection of HCE (5 mg/kg) into the air pouch caused a marked cell migration which was constituted basically of macrophage (Fig. 4). 4. Discussion In some endemic regions in the northeastern region of Brazil, alternative medicine has been used to treat the ulcerations caused by Leishmania species. Franc¸a et al. (1996) showed that Chenopodium ambrosioides is one of the main plant specie used
Fig. 3. Effect of intralesional and oral treatments with HCE from Chenopodium ambrosioides on the NO production by cells from peritoneum or draining lymph node. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group. # p < 0.05 when compared to IL group.
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Fig. 4. Effect of HCE from Chenopodium ambrosioides on neutrophil and mononuclear cells migration into air pouch. The HCE at the doses of 5 mg/kg was injected in the air pouch. After 24 h the pouches were washed and the cell counting was performed. The data represent the mean ± S.E.M. from 8 mice/group. * p < 0.05 when compared to the control group.
constitutes both the host and the effector cell against infection by Leishmania parasites and by other intracellular microorganisms. Thus, macrophage activation is fundamental for the infection control (Liew et al., 1990; MacMicking et al., 1997). We have previously shown that the HCE from the leaves of Chenopodium ambrosioides activates macrophages (Cruz et al., 2007) and also presents a moderate specific leishmanicidal effect in vitro against the promastigotes from Leishmania amazonensis (Bezerra et al., 2006). Therefore, it was reasonable to suppose that the use of Chenopodium ambrosioides in the treatment of Leishmania ulcerations could be really effective in the control of the infection. To test this hypothesis we infected the mice with Leishmania amazonensis, a New World Leishmania specie, which is one of the major agents of diffuse cutaneous leishmaniasis, and usually unresponsive to treatment (Fournet et al., 1994). The C3H/HePas mice were chosen because they are susceptible to the infection by the Leishmania amazonensis promastigotes and develop chronic cutaneous lesions when infected in the footpad (Vanloubbeeck and Jones, 2004). The protocol of treatment was similar to that described by Fournet et al. (1996) which used the oral and intralesional treatments after the 4th week of infection, when the lesion is already established. It is important to emphasize that this protocol permits the reproduction of the methods of treatment used by the population in the endemic areas (Franc¸a et al., 1996). We showed here that the intralesional and oral treatments with HCE induced disparity effects in almost all parameters. The intralesional treatment decreased the parasite load in the footpad lesion, in the spleen and in the lymph node, however, it induced an increase in the infected paw thickness when compared to both the control and the Sb groups (Fig. 1A). On the other hand, the oral treatment decreased the thickness of footpad but had no effect in the parasite load (Fig. 1B). These results seem controversial but are in according to Oliveira et al. (2004) and Teixeira et al. (2006). These authors showed that the reduction of the parasite load does not necessarily reduce the footpad thickness that can be influenced by leukocyte infiltration in the infection site. The increase in the paw thickness induced by HCE was probably a result of the association between the infection
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and a pro-inflammatory effect induced by the HCE treatment. It is likely that the HCE injected in the lesion induce a cellular influx. This hypothesis was reinforced by the fact that the injection of the HCE in the air pouch induced an intense cellular infiltration, which was constituted basically of macrophages. The ability of the HCE to recruit macrophages, a fundamental cell in the control of Leishmania, is important to explain the increase of the footpad size and the concomitant decrease of the parasite load observed after the intralesional treatment. We have previously shown that the same pattern of inflammation was observed when the site of injection was the peritoneal cavity. Beside this, in vivo and in vitro treatments with Chenopodium ambrosioides induced NO production (Cruz et al., 2007). Since the macrophage activation is important in the control of Leishmania infection, mainly due to the NO production by these cells, we investigated here if the Chenopodium ambrosioides treatment by intralesional or oral routes could, in association to the infection, induce the production of this metabolite. It was noted that the cells from the draining lymph nodes, but not from the peritoneum, spontaneously produced NO. This production was likely due the in vivo pre-stimulation by the Leishmania amastigotes present in the draining lymph node. On the other hand, the NO production in the lymph node cells cultures was not altered after the oral treatment with HCE; nevertheless it was significantly increased when the mice were treated with HCE by the intralesional route. This result confirm that the intralesional treatment induce a pro-inflammatory stimulus which induce the migration and activation of NO-producers macrophages. Interestingly, the intralesional treatment also induced a weak NO production by the peritoneal cells, what was not observed after the oral treatment. The NO production by macrophages is important to control the parasite growing (Liew et al., 1990). Despite the NO production induced by HCE not being so intense, it seems to be enough to control the infection. Recently, we demonstrated that NO production is associated to the resistance to some infections in mice, but the overproduction of this molecule can be associated to the susceptibility (Nascimento et al., 2002). Besides, we have demonstrated in another study with the canine leishmaniasis that small levels of NO are related to the asymptomatic disease, while high levels of this molecule are associated to the progression of infection, a result probably related to an immunosuppressive effect of NO (Silva et al., unpublished data). These results are encouraging especially because they were obtained with the crude extract in a small dose (5 mg/kg). Gadano et al. (2002) described that Chenopodium ambrosioides can be genotoxic in vitro. However, we have previously shown that this dose has no toxic effects in mice when used by oral, intraperitoneal or topical route (unpublished data). Beside this we have shown that this dose was an adequate amount to activate macrophages in vivo (Cruz et al., 2007), a crucial cell in the control of Leishmania. In fact, we observed here that the treatment of infected mice with this dose did not alter the weight of liver and kidney and had no pathological alterations in these organs (data not shown).
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Our results corroborate with the results obtained by Monzote et al. (2006) which showed that an essential oil from Chenopodium ambrosioides has a significant effect on macrophages infected in vitro with Leishmania amazonensis and that the in vivo treatment with this oil, by the intraperitoneal route, inhibits the progression of infection in experimentally infected mice. Summed up, the results obtained here suggest that the treatment with HCE by intralesional route affects not only the regulatory mechanisms that control Leishmania amazonensis dissemination but also seems to have a direct leishmanicidal effect. Nevertheless, more studies are necessary to totally exclude the toxic effects of Chenopodium ambrosioides and to validate its use as an accessible alternative topical treatment for Leishmania infection for the population from endemic areas. Acknowledgments We thank Mr. Wickliff Eric for revising the manuscript. We are grateful to Dr. Aldina Barral from FIOCRUZ/BA by the donation of Leishmania amazonensis promastigotes and also to Dr. Ana L´ucia Abreu Silva for helpful suggestions. This work was supported by CNPq (Proc. PNOPG 550433/20015 and 620081/2004-0 ACT). F.J.P., G.C.C., S.M.S., L.A.S., R.N.M.G. and F.R.F.N. were supported by fellowship from CNPq. P.V.S.P., J.B.F and M.C.G.M. were supported by fellowship from FAPEMA. W.C.A.F. was supported by fellowship from PET/SESu/MEC. References Akendengue, B., Ngou-milama, E., Laurens, A., Hocquemiller, R., 1999. Recent advances in the fight against leishmaniasis with natural products. Parasite 6, 3–8. Almeida, M.C., Vilhena, V., Barral, A., Barral-Netto, M., 2003. Leishmanial infection: analysis of its first steps. A review. Mem´orias do Instituto Oswaldo Cruz 98, 861–870. Bezerra, J.L., Costa, G.C., Lopes, T.C., Carvalho, I.C.D.S., Patr´ıcio, F.J., Sousa, S.M., Amaral, F.M.M., Rebelo, J.M.M., Guerra, R.N.M., Ribeiro, M.N.S., Nascimento, F.R.F., 2006. Avaliac¸a˜ o da atividade leishmanicida in vitro de plantas medicinais. Revista Brasileira de Farmacognosia 16, 631–637. Buffet, P.A., Sulahian, A., Garcia, Y.J.F., Nassar, N., Derouin, F., 1995. Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrobial Agents and Chemotherapy 39, 2167–2168. Conway, G.A., Slocumb, J.C., 1979. Plants used as abortifacients and emmenagogues by Espanish New Mexicans. Journal of Ethnopharmacology 1, 241–261. Costa, J.M.L., Viana, G.M.C., Saldanha, A.C.R., Nascimento, M.D.S.B., Alvim, A.C., Burattini, M.N., Silva, A.R., 1995. Visceral leishmaniasis in the state of Maranh˜ao, Brazil: evolution of an epidemic. Cadernos de Sa´ude P´ublica 11, 321–324. Costa, J.M.L., 1998. Estudo da leishmaniose cutˆanea difusa no Estado do Maranh˜ao, Brasil: avaliac¸a˜ o terapˆeutica e correlac¸a˜ o do perfil imunol´ogico entre pacientes e seus familiars. Revista da Sociedade Brasileira de Medicina Tropical 31, 401–403. Costa, J.M.L., Balby, I.T.A., Rocha, E.J.S., Silva, A.R., Rebˆelo, J.M.M., Ferreira, L.A., Gama, M.E.A., Branco, M.R.F.C., Burattini, M.N., Soares, N.J.S., 1998. Estudo comparativo da leishmaniose tegumentar Americana em crianc¸as e adolescentes procedentes das a´ reas endˆemicas de Buriticupu (Maranh˜ao) e Corte da Pedra (Bahia), Brasil. Revista da Sociedade Brasileira de Medicina Tropical 31, 279–288.
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