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Intramuscular and topical treatment of cutaneous leishmaniasis lesions in mice infected with Leishmania amazonensis using coumarin (--) mammea A/BB.



(-) Mammea A/BB

Calophyllum brasiliense

Leishmania amazonensis

Topical treatment

Intramuscular treatment


Treatment of cutaneous leishmaniasis remains limited to a few available options. Recent studies showed in vitro antileishmanial activity of mammea A/BB, a coumarin isolated from leaves of Calophyllum brasiliense. Moreover, the dichloromethane crude extract and hexane fraction from this plant demonstrated in vivo activity in mice infected with Leishmania amazonensis. We evaluated the antileishmanial activity of (-) mammea A/BB in the L. amazonensis BALB/c mice model. The animals were given intramuscular and topical treatment with (-) mammea A/BB for 30 consecutive days. The results demonstrated that 18 mg/kg/d intramuscularly or 0.2% topically of (-) mammea A/BB significantly reduced the size of skin lesions in footpads of mice compared with those in the control group (p <0.05). The activity of Glucantime[degrees] (corresponding to 27 mg/kg/d of pentavalent antimony) administered intramuscularly was similar to that of (-) mammea A/BB (p < 0.05) by both routes of administration. The histopathological evaluation showed no changes in the organs analyzed. These results indicate that the coumarin obtained from C. brasiliense is the antileishmanially active compound and can be used to control the development of cutaneous leishmaniasis lesions caused by L. amazonensis.

[C] 2012 Elsevier GmbH. All rights reserved.


Cutaneous leishmaniasis (CL) usually produces skin ulcers on the exposed parts of the body and they leave permanent scars and may cause serious disability (WHO 2011). In the Americas, CL is caused mainly by Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis, and Leishmania (Viannia) guyanensis, and requires treatment (Goncalves et al. 2005). Intralesional or systemic antiparasitic pentavalent antimonials such as sodium stibogluconate or meglumine antimoniate are the gold standard for the treatment of these diseases. Intramuscular pentamidine may be used in individuals intolerant to antimonial treatment or in cases of systemic antimony resistance. Liposomal amphotericin B may be an alternative to antimonials for the treatment of CL with mucosal involvement in South America. Oral miltefosine is promising for the treatment of CL, but more studies are needed to evaluate its effectiveness against different species of Leishmania (Minodier and Parola 2007; Mitropoulos et al. 2010).

Much emphasis has been given to the development of alternative therapeutic approaches. A compound for topical treatment of CL is an attractive option because it would be easy to apply and can avoid the problems with systemic toxicity of antileishmanial drugs. Paromomycin (Armijos et at. 2004) and imiquimod (Arevalo et al. 2001) can be applied topically in localized leishmaniasis.

In recent years, natural products have been a good source for researchers in search of bioactive substances. Although prospects for the cure of leishmaniasis are still uncertain, the development of new extraction techniques and identification of new and powerful drugs from plants are contributing to this effect (Carvalho and Ferreira 2001). Certain molecules isolated from natural sources represent a major breakthrough in the search for new antiprotozoal drugs, since there is urgent need for a new era of innovative medicines (Rocha et at. 2005).

Some species of the genus Colophyllum (Clusiaceae) are used against several diseases. Researchers have shown that these plants have cytotoxic activity, inhibition of HIV-1 reverse transcriptase, antisecretory and cytoprotective properties, antinociceptive, molluscicidal, and antimicrobial effects, due to the presence of coumarins, xanthones, flavonoicls, and triterpenes (Noldin et at. 2006). The compound mammea A/BB is a coumar-intype mammea purified from leaves of Calophyilum brasiliense Camb. Previous studies showed in vitro activity against L. amazonensis (Brenzan et al. 2007) and Trypanosoma cruzi (Reyes-Chilpa et al. 2008).

The present study experimentally evaluated the in viva activity of(-) mammea A/BB, in a BALB/c murine model infected with L. amazonensis josefa strain.

Materials and methods

Plant material

The leaves of C. brasiliense were collected and identified by Dr. Maria Claudia M. Young on Cardoso Island, July 2000, in the state of Sao Paulo, Brazil. After authentication, a voucher specimen (SP 363818) was deposited at the Herbarium of the Instituto de Botanica de Sao Paulo, Sao Paulo, Brazil. The botanical material was oven-dried, ground in a knife grinder and then stored in a dry dark location at room temperature.

Extraction and purification of compound (-) mammea A/BB

Extraction from leaf powder (7590 x g) was done after exhaustive maceration in ethanol:water (90:10) at room temperature. The extract was filtered, and evaporated under vacuum at 40C to remove the organic solvent and to obtain an aqueous extract and a residue. A dark-green residue from the crude extract was solubilized in dichloromethane, and the solvent was then completely evaporated at room temperature to yield a dichloromethane extract (238 g). Next, this extract was subjected to chromatography in a vacuum silica-gel column 60 (70-230 mesh) with hexane, hexane-dichloromethane (50:50), dichloromethane, dichloromethane-ethyl acetate (90:10-50:50), ethyl acetate, methanol, and finally methanol-water (90:10). Subsequently, the hexane fraction (38.5 g) was further chromatographed on a silica-gel column chromatograph and eluted with hexane, hexane-dichloromethane (98:2-50:50), dichloromethane, dichloromethane-ethyl acetate (98:2-50:50), ethyl acetate, and methanol. This procedure yielded the compound (-) mammea A/BB (500 mg) as described in previous studies (Brenzan et al. 2007, 2008).

Structure elucidation

The structure of the isolated compound was elucidated by chromatography-mass spectrometry (Micromass Quattro LC); nuclear magnetic resonance (NMR; Gemini 2000 BB; Varian), 1H NMR (300 MHz), and 13C NMR (75.5 MHz), DEPT, COSY (400 MHz), HMBC, and HSQC analysis in CDC13; infrared analysis (Bomem-MV 100; Hartmann & Braun-Michelson); and UV analysis (CARY 1E UV-Vis; Varian). The ap was obtained with a Perkin-Elmer Model 241 polarimeter at 20C at 589 nm, using CH2C12, and by comparison with literature data (Gasparotto-Jimior et al. 2005).


The MHOM/BR/75/Josefa strain of L. amazonensis, originally isolated from a human case of diffuse CL by C.A. Cuba-Cuba (University of Brasilia, Brasilia, Distrito Federal, Brazil) was used in the present study. The promastigotes were grown at 28 [degrees]C in Warren's medium (brain-heart infusion plus hemin and folic acid) supplemented with 10% heat-inactivated fetal bovine serum in a tissue flask. Parasites were centrifuged (2000 x g, 10 min), and washed three times in phosphate buffered saline (PBS -0.01 M, pH 7.2) for use.


The in vivo assay was carried out in male BALB/c mice weighing approximately 20 g and 4 weeks of age. This study was conducted according to ethical standards, and the experimental protocol (no. 013/2010) was approved by the Animal Ethics Committee of the Universidade Estadual de Maringa.


Animals were infected by subcutaneous inoculation with [10.sup.7] infective promastigotes of L. amazonensis (late-log phase of growth) in the left hind footpad. Footpad swelling was monitored by serial weekly measurements, for 30 days, of footpad thickness with the help of a digital pachymeter. Animals were kept under conventional conditions with free access to food (Nuvilab[R] Cr1) and water. The groups were housed in groups of five in plastic cages in controlled environmental conditions (12:12 h light/dark cycle and 22 [+ or -] 2[degrees]C room temperature).

In vivo evaluation of(-) mammea A/BB

After 8 weeks, lesions of measurable size were developed. Cages were randomly sorted to receive different treatments. The six groups were distributed as follows: group I: non-infected control, group II: infected control, group III: reference drug n-methylglucamine antimoniate (Glucantime[R], Aventis Pharma Ltda) dissolved in PBS and administered intramuscularly (100 mg/kg/d, corresponding to 27 mg/kg/d of pentavalent antimony), group IV: (-) mammea A/BB dissolved in dimethylsulfoxide (DMSO) and PBS administered intramuscularly (18 mg/kg/d), group V: (-) mammea A/BB 0.2% in a non-ionic base administered topically, group VI: DMSO (9%) control, administered intramuscularly. Treatments were administered daily. The final result was expressed as an increase in footpad thickness measured with a pachymeter, which is expressed as the degree of swelling, subtracting the value of the paw inoculated with the parasites from the value of the control footpad. The lesion size and the weight of mice were measured every week during a period of 30 days.

Brenzan et al. (2010) showed that each mg of dichloromethane extract contained 20.6 [+ or -] [micro]0.9 [micro]g of (-) mammea A/BB. Combined with data from the study of Honda et al. (2010), a correlation was performed to estimate the concentration to be used in the topical treatment in this study. For intramuscular treatment, the concentration was based on the recommendation of the manufacturer of Glucantime[R] for a concentration of pentavalent antimony of 10-20 mg/kg/d for CL.


After 30 days, the mice were killed in a [CO.sub.2] chamber. Liver, spleen, lung, testicles, esophagus, kidney, heart, duodenum, and stomach were washed in PBS, fixed in Bouin's solution, and processed for histopathology by paraffin inclusion. Blocks were cut at 7 [micro]m thickness in a microtome (Leica Microsystems Inc., Germany), mounted on slides and stained with hematoxylin-eosin.

Statistical analysis

For the statistical analysis in biological assays, means [+ or -] standard deviation (SD) were calculated by the Microsoft Office Excel 2007 software and the data were processed using Statistics 8.0[R]. Multiple comparisons between groups were made with a one-way analysis of variance (ANOVA) followed by Dunnet and Tukey tests. p values of less than 0.05 were considered significant and are represented by an asterisk.

Results and discussion

So far, there is no ideal treatment for leishmaniasis. Plant-derived substances of various structural classes have been extensively demonstrated to show antileishmanial activity and are promising leads for the development of new medicines (Polonio and Efferth 2008).

Previously, Brenzan et al. (2007) reported that the compound (-) mammea A/BB isolated from C. brasiliense showed in vitro activity against promastigote and amastigote forms of L. amazonensis, inhibiting 50% growth at concentrations of 3.0 and 0.88 [micro]/ml, respectively. Later, Honda et al. (2010) demonstrated that mice infected with L. amazonensis had a decrease in footpad lesions when treated topically with 10% dichloromethane extract or intraperitoneally with 100 mg/kg and 200 mg/kg of the hexane fraction of C brasiliense. Taken together, these studies encouraged us to apply the isolated compound (-) mammea A/BB in the treatment of mice experimentally infected with leishmaniasis.

The infection with L. amazonensis promastigotes in the footpad of BALB/c mice induced a progressive increase in the lesion size in all the mice. After 8 weeks, the treatment was started and untreated animals (Fig. 1B) and the DMSO controls (Fig. 1D) developed larger lesions with progressive disease. In contrast, animals treated with (-) mammea A/BB, either intramuscularly (Fig. 1E) or topicany (Fig. 1F), showed control of the Leishmania infection. Animals treated with the reference drug Glucantime[R] showed similar results (Fig. 1C). Non-infected control group presented normal footpad (Fig. 1A).

The untreated animals developed a characteristic lesion that may present a secondary bacterial infection. No occurrence of secondary bacterial infection was observed in the treated animals. This can be due to the antibacterial activity of mammea-type coumarin derivatives. Mammea A/BA, mammea A/AA, mammea B/BB and mammea B/BA were active against methicillin-sensitive and - resistant Staphylococcus aureus (Ouahouo et al. 2004; Yasunaka et al. 2005). On the other hand, mammea A/BA + A/BB, and mammea C/OA + C/OB also reduced the growth of S. aureus, as well as S. epidermidis and Bacillus subtilis (Reyes-Chilpa et al. 2004).

This isolated compound from C. brasiliense showed therapeutic potential, and a significant difference (p < 0.05) in the average lesion size was observed in mice treated with Glucantime[R] (1.36 [+ or -] 0.81 cm), (-) mammea A/BB intramuscularly (1.64 [+ or -] 0.8 cm) and (-) mammea A/BB topically (1.32 [+ or -] 0.57 cm), when compared with untreated animals (4.08 [+ or -] 1.31 cm). No significant difference was observed when (-) mammea A/BB intramuscularly or topically was compared to the positive control Glucantime[R] (Fig. 2).

With respect to animal average weight during the experiment, the treated group (groups III, IV and V) showed no significant difference (p <0.05) when compared with the untreated group (group II).

An incisional biopsy of all animals was taken for pathologic evaluation. All organs appeared healthy. The histopathological evaluation of liver, spleen, lung, testicles, esophagus, kidney, heart, duodenum, and stomach did not reveal any distinct compound-related changes. No changes in behavior were observed in all groups of mice.

Taken together, the results of all studies involving antileishmanial activity of C. brasiliense suggest that the active compound in the dichloromethane crude extract is (-) mammea A/BB. Natural products are undoubtedly valuable as a source of new medicinal agents for infectious diseases. Few compounds of natural origin have been proven to be effective against CL infection through in vivo studies. Intralesional injections of (+) usnic acid reduced significantly the weight lesions and the parasite loads in BALB/c mice footpads infected with L. amazonensis (Fournet etal. 1997). A significant decrease in the parasite burden and lesion size was observed in the L. amazonensis-infected mice treated topically or intrarectally with limonene (Arruda et al. 2009).

An alternative approach to a dermatological problem such as CL is a topical formulation. The advantages of topical treatment are the few adverse effects, better compliance by the patient, and reduced costs. Development of topical drugs is underway to investigate some antileishmanial drugs by increasing the absorption of the vehicle and their effect on percutaneous absorption and retention or drug release at the sites of infection in the dermis (Gamier and Croft 2002).


Treatment of leishmaniasis remains a challenge that urgently requires a solution and further research to obtain more potent and selective drugs. Summed up, the results obtained here suggest that it is important that in vitro tests be validated in animal studies. Therefore, it is reasonable to assume that the use of (-) mammea A/BB in the treatment of leishmaniasis ulcerations is truly effective in controlling L. amazonensis dissemination. The potent leishmanicidal activities of certain chemically defined molecules isolated from natural sources have become increasingly important in the search for new antiprotozoal agents at a time when there is an urgent need for a new group of innovative medicines.


We thank Adriana Oliveira dos Santos and Marco Antonio Costa for excellent technical assistance. This study was supported through grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico--CNPq, Capacitacao de Aperfeicoamento de Pessoal de Nivel Superior--CAPES, Financiadora de Estu-dos e Projetos--FINEP, PRONEX/Fundacao Araucaria, INCT_if, and Programa de Pos-graduacao em Ciencias Farmaceuticas da Univer-sidade Estadual de Maringa.


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* Corresponding author at: Departamento de Ciencias Basicas (la Saude, Labo-ratorio de Inovacao Tecnologica no Desenvolvimento de Farmacos e Cosmeticos, Bloco B-08, Universidade Estadual de Maringa, Av. Colombo 5790, CEP 87020-900, Maringa, Parana, Brazil. Tel.: +5544 3011 5012; fax: +5544 3011 5046.

E-mail addresses:, (C.V. Nakamura).

0944-7113/$--see front matter C 2012 Elsevier GmbH. All rights reserved.

Tatiana Shioji Tiuman (a), (b), Mislaine Adriana Brenzana (a), (b), Tania Ueda-Nakamuraa (a), (b), Benedito Prado Dias Filhoa (a), (b), Diogenes Aparicio Garcia Cortez (a), Celso Vataru Nakamura (a), (b) *

(a) Programa de Pos-Graduacao em Ciencias Farmaceuticas, Universidade Est-actual de Maringci, Av. Colombo 5790, 87020-900 Maringo, Parana, Brazil

(b) Departmento de Ciencias Basicas da Saude, Laborateirio de InovacCio Tecnologica no Desenvolvitnento de MI-mac:0s e Cosmeticos, Bloco 8-08, Universidade Estadual de Maringd, Av. Colombo 57.90, CEP 87020-900, Maringei, Parand, Brazil
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Title Annotation:Short communication
Author:Tiuman, Tatiana Shioji; Brenzan, Mislaine Adriana; Ueda-Nakamura, Tania; Filho, Benedito Prado Dias;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Oct 15, 2012
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