MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 103(5) August 2008
PAGES: 443-449 DOI: S0074-02762008000500006 Full paper
Brazilian flora extracts as source of novel antileishmanial and antifungal compounds

André Gustavo TemponeI, *; Patrícia SartorelliII; Denise TeixeiraI; Frederico O PradoI; Ivete ARL CalixtoI; Harri LorenziIII; Márcia SC MelhemI

ILaboratório de Toxinologia Aplicada, Serviço de Parasitologia, Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, 8º andar, 01246-000 São Paulo, SP, Brasil
IIDepartamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Diadema, SP, Brasil
IIIInstituto Plantarum de Estudos da Flora, Nova Odessa, SP, Brasil

Abstract

Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a protozoan parasitic disease found mainly in developing countries, and it has toxic therapies with few alternatives. Fungal infections have been the main cause of death in immunocompromised patients and new drugs are urgently needed. In this work, a total of 16 plant species belonging to 11 families, selected on an ethnopharmacological basis, were analyzed in vitro against Leishmania (L.) chagasi, Leishmania (L.) amazonensis, Candida krusei, and C. parapsilosis. Of these plant species, seven showed antifungal activity against C. krusei, five showed antileishmanial activity against L. chagasi and four against L. amazonensis, among them species of genus Plectranthus. Our findings confirm the traditional therapeutic use of these plants in the treatment of infectious and inflammatory disorders and also offer insights into the isolation of active and novel drug prototypes, especially those used against neglected diseases as Leishmaniasis.

Medicinal plants have been the basis for the treatment of various diseases in traditional methods. In Brazil, populations in rural areas rely on traditional medicines for the treatment of many infectious diseases. Some species are included in prescriptions for therapeutic purposes like the healing of wounds, inflammation due to microbial or parasitic infections, skin lesions, and ulcers (Lorenzi & Matos 2002). In some cases, the plant may be used for different purposes. For instance, the species of genus Plectranthusis traditionally used to treat a range of ailments including digestion, skin, infection, and respiratory problems (Lukhoba et al. 2006).

The Brazilian Atlantic forest (BAF) is one of the major biodiverse areas of the world, providing therapeutics for ancient diseases (Tempone et al. 2005). Plants used in folk medicine have offered natural products for the treatment of diseases caused by protozoan parasites. The discovery of artemisinin, a sesquiterpene lactone produced by Artemisia annua, as a pharmaceutical for the treatment of malaria promoted an interest in the discovery of new compounds from plants with antiprotozoal activity, especially those to treat against Leishmania parasites (Chan-Bacab & Peña-Rodrigues 2001). Leishmaniasis is a protozoan tropical disease that can manifest as a single cutaneous ulceration or can become a progressive and fatal disease (Singh & Sivakumar 2004). Besides the limited and toxic chemotherapeutic arsenal that is composed mainly of the pentavalent antimonials, amphotericin B (AmB) and pentamidine (PMD), the relapses due to resistant parasites demonstrate the urgent need for new drug candidates (Balaña-Fouce et al. 1998).

In recent decades, an increased incidence of fungal infections has been observed as a consequence of the growing number of immunocompromised patients, particularly those with AIDS or those taking anticancer drugs (Rahalinson et al. 1994). The expanding pool of high-risk patients susceptible to the opportunistic pathogen indicated that the yeast genus Candida is the most important invasive agent (Sobel 2006). Amb represents the main therapy for systemic fungal infections, but its use is highly limited as a result of renal toxicity and several other adverse effects (Zachino 2001). In some cases, azole-drugs, particularly fluconazole (FCZ), represent a less toxic alternative therapy agent although azole-resistant strains have been reported. Brazilian flora has been an astonishing source of biologically active compounds. Amazon forest plants have been showing promising results against Candida spp. (Carneiro et al. 2008). In this work, we have evaluated the antileishmanial and antifungal activities of 16 methanolic plant extracts from the BAF. Plants were selected from those that have traditional uses against cutaneous and infectious diseases such as ulcers, leishmaniasis, skin lesions, diarrhea, malaria, and inflammations, and those that have antimicrobial properties.

 

MATERIALS AND METHODS

Plant material - The plants were collected at Instituto Plantarum de Estudos da Flora, Brazil, in 2006. The vouchers of the species studied are deposited in the Herbarium Plantarum (HPL) and include the following: Plectranthus amboinicus (Lour.) Spreng. (voucher: H Lorenzi 3.411), Aristolochia cymbifera Mart. & Zucc. (voucher: H Lorenzi 2.115), Plectranthus barbatus Andrews (voucher: H Lorenzi 771), Lippia alba (Mill.) N.E. Br (voucher: H Lorenzi 1.713), Hydrocotyle bonariensis Lam. (voucher: H Lorenzi 3.516), Plectranthus neochilus Schlechter (voucher: H Lorenzi 1.441), Justicia pectoralis var. stenophylla Leon. (voucher: H Lorenzi 1.702), Herreria salsaparilha Mart. (voucher: H Lorenzi 3.391), Mentha X piperita L. (voucher: H Lorenzi 1.364), Eleutherine bulbosa (Mill.) Urb. (voucher: H Lorenzi 778), Baccharis trimera (Less.) DC. (voucher: H Lorenzi 432), Calamintha adscendens Willk. & Lang. (voucher: H Lorenzi 1701), Albizia inundata (Mart.) Barneby & J.W. Grimes (voucher: H Lorenzi 1692), Bauhinia forficata Link. (voucher: A Amaral Jr. 390), Cymbopogon citratus (DC.) Stapf. (voucher: H Lorenzi 3.462), and Plectranthus grandis (Cramer) R.H. Willemse (voucher: H Lorenzi 3.465).

Extract preparation - The dried (air incubator 55°C) and powered leaves (100 g) from plants were extracted with methanol (3 x 200 ml) through a maceration process for 48 h/ 25°C. The crude extracts were obtained after evaporation under reduced pressure at 45ºC in a rotating evaporator. All extracts were kept at 4°C until used in biological assays.

Parasites- Leishmania (L.) chagasi (strain MHOM/BR/1972/LD) promastigotes were maintained in M-199 medium supplemented with 10% calf serum and 2% human male urine (Singh et al. 2000) at 24°C. L. amazonensis (strain PH8) was maintained in RPMI-1640 medium, supplemented with 20% calf serum at 24°C.

Determination of the 50% effective concentration (EC50) - Promastigotes were used for all experiments, and the stationary phase was reached at 2-3 x 107 parasites/ml. The antileishmanial activity against promastigotes was determined using 96-well microplates (Sartorelli et al. 2007). Briefly, 1 x 106 promastigotes dissolved in methanol and diluted in M-199 medium (L. chagasi) or RPMI-1640 (L. amazonensis) medium were seeded with different extracts in the concentration range of 3.9 to 500 µg/ml. PMD was used as the standard drug. Plates were incubated at 24°C for 48 h and the viability of parasites was assayed colorimetrically by the mitochondrial oxidation of MTT (3-[4,5-dimethylthiazol-2-y1]-2,5diphenyl-tetrazolium bromide) (Tada et al. 1986). The optical density was detected at 570 nm with a MULTISKAN MS microplate reader and correlated with the number of living promastigotes, which were adequately standardized for each plate. Control wells without any sample were established as 100% viable. Methanol was incubated with parasites at 0.5%/well and used as an internal control.

Antifungal activity assay - In order to evaluate the susceptibility of the FCZ-resistant yeast Candida krusei (ATCC 6528) and the azole-susceptible Candida parapsilosis (ATCC 22019) to the extracts obtained, we developed an antimicrobial in vitro study. Candida spp. were maintained in RPMI 1640 medium (Sigma Chemicals, USA) without sodium bicarbonate, supplemented with L-glutamine (Gibco, Invitrogen) and 2% glucose at pH 7.0. Two-fold dilutions of extract in concentrations ranging from 3.90 to 500 µg/ml were dissolved in RPMI 1640 in 96-well microplates. Starting inoculum suspensions were obtained by the spectrophotometric method of inoculum preparation adjusted to 1-5 x 106cfu/ml and then diluted in test medium to 1-5 x 105cfu/ml. A 100 µl yeast inoculum was added to each well and the plates were incubated for 24 h and 48 h at 35°C in a humid atmosphere. After agitation, plates were read spectrophotometrically with an automatic plate reader (MULTISKAN FLOW, Sweden) at 492 nm. Amb was used as the standard drug. The MTT method was also used to assay the viability of yeasts in order to evaluate the fungicidal activity of C. krusei (Hjertstedt et al. 1998). Methanol was incubated with yeasts at 0.5%/well and was used as an internal control.

Statistical analysis - Data represent the mean ± SD of duplicate samples from two independent assays. The EC50 values were calculated using sigmoid dose-response curves in Graph Pad Prism 3.0 software, and the 95% confidence intervals were included in parentheses.

 

RESULTS AND DISCUSSION

In our search for plants with antileishmanial and antifungal activities, we tested methanolic crude extracts of native plants from the BAF. Our methodology was focused on plants traditionally used in Brazil and other countries for their anti-inflammatory and antimicrobial properties, as well as their specific antileishmanial and antifungal activities. Table I describes the 16 selected plants and their use in folk medicine. Many nonspecific symptoms such as fever, intestinal colic, and skin illness could be frequently described as microbial or parasitic in origin. Thus, many traditionally used plants may have an effect on a nonspecific target, thus contributing only as an anti-inflammatory. Laboratory in vitro testing models using cultured parasites, bacteria, or fungi, could be a useful and very important tool to confirm a specific activity, as well as an ethnopharmacological use.

As show in Fig. 1, five crude extracts (P. amboinicus, A. cymbifera, P. barbatus, L. alba and H. bonariensis) killed 100% of L. chagasi promastigotes at their highest concentration (500 µg/ml). L. chagasi is the etiologic agent of Visceral Leishmaniasis in Brazil, a systemic disease that kills 100% of untreated patients (Rath et al. 2003). From these five active extracts, four presented significant EC50 values in the concentration range of 45 to 89 µg/ml (Table II), showing very promising possibilities for future fractionation. These data suggest that very active compounds might be present in the crude extracts, and these may be a pool of a dozen independent secondary metabolites. Further isolation of the active compound could result in an individual prototype substance probably with a small EC50 value and better parasite selectivity. Based on 95% confidence intervals, P. barbatus and L. alba presented similar EC50 values against parasites, as did P. amboinicus and P. barbatus. To our knowledge, this data corroborates, for the first time, the ethnopharmacological use of P. amboinicus for Leishmaniasis, having an EC50 value of 45.14 µg/ml. This species has been used in the treatment of skin ulcerations caused by Leishmania spp. among the rural population of the cocoa-producing coastal area in the state of Bahia, Brazil (Franca et al. 1996,) and has also been used to treat inflammatory disorders (Lorenzi & Matos 2002).

 

 

A. cymbifera, a member of the Aristolochiaceae family, has been widely used to treat fevers, which are a very common and unspecific symptom (Lorenzi & Matos 2002). In our assays, it has presented a high antileishmanial activity with an EC50 of 89.17 µg/ml (Table II), and it has a fungicidal activity with an EC50 of 119.7 µg/ml (Table III). To date, only antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa by A. cymbifera has been described in the literature (De Barros Machado et al. 2005). Our results expand the scientific knowledge by demonstrating promising activity against human pathogenic fungi and a protozoan parasite. The traditional uses of P. barbatus in Kenya for the treatment of infectious and inflammatory diseases (Matu & van Staden 2003) have led our research to the finding of an antileishmanial activity with a very promising EC50 value of 54.46 µg/ml against L. chagasi (Table II). Despite the cellular similarity among Leishmania species, no activity was found against L. amazonensis, one of the etiologic agents of Cutaneous Leishmaniasis (CL) in Brazil. CL affects most poor communities all over the world. In some circumstances, the parasites migrate to mucosal areas, dramatically destroying the local tissue and contributing to a progressive and disfiguring inflammatory skin disease.

 

 

From the 16 plant extracts studied against Leishmaniasis, five presented activity against L. chagasi, and three distinct extracts showed an activity against L. amazonensis. P. grandis showed a marked antileishmanial activity (EC50 of 188.0 µg/ml) (Fig. 2), corroborating the ethnophamacological use of this genus against skin illness, infection, and respiratory problems (Lukhoba et al. 2006). C. citratus has been traditionally used to treat diarrhea and fever and it led our research to the discovery of an antiparasitic activity with an EC50 of 113.1 µg/ml (Table II). The medicinal use of B. trimera against the worldwide protozoan parasitic disease, malaria, as well as intestinal worms (Lorenzi & Matos 2002), resulted in our finding of an antileishmanial activity with an EC50 of 374.3 µg/ml. We also determined fungicidal activity against the FCZ-resistant C. krusei with an EC50 of 133.1 µg/ml (Fig. 3).

 

 

 

 

The traditional use of L. alba to treat bacterial respiratory infections (Caceres et al. 1991), as well as intestinal colic (Matos 1996), guided our research in the finding of new extracts against intracellular protozoan parasites and pathogenic yeasts. Our data confirmed the potent activity of L. alba, which was included among the four most active extracts against L. chagasi (EC50 of 62.67 µg/ml). Our work reports for the first time a fungicidal anti-Candida activity with an EC50 of 105.9 µg/ml. This data corroborates the anti-Candida activity of the essential oil obtained from leaves of L. alba (Duarte et al. 2005). In the search for plants used to treat skin illness, we studied the methanolic extract of H. bonariensis, which has been used in folk medicine to treat psoriasis, an inflammatory skin disorder (Natarajan & Paily 1973). We found activity against L. amazonensis (EC50 of 302.5 µg/ml), as well as L. chagasi promastigotes (EC50 of 235.0 µg/ml) in which 100% of parasites were killed at the highest concentration (500 µg/ml) (Fig. 1). Despite the elevated EC50 values when compared to other active plant extracts, H. bonariensis also presented a marked antifungal activity (EC50 of 31.63 µg/ml) (Table III) against the FCZ-resistant strain, Candida krusei. H. bonariensis has a fungicidal, rather than fungistatic activity, as demonstrated by the lack the oxidative mitochondrial activity assayed with MTT (data not shown). Candidiasis are fungal infections that commonly affect the human skin. Recently, due to HIV and other immunocompromised patients, candidiasis have been largely reported as a challenging invasive systemic infection. Many plants have been used in folk medicine to treat fungal infections (Lorenzi & Matos 2002). Besides the use of H. bonariensis in traditional medicine against skin illness, which could be frequently caused by pathogenic fungi, our data extend this knowledge to novel antiparasitic and antifungal activities.

Some other extracts showed a marked inhibition of the FCZ-resistant yeast, C. krusei. No antifungal activity of any tested extracts was found against the azole-susceptible C. parapsilosis. Besides the investigation of the growth inhibition at 492 nm (Clancy & Nguyen 1997), we extended our assays in order to demonstrate the potential killing effect of these plants, as most of the current drugs used in clinical therapy are fungistatic, rather than fungicidal, compounds. This characteristic limits the use of such agents by HIV-patients, because its prophylactic use should be limited to selected high-risk patients to limit the risk of the emergence of azole-resistant strains (Charlier et al. 2006).

Among the most effective tested extracts, P. neochilus demonstrated, for the first time in the literature, a marked a marked EC50 value of 20.51 µg/ml, killing 100% of yeast (C. krusei) at the highest concentration (Fig. 3). P. neochilus have also been traditionally used to treat skin and infective problems (Wellsow et al. 2006). Species of genus Albizia are used in African traditional medicine to treat microbial infection (Runyoro et al. 2006). In addition, species of Calamintha are traditionally used as antimicrobial agents in popular medicine (Panizzi et al. 1993). Despite the lack of antileishmanial activity detected for A. inundata and C. adscendens, our results show an antifungal activity against Candida albicans and a fungicidal activity with an EC50 value of 148.5 µg/ml at 492 nm and 196.3 µg/ml by MTT- 570 nm (data not shown) for A. Inundata. The anti-Candida activity of A. inundata has been recently described and our results corroborate those found in literature for the genus Albizia (Runyoro et al. 2006).

Despite the broad traditional use of J. pectoralis var. s. stenophylla (Chariandy et al. 1999, Leal et al. 2000), H. salsaparilha (Rodrigues & De Carvalho 2001), Mentha X piperita (McKay & Blumberg 2006), and E. bulbosa (Villegas et al. 1997) in closely related diseases, such as skin illnesses, our assays demonstrated neither antileishmanial nor anti-Candida activities. Other pathogenic species from the same genus could be susceptible to these same extracts. Continuous investigations must be done in order to find novel potent molecules against these important human diseases. Furthermore, Leishmaniasis is a tropical neglected disease and investments for the search of novel drugs are urgent. The use of ethnopharmacological information could significantly slow the time and cost of the research, allowing the study of new drug prototypes for drug design studies.

 

ACKNOWLEDGEMENTS

To Instituto Plantarum de Estudos da Flora for plant collections.

 

REFERENCES

Balaña-Fouce R, Reguera R.M, Cubría C, Ordóñez D 1998. The pharmacology of Leishmaniasis. Gen Pharmacol 30: 435-443.

Betoni JE, Mantovani RP, Barbosa LN, Di Stasi LC, Fernandes Junior A 2006. Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Mem Inst Oswaldo Cruz 101: 387-390.

Caceres A, Alvarez AV, Ovando AE, Samayoa BE 1991. Plants used in Guatemala for the treatment of respiratory diseases. Screening of 68 plants against gram-positive bacteria. J Ethnopharmacol 31: 193-208.

Carneiro ALB, Teixeira MFS, Oliveira VA, Fernandes OCC, Cauper GSB, Pohlit AM 2008. Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity. Mem Inst Oswaldo Cruz 103: 31-38.

Clancy CJ, Nguyen H 1997. Comparision of photometric method with standardized methods of antifungal susceptibility testing of yeasts. J Clin Microbiol 35: 2878-2882.

Chan-Bacab MJ, Peña-Rodriguez LM 2001. Plant natural products with leishamanicidal activity. Nat Prod Rep 18: 674-688.

Chariandy CM, Seaforth CE, Phelps RH, Pollard GV, Khambay BP 1999. Screening of medicinal plants from Trinidad and Tobago for antimicrobial and insecticidal properties. J Ethnopharmacol 64: 265-270.

Charlier C, Hart E, Lefort A, Ribaud P, Dromer F, Denning DW, Lortholary O 2006. Fluconazole for the management of invasive candidiasis: where do we stand after 15 years? J Antimicrob Chemother 57: 384-410.

Di Stasi LC, Oliveira GP, Carvalhaes MA, Queiroz M Jr, Tien OS, Kakinami SH, Reis MS 2002. Medicinal plants popularly used in the Brazilian Tropical Atlantic Forest. Fitoterapia 73: 69-91.

De Barros Machado T, Leal IC, Kuster RM, Amaral AC, Kokis V, de Silva MG, dos Santos KR 2005. Brazilian phytopharmaceuticals-evaluation against hospital bacteria. Phytother Res 19: 519-525.

Duarte-Almeida JM, Negri G, Salatino A 2004. Volatile oils in leaves of Bauhinia (Fabaceae-Caesalpinioidae). Biochem Syst Ecol 32: 747-753.

Duarte MC, Figueira GM, Sartoratto A, Rehder VL, Delarmelina C 2005. Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol 97: 305-311.

Franca F, Lago EL, Marsden PD 1996. Plants used in the treatment of leishmanial ulcers due to Leishmania (Viannia) braziliensis in an endemic area of Bahia, Brazil. Rev Soc Bras Med Trop 29: 229-2321.

Gene RM, Cartana C, Adzet T, Marin E, Parella T, Canigueral S 1996. Anti-inflammatory and analgesic activity of Baccharis trimera: identification of its active constituents. Planta Med 62: 232-235.

Hjertstedt J, Hahn BL, Kos WL, Sohnle PG 1998. Comparison of fungal viability assays using Candida albicans yeast cells undergoing prolonged incubation in the absence of nutrients. Mycoses 41: 487-492.

Leal LK, Ferreira AA, Bezerra GA, Matos FJ, Viana GS 2000. Antinociceptive, anti-inflammatory and bronchodilator activities of Brazilian medicinal plants containing coumarin: a comparative study. J Ethnopharmacol 70: 151-159.

Lorenzi H, Matos FJA 2002. Plantas medicinais no Brasil: nativas e exóticas cultivadas. 1st ed., Instituto Plantarum, Nova Odessa, 511 pp.

Lukhoba CW, Simmonds MS, Paton, AJ 2006. Plectranthus: a review of ethnobotanical uses. J Ethnopharmacol 103: 1-24.

Matos FJA 1996. As ervas cidreiras do Nordeste do Brasil- Estudo de três quimiotipos de Lippia alba (Mill.) N.E. Brown (Verbenaceae). Parte I- Farmacognosia. Rev Bras Farmacogn 77: 65-67.

Matu EN, Van Staden J 2003. Antibacterial and anti-inflammatory activities of some plants used for medicinal purposes in Kenya. J Ethnopharmacol 87: 35-41.

McKay DL, Blumberg JB 2006. A review of the bioactivity and potential health benefits of peppermint tea (Mentha X piperita L.). Phytother Res 20: 619-633.

Natarajan S, Paily PP 1973. Effect of topical Hydrocotyle asiatica in psoriasis. Indian J Dermatol 18: 82-85.

Panizzi L, Flamini G, Cioni PL, Morelli I 1993. Composition and antimicrobial properties of essential oils of four Mediterranean Lamiaceae. J Ethnopharmacol 39: 167-170.

Rahalison L, Hamburger M, Monod M, Frenk E, Hostettmann K 1994. Antifungal tests in phytochemical investigations: Comparison of bioautographic methods using phytopathogenic and human pathogenic fungi. Planta Med 60: 41-44.

Rath S, Trivelin LA, Imbrunito TR, Tomazela DM, de Jesús MN, Marzal PC, Andrade Junior HF, Tempone AG 2003. Antimonials employed in the treatment of Leishmaniasis: the state of the art. Quim Nova 26: 550-555.

Rodrigues VEG, de Carvalho DA 2001. Levantamento etnobotânico de plantas medicinais no domínio do cerrado na região do alto Rio Grande - Minas Gerais. Rev Bras Cienc Avic 25: 102-123.

Runyoro DK, Matee MI, Ngassapa OD, Joseph CC, Mbwambo ZH 2006. Screening of Tanzanian medicinal plants for anti-Candida activity. BMC Complement Altern Med 30: 6-11.

Sartorelli P, Andrade SP, Melhem MS, Prado FO, Tempone AG 2007. Isolation of antileishmanial sterol from the fruits of Cassia fistula using bioguided fractionation. Phytother Res 21: 644-647.

Singh S, Mohapatra DP, Sivakumar R 2000. Successful replacement of fetal calf serum with human urine for in vitro culture of Leishmania donovani. J Commun Dis 32: 289-294.

Singh S, Sivakumar R 2004. Challenges and new discoveries in the treatment of leishmaniasis. J Infect Chemother 10: 307-315.

Sobel JD 2006. The Emergence of non-albicans Candida species as causes of invasive candidiasis and candidemia. Curr Infect Dis Rep 8: 427-433.

Tada H, Shiho O, Kuroshima K 1986. An improved colorimetric assay for interleukin 2. J Immunol Methods 93: 157-165.

Tempone AG, Borborema SE, de Andrade HF Jr, de Amorim Gualda NC, Yogi A, Carvalho CS, Bachiega D, Lupo FN, Bonotto SV, Fischer DC 2005. Antiprotozoal activity of Brazilian plant extracts from isoquinoline alkaloid-producing families. Phytomedicine 12: 382-390.

Villegas LF, Fernandez ID, Maldonado H, Torres R, Zavaleta A, Vaisberg AJ, Hammond GB 1997. Evaluation of the wound-healing activity of selected traditional medicinal plants from Peru. J Ethnopharmacol 55: 193-200.

Wellsow J, Grayer RJ, Veitch NC, Kokubun T, Lelli R, Kite GC, Simmonds MS 2006. Insect-antifeedant and antibacterial activity of diterpenoids from species of Plectranthus. Phytochemistry 67: 1818-1825.

Zacchino S 2001. Estratégias para a descoberta de novos agentes antifúngicos. In: RA Yunes, JB Calixto, Plantas medicinais sob a ótica da química medicinal moderna, Argos Editora Universitária, Chapecó, p. 435-473.

Received 23 January 2008
Accepted 8 July 2008
Financial support: Fapesp (01/05023-1), Instituto Adolfo Lutz

* Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it. ; This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

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