title: Antileishmanial activity and trypanothione reductase effects of terpenes from the Amazonian species Croton cajucara Benth (Euphorbiaceae).
abstract: BACKGROUND
  Leishmaniasis comprises several infectious diseases caused by protozoa parasites of Leishmania genus. In recent years, there has been a growing interest in the therapeutic use of natural products to treat parasitic diseases. Among them Croton cajucara Benth. (Euphorbiaceae) is a plant found in the Amazonian region with a history of safe use in folk medicine.
  
  
  OBJECTIVE
  The purpose of this study was to investigate the effects of clerodane diterpenes, trans-dehydrocrotonin (DCTN), trans-crotonin (CTN) and acetylaleuritolic acid (AAA) obtained from powdered bark of C. cajucara against promastigotes, axenic and intracellular amastigotes of Leishmania amazonensis. Furthermore, the effects of DCTN and CTN on the trypanotiona reductase enzyme were also investigated. The extraction of the terpenes was carried out as previously reported (Maciel et al., 1998; 2003).
  
  
  METHODS
  The effect of the isolated compounds (DCTN, CTN and AAA) from the bark of C. cajucara was assessed in vitro against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis by counting of remaining parasites in a Neubauer chamber in comparison to pentamidine used as standard drug. The action of natural products on trypanothione reductase was assessed using soluble protein fraction of promastigotes. The assays were performed by incubation with HEPES, EDTA, NADPH and trypanothione disulfide to quantify the NAPH consumption by TryR.
  
  
  RESULTS
  The results showed very high efficacy, especially of the diterpene DCTN, against promastigotes (IC50 = 6.30 ± 0.06 µg/ml) and axenic amastigotes (IC50 = 19.98 ± 0.05 µg/ml) of L. amazonenesis. The cytotoxic effect of the best active natural product was evaluated on mouse peritoneal infected macrophages (IC50 = 0.47 ± 0.03 µg/ml in 24 h of culture), and the treatment revealed that DCTN never reaches toxic concentrations while reducing the infection and, most importantly, with no toxicity (>100 µg/ml with 0% of macrophage kill) when compared to pentamidine (37.5 µg/ml with 100% of macrophage kill). Furthermore, all of the natural products assayed on the trypanothione reductase enzyme inhibited the enzyme activity compared to the control.
  
  
  CONCLUSIONS
  Clerodane diterpenes from C. cajucara showed promising in vitro antileishmanial effects against L. amazonensis, specially the DCTN with no macrophage toxicity up to the assayed concentration. In addition, the action on trypanothione reductase enzyme revealed a possible mechanism of action.
authors.affiliation: Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brazil; Biomanguinhos, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Gerson S
authors.initials: GS
authors.lastname: Lima
authors.affiliation: Laboratório de Bioquímica de Tripanossomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Denise B
authors.initials: DB
authors.lastname: Castro-Pinto
authors.affiliation: Laboratório de Bioquímica de Tripanossomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Gerzia C
authors.initials: GC
authors.lastname: Machado
authors.affiliation: Universidade Potiguar Laureate International Universities, Programa de Pós-graduação em Biotecnologia, Campus Salgado Filho, Natal-RN, Brazil; Universidade Federal do Rio Grande do Norte, Instituto de Química, Campus Lagoa Nova, Natal-RN, Brazil.
authors.forename: Maria A M
authors.initials: MA
authors.lastname: Maciel
authors.affiliation: Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brazil. Electronic address: echevarr@ufrrj.br.
authors.forename: Aurea
authors.initials: A
authors.lastname: Echevarria
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D029802:Croton / Q000737:chemistry*; D004224:Diterpenes; D045785:Diterpenes, Clerodane / Q000302:isolation & purification / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007891:Leishmania / Q000187:drug effects*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000469:parasitology; D008519:Medicine, Traditional; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D024301:Plant Bark / Q000737:chemistry; D014315:Triterpenes
chemical_list: D000981:Antiprotozoal Agents; D004224:Diterpenes; D045785:Diterpenes, Clerodane; D014315:Triterpenes; C103060:dehydrocrotonin; C403585:trans-crotonin; C014947:acetylaleuritolic acid; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase
keywords: Antiprotozoal activity; Croton cajucara; Leishmania amazonensis; Macrophage; Terpenes; trans-Deydrocrotonin