Introduction
In the majority of countries that cope with high malaria infection, medicinal plants contribute significantly to primary health care due to cultural traditions, and lack of access and affordability of pharmaceuticals. In addition, natural products are potential sources of new and selective agents for the treatment of important tropical diseases caused by protozoans (Karioti et al., 2009; Froelich et al., 2007; Luize et al., 2005). For example, Zanthoxylum chiloperone var. angustifolium Engl. (syn. Fagara chiloperone Engl. Ex Chod. & Hassl.), Rutaceae, is a dioic tree indigenous to the central and southern continent of South America, which is called "tembetary hu" and "mamicão" (Spichiger & Stutz de Ortega, 1987; Tabanez et al., 2005). A decoction of Z. chiloperone root and stem bark has been used in traditional medicine to treat malaria and for its emmenagogue and antirheumatic properties (Ferreira et al., 2007; Milliken, 1997). Recent studies have shown that the crude extract of the stem bark and leaves have activity against Trypanosoma cruzi (Ferreira et al., 2011; Ferreira et al., 2002) and antifungal activity in vitro (Thouvenel et al., 2003). Further investigations demonstrate that canthinone type alkaloids, canthin-6-one and 5-methoxycanthin-6-one, are antifungal (Soriano-Agatόn et al., 2005; Thouvenel et al., 2003) and effective in vivo against Leishmania amazonensis and Trypanosoma cruzi (Ferreira et al., 2011; Ferreira et al., 2007; Ferreira et al., 2002). Canthin-6-one has been suggested to be an inexpensive and safe treatment for use in long-term oral treatment as well as a good candidate against drug resistant strains of T. cruzi.
Other compounds were isolated from species of Zanthoxylum including the pyranocoumarin avicennol and alkylamides, such as the sanshools (Jang et al., 2008; Soriano-Agatόn et al., 2005; Xiong et al., 1997; Yang, 2008). To date, there is a paucity of research on the biological activity of avicennol, which has been previously identified in Z. elephantiasis Macfad (Gray et al., 1977). Recent work with avicennol reports an induction of UDP-glucuronosyltransferases (UGT), specifically UGT1A1, which detoxifies xenobiotics (Chlouchi et al., 2007).
The alkylamides are a class of compounds which are well known in Asia, North America and Europe due to the tingling sensation on the tongue after ingestion of Sichuan pepper and Echinacea purpurea (Spelman et al., 2010). While many Zanthoxylum spp. are known to contain alkylamides, to the best of our knowledge there are no reports of the occurrence or identity of alkylamides in Z. chiloperone. Notably, these compounds have also been shown to be antiparastic (Lozano et al., 1984), as well as insecticidal (Jacobson, 1948). Recent reports have shown in vitro and in vivo anti-plasmodial activity of alkylamides identical to, or similar to, the alkylamides occurring in other Zanthoxylum spp. (Spelman et al., 2010).
One of the mechanisms of action of antimalarial drugs is based on their interference with the heme detoxication pathway (Weissbuch & Leiserowitz, 2008). As a toxic, non-peptidic by-product of hemoglobin digestion, free heme must be sequestrated in the digestive vacuole. The biomineralization process involves the heme dimerization and the subsequent aggregation of dimers into hemozoin, an insoluble pigment. Antimalarial drugs are known to form adducts with heme, thus impairing heme polymerization and leading to the death of parasites at the trophozoite stage. Artemisinin for instance has been showed to form a covalent complex between heme and the alkylating species generated by the endoperoxide ring opening through Fe(II)-mediated catalysis (Robert et al., 2002). The free heme in its commercial form (hemin or hematin) is known to be suited for testing heme polymerization inhibition (Tekwani & Walker, 2005). Most of the protocols are based on spectrophotometry or radioactivity monitoring. (Garavito et al., 2007; Kurosawa et al., 2000; Rush et al., 2009). A novel method based on mass spectrometry can be used as an alternative method to detect heme-drug adducts (Figadère et al. 2010). It has been used to detect adducts between Fe(III)-heme and quinine, artemisinin and its derivatives. (Bilia et al., 2002; Pashynska et al., 2004). Our laboratory used mass spectrometry and in-source collision-induced dissociation (CID) to assess the stability of the adducts. In our method, heme is incubated with the compound and analyzed by mass spectrometry using an increasing fragmentor voltage to dissociate the formed adduct. A relative assessment of the adduct stability can be obtained and compared to one reference compound, like quinine.
Thus far there has been limited exploration of Zanthoxylum chiloperones chemistry and biological activity. The experiments continued herein describe the isolation of two alkaloids and a pyranocoumarin, the identification of an alkylamide and the anti-malarial activity of the crude extracts and the isolated compounds. In addition, using mass spectrometry and in-source CID to study the formation and stability of the heme adducts with avicennol and the canthinone alkaloids, we propose a possible basis for the antiplasmodial activity of these Zanthoxylum compounds.