Leishmanicidal properties may reside in phytochemicals such as flavonoids, which are hence strong candidates for use in combination therapy against these infections. Flavonoids are abundant in fruits, vegetables, and saps of plants and are demonstrated to have anticarcinogenic, selleckchem antimicrobial, and antiparasitic activity [7, 8]. Flavonoid analogues derived from Consolida oliveriana exerted a significant effect on the in vitro growth of species of Leishmania spp. [9].In this work, the inhibitory effects of flavonoids from aerial parts of Delphinium staphisagria L. (Ranunculaceae) on the extracellular and intracellular stages of L. infantum and L. braziliensis were investigated. In addition, the cytotoxic effects of these compounds against a host cell line were assessed.

We also used 1H-NMR spectroscopic analysis to determine the nature and percentage of the excretion metabolites and to elucidate any inhibitory effect that the compounds have on the glycolytic pathway. Finally, the effects of the compounds on the ultrastructure were studied.2. Material and Methods2.1. Plant MaterialAerial parts of the Delphinium staphisagria were collected and processed as described previously D��az et al. [10]. Nine flavonoids (1�C9) were isolated, derivatized, and identified (Figure 1) [10].Figure 1Flavonoid compounds investigated.2.2. Parasite Strain and CultureL. infantum (MCAN/ES/2001/UCM-10) and L. braziliensis (MHOM/BR/1975/M2904) were cultivated in vitro in trypanosomes liquid medium (MTL) with 10% inactive fetal bovine serum and were kept in an air atmosphere at 28��C, in Roux flasks (Corning, USA) with a surface area of 75cm2, according to the methodology described by Gonz��lez et al.

[4].2.3. Cell Culture and Cytotoxicity TestsJ774.2 macrophages (ECACC number 91051511) were originally obtained from a tumour in a female BALB/c rat in 1968. The cytotoxicity test for macrophages was performed according to the methodology of Gonz��lez et al. [4]. After 72 hours of treatment, cell viability was determined by flow cytometry. Thus, 100��L/well of propidium iodide solution (100mg/mL) was added and incubated for 10min at 28��C in darkness. Afterwards, 100��L/well of fluorescein diacetate (100ng/mL) was added and incubated under the same conditions. Finally, the cells were recovered by centrifugation at 400g for 10min and the precipitate washed with phosphate buffered saline (PBS).

Flow cytometric analysis was performed with a FACSVantage flow cytometer (Becton Dickinson). The percentage viability was calculated in comparison with the control culture. The IC50 was calculated using linear regression analysis from the Kc values of the concentrations employed.2.4. In Vitro Activity Assay2.4.1. Promastigote Forms, Assay The compounds obtained were dissolved GSK-3 in the culture medium, at dosages of 100, 50, 25, 10, and 1��M.