In vitro trypanocidal activity of Smallanthus sonchifolius. Identification of active sesquiterpene lactones by bioassay-guided fractionation

ULLOA J, FRANK FM, CAZORLA SI, MALCHIODI E, MARTINO V, CATALÁN C, MUSCHIETTI L.

Congreso; XXI Congresso Italo-Latinoamericano di Etnomedicina XXI Congreso Italo-Latinoamericano de etnomedicina.XXI Italo-Latinamerican Congress of Etnomedicinesilae; 2012

IntroductionChagas disease or American Trypanosomiasis is an endemic disease in Latin America caused by the parasite Trypanosoma cruzi.  It affects nearly ten million people worldwide with an annual estimated number of 10,000 death cases. The therapy is based on benznidazole and nifurtimox, two drugs developed forty years ago that present limited efficacy, multiple side effects and are not well tolerated. Therefore, the discovery of new and safer drugs is an urgent need. We previously reported the trypanocidal activity of psilostachyin and psilostachyin C isolated from Ambrosia species. (Sülsen et al, 2008, 2010, 2011). This study was designed to evaluate the potential of Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson (Asteraceae) as antitrypanocidal leads. This perennial herb, from the Andean region of South America, is commonly known as ?yacón? and traditionally used as an anti-diabetic herbal tea.        Method.Air-dried and ground leaves (clone LIEY 97-1; 500 g) were extracted by soaking in dichloromethane for 30 min. The procedure was repeated twice and the filtrates combined and taken to dryness under vacuum to obtain the organic extract (OE). Twenty grams of OE was further chromatographed on a silica gel 60 column eluted with hexane, hexane/ethyl acetate (1:1), ethyl acetate, and methanol to obtain 10 final factions (F1A?F5B). OE and each fraction were tested for trypanocidal activity on T. cruzi epimastigotes by a [3H] thymidine uptake assay at 100 and 10 µg/ml and evaluated at 72 h. Benznidazole (5 to 20 µM; Roche) was used as positive control. Further purification of fractions F2B and F3A by chromatographic techniques, led to the isolation of three compounds that were identified by spectroscopic techniques (1HNMR, EI-MS and IR). The trypanocidal activity of pure compounds was evaluated as above and IC50 values estimated.   Results.At 10 μg/ml, Smallanthus sonchifolius OE inhibited the growth of T. cruzi epimastigotes by 95.14±0.51% at 72 h of incubation. Amongst the tested fractions, obtained by bioassay-guided fractionation of OE, F2B and F3A showed the highest in vitro inhibitory activity: 92.67±0.51% and 83.87±1.14 % respectively at the lower concentration tested. From these fractions, three sesquiterpene lactones were isolated and identified as enhydrin, uvedalin and polymatin B. Enhydrin (IC50= 0.43 μg/ml) and uvedalin (IC50= 0.58 μg/ml) showed a marked in vitro trypanocidal activity.  Conclusions.According to these results enhydrin and uvedalin may be considered promising antitrypanosomal lead molecules. Further investigations will involve the evaluation of in vitro activity against trypomastigotes and intracellular amastigotes and the determination of their cytotoxicity and selectivity.