Antiprotozoal activity against Entamoeba histolytica of furocoumarins isolated from Ruta chalepensis

Amoebiasis caused by Entamoeba histolytica, a protozoan of the family Endomoebidae is associated with high morbidity and mortality and is therefore considered as the third parasitosis of medical importance after malaria and schistosomiasis [1, 2]. Currently metronidazole is the therapeutic drug of choice for the treatment of amoebiasis, but is experiencing drug resistance by E. histolytica, resulting in the need for increased doses to overcome the infection and thus causing unpleasant side effects. For this reason new, more effective and safer antiprotozoal agents are urgently required [3].

We reported shortly the antiamoebic activity in vitro of the methanolic extract of Ruta chalepensis (Rutaceae) [4] and we are now reporting the bioguided isolation of compounds with amebicide activity from this plant. Partition of the methanolic extract of Ruta chalepensis by extraction with n-hexane led to a residue with good activity against E. histolytica (93.0% growth inhibition). Chromatography of this hexane residue over a silica gel column led to the isolation of chalepensin (1), whereas the following work up of the methanol residue by partition between methanol and ethyl acetate followed by chromatography of the EtOAc residue (84.8% growth inhibition) over a silica gel column afforded chalepin (2) and rutamarin (3) as well as the isoquinoline alkaloid graveoline (4). Identification of the isolated compounds was based on spectroscopic/spectrometric analyses and comparison with literature data.

These compounds were tested for antiprotozoal activity against E. histolytica tropohzoites using in vitro tests. Vials were incubated for 72h. The inhibition percentage was estimated as the number of dead cells compared with the untreated controls. IC₅₀ of these compounds was determined by using a Probit analysis with a 95% confidence level.

The isolated furocoumarins (Figure 1) from R. chalepensis displayed more than 90% growth inhibition against E. histolytica at a concentration of 150 µg/mL, with IC₅₀ values ranging from 6.54 to 38.71 µg/mL, far less effective than metronidazole (IC₅₀ 0.205 µg/mL), but these IC₅₀ values are suitable as selection criterion for further investigation of this plant as source of potential antiprotozoal agents. Graveoline did not show amebicide activity.

Fig. 1: Structure and IC₅₀ against E. histolytica of compounds isolated from Ruta chalepensis

Acknowledgements: The authors would like to thank the DAAD (Germany), who financed a stay of R.Q.L. at the University of Göttingen, and to CONACYT (Mexico) for doctoral fellowship awarded to A.F.B.R.

Keywords: neglected diseases, amoebiasis, Mexican medicinal plants, antiprotozoal agents.

References:

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