Identification of cytotoxic constituents of Narthecium ossifragum using bioassay-guided fractionation.

Narthecium ossifragum, a member of the Liliaceae family, contains phytochemicals that have hepatotoxic and nephrotoxic activity in several ruminant species. 3-Methoxyfuran-2(5H)-one has previously been isolated as the principal nephrotoxin, and its toxicity has been confirmed in vivo. However, previous investigations into the nephrotoxicity of N. ossifragum both in vivo and in vitro indicate that other phytochemical factors might contribute to the nephrotoxicity of the plant. In this study, the cytotoxicity in renal tubular cells (LLC-PK1) was measured using an aqueous extract from the plant and fractions from chromatographic separation to identify the cytotoxic constituents of the extract. In an iterative process two different groups of compounds were identified as the major cytotoxic principles in LLC-PK1 cells: steroidal saponins (primarily di- and trisaccharides of sarsasapogenin) and 5-hydroxy-4-methoxyfuran-2(5H)-one. Up to a concentration of 880 microg/mL (7.7 M) 3-methoxyfuran-2(5H)-one was not cytotoxic. The cytotoxicity of the saponins was abolished upon hydrolysis, indicating that the carbohydrate moiety of the molecule is a prerequisite for toxicity on the cellular level. The results of the present study have two important implications: first, the results question the direct involvement of 3-methoxyfuran-2(5H)-one in the nephrotoxicity of N. ossifragum; second, the findings should induce future investigations into the possible role of saponins in N. ossifragum-related nephrotoxicosis observed in ruminants that graze on this plant.