Comparative cytotoxicity of Astragalus pehuenches and Astragalus illini extracts: Fractionation and evidence of toxicity beyond swainsonine.

Astragalus species are major causes of livestock poisoning, traditionally attributed to swainsonine (SW). This study compared Astragalus pehuenches and Astragalus illini to determine whether cytotoxicity differences could be explained solely by SW or might involve other constituents. Alkaloid-enriched extracts (AEEs) were evaluated using phytochemical analyses and SW-normalised cytotoxicity assays, followed by A. illini extract fractionation. SW was quantified by HPLC-MS/MS, selenium and nitro compounds were assessed, and saponin-equivalent content was estimated. Although A. pehuenches contained more SW than A. illini (4.70 vs. 1.49 μg SW/mg AEE), the A. illini extract caused a greater reduction in C6 glioma viability under SW-normalised exposure. CC₅₀ values were 802.79 and 447.74 μM SW equivalents for A. pehuenches and A. illini AEEs, respectively. Neither extract induced detectable LDH release. Qualitative AO/EB and lectin staining showed changes in acidic vesicular staining patterns and intracellular glycoconjugate staining patterns consistent with SW-associated cellular alterations. However, because equivalent SW exposure required different total extract concentrations, their intrinsic cytotoxic potencies could not be directly compared. Fractionation of the A. illini extract showed that cytotoxicity did not parallel SW concentration, while some of the most cytotoxic fractions contained abundant saponin-related compounds. Non-SW metabolites, potentially including saponins, may be associated with the observed cytotoxicity; however, their specific contributions remain unconfirmed. These findings support integrated phytochemical and biological analyses for investigating cytotoxicity associated with constituents other than SW in Astragalus extracts. Future studies should structurally characterise candidate compounds and test their individual effects to establish their causal roles in the observed cytotoxicity.