Saponin-enriched extracts were prepared following the protocol of
Gurfinkel & Rao (2001) with slight modifications. 10 g of milled powder
was extracted in methanol (40 mL) for 4 h at 50 °C with gentle agitation
in a water bath. Every 30 min, the suspensions were thoroughly mixed.
The saponin-containing methanol extracts were filtered and the sample
residue was washed with additional methanol, obtaining a final volume
of 50 mL. Afterwards, a clean-up procedure was performed to separate
the saponins from accompanying compounds (e.g. sugar, proteins). To
this end, a 10 mL aliquot of the methanol extract was mixed in a 1:2
ratio with 0.4 M ammonium sulfate and the solution was left on a shaker
for at least 20 h. The resulting precipitate containing the non-saponin
constituents was removed by centrifugation (3.225 ×g, 30 min) at
room temperature. The precipitate was washed with a 1:2 (v/v) mixture
of methanol and 0.4 M ammonium sulfate, centrifuged and the supernatants
were combined (total volume 20 mL)