Bioassay-guided isolation of saponins from Apodytes dimidiata

In an in vitro screening programme for antiprotozoal activity, a crude extract of Apodytes dimidiata (Icacinaceae) showed promising activity against Leishmania infantum [1]. Because of the reported molluscicidal activity, the presence of saponins was suspected, and subsequently confirmed by phytochemical screening. The anti-leishmanial constituents were isolated by bioassay-guided isolation.

Dried and mixed leaves of Apodytes dimidiata were extracted with 80% methanol. The crude extract was extracted consecutively with petroleumether, dichloromethane and n-butanol (n-BuOH). The n-BuOH extract showed a high in vitro activity against Leishmania infantum (IC₅₀=0.4µg/mL) and was also active against Trypanosoma brucei (IC₅₀=1µg/mL). In order to isolate the active compounds, further fractionation was performed on the n-BuOH extract by silicagel column chromatography. Elution of the column started with EtOAc, and proceeded with mixtures of EtOAc and the organic phase of n-BuOH – HAc – H₂O 4:1:5. Fractions were evaluated by TLC sprayed with anisaldehyde – sulphuric acid, and combined according to their chromatographic pattern. Fractions 67–96 showed activity against Leishmania infantum and Trypanosoma brucei. The active fraction was separated by repeated semi-preparative HPLC (C18 Vydac Column 5µm(250×10mm)) using gradient elution (32 to 56% B in 45min (A=0.05% HAc; B=ACN + 0.05% HAc), 3 mL/min, 210 nm detection). The first fraction resulting from this separation was again separated by semi-preparative HPLC employing a modified gradient (32% to 34% B in 60min. (A=0.05% HAc; B=ACN + 0.05% HAc), 3 mL/min, 210 nm detection). Six saponins from the oleanane type were isolated and structure elucidation was performed using mass spectrometry and NMR spectroscopy.

References: [1] Maes, L. (2003) Ph.D. Thesis, Lead Identification and Development of a New Anti-leishmanial saponin PX-6518, isolated from the Vietnamese plant Maesa balansae, University of Antwerp, Belgium