Phytochemical screening of Pulsatilla species and investigation of their biological activities

Abstract

The present study aimed to identify biologically active secondary metabolites from the rare plant species, <em>Pulsatilla patens</em> subsp. <em>patens</em> and the cultivated <em>P. vulgaris</em> subsp. <em>vulgaris</em>. Chromatographic fractionation of the ethanolic extract of the roots of <em>P. patens</em> subsp. <em>patens</em> resulted in the isolation of two oleanane-type glycosides identified as hederagenin 3-<em>O</em>-β-d-glucopyranoside (2.7 mg) and hederagenin 3-<em>O</em>-β-d-galactopyranosyl-(1→2)-β-d-glucopyranoside (3.3 mg, patensin). HPLC analysis of the methanolic extract of the crude root of <em>P. patens</em> subsp. <em>patens</em> and <em>P. vulgaris</em> subsp. <em>vulgaris</em> revealed the presence of <em>Pulsatilla</em> saponin D (hederagenin 3-<em>O</em>-α-l-rhamnopyranosyl(1→2)-[β-d-glucopyranosyl(1→4)]-α-l-arabinopyranoside). Chromatographic analysis using GC-MS of the silylated methanolic extracts from the leaves and roots of these species identified the presence of carboxylic acids, such as benzoic, caffeic, malic, and succinic acids. The extracts from <em>Pulsatilla</em> species were tested for their antifungal, antimicrobial, and antimalarial activities, and cytotoxicity to mammalian cell lines. Both <em>P. patens</em> subsp. <em>patens</em> and <em>P. vulgaris</em> subsp. <em>vulgaris</em> were active against the fungus <em>Candida glabrata</em> with the half-maximal inhibitory concentration (IC₅₀) values of 9.37 µg/mL and 11 µg/mL, respectively. The IC₅₀ values for cytotoxicity evaluation were in the range of 32–38 μg/mL for <em>P. patens</em> subsp. <em>patens</em> and 35–57 μg/mL for <em>P. vulgaris</em> subsp. <em>vulgaris</em> for each cell line, indicating general cytotoxic activity throughout the panel of evaluated cancer and noncancer cells.