Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by high-performance liquid chromatography: Novel ecdysteroids from S. pseudotites

Many species in the genus Silene (Caryophyllaceae) have previously been shown to contain ecdysteroids and this genus is recognised as a good source of novel ecdysteroid analogues. We have used ecdysteroid-specific radioimmunoassays and the microplate-based Drosophila melanogaster BII cell bioassay for ecdysteroid agonist and antagonist activities to identify further phytoecdysteroid-containing species in this genus. The main ecdysteroid components from 10 Silene species (S. antirrhina, S. chlorifolia, S. cretica, S. disticha, S. echinata, S. italica, S. portensis, S. pseudotites, S. radicosa, S. regia) were isolated and identified, mainly by normal-phase and reversed-phase high-performance liquid chromatography. The amount of each ecdysteroid was determined by comparing chromatogram peak areas with those for reference 20-hydroxyecdysone (20E) on reversed-phase HPLC. 20E is the most abundant ecdysteroid in each of the Silene extracts. Polypodine B, 2-deoxy-20-hydroxyecdysone and ecdysone are also common ecdysteroids in these Silene species, but the proportions of these ecdysteroids vary between the Silene species. HPLC proved to be a quick and effective way to screen Silene species, determine ecdysteroid profiles and, hence, identify extracts containing novel analogues. An extract of the aerial parts of S. pseudotites was found to contain several new ecdysteroids. These have been isolated and identified spectroscopically (by NMR and mass spectrometry) as 2-deoxyecdysone 22β-d-glucoside, 2-deoxy-20,26-dihydroxyecdysone and 2-deoxypolypodine B 3β-d-glucoside. Additionally, (5α-H)-2-deoxyintegristerone A (5α-2H 91%, 5α-1H 9%) was isolated as an artefact. This study contributes to the understanding of ecdysteroid distribution in Silene species and provides further information on the chemotaxonomic significance of ecdysteroids in Silene species.