Steroidal Glycosides with Antiproliferative Activities from Digitalis trojana Hasan Kirmizibekmez, 1 * Milena Masullo, 2 Michela Festa, 2 Anna Capasso 2 and Sonia Piacente 2 1 Department of Pharmacognosy, Faculty of Pharmacy, University of Yeditepe, 34755 Kayisdagi, Istanbul, Turkey 2 Department of Pharmacy, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy The phytochemical investigation of Digitalis trojana led to the isolation of two cardiac glycosides (1, 2), one pregnane glycoside (3), three furostanol type saponins (4–6), along with three cleroindicins (7–9), four phenylethanoid glycosides (10–13), two flavonoids (14, 15) and two phenolic acid derivatives (16, 17). The structure elucidation of the isolates was carried out by NMR experiments as well as ESI-MS. The cytotoxic activity of compounds 1–13 against a small panel of cancer cell lines, namely MCF-7, T98G, HT-29, PC-3, A375 and SH-SY5Y, was investigated. Compounds 1–6 showed antiproliferative activity against human breast MCF-7 and colon HT-29 cancer cell lines with IC 50 values ranging from 8.3 to 50 mM. In order to understand the mechanism involved in the cell death, the active compounds were tested as pro-apoptotic agents using propidium iodide staining by flow cytometry method. No significant increase was observed in the apoptosis of the MCF-7 and HT-29 cancer cells. Moreover, the effects of the active compounds on cell proliferation were assessed on the same cancer cell lines by cell cycle analysis of DNA content using flow cytometry. No significative changes were observed in the cell cycle of MCF-7, while significant changes in G 2 /M cell cycle phase of HT-29 cells were observed after treatment with digitalin (1), cariensoside (3) and 22-O-methylparvispinoside B (6) at 10 mM. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Digitalis trojana; cardiac glycosides; pregnane glycosides; furostanol saponins; antiproliferative activity; cytostatic activity. INTRODUCTION Natural products, particularly those derived from medicinal plants, have long been and continue to be a source of anticancer drugs (Harvey and Cree, 2010). It is reported that more than two thirds of the anticancer drugs approved between the 1940s and 2006 are either natural products or derivatives (Efferth, 2010). Many reports occur in literature on the antiproliferative activ- ity of natural products and their mechanism of action (Perrone et al., 2005; Argyropoulou et al., 2012). The genus Digitalis (Plantaginaceae) consists of about 36 species, nine of which grows wild in the flora of Turkey. Digitalis trojana IVAN, one of the nine species of the genus Digitalis (Plantaginaceae) growing wild in Turkey, is a biennial or perennial plant endemic to Ida Mountains (Kazdaglari), Turkey (Davis, 1978). The fox- glove genus (Digitalis) contains medicinally important species from which very important drugs, i.e. digoxin and digitoxin are extracted. In addition to cardiac glycosides, steroidal saponins, pregnane glycosides and phenylethanoid glycosides constitute the main com- pounds occurring in the chemical profile of the genus Digitalis (Kirmizibekmez et al., 2002, 2009a; Perrone et al., 2012). There is growing interest on the cytotoxic activities of cardiac glycosides. They have been shown to inhibit cell growth and angiogenesis and to induce apoptosis in multiple cancer cell lines (Khan et al., 2009). One of the basic mechanisms underlying the cytotoxic activity of these compounds is their ability to inhibit Na + /K + –ATPase activity (Piacente et al., 2009). Thus, cardiac glycosides are regarded as promising anticancer drug candidates. There are also several reports on the cytotoxic activities of steroidal saponins and pregnane glycosides (Perrone et al., 2012; Yokosuka et al., 2009; Liu et al., 2009). In our previous investigation on Digitalis ciliata, significant changes in G 2 /M cell cycle phase of MCF-7, A549 and HT-29 cell lines were observed after treatment with glucogitoroside (2), also reported in D. trojana, which induced an accumulation of cells in G 2 /M phase in MCF-7 cells at 10 mM (Perrone et al., 2012). Along with cardiac glycosides, steroidal saponins and pregnane glycosides are among the chemi- cal constituents of the genus Digitalis. As a part of our ongoing investigation on the chemistry and bioactivity of the genus Digitalis, we herein describe the isolation of secondary metabolites from the aerial parts of a Turkish endemic species D. trojana, as well as the evaluation of their antiproliferative activities. MATERIAL AND METHODS General experimental procedures. TLC analyses were carried out on silica gel 60 F 254 precoated plates (Merck, Darmstadt), detection by 1% vanillin/H 2 SO 4 . For medium-pressure liquid chromatographic (MPLC) separations, Combi Flash Companion (Isco), were used with Redi step columns packed with LiChroprep C 18 (130 g and 43 g, Teledyne Isco) or SiO 2 (4 g, 12 g Teledyne Isco). Silica gel 60 (0.063–0.200 mm; Merck, * Correspondence to: Dr. Hasan Kirmizibekmez, Department of Pharmacognosy, Faculty of Pharmacy, University of Yeditepe, 34755. Kayisdagi, Istanbul, Turkey. E-mail: hasankbekmez@yahoo.com PHYTOTHERAPY RESEARCH Phytother. Res. 28: 534–538 (2014) Published online 31 May 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5012 Copyright © 2013 John Wiley & Sons, Ltd. Received 13 November 2012 Revised 1 May 2013 Accepted 4 May 2013