Structure elucidation and hepatotoxicity evaluation against HepG2 human cells of neo-clerodane diterpenes from Teucrium polium L. Antonio Fiorentino, Brigida D’Abrosca ⇑ , Severina Pacifico, Monica Scognamiglio, Grazia D’Angelo, Marialuisa Gallicchio, Angela Chambery, Pietro Monaco Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, via Vivaldi 43, I-81100 Caserta, Italy article info Article history: Received 16 June 2011 Received in revised form 29 June 2011 Accepted 6 July 2011 Available online 22 August 2011 Keywords: Teucrium polium L. Lamiaceae neo-Clerodane Teupolins Spectroscopic analysis HepG2 human hepatoblastoma cells Hepatotoxicity abstract Seven neo-clerodanes (teupolins VI–XII) and eleven known compounds were isolated and characterized from leaf extracts of Teucrium polium L., a medicinal plant used in traditional and herbal medicine for its hypolipidemic, hypoglycemic, antioxidant and antiproliferative properties. The structures of these com- pounds were elucidated by 1D ( 1 H, 13 C and DEPT) and 2D (COSY, TOCSY, HSQC, HMBC) NMR experiments and by mass spectrometry analysis. The complete stereostructure of each compound was defined with a NOESY experiment. Because the overexploitation of herbal remedies containing T. polium extracts has resulted in several cases of hepatitis, the hepatotoxic activity of pure metabolites against the human hepatoblastoma cancer cell line HepG2 was assessed by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide) test. All of the compounds showed low toxicity values at the highest concentration tested (200 lM). Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Teucrium polium L., commonly known as golden germander, is a steno-Mediterranean perennial shrub species belonging to the Lamiaceae family. This medicinal plant and other species belonging to the genus Teucrium have been used for over two thousand years in traditional and herbal medicine. T. polium has been utilized in Mediterranean countries to treat many diseases such as abdominal pain, indigestion and diabetes (Al-Khalil, 1995; Said et al., 2002; Ljubuncic et al., 2006). Several studies on aqueous and alcoholic extracts of golden germander revealed its antimicrobial (Darab- pour et al., 2010), hypoglycemic (Esmaeili and Yazdanparast, 2004), antinociceptive (Baluchnejadmojarad et al., 2005), antioxi- dant (Hasani et al., 2007; Yazdanparast and Ardestani, 2009), and antiproliferative (Kandouz et al., 2010) properties. The use of herbal remedies containing T. polium extracts with- out any scientific guidance has resulted in several cases of hepato- toxicity (Mattei et al., 1995; Mazokopakis et al., 2004; Savvidou et al., 2007; Abu Sitta et al., 2009). Other Teucrium species includ- ing other plants widely used in folk medicine have been demon- strated to be responsible for liver injuries (Stickel et al., 2000). In France, all preparations containing germander as an herbal medi- cine were prohibited (CFR 172-510, 1992). An analogous decision was made in Italy (Ministero della Sanità, G.U. 181, 1996), and Belgium prohibited the use of Teucrium chamaedrys and T. polium or their preparations in foodstuffs (Arrêté Royale du 29 Août 1997). The hepatotoxicity of Teucrium has been ascribed to its neo-clerodane diterpenes, including teucrin A and teuchamaedryn A. The furan ring of these diterpenoids is oxidized by CYP3A4 to produce a reactive epoxide (Zhou et al., 2004, 2007). In 2003, the Directorate-General for Health and Consumer Protection of the Scientific Committee on Food of the European Commission estab- lished the daily intakes of Teucrin A and ruled that germander preparations containing Teucrin A should not be used other than in flavored alcoholic beverages and that more data on the current intakes of Teucrin A via alcoholic beverages were needed. In the study of new bioactive secondary metabolites from plants of Mediterranean area, we carried out the phytochemical investi- gation of several species of Mediterranean macchia (Fiorentino et al., 2008; Pacifico et al., 2009, 2010). We studied T. polium and recently reported that abeo-abietane diterpenes from golden ger- mander provide protective effects against oxidative stress (Fioren- tino et al., 2010). In the current work, we describe the structural elucidation of eighteen neo-clerodane diterpenes isolated from the aerial por- tions of T. polium. We also evaluated the cytotoxicity of all furane diterpenes toward HepG2 human hepatoblastoma cells. HepG2 cells provide a useful model to study the function of CYP3A4 and its role in liver drug metabolism (Elizondo and Medina-Dıaz, 2003). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.07.006 ⇑ Corresponding author. Tel.: +39 0823 274564; fax: +39 0823 274571. E-mail address: brigida.dabrosca@unina2.it (B. D’Abrosca). Phytochemistry 72 (2011) 2037–2044 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem