Elicited Teucrium chamaedrys cell cultures produce high amounts of teucrioside, but not the hepatotoxic neo-clerodane diterpenoids Fabiana Antognoni a,⇑ , Carmelina Iannello a , Manuela Mandrone a , Monica Scognamiglio b , Antonio Fiorentino b , Pier Paolo Giovannini c , Ferruccio Poli a a Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Irnerio 42, 40126 Bologna, Italy b Dipartimento di Scienze della Vita, Laboratorio di Fitochimica, Seconda Università degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy c Dipartimento di Chimica, Laboratorio di Chimica Organica, Università degli Studi di Ferrara, Via Luigi Borsari 46, 44100 Ferrara, Italy article info Article history: Received 10 February 2012 Received in revised form 22 May 2012 Available online 5 July 2012 Keywords: Teucrium chamaedrys Lamiaceae NMR analysis Cell cultures Elicitation neo-Clerodane diterpenoids Phenylethanoid glycosides abstract Teucrium chamaedrys, one of the most common and investigated species of the genus Teucrium, has been used for centuries in traditional medicine for many purposes. Its phytochemical components comprise, among others, phenylethanoid glycosides (PGs) and neo-clerodane diterpenoids. Several reports have demonstrated a wide range of beneficial biological and pharmacological activities of the phenylethanoid components, while the diterpenes were shown to be strongly hepatotoxic. In this work, in vitro cultures were established from leaf explants of T. chamaedrys. Both solid (callus) and liquid (cell suspension) cultures maintained the capacity to produce PGs, with teucrioside (TS) representing the most abundant one. Cell suspensions had a lower TS content than that found in leaf extracts, but higher than that of calli. An NMR-based metabolomics approach was used to compare the product profile of intact plants vs. cell suspension cultures, and results showed that neo-clerodane diterpenes, present in the intact plant, were not detected in cell cultures. Several elicitors were supplied to cell cultures with the aim of increasing TS production, and elicitation was tested at different growth phases and by exposing cells for different peri- ods. Methyl jasmonate and fungal mycelia from Trichoderma viridae and Fusarium moniliforme were able to significantly increase TS production if supplied at the early-exponential growth phase for 24 h. Based on the proposed link between proline and the phenylpropanoid pathways, proline accumulation in cell cultures was followed throughout a 14-day culture period, showing that it strictly reflected that of TS production. Moreover, exogenously supplied proline, and its analogue hydroxyproline, turned out to be very effective in increasing teucrioside production. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The genus Teucrium, belonging to the Lamiaceae family, is rep- resented by about 100 species, distributed throughout the world, but mainly abounding in the northern temperate and subtropical regions of the Eastern Hemisphere. Various species of this genus have been used for over 2000 years in traditional medicine for their diuretic, diaphoretic, tonic, antispasmodic and cholagogic proper- ties (Ulubelen et al., 2000); more recently, the interest towards Teucrium species has increased, due to a pronounced anticancer activity demonstrated for plant extracts and isolated compounds of these plants (Stankovic et al., 2011). Moreover, extracts from some species were shown to potentiate the cytotoxic and proapop- totic effects of anticancer drugs vincristine, vinblastine and doxo- rubicin, against a panel of cancer cell lines (Rajabalian, 2008). One of the most common and highly investigated species in the genus is Teucrium chamaedrys, commonly called germander. It is native to Europe, and used in the treatment of digestive and respi- ratory disorders, abscesses, gout (Stankovic et al., 2010), and, externally, as an astringent infusion on the gums and in the treat- ment of wounds (Chiej, 1984). Hydroalcoholic extracts of this plant are currently used as approved substances in the preparation of fla- vored wines, bitters and liqueurs. Phytochemical constituents comprise diterpenes (in particular neo-clerodane diterpenoids), monoterpenes and other classes of compounds including saponins, glycosides (iridoids and phenylethanoids), and flavonoids (Bedir et al., 2003; Pacifico et al., 2009). neo-Clerodane diterpenoids con- stitute a large group of natural toxic compounds, and in particular two of them, teucrin A and teuchamaedryn A, are considered to be responsible for several cases of hepatotoxicity associated to the use of this plant in human beings (Gori et al., 2011). Phenylethanoid glycosides (PGs) are the main phenolic components in Teucrium species, and several reports have demonstrated the wide range of biological and pharmacological activities of these compounds 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.05.027 ⇑ Corresponding author. Tel.: +39 0512091291; fax: +39 051242576. E-mail address: fabiana.antognoni@unibo.it (F. Antognoni). Phytochemistry 81 (2012) 50–59 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem