Journal of Ethnopharmacology 94 (2004) 175–184 Investigation of Lepechinia graveolens for its antioxidant activity and phenolic composition Irene Parejo a , Elisabetta Caprai a , Jaume Bastida a , Francesc Viladomat a , Olga Jáuregui b , Carles Codina a,∗ a Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Catalonia, Spain b Serveis Cientificotècnics, Universitat de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Catalonia, Spain Received 26 January 2004; received in revised form 26 May 2004; accepted 30 May 2004 Abstract A bioguided separation of Lepechinia graveolens (Reg.) Epling. (Lamiaceae) for antioxidant activity was carried out. The ethyl acetate fraction (EAF) from the methanolic extract was chromatographed on Sephadex LH-20 affording five active fractions. The radical scavenging activity of each fraction, as well as that of the isolated compounds, was tested using three different methods. The major isolated antioxidant compounds were identified as luteolin-7-O-glucuronide, rosmarinic acid, and rosmarinic acid methyl ester by means of 1 H and 13 C NMR and mass spectrometry. Twenty other minor phenolic compounds were determined by liquid chromatography-negative electrospray ioniza- tion tandem mass spectrometry. They included hydroxycinnamic acid derivatives, flavonoids and phenolic diterpenes. The major phenolic compound was found to be rosmarinic acid, which was quantitatively determined by high-performance liquid chromatography (HPLC)-diode array detector (DAD). The content of rosmarinic acid was 2.8% (referred to dry weight), this amount being around 30% of the total phenolic content (TPH) of the crude extract. It can be concluded that the antioxidant activity of Lepechinia graveolens was mainly due to rosmarinic acid, which allows this plant species to be considered as a potential new source of this well known natural antioxidant. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Lepechinia graveolens; Antioxidant activity; Phenolic composition; Rosmarinic acid; Bioguided isolation; HPLC 1. Introduction Plants of the genus Lepechinia Willd. are mainly dis- tributed in South America and Mexico, and are commonly used in folk medicine as hot extracts and decoctions. Thus, for example, Lepechinia caulescens, Lepechinia chamaedry- oides, Lepechinia salviae and Lepechinia spicata are used as antidiabetics, emmenagogues, abortives and for uterine tumors (González and Silva, 1987; Román-Ramos et al., 1991; Alarcón-Aguilara et al., 1998; Rodr´ ıguez-López et al., 2003). Some of these plants are reported to contain different types of terpenoids and flavonoids (Jonathan et al., 1989; Bruno et al., 1991; Delgado et al., 1994; Velasco-Negueruela et al., 1994; Ahmed et al., 1996; Eggers et al., 1999). Several plants belonging to the family Lamiaceae, such as rosemary, oregano, sage, thyme and basil, are well known for ∗ Corresponding author. Tel.: +34 93 4024493; fax: +34 93 4029043. E-mail address: ccodina@farmacia.far.ub.es (C. Codina). their antioxidant activity and phenolic composition (Miura et al., 2002; Pizzale et al., 2002; Jayasinghe et al., 2003; Matsingou et al., 2003). The compounds responsible for the antioxidant activity were found to be mainly phenolic diter- penes, such as carnosic acid and carnosol, and other phe- nolic substances like rosmarinic acid and hydroxycinnamic acids (Malenˇ ci´ c et al., 2000; Exarchou et al., 2002). On the basis of these previous results and the antioxidant activity exhibited by the volatile fraction of Lepechinia schiedeana (Stashenko et al., 1999, 2002), antioxidant substances were also expected to be found in Lepechinia graveolens, a species from this plant family not hitherto studied from this point of view. According to ethnobotanical studies undertaken in Bo- livia, Lepechinia graveolens (white sage) is used in folk medicine as a tea infusion for the treatment of stomach ailments. Concerning its chemical composition, this plant species has been studied only for essential oils (De Riscala and Retamar, 1973). The purpose of this study was to evalu- ate the antioxidant activity of Lepechinia graveolens and to 0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.05.017