ANTI-OXIDANTS FROM BACCHARIS SPP. 307 Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 16: 307–314 (2005) PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 16, 307–314 (2005) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.826 Copyright © 2005 John Wiley & Sons, Ltd. Received 5 March 2004 Revised 25 June 2004 Accepted 29 June 2004 Isolation and On-line Identification of Anti- oxidant Compounds from Three Baccharis Species by HPLC-UV-MS/MS with Post-column Derivatisation Cláudia A. Simões-Pires, 1 Emerson F. Queiroz, 2 Amélia T. Henriques 1 and Kurt Hostettmann 2 * 1 Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre-RS, Brazil 2 Laboratory of Pharmacognosy and Phytochemistry, Geneva-Lausanne School of Pharmacy, University of Geneva, Quai Ernest-Ansermet 30, CH1211 Geneva 4, Switzerland The aqueous extracts of aerial parts of Baccharis trimera (Less.) DC., B. crispa Spreng. and B. usterii Heering (Asteraceae) displayed significant radical scavenging activity in a diphenylpicrylhydrazole (DPPH)/ TLC assay. In order to rapidly identify the active principles, the crude extracts were analysed by HPLC-UV, and an HPLC- micro-fractionation of the extract of B. usterii was performed. Six quinic acids derivatives (1–6) were isolated from B. usterii by MPLC. The fractions were monitored by DPPH/TLC assay and a series of radical-scavenging quinic acid derivatives could be identified. The comparison of the HPLC profiles of the extracts of B. usterii, B. trimera and B. crispa was performed. In order to obtain complementary on-line structural information for all peaks of interest, HPLC-MS/MS together with HPLC-UV involving post-column addition of UV shift reagents was undertaken on the crude extract. The interpretation of these data permitted the on-line identification of known compounds, some of which are reported for the first time in this plant. Copyright © 2005 John Wiley & Sons, Ltd. Keywords: HPLC-MS; HPLC-UV/PAD; post-column derivatisation; DPPH assay; bioautographic screening; quinic acid derivatives; Baccharis trimera; Baccharis crispa; Baccharis usterii; Asteraceae. * Correspondence to: K. Hostettmann, Laboratory of Pharmacognosy and Phytochemistry, Geneva-Lausanne School of Pharmacy, University of Geneva, Quai Ernest-Ansermet 30, CH1211 Geneva 4, Switzerland. Email: kurt.hostettmann@pharm.unige.ch Contract/grant sponsor: Swiss National Science Foundation; Contract/grant number: 2000-063670.00. INTRODUCTION The genus Baccharis belongs to the sub-tribe Asterinae, which has the largest number of species (more than 400) within the tribe Astereae of the family Asteraceae (Bremer, 1984). The present study refers to the species Baccharis trimera (Less.) DC., B. crispa Spreng. and B. usterii Heering, which all belong to the section Caulopterae DC (Giuliano, 2001) and are popularly known in Brazil as Carquejas. In spite of the large number of publications concern- ing species of Baccharis, there remains some controversy concerning the correct nomenclature and synonyms of the members, especially those in the section Caulopterae. In the absence of flowers, the botanical differentiation of these species is difficult to achieve, and this presents a particular problem with respect to B. crispa and B. trimera, both of which are used as medicinal plants often without correct identification (Gianello et al., 2000). Furthermore, some pharmacological and phyto- chemical studies claimed to have been conducted on B. genistelloides Persoon, collected in Bolivia (Suttisri et al., 1994; Abad et al., 1999; Gonzales et al., 2000), Brazil (Bauer et al., 1978; Soicke and Leng-Peschlow, 1987; Melo et al., 2001) and Chile (Daily et al., 1984), are more likely to refer to B. trimera since the identification of B. genistelloides was based on a specimen collected in Ecuador as Conyza genistelloides Lam., and this species occurs only in Colombia, Ecuador and Peru at altitudes greater than 3000 m (Cuatrecasas, 1967). Such misunder- standings of the nomenclature probably arise from descriptions in Flora Brasiliensis (Baker, 1882), in which six varieties of B. genistelloides Persoon were described, including Baccharis genistelloides var trimera Baker. Carquejas have been extensively used in Latin America for their digestive, diuretic (Correa, 1984) and anti-inflammatory properties (Di Stasi et al., 2002), and for their protective effect against liver disorders (Toursarkissian, 1980). Several phytochemical studies have been conducted on species belonging to the section Caulopterae, particularly with respect to the flavon- oids and their anti-inflammatory, analgesic and anti- hepatotoxic activities (Soicke and Leng-Peschlow, 1987; Gene et al., 1992). Nevertheless, there are no reports of the HPLC analysis of the aqueous extracts of these plants, even though this is the main form in which these plants are employed in folk medicine. In a preliminary biological screening, the aqueous extracts from the aerial parts of B. trimera, B. crispa and B. usterii were inactive in anti-fungal and anti- bacterial assays. However, the same extracts showed radical scavenging activity in a diphenylpicrylhydrazole (DPPH) assay (Cuendet et al., 1997; Cavin et al., 1998). Activity-guided isolation was undertaken for B. usterii using HPLC-UV micro-fractionation and medium pres- sure liquid chromatography MPLC, and six caffeoyl quinic acid derivatives could be identified.