Elucidation of the flavonoid and furocoumarin composition and radical-scavenging activity of green and ripe chinotto (Citrus myrtifolia Raf.) fruit tissues, leaves and seeds Davide Barreca, Ersilia Bellocco, Corrado Caristi, Ugo Leuzzi, Giuseppe Gattuso ⇑ Dipartimento di Chimica Organica e Biologica, Università di Messina, Viale F. Stagno d’Alcontres 31, 98166, Messina, Italy article info Article history: Received 1 April 2011 Received in revised form 4 May 2011 Accepted 24 May 2011 Available online 30 May 2011 Keywords: Citrus myrtifolia Raf. HPLC–DAD–ESI-MS/MS Flavonoids Furocoumarins Antioxidant activity abstract Citrus myrtifolia Raf. (chinotto) flavedo, albedo and carpel membranes from green and ripe fruits, as well as seeds and leaves, were investigated for the first time for their flavonoid and furocoumarin composi- tion. Twenty-three unique compounds distributed in the different fruit/plant parts were identified and quantified. All samples analysed contained flavanone O-glycosides, flavone C- and O-glycosides and fur- ocoumarins; flavedos and albedos also contained significant amounts of polymethoxyflavones. Flavedo and albedo extracts were found to be richest in flavonoids and furocoumarins, containing up to 1.95 g/ kg fresh weight. Flavedo, albedo and carpel membranes from ripe fruits were found to be richer than the corresponding tissues from unripe fruits. The remarkable radical-scavenging activity of all the extracts was tested by DPPH  , ABTS + and FRAP methods, revealing that (i) they were particularly efficient in quenching ABTS + radical cations (up to 9.8 mM Trolox equivalents), and (ii) flavedo and albedo extracts, on average, showed the highest antioxidant capacity. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Fresh citrus fruit and citrus-derived products have always been essential components of the human diet. Traditional folklore worldwide has always associated high citrus intake with general well-being and disease prevention. Indeed, a large number of sci- entific investigations, over the past decades, suggested that such a connection exists, linking individual compounds (or classes thereof), present in different parts of citrus fruits, to specific bene- ficial effects (Patil, Jayaprakasha, Murthy, & Vikram, 2009). Among the many phytochemicals, phenolics, and particularly flavonoids, have attracted the attention of researchers for the protection they provide against some types of carcinogenesis and cardiovascular diseases (Benavente-García & Castillo, 2008; Espín, García-Conesa, & Tomás-Barberán, 2007). As a consequence, considerable efforts have been devoted to the characterisation of the flavonoid fraction of the most common species, as well as that of many exotic Citrus taxons, and to the isolation of the most representative flavonoid derivatives (Berhow, Tisserat, Kanes, & Vandercook, 1998; Gattuso, Barreca, Gargiulli, Leuzzi, & Caristi, 2007a; Kawaii, Tomono, Katase, Ogawa, & Yano, 1999; Nogata et al., 2006). However, the flavonoid composition of many Citrus plants has not yet been unravelled. Citrus myrtifolia Raf., commonly known as chinotto or myrtle- leaved orange, is a species belonging to the Rutaceae family, sub- family Aurantioideae, which originates from a mutation of C. aur- antium (sour orange) (Tanaka, 1961). Its wide diffusion is due to its use as an ornamental plant (it is a bushy evergreen tree whose fruits stand on the tree for several months), although it is grown in Italy and Southern France for its applications in food industry. Con- centrated chinotto juice is a key ingredient in the production of soft drinks and liqueurs, whereas the whole fruit is used in the con- fectionery industry. However, despite its industrial applications, chinotto has enjoyed scarce attention. Very few papers in the liter- ature have dealt with the composition of the various parts of its fruit and leaves. In particular, four flavonoid glycosides (Camarda, Di Stefano, Del Bosco, & Schillaci, 2007; Cautela, Pirrello, Esposito, & Minasi, 2004) and ten carotenoids (Fanciullino et al., 2006) had been described for the juice, along with the composition of the essential oil fraction obtained from peel and leaves (Lota, de Rocca Serra, Jacquemond, Tomi, & Casanova, 2001). We recently reported on the flavonoid and furocoumarin distribution in the juice from green and ripe chinotto fruits (Barreca, Bellocco, Caristi, Leuzzi, & Gattuso, 2010a), identifying eleven derivatives and confirming, by comparing the flavonoid fingerprints (Robards, Li, Antolovich, & Boyd, 1997), its close taxonomical relations with C. aurantium L. (Barreca, Bellocco, Caristi, Leuzzi, & Gattuso, 2011a) and 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.130 Abbreviations: ABTS, 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); FRAP, ferric-reducing antioxidant power; DPPH, 2,2-diphenyl-1-picrylhydrazyl; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. ⇑ Corresponding author. Tel.: +39 0906765241; fax: +39 090392840. E-mail address: ggattuso@unime.it (G. Gattuso). Food Chemistry 129 (2011) 1504–1512 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem