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J. Agric. Food Chem. 2007, 55, 1356−1363
Identification of New Flavonoid Glycosides and Flavonoid Profiles To Characterize Rocket Leafy Salads (Eruca vesicaria and Diplotaxis tenuifolia) ASCENSIOÄ N MARTIÄNEZ-SAÄ NCHEZ, RAFAEL LLORACH, MARIÄA I. GIL, FEDERICO FERRERES*
AND
Research Group on Quality, Safety and Bioactivity of Plant Foods, Food Science and Technology Department, CEBAS-CSIC, P.O. Box 164, Espinardo, Murcia E-30100, Spain
“Rocket” is a collective name used to term some species within the Eruca and Diplotaxis genera, whose leaves are characterized by a more or less pungent taste. Different approaches have been carried out to differentiate both genera that have similar leaf morphologies. Following our research in flavonoid profiling of the Brassicaceae family using high-performance liquid chromatography/ ultraviolet-diode array detection/electrospray ionization mass spectroemtry, we have investigated Eruca vesicaria and Diplotaxis tenuifolia leaf samples as new ingredients of fresh salads. The MS/ MS study allowed the identification of new naturally occurring quercetin mono- and diacyl-tri-Oglucosides and the elucidation of the flavonoid glycosylation and acylation patterns. Important differences between flavonoid profiles of E. vesicaria and D. tenuifolia were observed. E. vesicaria contained kaempferol derivatives as principal compounds whereas D. tenuifolia instead accumulated quercetin derivatives. The exhaustive study of the profiling of these species could help further studies concerning the bioavailability of these flavonoids for epidemiological or clinical intervention studies because these species have considerable potential as healthy leafy salads because of the bioactive phytochemicals. KEYWORDS: Eruca vesicaria; Diplotaxis tenuifolia; Brassicaceae; crucifers; fresh-cut; HPLC/MS; polyphenols; vegetables
INTRODUCTION
In the past few years, an important increase in the cultivation of wild plant species has been carried out for the commercial production of traditional Mediterranean green salads. “Rocket” is a collective name used to term some species within Brassicaceae whose leaves are characterized by a more or less pungent taste. Although different species are referred under the common name of rocket, the main ones are those belonging to Eruca Miller and Diplotaxis DC. genera. The main species used for human consumption include Eruca Vesicaria (L.) Cav. and Diplotaxis tenuifolia (L.) DC (1). From the botanical point of view, these plants belong to tribe Brassiceae subtribe Brassicinae, which includes Brassica, Coincya, Erucastrum, Hirschfeldia, Raphanus, Sinapidendron, Sinapis, and Trachystoma. The Brassicinae subtribe is defined primarily on the basis of elongated (siliquose) dehiscent fruits, the presence of median nectaries, and usually seeded beaks (2). It is remarkable that the leaf morphology has not been used to differentiate these species. Both genera are used as new ingredients for green leafy salads. At optimum commercial maturity (young leaves), both * To whom correspondence should be addressed. Tel: +34-968-396324. Fax: +34-968-396213. E-mail:
[email protected].
plants are very similar, and this is the cause of misidentification. Different approaches have been carried out to find new tools to identify and differentiate both genera (1). In fact, flavonoids have been used as chemotaxonomy markers for the Diplotaxis genera (3). In the past few years, different studies on metabolite profiling, mainly based on polyphenols, of members of the Brassicaceae family have been used to differentiate between crucifer species (4-7). Thus, the presence of isorhamnetin glucoside allows the differentiation of the Brassica oleracea from Brassica rapa (6). Recently, Bennett et al. have investigated the ontogenic profiling of secondary metabolites of four rocket species including E. satiVa and D. tenuifolia (4). They have identified and quantified the major phytochemicals including glucosinolates and also phenolics and flavonoids of different rocket tissues (seeds, roots, leaves, and flowers). They observed that all rocket tissues, except roots, contained significant levels of polyglycosilated flavonoids and separated them into simple and acylated mono-, di-, and triglycosides (4). In addition, Weckerle et al. (8) identified three quercetin 3,3′,4′-tri-O-β-Dglucopyranosides in E. satiVa leaves as the main flavonoids. These previous reports on leaf flavonoids (4, 8) did not show the complete characterization of the rocket flavonoids. The complex flavonoid pattern found in rocket species has limited
10.1021/jf063474b CCC: $37.00 © 2007 American Chemical Society Published on Web 01/19/2007
Flavonoid Profiling of Rocket Species the interest in the studies of bioavailability and biological effects of phenolics and flavonoids of fresh rocket leaves. Among the biologically active compounds in fresh cut salads, polyphenols are an important group (both qualitative and quantitative) as they are closely related with health-promoting activities such as prevention of cardiovascular diseases and cancer (9). Metabolite profiling (or metabolome analysis) represents a tool in studies of plant taxonomy, physiology, and phytochemistry. The use of high-performance liquid chromatography/ultravioletdiode array detection/electrospray ionization mass spectroemtry (HPLC/UV-DAD/ESI-MSn) for metabolite profiling of flavonoids has been recently discussed in several papers (10, 11). The main objective of this work was to characterize two rocket species, E. Vesicaria (L.) Cav. and D. tenuifolia (L.) DC., based on the flavonoid profiling as a tool for qualitative and quantitative analysis of healthy salad phytochemicals. MATERIALS AND METHODS Plant Material. D. tenuifolia (wild rocket) and E. Vesicaria (salad rocket) were cultivated in fields located in La Aparecida (Cartagena, Murcia, Spain) and supplied by Agrolito S.L. (Torre Pacheco, Murcia, Spain). At optimum commercial maturity, uniform size plants, free from decay and/or mechanical damage, were selected at random and immediately transported to the laboratory (30 km). Approximately, three replicates of 80 g of each were weighed, frozen at -70 °C, and freezedried. The dried samples were ground into a fine powder for further analysis. The botanical identification was carried out at the Department of Botany (University of Murcia, Spain), and a voucher specimen was deposited in the University Herbarium. Wild rocket and salad rocket corresponded to the botanical names of D. tenuifolia (L.) DC. and E. Vesicaria (L.) Cav. respectively, but the last one was also accepted as Eruca satiVa Miller. Phenolics Extraction. The freeze-dried sample (0.5 g) was homogenized with 5 mL of methanol-water (v:v) using an Ultra-Turrax homogenizer. The extract was then centrifuged (10500g) for 5 min, and the supernatant was filtered through a 0.45 µm membrane filter (Millex-HV 13MM, Millipore, Bedford, MA). Isolation of Desacylated Flavonoid Glycosides. In order to study the structures of the desacylated flavonoid glycosides, a previous saponification step of the raw extracts was done before the isolation of the main compounds. A freeze-dried sample (20 g) was macerated overnight at room temperature with water (200 mL). The resulting extract was centrifuged (10500g) for 5 min, and the supernatant was collected and filtered. The supernatant was saponificated with 4 N NaOH (200 mL) for 16 h. Finally, the extract was acidified with concentrated HCl until pH 1-2. The acidified extract was mixed with the nonionic polymeric resin Amberlite XAD-2 (enough to fill a column of 3 cm × 50 cm) using a magnetic stirrer for 4 h to allow flavonoid adsorption on the resin particles as described by Llorach et al. (12). The resin particles were then poured into the glass column and washed with distilled water (1500 mL). Flavonoids were eluted with methanol (300 mL), and an aliquot was analyzed by HPLC/UV-DAD/ESI-MSn. The extract was evaporated to dryness under reduced pressure (50 °C) and redissolved in water. The resulting extract was fractionated by the semipreparative HPLC on a Spherisorb ODS-2 column (250 mm × 10 mm, 5 µm particle size) (Tecnokroma, Barcelona, Spain), with different isocratic mixtures of methanol and water. The purity of the isolated compounds was verified by HPLC/UV-DAD/ESI-MSn. Finally, these compounds were freeze-dried and stored. Acid Hydrolysis. Total acid hydrolysis was carried out by adding 1 mL of 4 N HCl to 1 mL of the hydroalcoholic phenolic extract, and this solution was kept in a stoppered test tube, incubated for 30 min at 85 °C, and directly analyzed by HPLC/UV-DAD/ESI-MSn. Mild acid hydrolysis was carried out by adding 5 mL of 2 N HCl to the isolated compounds. Kaempferol-3-sophoroside (
