1. Iron deficiency enhances bioactive phenolics in lemon juice Mellisho, C.D. a ; González-Barrio, R. b ; Ferreres, F. b ; Ortuño, M.F. a ; Conejero, W. a ; Torrecillas, A. a,c ; García-Mina. J.M. d ; Medina, S. b ; Gil-Izquierdo, A. b* a Department of Irrigation, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo (Murcia) Spain. b Research Group on Quality, Safety and Bioactivity of Plant Foods, Food Science and Technology Department, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo (Murcia) Spain. c Unidad Asociada al CSIC de Horticultura Sostenible en Zonas Aridas (UPCT-CEBAS), Paseo Alfonso XIII 48, E-30203 Cartagena (Murcia), Spain. d Agricultural Chemistry Department, University of Navarra, Fac Sci, Navarra, 31080, Spain and CIPAV (TAI, Roullier Group), Navarra 31160, Spain. *email author: angelgil@cebas.csic.esangelgil@cebas.csic.es Introduction Citrus fruit is one of the most important horticultural crops, with a worldwide agricultural production of over 100 millon metric tons per year [1]. Citrus juices and particularly lemon juice, show a high content of flavanoids (800-1500 mg L -1 ), especially flavanone and flavone glycosides and other phenolic compounds such as hydroxicinnamic acid and flavonols although at a lower concentration [1,2]. These compounds have been demonstrated by in vitro and in vivo assays to have beneficial properties for the human health, may contribute to the prevention of cardiovascular disease [3] and the administration of citrus juice was shown to limit tumour burden in animal models of carcinoma [4]. In the Mediterranean zone, soils have high calcium carbonated resulting lemon trees suffers Fe-deficiency that it results in yellowing of leaves and ultraestructural disorganization as well as in increases of organic acids and phenolics [5]. The goal of our study was to describe the phenolics status of lemon juice obtained in fruits harvested from lemon trees with different iron nutritional status. Three differents types of Fe chelates were used in the experiment. One synthetic chelate (Fe(III)-EDDHA) and two complexes derived from natural polymers of humic and lignine nature [6]. Material and methods Plant material and treatments. The experiments was performed with adult lemon tree (Citrus limon (L.) Burm. Fil, cv. Fino 95 grafted on Citrus macrophylla L. rootstock). The orchard is located in Fuente Librilla (Mula (Murcia) Spain). Four treatment were applied in order to achieve different Fe nutritional levels in lemon tress: - Treatment 0: Control. Receive any Fe compound. - Treatment 1: Fe(III)- EDDHA, 6% of chelated Fe. - Treatment 2: NC1 (natural complexes (humic)). 2,5 % - Treatment 3: NC2 (natural complexes (lignine)). 2,5 % SPAD and iron measurements in leaves. Chlorophyll content was estimated in the field (30 leaves per tree) using SPAD-502 chlorophyll Meter (Minolta Camera Co., Ltd., Osaka, Japan). Total and HCl-soluble iron were quantified by inductively coupled plas ma emission optical spectrometry (Iris Intrepid II, Thermo Electron Corporation, Franklin, USA). Lemon juice sample preparation. A domestic squeezer (Braun, Germany) was used to obtain the juice and were centrifuged at 12000 g for 5 min. 20 μL of filtered samples was injected HPLC for analysis. LC/PDAD/ESI-MS n qualitative analysis. (Agilent Technologies, Waldbrom, Germany). 250 x 4 mm, 5 μm, RP-18 LiChroCART column. Phase (A): H 2 O/ 1 % HCOOH, and (B): MeOH. Flow rate: 1 mL min -1 and injection volume: 20 μL. LC/PDAD quantitative analysis (Merck, Damstadt, Germany). Phase (A): H2O/ 5 % HCOOH, and (B): MeOH. Flow rate: 1 mL min -1 and injection volume: 30 μL. Results and discussion In the qualitative analysis of phenolics in lemon juice from Fe (III) deficient lemon trees with and without Fe (III) chelates treatment were found 17 compounds (Table 1, Figure 1). Table 1. R t ; UV, -MS, -MS2 [M-H] - data of phenolic compounds in lemon juice. Figure 1. Chromatogram of lemon juice phenolics recorder at 330 nm. Figure 2. Total phenolics in lemon juices (supernatant and hazel) of lemon fruits collected. The quantification of phenolics in lemon juice was carried out taking into account the toal sum of flavanones, flavones, flavonols and hydroxycinnamic acids. In this way, these group of phenolic were evaluated to follow their content in lemon juice from lemon collected from Fe deficient lemon trees with (Control, T0) or without Fe (III) chelates fertilisation (T1, T2, T3) (Figure 2). Conclusions 1. Phenolics content in lemon juice was higher in fruits from trees with ferric chlorosis than in lemons from Fe treated trees. 2. Iron deficiency in lemon trees can induce negative effects on fruit yield but not on lemon nutritional quality regarding phenolics in relation to the Fe (III) chelates treated lemon trees. 3. Diosmetin-6,8-di-C-glucoside can be selected as marker on correction of iron deficiency in lemon trees. References [1]. Gil-Izquierdo, A., Riquelme, M.T., Porras, I., Ferreres, F. (2004). Journal of Agricultural and Food Chemistry. 52, 324-331. [2]. Gil-Izquierdo, A., Gil, M.I., Tomás-Barberán, F.A., Ferreres, F. (2003). Journal of Agricultural and Food Chemistry. 51, 3024-3028. [3]. Wilcox, L.J., Borradaile, N.M., de Dreu, L.E., Huff, M.W. (2001). Journal of Lipid Research. 42, 725-734. [4]. So, F.V., Guthrie, N., Chambers, A.F., Moussa, M., Carroll, K.K. (1996). Nutrition and Cancer. 26, 167- 181. [5]. Briat, J.F., Fobis-Loisy, I., Grignon, N., Lobréaux, S., Pascal, N., et al. (1995). Biology of the Cell. 84, 69-91. [6]. García-Mina, J.M., Zamarreño, A.M., Bacaicoa, E., Baigorri, R., Fuentes, M. (2009). US2008221314-A1; EP1997793-A2. Acknowledgements Authors are grateful to CONSOLIDER-INGENIO 2010 “Fun-C-Food”. S. Medina and C. D. Mellisho are holders of a JAE-CSIC and FPI fellowship, respectively. M.F. Ortuño is holder of a “Ramón y Cajal” contract.