ࡱ> TVSXy%` `6bjbj"x"x@@@,{jWWWWffffPPP8\fn(,,BBnX$|jllllll$YhfՏՏՏWWBBՏ W BfBjՏj*+b(f$B Pl{$P*`&D0y$$rfz=pՏՏՏՏfffffffffWWWWWW Bioactive compounds in tomatoes: effect of organic vs conventional management in Parma in 2006 Nobili, F., Finotti, E.1, Foddai, M.S.1, Azzini, E.1, Garaguso, I.1, Raguzzini, A. 1, Tisselli, V., Piazza, C., Durazzo, A.1 & Maiani, G.1 Key words: organic tomato, nutritional quality, carotenoids, polyphenols, TEER Abstract External and internal factors such as variety, season, location, ripening, growing conditions, technological and domestic processes could affect the content of bioactive compounds in food. The aim of this study was to evaluate the influence of different agronomical practices (organic vs conventional) on the nutritional quality of tomatoes. Fresh tomatoes (cv. Perfectpeel), cultivated under organic and conventional practices were analysed for vitamin C, lycopene, (-carotene, chlorogenic acid, caffeic acid, coumaric acid, naringenin, rutin, quercetin, Total Antioxidant Capacity (TAC) and Ferric Reducing Ability of Plasma (FRAP). CaCO2 monolayer cell cultures were used for testing membrane damage by Trans Epithelial Electrical Resistance(TEER). Results showed that for lycopene, naringenin and rutin no significant differences were observed. For (-carotene and coumaric acid significantly higher values were found in organic samples. Values of vitamin C, chlorogenic acid, caffeic acid, quercetin and TAC were significantly higher in conventional tomato, but the FRAP values were significantly higher in organic tomato. The observed TEER values were not significant different between organic and conventional tomato. Introduction Several external and internal factors could affect the composition and content of bioactive compounds in food, such as agricultural factors (e.g. genotype, variety, season, geographic location/climate, stage of maturity, growing conditions), technological processes, and domestic treatments. The aim of this study was to evaluate the influence of different crop management practices (organic vs conventional) on the nutritional quality of tomato. The physiological properties of phytochemicals and their potential beneficial effects on human health are usually related to their antioxidant activities, that may protect tissues against oxygen free radicals, mutagenesis and lipid peroxidation (Manach et al. 2005). Materials and methods Samples: tomato fruits, cultivar Perfectpeel, were purchased as fruits from Stuard company (Parma, Italy), that has several years experience in cultivation of organic tomatoes. Organic and conventional products were cultivated in the same growing conditions: season, location, ripening and plant age. The tomato fruits were derived from field cultivation. The cultivation site was on a clay soil in the Emilia-Romagna Region, in particular the Po Valley, near Parma. Organic and conventional tomatoes were cultivated in two distinct plots (4 samples, 5 kg for each). Ripening stage of fruits was 100%. Analysis of nutritional parameters: Vitamin C, lycopene, (-carotene, chlorogenic acid, caffeic acid, coumaric acid, naringenin, rutin, quercetin, total antioxidant capacity (TAC) and Ferric Reducing Ability of Plasma (FRAP)- were evaluated. CaCO2 monolayer was used for testing Trans Epithelial Electrical Resistance (TEER). Lycopene and (-carotene were extracted from food matrix as described by Sharpless et al. (1999). The extracts were analyzed by HPLC system and the detection wavelength was set at 450 nm according to Maiani et al. (1995). Determination of polyphenols such as flavonoids (naringenin, quercetin and rutin) and hydroxycinnamic acids (caffeic acid, chlorogenic acid, coumaric acid) from food matrix was carried out as described by Hertog et al. 1992. Quantitative analysis was performed using an ESA-HPLC system with colorimetric array detection. Total ascorbic acid (AA+DHAA) was extracted and quantified as described by Margolis et al. (1997); chromatographic separation was carried using ESA-HPLC, equipped with colorimetric array detector (Serafini et al. 2002). Total Antioxidant Capacity (TAC) was evaluated using two different assays, TEAC (Trolox Equivalent antioxidant capacity) and FRAP (Ferric Reducing-Antioxidant Power) assays. The procedure extraction reported by Pellegrini et al. (2003), was carried out for TEAC and FRAP determinations. The TEAC assay measures the ability of antioxidants to quench radical cation in both lipophilic and hydrophilic environments in accordance to Re et al. (1999). The FRAP assay evaluates the reducing power of the sample according to Benzie and Strain et al. (1996). For Trans Epithelial Electrical Resistance (TEER) evaluation, the tomato extract was tested on a CaCo2 cell line in order to test the changes in tight junction permeability by TEER and the phenol red passage (Delie et al. 1997). Different increasing concentrations have been added on monolayer culture Caco-2 (cells from human adenocarcinoma). In the experiment the cells were seeded onto polycarbonate filter cell culture chamber inserts (diameter 6.5 mm; area 0.33 cm2; pore diameter 0.4 (m), at density of 1.5x105 cells per filter and placed in a multiwells Falcon; the filter divided the chamber in two parts: apical and basal that represent the lumen and the basal area of the gastroenteric system. In the two chambers, we have measured the TEER for assessment of tight-junction permeability; this test gives us information about cell damages. At the end of experiment, the permeability was also tested by phenol red: the amount of phenol red detected in the basal chamber confirmed the TEER test. These parameters were used to detect the early intestinal barrier function in vivo damages. Each analysis was performed in triplicate. Data are given as the mean and standard deviation. Statistical analysis was performed using the Statistica for Windows statistical package (release 45; StatSoft Inc., Vigonza PD, Italy). Results Tab 1 shows phenolics, vitamin C and carotenoids contents (mg/Kg) and TEAC ((Mol/100g) and FRAP (mMol/Kg) values respect to organic and conventional tomato. For lycopene, naringenin and rutin content there were no significantly different between organic and conventional tomatoes, while (-carotene and coumaric acid levels were significantly higher in the organic samples. In contrast, values of vitamin C, chlorogenic acid, caffeic acid, quercetin and TEAC were significantly higher in conventional tomatoes, but the FRAP values were significantly higher in organic tomatoes. Figure 1 shows the antioxidant/pro-oxidant results obtained using vitro models expressed as TEER (Ohm/cm2) by time in conventional and organic samples respectively for three different concentrations of tomato polyphenolic extract. The observed TEER values were not significant different between organic and conventional tomato, but we have observed, in both samples, a decrease of tight-junction permeability at 17uM of polyphenol concentrations. Tab. 1: Mean values(sd of tested nutritional parameters OrganicConventionalStudent T-testVitamin C mg/100g17.731.4120.291.49P<0.03Lycopene mg/Kg2.130.452.400.50n.s.(-Carotene mg/Kg0.610.090.470.12P<0.03Chlorogenic acid mg/Kg2.820.923.520.74P<0.02Caffeic acid mg/Kg3.290.333.610.71P<0.006Coumaric acid mg/Kg3.350.482.790.42P<0.02Naringenin mg/Kg41.5217.7134.5611.16n.s.Rutin mg/Kg36.6617.7237.066.68n.s.Quercetin mg/Kg17.9210.9033.906.31P<0.0004TAC (TEAC) (Mol/100g3527148534P<0.0001FRAP mMol/Kg4.060.483.151.35P<0.0001Each value is the mean of three determinations, S.D.  EMBED Excel.Chart.8 \s Figure 1 BIO and CONV tomato polyphenols extract at different concentration, each value is the mean of three determinations, S.D. Discussion The TEER results show that conventional and organic samples have same behaviour, and the monolayer system is able to resist until 17 M of tomato polyphenols extract. The main polyphenols in above mentioned extracts were quercetin (33.906.31 mg/kg in conventional extract; 17.9210.9 mg/kg in organic extract), chlorogenic acid (3.520.74 mg/kg in conventional extract; 2.820.92 mg/kg in organic extract) and caffeic acid (3.610.71 mg/kg in conventional extract; 3.290.33 mg/kg in organic extract). Even if statistical differences were found between organic and conventional extracts in several target compounds, no difference in biological effect was observed in the cell model. Further researches are required to explain this intrinsic mechanism of action. Conclusions As reported in literature data, there was no unidirectional trend of nutritional parameters towards organic or conventional product. In conclusion, focus points reached are: - the production system could affect the antioxidant content and so the food quality; - the ripening stage could affect the bioactive molecule content; - phenols, present in extract at high concentrations, could exert a pro-oxidant effect; - modifications and alterations of monolayer, after phenolic extract exposition, could induce cellular damage by permeability cell changes. Acknowledgments This research was supported by MUR-FISR Simbio-Veg project (2005-08) References Benzie I.F.F., Strain J.J. (1996): The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Analitical Biochem. 239: 70-76. Delie F., Rubas W.A. (1997): A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model. Crit Rev Therap Drug Carrier Syst. 14: 221-287. Hertog M.G.L., Hollman P.C.H., Venema D.P. (1992): Optimization of a quantitative HPLC determination of potential anticarcenogenic flavonoids in vegetable and fruit. J Agric Food Chem. 40: 1591-1598. Maiani G., Pappalardo G., Ferro-Luzzi A., Raguzzini A., Azzini E., Guadalaxara A., Trifero M., Frommel T., Mobarhan S. (1995): Accumulation of beta-carotene in normal colorectal mucosa and colonic neoplastic lesions in humans. Nutr Cancer. 24: 2331. Manach C., Williamson G., Morand C., Scalbert A., Remesy C. (2005): Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr. 81: 230S242S. Margolis S.A., Schapira R. (1997): Liquid chromatographic measurement of L-ascorbic acid and D-ascorbic acid in biological samples.  HYPERLINK "javascript:AL_get(this,%20'jour',%20'J%20Chromatogr%20B%20Biomed%20Sci%20Appl.');" J Chromatogr B Biomed Sci Appl. 690:25-33. Pellegrini N., Serafini M., Colombi B., Del Rio D., Salvatore S., Bianchi M. Brighenti F. (2003): Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. J Nutr. 133:2812-2819. Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. (1999): Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 26:1231-1237. Serafini M., Bugianesi R., Salucci M., Azzini E., Raguzzini A., Maiani G. (2002): Effect of acute ingestion of fresh and stored lettuce (Lactuca sativa) on plasma total antioxidant capacity and antioxidant levels in human subjects. Br J Nutr. 88:615-623. Sharpeless K. E., Arce-Osuna M., Thomas J.B., Gill L.M. (1999): Value assignment of retinol rettinyl palmitate, tocopherol, and carotenoids concentration in standard reference material 2383 (baby food composite). 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