Methanol (MeOH) and ethyl acetate (EtOAc) extracts of

1. ORGANIC AND CONVENTIONAL KIWIFRUIT: MYTHS VS. REALITY, ANTIOXIDANT, QUENCHING, ANTIPROLIFERATIVE AND HEALTH EFFECTS Yong Seo Park1, Myang Hee Im2, Hanna Leontowicz3, Maria Leontowicz3, Milan Suhaj4, Elena Katrich5, Moshe Weisz5, Zeev Tashma5, Shela Gorinstein5# 1Department of Horticultural Science, Mokpo National University, Jeonnam, South Korea; 2Regional Crop Research Institute, Mokpo National University, Jeonnam, South Korea; 3Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Warsaw, Poland; 4Food Research Institute, Bratislava, Slovakia; 5The Institute for Drug Research, The Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy, Jerusalem, Israel #This research is dedicated to the memory of my dear brother Prof. Simon  Trakhtenberg, who encouraged and supported me and our research group during all  his life. 1. Background Fruits with high content of bioactive compounds are effective in prevention and treatment of atherosclerosis (1-4). In this investigation the bioactivity of conventionally and organically grown kiwifruit’s cultivars ‘Hayward’ (‘KHaC', ‘KHaO') and 'Bidan' (‘KBiC', ‘KBiO') were studied. The main peaks (m/z) with Relative Abundance (RA, %), using different extraction procedures MeOH and EtOAc, for ‘KBiCMeOH' and ‘KBiOMeOH' (Fig. 4A and 4B) were at: 111(100, 100), 129(15, 15), 175(55, 55), 191(75, 75), 391(40, 80); for ‘KBiCEtOAc' and ‘KBiOEtOAc' (inserts in 4A and 4B): 111(12, 28), 217(45, 40), 311(30, 28), 325 (50,33), 339(43,30), 391(100,100). The main m/z peaks for ‘KHaCMeOH' and ‘KHaOMeOH' were at: 111(38, 43), 191(100, 100) and 391(47, 90); ‘KHaCEtOAc' and ‘KHaOEtOAc' - 111(12, 12), 217(22, 22); 311 (15, 12), 325(20, 18), 339(12, 12), 391(100, 100). The difference between conventional and organic 'Hayward' and 'Bidan' cultivars was only in RA of peaks, but between the cultivars was also in the shift of the peaks. A 2. Materials and methods Methanol (MeOH) and ethyl acetate (EtOAc) extracts of conventionally and organically grown in Korea (Fig.1) kiwifruit’s cultivars ‘Hayward’ (‘KHaC', ‘KHaO') and 'Bidan‘ (‘KBiC', ‘KBiO') were investigated. FTIR,3D-FL spectroscopy,ESI-MS, DSC and radical scavenging assays were used for characterization of bioactive compounds and the levels of their antioxidant activities (1-4). B Fig. 4.ESI-MS spectra of (A): methanol fraction (MeOH) of ‘KBiC’ with an insert of ethyl acetate (EtOAc) fraction of ‘KBiC’; (B), MeOH fraction of ‘KBiO’ with an insert of EtOAc fraction of ‘KBiO’ in negative ion mode. Fig. 1.Kiwifruit orchard Heanam County, Jeonnam province, Korea, 2011 (S.Gorinstein, Y.S. Park) 3. Results The significantly highest levels of bioactive compounds were estimated in methanol extract of ‘KBiO' (P < 0.05) than in other investigated kiwifruit samples, but the difference between the organic and conventional kiwifruit was not significant (Fig. 2). B C A A B Fig. 5.DSC curves of EtOAc fractions of (A): ‘KHaO’ (a), ‘KHaC’ (b), ‘KBiO’ (c), ‘KBiC (d)’; MeOH fractions (B): ‘KHaO’ (a), ‘KHaC’ (b), ‘KBiO’ (c), ‘KBiC’ (d); MeOH extracts (C): ‘KHaC’ (a), ‘KHaO’ (b), ‘KBiC’ (c), ‘KBiO’ (d). The pans were heated in the calorimeter at 10 oC min over the range 25-200 oC. Fig. 2. A, B, three-dimensional fluorescence (3D-FL) spectrum of methanol extracts of conventional and organic kiwifruits ‘Bidan’ (0.25 mg/mL) Endothermic peaks were at the same temperature [EtOAc and MeOH fractions (Fig.5A and 5B)] with nearly 3-fold and 14-fold more heat was required to soften the ‘KHaC’ that the ‘KHaO’. MeOH extract 2-fold more heat required for ‘KHaO’ (Fig 5C). The endothermic peaks for ‘KBiC’ and ‘KBiO’ were at the same temperature with the same number of peaks for EtOAc, but the enthalpy of transition was different and as in case of 'Hayward' 1.8-fold more heat required for conventional. In MeOH fractions and extracts the temperature peaks were similar, but 3.3-fold and 5.2-fold more heat required for organic 'Bidan'. A B 4. Discussion Fig. 3.Sun ray icon plot of organically (O) and conventionally (C) grown ‘Hayward’ kiwifruit treated with ethylene (0,6,12,18,24 hrs) were compared by the examined variables (fruit firmness, sensory value, soluble solid content, total tartaric acidity , vitamin C, antioxidant activity, electron donating ability , nitrite scavenging activity , angiotension-1 converting enzyme , total phenolics). The DSC thermograms of the phenolic extracts showed only one endothermic peak at different temperatures, depending on investigated samples and year of collection. The kiwifruit extracts decreased the proliferation of both Calu-6 and SNU-601 for human pulmonary carcinoma and gastric carcinoma cells, and the effect was concentration dependent. The proliferativity for concentrations of 1000µg/ml for ‘KBiO' methanol extract was lower than for other investigated samples. The interaction between drugs and human serum albumin plays an important role in the distribution and metabolism of drugs. The complexation reaction between flavonoids, and kiwifruit extracts, and bovine serum albumin (BSA) showed that kiwifruit polyphenol extracts have strong ability to quench the intrinsic fluorescence of BSA and comparable with quercetin (1-4). Each ray represents a different variable; the middle of the ray comprises the mean value of the variable. Values for each parameter are connected by a cord. This plot clearly demonstrates the found differences between organic and conventional kiwifruit [Principal Component Analysis (PCA), Fig.3]. The proliferativity (%) at 1000 µg/mL (Fig. 6A, B) for 'KHaO' on Calu-6 is 54.54%, and on SNU-601 is 60.08%, 'KHaC' for Calu-6 (56.23%) and SNU-601 (61.56%); for 'KBiO' on Calu-6 is 48.84%, on SNU-601 is 53.36% (Fig. 6C, D), 'KBiC' for Calu-6 (47.25%) and SNU-601 (52.28%). Our results show that antioxidant activity of the studied samples correlates with their antiproliferative activity. Significantly different values between the organic and conventional samples were not found. 5. Conclusion . Relatively high contents of bioactive compounds, positive antioxidant and antiproliferative properties of two cultivars of kiwifruit justify their use as a source of valuable antioxidants. 6.References Fig. 6.Cell viability (% of Control) of human cancer cells of the Calu-6 and Snu-601 lines in the presence of MeOH extracts: A, ‘KHaC’; B, ‘KHaO’; C, ‘KBiC’; D, ‘KBiO’. 1. Gorinstein et al., 2010. Phytochemical Analysis, 21, 355-362. 2. Park et al., 2010. Plant Foods for Human Nutrition, 65, 186–191. 3. Im et al., 2012. International Journal of Food Properties, 15, 49–59. 4. Leontowicz et al., 2008.Food and Chemical Toxicology,46, 581–589. 5. Our publications- http://www.bashanfoundation.org/shela/shelapub.html