Article pubs.acs.org/JAFC
Organic and Conventional Kiwifruit, Myths versus Reality: Antioxidant, Antiproliferative, and Health Effects Yong Seo Park,† Kyung-Sik Ham,‡ Seong-Gook Kang,‡ Yang-Kyun Park,‡ Jacek Namiesnik,§ Hanna Leontowicz,# Maria Leontowicz,# Aviva Ezra,⊗ Simon Trakhtenberg,△,⊥ and Shela Gorinstein*,⊗ †
Department of Horticultural Science and ‡Department of Food Engineering, Mokpo National University, Muan, Jeonnam, South Korea § Department of Analytical Chemistry, Gdansk University of Technology, Gdansk, Poland # Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Warsaw, Poland ⊗ The Institute for Drug Research, School of Pharmacy, The Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel △ Kaplan Medical Center, Rehovot 76100, Israel ABSTRACT: Comparison between organic and conventional kiwifruit cultivars ‘Hayward’ and ‘Bidan’, which was done by four radical scavenging assays, ESI-MS, and DSC measurements, showed significant differences between the cultivars. Such results were not estimated in kiwifruit growing under organic and conventional conditions. The extraction of bioactive compounds was done by two different methods: sequential extraction with ethyl acetate followed by methanol and maceration with methanol and ethyl acetate. The highest yield of polyphenols was found in the new cultivar ‘Bidan’ in comparison with the classic ‘Hayward’, by direct extraction with methanol. This is the first investigation of ‘Bidan’ kiwifruit cultivar, grown under organic conditions and compared with ‘Hayward’ organic. High contents of bioactive compounds and antioxidant and antiproliferative properties of the two kiwifruit cultivars justify their use as sources of valuable antioxidants. It is necessary to continue this study as a long-term experiment to eliminate the influence of seasonality. KEYWORDS: conventional and organic kiwifruit, bioactives, antioxidativity
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INTRODUCTION Coronary artery disease is the most important adult health problem in the world. In this connection the antioxidant properties of kiwifruit have received attention in regard to their possible health-promoting effects.1,2 There is a great variety of kiwifruit, which belongs to the genus Actinidia (A. melanandra and A. arguta var. purpurea) with different colors and compositions3 and Actinidia deliciosa.4−8 The kiwifruit cultivar A. deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson var. deliciosa ‘Hayward’ is a commercially important cultivar, which has a relatively long storage life.5 ‘Bidan’ is less consumed and belongs to the A. deliciosa species.9 Nowadays, there is interest in the possible differences between organically and conventionally grown fruits and vegetables.10−12 Organic foods are thought to have higher antioxidant capacity, because this form of agricultural management could induce synthesis of secondary compounds such as polyphenols.12 Some differences were shown between organic and conventionally produced sweet oranges of Valencia variety.10 The agronomic environments in which tomatoes are cultivated potentially affect the levels of antioxidants and other metabolites in commercial products.11 Whether or not all foods marketed to consumers as organic meet specified standards for the use of that descriptor, or are nutritionally different from conventional foods, is uncertain.12−14 The comparison between organic and conventional fruits is important, taking into consideration that, as mentioned above, few papers have studied this aspect. Therefore, we decided to © 2012 American Chemical Society
compare the bioactive compounds of organic and conventional kiwifruit cultivars using different extraction procedures and recent analytical methods.15−19 To obtain reliable data, four generally accepted antioxidant assays were used: ABTS, DPPH, FRAP, and CUPRAC. Additionally, ESI-MS and DSC measurements20−22 were made, and antiproliferative properties were investigated.23 As far as we know no results of such investigations have been published.
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MATERIALS AND METHODS
Chemicals. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), tris(hydroxymethyl)aminomethane (Tris), 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Folin−Ciocalteu reagent (FCR), lanthanum(III) chloride heptahydrate, FeCl3·6H2O, CuCl2·2H2O, and 2,9-dimethyl1,10-phenanthroline (neocuproine) were purchased from Sigma Chemical Co., St. Louis, MO, USA. 2,4,6-Tripyridyl-s-triazine (TPTZ) was purchased from Fluka Chemie, Buchs, Switzerland. All reagents were of analytical grade. Deionized and distilled water was used throughout. Samples. The samples of kiwifruit cultivars ‘Hayward’ and ‘Bidan’ at their maturity stage, according to the acidity, firmness, and soluble solid content, were grown in conventional and organic conditions in an Received: Revised: Accepted: Published: 6984
March 12, 2012 June 18, 2012 June 19, 2012 June 19, 2012 dx.doi.org/10.1021/jf3010614 | J. Agric. Food Chem. 2012, 60, 6984−6993
Journal of Agricultural and Food Chemistry
Article
orchard in Heanam County, Jeonnam province, Korea, 2011 (Figure 1). Chemical fertilizers, pesticides, and fungicides were regularly used in the
sequential extraction with ethyl acetate (and then with methanol) and maceration separately with methanol and ethyl acetate. The sequential extraction was done in the following way. For proanthocyanidin and flavonoid identifications, 1 g of the kiwifruit powder was extracted three times with 25 mL of acetone/water/acetic acid (AWA; 70:29.5:0.5) for 45, 45, and 30 min, respectively, using an orbital platform shaker at 300 rpm. The mixture was centrifuged, the supernatant was collected, and the acetone was evaporated under reduced pressure to yield a slurry (8 mL). To obtain the proanthocyanidins, the slurry was liquid−liquid extracted with ethyl acetate, and the ethyl acetate fraction was analyzed using ESI-MS. For flavonoid identification and quantification, the slurry was diluted four times with methanol and filtered through a Sartorius Minisart 45 μm porosity and analyzed using ESI-MS.19 Kiwifruit samples were extracted directly with methanol or ethyl acetate (concentration = 25 mg/mL) at room temperature twice for 3 h during maceration. Determination of Bioactive Compounds and Antioxidant Activity. The polyphenols were determined by Folin−Ciocalteu method24 with measurement at 750 nm with a spectrophotometer (Hewlett-Packard, model 8452A, Rockville, MD, USA). The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (DW). Total flavonoid content was determined by an aluminum chloride colorimetric method with some modifications.25 The absorbance was measured immediately against the blank at 510 nm in comparison with the standards. The total flavanols amount was estimated using the pdimethylaminocinnamaldehyde (DMACA) method, and then the absorbance at 640 nm was read. (+)-Catechin served as a standard for flavonoids and flavanols, and the results were expressed as catechin equivalents (CE).15,25 The limit of concentration of catechin standard curves for flavonoids was from 10 to 100 μg/mL and for flavanols was from 0.6 to 25 μg/mL. Determination of Antioxidant Activity (AA) [Micromoles of Trolox Equivalents (TE) per Gram of DW]. The AA was determined by four complementary assays: (1) The 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+) method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants.17 The ABTS•+ radical cation was generated by the interaction of ABTS (7 mmol/L) and K2S2O8 (2.45 mmol/L). This solution was diluted with water until the absorbance in the samples reached 0.7 at 734 nm. (2) The ferric reducing/antioxidant power (FRAP) assay measures the ability of the antioxidants in the investigated samples to reduce ferric tripiridyltriazine (Fe3+-TPTZ) to a ferrous form (Fe2+). FRAP reagent (2.5 mL of a 10 mmol ferric tripiridyltriazine solution in 40 mmol of HCl plus 2.5 mL of 20 mmol of FeCl3·H2O and 25 mL of 0.3 mol/L acetate buffer, pH 3.6) of 900 μL was mixed with 90 μL of distilled water and 30 μL of kiwifruit samples as the appropriate reagent blank. The absorbance was measured at 595 nm.18 (3) The cupric reducing antioxidant capacity (CUPRAC) assay is based on utilizing the copper(II) neocuproine [Cu(II)-Nc] reagent as the chromogenic oxidizing agent. To the mixture of 1 mL of copper(II) neocuproine and NH4Ac buffer solution were added acidified and nonacidified water extracts (or standard) solution (x, in mL) and H2O [(1.1 − x) mL] to make the final volume of 4.1 mL. The absorbance at 450 nm was recorded against a reagent blank.16 (4) Scavenging free radical potentials were tested in a solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH). In its radical form, DPPH has an absorption band at 515 nm, which disappears upon reduction by an antiradical compound. DPPH solution (3.9 mL, 25 mg/L) in methanol was mixed with the sample extracts (0.1 mL), and then the reaction progress was monitored at 515 nm until the absorbance was stable.17 MS Analysis. A TSQ Quantum Access Max (Thermo Fisher Scientific, Basel, Switzerland) mass spectrometer was used. Analytes were ionized by electrospray ionoization (ESI) in negative mode. The vaporizer temperature was kept at 100 °C. Settings for the ion source were as follows: spray voltage, 3000 V; sheath gas pressure, 35 AU; ion sweep gas pressure, 0 AU; auxiliary gas pressure, 30 AU; capillary temperature, 200 °C; skimmer offset, 0 V.
Figure 1. Two kiwifruit cultivars grown in Korea: ‘Bidan’ organic and conventional; ‘Hayward’ organic and conventional. kiwifruit orchard for growing conventional fruits. Under organic growing conditions, no chemical fertilizers were applied during cultivation; only organic fertilizers such as manure or humus were used. The samples were cleaned with tap water and dried. For the investigation five replicates of five fruits each were used. Their edible parts were prepared manually without using steel knives. The peeled fruits were weighed, chopped, and homogenized under liquid nitrogen in a high-speed blender (Hamilton Beach Silex professional model) for 1 min. A weighed portion (50−100 g) was then lyophilized for 48 h (Virtis model 10-324), and the dry weight was determined. The samples were ground to pass through a 0.5 mm sieve and stored at −80 °C until the bioactive substances were analyzed. Extraction of Phenolic Compounds for Antioxidant Activities, Mass Spectrometry (MS), and Differential Scanning Calorimetry (DSC). The extracts from kiwifruit were prepared in the same way for all tests (bioactive compounds; antioxidant and anticancer assays). To find the best extraction of polyphenols two different methods were used: 6985
dx.doi.org/10.1021/jf3010614 | J. Agric. Food Chem. 2012, 60, 6984−6993
Journal of Agricultural and Food Chemistry
Article
Table 1. Bioactive Compounds (per Gram Dry Weight) in Fractions Obtained by Sequential Extraction with EtOAc and MeOH and in Extracts by Maceration with EtOAc and MeOH of Conventionally and Organically Grown Kiwifruit Cultivarsa indices extracts and fractions of kiwifruit cultivarsb
POL, mg GAE
FLAVAN, μg CE
TAN, mg CE
FLA, mg CE
KHaC, EtOAc extract KHaO, EtOAc extract KBiC, EtOAc extract KBiO, EtOAc extract KHaC, EtOAc fraction KHaO, EtOAc fraction KBiC, EtOAc fraction KBiO, EtOAc fraction
1.10 ± 0.1 e 1.27 ± 0.2 e 2.26 ± 0.2 d 3.80 ± 0.4 d 0.73 ± 0.1 f 0.75 ± 0.2 f 0.83 ± 0.2 f 0.89 ± 0.4 f
25.47 ± 2.3 c 21.23 ± 2.5 cd 93.41 ± 8.5 b 97.66 ± 9.3 b 4.63 ± 0.3 e 3.84 ± 0.5 e 16.98 ± 1.5 d 16.49 ± 1.3 d
2.69 ± 0.3 d 2.72 ± 0.3 d 8.00 ± 0.9 b 10.80 ± 1.2 b 0.18 ± 0.1 f 0.22 ± 0.3 f 0.42 ± 0.3 e 0.38 ± 0.1 e
8.48 ± 0.9 b 10.80 ± 1.1 b 67.39 ± 4.6 a 66.38 ± 4.7 a 1.64 ± 0.3 cd 1.31 ± 0.6 d 8.54 ± 0.7 b 8.72 ± 0.7 b
KHaC, MeOH extract KHaO, MeOH extract KBiC, MeOH extract KBiO, MeOH extract KHaC, MeOH fraction KHaO, MeOH fraction KBiC, MeOH fraction KBiO, MeOH fraction
7.08 ± 0.8 c 7.98 ± 0.7 c 64.40 ± 5.6 a 60.86 ± 4.8 a 1.96 ± 0.2 e 1.81 ± 0.3 e 18.02 ± 1.6 b 18.87 ± 1.7 b
142.67 ± 9.1 a 127.00 ± 7.1 ab 28.03 ± 2.8 c 29.72 ± 2.7 c 31.85 ± 3.9 c 43.06 ± 4.3 bc 4.46 ± 0.7 e 4.35 ± 0.5 e
4.48 ± 0.8 c 5.12 ± 0.9 c 19.07 ± 1.8 a 17.87 ± 1.7 a 0.26 ± 0.1 f 0.31 ± 0.1 f 1.92 ± 0.3 de 1.52 ± 0.4 de
1.87 ± 0.4 c 1.48 ± 0.2 cd 2.13 ± 0.2 c 2.05 ± 0.3 c 0.03 ± 0.01 e 0.03 ± 0.01 e 0.04 ± 0.03 e 0.02 ± 0.01 e
Values are the mean ± SD of five measurements. Values in columns for every bioactive compound with different letters are significantly different (P < 0.05). bAbbreviations: KHaC, kiwifruit ‘Hayward’ conventional; KHaO, kiwifruit ‘Hayward’ organic; KBiC, kiwifruit ‘Bidan’ conventional; KBiO, kiwifruit ‘Bidan’ organic; POL, polyphenols; CE, catechin equivalent; GAE, gallic acid equivalent; FLAVAN, flavanols, TAN, tannins, FLA, flavonoids. Two extraction methods were used: sequential separation with EtOAc (EtOAc fraction), followed by MeOH (MeOH fraction) and maceration by direct extraction with MeOH (MeOH extract) and EtOAc (EtOAc extract). a
DSC Measurements. The extent of the stability of extracted polyphenol fractions in different kiwifruit samples was estimated on a differential scanning calorimeter (DSC1, STAR System, Mettler Toledo). Portions (4−6 mg) of lyophilized kiwifruit samples were transferred into preweighed aluminum pans and sealed. An empty pan was used as the reference. A number of unique temperatures such as onset (Ti), left limit, right limit, and T peak (Tp, °C) and endothermal enthalpy changes (ΔH, J/g) were computed from the thermograms. The equipment was previously calibrated with standard reference of indium.20−22 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. The antiproliferative activity of methanol extracts of the studied kiwifruit samples on human cancer cell lines (Calu-6 for human pulmonary carcinoma and SNU-601 for human gastric carcinoma) was measured using the MTT assay. The cell lines were purchased from Korean Cell Line Bank (KCLB) for MTT assay. Cells were grown in RPMI-1640 medium at 37 °C under 5% CO2 in a humidified incubator. Serum (10 g/100 g) and antibiotics (1 g/100 g) were added to the RPMI-1640 medium. Cells were trypsinized and then centrifuged to harvest. Cells were harvested, counted (3 × 104 cells/mL), transferred into a 96-well plate, and incubated for 24 h prior to the addition of test compounds. Serial dilutions of test samples were prepared by dissolving compounds in methanol followed by dilution with RPMI-1640 medium to give final concentrations at 10, 30, 100, 300, and 1000 μg/mL. Stock solutions of test samples were prepared for cell lines of 90 μL of medium and 10 μL of samples and incubated for 72 h. MTT solution at 5 mg/mL was dissolved in 1 mL of phosphate buffer solution (PBS), and 10 μL of it was added to each of the 96 wells. The wells were wrapped with aluminum foil and incubated at 37 °C for 4 h. The solution in each well containing media, unbound MTT, and dead cells was removed by suction, and 150 μL of DMSO was added to each well. The plates were then shaken, and the optical density was recorded using a microplate reader at 540 nm. The cytotoxicity was obtained by comparing the absorbance between the samples and the control.23 Statistical Analyses. To verify the statistical significance, means ± SD of five independent measurements were calculated. Differences between groups were tested by two-way ANOVA. In the assessment of the antioxidant activity, Spearman correlation coefficients (R) were
used. Linear regressions were also calculated. P values of
