Three-Year Comparative Study of Polyphenol ... - ACS Publications

Article pubs.acs.org/JAFC

Three-Year Comparative Study of Polyphenol Contents and Antioxidant Capacities in Fruits of Tomato (Lycopersicon esculentum Mill.) Cultivars Grown under Organic and Conventional Conditions Dea Anton,*,† Darja Matt,† Priit Pedastsaar,† Ingrid Bender,‡ Renata Kazimierczak,§ Mati Roasto,† Tanel Kaart,∥ Anne Luik,⊥ and Tõnu Püssa† †

Department of Food Hygiene, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 56/3 Tartu 51014, Estonia ‡ Department of Jõgeva Plant Breeding, Estonian Crop Research Institute, Aamisepa 1, 48309 Jõgeva alevik, Jõgevamaa, Estonia § Department of Functional, Organic Food and Commodities, Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences, Nowoursynowska 166, 02-786, Warsaw, Poland ∥ Department of Animal Genetics and Breeding, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 46, Tartu 51006, Estonia ⊥ Department of Plant Protection, Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 5 D, Tartu, 51014, Estonia ABSTRACT: In the present study, four tomato cultivars were grown under organic and conventional conditions in separate unheated greenhouses in three consecutive years. The objective was to assess the influence of the cultivation system on the content of individual polyphenols, total phenolics, and antioxidant capacity of tomatoes. The fruits were analyzed for total phenolic content by the Folin−Ciocalteau method and antioxidant capacity by the DPPH free radical scavenging assay. Individual phenolic compounds were analyzed using HPLC-DAD−MS/MS. Among 30 identified and quantified polyphenols, significantly higher contents of apigenin acetylhexoside, caffeic acid hexoside I, and phloretin dihexoside were found in all organic samples. The content of polyphenols was more dependent on year and cultivar than on cultivation conditions. Generally, the cultivation system had minor impact on polyphenols content, and only a few compounds were influenced by the mode of cultivation in all tested cultivars during all three years. KEYWORDS: tomato, Lycopersicon esculentum Mill., organic cultivation, conventional cultivation, polyphenols, antioxidants, HPLC−MS/MS, principal component analysis

■

compounds.5 Tomato polyphenols, mainly phenolic acids, are present in the fruits both in free soluble form and in insoluble form when bound to a fiber. Numerous flavonoids have been identified in different tomato varieties. Most of these structures belong to the flavonols, flavanones, flavones, and chalcones.6 Several authors7,8 have reported that naringenin chalcone is one of the main flavonoid compounds in fresh tomatoes. However, some researchers consider prunin (naringenin 7-glucoside) as a main phenolic constituent in fresh tomatoes. 9,10 The production of photoprotective compounds such as flavonols in the skins of tomato fruits may afford protection against UVB-induced oxidative damage.11 Current knowledge in the biological activity of flavonoid compounds clearly shows that the positive effect on the human body is mainly due to antioxidant properties. Research has provided many scientific papers on the activity of flavonoids against tumors, cardiovascular diseases, atherosclerosis, osteoporosis, inflammation, and allergies.12−14 Organic food is for the general

INTRODUCTION The quality of organic vegetables, including higher content of low-molecular bioactive compounds, greatly interests plant breeders, scientists, and consumers. There is also a growing interest for identifying alternative natural and safe sources of food antioxidants, especially of the plant origin.1 Tomato is one of the most popular and extensively consumed vegetables worldwide with an annual global production of over 159 million tons.2 Tomato fruits are an excellent source of phytochemicals with antioxidative properties, e.g., polyphenols, carotenoids, and vitamins C and E. Regular consumption of tomatoes and tomato products has been associated with a reduction of incidents of chronic degenerative pathologies such as certain types of cancer and cardiovascular diseases. These beneficial properties may be attributed to the presence of key metabolites and the interactions among them.3 Polyphenols comprise a variety of chemical structures with subtle biological properties and in general are important in conferring antioxidative benefits.4 These plant secondary metabolites are produced through the phenylpropanoid pathway and they function in different plant defense mechanisms. Environmental stresses like nutrient deficiency, wounding, pathogens, and UV radiation are known to activate the biosynthesis of phenylpropanoid © 2014 American Chemical Society

Received: Revised: Accepted: Published: 5173

February 18, 2014 May 6, 2014 May 8, 2014 May 8, 2014 dx.doi.org/10.1021/jf500792k | J. Agric. Food Chem. 2014, 62, 5173−5180

Journal of Agricultural and Food Chemistry

Article

of Maike, Malle F1, and Valve and 20 fruits of Gartenfreude were harvested. Fruits equal in size and ripeness were carefully selected to form the best representative samples for each cultivar. Four tomato cultivars, Maike, Malle F1, Valve, and Gartenfreude, were selected for the tests. The first three cultivars have been bred in Estonia, at the Jõgeva Plant Breeding Institute (registered as new cultivars in 2003, 2004, 2003, respectively). In the previous years these cultivars have shown good results in yields and disease resistance. All the cultivars are early ripening type; the first fruits ripen within 105− 115 days after germination, and the ripe fruits have red color. Maike is a determinate (with limited growth) cultivar with multibranched bunch and round, smooth fruits with average weight of 60−65 g. Malle F1 is an indeterminate (with unlimited growth) cultivar with uniform, medium-sized, slightly flattened fruits weighing about 80−90 g. Valve is a semideterminate cultivar with a single or multibranched bunch and with round or slightly elongated fruits weighing about 95 g. Gartenfreude is an indeterminate, cherry-type, high-yield cultivar with round-shaped fruits that are 2 cm in diameter and of an average weight of 15−20 g. Weather Conditions in the Years 2008−2010. In the early summer of 2008 (May, June), the weather was warm, dry, and windy with very low relative air humidity. The end of June and all of July and August were rainy and cool. September was cloudy and the total sum of active air temperature (>10 °C) was 155 °C, which is 59 °C lower than the long period average (LPA, 80 years). In 2009, the second half of May was cold and rainy, and low temperatures (even frosts) at night did not support plant growth. The first half of June was unusually warm (over 29 °C), but the second half was cold and very rainy (120−240% of normal). In the beginning of July, the temperature varied a lot, from 29 °C at daytime to frosts at night. August was cooler and rainier than usual; inversely, September was extremely warm with a total sum of active air temperature of 329 °C (LPA, 218 °C) with little rain. May 2010 was extraordinarily warm, but temperatures in June were below the average level. Inversely, July and half of August were extremely warm. Weather favored the plant species that require a lot of heat. In the second half of August and in September, the temperatures dropped but still remained higher than average (the total sum of active air temperature was 241 °C, which was 23 °C more than the LPA).25 Sample Preparation and Extraction. Organically and conventionally grown tomato fruits, three samples of each cultivar, were taken to the laboratory and kept at room temperature overnight. The next day tomato fruits were washed, dried, sliced into segments, and divided into three groups: for hot air-drying at 45 °C in a ventilated drier Binder FED 53 (Binder GmbH, Tuttlingen, Germany), homogenization, and freeze-drying for previous and further experiments. In this study, the air-dried material was ground into fine powder using a Foss Knifetec 1095 (Foss Tecator, Höganäs, Sweden) laboratory mill, passed through a sieve, and additionally ground in a mortar. Samples were extracted without hydrolysis of glycosides. For extraction, 0.5 g of tomato powder was weighed and 5 mL of 80% (v/v) aqueous methanol (Romil Ltd., Cambridge, UK) was added. The samples were placed into a Biosan Multi RS 60 (BioSan, Riga, Latvia) rotator for 5 h and left at room temperature overnight. The next day the samples were centrifuged at 20 °C at 3220g for 10 min using an Eppendorf 5810 R (Eppendorf AG, Hamburg, Germany) centrifuge, and supernatant was poured into another tube. The samples were reextracted with an additional 5 mL of 80% (v/v) aqueous methanol. After centrifugation, two supernatants were mixed and filtered through a 0.45 μm syringe filter, 20 μL of (0.8 mg mL−1) daidzein methanol solution as internal standard was added, and samples were stored at −40 °C prior the chromatographic analysis. Chromatographic Analysis. Single phenolic compounds were detected, identified, and quantified using tandem liquid chromatography with diode array detection−mass spectrometry (HPLC-DAD− ESI-MS/MS) in the negative ion mode at an Agilent 1100 Series (Agilent Technologies, Santa Cruz, CA) system, consisting of a binary pump, vacuum degasser, autosampler, thermostated column department, diode array detector, and ion trap analyzer with electrospray ionization (ESI). For the separation of compounds, a reversed-phase

public associated with improved nutritional properties, which led to increasing demand for organic vegetables.15 However, there is a need for better scientific data to prove whether the hypotheses like “organic tomatoes contain more healthpromoting compounds” or “some tomato cultivars are more suitable for cultivation in organic conditions than others” are true. Oliveira et al.16 have found significantly higher content of vitamin C, soluble solids, and total phenolics in organically cultivated tomato fruits. At the same time, the authors also state that the dimensions of organic fruits were smaller. Rembialkowska17 has shown in a review article that organic plant products contain more vitamin C, phenolic compounds, and minerals (Fe, Mg, P). Similar conclusions, pointed out by Hunter et al.,18 showed higher contents of micronutrients in organic vegetables, cereals, and legumes. The higher contents of phenolic compounds in organic agricultural crops has also been confirmed by Brandt et al.,19 Lima and Vianello,20 and Lairon.21 However, opposing views have been reported by Rosen22 and Dangour et al.,23 according to whom there is no difference in the chemical composition of organic and conventional food. Many factors may determine the level of secondary plant metabolites, polyphenols, and antioxidants in the plant food. Some factors depend on the cultivation conditions, plant genetics, agricultural practices, meteorological factors, soil fertility, pest-related factors, harvest time, and maturity of the crops.24 The aim of this study was to determine the differences in polyphenols contents, antioxidant capacities, and total phenolic contents in four tomato cultivars grown in organic and conventional conditions; to get more information about the polyphenolic composition and their antioxidant capacity in fruits of tomato cultivars bred in Estonia; and to evaluate the influence of cultivars, cultivation systems, and weather conditions in the growing years.

■

MATERIALS AND METHODS

Plant Material. The experiment was carried out in three consecutive years (2008−2010). Plants of four tomato cultivars were grown at the Jõgeva Plant Breeding Institute (58°44′N, 26°24′E), in two unheated plastic greenhouses, one for conventional and another for organic cultivation. Greenhouses with similar size and construction were located next to each other. Soil at the site was classified as a soddy−podzolic sandy loam. Every autumn soil was fertilized with cattle manure (60 t ha−1) and enriched with peat (40 t ha−1) incorporated into soil. For basic fertilization, the conventional experimental area was fertilized with a compound fertilizers including 90 kg ha−1 N, 60 kg ha−1 P, and 170 kg ha−1 K (plus other macro- and micronutrients).The seeds were sown in the heated greenhouse in March, transplanted twice, and planted in the middle of May in unheated greenhouses in double rows, six plants per plot, three replications per cultivar in different parts of greenhouses. Substrate for conventionally cultivated seedlings and transplants was a peat-based mixture fertilized with 2 kg m−3 PeatCare 11−25−24 (Kemira GrowHow) and 0.5 kg m−3 magnesium sulfate, mixed with dolomite lime (7 kg m−3). Substrate for organically cultivated seedlings and transplants was peat mixed with dolomite lime (7 kg m−3) and enriched with chicken manure pellets (1 L of pellets per 50 L of peat). During the growing period, once or twice a week the plants were irrigated through tubes installed in the soil. The conventional experimental area was fertilized with 50 kg ha−1 N and 60 kg ha−1 K. The organic experimental area was fertilized with humic acids solution (fertilizer Humistar diluted by 1:50), resulting in a total supply of 0.6 kg ha−1 K, and with fermented nettle infusion 1:5 three times during the growing period. Tomatoes were grown without any pesticides in the organic and conventional area. Red-ripe tomato fruits were harvested in the beginning of September. Six fruits per replication 5174

dx.doi.org/10.1021/jf500792k | J. Agric. Food Chem. 2014, 62, 5173−5180


Article

Figure 1. Phenolic compounds identified by HPLC−MS/MS in tomato fruit in negative ionization mode. MS-base peak chromatogram of the conventionally grown cultivar Gartenfreude (2010). Peak numbers designate identified compounds: 1, 2, 3, 4, and 5, caffeic acid hexosides; 6, homovanillic acid hexoside; 7, caffeic acid; 8, p-coumaroyl hexoside; 9, chlorogenic (3-caffeoylquinic) acid; 10, cryptochlorogenic (4-caffeoylquinic) acid; 11, neochlorogenic (5-caffeoylquinic) acid; 12 and 13, apigenin hexosides; 14, p-coumaric acid; 15, naringenin dihexoside; 16, rutin hexoside; 17, apigenin acetylhexoside; 18, ferulic acid; 19, eriodictyol hexoside; 20, quercetin dihexoside; 21, quercetin rutinoside (rutin); 22, kaempferol rutinoside pentoside; 23, phloretin dihexoside; 24, naringenin glucoside; 25, dicaffeoylquinic acid I; 26, dicaffeoylquinic acid II; 27, dicaffeoylquinic acid III; 28, kaempferol rutinoside; 29, naringin; 30, caffeic acid derivative; 31, dihydrocaffeic acid dihexoside; 32, eriodictyol; 33, quercetin; 34, naringenin; 35 tricaffeoylquinic acid; 36, naringenin chalcone; D, daidzein (internal standard). (RP) Zorbax 300SB-C18 column (2.1 × 150 mm, 5 μm; Agilent Technologies) was used. The mobile phase gradient was formed of 0.1% formic acid (Fluka Chemie GmbH) (solvent A) and acetonitrile (Romil Ltd., Cambridge, UK) (solvent B), and stepwise gradient elution program was from 1% solvent B up to 28% in 1−45 min, in 45−55 min solvent B concentration was raised up to 95% and remained there for 5 min. Subsequently, the solvent B content was decreased to the initial conditions and for 13 min the column was reequilibrated. The total run time was 73 min. The column flow was 0.3 mL min−1 and the thermostat was regulated at 35 °C. The injection volume of samples was 4 μL. The conditions of MS/MS detection were as follows: m/z interval, 50−1000 amu; target mass, 400 amu; number of fragmented ions, two; maximal collection time, 100 ms; compound stability, 100%; drying gas (N2 from agenerator) speed, 10 L min−1; gas temperature, 350 °C; gas pressure 30 psi; collision gas (He) pressure, 6 × 10−6 mbar. Data were collected and analyzed by an Agilent 2D ChemStation Software with a ChemStation Spectral SW module. Phenolic compounds were tentatively identified by their retention time, mass to charge ratio (m/z), UV spectra, and MS fragmentation spectra reported in the literature. Eight commercially available standards: caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, rutin, naringin, quercetin, and naringenin (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were used to confirm the identification. Determination of Total Phenolic Content. The content of total phenolics was determined spectrophotometrically with the Folin− Ciocalteau reagent (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) according to the method described by Waterhouse.26 The absorbance was measured at 765 nm using an AnalyticJena Specord 200 spectrophotometer (AnalyticJena AG). Gallic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used as the standard, and the results were expressed as gallic acid equivalent (GAE) milligrams per gram of dry matter. Antioxidant Capacity Assay. The DPPH (2,2-diphenyl-1picrylhydrazyl) free radical scavenging assay was performed according to the method described by Helmja et al.27 and Lee et al.28 with some modifications. The absorbance was measured at 515 nm using an AnalyticJena Specord 200 spectrophotometer (AnalyticJena AG). A

solution of DPPH (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in methanol (23.7 mg L−1) was freshly prepared, 100 μL of tested extracts was mixed with 3900 μL of DPPH solution, and the absorbance results were recorded immediately after mixing and after every 10 min during a 60 min period until a steady state of the reaction was registered. The reference cuvette (blank) contained 80% (v/v) aqueous methanol. The percentage inhibition was calculated by the formula: %inhibition = [(Abs0 − Abssample,60min )/Abs0 ] × 100 As a reference compound and a positive control, tert-butyl-1hydroxytoluene (BHT, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used. The results were expressed as millimole of BHT equivalents per gram of dry matter. Statistical Analysis. Four tomato cultivars grown conventionally and organically were analyzed in triplicate. MS extracted ion chromatogram (EIC) peak areas were used in statistical analyses to compare the polyphenols contents of organically and conventionally cultivated tomato fruits. The principal component analysis was applied to find the common patterns in single phenolic compounds. Two- and three-way ANOVA was applied to test the statistical significance of cultivar, year, and cultivation method effect on single polyphenols, on the total content of polyphenols evaluated as a sum of contents of single polyphenols, and on the total phenolic content and antioxidant capacity. Pearson correlation analysis was used to estimate the relationships between single polyphenols, total phenolic content, and antioxidant capacity. All statistical analyses were conducted using R statistical software (version 2.14.1) and STATISTICA software (version 10.0). All image manipulations were done using R statistical software, open source Inkscape Vector Graphics Editor (version 0.48.3.1), and MS Excel 2010.

■

RESULTS AND DISCUSSION Several previous studies have compared29−32 qualitative characteristics in relation to different tomato cultivars and growing conditions. In our study, we focused on identified phenolic acids and flavonoids reported by Helmja et al.,27 Moco 5175



Article

Figure 2. Principal component analysis (PCA) biplot expressing the relation of the first two principal components with polyphenols (black rhombus according to the primary axes) and average scores by cultivars, years, and cultivation methods (arrows according to the secondary axes).

et al.,33 Martı ́nez-Valverde et al.,34 Gómez-Romero et al.,3 and Slimestad and Verheul.35 Differently, in sample preparation we did not use hydrolysis, and therefore, the main identified compounds are glycosylated. Six identified aglycons, which showed very low levels, were excluded from the statistical analysis. Single Polyphenols. Kähkönen et al.36 reported that synthesis of phenolic compounds in plants is greatly influenced by the internal factors like genetics, nutrients, and enzymes, as well as by the external factors like sunlight, temperature, and humidity. Since in our experiment the greenhouses were situated close to each other, the same degree of illumination, hours of daylight, temperature, and humidity for all plants could be expected. When analyzing the chemical constitution of tomato fruits, particular attention was paid to polyphenols, which are naturally present in plants, fruits, berries, and vegetables. In tomatoes, polyphenols comprise basically phenolic acids and flavonoids, and 36 phenolic compounds (numbered peaks in Figure 1) were identified tentatively or by comparison with authentic standards. Unmarked peaks in Figure 1 may belong to other classes of compounds (e.g., amino acids, fatty acids) eluting at the same retention time as phenolic acids and flavonoids. Some compounds had several peaks or isomers that eluted at different retention times. Following the list of phenolic compounds described by Gómez-Romero et al.,3 there were several compounds in our cultivars that we could not detect, or it was not possible to identify them by fragmentation because of their very low contents. In our samples, phenylacetic acids were represented by homovanillic acid, and hydroxycinnamic acids that formed the main phenolic acids subgroup were represented by caffeic, chlorogenic, p-coumaric and caffeoylquinic acids and their derivatives. Mattila and Kumpulainen37 reported that the most common hydroxycinnamic acid derivatives frequently occur in tomatoes as simple esters with quinic acid or glucose. Chlorogenic acids and related compounds are the main phenolic compounds besides flavonoids in tomatoes. Chlorogenic acids are good antioxidants, but they may be also responsible for a somewhat astringent taste of tomato fruits.35 Identified flavonoid glycosides belong to the subgroups of flavones (apigenin), flavonols (quercetin, kaempferol), flavanones (eriodictyol, naringenin), dihydrochalcones (phloretin),

and chalcones (naringenin chalcone). Caffeic acid hexoside, chlorogenic acid, and quercetin rutinoside (rutin) were the main phenolic constituents in our tested tomato fruits. Chalcones and dihydrochalcones are precursors in the flavonoids biosynthesis pathway and are widely investigated for their chemopreventive and antitumor activity,38 but there is limited knowledge about their contents in tomatoes. In 2008, Slimestad et al.39 described for the first time a compound representing dihydrochalcones in tomatoes, phloretin dihexoside, as dihydrochalconaringenin (phloretin) 3′,5′-di-C-βglucopyranoside and confirmed the structure by NMR. A powerful antioxidant but unstable naringenin chalcone has been found in great amounts in fresh tomatoes,39 but in our study we found quite low amounts of naringenin chalcone. Most probably, during sample drying at 50 °C and the extraction procedure this polyphenol was converted into other compounds. Principal Component Analysis (PCA). The principal component analysis of the whole data set shows that the most common pattern in phenolic compounds reflects the differences between years (Figure 2). All detected compounds except apigenin hexosides (black rhombus on the left side of Figure 2) show the higher values for the year 2010 and lower values for 2008 and 2009. The second principal component mainly distinguishes the cultivars Malle F1 and Maike from Gartenfreude. The difference between cultivation methods is only fractional. On the basis of the results, we need to bring out the fact that some phenolic compounds had higher concentrations only in one cultivar in all three years either in conventional or organic fruits, e.g., eriodictyol hexose in cultivar Maike organic fruits and in cultivar Valve conventional fruits. Obviously, these compounds were more influenced by cultivation mode than by weather conditions. In all three years, but only in one cultivar, Maike had four compounds with higher concentrations for the organic fruits and five compounds (out of 30) for conventionally grown fruits. Cultivar Malle F1 showed higher concentrations for 17 compounds in organic fruits and none of the compounds in the conventional fruits. Cultivar Gartenfreude had six compounds with higher concentrations in favor of the organic and four in the conventional cultivation method. Cultivar Valve had higher contents of two compounds in 5176



Article

Figure 3. Biplots of principal component analysis (PCA) scores expressing the differentiation of polyphenols in organically and conventionally grown tomato cultivars: Maike (A), Malle F1 (B), Valve (C), and Gartenfreude (D).

cultivation modes an extremely low amount of apigenin acetylhexoside was found in fruits of cultivars Gartenfreude and Valve (P < 0.001), compared with cultivars Maike and Malle F1. Also Gómez-Romero et al.3 found that the presence of apigenin acetylhexoside was greatly cultivar-dependent. Impact of the Growing Year on the Content of Polyphenols. While assessing the influence of the year we can report that the content of all polyphenols was affected by the year (P < 0.001). Hallmann32 also pointed out that growing season has a large impact on the variability of content of phenolic compounds. As the weather conditions varied between the years greatly, it also impacted the content of polyphenols, and all three years were significantly different. The levels of detected compounds (except apigenin hexosides) as well as the total content of polyphenols in all cultivars were the highest in 2010 (P < 0.001) due to very good weather conditions in September and the lowest levels were in 2009 (P < 0.001), which can be explained by the cold and rainy weather during the harvesting period. Despite the fact that the growing period in 2008 was rainy and suitable for spreading gray mold, the percentage of damaged fruits in all cultivars was low. We suppose that the cultivars themselves had good resistance characteristics or the suitable concentration of phenolic compounds, with higher content in 2008 compared with year 2009. Impact of Cultivation Method on the Content of Polyphenols. In the current study under these growing conditions the cultivation method had generally minor impact on the content of phenolic compounds (Figure 2). We found

organically grown fruits and eight compounds in conventionally grown fruits. When the PCA was applied separately to all four studied tomato cultivars, these otherwise minor differences between cultivation methods could easily be observed (Figure 3). It is interesting to note that the effect of the cultivation method was cultivar dependent. When differences between the organic and conventionally grown Malle F1 were clearly distinguishable on the PCA biplot, the phenolic composition of cultivar Maike showed no difference. Impact of Cultivars on the Content of Polyphenols. While using three-way ANOVA analysis we observed that the content of almost all polyphenols, except one isomer of apigenin hexoside and caffeic acid hexoside, was statistically significantly affected by cultivar (P < 0.001). Fruits of cultivar Gartenfreude had the highest contents of 19 compounds out of 30, while the fruits of cultivar Valve had the lowest contents of 18 compounds out of 30. The total content of polyphenols of cultivar Gartenfreude was the highest but had no statistically significant differences from Maike and Malle F1. Cultivar Valve differed from other cultivars Gartenfreude (P < 0.001), Maike (P < 0.05), and Malle F1 (P < 0.05), having the lowest total content of polyphenols. Martıń ez-Valverde et al.34 also concluded that the concentration of various phenolic compounds as well as the antioxidant capacity was significantly influenced by the tomato cultivar. In our study, all identified compounds were found in all tested cultivars in lower or higher amounts. It is essential to mention that in all three consecutive years and in both 5177



Article

that some compounds had higher content in all cultivars only in one year. In 2008, the higher contents of naringenin chalcone (P < 0.01), naringenin glucoside (P < 0.001), phloretin dihexoside (P < 0.05), and neochlorogenic acid (P < 0.05) were found in all organic fruits and quercetin dihexoside (P < 0.05) in conventional fruits. In 2010, the same findings were observed for caffeic acid derivative (P < 0.05), dihydrocaffeic acid dihexoside (P < 0.01), phloretin dihexoside (P < 0.05), apigenin acetylhexoside (P < 0.01), chlorogenic acid (P < 0.05), p-coumaroyl hexoside (P < 0.05), homovanillic acid hexoside (P = 0.01), caffeic acid hexoside I (P < 0.05). The exception is naringenin dihexoside, which was found in higher content in conventionally grown fruits (P < 0.01). Considering the effects of cultivar and year with three-way ANOVA, we found that there still exist several compounds showing statistically significant differences between cultivation methods generally. We can highlight three compounds (with statistical significance P < 0.001), phloretin dihexoside, caffeic acid hexoside I, and apigenin acetylhexoside, whose contents were higher in the organic fruits irrespective of years and cultivars. The results are displayed as a volcano plot (Figure 4).

The means and standard deviations of triple analysis are shown in Table 1. The total phenolic contents were the highest in conventional Gartenfreude in 2010 and the lowest in organic Maike in the same year, ranging from 5.70 to 8.54 mg GAE g−1 DW, respectively. Compared with others, cultivar Gartenfreude had higher content of total phenolics in all three years (P < 0.001). Comparing total phenolics contents in organic or conventional fruits of cultivars Maike, Gartenfreude, and Valve, there was no statistically significant difference in growing systems. Statistically higher total phenolics content was found only for cultivar Malle F1 in 2008 in conventional cultivation and in 2010 in the organic system (P < 0.005 and P < 0.05, respectively). According to the three-way ANOVA, the total phenolics contents were affected only by cultivar (P < 0.001). Free Radical Scavenging Capacity. Antioxidant capacity results, presented in Table 1, show the highest DPPH radical scavenging capacity in three consecutive years (P < 0.001) in fruits of the cherry-type cultivar Gartenfreude. These results can be explained by the highest levels of several polyphenols and other compounds having high antioxidant capacity. None of the cultivars showed higher radical scavenging results either in organic or conventional system in all three years. Obviously, the antioxidant capacity of tomato extracts is not clearly influenced by cultivation mode, although the results were slightly in favor of the conventional cultivation. In the three year experiment, the free radical scavenging capacity depended first of all on cultivar (P < 0.001). The descending order of cultivars by the antioxidant capacity was again Gartenfreude, Malle F1, Maike, and Valve. Although in the organic fruits of cultivar Malle F1 17 polyphenols had higher contents in all three years, the radical scavenging tests did not confirm better results in favor of organic fruits of this cultivar. Polyphenols provide only part of the total antioxidant capacity and the remaining is the result of lycopene, vitamin C and other compounds. Nevertheless, a strong positive correlation between total phenolics content and free radical scavenging capacity with r = 0.80 (P < 0.001) shows the leading role of polyphenols in the overall scavenging effect. The polyphenols having the strongest statistically significant (P < 0.001) positive correlation with free radical scavenging capacity of tomato samples were rutin hexoside (r = 0.75), dihydrocaffeic acid dihexoside (r = 0.72), eriodictyol hexoside (r = 0.64), homovanillic acid hexoside (r = 0.61), naringenin glucoside (r = 0.59), caffeic acid hexoside II (r = 0.59), phloretin dihexoside (r = 0.53), and dicaffeoylquinic acids (r = 0.53). In summary, it was found that the growing year and cultivar had a major impact on the variations in the content of individual polyphenols, total phenolic contents, and free radical scavenging capacity of tomato fruits. The cultivation method, organic or conventional, affected some of the polyphenolic constituents of tomato fruits, but the direction of the effect was usually dependent on the cultivar. The contents of several polyphenols in single years were significantly influenced by the cultivation system. Especially three of them, apigenin acetylhexoside, phloretin dihexoside, and caffeic acid hexoside I, were positively influenced by the organic cultivation method in the fruits of all cultivars in all three years. The results indicate that the cherry-type tomato Gartenfreude clearly differed from the other cultivars, having the highest free radical scavenging capacity, total phenolics, and several individual polyphenols contents. This phenomenon can be explained by smaller dimensions of the fruits of this cultivar

Figure 4. A volcano plot, where the X-axis represents the difference between organic and conventional cultivation methods and the Y-axis represents the statistical significance of the difference evaluated according to the three-way ANOVA considering effects of cultivar, year, and cultivation method. Every individual compound is displayed as a point. Negative values on the X-axis represent compounds that accumulate better under organic practices and vice versa. Statistically significantly (P < 0.001) different compounds are presented with names.

Comparing organic and conventional fruits of different cultivars grown in 2008−2010, organically cultivated cultivars Gartenfreude and Malle F1 had statistically significantly higher total contents of polyphenols than the organically cultivated cultivar Valve (P < 0.01, P < 0.05, respectively). Contrary, fruits of the cultivar Valve had the best results in the conventional growing system. This finding can be used in selection of tomato cultivars for cultivation systems. Total Phenolics Content. Different methods can be used to determine the antioxidant capacity. In our study we used total phenolics assay by the Folin−Ciocalteu reagent and free radical scavenging method with DPPH. Both methods are the most frequently used techniques based on electron transfer from the antioxidant and color change of the oxidant in the reaction.40 5178



Article

Table 1. Total Phenolics Content (mg GAE g−1 DW) and Antioxidant Capacity (mmol BHT equiv g −1 DW) of Four Tomato Cultivars Grown in Organic and Conventional Conditions in 2008−2010a Maike organic 2008 total phenolics

Malle F1

conventional

6.17 ± 0.33

6.23 ± 0.27 ns

antioxidant capacity

66 ± 6.6

67 ± 6.3 ns

2009 total phenolics

6.39 ± 0.6

6.70 ± 0.47

organic

2010 total phenolics

47 ± 5.1

8.06 ± 0.57

7.17 ± 0.39

8.07 ± 0.67

6.35 ± 0.63 ns

antioxidant capacity

50 ± 12.9

48 ± 11.5 ns

a

7.44 ± 0.21

54 ± 6.4

Valve

conventional

organic

conventional

8.21 ± 0.1

6.68 ± 0.25

ns 98 ± 3.3

101 ± 6.4

58 ± 3.3

56 ± 5.6 ns

7.55 ± 0.14

5.8 ± 0.45

ns 57 ± 2.6

81 ± 5.8

ns

76 ± 6.1

7.15 ± 0.9

38 ± 2.6

51 ± 8.7 ns

8.54 ± 0.1

6.04 ± 0.27

ns 75 ± 6.3

6.84 ± 0.47 ns

86 ± 7.4 ns

6.8 ± 0.86 ns

ns

7.69 ± 0.28 6.51 ± 0.47 P < 0.05 75 ± 7.5 52 ± 12.8 P = 0.05

6.26 ± 0.19 ns

ns

ns

66 ± 10.6 P = 0.05

5.7 ± 0.49

organic

6.55 ± 0.04 6.95 ± 0.09 P < 0.005 52 ± 3.9 64 ± 5.5 P < 0.05

ns antioxidant capacity

Gartenfreude

conventional

45 ± 9.7

64 ± 7.3 P = 0.05

ns, statistically not significant. (8) Slimestad, R.; Verheul, M. Properties of chalconaringenin and rutin isolated from cherry tomatoes. J. Agric. Food Chem. 2011, 59, 3180−3185. (9) Hunt, G. M.; Baker, E. A. Phenolic constituents of tomato fruit cuticles. Phytochemistry 1980, 19, 1415−1419. (10) Minoggio, M.; Bramati, L.; Simonetti, P.; Gardana, C.; Iemoli, L.; Santangelo, E.; Mauri, P. L.; Spigno, P.; Soressi, G. P.; Pietta, P. G. Polyphenol pattern and antioxidant activity of different tomato lines and cultivars. Ann. Nutr. Metab. 2003, 47, 64−69. (11) Guidi, L.; Lorefice, G.; Pardossi, A.; Malorgio, F.; Tognoni, F.; Soldatini, G. F. Growth and photosynthesis of Lycopersicon esculentum (L.) plants as affected by nitrogen deficiency. Biol. Plant. 1998, 40, 235−244. (12) Miller, N. J.; Ruiz−Larrea, M. B. Flavonoids and other plant phenols in the diet: Their significance as antioxidants. J. Nutr. Envir. Med. 2002, 12, 39−51. (13) González-Gallego, J.; Sánchez-Campos, S.; Tuñoń , M. J. Antiinflammatory properties of dietary flavonoids. Nutr. Hosp. 2007, 22, 287−293. (14) Grassi, D.; Desideri, G.; Ferri, C. Flavonoids: Antioxidants against atherosclerosis. Nutrients 2010, 2, 889−902. (15) Raigón, M. D.; Rodríguez-Burruezo, A.; Prohens, J. Effects of organic and conventional cultivation methods on composition of eggplant fruits. J. Agric. Food Chem. 2010, 58, 6833−6840. (16) Oliveira, A. B.; Moura, C. F. H.; Gomes-Filho, E.; Marco, C. A.; Urban, L.; Miranda, M. R. A. The impact of organic farming on quality of tomatoes is associated to increased oxidative stress during fruit development. PLoS One 2013, 8 (2), e56354. (17) Rembiałkowska, E. Review: Quality of plant products from organic agriculture. J. Sci. Food Agric. 2007, 87, 2757−2762. (18) Hunter, D.; Foster, M.; McArthur, J. O.; Ojha, R.; Petocz, P.; Samman, S. Evaluation of the micronutrient composition of plant foods produced by organic and conventional agricultural methods. Crit. Rev. Food Sci. Nutr. 2011, 51, 571−582. (19) Brandt, K.; Leifert, C.; Sanderson, R.; Seal, C. J. Agroecosystem management and nutritional quality of plant foods: The case of organic fruits and vegetables. Crit. Rev. Plant. Sci. 2011, 30, 177−197. (20) Lima, G. P. P.; Vianello, F. Review on the main differences between organic and conventional plant-based foods. IJFST 2011, 46, 1−13. (21) Lairon, D. Nutritional quality and safety of organic food. A review. Agron. Sustainable Dev. 2010, 30, 33−41. (22) Rosen, J. D. A review of the nutrition claims made by proponents of organic food. Compr. Rev. Food Sci. Food Saf. 2010, 9, 270−277.

and accumulation of the polyphenols mainly in the peel. The cultivars Malle F1 and Gartenfreude showed higher overall polyphenols content when grown organically. These cultivars are evidently more reasonable for organic cultivation; cultivar Valve, on the contrary, is more appropriate for conventional cultivation, accumulating more phenolic compounds under these cultivation conditions. For the cultivar Maike, the weather conditions in growing years affected the chemical composition more than the cultivation method.

■

AUTHOR INFORMATION

Corresponding Author

*Phone +372 731 3921. Fax +372 731 3432. E-mail: dea. [email protected]. Funding

This study was supported by the Estonian Scientific Council Grant No. 9315 and European Social Fund’s Doctoral Studies and Internationalization Programme DoRa, which is carried out by Foundation Archimedes. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Gülçin, I. Antioxidant activity of food constituents: An overview. Arch. Toxicol. 2012, 86, 345−391. (2) FAOSTAT 2011; http://faostat.fao.org (accessed July 24, 2013). (3) Gómez-Romero, M.; Segura-Carretero, A.; Fernández-Gutiérrez, A. Metabolite profiling and quantification of phenolic compounds in methanol extracts of tomato fruit. Phytochemistry 2010, 71, 1848− 1864. (4) Slimestad, R.; Verheul, M. J. Content of chalconaringenin and chlorogenic acid in cherry tomatoes is strongly reduced during postharvest ripening. J. Agric. Food Chem. 2005, 53, 7251−7256. (5) Dixon, R. A.; Palva, N. L. Stress-induced phenylpropanoid metabolism. Plant Cell 1995, 7, 1085−1097. (6) Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: Food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727−47. (7) Krause, M.; Galensa, R. Analysis of enantiomeric flavanones in plant extracts by high-performance liquid chromatography on a cellulose triacetate based chiral stationary phase. Chromatographia 1991, 32, 69−72. 5179



Article

(23) Dangour, A. D.; Lock, K.; Hayter, A.; Aikenhead, A.; Allen, E.; Uauy, R. Nutrition-related health effects of organic foods: A systematic review. Am. J. Clin. Nutr. 2010, 92, 203−210. (24) Benbrook, C. Elevating Antioxidant Levels in Food through Organic Farming and Food Processing. An Organic Center State of Science Review; 2005; http://organic.insightd.net/reportfiles/Antioxidant_ SSR.pdf; (accessed July 28, 2013). (25) Valdmaa, U.; Keppart, L. Agricultural sector review, 2008, 2009, 2010. www.agri.ee (accessed July 10, 2013). (26) Waterhouse, A. L. Determnation of Total Phenolics. Current Protocols in Food Analytical Chemistry; Wrolstad, R. E., Ed.; Wiley: New York, 2001; pp I.1.1−1.1.8. (27) Helmja, K.; Vaher, M.; Püssa, T.; Raudsepp, P.; Kaljurand, M. Evaluation of antioxidative capability of the tomato (Solanum lycopersicum) skin constituents by capillary electrophoresis and highperformance liquid chromatography. Electrophoresis 2008, 29, 3980− 3988. (28) Lee, J. H.; Renita, M.; Fioritto, R. J., St; Martin, S. K.; Schwartz, S. J.; Vodovotz, Y. Isoflavone characterisation and antioxidant activity of Ohio soybeans. J. Agric. Food Chem. 2004, 52, 2647−2651. (29) Mitchell, A. E.; Hong, Y. J.; Koh, E.; Barrett, D. M.; Bryant, D. E.; Denison, R. F.; Kaffka, S. Ten-year comparison of the influence of organic and conventional crop management practices on the content of flavonoids in tomatoes. J. Agric. Food Chem. 2007, 55 (15), 6154− 6159. (30) Chassy, A. W.; Bui, L.; Renaud, E. N. C.; Van Horn, M.; Mitchell, A. E. Three-year comparison of the content of antioxidant microconstituents and several quality characteristics in organic and conventionally managed tomatoes and bell peppers. J. Agric. Food Chem. 2006, 54, 8244−8252. (31) Caris-Veyrat, C.; Amiot, M. J.; Tyssandier, V.; Grasselly, D.; Buret, M.; Mikolajczak, M.; Guilland, J. C.; Bouteloup-Demange, C.; Borel, P. Influence of organic versus conventional agricultural practice on the antioxidant microconstituent content of tomatoes and derived purees; consequences on antioxidant plasma status in humans. J. Agric. Food Chem. 2004, 52, 6503−6509. (32) Hallmann, E. The influence of organic and conventional cultivation systems on the nutritional value and content of bioactive compounds in selected tomato types. J. Sci. Food Agric. 2012, 92, 2840−2848. (33) Moco, S.; Bino, R. J.; Vorst, O.; Verhoeven, H. A.; de Groot, J.; van Beek, T. A.; Vervoort, J.; Ric de Vos, C. H. A liquid chromatography−mass spectrometry-based metabolome database for tomato. Plant Physiol. 2006, 141, 1205−1218. (34) Martínez-Valverde, I.; Periago, M. J.; Provan, G.; Chesson, A. Phenolic compounds, lycopene and antioxidant activity in commercial varieties of tomato (Lycopersicum esculentum). J. Sci. Food Agric. 2002, 82, 323−330. (35) Slimestad, R.; Verheul, M. Review of flavonoids and other phenolics from fruits of different tomato (Lycopersicon esculentum Mill.) cultivars. J. Sci. Food Agric. 2009, 89, 1255−1270. (36) Kähkonen, M. P.; Hopia, A. I.; Vuorela, H. J.; Rauha, J. P.; Pihlaja, K.; Kujala, T. S.; Heinonen, M. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 1999, 47, 3954−3962. (37) Mattila, P.; Kumpulainen, J. Determination of free and total phenolic acids in plant-derived foods by HPLC with diode-array detection. J. Agric. Food Chem. 2002, 50, 3660−3667. (38) Szliszka, E.; Czuba, Z. P.; Mazur, B.; Paradysz, A.; Krol, W. Chalcones and dihydrochalcones augment TRAIL-mediated apoptosis in prostate cancer cells. Molecules 2010, 15, 5336−5353. (39) Slimestad, R.; Fossen, T.; Verheul, M. J. The flavonoids of tomatoes. J. Agric. Food Chem. 2008, 56, 2436−2441. (40) Huang, D.; Ou, B.; Prior, R. L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 1841−1856.

5180


Three-Year Comparative Study of Polyphenol ... - ACS Publications

Recommend Documents