Bioanalytical Characterization of Apple Juice from 88 Grafted andNongrafted Apple Varieties Grown in Upper AustriaPeter Lanzerstorfer,†,‡ Jurgen Wruss,†,‡ Stefan Huemer,† Andrea Steininger,† Ulrike Muller,†

Markus Himmelsbach,§ Daniela Borgmann,∥ Stephan Winkler,∥ Otmar Hoglinger,†

and Julian Weghuber*,†

†University of Applied Sciences Upper Austria, Wels, Austria§Institute for Analytical Chemistry, Johannes Kepler University, Linz, Austria∥University of Applied Sciences Upper Austria, Hagenberg, Austria

*S Supporting Information

ABSTRACT: The compositional characteristics of untreated pure juice prepared from 88 apple varieties grown in the region ofEferding/Upper Austria were determined. Many of the analyzed varieties are noncommercial, old varieties not present in themarket. The aim of the study was to quantitate the mineral, phosphate, trace elements, and polyphenolic content in order toidentify varieties that are of particular interest for a wider distribution. Great variations among the investigated varieties could befound. This holds especially true for the total polyphenolic content (TPC) ranging from 103.2 to 2,275.6 mg/L. A cleardependence of the antioxidant capacity on the TPC levels was detected. Bioinformatics was employed to find specificinterrelationships, such as Mg2+/Mn2+ and PO4

3−/K+, between the analyzed bio- and phytochemical parameters. Furthermore,special attention was drawn on putative effects of grafting on the phytochemical composition of apple varieties. By grafting 27different apple varieties on two trees grown close to each other, it could be shown that the apple fruits remain their characteristicphytochemical composition. Finally, apple juice prepared from selected varieties was further characterized by additionalbiochemical analysis including cytotoxicity, epidermal growth factor receptor (EGFR) inhibition, and α-amylase activity tests.Cytotoxicity and inhibition of EGFR activation were found to be dependent on the TPC, while α-amylase activity was reduced bythe apple juices independent of the presence of polyphenolic substances. Taken together selected apple varieties investigatedwithin this study might serve as preferable sources for the development of apple-based food with a strong focus on healthbeneficial effects.

KEYWORDS: old apple varieties, polyphenolics, antioxidant activity, phytochemical composition, grafting

■ INTRODUCTION

Apples are one of the most widely cultivated fruits in Europe,especially in Germany and Austria.1,2 Within these countrieseach citizen monthly consumes on average 2.2 and 1.6 kg ofapples, respectively. According to the Food and AgricultureOrganization of the United Nations (FAO) the total worldapple production (2011) was ∼75 million tons. Austria ranks23rd among the producing countries (∼half a million tons peryear).From a medical perspective a high intake of these fruits is

desirable: the consumption of apples has been associated withlowered risks of cancer and cardiovascular and neuro-degenerative diseases.3−5 Oxidative processes involving reactiveoxygen species (ROS) and free radicals are considered to beone of the causes for these diseases. Apples are rich inphytochemicals including polyphenols that are known for theirantioxidant capacity and thus reduce the detrimental effects ofROS and free radicals.6 In addition, polyphenols have beenfound to modulate the Nrf2/ARE pathway, thereby protectingfrom oxidative damage.7 Numerous studies showed that thecontent of polyphenols and other health promoting phyto-chemicals in apples depend on maturity, location of production,and agricultural practices, as well as numerous environmental

factors.8 Consequently, every variety has its own typicalphytochemical composition.There is a great number of old and nonprevalent apple

varieties that are characterized by a high content ofphytochemicals.9,10 However, only a very limited number ofapple varieties actually dominate the market in Europe: morethan 70% of the total aggregate harvest is made up by the applevarieties Golden Delicious, Jonagold, and Red Delicious (includingoffspring varieties).11 In light of the enormous amount ofdifferent apple varieties (more than 20,000 worldwide12) thislow number is surprising. The reason for that is mainly thenecessity to meet various marketing schemes and customeracceptance for size, color, or sweetness. In addition, the storagestability of many apple varieties is unfavorable for large scalecultivation. In any case, information about the variety-typicalapple phytochemical composition, including, but not restricted,to polyphenolics, is essential for the development of consumer-relevant products with particular nutritional functionalities.

Received: August 2, 2013Revised: January 9, 2014Accepted: January 11, 2014Published: January 11, 2014

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This holds true especially for noncommercial, old varieties thatare only grown in restricted geographic regions.In this study the phytochemical composition of apple juices

derived from 88 mostly noncommercial old varieties of applescultivated in Upper Austria was determined. Information on thevast majority of the analyzed juices, especially concerning thepolyphenolic content, is still lacking. The results of our studywill help to identify available local apple varieties that are ofparticular interest for the development of apple-based food witha strong focus on health beneficial effects.

■ MATERIALS AND METHODSMaterials and Reagents. (+)-Catechin, α-amylase from porcine

pancreas, AAPH (2,2′-azobis(amidinopropane) dihydrochloride),ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), acetoni-trile, AG1478, bovine catalase, epidermal growth factor (EGF),fluorescein sodium salt, Folin−Ciocalteu reagent, in vitro toxicologyassay kit, methanosulfonic acid, phenolphthalein, potassium persulfate,potassium phosphate, nitric acid, rosmarinic acid, sodium bicarbonate,sodium hydroxide, trifluoroacetic acid (TFA), and Trolox [(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid] were pur-chased from Sigma Aldrich (Taufkirchen, Germany). Standards forK+, Mg2+, and Ca2+ (Certipur, cation multielement standard III) andantimony tartrate solution were from Merck-Millipore (Vienna,Austria). Potassium dihydrogen orthophosphate, ascorbic acid,methanol, and ammonium molybdate were purchased from VWR(Vienna, Austria). Sulfuric acid was obtained from Fisher Scientific(Vienna, Austria). Chlorogenic acid, epicatechin, epigallocatechin,epigallocatechin gallate, epicatechin gallate, procyanidin B1, procyani-din B2, and phloridzin were purchased from Extrasynthese (Genay,France). 96-Well plates were from Greiner Bio-One (Kremsmunster,Austria). Fetal calf serum, penicillin/streptomycin, and DMEM highglucose medium were purchased from PAA (Pasching, Austria). Aprotein phosphorylation analysis kit termed “cell-based Delfia assay”including a europium-labeled anti-phosphotyrosine-antibody wasobtained from Perkin-Elmer (Rodgau, Germany). The Syto62 cellstain reagent was from Life Technologies (Darmstadt, Germany).Apple Variety Selection and Preparation. All apple varieties

were grown under organic conditions in the Eferding region of UpperAustria. This region termed Eferdinger Becken is a plain landscape

area near the Danube river and has the most moderate climate inUpper Austria. The soil in this region consists mainly of loose orclayey sediments and is low or free of lime. The apples were collectedin September and October 2010 and immediately processed to applejuice. Apples were washed and the juice was pressed out using aconventional juice maker (Kenwood JE 850 XXL). At least 10 applesharvested from three to five individual trees were processed togenerate a combined juice for each variety to account for differenceswithin apples from the same variety. The samples were stored at <−60°C without addition of ascorbic acid. Analyzed apple varieties areshown in Table 1.

Total Phenolic Content (TPC). Total phenolic content wasdetermined using Folin−Ciocalteu reagent as described previously13

with small modifications. In short, apple juice was centrifuged at10,000 rpm for 10 min and only the supernatant was used for totalphenolic quantitation, since the cloudiness of apple juice has beenreported to influence the obtained TPC values.14 Deionized water (1.4mL) was mixed with 16.7 μL of apple juice supernatant and 83.3 μL ofFolin−Ciocalteu reagent. The mixture was allowed to stand for 3−6min at room temperature followed by addition of 167 μL of sodiumbicarbonate solution (200 g/L). After 70−75 min incubation at roomtemperature in the dark absorbance was measured at 750 nm. Totalphenolic content was expressed as (+)-catechin equivalents in mg/Lapple juice. Each juice was measured in triplicate.

Trolox Equivalent Antioxidant Capacity (TEAC). Totalantioxidant capacity of different apple juices was measured using theABTS decolorization assay.15 Apple juices were diluted 1:10 withdeionized water and analyzed by mixing 500 μL of diluted ABTSradical solution with 10 μL of diluted juice. After 5 min incubation at30 °C the absorbance was measured at 734 nm. Results werequantitated based on a dilution series of Trolox ranging from 6.25 μMto 1 mM. Each juice was measured in triplicate.

Oxygen Radical Absorbance Capacity (ORAC) Measure-ments. The ORAC assay was performed as described previouslywith slight modifications.16 Apple juices were centrifuged (5 min;10,000 rpm) prior to measurement. Supernatant was further diluted(1:200) with phosphate buffer (10 mM, pH 7.4). In short, 150 μL offluorescein (10 nM) was pipetted into each well and 25 μL of thestandard (Trolox) or diluted apple juice was added. The plate wasincubated at 37 °C for 30 min in the dark followed by addition of 25μL of AAPH solution (240 mM) per well. The decrease influorescence of fluorescein was determined by collecting readings at

Table 1. 88 Apple Varieties under Study Including 27 Apple Varieties, Which Were Grafted between the Years 2003 and 2008on Two Individual Trees Termed “F” and “L”

1 Alkmene 23 Hauxapfel 45 Roter Griesapfel L224 London Pepping2 Ananasrenette 24 Herrenapfel 46 Roter James Grieve L220 Magna Super3 Berneder 25 Jonathan 47 Roter Passamaner L203 Mostzigeuner4 Bismarck 26 Kammerapfel 48 Roter Stettinger L209 Rajka5 Boikenapfel 27 Kanada Renette 49 Samareiner Rosmarien L200 Rheinischer Winterrambour6 Carpetin 28 Kleiner Feiner 50 Schieblers Taubenapfel L226 Ribston Pepping7 Champagner Renette 29 Lesans Kalvill 51 Schmidberger Renette L217 Roter Herbstkalvill8 Damason Renette 30 Liberty 52 Schoner von Wiltshire L207 Seelander Reinette9 Deans Kuchenapfel 31 Maschanzker 53 Sommerrambour L202 Zuccalmanglios Renette10 Dulmaner Rosenapfel 32 Odenwalder 54 Spitzapfel F213 Christkindler11 Fasslapfel 33 Pilot 55 Spitzling F221 Discovery12 Florianer Rosmarin 34 Pinova 56 Steirischer Maschanzker F218 Florina13 Geheimrat Oldenburg 35 Piros 57 Weißer Passamaner F215 Freyperg14 Gelber Bellefleur 36 Plankenapfel 58 Weißer Winter-Taffetapfel F225 Gruter Edelapfel15 Gelber Edelapfel 37 Pom. Kongress 59 Winter-Goldparmane F223 Roter von Siemonffi16 Glasapfel 38 Prinzenapfel 60 Zabergau Renette F204 Royal Gala17 Glockenapfel 39 Relinda 61 Zuccalmaglios Renette F222 Sponheimer Flurapfel18 Goldrenette Freiherr von Berlepsch 40 Retina L214 Berlepsch F212 Staubli 219 Graue Herbstrenette 41 Rewena L205 Blenheim F216 Topaz20 Gruner Boskoop 42 Rheinischer Krummstiel L201 Dr. Seeligs Orangenpepping F219 Wachsrenette21 Harberts Renette 43 Riesenboikenapfel L211 Gewurzluiken F210 Weißer Winterkalvill22 Hausapfel 44 Roter Boskoop L206 Hallauer Maienapfel F208 Winterzitrone

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excitation of 485 nm and emission of 520 nm every minute for 90 minon a plate reader (POLARstar omega, BMG LABTECH, Ortenberg,Germany). The ORAC value was calculated using the ORAC plugin ofthe Omega MARS plate reader software. Each juice was measured intriplicate.Mineral Nutrients, Phosphate, and Trace Elements. The

minerals K+, Mg2+, and Ca2+ were quantitated by ion chromatography(Dionex ICS1000, Thermo Fisher Scientific, Vienna, Austria). AnIonpac CS 12A 4 mm column was used for the separation of thedifferent cations. The mobile phase consisted of 20 mMmethanosulfonic acid, the flow rate was 1 mL/min, and sampleinjection volume was 25 μL.Phosphate concentrations were measured using a phosphomolyb-

date method.17 The apple juices were passed through a 0.45 μm filter.500 μL of apple juice, 2 mL of ddH2O water, 100 μL of ascorbic acidsolution (10 g of ascorbic acid in 100 mL of ddH2O), and 200 μL ofammonium molybdate solution were mixed and filled up to 5 mL withddH2O. As a phosphate standard, potassium dihydrogen orthophos-phate was used. Absorbance was measured at 880 nm.The trace elements Mn2+, Cu2+, and Fe2+ were quantitated by

inductively coupled plasma mass spectrometry (ICP-MS). Prior tomeasurement 1 mL of apple juice was mixed with 3 mL of HNO3 andmicrowave-digested using the MLS Ultraclave-IV. The resulting clearsolution was diluted 1:10 with ddH2O. ICP-MS measurements wereperformed using an Agilent ICP-MS 7500 cx spectrometer (AgilentTechnologies, Santa Clara, CA, USA) operated in He mode (Mn2+ andCu2+) or H2 mode (Fe

2+). Each juice was measured in triplicate.Identification and Quantitation of Polyphenols by HPLC.

RPC-MS analysis was performed on an Agilent 1100 HPLC systemequipped with a vacuum degasser, a quaternary pump, an autosampler,and a UV−vis diode array detector (all from Agilent Technologies,Santa Clara, CA, USA). Separations were carried out using an ODSHypersil column (250 mm × 4.6 mm inner diameter; 1.8 μm particlesize; Thermo Fisher Scientific, Austria). Analytes were separated bygradient elution with 0.1% (v/v) formic acid (A) and acetonitrilecontaining 0.1% (v/v) formic acid (B) at a flow rate of 1 mL/min. Thelinear gradient elution program was as follows: starting conditions97.5% A and 2.5% B; the proportion of B was increased to 10% at 20min, 20% at 32 min, 50% at 45 min, and 80% at 50 min. The columnwas thermostatted at 40 °C, and the injection volume was 20 μL.MS measurements were done on a 6520 quadrupole/time-of-flight

(Q-TOF) instrument equipped with an electrospray ionization source(Agilent Technologies, Santa Clara, CA, USA). Results were obtainedusing the following settings: MS capillary voltage 3750 V, fragmentorvoltage 180 V, drying-gas (nitrogen) flow rate 12 L/min, drying-gastemperature 350 °C, and nebulizer pressure 60 psi. Scanning massrange was from m/z 70 to 3200 with an acquisition rate of 1.0 spectra/s in the negative MS mode.Quantitation of identified polyphenols was done using UV

absorption by reference substances of known concentrations preparedin deionized water. For reversed phase chromatography (RPC)analysis a Jasco LC-2000 Plus Series system comprising a quaternarypump with build-in degasser, an autosampler, a temperature controlledcolumn compartment, and a diode array detector (DAD) equippedwith Chrompass software (all from Jasco Corporation, Tokyo, Japan)was used. Separation was performed on a Hypersil ODS C18 column(250 mm × 4.6 mm inner diameter, 5 μm particle size; Thermo FisherScientific, Vienna, Austria). Column temperature was set to 40 °C, andelution was carried out at 1 mL/min. The injection volume for allsamples was 20 μL, and eluted substances were detected usingmultiple UV wavelengths from 200 to 350 nm. The followingconditions were used for RPC analysis: Mobile phase A contained0.1% TFA in water. Mobile phase B contained acetonitrile, water, andTFA in the ratio 50:50:0.1 (%). Mobile phase C contained 0.1% TFAin acetonitrile. The starting conditions were 95% A and 5% B. Elutionwas performed with a linear gradient: The proportion of B wasincreased to 20% at 20 min, 40% at 32 min, and 100% at 45 min. Limitof detection (LOD) was defined as signal-to-noise ratio of 2:1 andlimit of quantitation (LOQ) as 4:1. For flavan-3-ols LOD of 0.1 mg/Land LOQ of 0.4 mg/L were defined with a linear range of 1−500 mg/

L. For hydroxycinnamic acids LOD of 0.05 mg/L and LOQ of 0.2 mg/L were defined with a linear range of 1−1,000 mg/L. For quercetinderivates LOD of 0.1 mg/L and LOQ of 0.3 mg/L were defined with alinear range of 0.1−100 mg/L. Apple juice samples were centrifugedfor 10 min at 15,000 rpm followed by 0.1 μm filtration to remove anyremaining solids before analysis.

Quantitation of Acid Content by HPLC. Malic and citric acidcontent was quantitated for each sample using a RPC methoddescribed previously.18 Quantitation was done using purified malic andcitric acid dissolved in deionized water. For RPC analysis a Jasco LC-2000 Plus Series system was used as described above. Separation wasperformed on a Sorbax SB-C18 column (75 mm × 4.6 mm innerdiameter, 3.5 μm particle size; Agilent Technologies, Santa Clara, CA,USA). Column temperature was set to 35 °C, and isocratic elution wascarried out at 0.5 mL/min. A 50 mM potassium phosphate bufferadjusted to pH 2.8 was used as mobile phase. Apple juice samples werecentrifuged for 10 min at 15,000 rpm followed by 0.1 μm filtration toremove any remaining solids before analysis. The injection volume forall samples was 2 μL, and eluted substances were detected at 215 nm.Limit of detection (LOD) was defined as signal-to-noise ratio of 2:1and limit of quantification (LOQ) as 4:1. For citric acid a LOD of 20mg/L and LOQ of 50 mg/L were defined with a linear range of 20−1,000 mg/L. For malic acid the LOD was defined at 50 mg/L and theLOQ at 100 mg/L with a linear range of 0.1−20 g/L.

Quantitation of Fruit Acids by Titration. Total acid content wasdetermined using the acidic titration method described in an OECDguideline for food production, which can be found on the OECD Website (www.oecd.org/agriculture). In short, apple juice was diluted 1:10with deionized water, mixed with 3 μL of phenolphthalein, and titratedwith 0.1 M sodium hydroxide until the point of neutrality was reached(indicator changes from colorless to pink). Results were expressed asg/L of malic acid.

Quantitation of Anthocyanin Pigment Content. Theanthocyanin content of apple juices was determined using a pHdifferential method.19 In brief, apple juices were diluted 1:5 either inpH = 1.0 buffer (0.025 M potassium chloride) or in pH = 4.5 buffer(0.4 M sodium acetate) adjusted with HCl and transferred to 10 mmcuvettes. Absorbance (A) of each diluted sample was determined at520 and 700 nm within 15 min after preparation for both pH values.The anthocyanin pigment concentration was calculated as cyanidin-3-glucoside equivalent. The quantitation limit for this method wasdetermined to be 0.1 mg/L. Each juice was measured in triplicate.

Sugar Content Measurements. The sugar content of analyzedapple juice samples was measured using a RHB-55 refractometer(PCE-group, Meschede, Germany). In short, a single drop ofundiluted apple juice was loaded onto the prism and the °Brix valuewas read from the graduation.

Amylase Activity. Inhibition of amylase activity by various applejuices was analyzed using a modified commercial amylase assay(Abcam, Cambridge, U.K.). The nitrophenol included in the assay wasused to generate a standard curve. The apple juice samples wereincubated with 1 mg/mL α-amylase for varying time periods in 96-wellplates. For α-amylase activity measurements, double distilled water wasused as a reagent blank, and rosmarinic acid (1.5 mg/mL), a known α-amylase inhibitor,20 served as a positive control. 50 μL of theindividual samples (apple juice diluted 1:10 in ddH2O) was mixed with100 μL of reaction reagent composed of 50 μL of assay buffer and 50μL of substrate mix (containing substrate and α-glucosidase).Absorption was measured using a plate reader (POLARstar omega,BMG LABTECH, Ortenberg, Germany) at 405 nm (absorptionmode) at 1 min timed intervals. Each juice was measured in triplicate.

Cytotoxicity. Cytotoxicity was analyzed using a resazurin-basedassay. Human adenocarcinoma HeLa (DSMZ, Braunschweig,Germany) and human hepatocarcinoma HuH-7 cells (kind gift fromJohn McLauchlan, University of Glasgow) were seeded in 96-wellplates (30,000 cells/well) in 200 μL of DMEM high glucose mediumsupplemented with 10% fetal calf serum and 100 μg/mL penicillin/streptomycin. Cells were grown to 90% confluency in a humidifiedatmosphere at 37 °C and 5% CO2. Test substances were added atvarying concentration followed by incubation at 37 °C for different

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time periods. Catalase was added for some experiments. The cells werewashed once with medium and incubated with 10% resazurin solutionin cell culture medium for two hours at 37 °C and 5% CO2. Finally,fluorescence intensity (544 nm excitation, 590 nm emission) wasmeasured using a plate reader (POLARstar omega, BMG LABTECH,Ortenberg, Germany). Data analysis was done using the OmegaMARSData analysis software package (BMG Labtech, Ortenberg, Germany).Each juice was measured in triplicate.EGFR Activity. The epidermoid carcinoma cell line A431 (DSMZ,

Braunschweig, Germany) was grown in DMEM high glucose culturemedium in 96-well plates for 16 h (50,000 cells/well). Complete cellculture medium was removed, and cells were starved (DMEM + 0.1%BSA without FBS) for 3 h followed by incubation with different applejuices or a known inhibitor of EGFR phosphorylation (AG1478) for 6h. Cells were then washed once with PBS and treated with EGF (170nM) for 5 min. Immediately afterward cells were fixed andpermeabilized by addition of 100 μL of −20 °C cold methanol for10 min. After an additional PBS washing step 100 μL of Delfia-Eu-anti-phosphotyrosine-antibody was added at a concentration of 300 ng/mL(diluted in Delfia Assay Buffer supplemented with 0.5% BSA). Theplate was incubated for 2 h at room temperature and then washed fourtimes with Delfia Wash Solution. 200 μL/well Delfia Enhancementsolution was added for 5 min, and the fluorescence intensity wasmeasured using the time-resolved fluorescence protocol on aPOLARstar Omega plate reader (BMG Labtech, Ortenberg,Germany) with the following settings: start time 400 μs, cycle time1 ms, excitation 355 nm, and emission 620 nm. Results werenormalized to the total cell number per well using Syto62 cell staining.Each juice was measured in triplicate.

■ RESULTS AND DISCUSSION

Physicochemical Properties of Investigated AppleVarieties. It is a common sense that different maturity offruits affects the accumulation of phytochemicals.8 Thus theripeness of the investigated apple varieties by quantitation ofthe sugar content (°Brix value) and the sample acidity(titratable acidity (TA), malic and citric acid) was determined.As shown in Table 2 a mean °Brix value of 12.9 ranging from8.0 to 18.9, and a mean titratable acidity of 0.8 (% as malicacid) ranging from 0.27 to 1.95 was found. These resultsindicate a high sugar:acid ratio being consistent with a ripe stateas specified by the FAO. The values for the analyzed applejuices can be found in Supplementary Table 1 in theSupporting Information. All samples are characterized by highsugar levels, and the vast majority have been found to have lowacidity. Some apple juice varieties, e.g., Zuccalmaglios Renetteor Geheimrat Oldenburg, have been demonstrated to haverather high, variety-specific acidity levels. Nevertheless, thosesamples were included in our study since the obtained °Brixvalues are within an acceptable range. Taken together, collected

data confirm a sufficient maturity of the apples at the time ofharvesting.

Quantitation of Polyphenols. A high variability for TPCwas found ranging from only 103.2 mg/L up to 2.3 g/L in somevarieties (Table 3). For the full list see Supplementary Table 1in the Supporting Information. The mean TPC of all 88analyzed varieties was determined to be 777.7 mg/L. A largestandard deviation of 447.2 mg/L emphasizes the significantdifferences of the polyphenol content among the applevarieties. Our results clearly showed that the TPC levels ofcertain, popular apple varieties are relatively low: For example,the TPC content of juice prepared from Royal Gala (F204) orTopaz (F216) apples grown in the Eferding region was foundto be only 185.8 and 257.4 mg/L, respectively. Gala apples areamong the most common sold apple varieties worldwide(number 2 on the US market), and Topaz apples are highlypopular especially in Central Europe. The great majority of theapple varieties (∼60%) that were investigated within this studyare used as dessert apples, 25% for the production ofnonalcoholic or alcoholic beverages, and 15% as industrialapples that can be, e.g., utilized for food production. However,the economic significance of most of these apple varieties israther low and limited to the region of Eferding in UpperAustria. Thus, attempts for a broader distribution of selecteddessert apple varieties with high TPC levels as characterized inthis study (e.g., Harberts Renette, Odenwalder, and Zuccalma-glios Renette) should be made to tap the full potential of thesevarieties.A drawback of the FC method is that other reducing reagents

present in the apple juice supernatant, such as ascorbic acid,might lead to an overestimation of obtained TPC values.21 Inaddition, being a major feature of this study, the assay is notsufficient to predict the antioxidant effect of apple juice, sincebiological effects are to be expected from different polyphenols.To address this question, the exact composition of thepolyphenol components has to be unraveled. Importantly,using the Phenol-Explorer,22 information on the polyphenoliccontent could only be extracted for two apple varieties that areincluded in this study, namely Royal Gala and Gru ner Boskoop.However, a direct comparison to our results appears difficultdue to application of juice extracts for these studies rather thananalyzing untreated apple juice.23,24

Fifteen polyphenolic compounds belonging to four differentmajor polyphenolic groups were identified in the apple juicesprepared from each variety: chlorogenic, caffeic, and 4-p-coumaroylquinic acid (hydroxycinnamic acids), phloretin-2′-O-xyloglucoside and phloridzin (dihydrochalcone derivates),

Table 2. Ripeness Parameters (°Brix, Titratable Acidity, Malic Acid, Citric Acid) of Apple Juice Prepared from 88 AppleVarieties

units mean SD % CV min max range

°Brix 12.9 2.0 15.2 8.0 18.9 10.9TA (% as malic) 0.80 0.31 36.90 0.27 1.95 1.68malic acid (mg/L) 7175.4 4261.4 59.4 127.5 17245.1 17117.6citric acid (mg/L) 115.9 91.6 79.0 28.4 522.6 494.2

Table 3. Total Phenolic Content (TPC) and Antioxidant Capacities of Apple Juices under Study

units mean SD % CV min max range

TPC (mg/L) 777.7 447.2 57.5 103.2 2275.6 2172.4TEAC (mmol/L) 3.3 1.6 48.3 0.8 7.8 6.9ORAC (mmol/L) 18.8 9.5 50.5 3.0 58.9 55.9

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procyanidin B1 and B2, (−)-epicatechin, epigallocatechin, andepicatechingallate (flavan-3-ols), and five quercetin derivates(flavonols). A representative HPLC-DAD diagram, indicatingretention times and maximal wavelengths of each compound, isshown in Supplementary Figure 1 in the SupportingInformation. Table 4 summarizes the content of selectedpolyphenolics of all 88 apple juice samples. In addition,obtained values for each variety can be found in SupplementaryTable 2 in the Supporting Information. Among the fiveinvestigated polyphenolic groups, the hydroxycinnamic acidgroup was found to be the most abundant one, withchlorogenic acid being the main compound in this polyphenolgroup. The ratio between chlorogenic acid and p-coumar-oylquinic acid content in different varieties has been reported tovary between 1.2 and 37.1.25 Those ratio limits are in goodagreement with the mean ratio of 20.3 of the apple juicesamples presented in this study. However, for some samplesmuch higher ratios up to 104.1 (L203) or 181.5 (L214) werefound, which was mainly caused by high levels of chlorogenicacid and at the same time a very low concentration of p-coumaroylquinic acid being detected in these varieties. The lowamounts of caffeic acid found in most samples are in goodagreement with other studies.26

Flavan-3-ols represent the second largest group of poly-phenols detected in the investigated apple juices. ProcyanidinB2 and epicatechin were the most abundant polyphenolicsubstances in this group with a mean concentration of 40.1 and13.3 mg/L, respectively. Similar amounts were reported in aprevious study analyzing untreated apple juices.9 Twodihydrochalcone derivates were found in most apple juices:With a mean content of 6.8 mg/L phloridzin and 12.8 mg/Lphloretin-2′-O-xyloglucoside, our results are within the range ofa previous study (∼1−25 mg/L each).27 Our measurements

also confirmed the presence of flavonols. However, thispolyphenol group was found in very low concentrations(∼2% of total polyphenol amount). Anthocyanin pigmentswere found only in a few juice samples at low concentrationsranging from 0.13 to 6.79 mg/L. This is in line with thepresence of these phytochemicals exclusively in applescharacterized by a red skin.28

In agreement with the results from TPC measurements, RPCanalysis unraveled great differences in the polyphenol contentand composition of apple juices from different apple varieties.For example, the amount of chlorogenic acid for some varietiesranged from 1.79 mg/L (Royal Gala, F204) up to 1209.7 mg/L(Harberts Renette). In general, the estimated TPC valuescorrelate with the concentration of polyphenols found byHPLC measurements. However, a high TPC level does notnecessarily correlate with the detected amounts of the analyzedsingle polyphenols. For example, the varieties F213, L202, andL203 are all characterized by a similar TPC value of about 1.000mg/L. However, the concentration of chlorogenic acid wasfound to vary between 76.4, 149.73, and 145.89 mg/L,respectively. Thus, our results clearly showed that juicesprepared from various apple varieties are highly diverse intheir content of total polyphenols and show great variations intheir individual polyphenol composition. Furthermore, whencomparing different apple varieties grafted on the same tree (Fand L series, respectively), it could be observed that thesevarieties retain their individual polyphenolic profile. Thedescribed differences are likely to depend mainly on geneticfactors, which is consistent with a study analyzing the geneticvariability of apples.29 In conclusion, grafting proves to be asuperior tool for a fast growth of selected apple varieties withvarying phytochemical concentration, without the need for thetime-consuming cultivation of the whole tree.

Table 4. Single Polyphenol Content (mg/L) of Apple Juices under Study

mean SD % CV min max range

Hydroxycinnamic Acidschlorogenic acid 216.3 223.8 103.5 +a 1209.2 1209.2caffeic acid 3.8 4.4 116.4 + 32.5 32.54-p-coumaroylquinic acid 11.7 13.3 114.7 + 55.5 55.5∑ hydroxycinnamic acids 231.8

Dihydrochalcone Derivatesphloretin-2′-O-xyloglucoside 12.8 10.1 78.8 + 54.3 54.3thloridzin 6.8 5.9 85.6 + 29.5 29.5∑ dihydrochalcone derivates 19.6

Flavan-3-olsprocyanidin B1 3.5 5.2 147.6 + 23.0 23.0procyanidin B2 40.1 48.3 120.4 + 338.1 338.1(−)-epicatechin 13.3 17.5 131.8 + 104.7 104.7epigallocatechin 3.3 6.6 200.8 + 38.2 38.2epichatechingallate 11.3 10.4 91.9 + 51.0 51.0∑ flavan-3-ols 71.5

Flavonolsquercetin-3-O-galactoside 0.7 2.9 396.7 + 26.4 26.4quercetin-3-O-xyloside 2.5 4.9 194.8 + 41.1 41.1quercetin-3-O-rhamnoside 2.4 3.3 137.2 + 25.2 25.2quercetin 3.0 5.9 196.0 + 49.2 49.2quercetin-3-O-rutinoside 0.3 1.8 580.6 + 15.7 15.7∑ flavonols 8.9∑ anthocyanins 1.01 1.41 139.41 0.13 6.79 6.66total polyphenol amount (HPLC) 331.8

a+, < limit of quantitation.

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Quantitation of Total Antioxidant Capacity. Severalstudies have shown that apples possess a strong antioxidantcapacity.6,30 Here two different methods (TEAC and ORAC)were used to measure the total antioxidant capacity of thedifferent apple juices. Table 3 summarizes the TEAC andORAC values derived from all samples. The antioxidantcapacity of these juices ranged from 0.8 to 7.8 mM (TEAC)and 3.0 to 58.9 mM (ORAC), which is in good agreement withsimilar studies.31 The full list of TEAC and ORAC values fromall apple juice samples can be found in Supplementary Table 1in the Supporting Information. Our results indicate the generaldependence of the antioxidant capacity on the totalpolyphenolic concentration as can be seen by the linearregression and correlation analysis. The coefficients ofdetermination (R2) are given in Figure 1. All R values(correlation coefficients) were positive at the P < 0.0001significance level, indicating that the values of antioxidantcapacities, assayed by the two different methods, were highlycorrelated (R values for TPC/ORAC, 0.69; TPC/TEAC, 0.87;ORAC/TEAC, 0.63). The regression coefficient value obtainedfor TPC and ORAC assay was lower compared with TEACassay, but significant in both systems. These results indicatedthat the two assays were suitable and reliable for assessing totalantioxidant capacities. Thus, despite the aforementionedlimitations of the FC method to determine the TPC, it canpossibly be considered as a first indicator of antioxidantcapacity of apple juice.It was observed that some varieties contained equal (TPC of

Lesans Kalvill similar to Samareiner Rosmarien) or even moretotal polyphenols than others (TPC Riesenboikenapfel >Glasapfel), but lower antioxidant capacity as determined byboth TEAC and ORAC measurements. This effect can beexplained by the different antioxidant activity of individualpolyphenols as reported in previous studies.32 Consequently,the antioxidant capacity is dependent on the phenoliccomposition of an individual juice.The low TEAC and ORAC values of juice prepared from

certain apple varieties, including Topaz, is consistent with other

studies analyzing the antioxidant capacity of a number of oldand new apple varieties in Poland.33 The Topaz apple is a goodexample of a new apple variety (introduced in the 1980s),which offers several advantages for agriculture includingresistance to apple scab, high yields, and good storageproperties. It is also a common apple variety for organicfarming. However, the low level of phenolic substances in thesevarieties has not been taken into consideration so far.Consequently, the distribution of apple varieties with a highercontent of polyphenols should be supported to promote theirpositive effects on human health.

Analysis of Minerals, Phosphate, and Trace Elements.The mean and range values of the major minerals K+, Mg2+, andCa2+ are indicated in Table 5, and results for individual varietiescan be found in Supplementary Table 1 in the SupportingInformation. The potassium concentration ranged from 620.4(Retina) to 2064.0 mg/L (Gruner Boskoop) with largevariations between individual varieties. Compared to potassium,the concentration of magnesium and calcium was significantlylower, ranging from 8.4 to 64.4 (Mg2+) and 6.7 to 57.5 (Ca2+)mg/L, respectively. Similar to the potassium concentration,remarkable variations for calcium and magnesium between thedifferent varieties could be observed. Collected data, especiallyfor potassium and copper, are in good agreement with otherstudies analyzing minerals and trace elements of different applevarieties.26,34

The PO43− concentration of juice prepared from 56 apple

varieties was found to range between 90.0 and 420.0 mg/L(Table 5), which is consistent with the work of Eisele et al.,who reported PO4

3− values ranging from 86.0 to 459.0 mg/L.26

The variation between individual apple varieties was found tobe in a similar range as observed for potassium.Finally, 52 apple juice samples were analyzed for their copper

and manganese content and 27 (F and L series) for iron,respectively. The observed variations between individualsamples, especially those for Mn2+ and Fe2+, were highlypronounced for these elements. The mean and range values forthe Cu2+, Mn2+, and Fe2+ content are summarized in Table 5.

Figure 1. Linear correlation regression analysis. Correlations between (A) TPC and ORAC; (B) TPC and TEAC; (C) ORAC and TEAC. TPC:total phenolic content. ORAC: oxygen radical absorbance capacity. TEAC: Trolox equivalent antioxidant capacity.

Table 5. Mineral Content of Apple Juices under Study

units mean SD % CV min max range

K+ (mg/L) 1082.7 23.5 23.5 620.4 2064.0 1443.6Mg2+ (mg/L) 30.7 1.2 45.0 8.4 64.4 56.1Ca2+ (mg/L) 23.7 0.8 41.8 6.7 57.5 50.9Cu2+ (μg/L) 320.3 9.7 35.9 109.8 572.2 462.5Mn2+ (μg/L) 307.9 18.4 70.8 59.5 688.5 629.0Fe2+ (μg/L) 268.3 10.5 46.5 130.0 670.0 540.0PO4

3− (mg/L) 209.7 5.3 33.9 90.0 420.3 330.3

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Taken together, our results show large variations between thedifferent apple varieties, which is in good agreement with aprevious study analyzing the concentration of various mineralsin juice prepared from 175 apple varieties.26 These variationscan also be observed for various apple varieties grafted on asingle tree (F and L series). Thus, apples harvested from graftedtrees retain not only their polyphenolic profiles but also theircharacteristic mineral, trace element, and phosphate concen-trations. From this point of view an intensified cultivation ofselected apple varieties identified in this work should beconsidered. However, the availability of many of the varietiesthat possess these positive compositions for large scalecultivation remains a limiting factor. Engrafting turned out tobe an excellent way to enhance growth rates and provideresistance to bacterial or fungal infections. By engrafting 27different apple varieties on two trees grown close to each other,it could be shown that the apple fruits in fact remain theirprimary ingredient characteristics. This fact is clearly of keyimportance for the promotion of selected apple varieties.Biological Effects of Selected Apple Juice Varieties.

Different assays were used to determine several biologicaleffects of apple juice varieties that were preselected by apronounced variation of different ingredients includingpolyphenolics, minerals, and trace elements. Juices from nineapple varieties grafted on a single tree (F series) were used forthese analyses. First, the cytotoxic effects of apple juice weredetermined on two different human cancer cell lines using aresazurin based assay. Apple juice has been reported to be astrong cancer chemopreventive.35 Several studies have alreadyshown the inhibitory effect on cell proliferation of culturedcancer cell lines.36 Two human cell lines were used forinvestigation of the growth inhibition of selected apple juices.As shown in Figure 2A a strong reduction of cell viability couldbe observed in both cell lines for several apple juices at a 1:5dilution. The observed inhibitory effect was clearly dependenton the apple juice concentration: no reduction in cell viabilitycould be observed at higher dilution rates >1:50 (Supple-mentary Figure 1 in the Supporting Information). Using applejuice in cell culture medium might lead to H2O2 formation andpronounced cytotoxic effects.37 Addition of catalase (100 U/mL) to prevent the formation of H2O2 further slightly reducedthese cytotoxic effects (data not shown). Interestingly, a clearcorrelation between TPC levels and the described cytotoxiceffects was found (Figure 2B). Our results show that certainapple juices reduce the viability of the analyzed human cancer

cell lines in a TPC dependent manner. Interestingly, Veeriah etal. showed that native apple extracts were about twice as potentas a composed mixture of low molecular weight applepolyphenols in inhibiting cancer cell growth.36 This indicatesthat other constituents, such as oligomeric procyanidines,substantially contribute to the potent antiproliferative proper-ties of polyphenol-rich apple juices. Thus, the usage of applejuices instead of extract appears straightforward.In a second experiment a putative apple juice dependent

inhibition of human α-amylase, a major digestive enzyme thatbreaks down long-chain carbohydrates, was evaluated. Salivaryand pancreatic α-amylases lead to the formation of maltose andother related oligomers by catalyzing the hydrolysis of α-1,4-linked glucose chains.38 Several studies indicated a beneficialhealth effect of bioactive substances from apples, e.g., a reducedrisk of chronic diseases including type 2 diabetes. In this regardthe inhibition of α-amylase activity by these substances is ofparticular importance.39 As shown in Figure 3 a 30−40%inhibition of α-amylase activity was observed when rosmarinicacid (1.5 mg/mL) was added, confirming the inhibitorypotential of this substance. All tested apple juices of the Fseries also showed an incubation-time-dependent inhibition ofα-amylase activity between ∼80% (24 h incubation) and ∼50%

Figure 2. Cytotoxicity of selected apple juice varieties. (A) Resazurin based cytotoxicity test. HeLa or HuH-7 cells were grown to 90% confluency in96-well plates and incubated with apple juice diluted in cell culture medium (1:5 dilution) for 6 h. Cell viability is normalized to a nontreated sample.Error bars are based on the standard error of the mean (n = 4). (B) Linear correlation regression analysis between TPC and viability. Correlationanalysis reveals a significant interrelation of the total phenolic content (TPC) and the cell viability.

Figure 3. Inhibition of α-amylase activity by selected apple juicevarieties. Rosmarinic acid (Rosm. acid; 1.5 mg/mL) or different applejuice samples diluted 1:10 were incubated with 1 mg/mL α-amylasefor 30 min or 24 h, respectively. Inhibition was measured by the use ofa commercial amylase activity assay containing α-glucosidase. Errorbars are based on the standard error of the mean (n = 3).

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(30 min incubation). In these experiments differences betweenindividual varieties ranging from 40 to 70% (30 min) and 50 to98% (24 h) inhibition of α-amylase activity could be observed.In contrast to the antioxidant capacity, the observed inhibitoryeffect was not dependent on the TPC levels of the apple juicevarieties as statistical analysis determining Kendall’s andSpearman’s rank correlation coefficient did not reveal anysignificant correlation (data not shown). These results are ingood agreement with a recent study excluding a positivecorrelation between α-amylase inhibitory activity and totalphenolic content.39 However, single polyphenols that havebeen shown to inhibit this enzyme (e.g., chlorogenic acid)40 arefound at high concentrations in our apple varieties.Finally, the effects of different apple juice varieties on the

activity of the epidermal growth factor receptor (EGFR) wereanalyzed. The EGFR plays a major role in cellular signaling:

insufficient signaling may lead to the development ofneurodegenerative diseases,41 while excessive EGFR signalingis associated with the development of a wide variety of tumors.Highly elevated EGFR signaling seems to be a critical factor inthe development and malignancy of these tumors.42 Severalstudies have indicated that apple polyphenols inhibit EGFRactivity in various cell lines.43−45 Thus, apples rich inpolyphenols are thought to prevent the formation of variouscancer types in the human body. In total nine apple juicevarieties of the F series were analyzed for a potentialphosphorylation inhibition of the EGFR. As shown in Figure4A the applied time-resolved-fluorescence based ELISA-assay iswell-suited to detect phosphorylation of the EGFR uponstimulation. AG1478 pretreatment for 4 h clearly inhibited thephosphorylation upon EGF stimulation. When cells wereincubated for 4 h with juice from different apple varieties (1:20;

Figure 4. Epidermal growth factor receptor (EGFR) activity test. (A) HeLa cells endogenously expressing high levels of EGFR were grown to 90%confluency in 96-well plates and starved in cell culture medium without serum for 3 h, followed by incubation with the EGFR inhibitor AG1478 orselected apple juice varieties (diluted 1:20 in starving medium) for a further 6 h. After addition of 170 nM EGF, receptor phosphorylation wasmeasured by the use of a fluorescent antibody in combination with time-resolved fluorescence. Results were normalized to EGF treated cells(positive control). AG1478 served as a negative control. Error bars are based on the standard error of the mean (n = 3). (B) Linear correlationregression analysis reveals a significant interrelation of the total phenolic content (TPC) and the EGFR phosphorylation level.

Figure 5. Graphical representation of modeling results and variable interactions using linear regression and random forests. All variables weremodeled using linear regression (LR) and random forest regression (RF, 50 trees). Each node represents a variable, and the number given below avariable indicates the average quality (R2) of the models. In order to estimate the influence of the variables on each other, all variables are mutuallyexcluded from the modeling process, and the resulting modeling quality decrease is calculated. If the exclusion of a variable a leads to a significantdecrease of the modeling quality for variable b, then a directed edge is shown; continuous lines indicate modeling influences of at least 40%, dashedlines indicate modeling influences of at least 15%, and weaker influences are not shown. As TPC and TEAC are very strongly correlated (correlationR2 0.7125), these two variables are always excluded from the modeling process in combination, which is indicated by the dashed circles around thesetwo variables.

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in the presence of catalase), a pronounced inhibition of EGFRphosphorylation could be observed. Our experiments indicatedremarkable differences in the degree of phosphorylationinhibition depending on the used apple varieties. Furtheranalysis showed that the observed inhibitory effects significantlycorrelated with the respective TPC levels (Figure 4B), which isconsistent with similar studies. Individual polyphenols such asthe procyanidin dimers B1 and B2 or phloretin and phloretin-2′-O-xylogucoside have been found to specifically affect theEGFR activity.43−45 Accordingly, apple juice varieties thatinhibited the EGFR activity to the highest extent (F208, F213,and F223) were especially rich in these polyphenols.In-Depth in Silico Analysis. The described dependence of

the antioxidant capacity on the TPC levels is in agreement withprevious studies.6 This research was extended by the use ofmathematical modeling that allowed us to identify variableinteraction networks based on the analysis of apple componentdata. Regression models that approximate selected targetvariables using other available parameters in this data set havebeen identified. The relevance of a variable in this context canbe defined via the frequency of its occurrence in modelsidentified by evolutionary machine learning methods or via thedecrease in modeling quality after removing it from the dataset.46 The following algorithms have been applied for the dataset generated in this study including the results for TPC,TEAC, ORAC, Mn2+, Mg2+, Ca2+, Cu2+, K+, and PO4

3−: linearregression and random forests.47 As shown in Figure 5A, linearregression confirms the relationship of the antioxidant capacity(TEAC and ORAC) and the TPC level. In addition, asignificant interrelationship of Mg2+ and Mn2+ was found.These findings were confirmed when nonlinear modeling usingrandom forest was applied (Figure 5B). The latter model alsoindicated a strong relevance of PO4

3− and Mg2+ on themodeling of K+. Even though importance in regression andcorrelation do not imply causality, this analysis implies thatapples that are rich in Mg2+ by trend also contain higher levelsof Mn2+. The same assumption holds true for K+ and PO4

3−.Mn2+ and Mg2+ are abundant elements and essential to all livingcells. For example Mg2+ plays a major role in manipulatingbiological compounds including DNA, RNA, and ATP. Inaddition, a great number of enzymes require Mg2+ for theirfunction. The same is true for Mn2+ ions, which are essentialcofactors for many enzymes. However, many of these enzymescan use Mg2+ as a replacement of Mn2+.48 Of special interest isthe function of Mn2+ enzymes to detoxify superoxide freeradicals in mitochondria.49 In analogy copper and zinc boundenzymes are necessary for detoxification in the cytosol.50 Thus,similar to polyphenols, Mn2+ and Cu2+ ions play a key role inpreventing the human organism from oxidative damage.

■ ASSOCIATED CONTENT

*S Supporting InformationFigure depicting HPLC elution profile of a representative applejuice sample. Figure depicting influence of selected apple juicesamples with varying total phenolic content on HuH-7 cellviability. Table presenting overview of the phytochemicalcomposition of juice prepared from 88 apple cultivars. Tablepresenting overview of single polyphenol content of juiceprepared from 88 apple cultivars. This material is available freeof charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +43(0) 50804 44403. E-mail: julian.weghuber@fh-wels.at.

Author Contributions‡P.L. and J.W. contributed equally to this work

FundingThis work was funded by the program “Regionale Wettbe-werbsfahigkeit OO 2007−2013” with financial means of theEuropean fund for regional development as well as the countryof Upper Austria.

NotesThe authors declare no competing financial interest.

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