Chemical profile of major taste- and health-related compounds of (Oblačinska) sour cherry

Research ArticleReceived: 21 July 2014 Revised: 12 March 2015 Accepted article published: 13 April 2014 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7212

Chemical profile of major taste- andhealth-related compounds of Oblacinska sourcherryHassan Omran S Alrgei,a Dragana C Dabic,b Maja M Natic,c Vera S Rakonjac,a

Dušanka Milojkovic-Opsenica,c Živoslav Lj Tešicc and Milica M Fotiric Akšica*

Abstract

BACKGROUND: Oblacinska sour cherry, an autochthonous cultivar, is the most planted cultivar in Serbian commercial orchards.Owing to its long cultivation under different agro-ecological conditions, it is a mixture of different clones. To obtain comprehen-sive information on Oblacinska sour cherry fruit, the total carbohydrate composition, total phenolic content, total anthocyanincontent, antioxidant activity and polyphenolic profiles of 39 clones were investigated.

RESULTS: Fructose was found to be the dominant sugar, followed by glucose, sorbitol and sucrose. Rutin and chlorogenic acidwere the most abundant polyphenols. Some clones contained pinobanksin, hesperetin and galangin, which, to best knowledge,is the first time these compounds have been reported in sour cherry fruit.

CONCLUSION: The data obtained showed huge variation within the germplasm studied, confirming the fact that Oblacinska sourcherry is not a cultivar but a population.© 2015 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: Oblacinska sour cherry; phenolic acids; anthocyanins; sugars

INTRODUCTIONSour cherry (Prunus cerasus L., Rosaceae) is a deciduous tree origi-nating from areas around the Black and Caspian Seas. It is segmen-tal alotetraploid resulting from a natural hybridization betweensweet cherry (Prunus avium L.) and ground cherry (Prunus fruticosaPall.).1 Owing to its rapid coming to bearing, minor agro-biologicalneeds, high and regular yields, high content of bioactive com-pounds, numerous uses in the food industry and good marketdemand, there is great interest in expanding cultivation of this fruitvariety.2

Cherry fruits are commonly used for home or industrial produc-tion of juices, canned fruit, brandy, jams, preserves, etc. Oblacinskasour cherry is an autochthonous cultivar and is the most plantedsour cherry cultivar in Serbian commercial orchards. Because ofits long cultivation under different agro-ecological conditions anddifferent propagation types (both by suckers and by seeds), it is amixture of different clones. General characteristics of this cultivarare low vigor and regular high yields (15–20 t ha−1). The fruit is of‘morello’ type, small to medium in size, with dark red and thin skin.The flesh is red, medium firm, juicy, quite sour, aromatic and of highquality.3

The first selection from a diverse gene pool was conducted bylocal farmers in order to obtain certain desirable traits. Later, aseries of authors who studied this cultivar noticed huge variabilityin the majority of pomological and technological traits.3 – 5 Despitethe fact that Oblacinska sour cherry is mostly used for processed

products such as pie filling, jam or liquor, the present breedingprograms are aimed at finding new clones that will be suitablefor table consumption and have a high content of antioxidantcompounds.

Free radicals, which are unstable molecules, and oxidativestress processes are mostly recognized as factors connectedwith chronic diseases, including tumors and heart disease. Onthe other hand, antioxidant compounds, which play an impor-tant role as promoters of human health, have recently attractedgrowing interest by producers and researchers. Cherries arean important source of natural antioxidant substances, namelypolyphenols. Their polyphenols include flavonoids (anthocyanins,flavan-3-ols and flavonols), hydroxycinnamic acids and hydroxy-benzoic acids.6 Polyphenols are secondary metabolites in fruitswhich are important in preventing diseases and maintaining

∗ Correspondence to: Milica M Fotiric Akšic, Faculty of Agriculture, University ofBelgrade, Nemanjina 6, 11080 Zemun, Serbia. E-mail: [email protected]

a Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080, Zemun,Serbia

b Innovation Center, Faculty of Chemistry Ltd, University of Belgrade, Studentskitrg 12-16, 11000, Belgrade, Serbia

c Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000, Bel-grade, Serbia

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www.soci.org HOS Alrgei et al.

healthy conditions, since all of them have hydrogen donor capa-bility and are able to function as antioxidants; moreover, theyhave powerful reducing capability. They contribute to bitterness,astringency, color, flavor, odor and oxidative stability7 and areinvolved in antioxidative defense of plants against biotic andabiotic stresses such as high and low temperatures, drought, alka-linity, salinity, UV stress and pathogens.8 Current epidemiologicalstudies strongly support a contribution of polyphenols in theprevention of cardiovascular diseases, cancers and osteoporosisas well as neurodegenerative diseases and diabetes mellitus.9

Anthocyanins function as color markers in sour cherries and alsoas compounds with strong antioxidant and anti-inflammatoryactivities.10 Total anthocyanin content differs according to thesour cherry cultivar and was reported to vary between 278and 804 mg kg−1 frozen fruit.10,11 The main anthocyanins foundin cherry are cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside,cyanidin-3-O-glucosylrutinoside, cyanidin-3-O-sophoroside,pelargonidin-3-O-glucoside, peonidin-3-O-rutinoside andcyanidin-3-O-arabinosylrutinoside.12,13

Polyphenols identified in sour cherries, besides anthocyanins,include quercetin, kaempferol, rutin, catechin, epicatechin andphenolic acids (neochlorogenic acid, chlorogenic acid and3-coumaroylquinic acid).14 The composition of these colorlessphenolic compounds depends on the cultivar and is regulated byenvironmental and postharvest factors.15 Like other polyphenolcomponents, they act as powerful antioxidants and anticanceragents.16 The antimicrobial effect of sour cherry polyphenols wasproved to reduce the growth of Salmonella and Escherichia coliO157:H7.17

Since the content of the above components in cherry fruit canvary considerably, which means that the biological activity andconsequently the health-promoting effect of sour cherry as a func-tional food can differ significantly, the aim of this study was tocharacterize different clones of Oblacinska sour cherry. A totalof 39 clones were selected to determine carbohydrate composi-tion, total phenolic content (TPC), total anthocyanin content (TAC)and antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl rad-ical (DPPH• test). To identify and quantify phenolics and estab-lish polyphenolic profiles, ultra-high-performance liquid chro-matography (UHPLC) coupled with hybrid mass spectrometry (MS)combining a linear trap quadrupole (LTQ) and OrbiTrap massanalyzer was used. Carbohydrate content was determined byhigh-performance anion exchange chromatography with pulsedamperometric detection (HPAEC-PAD).

MATERIALS AND METHODSPlant materialThe 39 Oblacinska sour cherry genotypes used in this study areplanted at the Experimental Station ‘Radmilovac’, which belongsto the Faculty of Agriculture, University of Belgrade. The collectionorchard was established in 1993. Planting distance was 4 m× 2 m.The soil is classified as Eutric Cambisol. The trees were trainedas spindle bush, under non-irrigated standard cultural practices.Fruits were picked at the commercial maturity stage (dry stemscar). The evaluations were carried out in triplicate, each repli-cate being composed of 20 fruits. All fruits were collected from asingle plant, randomly from all cardinally oriented branches withdifferent directions around the canopy. The designation of geno-types contains the row number (Roman numeral or letter ‘D’) andthe tree number within a row (Arabic numeral). All samples werestored in a freezer at −20 ∘C.

Reagents and standardsAcetonitrile and formic acid (both of MS grade), methanol andethyl acetate (both of HPLC grade), sodium carbonate, potas-sium chloride, acetic acid, hydrochloric acid , sodium acetate andFolin–Ciocalteu reagent were purchased from Merck (Darmstadt,Germany). Ultrapure water (0.055 μS cm−1) from a TKA MicroP-ure water purification system (Thermo Fisher Scientific, Bremen,Germany) was used to prepare standard solutions and blanks.Syringe filters (13 mm, PTFE membrane 0.45 μm) were purchasedfrom Supelco (Bellefonte, PA, USA). The solid phase extraction (SPE)cartridges used for extraction and concentration of samples wereStrata C18-E cartridges (500 mg, 3 mL) obtained from Phenomenex(Torrance, CA, USA).

DPPH, cis,trans-abscisic acid, trans-resveratrol and phenolicstandards (protocatechuic acid, gallocatechin, aesculin, epigallo-catechin, p-hydroxybenzoic acid, gentisic acid, chlorogenic acid(5-O-caffeoylquinic acid or 5-CQA), catechin, caffeic acid, epicat-echin, gallocatechin gallate, rutin, p-coumaric acid, ellagic acid,ferulic acid, naringenin, apigenin, hesperetin, gallic acid, catechin3-gallate, naringin, epigallocatechin gallate, myricetin, quercetin,resveratrol, kaempferol, pinobanksin, chrysin, pinocembrin andgalangin) were purchased from Fluka AG (Buch, Switzerland).Sugar standards (trehalose, rhamnose, arabinose, glucose, fruc-tose, ribose, sucrose, turanose, isomaltotriose and maltose) werepurchased from Tokyo Chemical Industry (TCI Europe, Zwijndrecht,Belgium). Sugar alcohol standards (glycerol, erythritol, galactitoland sorbitol) were obtained from Sigma Aldrich (Steinheim,Germany).

Preparation of sample extractsSample extracts were prepared according to a slightly modifiedmethod.18 A mortar and pestle was used for sample homogeniza-tion. A 5 g sample of homogenized fruit was mixed with 50 mL ofmethanol containing 1 mL L−1 HCl in an ultrasonic bath for 1 h atroom temperature. The extract was placed in the dark at 4 ∘C for24 h, filtered and the clear supernatant was collected. The extrac-tion was repeated twice. All fractions were collected and evapo-rated to dryness by rotary evaporation under reduced pressureat 40 ∘C. The dried extract was re-dissolved in methanol/water(60:40 v/v) to 50 mL and this solutions was used for further anal-ysis. The extracts were filtered through 0.45 μm membrane filters(Supelco) before further fractionation and analysis.

Fractionation by SPE was used to separate anthocyanins fromnon-anthocyanin polyphenolics. C18 cartridges were precondi-tioned with ethyl acetate, followed by methanol and 0.01 mol L−1

HCl. Filtered extracts were passed through the cartridges, whichwere then washed with acidified water to remove all sugarsand other water-soluble compounds. Non-anthocyanin polyphe-nolic compounds were eluted with ethyl acetate, while acidicmethanol was used to elute the adsorbed anthocyanins from thecartridges. Anthocyanin and non-anthocyanin fractions were usedfor UHPLC/MS/MS OrbiTrap analysis. The aqueous fraction fromSPE was mixed with water (1:1 v/v) and used for sugar and sugaralcohol analysis by HPAEC-PAD.

Preparation of standard solutionsA 1000 mg L−1 stock solution of a mixture of all phenolic standardsand cis,trans-abscisic acid was prepared in methanol. Dilution ofthe stock solution with methanol yielded the working solutionat concentrations of 0.025, 0.050, 0.100, 0.250, 0.500, 0.750 and1.000 mg L−1. All stock and working solutions were stored in a

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freezer at −20 ∘C and were stable for at least 3 months. Calibrationcurves were obtained by plotting the peak areas of the compoundsidentified relative to the peak area against the concentration of thestandard solution. The calibration curves revealed good linearity,with R2 values exceeding 0.99 (peak areas versus concentration).

The carbohydrate content of cherry fruit samples was evaluatedfrom calibration curves of standard solutions of sugars and sugaralcohols. Under these chromatographic conditions, the last com-pound is detected after approximately 25 min and the analysis isended at 30 min.

Determination of total phenolic contentThe amount of total phenolics in extracts was determined accord-ing to the Folin–Ciocalteu procedure with some modification.19

Briefly, 0.5 mL of sample extract and 0.5 mL of ultrapure water weremixed with 2.5 mL of Folin–Ciocalteu reagent (100 mL L−1) andincubated for 5 min at room temperature. Next, 2 mL of sodiumcarbonate (75 g kg−1) was added. After 1 h at room tempera-ture, the absorbance at 765 nm was measured using a Cintra 6UV–visible spectrophotometer (GBC, Dandenong, Australia). Gal-lic acid was used as a standard at concentrations of 20–100 mg L−1.A mixture of water and reagent was used as a blank. TPC wasexpressed as mg gallic acid equivalent (GAE) g−1 frozen weight(FW).

Determination of total anthocyanin contentTAC was determined using the pH differential method.20

Methanol and acetone extracts were diluted with buffers ofpH 1.0 (hydrochloric acid/potassium chloride, 0.025 mol L−1) andpH 4.5 (acetic acid/sodium acetate, 0.4 mol L−1). The absorbancesof the extracts at 510 and 700 nm were measured against a blank.TAC was calculated and expressed as mg cyanidin 3-glucosideequivalent g−1 FW using the formulae

TAC =(

Atotal × MW × DF × 1000)∕ (𝜀 × l) (1)

Atotal =(

A510 –A700

)pH 1.0

–(

A510 –A700

)pH 4.5

(2)

where Atotal is the absorbance calculated by Eqn (2), MW is themolecular weight (MW= 449.2 g mol−1 for cyanidin 3-glucoside),DF is the dilution factor, l is the cuvette path length (l = 1 cm in thisstudy) and 𝜀 is the molar absorptivity (𝜀= 26 900 L mol−1 cm−1 forcyanidin 3-glucoside).

Antioxidant activity (DPPH• test)The antioxidant activity of extracts was evaluated using DPPH• bya slightly modified literature method.21 A 0.1 mL aliquot of extract(previously diluted 10 times) was mixed with 4 mL of methanolsolution of DPPH• (71 μmol L−1) and left to stand for 60 min in thedark (until stable absorbance values were obtained). The reductionof DPPH• was measured by continuously monitoring the decreasein absorbance at 515 nm. Antioxidant activity was calculated as thepercentage of DPPH• discoloration using the equation

DPPH (%) =[(

ADPPH –Asample

)∕ADPPH

]× 100 (3)

where ADPPH is the absorbance of the methanol solution of DPPH•

and Asample is the absorbance in the presence of extract. Theassays were carried out in triplicate and the results reported asmean values. A Trolox calibration curve was plotted as a functionof the percentage inhibition of DPPH•. Antioxidant activity wasexpressed as μmol Trolox equivalent (TE) g−1 FW.

Identification of polyphenolic compounds using LC/MS/MSanalysisChromatographic separations were performed using a UHPLCsystem comprising a quaternary Accela 600 pump and Accelaautosampler (Thermo Fisher Scientific). The UHPLC system wascoupled to a linear ion trap/OrbiTrap hybrid mass spectrome-ter (LTQ OrbiTrap MS) equipped with a heated electrospray ion-ization probe (HESI-II, Thermo Fisher Scientific). Separation ofpolyphenolics was performed on a Hypersil gold C18 column(100 mm× 2.1 mm, 1.9 μm) from Thermo Fisher Scientific. The MSwas operated in both negative and positive ionization modes. Forboth polarities, the capillary temperature was set at 300 ∘C and thesheath and auxiliary gas (N2) flow rates were 25 and 5 arbitraryunits respectively.

For determination and quantification of non-anthocyanins innegative ionization mode, the mobile phase consisted of (A)water+ 1 mL L−1 formic acid and (B) acetonitrile+ 1 mL L−1 formicacid. A linear gradient program at a flow rate of 0.3 mL min−1 wasused: 0.0–1.0 min, 5% B; 1.0–12.0 min, 5–95% B; 12.0–12.2 min,95–5% B; then 5% B for 3 min. The injection volume was 5 μL.HESI/source parameters were as follows: source voltage 5 kV, cap-illary voltage −35 V, tube lens voltage −110 V.

For determination of anthocyanins in positive ionization mode,the mobile phase consisted of (A) water+ 1 mL L−1 formic acidand (B) acetonitrile. A linear gradient program at a flow rate of0.3 mL min−1 was used: 0.0–2.0 min, 5% B; 2.0–12.0 min, 5–95% B;12.0–12.2 min, 95–5% B; then 5% B for 3 min. The injection volumewas 7 μL. HESI/source parameters were as follows: source voltage4.5 kV, capillary voltage 40 V, tube lens voltage 80 V.

Xcalibur software 2.1 (Thermo Fisher, Bremen, Germany) wasused for instrument control, data acquisition and data analysis.Fragmentation mechanisms and characteristic fragments wereconfirmed using Mass Frontier software 6.0 (Thermo Fisher, Bre-men, Germany). The generated MS/MS spectra were processedby ToxID software 2.1.1 (Thermo Fisher, Bremen, Germany). Aninternet database of accurate MS data, ChemSpider, was used asa reference library to identify compounds of interest. MS spectrawere acquired by full-range acquisition covering m/z 100–1000.For fragmentation study, a data-dependent scan was performedby deploying collision-induced dissociation (CID). The normalizedcollision energy of the CID cell was set at 35 eV.

Analysis of carbohydrates using HPAEC-PADChromatographic separations were performed using an ICS3000 DP LC system equipped with a quaternary gradient pump(Dionex, Sunnyvale, CA, USA). Carbohydrates were separated ona CarboPac® PA100 pellicular anion exchange column (4 mm×250 mm; Dionex) at 30 ∘C. The mobile phase consisted of (A)600 mmol L−1 sodium hydroxide, (B) 500 mmol L−1 sodium acetateand (C) ultrapure water. A linear gradient program at a flow rate of0.7 mL min−1 was used: 0–5 min, 15% A/85% C; 5.0–5.1 min, 15%A/2% B/83% C; 5.1–12.0 min, 15% A/2% B/83% C; 12.0–12.1 min,15% A/4% B/81% C; 12.1–20.0 min, 15% A/4% B/81% C; 20.0–20.1min, 20% A/20% B/60% C; 20.1–30.0 min, 20% A/20% B/60% C.Before analysis, the system was preconditioned at 15% A/85% Cfor 15 min. Each sample (25 μL) was injected with an ICS AS-DV 50autosampler (Dionex). The electrochemical detector consisted ofAu as working electrode and Ag/AgCl as reference electrode.

Statistical analysisAll data presented in the tables are the mean of three biologicalreplicates± standard deviation. Tukey’s test was used to detect

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differences (P ≤ 0.05) between mean values. Statistical analyseswere performed by NCSS software (www.ncss.com). Principalcomponent analysis (PCA) was applied to visualize the structure ofdata, identify important variables and confirm the presence of out-liers. PCA was carried out using a demo version of the PLS_Toolboxsoftware package for MATLAB 7.8.0 (Eigenvector Research, Inc.,Wenatchee, WA, USA). All data were group-scaled, i.e. groups ofvariables were scaled to grand standard deviation, prior to PCA.The singular value decomposition (SVD) algorithm and a 0.95 con-fidence level for Q and T 2 Hotelling limits for outliers were chosen.

RESULTS AND DISCUSSIONTotal phenolic content, total anthocyanin contentand antioxidant activitySamples were characterized by determination of TPC, TAC andantioxidant activity (DPPH• test) as presented in Table 1. Althoughno evident correlation was found among TPC, TAC and DPPH•

data (r < 0.4), these tests could be treated as preliminary, givingvaluable information on the differences among clones. Significantcorrelations among these tests would indicate that phenolicsare contributing to the antioxidant properties. Poor correlationcoefficients could be an indication that apart from polyphenoliccompounds the presence of other phytochemicals contributes tothe antioxidant activity.

TPC values ranged between 1.601 and 4.619 mg GAE g−1 FWof cherry sample, which is in good agreement with previouslypublished results.22,23 Considering all TAC values obtained forinvestigated genotypes, it is evident that the lowest content oftotal anthocyanins was found in clone IX/1 (0.498 mg cyanidin3-glucoside g−1 FW), while clone IV/5 had the highest contentof total anthocyanins (1.770 mg cyanidin 3-glucoside g−1 FW).Despite the fact that the average content of total anthocyaninsin all examined Oblacinska sour cherry clones coincides with thefindings from the literature, which cover a wide range of values,10

many accessions in our study showed much higher TAC. In fact,more than half of the genotypes in this study had TAC values over1.0 mg cyanidin 3-glucoside g−1 FW, which is quite substantial.The discrepancies may be due to the different cultivars studied.DPPH• values ranged from 2.224 to 19.650 μmol TE g−1 FW andwere similar to those obtained for some P. avium, P. cerasus, Prunustomentosa and Prunus pseudocerasus cultivars and the hybridrootstock ‘Colt’ (P. avium× P. pseudocerasus).24

Polyphenolic profileNon-anthocyanin profileUHPLC/OrbiTrap MS was utilized in order to obtain a comprehen-sive picture of individual phenolic compounds in both a qualitativeand quantitative sense. A total of 31 compounds were quantifiedby comparing retention times and MS spectra with available stan-dards, and these data are presented in Table 2 and Table S1 (‘Sup-porting information’).

Our study of the content of phenolic compounds in Oblacinskasour cherry showed that rutin and chlorogenic acid were the mostabundant phenolic compounds. Results regarding the content ofrutin in sour cherry samples ranged from 3.62 (XV/1) to 45.61 (D1)mg kg−1 FW, while chlorogenic acid, the most widespread naturalplant dietary antioxidant, varied from 8.10 (IV/5) to 36.76 (VII/2P)mg kg−1 FW, in line with previous studies.25,26

Most of the quantified flavonoids, such as naringin, myricetin,quercetin, kaempferol, chrysin and pinocembrin, were found only

in a few sour cherry samples (Table S1). Also, gallic acid wasfound only in five samples (XIII/4, D10, D4, XV/1 and II/2). Catechingallate was found only in two samples (III/14 and IX/1), whileepigallocatechin gallate was found in six samples (XIII/4, D7, IX/P,II/10, D8 and IX/1). The phytoalexin stilbene trans-resveratrol wasfound only in four samples (XIV/3, X/2, VIII/1 and XV/1).

To best knowledge, this is the first report on the presence ofpinobanksin, hesperetin and galangin in sour cherry samples,identified and quantified using standards (Table 2). Only as anexample, extracted ion chromatograms and MS/MS spectra ofthese three flavonoids are depicted in Fig. S1 (‘Supporting infor-mation’). Hesperetin was found in 15 samples, galangin was iden-tified in clones IX/1, V/9 and XV/1 (0.23, 7.04 and 2.58 mg kg−1

respectively), while pinobanksin was present only in clone V/9(2.25 mg kg−1).

Accurate mass measurement spectrometry techniques havebeen demonstrated to be a reliable tool for obtaining useful struc-tural information on compounds present in complex samples. Inthe absence of standards, identification of non-anthocyanins wasbased on the search for the [M−H]− deprotonated molecule andits MS/MS fragmentation. The main chromatographic and MS/MSdata of the identified compounds (ten hydroxycinnamic acid estersand two flavonol glycosides) are summarized in Table 3. The resultsshowed that the investigated clones were rich in quinic acid estersof hydroxycinnamic acids, in accordance with previous findings.17

Compounds with a quasi-molecular ion at m/z 337 andretention times of 4.94 and 5.52 min (Fig. 1A) were marked ascoumaroylquinic acid isomers. The isomer at 4.94 min producedan MS/MS base peak at m/z 163 ([p-coumaric acid−H]−), whichis characteristic of 3-O-p-coumaroylquinic acid (3-p-CoQA). Thesecond isomer (5.52 min) produced an MS/MS base peak atm/z 173 ([quinic acid−H2O−H]−), which is characteristic of4-O-p-coumaroylquinic acid (4-p-CoQA). The fragmentation path-way for both derivatives of coumaroylquinic acid can be found inthe literature.27,28

Two isomers of methyl esters of coumaroylquinic acid (5.71and 5.98 min, Fig. 1B) were detected at m/z 351 and showedsimilar fragmentation (MS/MS base peak at m/z 145 ([p-coumaricacid−H2O−H]−) by the loss of a methyl quinate moiety (206 Da)and secondary peaks at m/z 119 ([p-coumaric acid−CO2 −H]−)and m/z 117 ([p-coumaric acid−H2O – CO−H]−). The derivativeat 5.69 min was marked as methyl 3-O-p-coumaroylquinate(M3-p-CoQ), while that at 5.97 min was marked as methyl4-O-p-coumaroylquinate (M4-p-CoQ). The fragmentation pathwayfor derivatives of methyl coumaroylquinate found in the literaturewas conclusive29 and showed that none of them could be methyl5-O-p-coumaroylquinate, which would give an MS/MS base peakat m/z 163 ([p-coumaric acid−H]−).

Regarding caffeoylquinic acids (m/z 353), two peaks werefound (Fig. 1C). Apart from the quantified 5-O-caffeoylquinic acid(5-CQA) at 5.01 min, 3-O-caffeoylquinic acid (3-CQA) at 4.45 minwas also found. MS/MS fragmentation and retention of both ofcaffeoylquinic acid isomers were in accordance with previousresults.28 Both isomers showed an MS/MS base peak at m/z 191,indicating that esterification takes place at the 3-O position (in3-O-caffeoylquinic acid) and 5-O position (in 5-O-caffeoylquinicacid). This is not the case for 4-O-caffeoylquinic acid, which, ifpresent, would give an MS/MS base peak at m/z 173.27

Additionally, the exact mass search for m/z 367 showed fivesignals (Fig. 1D), which could be assigned to both feruloylquinicacids and methyl caffeoylquinic acid esters. As feruloylquinic acidisomers are characterized by an MS/MS base peak at m/z 193,28 all



Table 1. Total phenolic content (TPC, mg GAE g−1 FW), total anthocyanin content (TAC, mg cyanidin 3-glucoside equivalent g−1 FW) and antioxidantactivity (DPPH•, μmol TE g−1 FW) of Oblacinska sour cherry clones

Clone TPC TAC DPPH•

I/1 2.293± 0.031 t 0.950± 0.023klmno 6.192± 0.243klmII/1 3.353± 0.007ghi 0.973± 0.065klmn 8.528± 0.819efghijII/2 2.596± 0.000rs 1.095± 0.027hij 6.323± 0.604jklmII/6 3.167± 0.020jklm 0.945± 0.067mno 4.870± 0.665 mII/10 3.319± 0.231hijk 1.233± 0.073efg 15.084± 0.419bII/16 3.137± 0.090lmn 0.773± 0.016qr 7.630± 0.334ghijklIII/9 4.267± 0.043bc 1.255± 0.012def 19.650± 1.148aIII/14 3.924± 0.078de 1.125± 0.022hij 8.566± 0.375efghijIII/4P 3.289± 0.057ijkl 0.987± 0.023klmn 5.731± 0.700 lmIII/PP 3.049± 0.016mn 0.881± 0.013nopq 8.199± 0.493fghijkIV/1 2.762± 0.015opqr 0.969± 0.048klmno 5.642± 0.235 lmIV/2 3.720± 0.105f 1.375± 0.085c 8.763± 0.705defghiIV/5 3.088± 0.120mn 1.770± 0.066a 11.236± 1.014cIV/8 2.945± 0.007no 1.296± 0.058cde 9.420± 0.868cdefghV/9 2.492± 0.023 s 1.169± 0.062fgh 6.368± 0.948jklmV/P 3.141± 0.022klmn 1.278± 0.044cde 7.352± 0.231hijklVII/2P 3.374± 0.022ghi 0.934± 0.003no 15.173± 0.795bVIII/1 3.517± 0.107 g 1.090± 0.016hij 5.653± 0.789 lmIX/1 3.792± 0.069ef 0.498± 0.002 t 8.255± 0.000fghijkIX/P 3.929± 0.008de 0.945± 0.052lmno 5.522± 0.259 lmX/2 3.473± 0.007gh 1.153± 0.113ghi 8.286± 0.706fghijkXII/13 4.619± 0.008a 1.055± 0.045jkl 5.422± 0.126 lmXIII/1 4.121± 0.015c 0.978± 0.019klmn 9.283± 0.238cdefghiXIII/4 3.745± 0.022f 1.129± 0.034hij 10.157± 0.356cdefXIII/P 4.097± 0.082 cd 1.342± 0.071 cd 8.413± 0.166efghijkXIV/3 3.334± 0.068ghij 1.285± 0.071cde 7.434± 0.115hijklXIV/5 3.425± 0.000ghi 0.979± 0.017klmn 9.883± 0.270cdefgXV/1 1.905± 0.027u 0.664± 0.007 s 2.224± 0.628nD1 3.889± 0.007ef 1.507± 0.031b 8.446± 0.689efghijkD2 2.784± 0.008opq 0.832± 0.002pq 8.018± 0.447fghijkD3 4.343± 0.079b 0.730± 0.006rs 10.262± 0.135cdefD4 1.601± 0.033v 0.649± 0.011 s 4.300± 0.119mnD6 2.855± 0.007op 0.872± 0.045opq 8.023± 0.000fghijkD7 2.666± 0.007qrs 1.269± 0.008cdef 10.042± 0.000cdefD8 3.414± 0.122ghi 1.039± 0.053jklm 14.508± 0.573bD9 2.141± 0.054 t 1.048± 0.071jkl 6.443± 0.635jklmD10 2.678± 0.008pqr 0.962± 0.013klmno 7.138± 0.231ijklD12 4.166± 0.000bc 1.057± 0.107ijk 11.012± 0.654 cdD13 2.306± 0.008 t 0.918± 0.050nop 10.618± 0.620cde

Values are mean± standard deviation of three replications. Different letters in the same column denote significant differences according to Tukey’stest (P < 0.05).

compounds were assigned as isomers of methyl caffeoylquinates(Table 3). As can be seen from Fig. 2, the isomers at 5.16, 5.32and 5.68 min produced an MS/MS base peak at m/z 161 ([caffeicacid−H2O−H]−) and MS/MS secondary peaks at m/z 193 ([methylcaffeate−H]−) and m/z 135 ([caffeic acid−CO2 −H]−). Studyingthe retention times and fragmentation patterns of these threecompounds (Table 3), we assumed that two of them (at 5.16and 5.32 min) could be isomers of methyl 3-O-caffeoylquinate(M3-CQ), while the third (at 5.68 min) could be identified as methyl4-O-caffeoylquinate (M4-CQ). The main difference between M3-CQand M4-CQ was observed in the spectra of their MS/MS secondarypeaks: the 3-O derivatives showed a more intense peak at m/z193, while the 4-O derivative showed a more intense peak atm/z 135, in accordance with the literature.28,29 The remaining two

derivatives at 5.89 and 6.15 min (Fig. 1D) could be assigned asmethyl 5-O-caffeoylquinate isomers (M5-CQ) with an MS/MS basepeak at m/z 179 [caffeic acid−H]− and MS/MS secondary peaksat m/z 191 ([quinic acid−H]−), m/z 161 ([caffeic acid−H2O−H]−)and m/z 135 ([caffeic acid−CO2 −H]−),29 as shown in Fig. 2.

Three glycosides were identified as flavonol 3-O-rutinosides(Table 3). Their MS spectra were characterized by an MS/MS basepeak resulting from the loss of 308 Da. Such fragmentation indi-cates that the glycoside most probably consisted of rhamnose andglucose units (rutinose). The fragmentation pathway of quercetin3-O-rutinoside (Q3R) quantified at 5.85 min using a standardsupports this assumption. Observing the MS/MS data and alsostudying the available literature,30 we have presumed that theother two identified glycosides (at 4.86 and 6.25 min) could also be



Tab

le2.

Con

tent

ofp

olyp

heno

licco

mp

ound

s(m

gkg

−1

)in

Ob

laci

nska

sour

cher

rycl

ones

Clo

nePr

AG

CA

ESEG

Cp-

HBA

GeA

ChA

CC

aAEC

GCG

RUT

p-C

oAEl

lAFe

AA

bA

NA

RA

PIPN

BH

ESG

AL

I/1

5.90

2.28

1.09

1.51

0.78

0.06

24.3

7–

0.50

––

16.1

10.

894.

550.

810.

10–

0.01

––

–II/

1–

2.11

1.01

1.53

0.73

0.08

25.7

43.

550.

2922

.16

3.75

18.6

00.

734.

080.

250.

32–

0.02

–0.

17–

II/2

5.75

–1.

171.

262.

060.

2025

.48

–0.

471.

43–

18.1

80.

489.

832.

000.

41–

––

––

II/6

5.09

2.44

1.42

0.95

–0.

0431

.16

2.59

0.28

13.2

94.

0022

.10

0.49

2.28

––

––

––

–II/

105.

682.

321.

13–

1.06

0.06

23.6

72.

640.

3512

.98

3.33

24.9

10.

5522

.80

1.20

0.19

0.06

0.04

–0.

09–

II/16

5.29

9.93

1.04

1.73

1.99

0.04

28.5

01.

400.

355.

40–

17.4

10.

51–

0.65

0.10

0.08

0.03

–0.

11–

III/9

5.52

12.6

21.

451.

311.

680.

0732

.50

4.35

0.27

23.7

83.

3331

.54

0.91

–0.

73–

––

––

–III

/14

5.82

15.2

90.

821.

640.

760.

0725

.96

2.11

0.28

9.91

3.16

18.7

51.

172.

300.

570.

250.

060.

04–

––

III/4

P5.

9819

.28

0.96

2.19

0.64

0.07

18.3

03.

710.

2819

.33

–15

.23

0.98

–0.

51–

––

––

–III

/PP

5.68

14.9

50.

871.

482.

940.

0624

.48

4.06

0.27

19.4

03.

3516

.26

0.54

––

––

––

––

IV/1

5.58

15.5

61.

061.

092.

190.

1421

.94

–0.

392.

26–

15.1

90.

796.

280.

920.

250.

160.

05–

––

IV/2

5.33

–0.

832.

051.

710.

0717

.07

2.90

0.32

13.8

0–

23.7

70.

46–

0.57

0.11

–0.

01–

0.12

–IV

/55.

212.

290.

801.

780.

870.

088.

102.

970.

2616

.53

3.33

25.5

91.

10–

3.62

0.09

–0.

01–

0.11

–IV

/85.

6111

.37

1.08

–0.

870.

1018

.97

2.32

0.45

16.8

13.

8121

.87

0.82

–2.

19–

0.03

0.01

––

–V/

96.

06–

1.10

1.37

0.35

0.06

19.6

3–

1.03

––

17.2

10.

923.

721.

240.

200.

240.

452.

254.

957.

04V/

P5.

5215

.13

0.84

0.97

3.62

0.05

23.8

71.

760.

314.

193.

1915

.25

0.68

2.46

1.49

0.18

0.12

0.04

––

–VI

I/2P

5.24

–1.

401.

591.

320.

0536

.76

2.11

0.44

12.0

62.

8620

.06

0.49

–0.

960.

140.

070.

04–

––

VIII/

16.

03–

1.44

1.57

1.19

0.12

30.1

3–

0.42

7.21

3.69

21.9

80.

88–

1.24

0.22

0.13

0.06

–0.

08–

IX/1

5.82

–0.

951.

321.

980.

0525

.12

1.69

0.33

7.22

3.72

26.7

20.

586.

131.

910.

170.

080.

05–

0.15

0.23

IX/P

5.89

–1.

301.

091.

970.

0431

.92

3.77

0.37

22.2

3–

15.5

41.

255.

191.

060.

170.

020.

01–

––

X/2

5.50

–1.

161.

23–

0.05

32.2

5–

0.40

5.79

3.39

20.9

70.

59–

1.78

0.18

0.11

0.04

–0.

08–

XII/

136.

238.

541.

151.

430.

810.

1029

.75

–0.

48–

3.66

21.4

90.

552.

420.

980.

360.

060.

02–

––

XIII

/15.

8713

.61

1.05

1.61

1.93

0.03

26.5

3–

0.40

12.8

63.

8620

.88

0.77

8.44

3.04

0.15

–0.

02–

––

XIII

/45.

823.

701.

211.

241.

010.

0428

.28

4.56

0.27

32.9

13.

3128

.96

1.37

2.15

2.93

0.38

–0.

01–

––

XIII

/P5.

49–

1.06

1.63

0.72

0.10

26.6

83.

270.

2720

.48

–22

.75

0.57

2.42

0.85

0.19

––

–0.

11–

XIV

/35.

36–

1.05

2.01

0.60

0.06

29.2

92.

620.

2819

.85

–17

.53

0.71

––

0.09

––

––

–X

IV/5

5.32

–1.

161.

271.

650.

0325

.05

4.85

0.27

28.8

73.

4720

.97

0.53

––

0.42

––

–0.

16–

XV/

16.

15–

0.89

2.01

5.27

0.12

14.4

1–

0.72

–3.

463.

621.

115.

73–

0.33

0.07

0.11

–0.

482.

58D

16.

1519

.75

1.21

1.64

0.82

0.08

26.6

9–

0.65

–3.

9745

.61

0.76

–1.

980.

230.

160.

06–

0.12

–D

25.

6920

.73

1.25

2.09

1.45

0.05

21.5

95.

170.

3020

.57

3.14

17.8

60.

71–

–0.

15–

––

––

D3

5.94

–1.

131.

131.

580.

0528

.61

2.59

0.27

18.9

53.

7214

.04

0.86

–1.

600.

12–

––

0.08

–D

46.

19–

0.89

2.45

1.11

0.19

12.9

7–

0.52

–3.

286.

870.

47–

–0.

15–

––

––

D6

5.60

–1.

171.

130.

370.

1118

.31

2.29

0.29

10.8

83.

3722

.19

0.45

–2.

450.

140.

030.

04–

––

D7

5.79

9.57

0.95

1.06

1.19

0.16

14.9

74.

210.

3713

.70

3.35

24.1

20.

8014

.71

1.09

0.16

–0.

04–

––

D8

5.62

–0.

951.

820.

860.

1021

.14

2.91

0.43

12.0

0–

20.1

20.

54–

1.49

0.13

0.04

0.03

––

–D

94.

91–

2.34

1.22

1.23

0.02

28.0

91.

610.

363.

253.

2815

.15

––

0.36

–0.

050.

02–

––

D10

6.07

3.21

0.97

1.22

2.24

0.07

21.1

72.

850.

3613

.68

3.50

21.4

31.

262.

322.

020.

19–

0.01

–0.

10–

D12

6.33

3.41

1.04

1.43

0.89

0.06

25.4

74.

900.

3429

.18

4.19

19.6

90.

754.

01–

0.12

–0.

01–

––

D13

5.95

–0.

901.

042.

190.

0423

.25

2.03

0.48

5.92

–12

.75

1.68

–1.

200.

290.

140.

06–

––

PrA

,pro

toca

tech

uic

acid

;GC

,gal

loca

tech

in;A

ES,a

escu

lin;E

GC

,ep

igal

loca

tech

in;p

-HBA

,p-h

ydro

xyb

enzo

icac

id;G

eA,g

entis

icac

id;C

hA,c

hlor

ogen

icac

id;C

,cat

echi

n;C

aA,c

affei

cac

id;E

C,e

pic

atec

hin;

GCG

,gal

loca

tech

inga

llate

;RU

T,ru

tin;p

-CoA

,p-c

oum

aric

acid

;EllA

,ella

gic

acid

;FeA

,fer

ulic

acid

;Ab

A,c

is,tr

ans-

absc

isic

acid

;NA

R,na

ring

enin

;API

,ap

igen

in;P

NB,

pin

oban

ksin

;HES

,hes

per

etin

,GA

L,ga

lang

in.



Tab

le3.

Hyd

roxy

cinn

amic

acid

este

rs,fl

avon

olgl

ycos

ides

and

anth

ocya

nins

inO

bla

cins

kaso

urch

erry

clon

es:t

arge

tcom

pou

nd,m

olec

ular

form

ula,

mea

nex

pec

ted

rete

ntio

ntim

e(t

R),

calc

ulat

edm

ass,

exac

tmas

s,m

ean

mas

sac

cura

cyer

ror(Δ

)and

MS/

MS

frag

men

tsw

ithm

osta

bun

dant

ion

inte

nsit

y

Com

pou

ndA

bb

revi

atio

nM

olec

ular

form

ula,

[M−

H]−

/M+

t R(m

in)

Cal

cula

ted

mas

s,m

/zEx

actm

ass,

m/z

Δ(m

Da)

MS/

MS

frag

men

ts,m

/z(p

eak

inte

nsit

y,%

)

Hyd

roxy

cinn

amic

acid

este

rs3-

O-C

affeo

ylqu

inic

acid

3-CQ

AC

16H

17O

9−

4.45

353.

0878

135

3.08

707

0.74

191

(100

),17

9(6

0),1

35(1

0)3-

O-p

-Cou

mar

oylq

uini

cac

id3-

p-C

oQA

C16

H17

O8−

4.94

337.

0928

933

7.09

207

0.82

191

(10)

,173

(10)

,163

(100

),11

9(1

0)5-

O-C

affeo

ylqu

inic

acid

a5-

CQA

C16

H17

O9−

5.01

353.

0878

135

3.08

688

0.93

191

(100

),17

9(1

0)M

ethy

l3-O

-caff

eoyl

quin

ate

isom

er1

M3-

CQC

17H

19O

9−

5.16

367.

1034

636

7.10

278

0.68

193

(30)

,161

(100

),13

5(1

0)M

ethy

l3-O

-caff

eoyl

quin

ate

isom

er2

M3-

CQC

17H

19O

9−

5.32

367.

1034

636

7.10

266

0.80

193

(20)

,161

(100

),13

5(1

0)4-

O-p

-Cou

mar

oylq

uini

cac

id4-

p-C

oQA

C16

H17

O8−

5.52

337.

0928

933

7.09

192

0.97

191

(5),

173

(100

),16

3(1

0)M

ethy

l4-O

-caff

eoyl

quin

ate

M4-

CQC

17H

19O

9−

5.68

367.

1034

636

7.10

281

0.65

193

(10)

,161

(100

),13

5(2

0)M

ethy

l3-O

-p-c

oum

aroy

lqui

nate

M3-

p-C

oQC

17H

19O

8−

5.71

351.

1085

435

1.10

760

0.94

145

(100

),11

9(5

),11

7(5

)M

ethy

l5-O

-caff

eoyl

quin

ate

isom

er1

M5-

CQC

17H

19O

9−

5.89

367.

1034

636

7.10

260

0.86

191

(20)

,179

(100

),16

1(1

0),1

35(3

0)M

ethy

l4-O

-p-c

oum

aroy

lqui

nate

M4-

p-C

oQC

17H

19O

8−

5.98

351.

1085

435

1.10

773

0.81

163

(5),

145

(100

),11

9(1

5),1

17(1

0)M

ethy

l5-O

-caff

eoyl

quin

ate

isom

er2

M5-

CQC

17H

19O

9−

6.15

367.

1034

636

7.10

260

0.86

191

(20)

,179

(100

),16

1(1

0),1

35(4

0)Fl

avon

olgl

ycos

ides

Kaem

pfe

rol3

-O-r

utin

osid

eK3

RC

27H

29O

15−

4.86

593.

1511

959

3.15

033

0.86

327

(10)

,299

(20)

,285

(100

),28

4(3

0)Q

uerc

etin

3-O

-rut

inos

idea

Q3R

C27

H29

O16

−5.

8560

9.14

611

609.

1456

30.

4834

3(1

0),3

01(1

00),

300

(20)

,271

(10)

,255

(10)

Isor

ham

neti

n3-

O-r

utin

osid

eI3

RC

28H

31O

16−

6.25

623.

1617

662

3.16

107

0.69

315

(100

),31

4(2

0),3

00(2

0),2

71(1

0),2

55(1

0)A

ntho

cyan

ins

Cyan

idin

3-O

-glu

cosy

l-rut

inos

ide

Cyn-

glu-

rut

C33

H41

O20

+5.

4975

7.21

857

757.

2136

24.

9528

7(1

00),

611

(5)

Cyan

idin

3-O

-sop

horo

side

Cyn-

sop

hC

27H

31O

16+

5.52

611.

1606

661

1.15

601

4.65

287

(100

)Cy

anid

in3-

O-a

rab

inos

yl-r

utin

osid

eCy

n-ar

ab-r

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Figure 1. Extracted ion chromatograms with retention times and exactmasses of hydroxycinnamic acid esters present in Oblacinska sour cherryclones: (A) coumaroylquinic acids; (B) methyl coumaroylquinates; (C) caf-feoylquinic acids; (D) methyl caffeoylquinates.

Figure 2. MS/MS spectra of five methyl caffeoylquinates identified inOblacinska sour cherry clones (parent ion at m/z 367.10 in negative ionmode).

O-diglycosides with sugar units at the C-3 position and 1–6 linkagebetween them. Thus kaempferol 3-O-rutinoside (K3R) at 4.86 minwith a molecular ion at m/z 593 produced an MS/MS base peak atm/z 285, which corresponds to the aglycone part of the molecule,while the molecular ion of isorhamnetin 3-O-rutinoside (I3R) at m/z623 and 6.25 min produced an MS/MS base peak at m/z 315 by theloss of two sugar units ([M – H – rhamnosyl−glucosyl]−).

Anthocyanin profileThe identification of the corresponding anthocyanins was basedon the search for the M+ molecular ion together with the interpre-tation of its MS/MS fragmentation. The exact mass search and the

study of the fragmentation pathways described in the literatureenabled us to obtain as much structural information as possible. Inthis way, the profile of 12 anthocyanins was established (Table 3).

In the absence of standards, ion intensities were used forsemi-quantitative comparison of anthocyanin contents in sourcherry samples. The total intensity of each peak from the MSspectra was obtained with ToxID software (Table S2 of ‘Support-ing information’). The most abundant anthocyanins were deriva-tives of cyanidin: cyanidin 3-O-glucosyl-rutinoside and cyanidin3-O-rutinoside, followed by cyanidin 3-O-glucoside and cyanidin3-O-sophoroside. Such a profile is in accordance with previousresults found for sour cherry cultivars Geremaand, Montmorencyand Balaton.15,31 Agreeable results where the main anthocyanincompound in sour cherry was cyanidin 3-O-glucosyl-rutinosidewere also found in the literature.10,32

MS/MS spectra of identified anthocyanins in sour cherry samplesare presented in Fig. S2 (‘Supporting information’). All identifiedanthocyanins were characterized by an MS/MS base peak at m/zof the aglycone part of the molecule. According to the literature,in fruits the most common glycosylation is at the 3-O positionfor anthocyanins,33 as is the case with sour cherry samples.31,32

Likewise, for two hexoses in the 3-O position, MS/MS spectra showonly a fragment ion of the aglycone.34

Carbohydrate profileIn fruit species, the carbohydrate composition influences the tasteand can vary with cultivar, age, maturity, ecological conditionsand technical and cultural practices.35 The carbohydrate compo-nents of Oblacinska sour cherry are presented in Table S3 (‘Sup-porting information’). Fructose was found to be dominant, fol-lowed by glucose, both identified as the major sugar componentsin almost all sour cherry accessions and also prevailing in sweetcherry.36,37 In addition, Oblacinska sour cherry clones store sor-bitol in their fruits, sometimes at very high levels,38 and sucrose,whose concentration was very low in the majority of the stud-ied clones, as with other minor soluble carbohydrates. The sumof the above four carbohydrate compounds constituted on aver-age 89.56% of the total carbohydrate content, which is quite simi-lar to previous findings.39 The highest levels of fructose and glu-cose were recorded in genotypes D3 (46.314 mg g−1) and II/10(48.726 mg g−1) respectively. Sucrose contents ranged from 0.094(II/2) to 4.325 (VII/2P) mg g−1, in line with previous results.40 Lowlevels of sucrose were due to high invertase activity during thefinal maturation stages, contributing less than 1% in the major-ity of samples.41,42 The only exceptions were genotypes III/P andVII/2P where the sucrose share of total sugars was 18.20 and 6.79%respectively, which is most likely due to early picking. Trehalose,arabinose, isomaltotriose, rhamnose, ribose, turanose and mal-tose together with sugar alcohols (glycerol, erythrol and galactitol)were detected in all genotypes but present only as minor sugarconstituents. To best knowledge, the presence of isomaltotriose insour cherry fruits has not been reported previously.

PCA resultsOwing to the large amount of data, PCA was used to distinguishOblacinska sour cherry clones according to their chemical traits.

PCA produced 11 principal components with eigenvaluesgreater than 1, explaining 77.91% of the total variability. Thecumulative variation explained by the first six componentswas only 60.0%, revealing high genetic diversity among theOblacinska sour cherry clones and suggesting the evaluation ofother chemical traits for a meaningful characterization.



A

B

Figure 3. PCA (A) score and (B) loading plots for Oblacinska sour cherry clones.

Figure 3 shows the projection of Oblacinska sour cherry cloneson the PC1/PC2 plane, based on the regression factor score values.In general, starting from negative towards positive values of PC1,the accessions were characterized by high values of apigenin,pinocembrin, pinobanksin, kaempferol, chrysin and hesperetinbut lower values of catechin, epicatechin, TPC and DPPH•. Startingfrom negative towards positive values of PC2, the Oblacinska sour

cherry clones showed increases in galactitol, sorbitol, arabinose,ribose, sucrose and maltose but decreases in fructose and glucose.The majority of clones are located in the central part of the scatterplot (Fig. 3A), suggesting the existence of chemically similar clonesregardless of the fact that Oblacinska sour cherry is a mixture ofclones. Clone III/9 was characterized by high levels of catechin,epicatechin, TPC and DPPH•, while in a previous reports5 this clone



stood out as the most promising one for production. In contrast,clones IX/P, XIII/1 and D9 showed low levels of glucose and fructosebut high levels of chlorogenic acid. Clone XV/1 was distinguishedby its high levels of apigenin, pinocembrin, kaempferol, chrysinand hesperetin. The score plot reveals three samples lying outsidethe Hotelling T 2 ellipse (VII/2P, XIII/P and V/9), suggesting that theywere outliers.

Two outliers (VII/2P and XIII/P) overlapped for low levels ofglucose and fructose and elevated contents of other minor sugars(Fig. 3B). In contrast, clone V/9 contained untypical polyphenols forsour cherry, such as apigenin, hesperetin, galangin, kaempferol,pinocembrin and pinobanksin.

CONCLUSIONThe general conclusion from the chemical analysis of Oblacinskasour cherry is that this cultivar is an excellent source of raw materialthat could be utilized for the preparation of high-quality functionalfood. However, since it is a mixture of different clones, not all havethe same composition. The highest values for TPC in XII/13 and TACin IV/5 distinguished these clones as having the largest quantitiesof bioactive compounds, while antioxidant capacity reached itshighest level in clone III/9.

Rutin and chlorogenic acid were the most abundant polyphenolsin Oblacinska sour cherry clones, while pinobanksin, galanginand hesperetin were quantified for the first time in sour cherry.The anthocyanin profile revealed 12 components, with cyanidinderivatives being dominant. The carbohydrate profile showed thatfructose was dominant, followed by glucose. The sum of fructose,glucose, sorbitol and sucrose, but in different ratios within theclones, constituted on average 89.56% of the total carbohydratecontent. To best knowledge, the presence of isomaltotriose in sourcherry fruit, which was found in all studied clones, has not beenreported previously.

The absence of correlation between flavan-3-ols (gallocatechin,epigallocatechin and gallocatechin gallate), hydroxycinnamicacids (chlorogenic acid and p-coumaric acid), hydroxybenzoicacids (protocatechuic acid and p-hydroxybenzoic acid), ellagicacid and cis,trans-abscisic acid within any of the six PCs sug-gests that those traits were genetically affected by independentgenes not showing pleiotropic effects (Table S4 of ‘Supportinginformation’).

In summary, the importance of a high content of phenolic com-pounds in functional food justifies extensive studies such as theone presented herein. High-resolution MS as a powerful tech-nique allowed the identification of characteristic polyphenoliccompounds and revealed wide diversity in the selected sour cherryclones. Apart from the fact that this study has given comprehen-sive information on the chemical characterization of Oblacinskasour cherry fruit, the knowledge of its quality traits could be a use-ful tool for choosing the appropriate clones to be grown in Serbianor similar conditions for producing food with a high content ofbioactive compounds.

ACKNOWLEDGEMENTSThis study was supported by the Ministry of Education, Scienceand Technological Development, Republic of Serbia (Projects No.TR 31063 and No. 172017). The authors acknowledge the supportof the FP7 Project AREA 316004 and the FP7 RegPot Project FCUBERA GA No. 256716. The EC does not share responsibility for thecontent of the article.

SUPPORTING INFORMATIONSupporting information may be found in the online version of thisarticle.

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Chemical profile of major taste- and health-related compounds of (Oblačinska) sour cherry

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