Author’s Accepted Manuscript

Phenolic Compounds and Antioxidant Capacity inDifferent-Colored and non-Pigmented Berries ofBilberry (Vaccinium myrtillus L.)

Nesrin Colak, Anja K. Primetta, Kaisu R. Riihinen,Laura Jaakola, Jiři Grúz, Miroslav Strnad, HülyaTorun, Faik Ahmet Ayaz

PII: S2212-4292(17)30114-1DOI: http://dx.doi.org/10.1016/j.fbio.2017.06.004Reference: FBIO202

To appear in: Food Bioscience

Received date: 24 March 2017Revised date: 26 May 2017Accepted date: 9 June 2017

Cite this article as: Nesrin Colak, Anja K. Primetta, Kaisu R. Riihinen, LauraJaakola, Jiři Grúz, Miroslav Strnad, Hülya Torun and Faik Ahmet Ayaz,Phenolic Compounds and Antioxidant Capacity in Different-Colored and non-Pigmented Berries of Bilberry (Vaccinium myrtillus L.), Food Bioscience,http://dx.doi.org/10.1016/j.fbio.2017.06.004

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1

Phenolic Compounds and Antioxidant Capacity in Different-Colored and non-

Pigmented Berries of Bilberry (Vaccinium myrtillus L.)

Nesrin Colak1, Anja K. Primetta

2, Kaisu R. Riihinen

2, Laura Jaakola

3,4, Jiři Grúz

5, Miroslav

Strnad5, Hülya Torun

6, Faik Ahmet Ayaz

1*

1Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey,

2Departments of Environmental Science and Biosciences, University of Eastern Finland, P.O.

Box 1627, FIN-70211 Kuopio, Finland,

3Climate Laboratory Holt, Department of Arctic and Marine Biology, UiT the Arctic

University of Norway, NO-9037 Tromsø, Norway,

4Norwegian Institute of Bioeconomy Research, NIBIO, Box 2284 NO-9269 Tromsø, Norway

5Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre

of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science,

Palacky University & Institute of Experimental Botany AS CR, Slechtitelu 11, CZ-783 71

Olomouc, Czech Republic,

6Biosystem Engineering, Faculty of Agriculture and Natural Sciences, Düzce University,

81620 Düzce, Turkey,

ncolak@ktu.edu.tr

anja.latti@gmail.com

rkaisu@yahoo.com

laura.jaakola@uit.no

jiri.gruz@gmail.com

miroslav.strnad@upol.cz

hlytrn95@hotmail.com

faa@ktu.edu.tr

*Corresponding author. Prof. Dr. Faik Ahmet AYAZ, PhD, Tel and Fax: 00 90 462 377 37

12,

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ABSTRACT

Bilberries and their products are popular worldwide and represent a very interesting source of

dietary antioxidants. Berries of eight different-colored and non-pigmented bilberry

(Vaccinium myrtillus L.) samples from Finland were evaluated in terms of antioxidant

capacity and total phenolic compounds (range, 220.06 – 3715.21 mg/100 g dw) and total

monomeric anthocyanin (range, 206.18 – 867.52 mg/100 g dw) contents. Delphinidin (range,

5914.93 - 18108.39 g/g dw) was the major anthocyanin moiety, while sinapic acid was the

major phenolic acid in the free form (range, 0.01 – 6.06 μg/g dw), and 4-coumaric acid in the

ester (range, 26.39 -110.78 μg/g dw), glycoside (range, 15.83 – 57.73 μg/g dw) and ester-

bound (range, 2.32 – 14.20 μg/g dw) forms. The white colored berry samples did not contain

any anthocyanins, but the colored berries did contain them. Antioxidant capacity was much

higher in colored (pink to blue/black) berry samples than in the white sample, and it was more

related to the total phenolic concentration rather than to the anthocyanin concentration. This is

the first time that these different-colored berry phenotypes of bilberry (Vaccinium myrtillus

L.) have been analyzed within the same study.

Abbreviations: 4-HBA, 4-Hydroxybenzoic Acid ; AAPH, 2,2’-Azobis(2-

methylpropionamidine)dihydrochloride; AC, Antioxidant Capacity; CaA, Caffeic Acid ;

DBC, 2,6-di-tert-butyl-p-cresol; DPPH, 2,2-Diphenyl-1-Picrylhydrazyl; FC, Folin-Ciocalteu ;

FeA, Ferulic Acid ; FRAP, Ferric Reducing Antioxidant Power; GaA, Gallic Acid; GaA,

Gallic Acid ; HBAs, Hydroxybenzoic Acids, ; HCAs, Hydroxycinnamic Acids ; ORAC,

Oxygen Radical Absorbance Capacity; PAs, Phenolic Acids; PCA, Principal Component

Analysis; p-CoA, p-Coumaric Acid; PAs, Proanthocyanidin; SaA, Salicylic Acid ; SiA,

sinapic acid.; SyA, Syringic Acid ; SyA, Syringic acid ; TACY, Total Monomeric

Anthocyanin; TPC, Total Phenolic Compounds; VaA, Vanillic Acid

Keywords: Bilberry, Vaccinium myrtillus, anthocyanin, phenolic acid, antioxidant

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1. Introduction

The current worldwide interest in the health-promoting properties and economic importance

of blueberries has been attributed to the high antioxidant capacity of their polyphenolics,

particularly anthocyanins (Naczk and Shahidi, 2006; Riihinen et al., 2008; Stevenson and

Scalzo, 2012; Colak et al., 2016a, 2016b). Anthocyanins are, therefore, regarded as useful

bioactive compounds in the prevention of chronic and degenerative diseases and as exhibiting

a wide range of protective effects with potential benefits for human and animal health, which

have been substantially reviewed in detail elsewhere (Li et al., 2015).

Studies have mainly focused on the anthocyanin composition in blueberries (Stevenson

and Scalzo, 2012), and to some extent on phenolic acids (Ayaz et al., 2005; Riihinen et al.,

2008; Colak et al., 2016b). In brief, the distinctive anthocyanin profile in ripe bilberries

includes 15 major characteristic anthocyanidin glycosides in different sugar moieties

(galactose, glucose and arabinoside) bonded to five aglycones (delphinidin 3-glycoside,

cyanidin, petunidin, peonidin and malvidin (Naczk and Shahidi, 2006; Chu et al., 2011;

Primetta et al., 2013; Colak et al., 2016a).

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Small, wild berries (white strawberry, blackberry, raspberry, current berry hybrids, etc.)

are considered a native genetic source for breeders and producers. These are already available

on the market, which is constantly in search of novel products (Zorenc et al., 2016). Berry

characteristics of rare colored bilberries were previously reported by Jaakola et al. (2002)

from Finland. Zorenc et al. (2016) recently investigated the phenolics (e.g. anthocyanin and

nonanthocyanin phenolics) by comparing wild bilberry types (albino and blue) that differ in

terms of fruit quality from Slovenia. However, limited information is available about the

phenolic composition and antioxidant capacity of different-colored berry phenotypes

(white/albino to pink) of wild bilberries. In the light of the growing interest in the potential

nutraceutical properties and health-protective effects of bilberries, it seems desirable to

characterize the phenolic profiles and antioxidant capacity of different colored and non-

colored berries of bilberry samples/phenotypes in order to contribute to cultivation policies

for breeders. The purpose of this study was to profile the anthocyanins and phenolic acids (in

free, ester, glycoside and ester-bound forms) using HPLC−DAD−ESI-MS/MS and UPLC-

MS/MS in eight bilberry (V. myrtillus L.) berries from Finland in conjunction with their

antioxidant capacity.

2. Materials and Methods

2.1.Chemicals, Reagents and Solvents

Cyanidin 3-glucoside was purchased from Extrasynthese (Genay, France). HPLC-grade

methanol and acetonitrile were acquired from Lab-Scan (Dublin, Ireland) and J. T. Baker

(Deventer, Holland), respectively. All solvents were analytical grade and purchased from

Riedel-deHaen (Seelze, Germany) and Merck (Darmstadt, Germany). Deionized water was

prepared using a Simplicity 185 system (Millipore, Bedford, MA). Standards of gallic, 3,5-

dihydroxybenzoic, protocatechuic, gentisic, 4-hydroxybenzoic, caffeic, vanillic, syringic, 3-

hydroxybenzoic, 4-coumaric, sinapic, ferulic, 3-coumaric, 2-coumaric, salicylic acids were

purchased from Sigma–Aldrich Fine Chemicals (St. Louis, MO, USA). Deuterium-labeled

standards of 4-hydroxybenzoicacid (2,3,5,6-D4) and salicylic acid (3,4,5,6-D4) were

purchased from Cambridge Isotope Laboratories (Andover, MA, USA). DPPH (2,2-Diphenyl-

1-Picrylhydrazyl), TPTZ (2,4,6-tripyridyl-s-triazine), AAPH (2,2’-Azobis(2-

methylpropionamidine) dihydrochloride) and fluorescein were analytical grade and purchased

5

from Sigma–Aldrich Fine Chemicals (St. Louis, MO, USA), and Na2CO3, FeCl. 6H2O and

Folin-Ciocalteu (FC)’ reagent were purchased from Merck (Darmstad, Germany).

2.2. Plant Material

Ripe berries of different colored and non-colored bilberry (Vaccinium myrtillus L.) samples

were handpicked at the time when they are typically harvested in Finland. In general, the

bilberry season in southern Finland starts in the 2nd

or 3rd

week of July. Berry sampling (n =

8) was performed during the summer of 2007 from six different locations (Turku, Artjärvi,

Ruokolahti, Kontiolahti, Rautavaara and Raahe) at northern latitudes (60˚23" – 64˚31"). The

color of the berries varied from rare white, pink and purple variants to normal dark blue.

There were also two bilberries with normally dark-colored berries, but without a wax layer,

which reflects radiation, so these appeared blue-black (not dark blue as is the case with

similarly dark-colored berries with a wax layer) (Table 1). The berries were uniformly

colored, except for the white berries which had some hints/spots of red and green when, the

surface was closely examined. The samples were immediately cooled to below 10 ˚C and

stored at –25 ˚C before freeze-drying within the following 2 months.

2.3. Determination locations and phenological observations of bilberry

Before visiting the collecting locations in order to check the maturity of bilberry due to

possible local differences, and to start picking the berries if ripe, the phenological

observations of V. myrtillus (e.g. flowering, maturing of berries) by the Finnish Forest

Research Institute (Metla; a part of the Natural Resources Institute of Finland (Luke) date 01.

01. 2015, Fig. 1) were followed. Hundreds of study plots of V. myrtillus plants for

phenological observations from south to north in Finland have been available since the

1960s.. In summer 2007, Metla reported that the first bilberries started to ripen in the

southeastern parts of Finland on the 12th of July. We picked the first bilberries in that part of

Finland 4 days later. There may be some local variation in terms of ripening but generally the

berries start to ripen from south to north. We also checked that the berries were fully ripe

considering the following criteria as earlier indicated by Jaakola et al. (2002) during the

ripening stages of bilberries (color variations, white and pink, etc.); the ripe berries were fully

expanded, no longer hard, but still firm and the skin intact, not overripe, and not softened, in

which case the skin would easily break. Additionally, in the case of the colored berry

phenotypes of the bilbberry, the flesh would leak juice and stain the fingers, as bilberries

easily do. The berries were uniformly colored. The blue berries were dark blue and the berries

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without light reflecting wax were black. Purple and pink berries were uniformly colored

purple and pink, respectively. The white berries were colored white, but these had

some hints/spots of red and green, if the wax was carefully inspected. The flesh was darkest in

the blue and blue-black berries, and the purple berries were also purple. The flesh then

gradually became lighter color, similarly to the skin of the berries.

There are only five native Vaccinium species (V. oxycoccus, V. microcarpum, V vitis-

idaea, V. uliginosum and V. myrtillus) in Finland. Of these, the bilberries ripen first, then

followed by bog bilberries (V. uliginosum), a plant which is visibly differ from V. myrtillus

and that grows in various types of location (Woody Flora of Finland 1992). The bushes of V.

myrtillus grow in thick, acidic humus, among mosses and abundant twig vegetation in

Myrtillus-type forests. The main tree species are spruce or pine, as described in the Finnish

forest classification (Cajander, 1949).

The purple, dark blue and blue-black colored bilberries were picked from one bush for

each color berry sample from different locations (see Fig. 1). The white and pink bilberries

were easily recognized due to their abnormal color, and the berries were picked from the same

genet. The collection sites of white berries of the two bilberries were approximately 200-300

meters apart.

The blue colored bilberries, blue-black (without wax) and purple colored bilberries were

picked from one bush. The white and pink bilberries were easily recognized, and the

berries were picked from the same genet, from various adjacent bushes. The collection

locations of white berries of two bilberry samples were approximately 200-300 meters apart

(Fig. 1). They are therefore apparently of different genetic origins due to the size of the genet

according to the provious literature (Albert et al., 2003).

2.4. Extraction

Extraction of phenolic compounds was performed with slight modification of the Giusti and

Wrolstad method (2001), as described elsewhere. First, 0.2 g dry deseeded berry sample was

pulverized using a mortar and pestle. The sample was then placed in 10 ml extraction solvents

(first deionized water, second 70% v/v aqueous acetone, and third 70% v/v aqueous

methanol). The homogenates of each solvent were combined and centrifuged at 8000 g for 30

min at +4°C using a Hermle Z 326K centrifuge (Wehingen, Germany). The sediment was re-

extracted with a small portion of the same solvents until the solution became colorless. After

centrifugation, all supernatants were concentrated using a rotary evaporator at 35 °C under

partial vacuum with N2 flush. The aqueous combined extract was finally dried using a freeze

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dryer (Christ, Alpha 1-2LD plus, Germany). The dry material was then diluted to 10 ml with

deionized water and used in the determination of phenolic compounds and, antioxidant

capacity and for phenolic acids analysis as described in sections 2.5, 2.6 and 2.7, respectively.

2.5. Determination of total phenolic compounds (TPC), monomeric anthocyanin (TACY) and

proanthocyanidin (PAs) contents

The TPC content was estimated using the Folin-Ciocalteu (FC) reagent method as described

by Slinkard and Singleton (1977). The reaction mixture was prepared by mixing 500 µl

aqueous extract, 975 µL 2% Na2CO3 and 25 µl FC reagent dissolved in deionized water. A

blank was prepared at the same time and in the same manner as the reaction mixture, but

replacing the sample extract with 500 µl deionized water. After allowing the reaction mixture

and the blank to cool at room temperature (25 C) for 30 min, the absorbance was measured

against a blank at 750 nm using a UV-VIS spectrophotometer (Thermo, Evolution 201,

England). In the same way, a different concentration of a standard solution of gallic acid (GA)

was prepared to produce a calibration curve. Based on the measured absorbance, the TPC

concentration was read from the calibration curve. The data were expressed as mg GA

equivalent per 100 grams dry weight (dw) of berries.

A pH differential method described by Giusti et al. (1999) was performed in order to

measure TACY. Absorbance was measured with a UV-VIS spectrophotometer (Thermo,

Evolution 201, England) at 520 and 700 nm in buffers. Data were calculated with the molar

extinction coefficient for cyanidin-3-glucoside using the equation: A x MW x DF x 103/ ε x l,

where A = (A520nm – A700nm) pH 1.0 – (A520nm – A700nm) pH 4.5; MW (molecular weight) =

449.2 g/mol for cyanidin-3-glucoside (cyd-3-glu); DF = dilution factor (1:10); l = path length

in cm; ε ( = 26900 molar extinction coefficient, in L · mol–1

· cm–1

, for cyd-3-glu; and 103

=

factor for conversion from g to mg and expressed as mg cyanidin/100 grams dw of berries.

PAs were extracted from freeze-dried samples using 70% aqueous acetone, as previously

described (Vasco, 2009), and the content was estimated following Toivanen et al.’ method

(2009). Briefly, aliquots of the extracts in acidified methanol were incubated at 70 ºC for 3

hours to depolymerize PAs to anthocyanidins. Dilutions of bilberry dimers and trimers isolate

were depolymerized similarly for the calibration curve (Määtta-Riihinen et al., 2005).

Absorbances were read at 520 nm before and after incubation for semiquantification of PAs

using a UV-VIS spectrophotometer (Thermo, Evolution 201, England).

2.6. Determination of antioxidant capacity (AC) in berries

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The ability of the aqueous extract to scavenge DPPH (2,2-Diphenyl-1-Picrylhydrazyl) free

radical was assayed colorimetrically using Blois (1958)’ standart method, as described

elsewhere. Briefly, 1 ml of freshly prepared methanolic DPPH solution (1 mg/30 ml) was

added to 100 µl of each extract. After 30 min incubation in the dark, the absorbance of the

reaction mixture was read at 520 nm using a UV-VIS spectrophotometer (Thermo, Evolution

201, England). The data were expressed as µmol Trolox equivalent per gram dw berries.

The Ferric Reducing Antioxidant Power (FRAP) assay was performed following the

procedure described by Benzie and Strain (1999) with some slight modifications. Briefly, 100

µl of each sample was mixed with 2900 µl freshly prepared FRAP reagent (300 mM acetate

buffer (pH 3.6), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) and 20 mM FeCl.6H2O in

proportions of 10:1:1, v/v/v). The reaction mixture was then incubated for 30 min at 37 °C,

and the absorbance of the reaction mixture was measured at 593 nm against a blank using a

UV-VIS spectrophotometer (Thermo, Evolution 201, England). The FRAP values were

expressed as µmol Trolox equivalents per 100 grams dw berries.

The procedure based on a report by Ou et al. (2001) and described elsewhere was used,

with slight modifications, to determine oxygen radical absorbance capacity (ORAC). In brief,

the reaction mixture including 150 μl of 250 nM fluorescein (fluorescein sodium salt) and

25 μl of diluted extracts was pipetted into each of the 96 working wells of the microplate.

Next, 25 μl of 250 mmol/l 2,2’-Azobis(2-methylpropionamidine)dihydrochloride (AAPH)

was added onto the mixture. After shaking the microplate for 5 sec, the fluorescence

(excitation and emission wavelengths 485 nm 510 nm, respectively) was read every 3 min for

90 min using Multiskan Ascent (Labsystems, Helsinki, Finland) instrument. Net area under

the curve was used to calculate antioxidant capacity expressed as µmol Trolox equivalents per

g dry weight berries.

2.7. Extraction and Determination of Phenolic Acids (PAs) by UPLC-MS/MS

For the extraction of PAs, the same extraction procedure as that described in section 2.3 was

followed. In contrast, however, the extraction solvents each contained the antioxidant DBC

(2,6-di-tert-butyl-p-cresol, 6 mg/100 ml) in order to avoid any possible oxidations. The

homogenates of aqueous, methanolic and acetone extracts were combined, centrifuged and

concentrated until dry. The PAs acids were further fractioned using a previously described

method (Ayaz et al., 2005; Gruz et al., 2008) to give the acids in free, ester, glycoside and

ester-bound forms.

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PAs in different forms were analyzed using the ACQUITY Ultra Performance LC™

system (Waters, Milford, MA, USA) linked to a Micromass Quattro micro™ API bench top

triple quadrupole mass spectrometer (Waters MS Technologies, Manchester, UK). Sample

solutions were injected into a reversed phase column (BEH C8, 1.7 μm, 2.1 × 150 mm,

Waters, Milford, MA) maintained at 30 °C. Analytes were quantified using deuterium-labeled

internal standards of 4-hydroxybenzoic (2,3,5,6-D4) and salicylic (3,4,5,6-D4) acids, as

described previously (Ayaz et al., 2005; Gruz et al., 2008), with some slight modifications.

2.8. Determination of Anthocyanins by LC-MS/MS

Chromatographic analyses were performed on a HP-1090 module system (Hewlett–Packard,

Waldbronn Analytical Division, Germany) equipped with a quaternary pump, an autosampler

and a diode array detector (DAD) (HP 1040 M). The phenolic compounds were separated on

a Gemini C18 column (150 mm x 4.6 mm i.d., 5µm) (Phenomenex, Torrance, CA, USA)

fitted with a 4mm x 3mm i.d. C-18 guard column. The anthocyanins were quantified using the

method previously described for bilberries (V. myrtillus) (Lätti et al., 2008, 2011; Primetta et

al., 2013), with some modifications. Naturally, new six-point external standard calibration

curves were generated. The linearity was checked and was acceptable (R2 > 0.998). In every

run series, two quality control standards were analyzed to monitör for any possible

fluctuations in response, as described previously (Lätti et al., 2008, 2011; Primetta et al.,

2013)

Statistical analysis

All extractions and analysis are presented as mean ± pooled standard deviation (n = 3). One-

Way ANOVA and the Multiple Range Test and Pearson correlation (r) were performed on

IBM SPSS Statistics V22.0 software. Linear regression (R) and linear correlation (r) analysis

were carried out on Microsoft Office Excel 2010. Differences at P < 0.01 or 0.05 were

considered significant. A statistical software package (XLSTAT version 2014.6) using

ADDINSOFT (Damrémont, Paris, FRANCE) was also used to perform Principal Component

Analysis (PCA). The values for anthocyanins and phenolic acids were auto-scaled. Sample

similarities were calculated based on Euclidean distance and the Ward hierarchical

agglomerative method.

3. Results and Discussion

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3.1. Antioxidant capacity (AC) of total phenolic compounds (TPC) and total monomeric

anthocyanins (TACY) contents in the berry samples

Table 1 shows the moisture (%) content and the concentrations (mg/100 g dw) of TPC and

TACY and AC values in the berries. TPC concentrations ranged from 220.06 to 3715.21. The

white berry (wh1) from location Raahe exhibited the lowest concentrations of TPC, and the

blue berry (blu1) from location Ruokolahti the highest. No measurable level of TACY was

determined in the two white berries (wh1 and wh2). Significantly (P < 0.05), the highest

TACY concentration was thus obtained from the blue bilberry sample (blu1, 867.52), and the

lowest was identified in the pink-colored berry (pin, 206.18, location Artjärvi). Similarly, the

blu1 berry had the highest AC values (µmol TE/g) obtained from FRAP (140.55), followed by

DPPH (132.78) and ORAC (122.69) (Table 1).

The linear regressions and correlations of AC values with TPC, TACY or PAs

concentrations in the berries and a clear correlation matrix of these are also given in Figure 2

(AC). The AC values exhibited strong positive correlations with TPC and TACY contents,

while PAs exhibited no linear correlation (ave. r = -0.414, Figure 2D). These values indicate

that AC is strongly related to TPC and moderately related to TACY contents. However, the

linear regressions were quite low (ave. r2 = 0.194) for PA concentrations (Figure 2D).

3.2. Phenolic acid composition in berries

Concentrations (g/g dw) of 10 PAs in the berry phenotypes are summarized in Table 2. In

general, the blu2 had the highest syringic acid (SyA) (6.06) concentration, followed by p-

coumaric acid (p-CoA) (4.75) in the free form, blu2 had the highest concentration of p-CoA

(8.96), followed by SyA and caffeic acid (CaA) (76.21 and 74.55) in the ester form, and p-

CoA (57.73) followed by CaA (33.58) in the glycoside and ester bound (11.18 and 4.06)

forms.

Phenolic acids in blueberries are known to exhibit intra- and interspecies variation. For

instance, the major PA in cranberry, bilberry and lingonberry is p-CoA, with ferulic acids

(FeA) being the major PAs in blueberries and highbush blueberries, GA in rabbiteye

blueberries and vanillic (VaA) and CaA in cranberries and lingonberries (as reviewed by

Ayaz et al., 2005 and Colak et al., 2016a, 2016b). Significant differences have been noted in

CaA concentrations in half-highbush and highbush blueberries, bog bilberry and cascade

huckleberry (range, 41.3 - 182 g/g fw) (Taruscio et al., 2004). FeA was the major PA

reported in evergreen and black-leaf huckleberries (ave. 109, and 21.7, respectively), p-CoA

11

in oval-leaf blueberry and wild cranberry, and p-hydroxybenzoic acid (p-HBA) in red

huckleberry (range 23.9 - 533 g/g fw). In bilberries, VaA (2.58 g/g fw) was reported as the

major PA in free form, p-CoA (74.3 g/g fw) in ester form and 3,4-dimethoxycinnamic acid

(54.15) in glycoside form (Colak et al., 2016b). Our findings concur with these studies in that

the different-colored berries of bilberry contained p-CoA (p- or 4-CoA) and CaA or SyA as

the major PAs in all four PA forms.

3.3. Anthocyanin composition in berries

Anthocyanin concentrations (g/g dw) in the berries are summarized in Table 3, and the

representative chromatograms of each color berry are shown in Figure 3. Since the berries of

wh1 and wh2 are white, they do not contain any anthocyanin. Indeed, the presence of 15

anthocyanins was determinded in the pink colored berries of the pin, but the concentrations of

most of the anthocyanins were not quantifiable due to their very low levels (i.e. < 10 g/g

dw). Table 3 therefore includes only the results for the remaining five berries. Among the

bilberries, blu1 had the highest total anthocyanin content (43605.34), followed by blunw1

(31975.52) and blunw2 (31570.06). Delphinidin was the major anthocyanidin glycoside in the

blu1 (18108.39, ave. 6036.13; 13.84%), blunw1 (14341.85, ave. 4780.6; 14.95%) and pur

(5914.93, ave. 1971.6; 15.82%) berry phenotypes, followed by glycosides of cyanidin

(12599.86, ave. 4199.9; 9.63%), petunidin (6729.39, ave. 2243.13; 5.14%) and malvidin

(4656.47, ave. 1552.16; 3.56%) in the blu1, and peonidin (2389.52, ave. 796.51; 3.11%) in

blu2 (Table 3). Concentrations of some of the anthocyanins in the berries were positively

strongly correlated with TPC and TACY contents and DPPH values, while

non-significant moderate or high correlations were observed among the berries (for detail see

Table 4).

Cyanidin 3-galactoside (8.34 mg/kg fw), cyanidin 3-arabinoside (4.09 mg/kg fw),

delphinidin 3-arabinoside (4.32 mg/kg fw) and petunidin 3-glucoside (3.77 mg/kg fw) have

recently been reported as the major anthocyanidins in albino berries (Zorenc et al., 2016). The

contents were significantly reduced 1.6-fold for flavanols, 2.1-fold for flavonols, 2.5-fold for

hydroxycinnamic acid derivatives and 4.7-fold for TPC compared to blue bilberry (Zorenc et

al., 2016). Similarly, Jaakola et al. (2002) did report absence of anthocyanins in white berries

of bilberry.

3.4. Principal Component Analysis (PCA)

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3.4.1. Polyphenols and antioxidant capacity

Total variation of AC values and TPC or TACY or PAs with the berry phenotypes was

89.73% (Fig. 4A). Potential associations between the antioxidant capacity values and

polyphenol concentrations (TPC, TACY, PAs) were determined only at the left lower and

upper plan on PC1, where they accounted for 74.12% of the variation. At the right lower plan,

all three antioxidant capacity values of DPPH, FRAP and ORAC were associated and

significantly correlated with the pur, blunw2 and blu1 berries. The remaining berries were

located at the left lower (wh1 and wh2) and upper (pin and blu2) plans on PC2 and were

associated, but not correlated with proanthocyanidin concentrations (Fig. 4A).

3.4.2. Anthocyanins

PCA was applied to confirm any relationship between each anthocyanin and AC values or

colored berries (Fig. 4A-F). PCA of delphinidin anthocyanin explained 96.38% (Fig. 4B),

cyanidin 99.05% (Fig. 4C), petunidin 98.94% (Fig. 4D), peonidin 96.73% (Fig. 4E) and

malvidin 97.75% (Fig. 4F) of total variation, where PC1 accounts for 65.33%, 95.83%,

85.74%, 95.20%, 90.24% and 90.71% of the variance and PC2 ranged the values between

3.14% and 24.86%. Total variation of individual anthocyanins in relation to TPC, TACY or

AC values was 90.18%. As shown in Figure 4A, blunw1 with anthocyanins pt-gal, dp-gal,

mv-ara, pt-ara and dp-ara and blu1 with anthocyanins pt-glu, mv-gal and dp-glu were strongly

associated and correlated with TPC and AC values, positive loadings for which appeared at

the right lower plan on PC1. Only blunw2 was associated and correlated with pn-glu, -ara and

-glu, cy-glu, -gal and –ara, and mv-glu and TACY, and also exhibited positive loadings at the

right upper plan on PC1. Low amounts in the pur and blu2 berries led to their inclusion in

another group, exhibiting a negative loading at the left lower plan and a positive loading at the

upper plan on PC2, but neither of the berries was associated or correlated with any

anthocyanins.

PCA also confirmed that anthocyanin type was strongly associated and correlated with (r =

0.888 – 0.997, P < 0.01 or P < 0.05, see Table 4) the berries. This was more apparent for

delphinidin (range, r = 0.906 – 0.997) and petunidin (range, r = 0.888 – 0.990), with close

association and strong correlation. Blunw1 and blu1 with delphinidin (Fig. 4B), blu2, blunw2

and blu1 with cyanidin (Fig. 4C), blunw2, blunw1 and blu1 with petunidin (Fig. 4D), blu1,

blu2 and blunw2 with peonidin (Fig. 4E), and blunw2, blu1 and blunw1 with malvidin were

clearly separated on the PCs. Positive strong significant correlations (P < 0.01 or P < 0.05)

between anthocyanins and total phenolic contents (TPC or TACY) or antioxidant capacity

13

values were only determined with DPPH, while the other two antioxidant capacity tests,

ORAC and FRAP, were not significantly correlated on the correlation matrix table (for detail

see Table 4).

3.4.3. Phenolic acids (PAs)

PCA confirmed that the phenolic acid profiles were closely associated and correlated with the

different-colored and non-colored berries, explaining 89.29% of total variation, where PC1

accounts for 65.28% of the variance and PC2 for 24.02% (Fig. 5A). Principal component 1

(PC1) led to a complete separation of wh2, pur, blunw2, blunw1, blu2 and blu1 berries with

phenolic acids p-CoA, CaA, VaA, GaA, PCA and SyA in the free forms (f1) and the blunw1,

wh2, blunw2, pur, pin, blu1, blu2 and wh1 berries with phenolic acids 4-HBA, FeA, SiA and

SaA in glycoside forms (f3), with positive loadings at the right lower and upper plans on PC1.

However, on PC2, the PAs in the free and ester-bound (f4) forms and particularly the PAs in

berries of wh1 in the ester form (f2) exhibited a negative loading at the left lower plan, while

and the PAs in berries of the pin bilberry sample in the ester form (f2) exhibited a positive

loading at the upper plan, and these were not associated or correlated with any berries of the

bilberries.

In particular, PCs of PA in the different forms were positively associated with the

different-colored and non-colored berries of the sampled bilberries, with total variation of

79.00% for the free form (Fig. 5B), 89.45% for the ester form (Fig. 5C), 83.94% for the

glycoside form (Fig. 5E) and 72.70% for the ester-bound form (Fig. 5E). PC 1 in figures 5B

and C separated phenolic acids in the free and ester forms with the blu2, blu1, pur, wh2,

blunw1 and blunw2 berries with positive loadings from the other berries accounting for

58.58% and 77.87% of the total variance. In contrast, in figures 5D and E, only SiA was

associated with the pin and wh1 berries at the left upper plan on PC2, accounting for 20.45%

and 27.86% of the variances, respectively. The majority of the phenolic acids in these two

forms (glycoside and ester-bound) were associated and correlated with the blunw1, blu1 and

blu2 berries on PC1, with positive loadings at the right lower and upper plan on PC1,

accounting for 63.49% and 44.84% of the total variance, respectively (Fig. 5D and E).

3.4.4. Cluster analysis

In order to better understand the similarities and differences in the profile of the anthocyanins

and phenolic acids in conjunction with TPC and AC values, a hierarchical cluster analysis

(HCA) was performed using these different berry samples. According to the HCA, the present

14

berries can be classified into two groups, cluster A (blunw1 and blu1) and cluster B (blu2 and

blunw2) and an outlier sample, the pur berry. As the results show, the blunw1 and blu1 berries

appear in one group (cluster A), characterized by high TPC, TACY and AC values, followed

by the berries of blu2 and blunw2 (cluster B) belonging to the second group (Fig. 4G). The

latter group, the pur berry, is distinct from the above and is not clustered. This may be due to

its low content in terms of TPC, anthocyanin composition and AC values.

HCA was also used to evaluate similarities and differences between the PAs and the

berries (Fig. 5F). Two major HCAs, referred to as clusters A and B, were obtained with the

three minor clusters (B1, B2 and B3). Ester-bound forms (f2) of PAs in the blunw1, blunw2,

blu1 and blu2 (cluster A) and wh1, wh2, pin and pur berries (cluster B2), glycoside forms of

phenolic acids in berries of the blu2, wh1, blu1, blunw1, pin, blunw2, wh2 and pur (cluster

B1), and free (f1) and ester-bound (f4) forms of PAs in the berries were capable of being

clustered. The results suggest that PAs did not cluster within a short hierarchical distance in A

and B1, followed by B2, which are significantly different from those other forms of PAs.

Interestingly, the PCA in Fig. 5A confirms this similarity, as well as the distinguishing

characteristics.

4. Conclusion

This study represents the first comparison of phenolic compounds and antioxidant capacity

in different-colored and non-pigmented ripe berries of the wild bilberry samples in Finland.

The study data revealed a wide variation and difference in the phenolic compounds between

the colored and non-colored berries at the based on total contents (TPC, TACY and PAs) and

at the identified and quantified anthocyanin and phenolic acid level. As the color of the

berries varied from non-pigmented (wh1, wh2) to colored turning to pink-purple (pin, pur) or

blue-black (blu1, blu2, blunw1, blunw2), the concentrations of anthocyanins, phenolic acids

or antioxidant capacity changed visibly, as did their total contents. Significant variation in the

antioxidant capacity values was generally dependent on the type of anthocyanin as well as the

content of TACY concentration. The anthocyanins and phenolic acid profiles were very

distinctive and correlative with delphinidin (glucosides) and p-coumaric, syringic or caffeic

acids, being the major representatives in the berries. Our findings are generally in agreement

with previous research regarding the composition of phenolic acids, anthocyanins and

antioxidant capacity and are compatible with some previously published results. Detailed

information concerning the content of phenolic compounds (anthocyanins, phenolic acids) in

15

the different berry phenotypes of the bilberry, as first revealed by the present study, will

make a valuable contribution further taxonomic, breeding and health benefit studies.

Conflict of interest

Authors declare no conflict of interest

Acknowledgments

We are most grateful to Pekka and Paula Hyvönen, Kalle Määttä, and Juha Ullgren for

gathering the wild berries.

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Fig. 1. Phenological observations of maturation of berries of bilberries in Finland in summer

2007. The last map shows the forecast of the maturation of the bilberries (Vaccinium myrtillus

L.) in Finland in Summer 2007 according the Finnish Forest Research Institute

(http://www.metla.fi/tiedotteet/2007/2007-06-29-marjasato-mustikka.htm#mustikkamarjat)

Fig. 2. Influence of total total phenolic compounds (TPC), total monomeric anthocyanins

(TACY) and proanthocyanidin (PROANT) contents on antioxidant capacity (AD) in

different berries of bilberry (V. myrtillus L.) samples and their bi-plot (PC1xPC2) of

scores and loadings for the PCA (E) and correlations (Pearson r) situated at the right

lower bottom. Antioxidant capacity values were regretted (R2) with TPC and TACY or

proanthocyanidin contents and their R2 values are shown in each (AD).

Abbreviations of bilberries: wh1 (white1), wh2 (white2), pin (pink), pur (purple), blu1

(normal blue), blunw1 (blue/black no wax), blunw2 (blue/black no wax) and blu2

(normal blue).

Fig. 3. HPLC-DAD chromatograms at 520 nm of (A) pin (pink berry, Artjarvi), (B) pur,

(purple berry, Turku), (C) blu1 (normally colored berry, blue, Ruokolahti), (D) blu2

18

(normally colored berry, blue, Rautavaara), (E) blunw1 (blue-black berry, without wax,

Kontiolahti) and (F) blunw2 (blue-black berry, without wax, Turku) bilberries (V. myrtillus)

from different locations in Finland. Fig. 4. Biplot (PC1xPC2) of scores and loadings for the PCA of all identified anthocyanins (A), delphinidin (B),

cyanidin (C), petunidin (D), peonidin (E), malvidin (F) and agglomerative hierarchical clustering (AHC) (G) in

different berries of bilberries (V. myrtillus): wh1 (white), wh2 (white), pin (pink), pur (purple), blu1 (normal

blue), blunw1 (blue/black berry no wax), blunw2 (blue/black berry no wax) and blu2 (normal blue berry).

Abbreviations for anthocyanins: dp-gal; delphinidin galactoside, dp-glu; delphinidin glucoside, cy-gal; cyanidin

galactoside, dp-ara; delphinidin arabinoside, cy-glu; cyaniding glucoside, pt-gal; petunidin-galactoside, cy-ara;

cyanidin-arabinoside, pt-glu; petunidin glucoside, pn-gal; peonidin-galactoside, pt-ara; petunidin-arabinoside,

pn-glu; peonidin-glucoside, mv-gal; malvidin-galactoside, pn-ara; peonidin arabinoside, mv-glu; malvidin

glucoside, mv-ara; malvidin arabinoside. TPC; total phenolic compounds, TACY; total monomeric anthocyanin,

ACY/TP; Total monomeric anthocyanin/ total phenolic compounds.

Fig. 5. Biplot (PC1xPC2) of scores and loadings for the PCA of all identified phenolic acids (A), in free

(B), ester (C), glycoside (D), ester-bound (E), agglomerative hierarchical clustering (AHC) (F)

in different berries of bilberry (V. myrtillus) samples; wh1 (white1), wh2 (white2), pin (pink), pur

(purple), blu1 (normal blue berry), blunw1 (blue/black berry no wax), blunw2 (blue/black berry no wax)

and blu2 (normal blue berry), GaA; gallic acid, PCA; protocatechuic acid, 4-OHBA; 4-hydroxybenzoic

acid, SaA; salicylic acid, VaA; vanillic acid, CaA; caffeic acid, SyA; syringic acid, 4-CoA; 4-coumaric

acid, FeA; ferulic acid, SiA; sinapic acid, f1; free, f2; ester, f3; glycosides, f4; ester-bound.

Table 1 Locations, color characteristics, total phenolic compounds (TPC) and monomeric

anthocyanin (TACY) contents in conjunction with antioxidant capacity of differently colored

bilberries (V. myrtillus). Values represent the mean ± SD of three separate extractions

and determinations. An analysis of variance (SPSS version 11.5, one-way ANOVA) was used

for comparisons among the means. Values with the same letter within a column are not

significantly different at P < 0.05.

__________________________________________________________________________________________

Sample Moisture (%) Berry color

Location (N)

__________________________________________________________________________________________

wh1 86.4 white (light green hint/spots) Raahe

(64 ˚ 31")

wh2 87.3 white (light red hint/spots) Raahe

(64 ˚ 31")

pin 86.0 pink

Artjärvi (60˚ 44")

pur 88.3 purple

Turku (60˚ 23")

19

blu1 86.3 blue

Ruokolahti (61˚ 17")

blu2 87.1 blue

Rautavaara (63˚ 19")

blunw1 86.1 blue/black (without wax layer)

Kontiolahti (62˚ 40")

blunw2 86.0 blue/black (without wax layer)

Turku (60˚ 23")

phenolics contents (TPC) and antioxidant capacity (AC)

TPC TACY DPPH

ORAC FRAP ACY/TPC

wh1 220.067.87a n.d. 8.411.38

a 14.401.23

a

19.530.72a 0

wh2 397.6812.37b n.d. 8.541.50

a 17.091.33

a

22.872.19a

0

pin 1144.909.56c 206.186.77

a 21.191.22

b 43.202.89

c 53.242.41

b 0.2

pur 1422.895.29d 245.527.51

b 41.381.48

c 62.912.41

f 95.651.83

c 0.2

blu1 3715.216.88h 867.524.45

f 132.782.56

g 122.693.86

g 140.552.25

e 0.2

blu2 1625.448.91e 426.259.79

c 46.131.17

d 34.061.22

b 55.121.74

b 0.3

blunw1 3182.255.21g 680.942.87

e 91.113.78

f 52.892.42

e

121.243.25d

0.2

blunw2 3132.577.95f 639.976.28

d 90.952.42

e 48.080.78

d

123.081.05d

0.2

___________________________________________________________________________________

TPC: Total phenolics expressed as milligrams of gallic acid per 100 g dw of berries.

TACY: tot al monomeric ant hocyanin cont ent expressed as milligrams of cyanidin 3 glucoside (MW =

449. 2 and extinction coeffiicient = 26.900) per 100 g dw of berries.

DPPH: µmol Trolox equivalent / g dw of berries.

ORAC: µmol Trolox equivalent / g dw of berries.

FRAP: µmol Trolox equivalent / g dw of berries

n.d.: below detect able levels.

Table 2 Phenolic acid composition (μg/g dw) in some differently colored bilberries (V.

myrtillus) from Finnish populations. _________________________________________________________________________________________________________________

__________________________________________________________________________________________

Phenolic acids**

Total*** ______________________________________________________________________________________________________

________________________________________________________________ _______________________

20

Sample* GaA p-CoA 4-HBA SaA

VaA CaA SyA

4-CoA FeA SiA HBAs HCAs PHAs _________________________________________________________________________________________________________________

_________________________________________________________________________________________

Free (f1)

wh1 nd 0.00 nd 0.00 nd 0.00

nd 0.00 nd 0.00 nd 0.00 nd 0.00

0.11 0.02 0.01 0.01 nd 0.00 - 0.11 0.11

wh2 nd 0.00 0.31 0.06 0.06 0.01 0.04 0.01 0.06 0.01

0.99 0.17 0.01 0.01 2.95 0.49 0.28 0.04 0.09 0.02 0.17 4.62 4.79

pin 0.00 0.00 0.35 0.08 0.01 0.02 0.11 0.02 0.02 0.03 0.28 0.05

0.08 0.02 0.89 0.16 0.08 0.01 0.02 0.02 0.22 1.62 1.84

pur 0.03 0.01 1.65 0.33 0.07 0.01 0.14 0.03 0.10 0.02 0.70 0.12

0.34 0.06 1.55 0.25 0.16 0.02 0.11 0.03 0.68 4.17 4.85

blu1 0.06 0.01 2.44 0.50 0.06 0.01 0.24 0.05 0.13 0.02 0.81 0.14

0.57 0.11 1.77 0.30 0.14 0.02 0.03 0.01 1.06 5.19 6.24

blu2 0.08 0.02 4.75 1.13 0.07 0.02 0.11 0.03 0.37 0.07 2.08 0.41

6.06 1.28 1.81 0.30 0.27 0.05 0.06 0.02 6.69 8.7 15.35

blunw1 0.06 0.01 0.95 0.19 0.04 0.01 0.05 0.01 0.06 0.09 0.54 0.09

0.46 0.09 1.54 0.30 0.09 0.01 0.06 0.01 0.67 3.45 4.12

blunw2 0.01 0.02 1.76 0.23 0.04 0.01 0.10 0.01 0.20 0.02 0.26 0.03

0.28 0.04 1.05 0.11 0.13 0.01 0.08 0.01 0.63 3.28 3.91

mean 0.04 1.74 0.05 0.11 0.13 0.81

7.8 1.46 0.14

0.06 Ester (f2)

wh1 0.06 0.00 1.54 0.01 0.41 0.00 0.06 0.00 0.11 0.00 27.21 0.39

0.09 0.00 35.01 0.38 1.20 0.01 2.07 0.19 0.73 67.03 67.76

wh2 0.18 0.02 2.42 0.30 0.64 0.08 0.03 0.02 0.15 0.01 42.58 3.40

0.23 0.01 45.72 3.80 1.10 0.12 1.66 0.01 1.23 93.48 94.71

pin 1.58 0.10 2.11 0.07 0.81 0.03 0.30 0.02 0.47 0.01 17.55 0.63

1.61 0.08 26.39 0.86 1.65 0.02 3.11 0.35 4.77 50.81 55.58

pur 6.04 1.37 9.36 2.41 0.66 0.17 0.12 0.03 2.80 0.52 16.57 2.92

10.37 1.69 33.76 6.06 1.68 0.34 2.07 0.21 19.99 63.44 83.43

blu1 41.29 5.23 26.21 2.53 2.01 0.19 0.34 0.04 22.03 1.67 74.55 6.27

76.21 7.41 86.96 7.03 3.75 0.22 4.44 0.71 141.88 195.91 337.79

blu2 29.41 0.23 24.93 0.95 1.62 0.06 0.19 0.00 8.58 0.23 54.15 1.01

53.59 0.30 110.78 2.44 3.32 0.15 2.78 0.16 93.39 195.96 289.35

blunw1 26.45 1.81 14.48 0.56 1.19 0.04 0.09 0.01 11.48 0.35 36.25 1.42

51.45 2.69 55.42 1.98 2.18 0.03 3.13 0.36 90.66 111.46 202.12

blunw2 14.51 3.03 14.58 1.73 0.87 0.10 0.14 0.01 17.82 1.54 20.71 1.62

31.87 2.07 42.40 3.45 2.83 0.29 3.91 0.01 65.21 84.43 149.64

mean 14.94 11.95 1.03

1.10 7.93 36.20 28.18 54.55 2.21

2.90

glycoside (f3)

wh1 0.06 0.00 0.78 0.04 1.11 0.06 0.68 0.05 3.66 0.15 16.12 0.80

0.12 0.01 32.09 1.49 8.63 0.21 11.66 1.04 5.63 69.28 74.91

wh2 0.11 0.01 1.33 0.09 0.82 0.05 0.62 0.05 2.30 0.12 12.59 0.75

0.12 0.01 28.43 1.61 4.25 0.15 4.52 0.61 3.97 51.12 55.09

pin 0.40 0.02 0.70 0.01 0.78 0.01 0.51 0.02 2.32 0.03 7.42 0.16

0.32 0.01 15.83 0.30 4.15 0.01 8.21 0.80 4.33 36.31 40.64

pur 1.15 0.17 3.35 0.40 0.60 0.07 0.77 0.11 3.03 0.28 9.14 0.93

0.71 0.08 22.35 2.20 4.34 0.33 4.89 0.87 6.26 44.07 50.33

blu1 4.59 0.15 4.96 0.02 0.89 0.00 1.12 0.03 4.87 0.02 13.51 0.17

3.96 0.10 32.39 0.30 3.47 0.04 4.98 0.41 15.43 59.31 74.74

blu2 8.22 1.14 7.14 0.78 1.93 0.21 1.28 0.16 4.50 0.38 33.58 3.16

6.35 0.68 57.73 5.24 5.92 0.41 5.04 0.85 22.28 109.41 131.69

blunw1 3.59 0.04 3.38 0.14 1.22 0.05 0.32 0.01 4.81 0.14 11.12 0.23

2.35 0.02 31.25 0.73 2.84 0.13 5.83 0.32 12.29 54.42 66.71

blunw2 2.29 0.15 3.99 0.38 0.74 0.07 0.43 0.03 3.45 0.24 8.43 0.52

1.72 0.08 23.34 1.50 4.38 0.38 8.21 0.12 8.63 48.35 56.98

mean 2.55 3.20 1.01

0.72 3.62 13.99 1.96 30.43 4.75

6.67

ester-bound (f4)

wh1 nd 0.00 0.88 0.07 0.12 0.01 0.04 0.00 0.15 0.01

3.07 0.22 0.01 0.02 7.87 0.54 0.44 0.02 0.38 0.06 0.32 12.64 12.96

21

wh2 0.00 0.00 2.47 0.22 0.24 0.02 0.06 0.01 0.22 0.02 7.10 0.55

0.04 0.00 14.20 1.06 0.61 0.03 0.25 0.04 0.56 24.63 25.19

pin 0.06 0.01 1.50 0.09 0.18 0.01 0.06 0.01 0.17 0.01 1.99 0.12

0.13 0.01 5.16 0.28 0.47 0.02 0.42 0.06 0.6 9.54 10.14

pur 0.01 0.01 1.15 0.18 0.18 0.03 0.06 0.01 0.15 0.02 0.52 0.07

0.13 0.02 2.32 0.30 0.33 0.04 0.12 0.02 0.53 4.44 4.97

blu1 0.30 0.06 2.51 0.40 0.29 0.05 0.10 0.02 0.77 0.10 3.48 0.49

1.92 0.30 9.37 1.30 0.47 0.05 0.27 0.06 3.38 16.83 20.21

blu2 0.26 0.05 3.24 0.55 0.32 0.05 0.10 0.02 0.53 0.06 4.06 0.54

1.77 0.26 11.18 1.47 0.45 0.05 0.22 0.05 2.98 18.42 21.40

blunw1 0.15 0.02 1.46 0.19 0.18 0.02 0.01 0.01 0.46 0.05 2.14 0.24

1.00 0.12 6.98 0.75 0.27 0.02 0.27 0.05 1.8 11.12 12.92

blunw2 0.07 0.01 1.31 0.12 0.21 0.02 0.04 0.00 0.48 0.03 1.15 0.09

0.64 0.06 4.22 0.33 0.40 0.02 0.34 0.05 1.44 7.42 8.86

mean 0.12 1.81 0.21

0.06 0.37 2.94 0.70 7.66 0.43 0.28

-_________________________________________________________________________________________________________________

__________________________________________________________________________________________

*Sample: wh1 (white), wh2 (white), pin (pink), pur (purple), blu1 (normal blue), blunw1 (blue/black no wax), blunw2

(blue/black no wax) and blu2 (normal blue).

**GaA; gallic acid, p-CoA; p-coumaric acid,4-HBA; 4-hydroxybenzoic acid, SaA; salicylic acid, VaA; vanillic

acid, CaA; caffeic acid, SyA; syringic acid, p-CoA; p-coumaric acid, FeA; ferulic acid, SiA; sinapic acid.

***HBAs; hydroxybenzoic acids, HCAs; hydroxycinnamic acids, PHAs; total phenolic acids, sum of individual phenolic acids or sum

of HBAs and HCAs.

Table 3. Anthocyanins (g/g dw, %) in different berries of bilberry (V. myrtillus) samples

from Finnish populations. _________________________________________________________________________________________________________________

__________________________________________________

Anthocyanins** pur % blu1

% blunw1 % blunw2

% blu2 %

_________________________________________________________________________________________________________________

__________________________________________________

dp-gal 1950.4 15.65 6822.14

15.65 4701.26 14.70 4038.84

12.79 2097.46 8.19

dp-glu 1724.36 13.84 4352.76

9.98 3801.24 11.89 3398.71

10.77 2493.91 9.73

dp-ara 2240.14 17.97 6933.49

15.90 5839.35 18.26 3368.91

10.67 1957.65 7.64

∑dp 5914.93 47.46 18108.39

41.53 14341.85 44.84 10806.46

34.23 6549.02 25.56

meandp 1971.6 15.82 6036.13

13.84 4780.6 14.95 3602.15

11.41 2183.0 8.52

cy-gal 1140.68 9.15 4537.98

10.41 2427.38 7.59 4008.32

12.70 3518.65 13.73

cy-glu 1211.87 9.72 3345.94

7.67 2266.27 7.09 3645.51

11.55 4735.70 18.49

cy-ara 1340.24 10.75 4715.94

10.82 2756.09 8.62 3243.98

10.28 3071.83 11.99

22

∑cy 3692.79 29.62 12599.86

28.9 7449.74 23.3 10897.81

34.53 11326.18 44.21

meancy 1230.9 9.87 4199.9

9.63 2483.2 7.77 3632.6

11.51 3775.39 14.73

pt-gal 411.67 3.30 1940.00

4.45 1432.65 4.48 1284.70

4.07 539.41 2.11

pt-glu 897.57 7.20 3047.38

6.99 2607.11 8.15 2383.68

7.55 1754.99 6.85

pt-ara 435.00 3.49 1742.01

3.99 1501.22 4.69 830.41

2.63 481.44 1.88

∑pt 1744.24 13.99 6729.39

15.43 5540.98 17.32 4498.79

14.25 2775.84 10.84

meanpt 581.41 4.66 2243.13

5.14 1846.99 5.77 1499.59

4.75 925.28 3.61

pn-gal 47.18 0.38 380.42

0.87 247.11 0.77 410.86

1.30 357.60 1.40

pn-glu 189.31 1.52 1035.40

2.37 716.98 2.24 1472.87

4.67 1913.08 7.47

pn-ara 1.54 0.01 95.39

0.22 62.49 0.20 189.71

0.60 118.84 0.46

∑pn 238.03 1.99 1511.21

3.46 1026.58 3.21 2073.44

6.57 2389.52 9.33

meanpn 79.34 0.66 503.73

1.15 342.19 1.07 691.14

2.19 796.51 3.11

mv-gal 185.46 1.49 1229.45

2.82 857.28 2.68 880.44

2.79 426.97 1.67

mv-glu 540.26 4.33 2576.18

5.91 1972.23 6.17 1991.36

6.31 1871.14 7.30

mv-ara 147.59 1.18 850.84

1.95 786.84 2.46 421.76

1.34 280.46 1.09

∑mv 847.31 7 4656.47

10.68 3616.35 11.31 3293.56

10.44 2578.57 10.06

meanmv 282.44 3.5 1552.16

3.56 1205.45 3.77 1097.85

3.48 859.52 3.35

∑overall 12463.29 100.00 43605.34

100.00 31975.52 100.00 31570.06

100.00 25619.12 100.00

-

_________________________________________________________________________________________________________________

___________________________________________

*wh1, wh2 (whites ); pin (pink); pur (purple); blu1, blu2 (normal blues); blunw1, blunw2 (blue/black no wax) **dp-gal; delphinidin galactoside, dp-glu; delphinidin glucoside, cy-gal; cyanidin galactoside, dp-ara;

delphinidin arabinoside, cy-glu; cyaniding glucoside, pt-gal; petunidin-galactoside, cy-ara; cyanidin-arabinoside,

23

pt-glu; petunidin glucoside, pn-gal; peonidin-galactoside, pt-ara; petunidin-arabinoside, pn-glu; peonidin-

glucoside, mv-gal; malvidin-galactoside, pn-ara; peonidin arabinoside, mv-glu; malvidin glucoside, mv-ara;

malvidin arabinoside.

Table 4. Pearson (r) correlation of anthocyanins, total phenolic compounds (TPC), total

monomeric anthocyanins (TACY) and proanthocyanidins contents in conjunction with

antioxidant capacity (DPPH, ORAC and FRAP) in some differently colored berries of

bilberries (V. myrtillus).

dp-

gal

dp-

glu

cy-

gal

dp-

ara

cy-

glu

pt-

gal

cy-

ara

pt-

glu

pn-

gal

pt-

ara

pn-

glu

mv-

gal

pn-

ara

mv-

glu

mv-

ara

TPC

TAC

Y

DPP

H

OR

AC

FR

AP

PRO

ANT

TAC

Y/TP

dp-

gal -

0.9

51*

0.6

27

0.9

55*

0.0

8

0.98

9**

0.8

18

0.91

7 0.5

0.95

5*

-

0.02

4

0.96

3**

0.2

28

0.78

6

0.93

2*

0.95

0*

0.95

8*

0.99

2**

0.7

82

0.8

49

-

0.352

-

0.506

dp-

glu - -

0.7

1

0.9

06*

0.2

67

0.97

8**

0.8

47

0.99

3**

0.66

3

0.93

0* 0.19

0.98

4**

0.4

13

0.90

2*

0.95

1*

0.97

1**

0.99

5**

0.95

5*

0.5

6

0.7

46

-

0.125

-

0.352

cy-

gal - - -

0.4

15

0.7

93

0.65

2

0.9

37*

0.76

9

0.96

0**

0.45

4

0.72

9

0.76

8

0.8

8

0.90

1* 0.5

0.64

6 0.76

0.68

5

0.3

6

0.2

99

-

0.022 0.162

dp-

ara - - - -

-

0.0

97

0.93

7*

0.6

73

0.85

6

0.30

4

0.99

7**

-

0.19

9

0.87

5

-

0.0

04

0.68

3

0.97

5**

0.88

8*

0.88

9*

0.91

5*

0.7

39

0.8

17

-

0.235

-

0.532

cy-

glu - - - - -

0.12

1

0.6

17

0.37

3

0.86

5

-

0.03

4

0.98

7**

0.28

3

0.7

84

0.65

2

0.06

6

0.12

9

0.30

6

0.13

5

-

0.1

72

-

0.3

25 0.475 0.702

pt-gal - - - - - -

0.8

12

0.95

1*

0.56

2

0.94

4*

0.03

6

0.98

5**

0.3

27

0.81

8

0.93

6*

0.98

6**

0.98

0**

0.99

3**

0.6

85

0.8

57

-

0.320

-

0.509

cy-

ara - - - - - - -

0.87

7

0.84

2 0.7

0.51

3

0.88

1*

0.5

63

0.94

7*

0.72

4

0.76

5

0.88

6*

0.83

8

0.5

98

0.4

57

-

0.056 0.021

pt-glu - - - - - - - - 0.7

0.88

8*

0.30

2

0.97

7*

0.4

96

0.94

3*

0.92

3*

0.94

9*

0.99

1**

0.92

8*

0.4

97

0.6

77

-

0.053

-

0.256

pn-

gal - - - - - - - - - 0.36

0.83

8

0.69

5

0.9

01*

0.88

8*

0.43

9

0.59

2

0.69

8

0.56

9

0.1

03

0.1

83 0.153 0.260

pt-ara - - - - - - - - - -

-

0.13

3

0.89

2*

0.0

51

0.72

9

0.99

0*

0.90

1*

0.91

1*

0.91

7*

0.6

94

0.7

92

-

0.174

-

0.485

pn-

glu - - - - - - - - - - -

0.20

3

0.9

21

0.57

9

-

0.02

4

0.06

8

0.22

4

0.04

1

-

0.3

15

-

0.3

78 0.501 0.711

mv-

gal - - - - - - - - - - - -

0.4

72

0.89

2*

0.90

0*

0.97

9**

0.99

5**

0.98

2**

0.6

23

0.7

86

-

0.258

-

0.393

pn-

ara - - - - - - - - - - - - -

0.63

3

0.12

9

0.41

7

0.44

9

0.33

5

-

0.1

82

0.0

97 0.041 0.203

mv-

glu - - - - - - - - - - - - - -

0.78

9

0.80

6

0.91

5*

0.80

6

0.3

86

0.4

23 0.141 0.061

mv-

ara - - - - - - - - - - - - - - -

0.90

2*

0.92

8*

0.89

8*

0.6

02

0.7

29

-

0.054

-

0.392

TPC - - - - - - - - - - - - - - - -

0.96

9**

0.97

0**

0.5

75

0.8

69

-

0.327 -0.54

TAC

Y - - - - - - - - - - - - - - - - -

0.96

9**

0.5

98

0.7

46

-

0.169

-

0.339

DPP

H - - - - - - - - - - - - - - - - - -

0.7

46

0.8

61

-

0.391

-

0.510

ORA

C - - - - - - - - - - - - - - - - - - -

0.6

84

-

0.581

-

0.489

FRA

P - - - - - - - - - - - - - - - - - - - -

-

0.693

-

0.876

PRO

ANT - - - - - - - - - - - - - - - - - - - - - 0.804

TAC

Y/TP - - - - - - - - - - - - - - - - - - - - - -

Abbreviations for anthocyanins: dp-gal; delphinidin galactoside, dp-glu; delphinidin

glucoside, cy-gal; cyanidin galactoside, dp-ara; delphinidin arabinoside, cy-glu; cyaniding

glucoside, pt-gal; petunidin-galactoside, cy-ara; cyanidin-arabinoside, pt-glu; petunidin

glucoside, pn-gal; peonidin-galactoside, pt-ara; petunidin-arabinoside, pn-glu; peonidin-

glucoside, mv-gal; malvidin-galactoside, pn-ara; peonidin arabinoside, mv-glu; malvidin

glucoside, mv-ara; malvidin arabinoside.

24

* Significant at P < 0.05.

** Significant at P < 0.01.

Highlights

Phenolics in wild-type colored and non-colored berries of Finnish bilberries

Noticeable difference in phenolics acid forms was found among the berries.

Syringic, ferulic, 4-coumaric and sinapic acids significantly differed among the berries.

The main anthocyanidin was delphinidin in the wild-type berries.

The wild-type berry samples have higher antioxidant capacity than non-colored.

Fig. 1

25

Fig. 2

Fig. 3

26

Fig. 4

Fig. 5