Journal of Applied Botany and Food Quality 90, 115 - 125 (2017), DOI:10.5073/JABFQ.2017.090.014
1 University of Bandırma Onyedi Eylül, Bandırma Vocational School, Department of Food Processing, Balıkesir, Turkey
2 University of Uludag, Agricultural Faculty, Department of Food Engineering, Bursa, Turkey
3 Kurtsan Pharmaceuticals Inc., Bandırma, Balikesir, Turkey
4 University of Balikesir, Engineering and Architechture Faculty, Department Food Engineering, Balikesir, Turkey
Influence of hot air drying on phenolic compounds and antioxidant capacity
of blueberry (Vaccinium myrtillus) fruit and leaf
Nurcan Değirmencioğlu1*, Ozan Gürbüz2, Gözde Erdem Karatepe3, Reyhan Irkin4
(Received October 20, 2016)
* Corresponding author
Summary
The present study was undertaken to assess the effects of hot air
drying on phenolic compositions, total phenolic (TP) content, total
anthocyanin (TA) content, as well as antioxidant capacities of
methanol extracts from blueberry (Vaccinium myrtillus) fruit and
leaf introduced in Kapıdağ region of Turkey climate conditions. A
total of twenty-two phenolic standards were screened by HPLC, total
phenols were measured by spectrophotometric methods, antioxidant
capacity was determined using DPPH, CUPRAC, ABTS, and FRAP
assays in the blueberry fruit and leaf extracts. Analysis by HPLC
revealed that fruit extracts have different phenolic profiles due
to drying process and contain syringic acid, myricetin, naringin,
(-)-epicatechin, and malvidine-3-O-glucoside chloride as the main
compounds. Leaf extracts had higher resveratrol concentrations than
fruit extracts. The TP and TA contents gradually increased when the
blueberry fruits were dried under hot air condition. The fresh and dried
blueberry fruit and leaf extracts showed similar antioxidant capacity
values. Significant relationships between antioxidant capacity and TP
were found.
Keywords Vac. myrtillus; blueberry; hot ait drying; fruit; leaf;
phenolics; antioxidant capacity
Introduction
Blueberry, a perennial shrub of the genus Vaccinium, family Erica-
ceae, became well known around the world due to high levels of phe-
nolic compounds (EhlEnfEldt and Prıor, 2001; Prıor et al., 2001;
Kım et al., 2010; Routray et al., 2014). These compounds have
been reported to have numerous valuable health benefits including
superb antioxidant, anti-hypertensive, anti-diabetic, anti-leukemia,
anti-obesity, anti-inflammatory, and anti-microbial activity, as well as
neuroactive properties, to protect against cancer and stroke (EhlEn-
fEldt and Prıor, 2001; DEng et al., 2014; Lı et al., 2013). Blue-
berries are considered to be one of the richest sources of phenolic
compounds and antioxidant phytochemicals among fruits and vege-
tables, and they contain significant levels of anthocyanins, flavonols,
flavonons, proanthocyanidins, and phenolic acids (CastrEjón et al.,
2008; Wang et al., 2012). Factors that have an impact on the total
phenolic content, total anthocyanins, and the antioxidant capacity of
blueberries fruit and leaves, include genetic differences, the cultivar
type, growing location and season, agronomic factors, the degree of
maturity at harvest, and postharvest storage conditions (EhlEnfEldt
and Prıor, 2001; DEng et al., 2014).
Drying, as a preservation method, is a very important aspect of food
processing. The main functions of drying are lowering the water ac-
tivity, inhibiting the growth of microorganisms, decreasing chemical
reactions, extending shelf life, allowing for room temperature sto-
rage, reducing transportation costs with regard to refrigeration, and
also enhancing visual and taste of cereals, confections, and baked
goods. Several drying techniques such as sun drying, convection oven
drying, freeze drying, microwave drying etc., have been employed
in an effort to achieve high quality dried blueberries (MEjıa-MEza
et al., 2008; Hamrounı-SEllamı et al., 2013). The selection of dry-
ing methods to be used is dependent on the use of the end product,
economic viability, availability of resources, and composition of the
biomaterial (Routray et al., 2014). Convective hot air drying is a
traditional, low cost technique that is widely used to lower the water
content of fresh products at present, nevertheless it requires relatively
long times and high temperatures, causes degradation of important
nutrients, has shrunken and toughened dried products with noticeable
browning, and allows for little rehydration ability (SEllaPPan et al.,
2002).
The aim of this study was to determine the influence of location, part
of plant (fruit or leaf), and oven drying on the phenolic compounds,
total phenol and anthocyanin contents, and antioxidant capacities of
blueberries (Vac. myrtillus).
Material and methods
Sample collection and preparation
Blueberry leaves and fruit grown at three different locations of
Erdek (sea level, Balikesir, Turkey) and Kapıdağ (altitudes of 650 m,
Balıkesir, Turkey) regions were randomly hand-picked during Octo-
ber and November, 2014, and transported to the laboratory within the
same day. Upon arrival, the fruit and leaves were hand selected and
separated into two lots of equal weight. One lot of fruit and leaves
were stored separately fresh, and the other lots were firstly dried at
50 °C for 2 h in an air oven type FN 055 (NUVE, İstanbul, Turkey)
then cooled. The two lots were then separately vacuum packaged (VC
999/K12NA packing machine, Verpackungssysteme AG, Herisau,
Switzerland) in FMXBK polyamide-polyethylene film (PO2=15 cm3/
m2/24 h at 23 °C and 75 % relative humidity; Flexopack S.A. Plastics
Industry, Koropi, Greece) and stored at -20 °C (SF 312, Dairei, To-
kyo, Japan) until further analysis. Extracts of Vac. myrtillus fruit and
leaf were prepared according to the method of described by EhlEn-
fEldt and Prıor (2001) and Prıor et al. (2001), with some modifica-
tions. Fresh and dried fruit and leave samples (2 g) were separately
extracted twice with 20 mL of methanol:formic acid (99.5/0.5, v/v,
for fruit), and acetone:formic acid (99.5/0.5, v/v, for leaf) mixture in
an ultrasonic bath at room temperature (20 °C) for 15 min. Extracts
then were separately centrifuged at 3500 rpm for 10 min at 4 °C in
a centrifuge (Sigma 3K30, UK). The supernatants were combined,
after removal of methanol and acetone with a rotary evaporator (Hei-
dolph Laborota 4001, Germany) under vacuum conditions at 40 °C,
the residual extracts were subjected to a liquid-liquid partition with
methanol:formic acid (99.5/0.5, v/v, for fruit) and acetone:formic
acid (99.5/0.5, v/v, for leaves), respectively, filtered through a nylon
filter membrane (Sigma Z290793, pore size 0.45 μm, diam. 47 mm),
transferred to vials, and stored -20 °C until further analysis.
116 N. Değirmencioğlu, O. Gürbüz, G.E. Karatepe, R. Irkin
Chemicals
Phenolic standards were obtained from Fluka (St. Louis, MO, USA)
(gallic acid; CAS:149-91-7, ferulic acid; CAS:537-98-4, resvera-
trol; CAS:501-36-0, (+)-catechin; CAS:154-23-4, (-)-epicatechin;
CAS:490-46-0, myricetin; CAS:529-44-2, kaempferol; CAS:520-
18-3, (-)-epigallocatechin; CAS:970-84-1, Sigma (St. Louis, MO,
USA) (quercetin; CAS:117-39-5, caffeic acid; CAS:331-39-5, sy-
ringic acid; CAS:530-87-4, p-coumaric acid; CAS:501-98-4, narin-
gin; CAS:10236-47-2, hesperidin; CAS:520-26-3, neohesperidin;
CAS:13241-33-3, rutin hydrate; CAS:207671-50-9, cyanidin-3-O-
glycoside chloride; CAS:7084-24-4, malvidine-3-O-glycoside chlo-
ride; CAS:7228-78-6), Aldrich (St. Louis, MO, USA) (vanillic acid;
CAS:121-34-6, trans ferulic acid; CAS:537-98-4, 3-hydroxy-4-
metoxy-cinnamic acid; CAS:637-73-5), HWI Analytik GmbH
(Ruelzheim, Germany) (chlorogenic acid; CAS:327-97-9). Calibra-
tion curves were made by diluting stock standards in methanol.
Determination of phenolic composition using HPLC
Phenolic compositions were analysed according to a previously
reported method with modifications in HPLC elution conditions
(SEllaPPan et al., 2002). The phenolic extracts, phenolic standards
and also all solvents were filtered through a nylon filter membrane
(Sigma Z290793, pore size 0.45 μm, diam. 47 mm) prior to HPLC
analysis and then analysed in a HPLC chromatography system (Shi-
madzu Class VP V.6.14 SP1, USA) equipped with Shimadzu Diode
Array Detector (SPD-M 10A), VP and reversed-phase C18 column
(Zorbax Eclipse XDB, Agilent, 4.6 mm, 150 mm, 5 μm). The tempe-
rature of the column oven was set at 40 °C. The wavelengths used
for the quantification of phenolic compounds by the detector were:
280 nm for syringic acid, gallic acid, (+)-catechin, neohesperidin,
caffeic acid, hesperidin, (-)-epigallocatechin, (-)-epicatechin, narin-
gin, vanillic acid; 320 nm for trans-ferulic acid, chlorogenic acid,
3-hydroxy-4-metoxy-cinnamic acid, resveratrol, p-coumaric acid,
ferulic acid; 360 nm for myricetin, rutin hydrate, kaempferol, querce-
tin; and 520 nm for cyanidin-3-O-glycoside chloride, malvidine-3-O-
glycoside chloride. A gradient elution was employed with mobil
phase consisting of methanol:water:formic acid (3.5/96.4/0.1, v/v/v,
solvent A) and acetonitril:formic acid (98/2, v/v, solvent B) as fol-
lows: the composition of B was increased from 0.5 % to 7.5 % after
31 min, increased to 10 % for 9 min, and increased to 14 % for 5 min,
increased to 18 % for 5 min, increased to 30 % for 10 min, increased
to 45 % for 5 min, and increased to 60 % for 5 min. The composition
was decreased to 40 % for 5 min. The injection volume was 20 μL,
the flow rate was 0.7 mL/min at room temperature, the duration of a
single run was 75 min. All phenolic acids were quantified using an ex-
ternal standard. The total phenolic extracts and standard compounds
were analyzed under the same analysis conditions and a 10 min equi-
librium time was allowed between injections. All standard and sam-
ple solutions were injected in triplicate.
Determination of total phenolic (TP) content
The total phenolic (TP) contents of fresh and dried blueberry fruit
and leave extracts were measured by the Folin-Ciocalteu method de-
scribed by SınglEton et al. (1999), with some modifications. Briefly,
an aliquot (0.5 mL) of appropriately diluted extracts, or standard so-
lutions of gallic acid, 1.5 mL of double distilled water and 2.5 mL
Folin-Ciocalteu reagent, were mixed within volumetric flasks at room
temperature. After 10 min, 0.25 mL of 7.5 % sodium carbonate (1:3
diluted with double distilled water) solution (m/v) was added and
mixed thoroughly. The absorbance of the solution was measured us-
ing a spectrophotometer (UVMecasys Optizen 3220) at 750 nm after
30 min in the dark at room temperature. Methanol was used as the
blank and gallic acid was used for calibration of the standard curve
(0-500 mg/L). The results were expressed as mg of gallic acid equi-
valents (GAE) per kg. Each extract was measured in triplicate.
Determination of total anthocyanin (TA) content
The total anthocyanin content of extracts obtained from blueberry
fruit and leaves were determined by means of the pH-differential
method as described by SEllaPPan et al. (2002). The absorbance was
measured using a spectrophotometer (UVMecasys Optizen 3220)
at 700 nm and at the wavelength of maximum absorption (520 nm)
against a blank and calculated as:
A = (A520 – A700) pH1.0 - (A520 – A700) pH4.5
Monomeric anthocyanin pigment concentration of extracts was
calculated as cyanidin-3-glucoside equivalent and each extract was
measured in triplicate.
Monomeric anthocyanin pigment (mg/L) =
A × MW × DF × 1000 (ε × 1)
where A = absorbance, MW = molecular weight (449.2), DF =
dilution factor, ε = molar absortivity (26900). The final concentration
of total anthocyanins (mg/kg) was calculated based on total volume
of extract and weight of sample.
Determination of antioxidant capacity by cupric ion reducing
antioxidant capacity (CUPRAC) assay
Determination of CUPRAC was conducted according to the method
by APaK et al. (2007). One mL 10 mmol/L CuCl2, 1 mL 7.5 mmol/L
neocuproine, 1 mL 1 M NH4Ac, × mL extract, and (4-×) mL H2O were
mixed. The tubes were stopped and after 30 min the final absorbance
was recorded using a spectrophotometer (UVMecasys Optizen 3220)
at 450 nm against a reagent blank. A standard curve was prepared
using different concentrations of Trolox. The calculations of the
antioxidant capacity of phenolic antioxidants were expressed as μmol
of Trolox equivalent (TE) per gram. Each extract was measured in
triplicate.
Determination of antioxidant capacity by DPPH (2,2-diphenyl-2-
picrylhydrazyl) free radical assay
The free radical scavenging capacity of the blueberry fruit and leave
extracts were determined by colorimetric method described by
Brand-Wıllıams et al. (1995). In brief, the appropriately diluted
extracts (× mL), methanol (4-× mL), and DPPH solution (3.9 mL,
50 μM) in methanol were incubated in a water bath at 37 °C for
30 min. After incubation, the absorbance was measured at 515 nm
with a spectrophotometer (UVMecasys Optizen 3220). The results
were calculated against methanol without DPPH and compared to a
different concentration of Trolox standard curve. Each extract was
measured in triplicate. DPPH values, derived triplicate analyses,
were expressed as μmol of Trolox equivalent (TE) per gram and were
calculated as follows:
DPPH radical scavenging capacity (%) = (1-[Asample/Acontrol]) × 100
Determination of antioxidant capacity by ABTS [2,2-azinobis(3-
ethylbenzothiazoline-6-sulphonic acid)] assay
The ABTS method is based on the deactivation of the antioxidant
radical cation ABTS·+. The ABTS method was performed as de-
scribed by RE et al. (1999). ABTS radical cation (ABTS+) was pro-
duced by reacting 7 mM ABTS solution with 2.45 mM potassium
persulfate (K2S2O8) aqueous solution and allowing the mixture to
stand in the dark at room temperature for 12-16 h before use. Dif-
ferent concentrations of fruit and leaf extracts were mixed with
1 mL of diluted ABTS·+ solution and the reduction of ABTS·+ radical
was measured by the decrease in absorbance at 734 nm after 6 min by
Bioactive compounds in fruits and leaves of blueberry 117
using the spectrophotometer UVMecasys Optizen 3220. To develop
a standard curve, a standard Trolox solution was diluted with ethanol
and added to 1 mL of the diluted ABTS·+ solution. The controls con-
tained the extraction solvent instead of the test samples. Each extract
was measured in triplicate. The scavenging capacity of ABTS free
radical was calculated as:
ABTS radical scavenging capacity (%) = (1-[Asample/Acontrol]) × 100
Determination of antioxidant capacity by FRAP assay
The FRAP assay was conducted according to BEnzıE and Straın
(1996). This method is based on an increase of the absorbance at
593 nm due to the formation of tripyridyl-S-triazine complexes with
Fe2+ [TPTZ-Fe(II)] in the presence of a reductive agent. The FRAP
reagent was prepared by mixing TPTZ solution (10 mmol/L) in hy-
drochloric acid (40 mmol/L) and FeCl3 solution (20 mmol/L) mixed
with 25 mL of acetate buffer (0.3 mol/L, pH=3.6). An appropriately
diluted sample extract (× μL) and FRAP reagent (1-× mL) were added
and, the mixture and extraction or solvent for the reagent blank were
incubated at 37 °C for 30 min. At the end of incubation, absorbance
was immediately measured using a spectrophotometer (Perkin Elmer
UV/VIS Lambda35) at 595 nm. Solutions of Trolox dissolved in ex-
traction solvent, ranging from 10-100 μmol/L were used for prepa-
ration of a calibration curve. FRAP values, derived from triplicate
analyses, and were expressed as μmol of Trolox equivalent (TE) per
gram. Each extract was measured in triplicate.
Statistical analysis
Statistical differences between the data sets were determined by
two-way Analysis of variance (ANOVA) using the SPSS statistical
package (SPSS 16.0, Chicago, IL). Differences between treatments
that are described subsequently as being significant, were determined
at least p<0.05. The least significant difference (LSD) test was used
to determine differences between means.
Results and discussion
Phenolic compositions
The data set for the contents of phenolic acids, flavonols, flavanones,
monomeric of flavan-3-ol derivatives, anthocyanins, and the stilbene
in the Vac. myrtillus fresh and dried fruit and leaf extracts are given
in Tab. 1-4. These compounds can act as antioxidants and may be
important components of functional foods. The dominant phenolic
acid was syringic acid in fresh fruit extracts grown in Erdek and
Kapıdağ regions (28.79-637.43 mg/kg FW, 339.13-995.15 mg/kg
FW, respectively), and their content was especially high (p<0.05) in
dried fruits. In fresh and dried Vac. myrtillus leaf extracts, syringic,
p-coumaric, gallic and vanillic acids were the most abundant phenolic
acids. Some research has reported that chlorogenic acid, referred to
as 5-O-caffeoylquinic acid (5-CQA), is considered a major colourless
phenolic acid in blueberry fruit and leaf (Harrıs et al., 2007; Kım
et al., 2010), and a more readily available sources of 5-CQA, even
compared to green coffee beans (Kım et al., 2010). Prıor et al.
(2001) found the level of chlorogenic acid in blueberries to be 60-
100 mg/g of fresh fruit, while Harrıs et al. (2007) detected this
compound 30 times more concentrated in the leaf extract than in
fruit. Nevertheless, in this study high chlorogenic acid levels in the
fruit and leaf extracts were not determined. On the other side, vanillic
acid, 3-hydroxy-4-metoxy cinnamic acid, and ferulic acid were
obtained in fresh fruit extracts, whereas in dried fruit extracts, higher
levels of vanillic acid, gallic acid, p-coumaric acid, ferulic acid, and
3-hydroxy-4-metoxy cinnamic acid were determined. Apart from
chlorogenic acid, the phenolic acids have been found to be present
in smaller concentrations, such as caffeic, p-coumaric and ferulic
acid (SEllaPPan et al., 2002). In addition, other phenols that may be
found include gallic, p-hydroxybenzoic, m-hydroxybenzoic, ellagic,
vanillic, protocatechuic, gentisic, syringic, sinapic and salicylic acids,
and catechin, epicatechin, myricetin, and kaempferol (SEllaPPan
et al., 2002; Harrıs et al., 2007). In our samples obtained from Erdek
and Kapıdağ, we found some of these components, and most phenolic
compounds detected in this study were consistent with previous
reports on blueberry fruit and leaves from different locations in the
world. It is believed that the significant qualitative and quantitative
differences (p<0.05) of phenolic compounds profiles which have
been confirmed in blueberries, are due to variations in genotypes,
locations, cultivation conditions, increased maturity, different parts
of plants examined, stresses, organically grown, extraction methods,
and all can have varying effects on the level of total anthocyanins,
total phenolics and antioxidant capacity (Xıaoyong and Lumıng,
2014). Also drying conditions can cause differences.
Some recent studies have been accomplished on the content of blue-
berry and bilberry native flavonols (SEllaPPan et al., 2002; Harrıs
et al., 2007; MozE et al., 2011; VrhovsEK et al., 2012). In this study,
fresh Vac. myrtillus fruit extracts contained low flavonols contents
(0.79-91.98 mg/kg FW), whereas dried fruit and leaf extracts con-
tained relatively high flavonols. While myricetin was found to be
the main flavonol compound in the leaves, kaempferol and querce-
tin were also detected, in agreement with other studies. Previous re-
search also determined that the green leaves of blueberry contained
a much larger amount of flavonoids (quercetin and kaempferol)
and hydroxycinnamic acid (p-coumaric and caffeic acid) than fruits
(RııhınEn et al., 2008). Nevertheless, in this study the concentra-
tions of myricetin were higher in the fresh leaves extracts than fresh
fruit extracts (p<0.05). Also in the dried fruit extracts, the concen-
trations of flavonols were considerably higher than in the dried leaf
extracts. As mentioned by JaaKola et al. (2004) and Oszmiański
et al. (2011), p-coumaric acid is the precursor of flavonoids, and the
increase in p-coumaric acid concentration in the leaves, growing
under high solar radiation, can also reflect the overall activation of
flavonoid biosynthesis. Tab. 1-4 shows that all the flavonol com-
pounds had some significant (p<0.05) level of variability due to alti-
tude, and due to the drying process. Rutin hydrate, which has been re-
ported in high amounts in buckwheat (MozE et al., 2011), was found
in both regions of Vac. myrtillus fruits under investigation, the dried
fruit extracts containing more rutin hydrate than fresh fruit extracts
and also compared to the fresh and dried leaf extracts. MozE et al.
(2011) first detected rutin in bilberry and blueberry samples (0.2 and
3.1 mg/100g FW, respectively), and also rutin (quercetin-3-O-rutino-
side) was the major phenolic compounds in leaves of rabbiteye blue-
berry cultivated in Japan (lı et al., 2013). Harrıs et al. (2007) deter-
mined this compound as 3.10 mg/100 g in Vac. corybocum L. fruits,
whereas our results were higher than their results. Bioflavonoids like
rutin and naringin have been proven to be efficacious antioxidants
and are widely distributed in fruits and vegetables. Rutin belongs to
the class of flavonols and naringin belongs to flavanones. It is well
noted and proven in several studies, that flavonols are very active
in conveying therapeutic benefit compared to flavonones (AKondı
et al., 2011). The flavonones (naringin, hesperidin, and neohesperi-
din) were detected in studied Vac. myrtillus extracts. Naringin is one
of the most abundant flavanone in fresh and dried fruit and leaf ex-
tracts in this study. It was also observed that naringin levels were
greater in samples harvested from high altitude compared to those
samples originating from areas at sea level. Some reports have indi-
cated that drying methods can affect phenolic contents and the anti-
oxidant capacity of plant materials due to drying time/temperature,
light intensity, packaging, and storage time etc. (Lu and Luthrıa,
2014).
The three flavanols were found in both regions of Vac. myrtillus, the
dried fruit extracts being a better source of (+)-catechin, (-)-epicate-
118 N. Değirmencioğlu, O. Gürbüz, G.E. Karatepe, R. Irkin
Tab. 1: Phenolic compounds concentrations of fresh Vac. myrtillus fruits grown in Erdek ve Kapıdağ regions (mg/kg)
Phenolic compounds E1FRF* E2FRF E3FRF K1FRF K2FRF K3FRF
Gallic acid 19.19 ± 2.12Cb** 36.82 ± 4.58Aa 30.29 ± 3.14Ba 10.00 ± 1.13Dk 8.10 ± 1.10Dl 0.87 ± 0.12Em
Vanillic acid 532.97 ± 25.16Aa 52.36 ± 3.25Cc 289.61 ± 5.12Bb 20.91 ± 1.95El 9.25 ± 1.63Fm 29.75 ± 1.98Dk
Caffeic acid 5.46 ± 1.23Aa 4.84 ± 1.10Bc 5.35 ± 1.00Ab 1.87 ±0.12Ck 1.86 ± 0.24Ck 1.73 ± 0.23Dl
Chlorogenic acid 1.25 ± 0.25Ec 4.37 ± 0.85Aa 1.71 ± 0.26Cb 1.42 ± 0.10Dl 1.50 ± 0.27CDl 2.71 ± 0.45Bk
Syringic acid 637.43 ± 3.45Ba 28.79 ± 4.85Fc 32.30 ± 1.26Eb 529.53 ± 6.52Cl 995.15±6.54Ak 339.13± 3.21Dm
p-Coumaric acid 7.63 ± 1.48Ca 1.81 ± 0.12Ec 4.04 ± 1.85Db 11.18 ± 2.13Bl 20.05 ± 2.31Ak 8.68 ± 1.00Cm
Ferulic acid 10.60 ± 2.69Da 6.06 ± 0.95Eb 4.13 ± 0.74Fc 20.54 ± 2.31Bl 24.87 ± 1.98Ak 14.77 ± 1.14Cm
Trans-ferulic acid 5.99 ± 1.78Da 1.89 ± 0.14Eb 5.45 ± 1.23Da 8.49 ± 2.85Cm 11.50 ± 2.14Bl 19.40 ± 1.56Ak
3-hydroxy-4-methoxy 50.63 ± 4.65Db 75.95 ± 4.65Ca 26.29 ± 2.32Ec 78.11 ± 3.12BCm 103.39 ± 6.54Ak 80.61 ± 3.24Bl
cinnamic acid
Myricetin 51.64 ± 2.98Db 55.12 ± 3.12Da 43.06 ± 2.96Ec 79.78 ± 2.96Cm 91.98 ± 3.45Ak 83.39 ± 3.59Bl
Quercetin 1.62 ± 0.12Ec 4.27 ± 1.00Bb 6.93 ± 1.85Aa 2.48 ± 0.62Dl 3.71 ± 0.56Ck 2.24 ± 0.46Dl
Kaempferol 1.72 ± 0.45ABa 0.94 ± 0.16Db 1.82 ± 0.15Aa 0.79 ± 0.10Em 1.01 ± 0.15Dl 1.67 ± 0.37Bk
Rutin hydrate 3.84 ± 1.10Da 1.31 ± 0.25Fc 2.07 ± 0.23Eb 13.30 ± 1.10Bl 24.01 ± 1.47Ak 7.64 ± 0.96Cm
Naringin 5.50 ± 1.14Dc 33.60 ± 3.74Bb 79.37 ± 3.41Aa 4.42 ± 0.65Em 6.97 ± 0.84Ck 6.13 ± 0.45Dl
Hesperidin 7.07 ± 1.56Cc 27.45 ± 2.85Bb 31.59 ± 2.16Aa 2.40 ± 0.13El 4.93 ± 0.16Dk 2.35 ± 0.78El
Neohesperidin 2.50 ± 0.45Bb 8.38 ± 2.14Aa 8.80 ± 1.62Aa 1.83 ± 0.14CDl 1.92 ± 0.11Ck 1.73 ± 0.15Cbm
(+)-Catechin 2.72 ± 0.41Dc 29.57 ± 2.48Aa 9.45 ± 1.64Bb 6.41 ± 0.64Ck 1.36 ± 0.12Em 2.63 ± 0.43Dl
(-)-Epicatechin 28.54 ± 3.41Da 10.43 ± 1.96Eb 8.63 ± 1.18Fc 50.63 ± 2.63Bl 80.23 ± 4.85Ak 46.04 ± 2.45Cm
(-)-Epigallocatechin 6.66 ± 1.58Dc 24.99 ± 3.11Bb 82.62 ± 3.98Aa 17.88 ± 1.84Cl 22.79 ± 2.41Bk 16.32 ± 1.47Cm
Cyanidin-3-O-glucoside 373.38 ± 15.69Ca 70.94 ± 4.23Eb 384.06 ± 4.85Ca 438.33 ± 5.23Bl 781.26 ± 6.52Ak 335.60 ± 6.21Dm
chloride
Malvidine-3-O-glucoside 3231.38 ± 25.89Ca 212.93 ± 6.52Fc 329.94 ± 6.12Eb 3644.61 ± 8.95Bl 4433.19 ± 9.96Ak 2433.10 ± 5.27Dm
chloride
Resveratrol 1.01 ± 0.05Aa 0.87 ± 0.05Bb 0.78 ± 0.05Bb 0.70 ± 0.06Bm 0.84 ± 0.06Bl 1.00 ± 0.00Ak
* E: Erdek, K: Kapıdağ, FR: Fresh, F: Fruit, 1-3: Codes of samples collected from different regions, ** Mean values (mg/kg)±standard deviation (N=3×2) with
different capital letters (A-F) in the same row are significantly different (p<0.05) according to collected from different region at fresh fruit. Mean values±standard
deviation (N=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at fresh
fruit.
Tab. 2: Phenolic compounds concentrations of dried Vac. myrtillus fruits grown in Erdek ve Kapıdağ regions (mg/kg (mg/kg)
Phenolic compounds E1DRF* E2DRF E3DRF K1DRF K2DRF K3DRF
Gallic acid 43.53 ± 4.78Cb** 28.11 ± 1.78Ec 57.63 ± 5.41Ba 52.29 ± 2.18Bl 10.43 ± 1.10Dm 72.59 ± 3.25Ak
Vanillic acid 244.73 ± 3.65Bb 352.64 ± 3.65Aa 123.76 ± 3.29Cc 42.04 ± 2.87Em 117.16 ± 2.85Ck 71.00 ± 4.58Dl
Caffeic acid 3.92 ± 1.85Db 2.19 ± 0.18Ec 6.99 ± 1.23Ca 3.80 ± 0.51Dl 35.14 ± 1.47Ak 31.88± 1.95Bm
Chlorogenic acid 1.19 ± 0.14Eb 0.94 ± 0.02Fb 1.66 ± 0.45Da 2.11 ± 0.11Cm 8.04 ± 1.45Ak 2.86 ± 0.10Bl
Syringic acid 1338. 96 ± 16.84Db 245.49 ± 1.85Fc 2971.61 ± 25.56Ca 3344.54 ± 28.62Bl 5627.47 ± 32.14Ak 304.33 ± 3.85Em
p-Coumaric acid 22.25 ± 2.14Eb 9.09 ± 1.63Fc 73.98 ± 3.45Da 390.96 ± 3.61Bl 557.22 ± 3.25Ak 137.30 ± 4.45Cm
Ferulic acid 11.92 ± 3.16Db 7.49 ± 1.74Ec 24.32 ± 2.89Ca 94.46 ± 3.54Ak 77.55 ± 1.85Bl 71.48 ± 6.18Bl
Trans-ferulic acid 1.54 ± 0.14Eb 1.06 ± 0.16Ec 2.52 ± 0.85Da 20.17 ± 4.52Ak 15.59 ± 3.85Cm 18.13 ± 2.85Bl
3-hydroxy-4-methoxy 89.40 ± 2.96Cb 30.19 ± 2.65Ec 144.22 ± 2.84Aa 98.70 ± 3.21Bl 145.96 ± 3.84Ak 71.86 ± 4.78Dm
cinnamic acid
Myricetin 122.64 ± 4.32Db 94.79 ± 2.85Ec 311.82 ± 9.12Aa 310.39 ± 9.85Ak 241.37 ± 5.45Bl 194.10 ± 6.59Cm
Quercetin 2.94 ± 0.56Db 1.13 ± 0.13Ec 4.67 ± 1.12Ca 9.67 ± 1.25Bl 11.35 ± 1.84Ak 4.36 ± 0.19Cm
Kaempferol 8.87 ± 1.12Db 3.82 ± 0.41Ec 21.49 ± 1.45Ca 70.19 ± 7.84Ak 21.42 ± 2.14Cm 28.07 ± 1.18Bl
Rutin hydrate 23.93 ± 1.45Eb 8.53 ± 0.45Fc 83.39 ± 3.12Ca 236.59 ± 6.10Ak 226.05 ± 6.95Bl 41.86 ± 2.14Dm
Naringin 119.57 ± 2.34Db 117.91 ± 2.95Db 288.42 ± 4.59Ca 343.55 ± 6.25Ak 326.60 ± 6.54ABkl 313.44 ± 5.61Bl
Hesperidin 2.69 ± 0.16Db 2.30 ± 0.16Db 3.55 ± 0.48Ca 12.66 ± 1.42Ak 12.27 ± 2.14Ak 8.46 ± 1.26Bl
Neohesperidin 1.23 ± 0.14DEb 0.94 ± 0.05Ec 1.66 ± 0.12Da 3.58 ± 0.95Bl 2.97 ± 0.23Cm 6.14 ± 1.14Ak
(+)-Catechin 1.70 ± 0.19Fc 5.63 ± 0.96Eb 32.39 ± 0.41Ba 7.21 ± 1.58Dm 23.91 ± 1.45Cl 47.75 ± 4.40Ak
(-)-Epicatechin 262.33 ± 2.64Db 61.12 ± 2.46Ec 661.53 ± 9.65Ca 2599.73 ± 29.41Bl 3675.69 ± 42.41Ak 622.78 ± 9.98Cm
(-)-Epigallocatechin 35.34 ± 1.97Eb 28.40 ± 1.85Fc 89.71 ± 5.12Da 195.44 ± 2.52Bl 254.97 ± 6.95Ak 138.53 ± 3.48Cm
Cyanidin-3-O-glucoside 2079.27 ± 6.45Db 444.10 ± 3.47Ec 5297.84 ± 23.14Ba 3649.42 ± 10.85Cl 6154.05 ± 58.42Ak 153.29 ± 3.87Fm
chloride
Malvidine-3-O-glucoside 3891.21 ± 5.23Db 1566.54 ± 5.42Ec 12933.81 ± 45.87Aa 6095.81 ± 19.84Cl 7985.69 ± 23.87Bk 1520.40 ± 27.95Em
chloride
Resveratrol 1.07 ± 0.12DEab 0.97 ± 0.04Eb 1.12 ± 0.14Da 1.37 ± 0.10Cm 2.18 ± 0.12Bl 2.58 ± 0.13Ak
* E: Erdek, K. Kapıdağ, DR: Dried, F: Fruit, 1-3: Codes of samples collected from different regions, ** Mean values (mg/kg)±standard deviation (N=3×2) with
different capital letters (A-F) in the same row are significantly different (p<0.05) according to collected from different region at dried fruit. Mean values±standard
deviation (N=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at dried
fruit.
Bioactive compounds in fruits and leaves of blueberry 119
Tab. 3: Phenolic compounds concentrations of fresh Vac. myrtillus leaves grown in Erdek ve Kapıdağ regions (mg/kg)
Phenolic compounds E1FRL* E2FRL E3FRL K1FRL K2FRL K3FRL
Gallic acid 12.09 ± 1.65Cb** 33.33 ± 2.14Ba 6.53 ± 0.96Ec 46.64 ± 3.58Ak 12.53 ± 1.26Cl 7.17 ± 1.12Dm
Vanillic acid 159.69 ± 6.85Ba 92.79 ± 6.51Cb 48.28 ± 3.48Ec 18.00 ± 2.14Fm 167.59 ± 15.20Ak 62.69 ± 9.87Dl
Caffeic acid 248.76 ± 9.78Aa 197.65 ± 12.85Bb 28.66 ± 2.14Dc 51.85 ± 4.59Ck 27.82 ± 3.85Dl 14.49 ± 2.15Em
Chlorogenic acid 9.07 ± 1.23Aa 3.16 ± 0.29Bc 3.41 ± 0.27Bb 2.27 ± 0.52CDl 2.20 ± 0.84Dl 2.49 ± 0.48Ck
Syringic acid 31.93 ± 2.14Cb 960.56 ± 15.26Aa 26.05 ± 3.56Dc 24.18 ± 3.65El 130.15 ±10.84Bk 24.09 ± 2.47El
p-Coumaric acid 123.44 ± 6.54Aa 69.26 ± 6.58Cb 50.35 ± 5.84Dc 92.83 ± 9.85Bl 129.87 ± 15.20Ak 36.23 ± 3.65Em
Ferulic acid 5.51 ± 1.10BCa 5.23 ± 0.95Db 5.33 ± 1.10CDab 5.59 ± 1.05BCl 5.68 ± 0.95Bl 6.50 ± 0.98Ak
Trans-ferulic acid 9.98 ± 1.18Bb 11.68 ± 1.84Aa 5.19 ± 0.56Cc 4.48 ± 0.96Dl 4.38 ± 0.28Dl 5.27 ± 0.82Ck
3-hydroxy-4-methoxy 68.30 ± 3.87Ba 35.45 ± 3.58Fc 59.22 ± 3.98Cb 49.34 ± 4.12Dl 86.71 ± 6.54Ak 41.80 ± 6.14Em
cinnamic acid
Myricetin 118.02 ± 6.41Ba 57.06 ± 4.50Dc 87.53 ± 6.52Cb 84.26 ± 9.23Cl 141.45 ± 8.59Ak 56.33 ± 10.20Dm
Quercetin 2.26 ± 0.25Db 0.99 ± 0.19Ec 9.32 ± 1.12Ca 9.97 ± 1.10BCm 11.83 ± 1.65Ak 10.84 ± 3.45ABl
Kaempferol 2.56 ± 0.39Cb 1.28 ± 0.25Fc 9.91 ± 1.48Aa 1.79 ± 0.27Dl 1.38 ± 0.29Em 8.78 ± 2.15Bk
Rutin hydrate 5.74 ± 0.48Dc 24.18 ± 1.17Aa 9.81 ± 2.00Bb 5.53 ± 0.52Dl 2.46 ± 0.54Em 8.95 ± 1.84Ck
Naringin 21.66 ± 1.85Aa 4.45 ± 0.98Dc 14.39 ± 2.58Bb 5.10 ± 0.29Dl 13.40 ± 2.87Ck 14.04 ± 2.14BCk
Hesperidin 13.48 ± 1.74Aa 7.50 ± 1.25Cc 9.56 ± 2.05Bb 6.31 ± 0.84Dl 5.89 ± 0.68Em 7.73 ± 0.96Ck
Neohesperidin 2.81 ± 0.69BCb 2.68 ± 0.62Cb 7.73 ± 2.14Aa 2.36 ± 0.26Dl 2.19 ± 0.39Em 3.12 ± 0.25Bk
(+)-Catechin 33.20 ± 2.98Ecc 47.15 ± 3.48Cb 61.71 ± 9.51Ba 78.83 ± 8.63Ak 44.65 ± 5.62Dl 22.51 ± 2.15Fm
(-)-Epicatechin 18.72 ± 2.45Ba 7.06 ± 1.18Db 5.60 ± 1.10Ec 2.55 ± 0.35Fm 14.00 ± 2.10Cl 20.55 ± 2.46Ak
(-)-Epigallocatechin 9.84 ± 2.12Cc 26.79 ± 3.15Bb 61.26 ± 8.56Aa 8.70 ± 0.95Dk 7.23 ± 1.98El 9.12 ± 1.10CDk
Cyanidin-3-O-glucoside 1.51 ± 0.23Ba 1.02 ± 0.20Cb 0.96 ± 0.18Db 0.98 ± 0.27CDl 0.97 ± 0.19CDl 23.61 ± 2.58Ak
chloride
Malvidine-3-O-glucoside 1.57 ± 0.15Ba 1.10 ± 0.14Cb 0.96 ± 0.12Db 1.05 ± 0.16CDl 0.00 ± 0.00Em 42.66 ± 5.00Ak
chloride
Resveratrol 1.57 ± 0.10Ec 4.11 ± 0.58Cb 6.29 ± 1.10Aa 3.91 ± 0.56Dm 5.89 ± 0.84ABk 5.31 ± 0.85Bl
* E: Erdek, K: Kapıdağ, FR: Fresh, L: Leaf, 1-3: Codes of samples collected from different regions, ** Mean values (mg/kg)±standard deviation (N=3×2) with
different capital letters (A-F) in the same row are significantly different (p<0.05) according to collected from different region at fresh leaf. Mean values±standard
deviation (N=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at fresh
leaf.
Tab. 4: Phenolic compounds concentrations of dried Vac. myrtillus leaves grown in Erdek ve Kapıdağ regions (mg/kg)
Phenolic compounds E1DRL* E2DRL E3DRL K1DRL K2DRL K3DRL
Gallic acid 176.95 ± 6.98Cc** 352.30 ± 3.59Aa 201.37 ± 3.29Bb 203.48 ± 4.36Bk 67.43 ± 3.69Em 110.28 ± 9.62Dl
Vanillic acid 271.17 ± 5.89Bb 1156.80 ± 18.29Aa 249.72 ± 4.62Cc 157.27 ± 2.48Dl 230.63 ± 13.52Ck 20.78 ± 2.98Em
Caffeic acid 27.05 ± 3.21Aa 5.28 ± 1.14Cb 28.24 ± 2.12Aa 8.61 ± 1.10Bk 4.65 ± 0.85Dl 3.28 ± 0.68Em
Chlorogenic acid 3.31 ± 0.45Ba 3.23 ± 0.58Ca 1.25 ± 0.26Eb 4.14 ± 0.87Ak 1.94 ± 0.34Dm 3.25 ± 0.52BCl
Syringic acid 202.99 ± 5.47Aa 84.36 ± 6.47Dc 113.56 ± 3.29Bb 95.71 ± 6.54Ck 47.56 ± 3.85El 32.46 ± 5.20Fm
p-Coumaric acid 15.93 ± 2.85Ba 11.71 ± 2.19Dc 13.18 ± 1.23Cb 8.51 ± 1.98Em 15.24 ± 2.18Bl 17.58 ± 2.16Ak
Ferulic acid 3.13 ± 0.95Eb 3.93 ± 0.48Ca 3.80 ± 0.48CDa 9.99 ± 2.57Ak 3.50 ± 0.84DEm 6.56 ± 1.05Bl
Trans-ferulic acid 12.29 ± 2.14Db 11.90 ± 2.43DEb 24.93 ± 1.15Aa 16.03 ± 2.18Bk 15.89 ± 2.17Cl 10.95 ± 2.84Em
3-hydroxy-4-methoxy 111.93 ± 4.58Cb 254.11 ± 4.56Aa 88.46 ± 3.58Ec 141.86 ± 9.84Bk 96.34 ± 9.27Dl 51.75 ± 9.13Fm
cinnamic acid
Myricetin 101.45 ± 4.32Db 237.56 ± 5.42Aa 94.76 ± 4.51Dc 152.97 ± 11.18Bk 140.59 ±12.17Cl 49.44 ± 7.68Em
Quercetin 4.63 ± 0.47Cb 2.07 ± 0.23Dc 8.10 ± 1.18Ba 4.27 ± 0.87Cl 2.75 ± 0.96Dm 11.38 ± 2.34Ak
Kaempferol 1.63 ± 0.28Dc 3.37 ± 0.43Aa 2.17 ± 0.18Bb 2.20 ± 0.59Bl 3.30 ± 0.95Ak 2.07 ± 0.56Bl
Rutin hydrate 12.27 ± 1.15Dc 79.69 ± 6.58Aa 16.63 ± 2.15Cb 21.79 ± 2.51Bk 11.98 ± 3.25El 8.08 ± 1.02Fm
Naringin 95.12 ± 5.21Bb 261.38 ± 5.53Aa 83.45 ± 6.41Cc 34.66 ± 3.26El 43.08 ± 8.26Dk 1.14 ± 0.38Fm
Hesperidin 82.06 ± 5.10Bb 134.01 ± 4.87Aa 60.14 ± 4.28Cc 36.93 ± 3.48Dk 7.45 ± 2.14Fm 9.74 ± 1.36El
Neohesperidin 6.52 ± 0.75Ba 3.18 ± 0.59Cb 6.06 ± 0.58Ba 17.24± 2.47Ak 2.63 ± 0.51Cm 16.20 ± 2.01Al
(+)-Catechin 17.50 ± 1.48Db 95.59 ± 3.57Aa 21.94 ± 2.43Bb 19.73 ± 2.52Cl 7.31 ± 2.18Em 22.71 ± 3.20Bk
(-)-Epicatechin 49.08 ± 3.47Ba 12.78 ± 2.15Db 10.06 ± 1.64Dc 84.06 ± 9.52Ak 37.31 ± 6.20Cm 49.38 ± 4.02Bl
(-)-Epigallocatechin 52.07 ± 3.65Ca 18.20 ± 2.39Ec 28.78 ± 3.28Db 67.26 ± 6.53Bl 29.84 ± 3.27Dm 197.80 ± 12.10Ak
Cyanidin-3-O-glucoside 0.00 ± 0.00Cb 1.00 ± 0.06ABa 1.01 ± 0.17ABa 1.06 ± 0.26Ak 1.01 ± 0.18ABkl 0.95 ± 0.09Bl
chloride
Malvidine-3-O-glucoside 1.09 ± 0.18ABa 0.00 ± 0.00Cb 1.14 ± 0.16ABa 1.20 ± 0.19Ak 1.11 ± 0.24ABkl 0.95 ± 0.12Bl
chloride
Resveratrol 3.32 ± 0.42Ba 1.54 ± 0.28Cb 1.50 ± 0.45Cb 8.89 ± 2.21Ak 8.55 ± 1.00Ak 3.85 ± 0.45Bl
* E: Erdek, K. Kapıdağ, DR: Dried. L: Leaf, 1-3: Codes of samples collected from different regions, ** Mean values (mg/kg)±standard deviation (N=3×2) with
different capital letters (A-F) in the same row are significantly different (p<0.05) according to collected from different region at dried leaf. Mean values±standard
deviation (N=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at dried
leaf.
120 N. Değirmencioğlu, O. Gürbüz, G.E. Karatepe, R. Irkin
Fig. 1: Total phenolic (TP) contents (mg GAE/kg) of fresh and dried Vac. myrtillus fruit and
leaf extracts (E: Erdek, K. Kapıdağ, FR: Fresh, DR: Dried, F: Fruit, L: Leaf, 1-3: Codes of
samples collected from different regions, Different letter(s) on bar indicate statistically
significant differences, p<0.05)
0,00
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30000,00
E1FRF E2FRF E3FRF K1FRF K2FRF K3FRF
m
g
G
A
E
/k
g
Fresh Vac.myrtillus fruit extracts
Total Phenol
d
d
d d
b
a
c
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E1DRF E2DRF E3DRF K1DRF K2DRF K3DRF
m
g
G
A
E
/k
g
Dried Vac.myrtillus fruit extracts
Total Phenol
de d
d
c
d
b
d
a
d
de
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E1FRL E2FRL E3FRL K1FRL K2FRL K3FRL
m
g
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A
E
/k
g
Fresh Vac.myrtillus leaf extracts
Total Phenol
c
c
e
a
d
b
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/k
g
Dried Vac.myrtillus leaf extracts
Total Phenol
d
c
b b
a a
chin, and (-)-epigallocatechin compared to fresh fruit and leaf and
also dried leaf extracts. The flavanol contents in fruit and leaf ex-
tracts were determined to be increasing or decreasing depending on
the altitude, the area, and the harvesting time where the samples were
collected as well as the drying process. Drying and the drying condi-
tions (oxygen, high temperature, length of time, without vacuum,
etc.) may cause polymerization reactions, reduce some polyphenols
and their antioxidant capacity, and induce the formation of new com-
pounds (KEndarı et al., 2012). As a stated by KEndarı et al. (2012)
catechin oxidation mechanism initially constructs a semiquinon,
which then converts into a quinon. The quinon compound can react
with amino acid, protein, or other polymers to produce proanthocy-
anidin. And then proanthocyanidin can degrade into catechin because
proanthocyanidin represents an oligomer or polymer from flavan-3-ol
(catechin/epicatechin). These flavanols have already been detected in
some berry fruit and leaf (SEllaPPan et al., 2002; Harrıs et al., 2007;
VrhovsEK et al., 2012; DEng et al., 2014).
In blueberry skin and flesh, delphinidin, cyanidin, petunidin, peoni-
din, and malvidin monoglycosides are the main anthocyanins. Be-
cause of the diversity in the glycosylation and acylation pattern, more
than 25 anthocyanins have been identified in blueberries (Harrıs
et al., 2007; MozE et al., 2011). The amounts and distribution of an-
thocyanins in the berries differ depending on their plant species, cul-
tivation conditions in which the fruit have been grown or stored (e.g.
light, temperature), and producing districts, due to genetic differences
among wild and cultivated varieties (EhlEnfEldt and Prıor, 2001).
Blueberry leaves are by-products of the blueberry industry. The
leaves contain in an excessively amount of polyphenols, which cre-
ates an opportunity for their use in the neutraceutical industry, more
so than the fruits (EhlEnfEldt and Prıor, 2001; Kım and Um, 2011;
Lı et al., 2013; DEng et al., 2014). But no anthocyanins have been
found in fresh green leaves (Harrıs et al., 2007; Lı et al., 2013). We
found fresh and dried Vac. myrtillus fruit extracts with high levels
of malvidin-3-O-glucoside chloride (212.93-4433.19 mg/kg FW and
1520.40-12933.81 mg/kg DW) and cyanidin-3-O-glucoside chloride
(70.94-781.26 mg/kg FW and 153.29-6154.05 mg/kg DW) collec-
ted from Turkey, respectively. As mentioned earlier, fresh and dried
blueberry leaf extracts have significantly less (p<0.05) anthocya-
nins than fruit extracts. Nevertheless, there were numerous reports
that anthocyanins were detected in blueberry leaves (Harrıs et al.,
2007; Kım and Um, 2011). These differences are possibly related to
the harvesting season of leaves. Within the anthocyanidin reductase
or leucoanthocyanidin reductase activity, the transcriptional control
favors the expression of the anthocyanidin synthase in sun-exposed
leaves. The decrease in proanthocyanidin content in favor of antho-
cyanin production has also been observed during fruit development
at the point when leaves turned red, activating the biosynthesis of
cyanidin glycosides (JaaKola et al., 2004; Lı et al., 2013). In a pre-
vious study reported by Lı et al. (2013) and RııhınEn et al. (2008)
anthocyanins were not detected in green leaves of blueberry and bil-
berry, but a low amount was detected in red leaves. Resveratrol is
a type of natural phenol and a phytoalexin produced naturally by
several plants in response to injury or when the plant is under at-
tack by pathogens such as bacteria or fungi (Frémont, 2000). The
occurrence of resveratrol in Vaccinium berries should not be sur-
prising, as its occurrence in the plant kingdom appears to be wide-
spread (Rımando et al., 2004). Food sources of resveratrol include
the skin of grapes, blueberries, raspberries, and mulberries (JasıńsKı
et al., 2013) and the variability in the resveratrol content in fruit such
as blueberries changes due to food processing or preparation. Re-
cently, resveratrol was reported by Lyons et al. (2003) at levels of
approximately 0.0002-0.0006 ng/g sample (Rımando et al., 2004).
The resveratrol contents of blueberries and the related bilberry, Vac.
myrtillus L., were cultivated in several different geographical regions
(Lyons et al., 2003). In our study, resveratrol was found in fresh fruit
and leaf extracts (0.70-1.01 mg/kg FW and 1.57-6.29 mg/kg FW, re-
spectively) from both regions. The previous data for trans-resveratrol
content (0.4 mg/100 g FW) in blueberry (MozE et al., 2011) agreed
with ours, but it has also been determined to be lower for blueberries
(Wang et al., 2008). Also, as stated by Lyons et al. (2003), blue-
berries and bilberries were found to contain resveratrol and the level
of this chemoprotective compound in these fruits was <10 % of that
reported for grapes. In grapes, the levels of resveratrol were found to
vary with the time of harvest, environmental and climatic conditions,
Fig. 1: Total phenolic (TP) contents (mg GAE/kg) of fresh and dried Vac. myrtillus fruit and leaf extracts (E: Erdek, K. Kapıdağ, FR: Fresh, DR: Dried, F: Fruit,
L: Leaf, 1-3: Codes of samples collected from different regions, Different letter(s) on bar indicate statistically significant differences, p<0.05)
Bioactive compounds in fruits and leaves of blueberry 121
and plant developmental stage (Rımando et al., 2004). These factors
possibility affected the differences in resveratrol contents of the Vac.
myrtillus extracts in this study.
Total phenol and total anthocyanin contents
The TP contents were significantly different among the fruit and leaf
Vac. myrtillus extracts (Fig. 1). The TP was 6152.05-25688.90 mg
GAE/kg in fresh fruit extracts, whereas in dried fruit were deter-
mined to be 14512.49-97214.25 mg GAE/kg. For the fresh and dried
blueberry leaf extracts, their TP contents were significantly (p<0.05)
higher than those in fresh and dried fruits. In the leaf tissues of 87
highbush blueberries, the mean values of polyphenol contents were
~ 30 times higher than observed in fruits on a FW basis (EhlEnfEldt
and Prıor, 2001). SKuPıEń et al. (2006) reported that the TP con-
tents in Vac. corymbosum L. leaf extracts was 111.5 mg/100 g dw
dried leaves. In addition, the TP of the leaf extracts of Vac. myrtil-
lus in Erdek and Kapıdağ regions were significatly higher (p<0.05)
than those of blackberry leaves (82.8-91.6 mg of GAE/g of DW) and
strawberry leaves (55.2 mg of GAE/g of DW) reported by Wang and
Lın (2000). Lı et al. (2013) and OszmıańsKı et al. (2011) indicated
that the polyphenol content of the blueberry leaves was much higher
than those of any other leaves of tested berries (blackberry, raspberry,
honeyberry, and strawberry). The differences in TP contents between
fruit and leaf extracts were statistically significant (p<0.05). Gene-
rally, the TP of plant extracts can be affected by solvent, its pola-
rity, its concentration, and/or extraction method method (DEng et al.,
2014; Xıaoyong and Lumıng, 2014). It is also well known that ge-
netic, agronomic or environmental factors play important roles in
phenolic composition and nutritional quality of crops (Yang et al.,
2009). When the TP contents of these extracts are compared with
white wines, these plants could contribute the same health benefit as
those wines in terms of polyphenols. A large variation was observed
among fruit and leaf extracts for TA content (Fig. 2), ranging from
2805.08 to 5973.69 mg/kg (in fresh fruit), from 2094.56 to 5975.14
mg/kg (in dried fruit), from 12.89 to 262.02 mg/kg (in fresh leaf), and
from 51.91 to 318.30 mg/kg (in dried leaf). Fresh and dried fruit sam-
ples are a good source of anthocyanin (Tab. 1-4), however, fresh fruit
samples recorded decreases/increases in TA content. The TA content
in blueberry fruit and leaf extracts in this study was comparable to
the quantity reported by EhlEnfEldt and Prıor (2001), SEllaPPan
et al. (2002), LohachoomPol et al. (2008), and Wang et al. (2015).
Results obtained from several studies suggest that the TP and TA con-
tents in blueberry fruit are influenced by the cultivar, harvest time,
the growing season, fruit mass, maturity, environmental growing
conditions, growing location, postharvest storage conditions, drying
process, different extraction methods, irridation, temperature, and
pathogen attacks (Routray et al., 2014).
Antioxidant capacity
Blueberry fruit and leaf show high antioxidant capacity, correlated
especially with their anthocyanin and other phenolic compounds con-
tent, and may be considered as one of the highest antioxidant sources
among fruits and vegetables (VrhovsEK et al., 2012). The values
found in the fresh Vac. myrtillus fruit extracts for antioxidant capa-
city (Fig. 3) were 8.37-23.26 μmol TE/g FW by CUPRAC, 8.56-
19.23 μmol TE/g FW by DPPH, 4.26-9.56 μmol TE/g FW by ABTS,
and 0.97-1.73 μmol TE/g FW by FRAP method, values lower/higher
than or close to those found in fresh leaf extracts (Fig. 4). Also in
blueberry fruits and leaves studied by Wang and Lın (2000), EhlEn-
fEldt and Prıor (2001), SEllaPPan et al. (2002), Lı et al. (2013),
and Wang et al. (2015) using the same methods, the Vac. myrtillus
fruit and leaf extract results obtained in this study were similar to
those studies. Therefore, as a reported by Wang and Lın (2000) and
ParK et al. (2012), because of their high antioxidant content, blue-
berry leaves can also be added to tea mixes to increase the antioxidant
capacity level in the beverages for greater benefits to human health.
Previous studies found a direct relationship between the antioxidant
capacity, and the TP and TAs contents in blueberry fruits and leaves
(EhlEnfEldt and Prıor, 2001; Lı et al., 2013). In this study, there
was a strong correlation between antioxidant capacity and TP content
Fig. 2: Total anthocyanin (TA) contents (mg/kg) of fresh and dried Vac. myrtillus fruit and leaf extracts (E: Erdek, K. Kapıdağ, FR: Fresh, DR: Dried, F: Fruit, L:
Leaf, 1-3: Codes of samples collected from different regions, Total anthocyanins were expressed as cyanidin-3-glucoside equivalents, Different letter(s)
on bar indicate statistically significant differences, p<0.05)
Fig. 2: Total anthocyanin (TA) contents (mg/kg) of fresh and dried Vac. myrtillus fruit and
leaf extracts (E: Erdek, K. Kapıdağ, FR: Fresh, DR: Dried, F: Fruit, L: Leaf, 1-3: Codes of
samples collected from different regions, Total anthocyanins were expressed as cyanidin-3-
glucoside equivalents, Different letter(s) on bar indicate statistically significant differences,
p<0.05)
0,00
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3000,00
4000,00
5000,00
6000,00
7000,00
E1FRF E2FRF E3FRF K1FRF K2FRF K3FRF
m
g/
kg
Fresh Vac.myrtillus fruit extracts
Total Anthocyanin
c
a
b
c
d
a
0,00
1000,00
2000,00
3000,00
4000,00
5000,00
6000,00
7000,00
E1DRF E2DRF E3DRF K1DRF K2DRF K3DRF
m
g/
kg
Dried Vac.myrtillus fruit extracts
Total Anthocyanin
b
a a
bc
c
b
0,00
50,00
100,00
150,00
200,00
250,00
300,00
350,00
E1FRL E2FRL E3FRL K1FRL K2FRL K3FRL
m
g/
kg
Fresh Vac.myrtillus leaf extracts
Total Anthocyanin
bc cd
a
d
b cd
0,00
50,00
100,00
150,00
200,00
250,00
300,00
350,00
E1DRL E2DRL E3DRL K1DRL K2DRL K3DRL
m
g/
kg
Dried Vac.myrtillus leaf extracts
Total Anthocyanin
a a
a
a
a
a
122 N. Değirmencioğlu, O. Gürbüz, G.E. Karatepe, R. Irkin
Fig. 3: Antioxidant capacities (μmol TE/g) of fresh and dried Vac. myrtillus fruit extracts (E: Erdek, K. Kapıdağ, FR: Fresh, DR: Dried, F: Fruit, 1-3: Codes of
samples collected from different regions, Different letter(s) on bar indicate statistically significant differences, p<0.05)
in the dried fruit and fresh leaf extracts, while weak correlation TA
content in fresh fruit extracts (Tab. 5, is available in Supplementary
material). These results indicated that the phenolics, rather than the
anthocyanins alone, play an important role in contributing to the
whole antioxidant capacity. The reason may be that anthocyanins
in fruit and leaves could transform into other types of phenolics,
which have higher levels of antioxidant capacity, via the drying and
sample preparation processes. As a reported by previously study, in
the dry heating experiment, all phenolics including anthocyanins
in the blueberry pomace were completely degraded to small
fragments that had no antioxidant capacity. Thus, the loss of the
anthocyanins and other phenolics straightforwardly linked to the
decrease of antioxidant capacity of the dry-heated pomace (BEnEr
et al., 2013).
Conclusion
The drying method affects the quality of the end products such as
color, texture, aroma, along with its chemical constituents (Routray
et al., 2014). Therefore, the control method chosen during this study
was fresh storage after vacuum packaging. Oven-dried fruit and
leaf samples retained higher/lower amounts of phenolic content, TP
and TA according to the degree of resistance to the drying process
of the phenolic compounds and the growing season, suggesting that
enviromental growing conditions, harvesting altitude, and length
and type of drying time used for the dried samples when compared
to the amount obtained from fresh samples. Also, anthocyanins,
flavonols, and proanthocyanidins are located mainly in the peel while
hydroxycinnamates are found in the flesh (Goldıng et al., 2001),
therefore, it is believe that they are affected by different degrees
Fig. 3: Antioxidant capacities (µmol TE/g) of fresh and dried Vac. myrtillus fruit extracts (E: Erdek,
K: Kapıdağ, FR: Fresh, DR: Dried, F: Fruit, 1-3: Codes of samples collected from different regions,
Different letter(s) on bar indicate statistically significant differences, p<0.05)
0,00
10,00
20,00
30,00
40,00
50,00
60,00
E1FRF E2FRF E3FRF K1FRF K2FRF K3FRF
µm
ol
T
E
/g
Fresh Vac.myrtillus fruit extracts
CUPRAC
c c d
b
a
c
0,00
10,00
20,00
30,00
40,00
50,00
60,00
E1DRF E2DRF E3DRF K1DRF K2DRF K3DRF
µm
ol
T
E
/g
Dried Vac.myrtillus fruit extracts
CUPRAC
b
ab a
d
cd c
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
E1FRF E2FRF E3FRF K1FRF K2FRF K3FRF
µm
ol
T
E
/g
Fresh Vac.myrtillus fruit extracts
DPPH
b
c d cd
a
cd
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
E1DRF E2DRF E3DRF K1DRF K2DRF K3DRF
µm
ol
T
E
/g
Dried Vac.myrtillus fruit extracts
DPPH
a a
b
d d
c
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00
E1FRF E2FRF E3FRF K1FRF K2FRF K3FRF
µm
ol
T
E
/g
Fresh Vac.myrtillus fruit extracts
ABTS
b b b
a a
b
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
20,00
E1DRF E2DRF E3DRF K1DRF K2DRF K3DRF
µm
ol
T
E
/g
Dried Vac.myrtillus fruit extracts
ABTS
c
ab
a
b
d
c
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
E1FRF E2FRF E3FRF K1FRF K2FRF K3FRF
µm
ol
T
E
/g
Fresh Vac.myrtillus fruit extracts
FRAP
c c
b
a ab
c
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
E1DRF E2DRF E3DRF K1DRF K2DRF K3DRF
µm
ol
T
E
/g
Dried Vac.myrtillus fruit extracts
FRAP
c
ab b
a
d d
Bioactive compounds in fruits and leaves of blueberry 123
Fig. 4: Antioxidant capacities (μmol TE/g) of fresh and dried Vac. myrtillus leaf extracts (E: Erdek, K. Kapıdağ, FR: Fresh, DR: Dried, L: Leaf, 1-3: Codes of
samples collected from different regions, Different letter(s) on bar indicate statistically significant differences, p<0.05)
from an applied drying method, while the antioxidant capacities of
the samples increase under the same conditions. If combined with
the other protective methods, oven-drying proved to be a suitable
method for Vac. myrtillus samples preservation because the phenolic
compounds and their functional properties were either increased or
at least decreased. Consequently, Vac.myrtillus fruit and leaf can be
recommended as an addition to food composition, to increase the
antioxidant capacity, because of their high antioxidant properties.
Acknowledgements
The authors are grateful to Balikesir University Scientific Research
Projects Unit (Project No: 2012-117) for the financial support of this
research.
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/g
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b b
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µm
ol
T
E
/g
Fresh Vac.myrtillus leaf extracts
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µm
ol
T
E
/g
Dried Vac.myrtillus leaf extracts
FRAP
d
a
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Address of the corresponding author:
E-mail: ndegirmencioglu@bandirma.edu.tr; nurcan.degirmencioglu@gmail.
com
© The Author(s) 2017.
This is an Open Access article distributed under the terms
of the Creative Commons Attribution Share-Alike License (http://creative-
commons.org/licenses/by-sa/4.0/).
I Supplementary material
Supplementary material
Tab. 5: Correlation coefficient of total phenolic content. total anthocyanin content and antioxidant capacity of fresh and dried Vac. myrtillus fruit and leaf
extracts
Table 5. Correlation coefficient of total phenolic content. total anthocyanin content and
antioxidant capacity of fresh and dried Vac. myrtillus fruit and leaf extracts
Total phenol
Total
anthocyanin
CUPRAC DPPH ABTS FRAP
Total phenol 1 -0.406 0.935** 0.323 0.984** 0.704
Total anthocyanin -0.406 1 -0.679 -0.774 -0.638 -0.632
CUPRAC 0.935** -0.679 1 0.545 0.987** 0.745
DPPH 0.323 -0.774 0.545 1 0.420 0.187
ABTS 0.948** -0.638 0.987** 0.420 1 0.811
F
re
sh
fr
ui
t e
xt
ra
ct
FRAP 0.704 -0.632 0.745 0.187 0.811 1
Total phenol
Total
anthocyanin
CUPRAC DPPH ABTS FRAP
Total phenol 1 0.572 0.997** 0.869* 0.949** 0.981**
Total anthocyanin 0.572 1 0.630 0.835* 0.777 0.642
CUPRAC 0.997** 0.630 1 0.900* 0.970** 0.984**
DPPH 0.869* 0.835* 0.900* 1 0.955** 0.919**
ABTS 0.949** 0.777 0.970** 0.955** 1 0.960**
D
ri
ed
fr
ui
t e
xt
ra
ct
FRAP 0.981** 0.642 0.984** 0.919** 0.960** 1
Total phenol
Total
anthocyanin
CUPRAC DPPH ABTS FRAP
Total phenol 1 0.861* 0.981** 0.738 0.957** 0.918**
Total anthocyanin 0.861* 1 0.932** 0.659 0.925** 0.930**
CUPRAC 0.981** 0.932** 1 0.782 0.978** 0.958**
DPPH 0.738 0.659 0.782 1 0.814* 0.814*
ABTS 0.957** 0.925** 0.978** 0.814* 1 0.992**
F
re
sh
le
af
e
xt
ra
ct
FRAP 0.918** 0.930** 0.958** 0.814* 0.992** 1
Total phenol
Total
anthocyanin
CUPRAC DPPH ABTS FRAP
Total phenol 1 0.474 0.793 -0.020 0.420 0.238
Total anthocyanin 0.474 1 0.607 0.576 0.971** -0.131
CUPRAC 0.793 0.607 1 0.048 0.444 0.231
DPPH -0.020 0.576 0.048 1 0.653 -0.863*
ABTS 0.420 0.971** 0.444 0.653 1 -0.250
D
ri
ed
le
af
e
xt
ra
ct
FRAP 0.238 -0.131 0.231 -0.863* -0.250 1
**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).