Scientia Horticulturae 216 (2017) 139146

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Scientia Horticulturae
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Content of phenolic compounds, ascorbic acid, and photosynthetic

pigments in Vaccinium macrocarpon Ait. dependent on seasonal plant
development stages and age (the example of introduction in Russia)
E.V. Berezina , A.A. Brilkina, A.P. Veselov
Department of Biochemistry and Physiology, Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod, Gagarin Avenue 23,
RU-603950 Nizhny Novgorod, Russia

a r t i c l e i n f o a b s t r a c t

Article history: American cranberry plants (Vaccinium macrocarpon Ait.) were introduced in Nizhegorodsky region (Rus-
Received 11 July 2016 sia) in 2008. The main goal was to evaluate inuence of seasonal plant development stages and age
Received in revised form 10 January 2017 on secondary metabolites accumulation in two cranberry cultivars. This article presents ndings from
Accepted 11 January 2017
the investigation of annual and seasonal dynamics of phenolic compounds (total soluble phenolic com-
pounds, avonoids, catechins, procyanidins, and anthocyanins) and ascorbic acid accumulation in V.
Keywords:
macrocarpon leaves and fruits. The analysis of photosynthetic pigments level in leaves was also conducted.
American cranberry
Antioxidant activity of leaf and fruit extracts was evaluated. Some chemical parameters of soil from V.
Flavonoid
Catechin
macrocarpon growing site were characterized. Young plants featured high secondary metabolites content
Procyanidin without clearly marked seasonality. Plants of reproductive age accumulated less phenolic compounds and
Anthocyanin demonstrated minimum of their concentration in leaf tissues during owering. Content of leaf photo-
Chlorophyll synthetic pigments during fruiting tended to decrease with plant age. There was no regularity in ascorbic
acid accumulation. We suggest that seasonal plant development stages and age have a considerable
effect on secondary metabolites content in tissues of introduced plants. The obtained results provide new
insight into accumulation dynamics of phenolic compounds, ascorbic acid and photosynthetic pigments
in American cranberry while being introduced in Russia.
2017 Elsevier B.V. All rights reserved.

1. Introduction research will denitely add extra dimension to the discussion on

Phenolic compounds play an important role in plant organ-
ism. They possess an antioxidant activity, defend photosynthetic
apparatus against high solar radiation, serve as pigments and
co-pigments, respiration substrates, endogenic growth regulators,
and participate in pollination (Jaakola, 2003; Zaprometov, 1993).
Phenolic compounds synthesis is a dynamic process and their com-
position in different plant parts is determined by both genetic and
environmental factors (Jaakola, 2003). Understanding the factors
affecting secondary metabolites accumulation in plants has fun-
damental and applied importance, and the regional aspect of the
Abbreviations: AAPH, 2,2 -azobis(2-amidinopropane) dihydrochloride; FW,
fresh weight; ORAC, oxygen radical absorbing capacity; TSPC, total soluble phenolic
compounds.
Corresponding author.
E-mail addresses: berezina.kat@gmail.com (E.V. Berezina), annbril@mail.ru
(A.A. Brilkina), veselov-ap@ya.ru (A.P. Veselov).
secondary metabolites role and plant nutraceutical properties.
Cranberry is an object of interest in different ecological and
botanical (Kool and Heijmans, 2009), physiological and biochemi-
cal (Pelletier et al., 2016; Zhou and Singh, 2002), cytogenetic (Suda,
2002; Sun et al., 2015), biotechnological (Polashock and Vorsa,
2002; Debnath and McRae, 2008), and biomedical (Wilson et al.,
2008; La et al., 2010) studies. In particular, phenolic prole and
antioxidant activity of cranberry fruits were analyzed by Celic et al.
(2008), He and Liu (2006), Namiesnik et al. (2014), Wang and
Stretch (2001). It should be noted that the most popular research
object is American cranberry that is endemic to North America
and doesnt grow in natural conditions on the territory of Europe
and Asia. There are several collections of American cranberry cul-
tivars in botanical gardens and forest experiment stations in the
Russian Federation (Gorbunov et al., 2005; Makeev and Makeeva,
2002; Mishukova, 2014) and there is a growing interest in cranberry
planting stock among practitioners.
Secondary metabolism serves as an indicator of plant chemi-
cal adaptation to growing conditions and it can change in new

http://dx.doi.org/10.1016/j.scienta.2017.01.020
0304-4238/ 2017 Elsevier B.V. All rights reserved.
140 E.V. Berezina et al. / Scientia Horticulturae 216 (2017) 139146
conditions. The aim of the present study was to evaluate inu-
ence of seasonal plant development stages and age on phenolic
compounds, ascorbic acid, and photosynthetic pigments accumu-
lation in leaves and fruits of two cranberry cultivars introduced in
Nizhegorodsky region (Russia).
2. Materials and methods
2.1. Plant material
The research objects were American cranberry plants (Vac-
cinium macrocarpon Ait., Ericaceae Juss.), cv. Stevens and Howes.
They were obtained from in vitro culture and planted in the Botani-
cal garden of Lobachevsky State University (Nizhegorodsky region,
european part of Russia) in late May 2008. The Botanical garden
(56 15 north latitude, 44 20 east longitude) is situated in the
coniferous-broad-leaved forest zone on the right bank of the Oka;
altitude is 182 m above the sea level (Geographical and . . ., 2013).
The climate is temperate continental with average annual temper-
ature of +3.1 C and precipitation of 675 mm. The soil is light-grey
forest, mechanical structure is middle-loamy (Geographical and . . .,
2013).
The biochemical research cycle included several seasonal plant
development stages in 20102012 and 2014. In 2010, when plants
were 2 years old, their leaves were collected during owering (June)
and fruiting (September); in 2011, 2012, and 2014 leaves were col-
lected during budding (end of April), owering (June) and fruiting
(September). Cv. Stevens reached the reproductive age in 2011
(3-year-old plants) and cv. Howes in 2012 (4-year-old plants),
that is why fruit biochemical analysis for both cultivars was con-
ducted in 2012 and 2014. Fully ripe berries were hand harvested in
September. Soil samples were collected in 2012 and 2014 during
budding, owering and fruiting. In 2012 fertilizing with KH2 PO4
was undertaken (about 4 g m2 ) every two weeks from the end of
May till the end of June.
The weather conditions were characterized with marked inter-
and intraseasonal contrasts (Supplementary Table 1) (Weather
archive in Nizhny Novgorod (2010, 2011, 2012, 2014) and Weather
in Nizhni Novgorod (2010, 2011, 2012, 2014)). The hottest
and driest research year was 2010, when precipitation was 1.5
(September)-17 (July) times less than long-term average values and
temperature prevalence constituted 10% (September)-32% (July).
All examinations were carried out in triplicate with at least 8
plants per replication. A total of 20 g leaves and 100 g fruit were col-
lected from each cultivar in the growing site over a one week period.
About 0.6 kg of soil per replication was collected for soil analysis.
All the samples were kept in room temperature and studied within
20 min after collection.
2.2. Chemicals
Rutin was obtained from Acros Organics (Geel, Belgium).
Catechin, 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH),
sodium uorescein, and Trolox were purchased from Sigma-
Aldrich (St. Louis, USA). Vanillin was purchased from Roth
(Karlsruhe, Germany). Ascorbic acid, sodium citrate, AlCl3 6H2 O,
K3 [Fe(CN)6 ], FeCl3 , and NaF were obtained from LenReactiv (St.
Petersburg, Russia). Other chemicals were obtained from Reachem
(Moscow, Russia). It was used deionized and distilled water.
2.3. Analytical procedures
2.3.1. Extraction of phenolic compounds
Samples of leaves (1 g) and berries (2 g) were extracted with
boiled 80% aqueous ethanol (81 C, 10 and 20 ml, respectively) for
10 min, homogenized and then centrifuged at 6010g for 15 min
using Z 36 HK centrifuge (Hermle, Germany). Leaf extracts were
diluted twofold with distilled water. Each ethanolic extract (single
biological replication) was used for the analyses of total soluble
phenolic compounds (TSPC), avonoids, catechins, procyanidins
and anthocyanins.
The quantitative analysis of TSPC, avonoids, catechins, pro-
cyanidins, anthocyanins as well as ascorbic acid, chlorophylls a
and b, carotenoids, and available phosphorus was performed with
UV-1700 spectrophotometer (Shimadzu, Japan).
2.3.2. Analysis of total soluble phenolic compounds (TSPC)
Content of TSPC was analyzed spectrophotometrically from the
aforementioned ethanolic extracts on the basis of the method,
described by Zaprometov (1971). Ethanolic extract (0.05 ml for
leaves and 0.1 ml for berries) was mixed with distilled water (9
and 8.95 ml, respectively), followed by addition of 0.5 ml of Folin-
Denis reagent. After shaking for 3 min, 1 ml of 7% Na2 CO3 was added
and the mixture was left to stand for 1 h at room temperature. The
absorbance was measured at 725 nm. The results were calculated
according to calibration curve for rutin and expressed as milligram
of rutin equivalents per gram of fresh weight (FW).
2.3.3. Analysis of avonoids
Content of avonoids was analyzed spectrophotometrically on
the basis of the method, described by Gunes et al. (2002). Leaf
or berry extract (0.05 or 0.1 ml) was mixed with distilled water
(7.35 or 7.3 ml, respectively), followed by the addition of 0.3 ml
of 5% NaNO2 . After 5 min, 0.3 ml of 10% AlCl3 6H2 O was added
and the mixture was left to stand for 6 min before 2 ml of 1
NaOH was added. The absorbance was measured immediately at
510 nm against control (contained distilled water instead of alu-
minium chloride) and the results were expressed as milligram of
rutin equivalents per gram of FW.
2.3.4. Analysis of catechins
Content of catechins was analyzed spectrophotometrically on
the basis of the method, described by Zaprometov (1971). Leaf or
berry extract (0.04 or 0.08 ml) was mixed with distilled water (0.36
or 0.32 ml, respectively) and 2 ml of vanillin reagent (0.1% vanillin
in concentrated HCl). The absorbance was measured after 15 min
at 510 nm against control (for leaves it was the mixture of distilled
water and vanillin reagent and for berries it was the mixture of
extract, distilled water and concentrated hydrochloric acid). The
results were calculated according to calibration curve for catechin
and expressed as milligram of catechin equivalents per gram of FW.
2.3.5. Analysis of procyanidins
Content of procyanidins was analyzed spectrophotometrically
on the basis of the method, described by Khishova and Buzuk
(2006). Leaf or berry extract (0.04 or 0.08 ml) was mixed with
80% ethanol (0.36 or 0.32 ml, respectively), 2.4 ml of acid butanol
(butanol:HCl = 19:1), and 0.12 ml of acid Mohrs salt (2% in 2 M
HCl). The mixture was heated on water bath at 95 C for 40 min
and then cooled to room temperature. The absorbance was mea-
sured at 550 nm. The results were calculated using formulae for
cyanidin chloride and expressed as milligram of cyanidin chloride
equivalents per gram of FW. The analysis was performed only in
2014.
2.3.6. Analysis of anthocyanins
Anthocyanins content in red leaves and berries was determined
by using the pH differential method (Srivastava et al., 2007). 0.3 ml
of extract (leaf extracts werent diluted) was mixed with 2.2 ml of
pH 1.0 (0.025 M KCl + HCl) or 4.5 (0.4 M CH3 COONa + CH3 COOH)
buffer. The absorbance was measured immediately at 510 and
700 nm against corresponding buffers. The results were calculated
 E.V. Berezina et al. / Scientia Horticulturae 216 (2017) 139146
using formulae for cyanidin-3-glucoside and expressed as mil-
ligram of cyanidin-3-glucoside equivalents per gram of FW.
2.3.7. Analysis of ascorbic acid
Content of ascorbic acid was analyzed spectrophotometrically
according to Polevoj and Schiparev (1996). Ascorbic acid was
extracted from 1 g of leaves and 2 g of berries using 7 ml of 0.1 M
citrate buffer ( = 3.7). The mixture was centrifuged at 20000g for
20 min at 4 C. Leaf extracts were diluted twofold with buffer.
2 ml of extract was mixed with 0.1 ml of 1% K3 [Fe(CN)6 ], fol-
lowed by addition of 0.1 ml of 2% NaF and 5 ml of distilled water.
Then 0.2 ml of 2% FeCl3 and 2.6 ml of distilled water were added.
The absorbance was measured after 5 min at 670 nm against con-
trol (contained distilled water instead of ferric chloride). The results
were calculated according to calibration curve for ascorbic acid and
expressed as milligram of ascorbic acid per gram of FW.
2.3.8. Analysis of photosynthetic pigments
Analysis of photosynthetic pigments (chlorophylls and
carotenoids) was performed as described by Gavrilenko and
Zhigalova (2003). Content of chlorophylls a and b and carotenoids
was analyzed spectrophotometrically (absorbance at 440.5, 644,
and 662 nm) from 100% acetone extracts and calculated using
formulae of Holm and Wettstein. Content of photosynthetic pig-
ments was expressed as milligram per gram of FW. Chlorophylls
a/b and carotenoids/chlorophylls ratios were also calculated and
expressed as the mean standard deviation.
2.3.9. Determination of antioxidant activity
Antioxidant activity was determined by oxygen radical absorb-
ing capacity (ORAC) assay (Held, 2005). The reaction mixture
contained 0.04 ml of 75 mM phosphate buffer (pH = 7.4), 0.05 ml of
16 nM sodium uorescein, 0.06 ml of 240 mM AAPH, and 0.05 ml
of diluted ethanolic extract (leaves 1:5000, berries 1:200).
Phosphate buffer was used as a blank. The reaction was started
by adding AAPH. Fluorescence was measured and recorded every
minute at emission of 520 nm and excitation of 485 nm for 60 min
using Synergy MX plate reader (BioTek, USA). Final results were cal-
culated using differences of areas under uorescence decay curves
between the blank and a sample. The results were expressed as Mol
of Trolox equivalents per gram of FW. The analysis was performed
only in 2014.
2.3.10. Soil analysis
The following soil chemical parameters were determined: soil
acidity, available phosphorus (P, according to Pejve), potassium
(K, according to Kirsanov) (Kurganova and Sitintsina, 2005), and
nitrates (N) according to GOST 26951-86 (1986). Soil acidity (pH in
deionized water and in 1 M KCl; 1:2.5 soil:water ratio) was analyzed
potentiometrically using pH-meter pH-150 M (Gomel Measuring
Equipment Plant, Belarus). Reserve acidity pH was calculated as
difference between pH (H2 O) and pH (KCl).
For P analysis, 4 g of air-dried soil was extracted with 20 ml
of 0.2 N HCl and equal volumes of extract and acid ammonium
orthomolybdate solution were mixed with stannous stick. The
absorbance was measured immediately at 730 nm and the results
were expressed as milligram 2 O5 per 100 g of air-dried soil.
For K analysis, 20 g of soil was extracted with 40 ml of 1 N NaCl.
9 different dilutions were made and 0.1 g of Na3 [Co(NO2 )6 ] was
added. After 30 min the rst dilution where precipitation took place
was taken into account and compared with the table given in the
guide (Kurganova and Sitintsina, 2005). The results were expressed
as milligram 2 O per 100 g of soil.
For N analysis, 20 g of soil was extracted with 50 ml of 1 N K2 SO4 .
Nitrates content was analyzed potentiometrically using nitrate
meter pNO3 -07 (Gomel Measuring Equipment Plant, Belarus) and
141
Fig. 1. Total soluble phenolic compounds (TSPC) content in V. macrocarpon leaves in
20102012 and 2014 growing seasons. Error bars indicate standard deviation from
mean values. For each cultivar, different letters next to the values denote signicant
differences (p < 0.05) between plant development stages within a year (ab) and
between years (for the same development stages) (AB).
the results were expressed as milligram N per 100 g of soil. This
analysis was performed only in 2014.
2.4. Statistical analysis
All examinations were carried out with three independent bio-
logical replicates and repeated three times (n = 9). The results
were analyzed with Microsoft Excel (Microsoft Corporation, USA)
and differences between them were determined using analysis of
variance (ANOVA). All results are expressed as means standard
deviation. Differences at p < 0.05 were considered statistically sig-
nicant.
3. Results
3.1. Bioactive compounds and antioxidant activity
The results represented in Fig. 1 show that TSPC content in
leaves of introduced American cranberry in a certain seasonal plant
development stage decreased from year to year (or had such a
tendency), though it was observed less during budding. During
this development stage in 2011 and 2012 there were signicant
(p < 0.05) differences registered between cultivars: maximum for
cv. Stevens was observed in 2011 and minimum in 2012 as
opposed to cv. Howes. TSPC content during owering was not
less than during other seasonal plant development stages till 2014.
In 2014 dynamics of TSPC accumulation during owering featured
minimum.
Maximal amount of TSPC was 151 (cv. Stevens, budding-2011)
and 131 (Howes, owering-2012) mg g1 FW. Minimal amount
was 4246 mg g1 FW as was observed in 2014 during owering.
The same character of accumulation during investigation
period was revealed for avonoidsthe largest class of pheno-
lic compounds. Fig. 2 demonstrates avonoids content gradually
decreasing as plants reached reproductive age. In contrast to TSPC
level, avonoids level in June 2010 was less than that in September
2010. Flavonoids rate in leaves TSPC was more than 50% with max-
imum of 9096% during fruiting-2010.
Catechins, the most reduced C6 C3 C6 -compounds, had the
same accumulation dynamics as avonoids (Supplementary Fig.
1). It is important to highlight that in contrast to other phenolic
compunds in leaves, for catechins content there was demonstrated
a surplus (more than 2-fold) during fruiting-2010 compared to
owering-2010: 24 and 10 mg g1 FW for cv. Stevens, 21 and
10 mg g1 FW for cv. Howes, respectively.
142 E.V. Berezina et al. / Scientia Horticulturae 216 (2017) 139146
Fig. 2. Flavonoids content in V. macrocarpon leaves in 20102012 and 2014 growing
seasons. Error bars indicate standard deviation from mean values. For each cul-
tivar, different letters next to the values denote signicant differences (p < 0.05)
between plant development stages within a year (ab) and between years (for the
same development stages) (AC).
Fig. 3. Ascorbic acid content in V. macrocarpon leaves in 20102012 and 2014 grow-
ing seasons. Error bars indicate standard deviation from mean values. For each
cultivar, different letters next to the values denote signicant differences (p < 0.05)
between plant development stages within a year (ac) and between years (for the
same development stages) (AD).
Procyanidins accumulation in American cranberry leaves during
2014 growing season is reected in Supplementary Fig. 2. Procyani-
dins amount varied from 3 to 8 mg g1 FW, and it was lower in
budding compared with that in fruiting. It was also lower in ow-
ering compared with that in fruiting (cv. Stevens) or had such a
tendency (cv. Howes).
Anthocyanins didnt signicantly contribute to phenolic prole
of leaves collected during owering. As a rule, in the periods of bud-
ding and fruiting their content was higher. The maximal content
was found in leaves collected during fruiting-2010 and budding-
2011: 0.07 mg g1 FW in cv. Stevens leaves and 0.04 mg g1 FW in
cv. Howes leaves.
The dynamics of ascorbic acid accumulation in V. macrocarpon
leaves was unstable though identical for both cultivars (Fig. 3).
Ascorbic acid content in 2010 during both owering and fruiting
was about 2 mg g1 FW and the highest of the whole investiga-
tion cycle. In 2011 and 2014, in contrast to 2010 and 2012 years of
investigation, minimal ascorbic acid content was observed during
fruiting. In 2012 minimum was registrated during owering.
The results of leaf photosynthetic pigments determination are
summarized in Table 1. In 2010 photosynthetic pigments concen-
tration during owering was lower than that during fruiting. In
2012 their concentration decreased by owering and then rose
again by fruiting. In 2014 photosynthetic pigments content was
observed to decrease from budding to fruiting. There was an excep-
tion for cv. Howes leaves with photosynthetic pigments content
in fruiting-2014 being not less than that in owering-2014.
The small amount of chlorophyll b during owering-2010 was
the cause of increased chlorophylls a/b ratio (3.133.96). In the
other years of investigation the ratio was 2.163.48. It is also
important to point out that the changes in green and orange pho-
tosynthetic pigments content were unidirectional.
Carotenoids/chlorophylls ratio changed a little during the inves-
tigation cycle and amounted to 0.480.74.
Phenolic compounds content in fruits in 2014 was lower than
that in 2012 (Table 2). Signicant ( < 0.05) differences between
cultivars were observed in 2012 and 2014 for catechins amount.
Ascorbic acid content in cv. Stevens berries in 2014 was higher
than that in 2012, and its content in cv. Howes berries had such a
tendency.
TSPC content in berries was 410 times less than that in leaves
(depending on seasonal plant development stage and cultivar),
avonoids content was 1467 times less, catechins 421, pro-
cyanidins 25, and ascorbic acid up to 6 (but in some cases
such data may be comparable). On the other hand berries contained
signicantly more (up to 31 times) anthocyanins than leaves.
Antioxidant activity of leaf and fruit extracts (cv. Stevens, 2014)
is shown in Supplementary Fig. 3. Leaves collected in the end of
April and in September 2014 exhibited the highest antioxidant
activity (without signicant ( < 0.05) differences between vegeta-
tion periods). Obviously, seasonal dynamics of antioxidant activity
and of phenolic compounds level in 2014 were similar with high
gures in budding and fruiting and lowered gures in owering;
the difference was 1.52.5 times. The dynamics of ascorbic acid
and of carotenoids accumulation had another character. Antioxi-
dant activity of leaf extracts was 613 times higher than that of fruit
extracts, which cant be explained with hypothetically remarkable
contribution of ascorbic acid to antioxidant activity.
3.2. Soil conditions
Soil substrate from V. macrocarpon growing site (Supplementary
Table 2) was characterized with almost neutral reaction and low
reserve acidity: pH (H2 O) was 7.197.45, pH (KCl) was 6.316.70,
and pH was 0.520.88.
The amount of potassium was no more than 6 mg 100 g1 of soil.
The amount of phosphorus in the end of April was 1.25 mg 100 g1
of air-dried soil. In the end of May-the end of June 2012 fertilizing
with KH2 PO4 was undertaken; after that P content was not less
than 10 mg 100 g1 of air-dried soil. N content in 2014 decreased
from 15.67 mg 100 g1 of soil in budding to 0.63 mg 100 g1 of soil
in fruiting.
4. Discussion
4.1. Bioactive compounds and antioxidant activity
Complex characterization of secondary metabolites qualitative
and quantitative changes can provide better understanding of age-
dependent metabolism alterations. Long-term investigations of
accumulation dynamics of secondary metabolites are exceedingly
important for introduced species and have fundamental and prac-
tical implications.
In the discussion of the obtained results it is necessary to under-
line that all data for American cranberry plants introduced in
Nizhegorodsky region correspond to metabolites content ranges
established for other heather species in Russia: lingonberry V. vitis-
idaea (Kychkina, 2009), European cranberry V. oxycoccos (Berezina
et al., 2015), bilberry V. myrtillus, native blueberry V. uliginosum
and introduced blueberry V. corymbosum, V. atrococcum (Brilkina
et al., 2014; Pavlova et al., 2012), and rhododendron Rhododendron
smirnowii, Rh. ledebourii, Rh. japonicum (Kostina 2009).
 E.V. Berezina et al. / Scientia Horticulturae 216 (2017) 139146 143

Table 1
Photosynthetic pigments content in V. macrocarpon leaves in 20102012 and 2014 growing seasons.

Cultivar Year Development stage Photosynthetic pigments content, mg g1 FW Photosynthetic pigments ratio

Chlorophylls a Chlorophylls b Carotenoids Chlorophylls a/b Carotenoids/chlorophylls

Stevens 2010 Flowering 0.35 0.00aA 0.09 0.00aA 0.26 0.00aA 3.96 0.00bA 0.59 0.00aA
Fruiting 0.38 0.03aA 0.13 0.01bA 0.29 0.02aA 2.90 0.26aA 0.55 0.05aA
2011 Budding 0.34 0.10aA 0.16 0.02aA 0.30 0.08aA 2.16 0.49aA 0.60 0.17aA
Flowering 0.50 0.10aA 0.17 0.04aAB 0.33 0.07aA 3.04 0.64aA 0.49 0.12aA
Fruiting 0.49 0.17aA 0.17 0.04aA 0.37 0.09aA 2.78 0.80aA 0.56 0.20aA
2012 Budding 0.49 0.04aAB 0.22 0.06abA 0.40 0.02aAB 2.25 0.59aA 0.58 0.06aA
Flowering 0.46 0.16aA 0.18 0.06aAB 0.34 0.13aAB 2.52 1.19aA 0.54 0.24aA
Fruiting 1.07 0.11bB 0.34 0.04bB 0.70 0.06bB 3.21 0.47aA 0.50 0.07aA
2014 Budding 0.72 0.20bB 0.30 0.10bA 0.61 0.18aB 2.54 1.10aA 0.59 0.25aA
Flowering 0.54 0.02abA 0.22 0.05abB 0.56 0.05aB 2.62 0.63aA 0.73 0.07aA
Fruiting 0.33 0.08aA 0.11 0.03aA 0.32 0.08aA 3.06 1.06aA 0.71 0.24aA
Howes 2010 Flowering 0.35 0.00aA 0.11 0.00aAB 0.25 0.00aAB 3.13 0.00aA 0.53 0.00aA
Fruiting 0.59 0.09bB 0.21 0.03bB 0.43 0.05bB 2.77 0.56aA 0.53 0.09aA
2011 Budding 0.35 0.03aA 0.16 0.04aA 0.29 0.01aA 2.18 0.35aA 0.56 0.07aA
Flowering 0.63 0.17aB 0.22 0.04aC 0.41 0.10aB 2.80 0.63aA 0.48 0.14aA
Fruiting 0.47 0.17aAB 0.20 0.06aB 0.38 0.12aAB 2.35 0.80aA 0.57 0.22aA
2012 Budding 0.52 0.04bB 0.24 0.01bA 0.42 0.03bB 2.22 0.21aA 0.55 0.06aA
Flowering 0.38 0.08aAB 0.17 0.01aBC 0.29 0.07aAB 2.28 0.50aA 0.53 0.15aA
Fruiting 0.67 0.00cB 0.19 0.00aB 0.46 0.00bB 3.48 0.00bA 0.53 0.00aA
2014 Budding 0.66 0.02bC 0.22 0.01bA 0.52 0.02bC 3.03 0.12aB 0.59 0.03aA
Flowering 0.20 0.05aA 0.06 0.03aA 0.20 0.05aA 3.17 1.50aA 0.72 0.25aA
Fruiting 0.21 0.03aA 0.09 0.02aA 0.20 0.03aA 2.55 0.67aA 0.67 0.14aA

Values are reported as the mean standard deviation. For each cultivar, different letters next to the values denote signicant differences (p<0.05) between plant development
stages within a year (ac) and between years (for the same development stages) (AC).

Table 2
Total soluble phenolic compounds (TSPC), avonoids, catechins, anthocyanins, procyanidins, and ascorbic acid content in V. macrocarpon fruits in 2012 and 2014 growing
seasons.

Metabolites (mg g1 FW) Stevens Howes

2012 2014 2012 2014

TSPC 18.03 1.64B 12.13 0.64A 14.29 1.89B 9.28 1.39A

Flavonoids 3.49 0.68B 1.54 0.28A 2.54 0.57B 0.95 0.11A
Catechins 2.57 0.18B 1.40 0.04A 1.90 0.11B 0.80 0.16A
Anthocyanins 0.23 0.04A 0.20 0.01A 0.31 0.01B 0.17 0.01A
Procyanidins 1.65 0.08 1.43 0.04
Ascorbic acid 0.20 0.01A 0.37 0.02B 0.28 0.04A 0.32 0.03A

Values are reported as the mean standard deviation. The dash () means that samples in 2012 were not analyzed for procyanidins. For each cultivar, different letters next
to the values denote signicant differences (p < 0.05) between years (AB).

Phenolic compounds accumulation in different plant organs is
connected with their function and seasonal plant development
stage and age. While analyzing changes in TSPC, avonoids, and
catechins content in leaves of introduced in Russia V. macrocarpon
plants it is necessary to point out certain differences in seasonal
accumulation dynamics connected with plant age. Phenolic com-
pounds content during certain seasonal plant development stages
decreased from year to year (or had the respective tendency) and
neared the data acquired for the native plant, European cranberry
(Berezina et al., 2015). It is possible that physiological changes in
virginile plants developing into reproductive ones is accompanied
by the decrease in secondary metabolites accumulation.
There are several uctuations in phenolic compounds content
that are linked with their functions in plant organism. High TSPC
content during budding can be connected with increased concen-
trations of lignin and tannin degradation products, and during
fruiting with accumulation of growth inhibitors of phenolic origin
(Artemkina and Gorbacheva, 2009; Zaprometov, 1993).
Phenolic compounds increase is assumed also to have a link
with immunity towards different stress factors. In the current study
this revealed itself especially in September 2010 when V. macro-
carpon leaves featured a considerable increase of catechins and
anthocyanins concentration and avonoid percentage in pheno-
lic complex (up to 9096%). Such changes may be considered as
manifestation of leaf adaptation capabilities, since abnormally high
temperature and drought were documented in 2010 from mid-June
till mid-September, and a state of emergency was declared in the
region.
Flavonoids are utilized in pollination and sexual reproduction
processes (Jaakola, 2003) which could be a reason for the decrease
of these compounds content in owering especially noticeable in
2014 during the nal transition to reproductive age.
Taking into consideration the similarity of accumulation
dynamics of TSPC and avonoids in leaves as well as avonoids high
percentage in phenolic prole it can be concluded that avonoids
level play primary role in determining phenolic compounds content
in cranberry leaves.
Leaf anthocyanins full photoprotective and antioxidant func-
tions. In owering leaf photosynthetic apparatus terminates its
formation and acquires resistance to photodestruction. That is
apparently why anthocyanins concentration in V. macrocarpon
plants during this development stage was low. By the time of
photosynthetic apparatus senescence and its resistance decrease,
anthocyanins content augments and starts catching consider-
ably more solar radiation in the green part of visible spectrum
(Pehkonen et al., 2008; Solovchenko, 2009). Lower temperatures
can also stimulate increased anthocyanins production (Parr and
Bolwell, 2000). On the other hand, increased anthocyanins con-
tent observed in September 2010 ought to have been caused
by high temperature and drought registered in Nizhegorodsky
144 E.V. Berezina et al. / Scientia Horticulturae 216 (2017) 139146
region (Supplementary Table 1). In case of noticeable avonols and
anthocyanins concentration, photosynthetic apparatus is almost
undamaged by environmental doses of visible and ultraviolet
spectra. Indirectly it can be conrmed with heightened chloro-
phylls content during fruiting-2010 compared to owering-2010
(Table 1). It is necessary to underline that increased chlorophylls
accumulation in September (fruiting) 2012 compared to June (ow-
ering), was documented when the weather was not hot and
droughty. Moreover, photoprotective pigments accumulation in
vacuoles (anthocyanins, avonols) can protect from photodynamic
activity of chlorophylls degradation products and other photosen-
sitizers present in cell.
Proanthocyanidins (including procyanidins) are most common
in woody plants than in herbaceous species (Zifkin et al., 2012).
They perform protective functions (against fungal infections in par-
ticular) and contribute to developing plant stress tolerance. At the
same time procyanidins due to their low concentration are assumed
not to take part in leaf tissues defense against excessive radiation
(Jaakola, 2003). According to Jaakola (2003), procyanidins content
in green bilberry (subshrub) leaves was 0.96 mg g1 , and in red
leaves it was only 0.4 mg g1 , which is considerably less in com-
parison with the data obtained in the current research of cranberry
(dwarf shrub) leaves.
Ascorbic acid as well as phenolic acids, avonoids, and
carotenoids play a photoprotective role. This compound indi-
rectly participates in photosynthesis (in particular, it hampers
chlorophyll degradation) and stimulates growth and owering
(Chupakhina, 1997). Besides, phenolic compounds are synergists of
ascorbic acid that is why comparing the character of these metabo-
lites accumulation in the research objects represents signicant
interest. It was established that their accumulation was similar only
in 2010 and 2011 growing seasons, and high amounts of pheno-
lic compounds and especially ascorbic acid in June and September
2010 seemed to be caused by hot and dry weather conditions (Sup-
plementary Table 1).
In the present investigation ascorbic acid content in leaves var-
ied a lot depending on seasonal plant development stage and age
but, upon the whole, the ascorbic acid content range was in accor-
dance with the range determined for blueberry in Nizhegorodsky
region (Pavlova et al., 2012). The increase in ascorbic acid content in
blueberry leaves, associated with fruiting (Pavlova et al., 2012), was
observed for cranberry only in 2012. For V. macrocarpon plants, low
ascorbic acid content in owering-2012 compared to fruiting-2012
can be possibly explained through its active expenditure during the
process of owering. But it is necessary to emphasize that fruiting
for blueberry is in July, while for cranberry it is in September, and
the functional signicance of this compound in different months
with different weather conditions is varied.
There was no decrease observed in photosynthetic pigments
concentration from owering to fruiting in 2010 and 2012. This may
indicate that young American cranberry plants were not adapted
enough to winter. Upon the whole, from year to year a tendency
was observed to increasing green photosynthetic pigments con-
tent which was also established for American cranberry cultivated
in Belarus (Rupasova et al., 1989).
If chlorophylls content in leaves is 0.53 mg g1 FW and
carotenoids content is 0.1-0.5 mg g1 FW then chlorophylls a/b
ratio is often 2.53 (Gavrilenko and Zhigalova, 2003). Optimal
chlorophylls a/b ratio is an indicator of plant tolerance to biotic
and anthropogenic factors (Kalchenko et al., 2007), provides pho-
tosynthetic apparatus effectual functioning and allows to preserve
considerable amounts of assimilates to form yield. In the present
investigation chlorophylls a/b ratio was more than 3 in the period
of owering in 2010 (and in 2012 for cv. Howes); that is assumed
to be a plant response towards hot and dry weather conditions
(Supplementary Table 1).
Numerous publications indicate the main biological active sub-
stances affecting edibility characteristics of berries are phenolic
compounds (Jaakola, 2003; Namiesnik et al., 2014). The main
avonoids in Vaccinium L. fruits are quercetin and cyanidin, as well
as myricetin, delphinidin, malvidin, and peonidin (Jaakola, 2003;
Pappas and Schaich, 2009).
According to our data phenolic compounds content in berries
was lower than in leaves (the only exception were anthocyanins).
The most perceptible difference was established for avonoids. The
same difference in leaf and fruit avonoids content was described
for lingonberry, European cranberry (Berezina et al., 2015), bilberry
and blueberry species (Brilkina et al., 2014; Pavlova et al., 2012). The
avonoids percentage in leaf phenolic prole was 5496% (depend-
ing on the year and vegetation period) and in fruit phenolic prole
was only 1121%, i.e. in berries non-avonoid constituents are
predominant and play a signicant role. These are, probably, phe-
nolic acids that along with citric, benzoic, and ascorbic acids are
responsible for sour taste and determine fruit tolerance towards
microorganisms (Pappas and Schaich, 2009; Pomology, 2014).
According to Wang and Stretch (2001), phenolic compounds
content in V. macrocarpon fruits was about 1.3 mg g1 FW (in gal-
lic acid equivalents); according to Namiesnik et al. (2014), about
22 mg g1 of dry weight (in gallic acid equivalents), avonoids
4 mg g1 and catechins 0.5 mg g1 (in catechin equivalents). The
aforementioned results are about 10 times lower than our results.
That in case of TSPC and avonoids can be due to different stan-
dards chosen for calibration. Our results for TSPC content were
similar to those for V. microcarpon in Finland described by Kylli
(2011). Comparing our results for TSPC content in American cran-
berry fruits with the results for other ericaceous species it can be
outlined that American cranberry accumulated more metabolites
than V. corymbosum (Dragovic-Uzelac et al., 2010), V. uliginosum
and V. atrococcum (Pavlova et al., 2012). Flavonoids content in fruits
of these species was the same.
Cranberries with total avanol content of 0.07 mg g1 FW are
considered as a moderate source of catechins (Pappas and Schaich,
2009). Catechins content in fruits of American cranberry introduced
in Belarus was 9.511.7 mg g1 of dry weight and it was found to
be 10 times less than in leaves (Rupasova et al., 1989). Our results
are in agreement with the data obtained in Belarus.
Anthocyanins content in American cranberry fruits cultivated
in the Botanical garden of Lobachevsky State University (Nizhe-
gorodsky region, Russia) and in Rutgers Blueberry and Cranberry
Research Center (New Jersey, USA) is comparable (Wang and
Stretch, 2001). It is comparable as well with anthocyanins content
in V. microcarpon fruits (Kylli, 2011) but lower than in V. myrtillus
(Jaakola et al., 2010), V. angustifolium (Brilkina et al., 2014) and V.
corymbosum (Dragovic-Uzelac et al., 2010).
Procyanidins are responsible for astringent and bitter taste of
immature fruits which helps to deter frugivores from consuming
fruits before they are ripe; thus though procyanidins and antho-
cyanins share a common biosynthetic pathway their biological
functions are opposite (Jaakola et al., 2010; Zifkin et al., 2012).
According to our ndings, procyanidins content in American cran-
berry fruits was 8 times bigger than that of anthocyanins. High
procyanidins accumulation in berries in comparison to that of
anthocyanins is, evidently, a biochemical characteristic property
of investigated species.
Ascorbic acid content in fruits of investigated V. macrocarpon
cultivars sometimes was not lower than its content in leaves.
According to Borges et al. (2010), ascorbic acid content in V. macro-
carpon fruits was 1.1 mM g1 FW, i.e. about 0.2 mg g1 FW, which
is in correspondence with our gures. The same level of fruit ascor-
bic acid accumulation is characteristic to lingonberry (Tyak et al.,
2002), blueberry (Gorbunov et al., 2005; Pavlova et al., 2012), and
European cranberry (Makeev and Makeeva, 2002).
 E.V. Berezina et al. / Scientia Horticulturae 216 (2017) 139146
Phenolic compounds content in plant tissues mostly determines
their antioxidant activity (He and Liu, 2006). According to Borges
et al. (2010), ascorbic acid contribution towards fruit antioxidant
activity is 23%, anthocyanins contribution 39%, procyanidins
12%, avonols 10%, and chlorogenic acid 2%.
On the basis of our ndings concerning ORAC obtained in 2014
it can be assumed that the major contribution towards antioxidant
activity is made by phenolic compounds, seasonal concentrations
changes of which were in accordance with antioxidant activity
dynamics of leaf extracts, in contrast to that of ascorbic acid and
carotenoids. Besides, ascorbic acid and carotenoids concentrations
were lower than phenolic compounds concentrations; antioxidant
activity of ascorbic acid is lower than that of some phenolic com-
pounds (Held, 2005); carotenoids are lipophilic compounds and
their antioxidant activity is crucial only for chloroplasts.
4.2. Soil conditions
As it was found out, the examined plants grew on alkales-
cent depleted soils. According to DeMoranville (2015), the optimal
soil pH for cranberry is 45.5. Besides, Russian soil in the regions
of natural European cranberry (V. oxycoccos) habitat is character-
ized with acid reaction (2.74.7) and considerable reserve acidity
(pH = 1.41.5) (Berezina et al., 2015; Timoshok, 2006).
The amount of mineral elements necessary for all plants, K and P
(till June 2012), in analyzed soil determined it as poor substrate. The
same was also documented for native European cranberry in Nizhe-
gorodsky region (Berezina et al., 2015). N content decreased from
budding to fruiting reected the element exhaustion from soil by
growing plants. It is known that phenolic compounds accumulation
in different plant organs is stimulated by mineral elements decit in
substrate, including N (Jaakola, 2003; Parr and Bolwell, 2000). That
could be one of the reasons for heather plants intensied ability to
synthetize phenolic compounds.
5. Conclusions
In the present investigation it was revealed that American cran-
berry introduced in the Botanical garden in Nizhegorodsky region
(Russia) is characterized with high phenolic compounds accumula-
tion level, and their content during budding, owering, and fruiting
might differ. TSPC content range was 41151 mg g1 FW, avonoids
32131 mg g1 FW, catechins 933 mg g1 FW, procyanidins
38 mg g1 FW. Plants age might signicantly inuence secondary
metabolites synthesis (not only phenolic compounds, but ascorbic
acid and photosynthetic pigments, too).
Long-standing character of the study (several years: 20102012
and 2014) gives ground to the conclusion that high level and
seasonal changes in phenolic compounds content are important
features of secondary metabolism of young American cranberry
plants introduced in this region. Thus, phenolic prole of V.
macrocarpon leaves was labile, its changes were determined by uc-
tuations in avonoid content, there was not always a decrease in
metabolites content during owering, and leaves of young plants
synthesized more metabolites than leaves of plants of reproductive
age. In the course of maturing in the conditions of Nizhegorod-
sky region the introduced specimen accumulated almost the same
quantity of secondary metabolites as native one.
It was established that TSPC content in V. macrocarpon leaves
was higher than in berries (about 10 times); the variations in
avonoids content were even more pronounced. But anthocyanins
content in berries was superior to their content in leaves which
might be due to their extremely high functional signicance in
berries compared to leaves.
145
Ascorbic acid accumulation in leaves was also unstable
(0.22.2 mg g1 FW) and not always in accordance with phenolic
compounds accumulation. Photosynthetic pigments level in Amer-
ican cranberry leaves tended to decrease during fruiting from year
to year, which was required to provide adequate plants adaptation
towards winter conditions.
The undertaken investigation has revealed that introduced cran-
berry plants had high and species typical content of secondary
metabolites, that is why American cranberry is objectively consid-
ered as valuable promising plant for commercial planting in Russia
and for broader usage in food industry and medicine.
This research did not receive any specic grant from funding
agencies in the public, commercial or non-for-prot sectors.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.
01.020.
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