Scientia Horticulturae 255 (2019) 30–36

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Cyanogenic glycosides and phenolics in apple seeds and their changes T

during long term storage
⁎
Mateja Senica , Franci Stampar, Robert Veberic, Maja Mikulic-Petkovsek
Department of Agronomy, Chair of Fruit Growing, Viticulture and Vegetable Growing, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana,
Slovenia

A R T I C LE I N FO A B S T R A C T

Keywords: Despite fruit seeds being considered fruit waste, they have good potential in human diet. The present research
Location investigated the levels of bioactive compounds (phenolics and cyanogenic glycosides) in apple seeds of the
Phenolics cultivar ‘Golden Delicious’ grown in four different orchards in the world and their change over the course of 8
Cyanogenic glycosides months of storage. Both phenolics and cyanogenic glycosides certainly respond to some environmental condi-
Storage
tions, mainly temperature, precipitation and altitude. Apple seeds from Azerbaijan, with the highest temperature
and the lowest annual precipitation, contained the highest cyanogenic glycosides contents (2.2 mg/g) and apple
seeds from Russia, with the highest altitude and the lowest temperatures, had the highest levels of phenolics
(6.2 mg/g). Additionally, their initial contents further altered over the course of 8 months of storage in a con-
trolled atmosphere.

1. Introduction
Apple is a deciduous fruit species, growing in colder regions all over
the world. It is one of the most consumed fruits worldwide and its
popularity has been gradually increasing, together with its use in nu-
trition, food and the drug industry (Gundogdu et al., 2018; Yildiz and
Çolak, 2018). Apples are today the third most widely produced fruit,
with more than 80 million tons being produced annually over the whole
world. That is slightly less than the combined lemon and orange pro-
duction (FAO, 2016). China, USA and Poland are the three top countries
in world apple production.
An apple is a symbol of wholesomeness: ‘An apple a day keeps the
doctor away’, and it is a rich source of vitamins, minerals and various
phenolic compounds. Phenolic compounds, determined as secondary
metabolism products of plants, are a group of substances with a very
wide range in plants. In recent years, the structures of thousands of
phenolic compounds have been determined (Siddiqui et al., 2018).
Phenolic compounds are densely available in fruits and vegetables, in
the seeds, flowers, leaves, boughs and bodies of plants (Okatan, 2018).
Phenolic compounds are classified into two groups: phenolic acids and
flavonoids. Flavonoids, which are polyphenolic antioxidants, are
available in the natural structures of herbal teas, fruits and vegetables.
Some phenolic compounds are very effective in giving fruits and ve-
getables their taste. In particular, they contribute to two significant
taste aspects: bitterness and sourness in the mouth. Others govern the
formation of colours in the yellow, yellow-brown, red-blue tones in
fruits and vegetables. They lead to various problems, such as enzymatic
browning in processing fruits and vegetables and transforming them
into new products. These aspects are very significant for fruits and
vegetables and the products that are obtained from them (Kirca et al.,
2007; Zorenc et al., 2016; Çolak and Alan, 2017; Okatan and Çolak,
2019; Zeb et al., 2018).
Apple seeds, considered to be fruit waste, usually represent a 2–4 %
share of the whole fruit (Xu et al., 2016). On that basis, from the entire
world production of apples, seeds represent one and a half million tons
of waste. However, apple seeds have good commercial potential as a
promising antioxidant for use in food or cosmetics (Vendruscolo et al.,
2008; Xu et al., 2016), so need not be considered as waste. Various
seeds have recently become very popular for use in human diet. They
are recommended for people with wheat allergies and celiac diseases,
and they are gluten-free food (Ballhorn, 2011). Additionally, some
authors have also reported that selected ingredients found in the seeds
of the Rosaceae family are promoted as an alternative cancer therapy
(Savic et al., 2016). Spirit-based beverages made from fruits are today
very attractive, with the seeds contributing a special aroma and flavor
(Śliwińska et al., 2016). Some people also eat whole apples, including
the seeds. Polyphenolics are the predominant ingredients in apple seeds
(Xu et al., 2016). These polyphenolics mainly consist of

⁎
Corresponding author.
E-mail address: mateja.senica@bf.uni-lj.si (M. Senica).

https://doi.org/10.1016/j.scienta.2019.05.022
Received 7 March 2019; Received in revised form 7 May 2019; Accepted 8 May 2019
Available online 14 May 2019
0304-4238/ © 2019 Published by Elsevier B.V.
M. Senica, et al. Scientia Horticulturae 255 (2019) 30–36

dihydrochalcones (phloridzin), hydroxycinnamic acids, flavanols and Table 1

flavonols (Bolarinwa et al., 2015; Ehrenkranz et al., 2005; Van der Sluis Location site and meteorological data for four studied countries.
et al., 2001; Xu et al., 2016). Phloridzin, a derivative of chalcone, is a Azerbaijan Russia Serbia Slovenia (Bilje)
phyotoalexin that provides resistance to some plant pathogens (Gosch (Xudat) (Nalchik) (Gibarac)
et al., 2010). Seeds also accumulate cyanogenic glycosides to deter
Latitude 41°37'49” N 43°35'38”N 45°06'15”N 45°53'38”N
various herbivores, (Ganjewala et al., 2010). These are nitrogen con-
Longitude 48°40'53”E 43°45'58”E 19°15'58”E 13°38'14”E
taining compounds and they can also provide nitrogen storage, which is Altitude (m) 74 465 93 55
required for seed germination (Ballhorn, 2011). They have many po- Average 356 724 631 1456
sitive effects on the plant regenerative cycle, although they are con- precipitation
sidered harmful for human consumption. After seeds containing cya- (mm)
Average relative 71.2 68 74.1 74.9
nogenic glycosides have been physically processed or damaged, they
humidity (%)
release hydrocyanic acid (HCN), which is toxic for humans. Ingestion of Average 13.0 9.6 11.3 11.8
some fruit seeds with cyanogenic glycosides can therefore result in temperature
acute or sub-acute health problems (Akyildiz et al., 2010). In the Ro- (°C)
saceae family, which includes apples, the main cyanogenic glycosides in
seeds are prunasin and amygdalin (Ballhorn, 2011; Senica et al.,
2017a). Additionally, a study by Senica et al. (2017a) found that the 2.2. Extraction of phenolics and cyanogenic glycosides from apple seeds
seeds of ‘Gala’, ‘Golden Delicious’ and ‘Granny Smith’ cultivars contain
the highest cyanogenic glycoside contents. Apple seeds were manually removed from the cores and crushed in a
Consumers expect their favorite apple cultivars, with firm and juicy mortar with liquid nitrogen and 0.5 g of seed paste was placed into a
flesh, to be available on the market throughout the entire year. Fruit 10 ml screw-cap tube. The samples were extracted with 2.5 ml of me-
must therefore be of the highest quality and should be delivered in an thanol/water (70:30, v/v, MeOH/H2O) according to the method of
unchanged or only slight changed state (Jeziorek et al., 2010). It is very Senica et al. (2017a). All treatments were performed in 7 repetitions.
important to extend the storage life of fresh fruit and apples can be kept
in a controlled atmosphere for more than 12 months. Ethylene triggers 2.3. HPLC-MS analysis of phenolics
ripening in many fruits and can be responsible for storage disorders that
can spoil products and result in fruit loss (Jeziorek et al., 2010). In a Phenolic compounds were first identified on a mass spectrometer
controlled atmosphere, the metabolic process of fruits can be slower (LCQ Deca XP MAX, Thermo Scientific). The mass spectrophotometer
and, consequently, fruit quality remains better for a longer time. had electrospray ionization (ESI) and was operated in negative ion
There is limited information about selected compounds in apple mode. Its main specifics were: capillary temperature 250 °C, source
seeds that could have potential uses in human health. This work had voltage 4 kV, sheath gas and auxiliary gas with 20 and 8 units, re-
three objectives. The first was to distinguish and identify beneficial spectively. The analysis was carried out using full scan data-dependent
phenolics and harmful cyanogenic glycosides in the apple seeds of the MSn scanning from m/z 115–1500; flow rate, 0.6 ml min−1; injection
cultivar ‘Golden Delicious’, which is one of the most widely consumed volume, 10 μl. The identification of phenolic compounds was confirmed
apple in Europe and Asia. The second objective of this study was to by comparing fragmentation patterns, their spectra and additionally by
evaluate the composition of both chemical groups in apple seeds from adding a standard solution to the sample for confirmation.
four different orchards in the world (Slovenia, Russia, Serbia and Phenolic analyses were carried out using the Accela HPLC system
Azerbaijan). The third objective was to follow up the changes of both (Thermo Scientific, San Jose, CA), equipped with a diode array detector
secondary metabolites over the course of 8 months of storage in a (DAD), controlled by CromQuest 4.0 chromatography workstation
controlled atmosphere. This research is the first to study the occurrence software. The specific parameters used for the analysis were: Gemini
of cyanogenic glycosides and phenolics in apple seeds from four dif- C18 column (150 × 4.6 mm 3 μm; Phenomenex, Torrance, USA) oper-
ferent locations and their variation during long storage. The detailed ated at 25 °C; detection wavelength, 280 nm and 350 nm; injection
study will contribute better knowledge and provide an important volume 10 μl; flow rate 0.6 ml/min; mobile phase A: 0.1% formic acid,
source of information for consumers, producers and the branch of the 3% acetonitrile (ACN) and 96.9% double distilled water (v/v/v); mo-
food industry that processes apples into various fruit products. bile phase B: 0.1% formic acid, 3% double distilled water and 96.9%
ACN (v/v/v). The gradients for HPLC analysis were described in a
previous study by Wang et al. (2002).
2. Materials and methods
2.4. MS analysis of cyanogenic glycosides
2.1. Plant material
The presence of cyanogenic glycosides was confirmed on a TSQ
Fruits from the apple cultivar ‘Golden Delicious’ were harvested at Quantum Access Max quadrupole mass spectrometer. The MS instru-
the technological ripeness stage from four different apple orchards ment was operated using an (ESI) source in positive ion mode. Its
(Azerbaijan, Russia, Serbia, Slovenia). The local and meteorological specifics were: capillary temperature 275 °C, corona voltage 4.7 kV,
specifics of the countries are described in Table 1. All apple trees were sheath gas 60 l/h, auxiliary gas 10 l/h. Mass spectra were scanned in a
the same age (6 years). After harvest, apple seeds from 40 apples from range from m/z 70–650. Separation of cyanogenic glycosides was per-
each orchard were removed from the apple core and used for extraction formed on a HYPERSIL GOLD aQ column (Thermo Scientific) at 25 °C
and further analysis. Other apples were stored for 8 months (240 days) with flow rate 0.8 ml/min. The sample injection volume was 10 μl.
in cold storage rooms. During this time, on three subsequent sampling Mobile phase A was 3% methanol in double distilled water and mobile
dates, the seeds from 40 apples were removed from the apple core and phase B was 97% methanol in double distilled water. The gradient
analyzed, as had been done for the first treatment. The first sampling elution is described in a study by Senica et al. (Senica et al., 2017a).
date was 100 days after harvest, the second was 180 days after harvest Data acquisition was performed using Xcalibur 2.2. software (Thermo
and the third was 240 days after harvest (end of cold storage). All ap- Fischer Scientific Institute, Waltham, MA, USA). Cyanogenic glycosides
ples were stored under the same conditions: in cold storage rooms with were analyzed in selected reaction monitoring (SRM) mode, which
a controlled atmosphere at 1 °C and 96% humidity. provides excellent sensitivity for the quantification of target com-
pounds, and additionally by comparison with MS data standards.

31
M. Senica, et al.
Contents of cyanogenic glycosides were expressed in μg per gram.
2.5. Chemicals
Methanol, acetonitrile, formic acid, and the following standards:
chlorogenic acid, neochlorogenic acid, p-coumaric acid, ferulic acid,
sinapic acid, quercetin-3-rutinoside, amygdalin and prunasin, were
purchased from Sigma-Aldrich (Steinheim, Germany). (+)Catechin,
kaempferol-3-glucoside, phloretin, phloridzin, procyanidin B2, quer-
cetin-3-glucoside and quercetin-3-rhamnoside were purchased from
Fluka Chemie (Buchs, Schwitzerland); apigenin from Merck
(Darmstadt, Germany) and quercetin-arabinopyranoside from Apin
Chemicals (Abingdon, UK). Ultra-pure water was produced with a
Millipore purification system and used for all aqueous solutions.
2.6. Statistical analysis
Results are presented as mean ± standard deviation of seven re-
plications. Significant differences among different treatments were
calculated by one-way analysis of variance (ANOVA). Differences in the
content of each individual component were estimated with the LSD test
and were considered significant at p < 0.05. All statistical analyses
were performed with R-Commander (R Formation for Statistical
Computing, Auckland, New Zealand).
3. Results
The apple seeds from the four different orchards significantly dif-
fered in both their phenolics and cyanogenic glycosides contents (Fig. 1;
Table 2 and 3). Furthermore, their contents additionally altered during
storage in a controlled atmosphere (Fig. 2; Tables 2 and 3).
3.1. Phenolics
We identified 23 different phenolic compounds in the apple seeds of
the cultivar ‘Golden Delicious’. All phenolics were classified into 6
phenolic groups: hydroxycinnamic acids, flavones, flavanols, flavonols
and dihydrochalcones. The sum of all analyzed phenolics in the apple
seeds from the four orchards at harvest time showed the following
order: Russia (6.22 mg/g) > Serbia (5.91 mg/g) > Slovenia (5.31 mg/
g) > Azerbaijan (4.24 mg/g) (Fig. 1). Apple seeds from Russia (6.2 mg/
g) had the highest sum of phenolics at harvest time, while at the end of
storage, seeds from Slovenia had the highest content (7.9 mg/g)
(Fig. 2). Apple seeds from Azerbaijan had the lowest contents of phe-
nolics immediately after harvest (4.2 mg/g), as well as after 8 months of
storage in a controlled atmosphere (5.3 mg/g) (Fig. 2).
The most abundant phenolic group was chalcone derivatives (di-
hydrochalcones). Their content ranged from 3.9 mg/g (Azerbaijan, 1st
sampling date; 100 days after storage) to 8.3 mg/g (Slovenia, 2nd
Fig. 1. The contents of phenolics and cyanogenic glycosides in apple seeds at
harvest from four different locations.
32
Scientia Horticulturae 255 (2019) 30–36
sampling date; 180 days after storage) (Table 2). Phloridzin was iden-
tified on the basis of the mother ion at m/z 435 and daughter ions at m/
z 273 (MS2) and m/z 167 (MS3). This phenolic compound contributed
80% of the most abundant phenolic group in this study. The second
most abundant group of all analyzed phenolics in this study was fla-
vanols. They had from 15- to 25-times lower content than dihy-
drochalcones (Table 2) and did not exceed 400 μg/g. The main com-
pound in the flavanol group was procyanidin, followed by catechin. The
groups of hydroxycinnamic acids and flavones did not reach a higher
content than 55.00 μg/g. The main phenolic in the group of hydro-
xycinnamic acids was neochlorogenic acid, while in flavones it was
apigenin hexoside. The contents ranged from 5.36 μg/g (Azerbaijan, 3rd
sampling date; 240 days in controlled storage) to 24.41 μg/g (Slovenia,
2nd sampling date; 180 days in controlled storage) (Table 1). There was
also the group of flavonols, including quercetin and kaempferol gly-
cosides, with the lowest contribution to total analyzed phenolics in
apple seeds. Their contents did not exceed 10 μg/g at harvest, although
they had slightly increased by the end of storage.
Phenolics in apple seeds from all investigated locations altered over
the course of 8 months of storage in a controlled atmosphere. Apple
seeds from Serbia had a 13% higher phenolic content after 240 days of
storage than immediately after harvest. In those from Slovenia, it was
almost 50% higher and in those from Azerbaijan there were 26% higher
values of phenolics at the end of storage than in the initial material. In
contrast, in those from Russia, 5% lower phenolic contents were de-
tected at the end of storage.
In general, apple seeds from Azerbaijan and Russia showed smaller
variations in the sum of phenolic contents than apple seeds from Serbia
and Slovenia during the 8 months of fruit storage (Fig. 2). All in-
vestigated phenolic groups in apple seeds from Slovenia, with the ex-
ception of flavonols, increased during the first 180 days of storage and
thereafter their contents decreased by half (Fig. 2). In Serbian seeds, the
contents of phenolics increased during the first 100 days of storage,
then decreased, before again increasing. In contrast, in those from
Azerbaijan, the groups of hydroxycinnamic acids, flavones and flava-
nols decreased over the first 100 days, increased again from 100 to 180
days and then again decreased to lower than they had been at harvest
(Table 2). Both phloridzin and the sum of all analyzed phenolic con-
tents in apple seeds from Azerbaijan increased until the end of storage
(Fig. 2; Table 1). The phenolic contents in apple seeds from Russia
changed very slightly over the 240 days of cold storage. The groups of
hydroxycinnamic acids, flavones and flavanols increased for the first
100 days and thereafter decreased.
3.2. Cyanogenic glycosides
We identified 3 different cyanogenic glycosides: amygdalin, neoa-
mygdalin and prunasin. The main cyanogenic glycoside in apple seeds
from Russia and Azerbaijan was prunasin (> 50%), from Serbia
amygdalin and prunasin, while in seeds from Slovenia amygdalin made
the highest contribution to total cyanogenic glycosides (54–67%).
Neoamygdalin had the lowest share of total cyanogenic glycosides
(1–3%) (Table 3).
Immediately after harvest and throughout the whole storage time,
apple seeds from Azerbaijan had the highest contents of cyanogenic
glycosides (2.2 mg/g at the beginning; 3.9 mg/g after 240 days of sto-
rage). Apple seeds from the other three orchards (Russia, Serbia, and
Slovenia) had in general lower cyanogenic glycosides contents (Fig. 1),
which additionally decreased in the last 60 days of cold storage (Fig. 2).
During the 8 months of fruit storage, the sum of cyanogenic gly-
cosides showed lower changes in their contents than phenolics. In seeds
from Azerbaijan, cyanogenic glycoside contents increased from the start
to the end of storage. In those from Slovenia, too, the contents increased
for the first 180 days but decreased in the last 60 days of storage
(Fig. 2). In seeds from Serbia, none of the analyzed cyanogenic glyco-
sides showed any significant differences during the first 6 months, but
M. Senica, et al. Scientia Horticulturae 255 (2019) 30–36

Table 2
The content of phenolic groups in apple seeds over the course of 8 months of storage from four different locations (μg/g ± standard error).
Location Sampling time Hydroxycinnamic acids Flavones Flavanols Flavonols Dihydrochalcones

ab a ab b
Azerbaijan 0. 26.85 ± 0.62 15.58 ± 0.20 280.60 ± 10.84 7.61 ± 0.77 3911.24 ± 38.36c
1. 19.87 ± 2.04b 10.88 ± 1.11b 207.74 ± 20.22b 5.33 ± 0.14bc 3869.48 ± 184.26c
2. 30.49 ± 3.41a 17.84 ± 1.37a 301.12 ± 39.56a 5.02 ± 0.63c 4394.73 ± 185.17b
3. 21.74 ± 2.36b 5.36 ± 0.09c 217.09 ± 21.29b 14.18 ± 0.58a 5070.02 ± 134.18a
Russia 0. 28.05 ± 2.77b 13.79 ± 0.53a 326.32 ± 20.43ab 5.26 ± 0.37a 5848.26 ± 168.08a
1. 44.72 ± 1.97a 18.53 ± 0.65b 394.12 ± 14.77a 5.75 ± 1.08a 5772.36 ± 102.10ab
2. 25.45 ± 2.78b 15.39 ± 1.46b 308.00 ± 34.37b 6.05 ± 1.23a 5448.12 ± 144.30b
3. 22.06 ± 2.34b 14.08 ± 1.10b 213.64 ± 20.64c 8.43 ± 1.86a 5769.76 ± 79.33ab
Serbia 0. 44.54 ± 2.40a 17.61 ± 1.54ab 370.04 ± 16.95a 6.62 ± 0.87b 5469.38 ± 315.82bc
1. 33.48 ± 4.01b 15.98 ± 1.11b 288.81 ± 24.70b 6.17 ± 0.64b 6033.65 ± 286.85ab
2. 41.55 ± 1.21ab 19.50 ± 0.60a 307.46 ± 12.00b 4.43 ± 0.65b 4957.58 ± 248.61c
3. 22.52 ± 2.32c 14.63 ± 1.10b 144.96 ± 14.59c 12.64 ± 1.19a 6455.81 ± 255.29a
Slovenia 0. 28.76 ± 4.38b 12.78 ± 1.63c 246.99 ± 51.33ab 6.62 ± 1.75c 5013.11 ± 269.04b
1. 29.45 ± 5.88b 14.57 ± 3.15bc 265.87 ± 69.83ab 14.00 ± 4.33bc 6679.33 ± 239.83a
2. 53.63 ± 3.03a 24.41 ± 0.84a 385.29 ± 65.53a 11.47 ± 0.97a 8263.61 ± 342.45a
3. 34.05 ± 1.48b 20.02 ± 1.50ab 169.94 ± 31.94b 27.80 ± 2.42a 7637.16 ± 331.07a

The average content ± standard error, in μg/g, different letters (a−c) in the same column for each location separately indicate significant differences in phenolic
contents between different sampling date of storage (p < 005) by Duncan range test.

their contents also decreased in the last 60 days of storage. Apple seeds
from Russia had the highest variations in cyanogenic glycosides during
the 240 days of storage in a controlled atmosphere. Cyanogenic gly-
cosides decreased during the first 100 days of storage, increased over
the next 80 days, and slightly but statistically significantly decreased in
the last 60 days.
play, which additionally impact on the contents of the selected com-
pounds in fruit. Fruit stored in a controlled atmosphere can be ad-
ditionally affected by temperature, humidity, time of storage and the
presence of oxygen, CO2 and ethylene (Łata et al., 2005).
4.1. Phenolics

4. Discussion Nalchik (Russia) has a temperate climate, with a significant amount

In the present study, significant differences were detected in phe-
nolics and cyanogenic glycosides contents in apple seeds from different
orchards. The significant diversity in the composition and contents of
analyzed secondary metabolites was closely related to the location of
the orchards (Xu et al., 2016). There are many factors that impact on
the accumulation of the selected secondary metabolites in fruit. Some of
the main factors are latitude, relative humidity, precipitation, tem-
perature and light intensity (Jaakola and Hohtola, 2010; Yang et al.,
2013). Previously reported factors influence not only the external parts
of the fruit but can also be reflected in the internal fruit quality. The
inner, hard part of the fruit pericarp consists of sclerenchyma, closely
adhering cells with a thick, rigid protective layer for seeds. This de-
creases with senescence and the breakdown of cells, and secondary
metabolites can be transferred from the external fruit into the seeds
(Leszczuk et al., 2018). After fruit harvest, further factors come into
of rainfall during the year. The orchard is located at the highest altitude
of all four included orchards (465 m; Table 1). Previous studies have
reported that some phenolics respond positively to higher altitude, ul-
traviolet radiation and lower temperatures (Jaakola and Hohtola, 2010;
Senica et al., 2017b; Yang et al., 2013). Some of the enzymes that
catalyze the initial step in the biosynthesis of polyphenolics have been
reported to be highly related to latitude and weather conditions during
fruit ripening (Yang et al., 2013; Łata et al., 2005). As a result of en-
zyme activity, phenolic contents vary in plants growing in different
environmental circumstances. This finding is supported by our study.
The highest phenolic contents immediately after harvest were de-
termined in apple seeds from Russia (Nalchik). These apple trees were
growing at a 5-times higher altitude and had the lowest annual tem-
perature of all the investigated orchards (Table 1). In contrast, apple
seeds from Azerbaijan had the lowest phenolics contents of all the
analyzed apple seeds in this study. Azerbaijan (Xudat) has a local steppe

Table 3
The content of cyanogenic glycosides in apple seeds from four different locations over the course of 8 months of storage (μg/g ± standard error).
Location Sampling time Amygdalin (μg/g) Neoamygdalin Prunasin

d c
Azerbaijan 0. 852.59 ± 27.12 24.11 ± 3.06 1316.17 ± 56.65c
1. 1024.72 ± 28.65c 42.04 ± 1.80b 1615.98 ± 64.10b
2. 1114.53 ± 95.88b 26.12 ± 3.80c 1506.62 ± 67.94b
3. 1466.15 ± 31.23a 60.62 ± 5.87a 2009.46 ± 96.75a
Russia 0. 651.36 ± 34.75b 27.50 ± 2.39a 1086.13 ± 29.91b
1. 577.34 ± 12.67b 15.16 ± 2.14b 914.37 ± 32.20c
2. 745.28 ± 58.25a 26.41 ± 9.30a 1120.17 ± 86.53b
3. 578.36 ± 25.87b 33.95 ± 3.53a 1543.48 ± 43.53a
Serbia 0. 795.35 ± 69.98a 15.99 ± 1.47b 900.02 ± 67.41c
1. 812.12 ± 12.40a 20.36 ± 1.67b 986.58 ± 23.07b
2. 834.13 ± 18.94a 21.77 ± 2.16b 1137.99 ± 16.21a
3. 521.66 ± 27.03b 32.18 ± 4.07a 435.21 ± 39.50d
Slovenia 0. 519.83 ± 40.92b 14.40 ± 3.83c 263.83 ± 28.31b
1. 910.27 ± 90.64a 31.58 ± 2.98a 857.65 ± 39.32a
2. 996.14 ± 74.07a 28.04 ± 2.42ab 778.02 ± 77.86ab
3. 499.66 ± 27.47b 23.02 ± 2.10b 219.30 ± 28.37c

The average content ± standard error, in μg/g, different letters (a−d) in the same column for each location separately indicate significant differences in cyanogenic
glycosides contents between different sampling dates during storage (p < 0,05) by Duncan range test.

33
M. Senica, et al.
Fig. 2. Changes of phenolic and cyanogenic glycoside contents in apple seeds from four different apple orchards (μg/g) over the course of 8 months in a controlled
atmosphere. 0.S.-harvest time; 1.S-1stsampling date during storage; 2.S.-2nd sampling date during storage; 3.S-3rd sampling date during storage.
climate, with little annual rainfall and also the highest annual average
temperature (13.0 °C; Table 1). The higher temperature negatively af-
fects the biosynthesis of phenolics and the initial material from this
orchard thus had lower contents. Serbia with a continental climate and
Slovenia with a Mediterranean had almost identical climatic conditions
and the apple seeds also had relatively similar phenolics contents.
Phloridzin is the main phenolic compound in apple seeds (Xu et al.,
2016). The group of dihydrochalcones contributed more than 80% of all
phenolics in this study. Phloretin, with much lower contents than
phloridzin, also belongs to the group of dihydrochalcones (Gosch et al.,
2010). Both chalcone derivatives show resistance to various pathogens,
such as bacteria, microbes and fungi, especially decisive for scab re-
sistance (Gosch et al., 2010; Muthuswamy and Rupasinghe, 2007;
Mikulic-Petkovsek et al., 2008; Slatnar et al., 2010). Some biological
processes may change in apple fruits with fruit senescence. Some en-
zymes (peroxidase and polyphenol oxidase) generate changes in the
contents of the selected compounds during storage (Gago et al., 2016;
Zupan et al., 2014; Łata et al., 2005; Muthuswamy and Rupasinghe,
2007). Reactions take place mainly in the peel and flesh, but also in
seeds. Apple seeds exhibit the highest ascorbate peroxidase and catalase
activity (Łata et al., 2005). With fruit senescence, the seeds are pre-
pared for germination and reactions to produce more protective agents,
such as phloridzin and phloretin, start to occur. Persic et al. (2018) and
Gleadow et al. (2016) also reported that phenolics increase in seeds
over the growing season and fruit senescence because of certain bio-
logical processes and the allocation of them into the seeds. It may be
similar in our study, in which, in general, synthesis of phenolics in-
creased in seeds. The apple seeds prepare for growth and selected
compounds protect the seeds against various microbial, fungal and
bacterial attacks. In our study, apple seeds from Azerbaijan, Serbia and
Slovenia had higher phenolic contents after 8 months of storage than at
harvest. In Russia, these contents slightly decreased from 100 to 180
days of storage, but thereafter again increased. Variations of phenolic
34
Scientia Horticulturae 255 (2019) 30–36
contents over the course of the 240 days of storage in a controlled at-
mosphere apparently occurred because of different enzyme activity
inside the fruit. The time of storage is essential for enzyme reactions in
fruits. With a longer storage time, their activity increased and, conse-
quently, phenolics decreased in some parts of the fruit (Gago et al.,
2016; Muthuswamy and Rupasinghe, 2007). Additionally, there is re-
spiration, which is a very important biological process in stored fruits.
The fruit respiration rate decreases when the amount of oxygen de-
creases and the concentration of ethylene and carbon dioxide increases
(Van der Sluis et al., 2001). Some flavonoids are reported to be quite
stable during storage in a controlled atmosphere (Awad and de Jager,
2003). In our study, quercetin and kaempferol derivatives from the
group of flavonols increased over the 8 months of storage, which is in
accordance with a study by Awad and de Jager (2003). Furthermore,
they reported that flavanols (catechin and procyanidin) stay relatively
constant during the first 6 months of cold storage (Awad and de Jager,
2003). In our study, too, the contents of flavanols did not change much
until 180 days of storage, but from 180 days to 240 days their contents
decreased.
4.2. Cyanogenic glycosides
Plants synthesize cyanogenic glycosides and allocate them to seeds,
which occurs in negative environmental circumstances. Previously
published studies have reported that the contents of cyanogenic gly-
cosides were higher in drought plants than in well-watered plants
(Brown et al., 2016; Vandegeer et al., 2013). Because of low pre-
cipitation in Azerbaijan, the apple seeds from there had the highest
levels of cyanogenic glycosides (Fig. 1). In Slovenia, on the other hand,
with the highest annual precipitation (1456 mm) (Table 1), apple seeds
had the lowest cyanogenic glycoside contents. Russia and Serbia, with
approximately 700 mm annual precipitation, had similar cyanogenic
glycoside contents. The soil may be an additional reason for the highest
M. Senica, et al.
cyanogenic glycoside contents in apple seeds from Azerbaijan. Xudat
lies close to the Caspian Sea and the orchard soil had a higher salt
content. It has been demonstrated that the contents of cyanogenic
glycosides increase in plants with high soil salinity (Gleadow et al.,
2016). According to a study by Jørgensen et al. (2005), cyanogenic
glycosides synthesize in the leaves and are then transported to other
plant parts. Zhao (2012) also reported that, during fruit development,
some cyanogenic glycosides synthesize in the cell tegument and are
afterwards transported to the seed cotyledon. In common with phe-
nolics, cyanogenic glycosides also play an important role as an effective
defense against herbivores (Gleadow and Woodrow, 2002) and can
represent a significant drain on resources that might otherwise be used
for growth (Simon et al., 2010). They can also participate in primary
metabolism, such as transport and storage of nitrogen and mitigation of
oxidative stress (Gleadow et al., 2016; Gleadow and Woodrow, 2002).
They are stored in seeds until the stage at which they are synthesized
into nitric compounds and carbohydrates, which are used for germi-
nation. In our study, their contents decreased after 180 days of storage,
while cyanogenic glycosides probably started detoxification into amide,
which is essential for germination. Some additional factors may also be
responsible for the decrease in cyanogenic glycosides in our study.
Temperature, oxygen and carbon dioxide are important for respiration
intensities during storage (Watkins and Nock, 2007). The higher the
respiration rate, the higher are CO2 concentrations. Gleadow et al.
(2009) reported that elevated CO2 concentrations show a higher protein
ratio and also higher cyanogenic glycosides contents. With ‘Golden
Delicious’, the respiration rate is most intensive in the first few months
after harvest, after which the respiration rate slows (Wei et al., 2010).
In our study, after 180 days of storage in a controlled atmosphere, the
contents of cyanogenic glycosides decreased in apple seeds from Russia,
Serbia and Slovenia, while in those from Azerbaijan, the content in-
creased across the whole 8 months of storage. With respiration during
storage, despite the carbon dioxide, heat and water also form certain
by-products, such as ethylene, esters, acids and alcohols, which ad-
ditionally affect the activity of some enzymes in the fruit (Wei et al.,
2010). This probably also causes differences in the chemical composi-
tion of the cyanogenic glycosides in apple seeds.
5. Conclusion
Both phenolics and cyanogenic glycosides respond greatly to en-
vironmental conditions. Phenolics achieved higher values in apple
seeds at the location with a higher altitude and lower temperature,
while cyanogenic glycosides had the highest values at the location with
a low precipitation rate. Furthermore, after 8 months storage in a
controlled atmosphere, their contents in apple seeds additionally
changed. Apples stored in a controlled atmosphere have a higher re-
spiration rate in the first months of storage, which means higher
ethylene and CO2 concentrations and, consequently, higher cyanogenic
glycosides contents. Additionally, apple seeds prepare for seed germi-
nation during storage and so accumulate higher cyanogenic glycosides
contents in the seeds, which affect nitrogen reserves. Both cyanogenic
glycosides and phenolics provide defense against various risks at the
beginning of germination and so both of them translocate from external
parts of the fruit into the seeds. Further investigation of the changes in
selected compounds under different storage conditions is needed. The
contents of beneficial phenolics were almost twice as high as harmful
cyanogenic glycosides. If the new limit of a lethal dose for cyanogenic
glycosides, which is 20 μg/kg of body weight, is taken into account,
then there is no danger of consumers suffering toxic effects of cyano-
genic glycosides after consuming apple seeds, if they are taken in
normal amounts per day. On the other hand, the seeds are also not so
rich in phenolics that a significant beneficial effect on human health can
be expected. Their contents are much higher in apple peel and flesh.
Further studies are therefore required to determine the contents in
different apple cultivars and especially under different storage
35
Scientia Horticulturae 255 (2019) 30–36
conditions. Further pharmacological studies are also needed to de-
termine their adverse and beneficial effects on human health.
Acknowledgement
The research is part of Horticulture Program No. P4-0013-0481
funded by the Slovenian Research Agency (ARRS). We would like to
thank the fruit producers in Russia, Azerbaijan, Serbia and Slovenia
who provided the plant material for this study.
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