Romanian Biotechnological Letters
Copyright © 2016 University of Bucharest
Vol. , No. x,
Printed in Romania. All rights reserved
ORIGINAL PAPER
Assessment of Polyphenols, Chlorophylls and Carotenoids during
Developmental Phases of Three Apple Varieties
Received for publication, June 13, 2015
Accepted, April 27, 2016
ELENA ANDRUŢA MUREŞAN1, SEVASTIŢA MUSTE1*, CRINA CARMEN
MUREŞAN1, ELENA MUDURA1, ADRIANA PĂUCEAN1, LAURA STAN2, ROMINA
ALINA VLAIC1, CONSTANTIN GHEORGHE CERBU3, VLAD MUREŞAN1
1
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Food
Science and Technology, Food Engineering Department, Cluj-Napoca, Romania
2
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Food
Science and Technology, Food Science Department, Cluj-Napoca, Romania
3
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of of
Veterinary Medicine, Department of Infectious Diseases, Cluj-Napoca, Romania
*Address correspondence to: University of Agricultural Sciences and Veterinary Medicine
Cluj-Napoca, Faculty of Food Science and Technology, Food Engineering Department, Calea
Florești 64, 400509, Cluj-Napoca, Cluj, Romania. Tel.: +40 264 596 384;
Email: [email protected]
Abstract
Variations of total polyphenol content, antioxidant capacity, chlorophyll pigments and
carotenoid pigments during fruit development of Golden Delicious, Starkrimson and Ionathan apple
varieties were investigated at 7, 15, 35, 65, 107 and 144 days after full bloom (DAFB). For all
varieties, the highest total polyphenol content was recorded for the apples harvested at 7 DAFB,
decreasing while the fruit was growing. Antioxidant capacity reaches the highest level at 15 DAFB and
then declines until the apples reached technological maturity. Total chlorophyll, chlorophyll a and b
recorded high values at 65 DAFB, when both the temperature and the light intensity are high (July).
After this harvesting time, chlorophyll content decreased. The concentration of carotenoids
accumulated in apple peel varied, however following a downward trend during fruit growth, regardless
of the variety or the crown position.These results provide a preliminary foundation for the further use
of apples from physiological falls in different foods and pharmaceuticals.
Keywords: Ionathan, Starkimson, Golden Delicious, spectrophotometry
1. Introduction
Clinical and epidemiological studies have established that a high consumption of
fruits and vegetables may reduce chronic and degenerative diseases that are the major causes
of death in developed countries (HENRÍQUEZ & al. [1]). Researchers showed interest in the
study of beneficial effects of pigments on human health as a response of food industry’s
increasing demand for their use as food supplements and as food additives (LEE & al. [2]).
Apples are a significant part of the human diet (WOLF & al. [3]) as a major source of
antioxidants, mainly phenolic compounds (BOYER & al.[4]). The content of polyphenols and
pigments found in apples is important because it contributes to the sensory quality of fresh
fruits and products made from apples (KHANIZADEH & al. [5]).
Several studies have reported that the apple peel contains higher levels of phenolic
compounds than the ones found in apple pulp (KHANIZADEH & al. [5], WOLF & al. [6],
CERBU & al. [7]).
Polyphenol content and antioxidant capacity of apple fruits are mostly influenced by
genetical factors such as the apple variety, the harvest quality or the fruit colour (red, yellow,
green or bicolored shell), but also by apple pulp which can be more or less colourful and
bright, by the fruit´s tissue and the variable content of sugars and acids (LEE & al. [8] or
VRHOVSEK & al. [9] or DROGOUDI & al. [10]). Moreover the polyphenol content and
antioxidant capacity can thus be influenced by environmental factors and technological
factors such as post-harvest factors, the harvest date, the fruit exposure in the crown of the
tree (i.e., sunny or shady places) but also by its processing and storage (McGHIE & al. [11]).
On the other hand it is well known that genetics plays an important role in controlling the
composition of polyphenols in apples and other fruits (KHANIZADEH & al.[12]).
Chlorophyll is the most common pigment found in plants. The degradation of
chlorophyll begins with fruit maturation due to the action of aging related hormones
(ethylene) (JACOB-WILK & al. [13]). The mechanism of chlorophyll disappearance is
complex and still not fully understood because of the rapidity and the formation of catabolic
colour (VICENTINI & al.[14]). MUSSINA & al. [15] showed in Granny Smith variety a
steady decline of chlorophyll during fruit development. The same pattern of chlorophyll
decline was observed by GREER [16] for the Royal Gala and Braeburn variety. REAY & al.
[17] showed that the total chlorophyll in Gala apples peaked at about 70-110 days after full
bloom in two successive seasons, after which it decreased in the ripening fruit.
The apple fruit carotenoid content varies depending on variety, stage of fruit
development and environmental factors (temperature and light). A very important aspect
linked to apples which are due to be consumed is that the majority of carotenoid pigments are
concentrated in the skin, contributing to the colour of the fruit, thus making it attractive;
however, apple pulp carotenoid concentration is low. There are some apples varieties that are
not intended for the consumers market, such as „Aotea" apples that have high concentrations
of carotenoids in the pulp (AMPOMAH-DWAMENA & al. [18]).
The aim of the current study was to assess the total polyphenol, antioxidant capacity,
chlorophyll and carotenoid pigments during fruit development phases for three apple
varieties: Ionathan, Starkrimson and Golden Delicious. The results will help to select the best
time to use the apples from physiological falls in different foods and pharmaceuticals.
2. Materials and Methods
Plant materials
A number of 80 apples from each studied cultivar (i.e., Starkrimson, Ionathan,
Golden Delicious) were harvested from the same orchard’s position of Reghin region Romania, at 7, 15 and 35 days after full bloom (DAFB) (each sample weighted between 50
and 1200g depending on the apples size at the harvesting time). Following the same protocol,
40 apples from each studied cultivar were harvested at 65, 107 and 144 DAFB (each sample
weighted between 1700 and 4000g depending on the apples size at the harvesting time). The
same samples were harvested from inside and periphery of the crown, respectively. Samples
were preserved at -16 °C until analysis.
Folin-Ciocalteu Method for Total Polyphenols Assessment
Total polyphenol content of whole apple was determined according to the method
described by CERBU & al. [7]. Absorbance was read at 750 nm with a Shimadzu UV-VIS
1700 spectrophotometer. The standard curve was carried out using concentrations of 0, 0.25,
0.50, 0.75, 1 mg/ml of gallic acid. Total polyphenol content in whole apple was expressed in
gallic acid equivalents, mg of GAE/100 g FW.
DPPH Method for Antioxidant Capacity Assessment
The antioxidant capacity was determined by assessing free radical scavenging effect
over 1,1-diphenyl l-2-picrylhydrazyl (DPPH) radical. This determination is based on the
method proposed by ODRIOZOLA-SERRANO & al. [19]. An amount of 10μl of the
methanol extract from the analysed samples, obtained according to the method described by
BUNEA & al. [20], was mixed with 3.9 ml of DPPH (0.025g /l) and 90 µl of distilled water.
The mixture was stirred and maintained properly in the dark for 30 min. The absorbance of
the samples was measured at 515 nm (Shimadzu 1700 UV-VIS) against a methanol blank.
Results were expressed as percent over standard DPPH absorbance.
𝐴𝐷𝑃𝑃𝐻 − 𝐴𝑃
𝑅𝑆𝐴 [%] =
× 100
𝐴𝐷𝑃𝑃𝐻
RSA [%] - Radical Scavenging Activity; ADPPH – DPPH absorbance; AP –sample absorbance.
Chlorophylls Assessment
Apple peel chlorophyll extraction was performed according to the method described
by LANCASTER & al. [21] with some modifications. Absorbance was measured at 645 and
663nm.
Chlorophyll content was calculated using ARNON [22] equations:
Ca (mg g-1) = [(12.7 × A663) - (2.6 × A645)] × ml acetone / mg sample
Cb (mg g-1) = [(22.9 × A645) - (4.68 × A663)] × ml acetone / mg sample
CT = Ca + Cb.
where: Ca – chlorophyll a; Cb – chlorophyll b; A663 – absorbance at 663 nm; A645 –
absorbance at 645 nm; CT– total chlorophyll.
Carotenoids Assessment
Extraction was performed using the procedure described by BUNEA & al. [23] with
some modifications. Apple skins were freeze-dried by using liquid N2. Carotenoids were
extracted from the freeze-dried apple skins using as solvents: methanol, ethyl acetate,
petroleum ether (1:1:1, v/v/v). Successive extractions were performed. The extracts were
combined, filtered and washed with distilled water, diethyl ether and a saturated solution of
NaOH. The ethereal phase was recovered and subjected to rotary evaporation at 35oC. The
remaining extract was dissolved in a known volume of methanol and stored at -18°C until it
was subjected to analysis.
Estimation of carotenoids was spectrophotometrically determined using Shimadzu
UV-VIS 1700 set at 450 nm. Estimation of carotenoids content was calculated with the
following formula:
𝐴 × 𝑉 × 103
𝑋 (𝑚𝑔 𝑜𝑓 𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑜𝑖𝑑s) =
2500 × 𝑙 × 100
A = absorbtion at λmax = 450 nm; V = sample volume (ml); 2500 = the molar absorption
coefficient (E1%); l = 1 cm – optical path length (BRITTON & al.[24]).
3. Results and Conclusions
Evolution of the total polyphenols content in apple fruit during development
It was found that the highest total phenolic content was recorded seven DAFB. Apples
total polyphenols content (TPC) was 1606.48 mg EAG/100g for Golden Delicious, 1192.58
mg EAG/100g for Ionathan and 1094.47 mg EAG/100g for Starkrimson variety. TPC of
analysed apples, statistically significant (p<0.05) decreased while fruit growing and was not
influenced by the fruit position in the tree crown (Figure 1). At the end of the experiment
(144 DAFB), the polyphenol content in apple fruits decreased to 1.35 mg EAG/100g in
Golden Delicious variety, 21.80 mg EAG/100g in Ionathan and 57.04 mg EAG/100g in
Starkrimson variety.
At 65 DAFB there is a massive decrease in polyphenol content due to increased
metabolic activity occurring in secondary metabolites biosynthesis and cell expansion phase.
For Fuji variety, ZHENG & al. [25] observed a similar decrease of total phenolic compounds
starting at early growth (25 DAFB). When comparing the three varieties studied, differences
were observed in TPC (Figure 1). These differences are due the distribution of phenolic
compounds in fruit tissue, depending on the variety (ALONSO [26]). Moreover, previous
research had shown that the position of the fruit in the tree crown, the tree´s position in the
orchard, the microclimate and the cultivar play an important role in the polyphenols content
of apple fruits (VOLZ & al. [27]). As discussed, our results confirmed that the variations in
the polyphenol content are caused by fruit maturity, harvesting time, variety and fruit position
in the tree crown (Figure 1).
Antioxidant capacity of apple fruit during development
For each variety, regardless of position in the crown, the highest antioxidant capacity
was recorded at 15 DAFB (Figure 2). Golden Delicious records statistically significant
differences (p<0.05) starting 15 DAFB when antioxidant capacity reaches the highest level.
During the growth and development of the fruit, antioxidant capacity decreased significantly
(p<0.05) by the end of the experiment regardless of the position of the fruit in the tree crown.
The same trend can be seen for the Ionathan and Starkrimson varieties (Figure 2). ZHENG &
al. [25] reported a decline of the antioxidant capacity in the case of Fuji during the growth
and the development of fruits similar to the decrease observed in our experiment.
80
120
160
1600
1400
1200
Variety
(a) Ionathan
(b) Golden Delicious
(c) Stark rimson
1000
800
600
(1) Inside of the crown
35
400
200
Antioxidant Capacity [%]
Total phenolic content [m g GAE / 100g]
1800
40
(2) Periphery of the crown
40
80
120
160
(2) Periphery of the crown
0
30
25
Variety
(a) Ionathan
(b) Golden Delicious
(c) Stark rimson
20
15
10
0
0
40
80
120
5
Figure 1. Changes of total
polyphenols content during fruit
growth for three apple varieties
(1) Inside of the crown
5000
40
80
120
160
(2) Periphery of the crown
4000
Variety
(a) Ionathan
(b) Golden Delicious
(c) Starkrimson
3000
2000
1000
0
160
Time [Days after full bloom]
Total carotenoid content [µg/100g]
0
0
(1) Inside of the crown
40
80
120
160
Time [Days after full bloom]
0
40
80
120
160
Time [Days after full bloom]
Figure 2. Changes of antioxidant
capacity during fruit growth for three
apple varieties
Figure 3. Changes of
carotenoids content during fruit
growth for three apple varieties
Evolution of carotenoid content during fruit development
The concentration of carotenoids accumulated in the apple peel generally decreased
depending on the time of harvest, regardless of the variety or the crown position (Figure 3).
Comparing the three studied varieties by the crown position, Starkrimson variety
accumulated the highest carotenoid content in both the apples harvested from inside of the
crown and those harvested from the periphery of the crown.
Evolution of chlorophyll content during fruit growth
Ionathan variety registered statistically significant differences (p<0.05) starting 15
DAFB, when the amounts of CT, Ca and Cb were lower than the ones at 7 DAFB. These
differences are observed both in the apples harvested from the periphery of the crown and
from those harvested from inside of the crown. At 65 DAFB, when the temperature and the
light intensity are high (July) there is a statistically significant increase in the CT, Ca and Cb
content. On the other hand at 107 DAFB, CT, Ca and Cb decreased significantly until the end
of the study. The same chlorophyll content pattern was observed for both Starkrimson and
Golden Delicious varieties (Figure 4, 5, 6). Similar amounts of CT, Ca and Cb were reported
by SUPARNA &. al. [28] for Pink Lady variety, which accumulated, for fruits harvested at
60 DAFB, 200 µg/g CT, 120 µg /g Ca and 55 µg/g Cb. Regarding the content of Cb, for
Ionathan variety values between 75.65 and 20.51 µg/g for apples harvested from inside of the
crown and between 75.68 and 26.14 µg/g for the apples harvested from the periphery of the
crown were recorded. Golden Delicious registered Cb values between 78.55 and 38.09 µg/g
for the apples harvested from inside of the crown and between 93.67 and 51.52 µg / g for the
apples harvested from the periphery of the crown. Starkrimson variety recorded Cb values
between 103.66 and 52.36 µg/g for the apples harvested from inside of the crown and
between 99.27 and 27.84 µg/g for the apples harvested from the periphery of the crown. At
the end of apple fruit development statistically significant differences (p<0.05) were noticed;
the amount of chlorophyll was influenced by the position in the crown and fruit variety.
There is a range of important factors that may influence the absorption of chlorophyll in the
apple peel: light, temperature, variety, fruit development, fruit position in the tree crown.
200
100
300
(2) Periphery of the crown
Variety
(a) Ionathan
(b) Golden Delicious
(c) Starkrimson
200
(1) Inside of the crown
200
100
150
80
100
50
(2) Periphery of the crown
200
Variety
(a) Ionathan
(b) Golden Delicious
(c) Starkrimson
150
100
100
Figure 4. Changes of total
chlorophyll content during fruit
growth for three apple varieties
60
40
20
(2) Periphery of the crown
100
Variety
(a) Ionathan
(b) Golden Delicious
(c) Starkrimson
80
60
40
50
0
20 40 60 80 100 120 140 160
Timp [Zile de la fenofaza înflorirea deplina]
Chlorophyll b content [µg/g]
(1) Inside of the crown
300
Chlorophyll a content [µg/g]
Continutul Clorofila Totala [µg/g]
(1) Inside of the crown
0
20
40
60
80
100 120 140 160
Time [Days after full bloom]
Figure 5. Changes of Chlorophyll a
content during fruit growth for three
apple varieties
20
0
20
40 60 80 100 120 140 160
Time [Days after full bloom]
Figure 6. Changes of Chlorophyll b
content during fruit growth for
three apple varieties
Effect of harvesting time, variety and position in the tree crown on the total
polyphenols, antioxidant capacity, total chlorophyll and carotenoids
For advanced interpretation of the obtained results, it was considered appropriate to
calculate the proportion between the total variability of main factors effects and their
interactions. This proportion was expressed by eta squared (η2), being currently used as the
estimate of effect size (MUREŞAN [29]).
Classical Eta squared value was calculated as follows:
η2 = SSfactor/SStotal
where: SSfactor was the variation attributed to the factor and SStotal represented total variance
(PIERCE & al.[30]).
Three-factorial analysis of variance was performed to assess the influence of
harvesting time, variety and fruit position in the tree crown over the total polyphenol content,
antioxidant capacity, total chlorophyll and carotenoid.
In the case total polyphenol main effects: Time, Variety and interaction between
Time, Variety and Position indicated highly significant differences (***; p<0.0001), while
position factor was statistically significant (*; p< 0.05). Consequently, one is interested in
debating the statistically significant interaction, in this case, the second degree interaction:
Time*Variety *Position. The main effects: Harvesting time, Variety and Position in the
crown explained 93.38 %, 1.94 %, 0.01 % of the total variability of total polyphenols,
indicating the importance of harvesting period (Table 1). Also, this statistical analysis showed
the low influence of the variety (explains 1.94 % of variance) and low importance of position
(explaining 0.01 % of variance). The greatest importance interaction was represented by
Variety*Harvesting time that recorded 4.40 % of the total polyphenols variability, while the
remaining interactions gathered together less than 0.3%.
Table 1. Analysis of variance - apples total phenolic content
Source
Variety
Position
Time
Int. Variety*Position
Int. Variety*Time
Int. Position*Time
Int.Variety*Position*Time
Error
Total
Sum of
Squares
344282.37
1046.16
16584166.22
773.67
781329.71
24873.08
17966.01
5465.26
17759902.51
Mean Square
F-value
P-value
172141.18
1046.16
3316833.24
386.83
78132.97
4974.61
1796.60
151.81
1133.903
6.891
21848.163
2.548
514.666
32.768
11.834
3.086E-33***
0.013*
1.326E-61***
0.092ns
1.010E-35***
1.915E-12***
1.126E-08***
Eta
Squared
0.0194
0.0001
0.9338
0.0000
0.0440
0.0014
0.0010
0.0003
* significant (p<0.05); ** very significant (p<0.01); *** highly significant (p<0.001); ns - no statistical
significance (p>0.05).
Three-factorial analysis of variance for the antioxidant capacity of apple samples
under study, including the experimental factors: Harvesting time, Variety and Position in the
crown, and the interactions between these, indicates highly significant significations
(***,p<0.0001) for all experimental factors as well as their interactions. Relative sizes of the
effects of the studied factors and their interactions over the antioxidant capacity are shown in
Table 2. The main effects: Harvesting time, Variety, Position in the crown, explained 80.59
%, 7.17 % respectively 0.63 % of the total variability in antioxidant capacity indicating the
great importance of Harvesting time. The statistical analysis also showed the influence of the
Variety (7.17% of variance explained) and the insignificance of Position (explaining 0.63 %
of variance).
The most important interaction is Variety * Harvesting time which recorded 10.31 %
from the variability of antioxidant capacity, while the remaining interactions gather together
less than 2%. Analysis of variance for total chlorophyll values showed highly significant
differences (***,p< 0.0001) for the Harvesting time, Variety and very significant differences
(**,p<0.01) for the Position in the crown. Second order interaction (Harvesting
time*Variety*Position) was highly significant (***,p<0.0001) which explained why
chlorophyll value depends on a combination between Harvesting time, Variety and Position
in the crown.
Table 2. Analysis of variance - apples antioxidant capacity
Source
Variety
Position
Time
Int. Variety*Position
Int. Variety*Time
Int. Position*Time
Int.Variety*Position*Time
Error
Total
Sum of
Squares
260.39
22.83
2927.30
13.09
374.53
4.99
28.23
1.11
3632.51
Mean
Square
130.19
22.83
585.46
6.54
37.45
0.99
2.82
0.03
F-value
P-value
4203.330
737.315
18901.232
211.416
1209.171
32.222
91.167
2.172E-43***
1.424E-25***
1.798E-60***
1.270E-20***
2.316E-42***
2.443E-12***
1.714E-22***
Eta
Squared
0.0717
0.0063
0.8059
0.0036
0.1031
0.0014
0.0078
0.0003
* significant (p<0.05); ** very significant (p<0.01); *** highly significant (p<0.001); ns - no statistical
significance (p>0.05).
Relative sizes of the studied factors effects and their interactions regarding total
chlorophyll content are shown in Table 3. Main effects, Harvesting time, Variety and Position
in the crown are explaining 85.26%, 5.55 % and 0.02 % of total chlorophyll variability
indicating the importance of Harvesting time. The most important interaction was the
Variety*Harvesting time interaction, recording 5.30% of total chlorophyll variability while
remaining interactions gathers together about 2%.
Table 3. Analysis of variance - apples total chlorophyll content
Source
Variety
Position
Time
Int. Variety*Position
Int. Variety*Time
Int. Position*Time
Int.Variety*Position*Time
Error
Total
Sum of
Squares
11999.67
39.03
184386.37
788.01
11471.47
3237.94
4136.47
206.51
216265.50
Mean
Square
5999.83
39.03
36877.27
394.00
1147.14
647.58
413.64
5.73
F-value
1045.888
6.804
6428.425
68.683
199.970
112.887
72.107
P-value
1.291E-32***
1.316E-02**
4.782E-52***
5.153E-13***
1.956E-28***
5.833E-21***
9.401E-21***
Eta
Squared
0.0555
0.0002
0.8526
0.0036
0.0530
0.0150
0.0191
0.0010
* significant (p<0.05); ** very significant (p<0.01); *** highly significant (p<0.001); ns - no statistical
significance (p>0.05).
Three-factorial analysis of variance for carotenoids extracted from the apple samples
under study, including experimental factors Harvesting time, Variety and Position in the
crown and the interactions between them, indicates highly significant differences
(***,p<0.0001 ) for all experimental factors as well as for their interactions.
Relative sizes of the studied factors effects and their interactions over carotenoids are
shown in Table 4. The main effects Harvesting time, Variety and Position of the crown,
explained 66.40%, 20.66% respectively 0.03% of the total variability carotenoids, indicating
the importance of Harvesting time and Variety. However the statistical analysis showed the
low influence of Position in the crown (explaining 0.03 % of variance). The most important
interaction is Position in the crown*Harvesting time which recorded 4.96% regarding
carotenoids variability, while the remaining interactions gathers together less than 8%.
Table 4. Analysis of variance - apples carotenoids content
Source
Variety
Position
Time
Int. Variety*Position
Int. Variety*Time
Int. Position*Time
Int.Variety*Position*Time
Error
Total
Sum of
Mean
Squares
Square
16623242.57 8311621.28
22917.34
22917.34
53414864.11 10682972.82
867180.07
433590.03
3845438.67
384543.86
3987258.01
797451.60
1671010.80
167101.08
16658.07
462.72
80448569.66
F-value
P-value
17962.367
49.527
23087.130
937.038
831.043
1723.384
361.125
1.020E-54***
2.931E-08***
4.916E-62***
9.006E-32***
1.921E-39***
8.808E-42***
5.571E-33***
Eta
Squared
0.2066
0.0003
0.6640
0.0108
0.0478
0.0496
0.0208
0.0002
* significant (p<0.05); ** very significant (p<0.01); *** highly significant (p<0.001); ns - no statistical
significance (p>0.05).
In conclusion, for all varieties, the highest total polyphenol content was recorded for
the apples harvested at 7 DAFB, decreasing while the fruit was growing. Antioxidant
capacity reaches the highest level at 15 DAFB and then declines until the apples reached
technological maturity. Total chlorophyll, chlorophyll a and b recorded high values at 65
DAFB, when both the temperature and the light intensity are high (July). After this harvesting
time, chlorophyll content decreased. The concentration of carotenoids accumulated in apple
peel varied, however following a downward trend during fruit growth, regardless of the
variety or the crown position.
Following the results, the best time to use the apples from physiological falls in
different foods and pharmaceuticals is when the total polyphenols, the antioxidant capacity or
chlorophyll a and b record the highest values, depending on the way the product was
designed and its main purpose.
4. Acknowledgements
Vlad Muresan and Romina Alina Vlaic thank for support from Euro pean Social
Fund, Human Resources Develop ment Operational Programme, project no. POSDRU/
159/1.5/S/132765.
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