EQOL Journal (2022) 14(2): 31-39
ORIGINAL ARTICLE
31
Chokeberry juice affects membrane lipid status and cellular
antioxidant enzymes in healthy women with aerobic training activity
Nevena Vidović
1
✉ • Ana Pantović
1
• Vuk Stevanović
1
• Ivana Šarac
1
• Kristina Robal
1, 2
•
Stevan Stevanović
1, 3
• Maria Glibetic
1
Received: 18
th
May, 2022 DOI: 10.31382/eqol.221204
Accepted: 29
th
August, 2022
© The Author(s) 2022. This article is published with open access.
Abstract
The present study examined the effects of aerobic
training alone or combined with chokeberry juice on
membrane lipid status and activities of antioxidant
enzymes in non-athlete women. Participants were
randomly assigned into the training group
performing aerobic training three times per week;
the chokeberry-training group followed the same
training regime and additionally consumed 100 ml
of chokeberry juice per day and the control group
neither trained nor consumed the juice.
Blood samples were collected at baseline and the
end of the eight-week-long intervention. Membrane
fatty acids' composition was analyzed by gas-liquid
chromatography, while the activities of antioxidant
enzymes were measured by spectrophotometry.
As a result, the n-3 fatty acids' content was
significantly higher in the chokeberry-training
(median (interquartile range) of 5.96 (1.65) %)
compared with the control group (5.12 (0.87) %),
while saturated fatty acids' content was lower in the
chokeberry-training (40.14±1.19 %) than in the
training group (42.59±2.29 %). We detected signifi-
cantly higher activity of superoxide dismutase in
the training (2224 (2170) U/gHb) compared with
the chokeberry-training (1252 (734) U/gHb) and
control group (1397 (475) U/gHb).
Our study indicates that supplementation with
chokeberry juice may induce favorable changes in
cell fatty acid composition and antioxidant
response in women performing aerobic training.
Keywords aerobic training • antioxidant enzymes
• chokeberry juice • fatty acids • healthy women.
Introduction
Moderate-intensity exercise has multiple health
benefits, and it is, thus, highly recommended for
the prevention of cardiovascular diseases. Still,
exercise, specifically intense or long-lasting can
lead to excessive production of free radicals and
reactive oxygen species (ROS) and negatively
affect pro-oxidant–antioxidant balance (He et al.,
2016). This, in turn, causes the oxidation of cell
components, such are lipids and fatty acids. Fatty
acids in cell membranes are highly susceptible to
oxidative damage, especially those with a high
number of unsaturated bonds (Jimenez, Winward,
Walsh & Champagne, 2020). The extent of
oxidative damage depends on exercise intensity
and duration, as well as on the host's defense
system. Moderate exercise induces the production
of low levels of ROS that can activate signaling
pathways involved in cellular responses beneficial
for health (Ristow & Zarse, 2010). In addition,
regular training can up-regulate antioxidant
defense and attenuate oxidative stress in diseases
✉
nevena.vidovic@imi.bg.ac.rs
1
University of Belgrade, Institute for Medical
Research, National Institute of Republic of Serbia,
Centre of Research Excellence in Nutrition and
Metabolism, Belgrade, Serbia
2
Harokopio University, School of Health Science
and Education, Department of Nutrition and
Dietetics, Athens, Greece
3
Special Hospital for Psychiatric Disorders
“Kovin”, Kovin, Serbia
EQOL Journal (2022) 14(2): 31-39
32
such are cardiovascular ones (Jackson, 2008).
Cardiovascular benefits of aerobic exercise can be
exerted in minutes-hours or even several days after
training (Bolli, 2000). Although some authors
indicated that antioxidant supplementation may blunt
these cardiovascular benefits, others underlined
inconsistency of data and varying effects of
antioxidants combined with training, depending on
compounds, doses, and characteristics of exercise
(Gliemann et al., 2013; Mankowski, Anton, Buford &
Leeuwenburgh, 2015). Thus, a variety of dietary
antioxidant supplements have been developed aiming
to ameliorate excessive production of free radicals
during exercise and upgrading the physical outcomes
of the training (Williams, Strobel, Lexis, & Coombe,
2006). Researchers have demonstrated the impact of
natural antioxidants in reducing exercise-induced
oxidative stress, as well as improving performance
and resistance to injury. Among these, polyphenols
take an important place, as they express the potential
to attenuate oxidative stress caused by both acute and
chronic exercise (McAnultya et al., 2004; Panza et al.,
2008). One of the promising polyphenol-rich foods is
chokeberry juice, with beneficial effects
demonstrated in human intervention trials,
specifically those on oxidative status (Broncel et al.,
2010; Kardum et al., 2014a; Pilaczynska-Szczesniak,
Skarpanska-Steinborn, Deskur, Basta &
Horoszkiewicz-Hassan, 2005). Additionally,
chokeberry acts health promoting not only in subjects
at high risk of cardiovascular disease but in healthy
subjects as well (Kardum et al., 2014b). Three
months-long supplementations with chokeberry juice
beneficially affected cellular antioxidant status and
membrane fatty acid composition in apparently
healthy females (Kardum et al., 2014b). The
beneficial effects of chokeberry extract
supplementation on redox status have been
demonstrated in a recent study with active handball
players as volunteers (Cikiriz et al., 2021). On the
other side, the lack of antioxidant effects has been
reported in a study that investigated the impact of
chokeberry juice supplementation on oxidative
balance in young footballers (Stankiewicz et al.,
2021).
Taking into account all these facts, the objective
of our study was to investigate the effects of a type of
aerobic training alone or in combination with
chokeberry juice consumption, on cellular oxidative
status, measured as membrane fatty acid composition,
and the activities of antioxidant enzymes, in healthy
non-athlete women.
Method
Subjects and study design
We included 28 healthy women with a mean age of
25.1±2.8 years, body height 168.2±5.8 cm, and body
weight of 60.4±9.7 kg and randomly assigned them
into three groups. The participants were non-athletes
assuming they undertook less than 30 minutes of
intense or 60 minutes of moderate activity per week
in the preceding three months. This study was
approved in advance by the Ethical Committee of the
Medical Clinical Center in Zemun (Belgrade, Serbia)
and was undertaken according to the Helsinki
Declaration. Each participant voluntarily provided
written informed consent before participating. The
exclusion criteria were: the presence of chronic
diseases, body mass index ≤ 18 or ≥ 30 kg/m2, food
allergy or intolerance to juice components, irregular
dietary pattern, pregnancy, or breast feeding.
The first subject group performed training; the
second group trained and consumed 100 ml of
chokeberry juice per day, and the third group was
defined as the control group neither training nor
consuming chokeberry juice. Both training and
chokeberry-training groups were instructed to do the
same training program.
Training
A commercial aerobic dance exercise program named
body combat (Les Mills, New Zealand) was
performed by participants 3 times a week, for 60
minutes, in the evening hours, at three exercising
facilities located in Belgrade (Serbia) certified by Les
Mills. Body combat is defined as a vigorous or
moderate-to-high-intensity aerobics class. More
precisely, it is a form of interval training with a
regular exchange of moderate and high-intensity
bouts, like a high interval intermittent training (Jung,
Bourne & Little, 2014).
The certified instructors performed the same
choreography in all three facilities and encouraged
the participants to exercise at the highest possible
intensity they could. The planned number of training
sessions was 24, and participants were allowed to
miss a maximum of 2 of them. When they were
unable to attend the training in their term, they could
do it in the scheduled session on the following day.
Chokeberry juice
Participants in the chokeberry-training group
consumed polyphenol-rich chokeberry juice and they
were advised to keep it in a refrigerator after opening
EQOL Journal (2022) 14(2): 31-39
33
it. The content of total phenolics was 648.4 mg of
gallic acid equivalents per 100 mL, while
proanthocyanidins, anthocyanins, and phenolic acids
were the main phenolic subclasses. The detailed
characterization and quantification of phenolic
compounds present in the juice have been previously
published by Tomic et al. (2016). The chokeberry
juice was donated from Rheapharm d.o.o., Belgrade,
Serbia.
Laboratory assays
Blood samples were collected after an overnight fast
at baseline and the end of the 8-week-long
intervention. Biochemical parameters were
determined on the same day the samples were
collected, using a clinical chemistry analyzer Cobas
c111 (Roche Diagnostics, Basel, Switzerland) and
Roche Diagnostics' kits according to the
manufacturer’s instructions. Erythrocytes were
isolated, washed out with cold isotonic saline, divided
into aliquots, and stored at -80ºC for further analysis
of the fatty acid profile and antioxidant enzyme
activities.
Analyses of fatty acid composition
Firstly, lipids were extracted using the organic
solvents chloroform and isopropanol as previously
described (Rose & Oklander, 1965). Further,
phospholipids were separated by a thin layer of
chromatography with a mixture of petroleum ether,
diethyl ether, and glacial acetic acid. According to the
modified procedure of Cristopherson and Glass
(1969), direct transesterification of fatty acid was
carried out, followed by the evaporation of hexane
extracts under a stream of nitrogen. The final residue
was dissolved in 10 µL of hexane and 1 µL was
injected into the Shimadzu chromatograph GC 2014.
The chromatograph was equipped with a flame
ionization detector and Rtx 2330 column (60m x
0.25mmID, 0.2μm, Restek, Bellefonte,
Pennsylvania).
Adequate separation of methyl esters was obtained
over 50 minutes with a temperature of 140°C held for
5 minutes, then increased to 220°C at a rate of
3°C/min and held at final temperature for 20 minutes.
The identification was made by comparing peak
retention times with standard mixtures and the
contents of fatty acids from C16:0 through C22:6n-3
were expressed as a percentage of total fatty acids
identified. The content of total saturated fatty acids
(SFA) was calculated by summing the contents of
C16:0 and C18:0 percentages, while the content of
monounsaturated fatty acids was calculated from
C16:1n-7, C18:1n-9, and C18:1n-7 percentages.
Finally, the content of total polyunsaturated fatty
acids (PUFA) was calculated from the percentages of
C18:2n-6, C20:3n-6, C20:4n-6, C22:4n-6, C20:5n-3,
C22:5n-3, and C22:6n-3.
Analyses of antioxidant enzymes
The activity of superoxide dismutase was determined
using a commercial kit Ransod (Randox, Crumlin,
UK), based on superoxide anion production in
xanthine-xanthine oxidase system and its further
reaction with 2-(4-iodophenyl)-3-(4-nitrophenol)-
phenyltetrazolium chloride, resulting in the formation
of red formazan dye, and absorbance measurement at
505 nm and 37°C for 3min.
The glutathione peroxidase activity was
determined with the use of the commercial kit Ransel
(Randox, Crumlin, UK). In the presence of cumene
hydroperoxide, glutathione peroxidase from samples
catalyzed the oxidation of glutathione, while
glutathione reductase further reduced oxidized
glutathione with the consumption of coenzyme
NADPH+H+.
The determination of catalase activity was based
on its ability to degrade hydrogen peroxide, as
previously described (Aebi, 1984). A decrease in
H2O2 absorbance was measured at 230 nm for 3
minutes and this change was used for the
determination of catalase activity.
The activities of enzymes were expressed in
U/gHb, and the cyanmethemoglobin method with
Drubkin’s reagent was applied for the assessment of
hemoglobin (Hb) concentration (Van Kampen &
Zijlstra, 1961).
Dietary intake
The participants were explicitly instructed to continue
with their normal lifestyle, particularly regarding
their diet. The training and control groups were
instructed not to include any antioxidant-rich food or
supplements that they haven’t habitually consumed.
The chokeberry-training group was allowed to
consume only chokeberry juice as additional
polyphenol-rich food, as explained in the study
protocol. We then evaluated participants' dietary
intake using a validated 24-hour dietary recall
questionnaire on three separate days (1 working day
at the beginning of the study, one weekend day during
the study, and one working day at the end of the
study). A well-trained and experienced interviewer in
dietary intake assessment collected information on
food type, preparation methods, recipes, and
EQOL Journal (2022) 14(2): 31-39
34
commercial products from each participant in a face-
to-face interview. For estimating the portion sizes, we
provided pictures of various foods, dishes, and
beverages, as previously reported (Gurinović et al.,
2016). Nutrient calculations were performed using
the Serbian Food composition database.
Statistical analysis
Before comparisons, the normal distribution was
evaluated by the Shapiro-Wilk test. For normally
distributed variables a student’s paired t-test was
applied, and data are presented as mean and standard
deviation (SD). Wilcoxon Signed Rank test was used
to compare non-normally distributed variables within
each group, and data are presented as the median and
interquartile range (IQ). To identify differences
between groups and other parameters at baseline,
one-way analysis of variance (ANOVA) and Kruskal-
Wallis tests were applied for normally and non-
normally distributed data, respectively. Finally, to
compare the intervention effects between the groups,
data were analyzed using an analysis of covariance
(ANCOVA), with baseline values as covariables, and
Bonferroni as a post hoc test. Analysis was performed
using the SPSS software (ver. 20.0) and p values
<0.05 were considered statistically significant.
Results
Characteristics of the subjects, dietary intake, and
biochemical parameters
Participants were well matched by age, body weight,
and height with no difference between study groups.
There were no significant changes in body weight at
the end of the intervention period (Table 1). By
comparing the dietary intakes of all three study
groups (as a mean of three 24 h dietary recalls), we
confirmed that participants had a uniform diet during
the intervention period.
The values of total cholesterol significantly
decreased in all three study groups: training
(p=0.007), chokeberry-training (p=0.034), and
control (p=0.002), compared with the baseline. A
Similar was observed for blood glucose, with a
significant decrease just in the training group
(p=0.017). The post-intervention level of low-density
lipoprotein (LDL) cholesterol was significantly
higher in the chokeberry-training group compared
with the training group (p=0.04). Still, the values of
the total, LDL, and high-density lipoprotein (HDL)
cholesterol, as well as fasting blood glucose and
triglycerides were in reference ranges, indicating that
the participants were in good cardiovascular health
(Table 1).
Table 1. Characteristics of subjects at baseline and after 8 weeks of intervention
Training
Chokeberry-training
Control
Baseline
8 weeks
Baseline
8 weeks
Baseline
8 weeks
25.7±2.8
24.5±2.4
25.1±3.5
170.8±4.9
166.0±5.7
167.7±7.3
60.9±6.6
60.2±5.3
60.8±11.8
60.9±11.8
59.1±11.1
58.6±11.0
1899±340
1685±306
1919±351
195±60
176.29±52.26
192.97±54.21
70.72±11.90
67.66±17.42
73.18±14.44
92.46±21.15
78.21±20.99
94.69±14.50
4.60 (0.58)
4.06 (0.65)*
4.58 (0.51)
4.24 (0.41)
4.46 (0.40)
4.09 (0.59)
0.61 (0.70)
0.66 (0.65)
0.64 (0.29)
0.71 (0.25)
0.71 (0.32)
0.63 (0.28)
4.80±0.79
4.29±0.77**
4.97±0.54
4.69±0.52*
4.49 ± 0.65
4.09±068**
2.16±0.58
2.08±0.52
2.29±0.48
2.50±0.48*;†
1.90±0.26
2.05±0.33*
1.73±0.23
1.73±0.19
1.74±0.39
1.71±0.32
1.70±0.37
1.64±0.40
280.3±51.3
268.2±46.3
228.5±62.6
233.7±53.3
252.1±68.1
266.2±31.8
14.5 (4.7)
14.0 (5.7)
11.7 (6.8)
14.2 (5.8)
11.6 (3.4)
11.0 (4.1)
20.5 (6.5)
19.6 (3.6)
18.1 (4.6)
19.0 (6.5)
18.1 (3.0)
16.4 (2.6)
Note: Data are presented as mean ± standard deviation or as median (interquartile range) *p<0.05** p<0.01 compared
with baseline; † p<0.05 compared with training group
ALT- alanine aminotransferase; AST – aspartate aminotransferase; chol – cholesterol; CHO – carbohydrate; HDL –
high-density lipoprotein; LDL – low-density lipoprotein; TG – triglycerides.
EQOL Journal (2022) 14(2): 31-39
35
Effects on fatty acid profile
The paired t-test revealed a significant decrease in the
levels of total SFA (p=0.026), palmitic acid
(p=0.047), and n-6/n-3 ratio (p=0.034) in the
chokeberry-training group (Table 2). In the same
group, there was a significant increase in the content
of total PUFA (p=0.009), n-3 PUFA (p=0.003),
docosahexaenoic (C22:6n-3; p=0.002), and
arachidonic acid (C20:4n-6; p=0.012). Between
groups comparisons at the end of the intervention
confirmed these findings. The chokeberry-training
group had a significantly higher content of total n-3
PUFA (p=0.019) and docosahexaenoic acid
(p=0.011) than the control group, while the n6/n3
ratio was significantly lower (p=0.049). When
compared with the training group, the chokeberry-
training group had significantly lower concentrations
of SFA (p=0.017) and palmitic acid (C16:0;
p=0.021). We found no significant differences in fatty
acid profile between groups at the baseline (Table 2).
Table 2. Fatty acid composition of membrane phospholipids at baseline and after 8 weeks of intervention
Variable
Training
Chokeberry-training
Control
Baseline
8 weeks
Baseline
8 weeks
Baseline
8 weeks
SFA (%)
42.78±2.32
42.59±2.29
41.95±1.68
40.14±1.19*;†
41.36±3.58
40.91±1.57
16:0
21.36 (2.46)
21.54 (1.46)
20.77 (1.79)
19.92 (0.78)*;†
20.56 (1.87)
20.25 (3.18)
18:0
21.35 (2.19)
20.71 (3.48)
20.81 (1.39)
20.36 (1.89)
19.86 (2.41)
20.59 (1.21)
MUFA (%)
14.69 (1.16)
14.70 (3.08)
14.50 (1.73)
14.76 (1.25)
15.19 (1.72)
15.00 (2.57)
16:1n-7
0.20 (0.08)
0.21 (0.09)
0.24 (0.06)
0.20 (0.09)
0.20 (0.07)
0.21 (0.08)
18:1n-9
12.88 (1.07)
12.82 (1.36)
12.62 (1.48)
12.91 (1.39)
13.25 (1.65)
13.20 (1.91)
18:1n-7
1.58 (0.17)
1.60 (0.29)
1.62 (0.09)
1.60 (0.29)
1.56 (0.27)
1.57 (0.52)
n-6 PUFA (%)
36.86±1.99
37.91±2.25
38.09±1.92
38.99±1.02
37.92±3.22
38.56±2.06
18:2n-6
14.20 (2.84)
14.02 (1.20)
13.71 (1.56)
13.53 (1.27)
14.19 (1.03)
13.89 (2.18)
20:3n-6
1.53 (0.46)
1.50 (0.36)
1.62 (0.32)
1.67 (0.24)
1.80 (0.49)
1.96 (0.34)
20:4n-6
17.18±1.48
17.57±1.60
18.27±1.04
19.07±1.43*
17.50±2.39
18.54±1.40
22:4n-6
4.46±0.68
4.53±0.55
4.60±0.82
4.84±0.63
4.72±0.63
4.72±0.42
n-3 PUFA (%)
5.37 (0.89)
6.20 (1.16)
5.42 (1.41)
5.96 (1.65)**; ‡
5.14 (2.24)
5.12 (0.87)
20:5n-3
0.22 (0.06)
0.22 (0.10)
0.18 (0.09)
0.24 (0.10)
0.20 (0.10)
0.19 (0.06)
22:5n-3
1.36 (0.42)
1.47 (0.28)
1.42 (0.45)
1.46 (0.47)
1.57 (0.13)
1.63 (0.30)
22:6n-3
3.72 (1.30)
1.57 (0.13)
1.63 (0.30)
4.40 (1.41)**;‡
3.48 (1.84)
3.42 (1.06)
Total PUFA (%)
42.05 (3.08)
4.46 (0.80)
3.74 (1.41)
45.31 (1.72)**
44.64 (5.19)
45.21 (2.46)
n-6/n-3 ratio
6.82±0.90
43.58 (2.28)
43.03 (2.14)
6.34±0.99*;‡
6.98±1.34
7.09±1.10
Note: Data are presented as mean ± standard deviation or as median (interquartile range)
*p<0.05** p<0.01 compared with baseline; † p<0.05 compared with training group; ‡ p<0.05 compared with control
group MUFA – monounsaturated fatty acids; PUFA – polyunsaturated fatty acids; SFA – saturated fatty acids