Vidnoe, г. Москва и Московская область, Россия
Vidnoe, г. Москва и Московская область, Россия
Vidnoe, г. Москва и Московская область, Россия
Moscow, г. Москва и Московская область, Россия
Introduction. Apple juice owes its beneficial properties to various biologically active compounds, e.g. antioxidants. Therefore, food science needs effective methods that would cover all the mechanisms of their effect on human metabolism. However, fruit juice production raises certain safety issues that are associated not only with production risks, but also with some natural components in the raw material. The Allium cepa test seems to be an effective solution to the problem. This plant bioassay has a good correlation tested on mammalian cell cultures. Study objects and methods. Onion roots (A. cepa) were treated with aqueous solutions of juices and sorbic acid to assess their antioxidant profile. The toxic effects on root tissues were described according to biomass growth, malondialdehyde (MDA) concentration, and proliferative and cytogenetic disorders. Results and discussion. The study revealed the optimal conditions for the A. cepa assay of the antioxidant properties of apple juice. The antioxidant activity was at its highest when the juice was diluted with water 1:9 and the onion roots were treated with sorbic acid. The lipid oxidation of the A. cepa roots decreased by 43%. A comparative analysis of three different juice brands showed that the difference in their antioxidant profiles was ≤ 3%. As for toxic side effects, the chromosome aberrations increased by six times in all samples. Conclusion. The research offers a new in vivo method for determining the antioxidant profile of apple juice. Three juice brands proved to have irreversible cytotoxic and genotoxic effects.
Apple juice, bioassay, antioxidant activity, side effects, Allium cepa test, biologically active substances
INTRODUCTION
Apple juice is one of the most popular fruit juices
in Russia. Therefore, domestic food industry needs
reliable methods for its nutritional value and risk
assessment. The beneficial properties of apple juice are
associated with various biologically active compounds.
Recent antioxidant studies show that apple juice is rich
in such antioxidants as polyphenols, e.g. quercetin,
phloretin, chlorogenic acid, and epicatechin. A fruit and
vegetable diet reduces oxidative stress, thus preventing
chronic diseases and slowing down aging. Apples and
apple products are known to reduce the risk of cancer,
cardiovascular diseases, asthma, and type II diabetes
[1]. The chemical composition of juices depends on the
variety of apples, their ripeness, climate, cultivation
method, etc. Apple juice production involves a wide
variety of apple cultivars but gives preference to winter
and autumn varieties because they are juicy, firmfleshed,
and rich in aromatic and phenolic substances.
Consumers see apple juice as a source of biologically
active compounds that are beneficial to human health.
As a result, the volume of its industrial production
keeps increasing. Food processing determines the
nutritional value of the finished product [2]. Crushing,
heat treatment, fermentation, and clarification of
apples affect the phytochemical composition of apple
juice. These processes decrease the amount of phenolic
compounds. After heat treatment and direct extraction,
fruit juice had 10% of the antioxidant properties of
fresh fruits. After pulp fermentation, this figure was 3%.
Pulp fermentation decreased the content of phloridzin,
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Foods and Raw Materials, 2022, vol. 10, no. 1
E-ISSN 2310-9599
ISSN 2308-4057
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Samoylov A.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 176–184
chlorogenic acid, and catechin by 31, 44, and 58%,
respectively. Most of the active compounds remained in
apple pomace [3].
Another study compared polyphenols in apple juice
after heat and high pressure treatments [4]. The phenolic
profile of the resulting apple juice changed significantly.
The epicatechin concentration was 0.42 mg/100 mL in
the raw juice; it decreased to 0.31 mg/100 mL at 25°C
and increased to 0.39 mg/mL at 65°C. Heat treatment
increased the amount of catechin and chlorogenic
acid, while pressure treatment decreased the amount
of polyphenols. The authors linked this phenomenon
to structural destruction because the rapid release of
carbon dioxide led to pressure gradient.
Various plant assays of antioxidants properties
receive more and more scientific attention each year.
Unfortunately, different antioxidant tests use different
terms and measurements [5]. Moreover, antioxidants
may respond differently to different radicals or their
sources. Phytochemical compounds are present in
numerous products and possess numerous mechanisms
of action on metabolic processes. Thus, the food
industry has a wide choice of adequate antioxidant
assessment methods [6]. Therefore, an objective analysis
of data on bioactive compounds needs specifically
tailored markers. Finally, the bioactivity of plant food
products depends on a whole complex of phytochemical
compounds. Lipid peroxidation is measured by the
levels of malondialdehyde (MDA), β-carotene, and diene
conjugates [6].
Other methods determine the total antioxidant
potential according to the concentration of free
radicals, e.g. 2,2-diphenyl-1-picrylhydrazyl radical
(DPPH), pre-generated radical cation 2,2’-azinobis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), iron
reducing/antioxidant (FRAP), ferrous complex and
xylenol orange (FOX), iron(III) thiocyanate complex
(FTC), alkyl carboxylic acid (ACA), etc.
These approaches make it possible to analyze the
level of antioxidant activity both in food products
and in living organisms after consumption. However,
bioassays seem to be the most informative and accurate
methods, since all nutritionally valuable substances
are bioavailable and bioactive. Testing food matrices
on laboratory animals or human cell lines is expensive
and labor-consuming. Therefore, plant assays are more
preferable.
Scientists compared the level of lipid peroxidation
in onion roots after their treatment with apple juice and
a model aqueous solution of fructose, glucose, sucrose,
D-sorbitol, and malic acid. After incubation, the content
of MDA in root tissues was 1.7 times higher in the
model solution than in the apple juice [7]. Such results
proved that the juice possessed some antioxidant activity,
which lowered the carbohydrate-induced lipid oxidation
almost to the control values, i.e. those of water.
Domestic regulations ban synthetic additives from
juice production. Unfortunately, these measures fail
to eliminate juice-related safety risks. Therefore, food
producers have to check raw materials for various
contaminants, such as heavy metals, pesticides,
and herbicides, as well as to monitor the safety of
technological production means, e.g. detergents,
lubricants, packaging material, etc. Moreover,
technological methods of juice processing require
exposure to high temperatures during pasteurization,
sterilization, etc., which can result in accumulation
of toxic compounds and adducts. For example, some
phytochemical compounds of plant products are known
to react with cellular macromolecules during storage,
thus causing cellular toxicity or even genotoxicity if they
react with DNA [7, 8].
Almost all higher plants contain such natural
mutagens as pyrolizidine alkaloids and some flavonoids
[9]. In fact, recent studies linked the consumption
of fruits and juices to cancer and asthma in children
[10–13]. Finally, juices are rich in carbohydrates, and
fructose and sucrose produce adverse metabolic effects
on human health [14, 15]. Food scientists have developed
numerous physicochemical assay methods for these
toxic agents. However, bioassays seem to be the only
method that gives an integrated assessment of their
synergetic effect.
In this regard, the Allium cepa test is especially
promising. This test is recommended by WHO experts
as a standard for cytogenetic monitoring. The A. cepa
assay is a popular method to define the bioindicator of
cyto- and genotoxicity of xenobiotics in food products
and their components [16]. The A. cepa test provides a
prompt comparative analysis of individual compounds
and their combinations. A. cepa cells share metabolic
mechanisms with all eukaryotes, but unlike animal and
human cell lines, they are not subject to transformation
and can be useful in detoxification modeling. This test
can screen biomarkers that determine the negative
potential of food matrix toxicants for metabolic
processes in onion root tissues [17].
Taking into account these indicators and the data
on antioxidant activity, plant bioassays can logically be
applied to various brands of apple juice [7]. However,
research databases seem to contain no publications on
the Allium-based comparative evaluation of various
domestic brands of apple juice. The present research
objective was to compare the antioxidant activity,
cytotoxicity, and genotoxicity of various domestic apple
juice brands.
STUDY OBJECTS AND METHODS
Preparation of bioassay solutions. The research
featured samples of processed and clarified apple juices
from four producers. The juices were purchased from a
retail chain and marked as A, B, C, and D. The juices
were within the expiration date, with intact packaging.
The juices were diluted with bottled water in ratios 1:5,
1:9, and 1:20. Sorbic acid (Thermo Fisher Scientific,
USA) simulated oxidative stress. Solutions of sorbic
acid (100 and 50 mg/L) included bottled water and
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were prepared in a water-bath by heating to 78°C with
constant stirring.
Bioassay. The bioassay featured peeled onion bulbs
of the same weight (5–7 g) and diameter (≥ 3 cm). The
onions were placed in 2-mL test tubes with bottled
water and left for two or three days, depending on the
experimental conditions, in a thermostat (24 ± 1°C)
in total darkness. After two days of preliminary
germination, the onions with a root length of ≥ 1 cm
were placed in experimental solutions with apple juice,
sorbic acid, or their mix. They were incubated in the
thermostat for the next 24 or 48 h. Bottled water was
used as a negative control. Ten onions were selected
from each group of experimental and control samples.
After preliminary three days of germination and two
days of treatment with solutions of different juices, some
onions were thoroughly washed and then incubated in
bottled water for another 48 h at 25 °C to be tested for
restorative germination. After the experiment, all roots
were cut off, dried with filter paper, and weighed. The
weight gain was determined as the arithmetic mean for
each solution.
Staining and microscopy. A 2% solution of
acetoorcein was used to stain the preparations of onion
apical root cells. The solution included 1 g of orcein
dye per 50 mL of 45% acetic acid. A 70% solution
of ethyl alcohol facilitated the long-term storage in
the refrigerator. The experiment involved the instant
pressure method. A root end of 2–4 mm in length was
cut off from the root and washed in distilled water.
The piece was placed in a drop of 45% acetic acid and
crushed with a glass spatula under a coverslip. The
cells were observed in interphase, prophase, metaphase,
anaphase, and telophase in an Axioskop 40 (Zeiss) light
microscope under 40× magnification (Fig. 1).
Cytogenetic indicators. The mitotic index, % was
calculated by the following formula:
Mitotic index = (1)
The chromosomal aberration analysis revealed
disorganization, adhesion, overlap, lagging, colchicine
mitosis, and a small percentage of bridging and
micronuclei formation (Fig. 2).
For a quantitative description, the index of
chromosome aberrations, % was calculated as follows:
Chromosome aberrations =
The cytogenetic studies revealed on average 10 000
cells per variant.
Concentration of malondialdehyde in the onion
root cells. The lipid peroxidation in root tissues was
determined by the amount of malonic dialdehyde
(MDA) interacting with 2-thiobarbituric acid (MDA
in fresh mass) [18]. During the experiment, 0.2–0.9 g
of onion roots were placed into a polymer 15-cm3
tube (weighing error ± 0.0001 g). After that, 1 cm3 of
trichloroacetic acid (Merck, Germany) with a mass
concentration of 200 g/dm3 was added to the sample.
The mix was stirred and diluted with 3 cm3 of the
same trichloroacetic acid solution. The tubes were
centrifuged for 15 min at 1000×g at 4°C. Then, 1 cm3
of the upper liquid layer was transferred to another tube.
After that, 4 cm3 of a thiobarbituric acid solution (0.5 g
of thiobarbituric acid (Diam, Russia)) was poured into
100 cm3 of trichloroacetic acid solution (200 g/dm3).
The tubes were placed in a 95°C water-bath for 30 min
followed by an ice bath. Next, the tubes were placed in
a centrifuge for 10 min at 1000×g at 20°C. The resulting
solutions were subjected to spectrophotometry in a
Cary WinUV 100 spectrophotometer (Varian, USA) at
wavelengths of 600 and 532 nm.
Statistical analysis. Statistical processing involved
Microsoft Excel 2016 and Statistica 12 software.
The root mass indicator was calculated using the
nonparametric Mann-Whitney test to compare two
means (P ≤ 0 .05). F isher’s t est ( P ≤ 0.05) quantified
the differences in data with a binomial distribution, i.e.
mitotic index and frequency of chromosome aberrations.
RESULTS AND DISCUSSION
The research tested the antioxidant effect of waterdiluted
apple juice on Allium cepa roots after sorbic
acid-induced oxidative stress. Antioxidants of plant
origin could delay or prevent lipid oxidation because
they inhibited the development and accumulation of free
radicals [19]. However, sorbic acid is known to trigger
the dose-dependent development of oxidative stress
and increase the malonic dialdehyde (MDA) content in
root tissues [20]. Concentrated solutions of apple juice
activated lipid oxidation during the A. cepa test [7].
Therefore, the initial task was to select the optimal
concentrations of sorbic acid and juice to obtain the
maximal antioxidant effect. The onion samples spent
48 h incubating in solutions of sorbic acid and apple
juice: 100 mg/L of sorbic acid was diluted with brand A
apple juice as 1:2, 1:5, and 1:9. After the incubation, the
Figure 1 Mitosis phases, from left to right: prophase, metaphase, anaphase, and telophase
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Figure 2 Chromosome aberrations: a) lagging in telophase; b) detachment in metaphase; c) lagging in anaphase; d), e), and
i) disorganization in metaphase; f) multipolar mitosis and disorganization in metaphase; g) disorganization in metaphase;
h) detachment in metaphase; j) k-mitosis
* statistically significant difference from control (P < 0.05); error bars determine the value of the standard deviation
Figure 3 Decrease in weight gain of onion roots after treatment with brand A juice and sorbic acid (100 mg/L)
0 20 100 120
Juice 1:2 + sorbic acid
Juice 1:5 + sorbic acid
Juice 1:9 + sorbic acid
Control
Pre-test control
40 60 80
Root weight gain, % of control
0 20 100 120
Juice 1:2 + sorbic acid
Juice 1:5 + sorbic acid
Juice 1:9 + sorbic acid
Control
Pre-test control
40 60 80
Root weight gain, % of control
0 20 100 120
Juice 1:2 + sorbic acid
Juice 1:5 + sorbic acid
Juice 1:9 + sorbic acid
Control
Pre-test control
40 60 80
Root weight gain, % of control
Figure 4 MDA in the roots treated brand A apple juice and sorbic acid (SA, 50 mg/L)
0
5
10
15
20
25
30
35
Control Juice
1:20+SA
Juice 1:9+SA Juice 1:5+SA Juice 1:9 SA
MDA concentration,
μmoL/g wet weight
0
5
10
15
20
25
30
35
Control Juice
1:20+SA
Juice 1:9+SA Juice 1:5+SA Juice 1:9 SA
MDA concentration,
μmoL/g wet weight
0
5
10
15
20
25
30
35
Control Juice
1:20+SA
Juice 1:9+SA Juice 1:5+SA Juice 1:9 SA
MDA concentration,
μmoL/g wet weight
a
e
i
b c d
f g h
j
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mass of the roots remained the same. In fact, they turned
yellow and mucous, which meant that the doses had an
acute toxic effect (Fig. 3).
In the next experiment, the treatment time and the
acid concentration were halved, and the juice samples
were diluted as 1:20, 1:9, and 1:5. Figure 4 shows that the
1:9 juice solution provided the maximal protective effect
under oxidative stress caused by a 50 mg/mL solution
of sorbic acid. In these samples, the level of MDA
was lower by 43% than in the samples with the same
concentration of sorbic acid.
The obtained data confirmed the results described in
[21], where apple juice in rats’ diet decreased the level
of MDA in their blood plasma. The phenolic compounds
and dietary fiber of apple juice proved to reduce the lipid
oxidation in humans as well [1, 22, 23].
The dose-dependent decrease in MDA was revealed
only in the first two, more diluted juice solutions
(Fig. 4). In 1:5 juice samples, this indicator increased
again. This effect was associated with carbohydrates,
which are known to have prooxidant properties
at this concentration during the A. cepa test [7].
Therefore, these data also confirmed that the maximal
antioxidant activity of apple juice depended not only
on its biologically active compounds, but also on the
concentration of carbohydrates.
Some recent research featured the effect of fructose
on the redox balance in the organs of the central
nervous system. Rat studies revealed an increase in
lipid oxidation of brain tissues after both short-term and
long-term intake of this carbohydrate [24]. These animal
models showed the same results as the abovementioned
plant bioassays for the prooxidant properties of apple
juice carbohydrates. Therefore, the A. cepa test proved
to be a reliable research method for the molecular
mechanisms of antioxidant and prooxidant properties of
apple juice.
Growth indicators demonstrated no significant
differences after the onions were treated with solutions
of juice and sorbic acid (Table 1). However, previous
research revealed that the increase in juice concentration
had an adverse effect on onion root cell proliferation
[7]. However, the decrease in the mitotic index against
the increase in the juice proportion was not dosedependent
(Table 1). Both juice concentrations, 1:20 and
1:9, had the same values of this indicator. Probably, the
maximal antioxidant status of the samples diluted 1:9
had protected the proliferative processes by reducing the
effects of oxidative stress.
Similar conclusions were reported in a publication
about the effect of antioxidants on bisphenolinduced
oxidative stress in mouse spermatozoa [25].
Antioxidants preserved the motility of these germ
cells, improved the fertilization process, and prevented
premature development of the resulting fetus.
The low values of the mitotic index meant a low
proportion of dividing cells in the experimental samples
with mixes of juice and sorbic acid (Table 1). Therefore,
no comparative analysis of chromosomal aberrations
was necessary.
The next stage featured the antioxidant potential of
various juice brands diluted 1:9 after 24 h of sorbic acidinduced
oxidative stress. Juice brands A, B, and C in
the mix reduced the level of MDA by 23, 26, and 26%,
respectively (Fig. 2). Juice brands B and C also revealed
some antioxidant activity; however, the differences
between the experimental samples in MDA values were
insignificant (3%). In the experimental mixes, the root
masses were very similar and minimal, while the values
of the mitotic index showed some statistically significant
differences (Table 2).
Phenolic compounds are mainly to be found in apple
peel and pulp cell walls [1, 26]. Therefore, the processed
and clarified juices had some residual differences
in antioxidant activity in relation to lipid oxidation.
Nevertheless, the bioassay was able to register a rather
high antioxidant activity even in these non-pulp juices.
The similar MDA values could also be explained by
the absence of the pulp as the main source of phenolic
compounds.
The results indicated an acute toxic effect (Fig. 3)
and an increase in the levelЦ of lipid oxidation (Fig. 5)
in the mixes of various juices and sorbic acid at
concentrations of 100 and 50 mg/L, respectively.
For some juice-containing drinks, domestic regulatory
documents state much greater permissible
concentrations of this preservative, ≥ 1 g/kg. Therefore,
sorbic acid can reduce the initial antioxidant potential
of these products, but not the content of phytochemical
compounds. These data are important if the production
Table 1 Root weight gain, mitotic activity, and frequency of chromosome aberrations in onion root meristem cells after incubation
in solutions of brand A juice, sorbic acid (SA), and their mixes
*SE – standard error, ** – values marked by the same letter have no significant statistic difference (P < 0.05)
Experiment Root weight gain, g/onion,
mean ± SE*
Mitotic index, %,
mean ± SE
Chromosome aberrations
per total cells, %, mean ± SE
Control 0.296 ± 0.048a** 8.70 ± 0.24a 0.26 ± 0.04a
Juice 1:20 + SA, 50 mg/L 0.189 ± 0.034ab 1.08 ± 0.11b 0.02 ± 0.02b
Juice 1:9 + SA, 50 mg/L 0.162 ± 0.029bc 1.01 ± 0.08b 0.08 ± 0.02c
Juice 1:5 + SA, 50 mg/L 0.138 ± 0.032bcd 0.40 ± 0.06c 0.04 ± 0.02bc
Juice 1:9 0.109 ± 0.012bcd 1.40 ± 0.09d 0.21 ± 0.03ad
SA, 50 mg/L 0.243 ± 0.021ab 5.86 ± 0.21e 0.18 ± 0.04d
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technology provides for this preservative. However, only
bioassay can determine how these effects interact.
Our experiments on the toxic potential of different
juice brands were aimed at a comparative assessment
of their side effects on the growth and the cytological,
cytogenetic, and biochemical parameters of onion
roots. We found no scientific publications that featured
the A. cepa test as a means of researching the toxic
0
5
10
15
20
25
30
35
40
Control SA Ju ice A J uice B Juice C Juice A* Juice B* Juice C*
MDA μmoL/g wet weight
0
5
10
15
20
25
30
35
40
Control SA Ju ice A J uice B Juice C Juice A* Juice B* Juice C*
MDA concentration, μmoL/g wet weight
0
5
10
15
20
25
30
35
40
Control SA Ju ice A J uice B Juice C Juice A* Juice B* Juice C*
MDA concentration, μmoL/g wet weight
Figure 5 MDA in roots treated with various apple juice brands
Note: Vertical error bars indicate the value of the standard deviation; * marks the incubation experiments with mixes of juices and sorbic acid
(SA, 50 mg/L)
Table 2 Root weight gain, mitotic activity, and frequency of chromosome aberrations in onion root meristem cells after incubation
in solutions of juices and their mixes with sorbic acid (SA)
* SE – standard error, ** – values marked by the same letter have no significant statistic difference (P < 0.05)
Table 3 Root weight gain, mitotic activity, and frequency of chromosome aberrations in onion root meristem cells before and after
restorative germination in juice solutions
effect of apple juice. The main task was to obtain data
on possible irreversible violations of these processes.
In case of complete or partial irreversibility after the
juice treatment, the detoxification systems of the plant
organism failed to cope with the load, and these negative
phenomena might progress in the future.
The previous experiments had a high toxic load
because of sorbic acid (Table 2). In this experiment,
Experiment Root weight gain, g/onion,
mean ± SE*
Mitotic index, %, mean ± SE Chromosome aberrations per
total cells, %, mean ± SE
Control 0.236 ± 0.030a** 8.75 ± 0.24a 0.29 ± 0.05a
SA, 50 mg/L 0.211 ± 0.034ab 5.17 ± 0.19b 0.18 ± 0.04b
Juice А 0.139 ± 0.016bc 2.55 ± 0.13c 0.05 ± 0.02c
Juice B 0.146 ± 0.016bcd 1.10 ± 0.09d 0.11 ± 0.03b
Juice C 0.184 ± 0.024abcde 0.99 ± 0.08d 0.01 ± 0.01d
Juice А +SA, 50 mg/L ЦЦ0.155 ± 0.021bcdef 0.48 ± 0.05e 0.020 ± 0.001cd
Juice B + SA, 50 mg/L 0.143 ± 0.030bcdefg 0.58 ± 0.07ef 0.02 ± 0.01e
Juice C + SA, 50 mg/L 0.171 ± 0.012abcdefg 0.56 ± 0.07ef 0.04 ± 0.02e
Experiment Root weight gain, g/onion,
mean ± SE*
Mitotic index, %, mean ± SE Chromosome aberrations per total cells, %,
mean ± SE
Before restorative germination
Control 0.799 ± 0.089a** 8.52 ± 0.27a 0.02 ± 0.01a
Juice А 0.561 ± 0.056ab 3.06 ± 0.19b 0.12 ± 0.04b
Juice C 0.540 ± 0.048bс 3.64 ± 0.18c 0.08 ± 0.03bc
Juice D 0.597 ± 0.060abc 3.95 ± 0.20c 0.15 ± 0.04abc
After restorative germination
Control 1.060 ± 0.082a 7.82 ± 0.28a 0.16 ± 0.04a
Juice А 0.791 ± 0.088ab 7.99 ± 0.25ab 0.43 ± 0.06b
Juice C 0.827 ± 0.094abc 8.08 ± 0.27ab 0.99 ± 0.10c
Juice D 0.944 ± 0.095abc 6.93 ± 0.25c 0.41 ± 0.06b
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Figure 6 shows the chromosome aberrations found in
the apical meristem of the onion roots after incubation
in juice solutions, as well as after incubation and
subsequent regeneration in bottled water. No statistically
significant differences (P ≤ 0.05) in chromosomal
disorders were revealed before or after restorative
germination in bottled water. However, juice A samples
demonstrated all kinds of aberrations after restorative
germination.
Thus, all the experimental samples revealed
irreversible significant genotoxic effects (Table 3),
represented mostly by chromosome disorganization
0
10
20
30
40
50
60
70
80
90
100
Control Juice A Juice C Juice D
After incubation in juice solutions
Control Juice A Juice C Juice D
% of total abberations revealed
1 2 3 4
After restorative germination
0
2
4
6
8
10
12
14
16
18
Control Juice A Juice C Juice D Control* Juice A* Juice C* Juice D*
MDA concentration, μmoL/g wet weight
Figure 6 Chromosomal aberrations in meristem cells of onion roots before and after restorative germination in juice solutions: 1)
disorders of chromosome segregation (overlap, lag); 2) anomalies of mitotic apparatus (adhesion, multipolar mitosis); 3) aberrations
of clastogenic character (bridges, fragments); 4) miscellaneous (fragmentation, agglutination, pulverization)
Note: Vertical error bars indicate the value of the standard deviation, * – marks incubation in acid solutions followed by germination in bottled
water
Figure 7 MDA in roots treated with aqueous solutions of juices
the treatment time with juice solutions reached 48 h.
After restorative germination, the average weight of
the roots was by 11–25% lower than in the control
samples, but this difference was not significant (Table 3).
Mitotic index had the same trend, except for the brand
D juice samples, although this indicator differed from
the control by only 11%. However, the cytogenetic
analysis showed a significant increase in chromosomal
aberrations in all the experimental groups, while the
maximal growth by more than six times was recorded in
the brand C juice samples.
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in metaphase, lagging in anaphase, detachment
in metaphase, and lagging in telophase (Fig. 6).
Disorganization of chromosomes in metaphase, for
instance, was a typical irreversible side effect of benzoic
acid on onion roots [20].
After restorative germination, MDA content was
higher in all the experimental variants by 21–51%
compared with the control values (Fig. 7). This
indicator also demonstrated the irreversible nature of
the identified adverse effects after exposure to juice
solutions.
Thus, the maximal negative effects after restorative
germination were recorded when analyzing the values
of the mitotic index and MDA in the D juice samples
and the level of chromosome aberrations in the C juice
samples. If the first two indicators differed from the
control only by tens of percent, the latter differed by
several times in all the experimental variants. In the
juice C samples, the level of cytogenetic disorders was
two times higher compared to samples A and D. This
biomarker requires more attention when assessing the
genotoxic potential of this product, both phytochemical
and technological.
CONCLUSION
The research featured a new bioassay method for
determining the antioxidant potential of processed
apple juice. The juice reduced the lipid oxidation in
onion roots to 40% after oxidative stress induced by
sorbic acid. The antioxidant potential in juice solutions
depended on the ratio of biologically active compounds
and carbohydrates.
The research included a comparative analysis of
three juice brands. Sorbic acid had a possible negative
effect on the quality of juice-containing products: even
50 mg/L reduced the antioxidant profile of the finished
product. When the concentration of sorbic acid reached
100 mg/L, its effect became toxic, and onion roots died.
No side toxic subchronic effects on the weight gain were
registered after onion roots were treated with three juice
brands. However, one of the three juices demonstrated
an irreversible decrease in the proliferative index by
11%.
The cytogenetic analysis of the root meristem
revealed the maximal adverse side effect: chromosomal
aberrations increased in all experimental groups. For
one brand, these disorders increased by more than six
times. In general, the Allium cepa bioassay of toxic
subchronic effects provided reliable results for side
effects in apple juice production.
CONTRIBUTION
The authors were equally involved in writing the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interests regarding the publication of this article.
1. Boyer J, Liu R. Apple phytochemicals and their health benefits. Nutrition Journal. 2004;3. https://doi.org/10.1186/1475-2891-3-5.
2. Yong W, Amin L, Dongpo C. Status and prospects of nutritional cooking. Food Quality and Safety. 2019;3(3):137-143. https://doi.org/10.1093/fqsafe/fyz019.
3. Van der Sluis AA, Dekker M, Skrede G, Jongen WMF. Activity and concentration of polyphenolic antioxidants in apple juice. 1. Effect of existing production methods. Journal of Agricultural and Food Chemistry. 2002;50(25):7211-7219. https://doi.org/10.1021/jf020115h.
4. Murtaza A, Iqbal A, Marszałek K, Iqbal MA, Ali SW, Xu X, et al. Enzymatic, phyto-, and physicochemical evaluation of apple juice under high-pressure carbon dioxide and thermal processing. Foods. 2020;9(2). https://doi.org/10.3390/foods9020243.
5. Khodos MYa, Kazakov YaE, Vidrevich MB, Brainina KhZ. Monitoring of oxidative stress in biological objects. Journal of Ural Medical Academic Science. 2017;14(3);262-274. (In Russ.).
6. Moon J-K, Shibamoto T. Antioxidant assays for plant and food components. Journal of Agricultural and Food Chemistry. 2009;57(5):1655-1666. https://doi.org/10.1021/jf803537k
7. Samoylov AV, Suraeva NM, Zaytseva MV, Rachkova VP, Kurbanova MN, Belozerov GA. Toxicity of apple juice and its components in the model plant system. Foods and Raw Materials. 2020;8(2):321-328. https://doi.org/10.21603/2308-4057-2020-2-321-328.
8. Samoylov AV, Suraeva NM. Modern trends in the assessment of food safety. Vsyo o Myase. 2021;(2):32-36. (In Russ.). https://doi.org/10.21323/2071-2499-2021-2-32-36.
9. Durnev AD, Oreshchenko AV. Pishcha: mutagenez i antimutagenez [Food: mutagenesis and anti-mutagenesis]. Storage and Processing of Farm Products. 1996;(3):14-18. (In Russ.).
10. Farvid MS, Chen WY, Michels KB, Cho E, Willett WC, Eliassen AH. Fruit and vegetable consumption in adolescence and early adulthood and risk of breast cancer: population based cohort study. BMJ. 2016;353. https://doi.org/10.1136/bmj.i2343.
11. Büchner FL, Bueno-de-Mesquita HB, Linseisen J, Boshuizen HC, Kiemeney LALM, Ros MM, et al. Fruits and vegetables consumption and the risk of histological subtypes of lung cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC). Cancer Causes and Control. 2010;21(3):357-371. https://doi.org/10.1007/s10552-009-9468-y.
12. Rietjens IM, Martena MJ, Boersma MG, Spiegelenberg W, Alink GM. Molecular mechanisms of toxicity of important food-borne phytotoxins. Molecular Nutrition and Food Research. 2005;49(2):131-158. https://doi.org/10.1002/mnfr.200400078.
13. DeChristopher LR, Uribarri J, Tucker KL. Intakes of apple juice, fruit drinks and soda are associated with prevalent asthma in US children aged 2-9 years. Public Health Nutrition. 2016;19(1):123-130. https://doi.org/10.1017/S1368980015000865.
14. Hallfrisch J, Ellwood K, Michaelis OE, Reiser S, Prather ES. Plasma fructose, uric acid, and inorganic phosphorus responses of hyperinsulinemic men fed fructose. Journal of the American College of Nutrition. 1986;5(1):61-68. https://doi.org/10.1080/07315724.1986.10720113.
15. Dragsted LO, Daneshvar B, Vogel U, Autrup HN, Wallin H, Risom L, et al. A sucrose-rich diet induces mutations in the rat colon. Cancer Research. 2002;62(15):4339-4345.
16. Silva de Sá I, Peron AP, Leimann FV, Bressan GN, Krum BN, Fachinetto R, et al. In vitro and in vivo evaluation of enzymatic and antioxidant activity, cytotoxicity and genotoxicity of curcumin-loaded solid dispersions. Food and Chemical Toxicology. 2019;125:29-37. https://doi.org/10.1016/j.fct.2018.12.037.
17. Samoilov AV, Suraeva NM. Prospects for the use of plant biotesting to search for metabolic biomarkers of the toxic potential of components of food matrices (review). Achievements of Science and Technology in Agro-Industrial Complex. 2021;35(4):65-71. (In Russ.). https://doi.org/10.24411/0235-2451-2021-10411.
18. Zhang H, Jiang Y, He Z, Ma M. Cadmium accumulation and oxidative burst in garlic (Allium sativum). Journal of Plant Physiology. 2005;162(9):977-984. https://doi.org/10.1016/j.jplph.2004.10.001.
19. Ciftci D, Ozilgen S. Evaluation of kinetic parameters in prevention of quality loss in stored almond pastes with added natural antioxidant. Journal of Food Science and Technology. 2019;56(1):483-490. https://doi.org/10.1007/s13197-018-3510-6.
20. Samoylov AV, Suraeva NM, Zaytseva MV, Rachkova VP, Kurbanova MN, Petrov AN. Comparative assessment of sorbic and benzoic acid via express biotest. Foods and Raw Materials. 2020;8(1):125-133. https://doi.org/10.21603/2308-4057-2020-1-125-133.
21. Breinholt VM, Nielsen SE, Knuthsen P, Lauridsen ST, Daneshvar B, Sorensen A. Effects of commonly consumed fruit juices and carbohydrates on redox status and anticancer biomarkers in female rats. Nutrition and Cancer. 2003;45(1):46-52. https://doi.org/10.1207/S15327914NC4501_6.
22. Meccariello R, D'Angelo, S. Impact of polyphenolic-food on longevity: An elixir of life. An overview. Antioxidants. 2021;10(4). https://doi.org/10.3390/antiox10040507.
23. Dhalaria R, Verma R, Kumar D, Puri S, Tapwal A, Kumar V, et al. Bioactive compounds of edible fruits with their anti-aging properties: a comprehensive review to prolong human life. Antioxidants. 2020;9(11). https://doi.org/10.3390/antiox9111123.
24. Spagnuolo MS, Iossa S, Cigliano L. Sweet but bitter: Focus on fructose impact on brain function in rodent models. Nutrients. 2020;13(1). https://doi.org/10.3390/nu13010001.
25. Rahman MS, Kang K-H, Arifuzzaman S, Pang W-K, Ryu D-Y, Song W-H, et al. Effect of antioxidants on BPA-induced stress on sperm function in a mouse model. Scientific Reports. 2019;9(1). https://doi.org/10.1038/s41598-019-47158-9.
26. Fierascu RC, Fierascu I, Avramescu SM, Sieniawska E. Recovery of natural antioxidants from agro-industrial side streams through advanced extraction techniques. Molecules. 2019;24(23). https://doi.org/10.3390/molecules24234212.