ISSN 2308-4057 (Print),
ISSN 2310-9599 (Online)

Bioassay of oxidative properties and toxic side effects of apple juice

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.
Ключевые слова
Apple juice , bioassay , antioxidant activity , side effects , Allium cepa test , biologically active substances

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, 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), 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/ 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.


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 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 recovery treatment. 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:

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:

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.


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Как цитировать?
Samoylov A.V., Suraeva N.M., Zaytseva M.V., Petrov A.N. Bioassay of oxidative properties and toxic side effects of apple juice. Foods and Raw Materials, 2022, вып. 10, том. 1, стр. 176-184
Кемеровский государственный университет
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2310-9599 (Online)
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