CC BY
39
Hepatoprotective effect of breads with extracts of plants growing in the Far East
Elena S. Smertina* , Lyudmila N. Fedyanina , Vladimir A. Lyakh
Far Eastern Federal University, Vladivostok, Russia * e-mail: [email protected] Received December 07, 2018; Accepted in revised form March 14, 2019; Published May 12, 2020
Abstract:
Breads with proven hepatoprotective properties can make a significant contribution to preventing liver disease. This work aimed to study hepatoprotective and antioxidant effects of breads enriched with water and ethanol extracts of polyphenol-containing viburnum (Viburnum sargentii Koehne L.), magnolia-vine (Schisandra chinensis L.), and grapes (Vitis amurensis L.). It was based on an experimental model of toxic hepatitis in mice intoxicated with carbon tetrachloride. Experimental groups of animals were fed on bread with extracts for 7 days and control groups had a bread-free diet. We analysed their body weight, liver lipid metabolism, "lipid peroxidation - antioxidant protection" system, and antiradical activity. The level of reduced glutathione and malonic dialdehyde was determined by micro-thin-layer chromatography. Superoxide dismutase, glutathione reductase, and glutathione peroxidase activity was measured to analyse the antioxidant system. The total content of common polyphenols in breads was determined by the colorimetric method with the Folin-Chocalteu reagent. The animals on a bread-free diet showed an impaired lipid metabolism and higher activity of liver enzymes. They had a 22% increase in liver weight and a 1.9 times depletion of antiradical protection (6.65 ± 0.15 Trolox units/mg protein vs. 13.15 ± 0.21 Trolox units/mg protein in the control; P < 0.001). We also registered a 2.5 times decrease in superoxide dismutase, a key enzyme of the antioxidant defence system. The animals fed on breads with the above extracts showed a statistically significant normalization of the parameters, compared to the bread-free group. We found that those breads had hepatoprotective and antioxidant effects on the animals, stabilizing their general condition and normalizing their biochemical parameters and antioxidant system.
Keywords: Bread, plant extracts, viburnum, magnolia-vine, grapes, polyphenols, antioxidant, hepatoprotector
Please cite this article in press as: Smertina ES, Fedyanina LN, Lyakh VA. Hepatoprotective effect of breads with extracts of plants growing in the Far East. Foods and Raw Materials. 2020;8(2):232-240. DOI: http://doi.org/10.21603/2308-4057-2020-2-232-240.
E-ISSN 2310-9599 ISSN 2308-4057
Research Article Open Access
^ Check for updates
DOI: http://doi.org/10.21603/2308-4057-2020-2-232-240 Available online at http://jfrm.ru/en
INTRODUCTION
Current environmental problems are increasingly exposing the world population to various stress factors such as foreign substances (xenobiotics). The liver is commonly known as the main barrier in the human body that neutralizes xenobiotics and toxic substances. Thus, preventing liver disease is highly important.
According to modern studies, free radical reactions play a significant role in the development of pathology during toxic liver damage. Reactive oxygen species activate the process of lipid peroxidation in hepatocyte cell membranes, causing liver dysfunction. Inhibiting such processes with various biologically active substances (BASs), including natural antioxidants, can have a preventative or therapeutic effect.
Foods with proven hepatoprotective and antioxidant properties can make a significant contribution to solving
this problem. Today, bread is a basic food product, and breadmaking is a socially important industry [1]. Bread is part of a daily diet for the vast majority of consumers in many countries. Thus, it can serve as a basis for preventative and dietetic specialized products that can contribute to the prevention of various liver diseases [2].
Introducing plant polyphenols into the product formulation is one of the ways to develop breads with antioxidant and hepatoprotective properties. Characterized by high antioxidant activity, plant polyphenols are a source of effective hepatoprotectors. They are able to eliminate increased lipid peroxidation during toxic hepatitis and improve the antitoxic function of liver cells [3].
In contrast to synthesized polyphenols, natural polyphenols have an extremely low toxicity and do not cause adverse reactions (allergies, addiction, or
Copyright © 2020, Smertina et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
accumulation). According to studies, plant phenolic compounds display a wide range of biological (including hepatoprotective) effects. This is due to their antioxidant properties and an ability to act as traps for free radicals of various types [4, 5].
Being plant secondary metabolites, phenolic compounds are contained in traditional plant products used by humans (fruits, vegetables, berries, etc.). Thus, they are evolutionarily adapted for the human body, form complexes with transition metal ions and block lipid peroxidation processes [6-8].
However, of all the plant materials studied (plants, fruits, vegetables, cereals, seeds, bark, etc.), only 10% are sufficiently rich in polyphenolic compounds and can be used as effective natural antioxidants [9, 10]. Both foreign and Russian studies have revealed a need for enriching breads with plant polyphenols to ensure their antioxidant and hepatoprotective properties. For this, we can use such innovative ingredients as berry and fruit extracts, pectin-rich fruits, blueberry paste, and bioflavonoids from larch, vegetables (onions, beets), or ground coffee [17-19].
We evaluated sensory and physicochemical properties of breads containing various concentrations of polyphenols. The bread showed an increased antioxidant activity. Preclinical studies in animals with various diseases are vital in justifying the choice of new ingredients, their efficacy and biological effects in foods with preventative properties. However, scientific literature lacks data on experimental studies in animals that confirm the preventive efficacy of hepatoprotective ingredients in breads. This determined the aim of our study.
The Far East, particularly the Ussuri taiga with its wild plants, is a valuable source of plant raw materials rich in various BASs and used in medicine, pharmacology, animal husbandry, and food industry. BASs are often used as physiologically functional ingredients for dietetic (medicinal and preventative) foods [20].
People have to consume enormous quantities of plants to obtain the required amount of macro- and microelements, as well as other BASs, which is often impossible for many reasons. In this regard, food products are enriched with individual components of plant materials and their extracts that have high biological activity.
Water and ethanol extracts of plant materials are concentrates of biologically active substances. They are effective in small amounts and therefore can be added to products in amounts similar to dried berries [21]. Low contents of biologically active physiologically functional ingredients ensure the preservation of traditional sensory characteristics in the products, which is an important factor for the consumer. Baking technology (with temperatures up to 200-220°C) can neutralize the alcohol component of water and ethanol extracts
and their specific sensory properties. This makes such extracts a promising ingredient for breads with dietetic and preventative properties.
For this study, we developed breads with three biologically active dietary supplements (BADSs): Caliphene, Eklikit, and Diprim. They are water and ethanol extracts developed at V.I. Ilyichov Pacific Oceanological Institute, Russia.
Caliphene was obtained from viburnum (Viburnum sargentii Koehne L.) processing waste with proven membrane- and hepatoprotective, antioxidant, antiradical, and other properties. The content of common polyphenols in it was 32.8 ± 2.4 g/L.
Eklikit was extracted from magnolia-vine (Schisandra chinensis L.) processing waste. Its polyphenol complex included proanthocyanidins, leucoanthocyanins, catechins, flavonols, organic acids, free amino acids, etc. The content of common polyphenols was 14.4 ± 1.7 g/L.
Diprim was made of grape (Vitis amurensis L.) stalks. Its main component was such polyphenolic compounds as catechins and their polymer forms, leucoanthocyanins, flavonoids, procyanidins, oligomeric tannins, and lignin. The content of common polyphenols was 35.4 ± 2 g/L [20].
Biomedical studies have found a multifactorial positive effect of Caliphene, Eklikit, and Diprim on the human body, both when used as a dietary supplement and as part of foods, including breads [3, 22-25].
The quality and safety evaluation of breads enriched with water and ethanol extracts showed their compliance with the regulatory documents of the Russian Federation and the Eurasian Economic Union [26, 27].
The preclinical tests of extract-containing breads in animals using standard pharmaceutical models demonstrated their stress-protective (or adaptogenic) and actoprotective effects [28].
We believe that hepatoprotective and antioxidant properties of breads containing water and ethanol extracts are determined by the extract chemical composition as reported in [29, 30], a significant content of polyphenols, and its stability in the products.
Our experimental study in animals with toxic hepatitis aimed to evaluate the hepatoprotective and antioxidant effects of breads enriched with polyphenol-containing functional ingredients, namely viburnum, magnolia-vine, and grapes water and ethanol extracts (hereinafter referred to as "extracts").
STUDY OBJECTS AND METHODS
The objects of our experimental studies were breads produced by the traditional method from wheat flour with the addition of water and ethanol extracts of viburnum, magnolia-vine, and grapes [31].
To determine the potential efficacy of introducing Caliphene, Eklikit, and Diprim, the total content of common polyphenols was measured by the colorimetric
method with the Folin-Chocalteu reagent using gallic acid as a standard [32].
We applied a standard experimental model of toxic hepatitis in mice through their intoxication with carbon tetrachloride, one of the strongest stimulants of lipid peroxidation. Carbon tetrachloride is an organ-specific toxin with a hepatotropic effect. Its toxicity is primarily associated with the prooxidant effect of free radicals that form during its metabolism, trichloromethyl and trichloromethyl peroxyl. These radicals initiate lipid peroxidation followed by a chain reaction of free radical oxidation, leading to profound disruption of the functional properties of liver cell membranes, their lysis, and death'.
The experiments were performed on white mice bred in the Pacific Institute of Bioorganic Chemistry, Russia.
For the experimental model of toxic liver damage by carbon tetrachloride, we used adult male mice with an average weight of 25.00 ± 1.56 g. The animals were kept in standard vivarium conditions in compliance with all the rules and recommendations of the European Convention for the Protection of Vertebrate Animals (Strasbourg, 1986).
The experimental model of carbon tetrachloride intoxication (toxic hepatitis) in animals followed the methodological guidelines for studying hepatoprotective activity of pharmacological substances [33, 34].
1.25 mL/kg of carbon tetrachloride (50% solution in olive oil) was injected into the dorsal nuchal fold of mice for 4 days. After that, the mice were given portions of breads containing Caliphene, Eklikit, and Diprim with a total complex of common polyphenols for 7 days. A grid with individual cells was placed in the cage to feed the animals and removed after feeding.
The study used three control groups and three experimental groups, 18 mice in each.
The control groups included:
- group 1: intact mice fed on the normal diet;
- group 2: mice with toxic hepatitis fed on the normal diet; and
- group 3: mice with toxic hepatitis 7 days after the toxicant withdrawal.
The experimental groups included:
- group 4: mice with toxic hepatitis fed on Diprim-containing bread for 7 days after the toxicant withdrawal;
- group 5: mice with toxic hepatitis fed on Caliphene-containing bread for 7 days after the toxicant withdrawal; and
- group 6: mice with toxic hepatitis fed on Eklikit-containing bread for 7 days after the toxicant withdrawal.
The biomaterial was obtained as follows. Blood samples were taken from the cervical vein of mice in all the groups and collected in test tubes with heparin to
: MUK 2.3.2.721-98 Opredelenie bezopasnosti i ehffektivnosti biologicheski aktivnykh dobavok k pishche [Guidelines for establishing safety and efficacy of dietary supplements]. Moscow: Ministry of Health of the Russian Federation; 1999. 89 p.
measure antioxidant protection. Mice blood serum was used to study lipid metabolism and some biochemical parameters. Mice livers were extracted, washed in a physiological solution, and used to evaluate weight and biochemical parameters of mice.
The antioxidant system was assessed by measuring the activity of superoxide dismutase, glutathione reductase, and glutathione peroxidase, the antiradical activity of blood, as well as the level of reduced glutathione and malondialdehyde [35-37].
The "lipid peroxidation - antioxidant protection" system was studied with biochemical methods. Common lipids were extracted from liver tissue and prepared according to Folch et al. [38]. Micro-thin-layer chromatography on silica gel was used to measure the quantity of phospholipid and neutral lipid fractions. The Russian KSK silica gel was used as a sorbent.
The chromatographic distribution of neutral lipids was performed by one-dimensional thin-layer chromatography on silica gel in a solvent system of "hexane:sulfuric ether:acetic acid" in a ratio of 90:10:1 (v/v/v). Lipid stains were identified using purified preparations of Russian origin. Neutral lipid fractions were quantified according to Amenta [40]. After chromatography, standards and samples were detected with iodine vapours.
The fractions (triacylglycerols, cholesterol, free fatty acids, cholesterol esters, and fatty acid esters) were transferred from the plates to a tube with a special spatula. The tubes were filled with 2 mL of bichromate reagent and heated on a boiling water bath for 15 min. After the samples cooled, 4 mL of distilled water was added to them. They were then stirred and centrifuged at 3000 rpm for 10 min. Optical density was measured on a spectrophotometer at a wavelength of 440 nm. The results were expressed as a percentage of the sum of all fractions.
The results were processed with Instat 3.0 (GraphPad Software Inc. USA, 2005). The parametric Student's t-test or the non-parametric Mann-Whitney U-test were used to determine the statistical significance of the differences depending on the distribution parameters. The differences were considered statistically significant at P < 0.05.
RESULTS AND DISCUSSION
As shown in Table 1, the content of polyphenols in, and the antiradical activity of, the breads containing Diprim, Caliphene, and Eklikit extracts are statistically significant and higher than in the control sample (bread without additives). The antiradical activity of breads with plant extracts is determined by polyphenolic structures.
The data confirmed the efficacy of adding Diprim, Caliphene, and Eklikit as functional ingredients to breads with preventative properties in preclinical studies.
Table 1 Antiradical activity and common polyphenols in the breads (M ± m)
Name of product Antiradical Common
activity, ^mol polyphenols,
trolox/g product mg/g product
Bread (control) 0.78 ± 0.013 120 ± 1.8
Bread + Diprim 0.97 ± 0.0123 388 ± 2.43
Bread + Caliphene 0.95 ± 0.0183 385 ± 2.63
Bread + Eklikit 0.94 ± 0.0153 384 ± 2.53
Table 2 Weight changes in carbon tetrachloride intoxicated mice and their correction with extract-containing breads (M ± m)
Mice groups
Mice weight, g
Mice liver weight, g
Group 1 control 25.40 ± 0.88 1.99 ± 0.06
Group 2 carbon tetrachloride 19.43 ± 0.573 2.55 ± 0.123
Group 3 deprivation (toxicant 21.52 ± 0.951 2.39 ± 0.083 withdrawal)
Group 4 deprivation + bread 25.87 ± 0.72c 2.09 ± 0.06b with Caliphene
Group 5 deprivation + bread 25.29 ± 0.89c 2.06 ± 0.08b with Diprim
Group 6 deprivation + bread 25.18 ± 0.98c 2.07 ± 0.09b
with Eklikit_
The differences are statistically significant at: 1 - P < 0.05; 2 - P < 0.01; 3 - P < 0.001 vs. control; a - P < 0.05; b - P < 0.01; c - P < 0.001 vs. group 3
Four days of carbon tetrachloride injections resulted in the mice developing toxic hepatitis with its characteristic symptoms. Their hair became dull and stuck together. They were apathetic and had a poor appetite. Their liver metabolic reactions were impaired, manifesting in:
- weakened antioxidant defences;
- activated lipid peroxidation;
- poor membrane permeability;
- imbalanced lipid metabolism;
- intensified lipolytic processes; and
- poor cholesterol metabolism (esterifying liver function).
Tables 2-4 present the indicators of metabolic processes in the control groups of mice (intact and mice with toxic hepatitis), compared with those in the experimental groups of mice (fed on breads with Diprim, Caliphene, and Eklikit extracts after carbon tetrachloride withdrawal).
After four days of carbon tetrachloride injections, the weight of the mice decreased to 19.43 ± 0.57 g, which was 24% (P < 0.001) lower than that of the intact control mice (25.40 ± 0.88 g). This confirmed the development of toxic hepatitis (Table 2).
We found a statistically significant normalization of the parameters, starting from day 4, in the mice fed on breads with plant extracts after carbon tetrachloride intoxication (groups 4-6, compared with the control). Their weight returned to the control values; their hair became smooth and fluffy; they began to eat well and move actively. Their liver weight was higher than that in the control group, but slightly lower than in the animals with toxic hepatitis. This decrease in liver weight of the animals fed on breads with Diprim, Caliphene, and Eklikit indicates the stability of the extracts and their hepatoprotective role in the product.
The development of toxic hepatitis in the control mice intoxicated with carbon tetrachloride was manifested in both the impaired biochemical parameters of their blood, indicative of free-radical processes in the body, and lipid metabolism in their livers. The activity of alanine aminotransferase (ALT), an enzyme marker of liver damage, in the blood serum of mice in this group, was almost 20 times as high (34.2 ± 1.80 ^mol/mL/h) as in the control group (1.72 ± 0.09 ^mol/mL/h; P < 0.001). This was due to the release of the enzyme from the liver cells (hepatocytes) into the blood caused by impaired membrane permeability (Table 3).
Hepatocyte membranes became more permeable as a result of lipid peroxidation. This was indicated by an increase in malondialdehyde (MDA) to 6.83 ± 0.09 ^mol/mL of blood plasma in the mice with toxic hepatitis, compared to 3.70 ± 0.10 ^mol/mL (P < 0.001) in the control group of intact mice (Table 3).
Membrane permeability was impaired by trichloromethine and chlorine (CC13 and Cr), the
Table 3 Biochemical blood parameters of carbon tetrachloride intoxicated mice and their correction with extract-containing breads (M ± m)
Mice groups ALT, ARA, trolox SOD, activity MDA,
^mol/mL/h units/mg protein units/mL blood ^mol/mL
Group 1 control 1.72 ± 0.09 13.15 ± 0.21 678.49 ± 6.47 3.70 ± 0.10
Group 2 carbon tetrachloride 34.24 ± 1.803 6.65 ± 0.153 276.56 ± 8.643 6.83 ± 0.093
Group 3 deprivation (toxicant withdrawal) 13.38 ± 1.203 8.85 ± 0.653 435.55 ± 2.753 5.62 ± 0.053
Group 4 deprivation + bread with Caliphene 1.90 ± 0.08c 12.80 ± 1.01c 683.43 ± 4.48c 3.72 ± 0.13c
Group 5 deprivation + bread with Diprim 1.83 ± 0.07c 12.54 ± 0.89c 685.74 ± 4.32c 4.03 ± 0.16c
Group 6 deprivation + bread with Eklikit 1.87 ± 0.07c 12.02 ± 0.75c 665.43 ± 4.78c 3.61 ± 0.14c
The differences are statistically significant at: 1 - P < 0.05; 2 - P < 0.01; 3 - P < 0.001 vs. control; a - P < 0.05; b - P < 0.01; c - P < 0.001 vs. group 3. Abbreviations: ARA - antiradical activity, SOD - superoxide dismutase, MDA - malondialdehyde
Table 4 Indicators of antioxidant liver and blood system of carbon tetrachloride intoxicated mice and their correction with extract-containing breads (M ± m)
Mice groups Reduced glutathione (^mol/g liver) Glutathione reductase (nmol/min/mL plasma) Glutathione peroxidase (nmol/min/mL plasma)
Group 1 control (intact) 4.70 ± 0.15 88.21 ± 4.26 139.0 ± 4.83
Group 2 carbon tetrachloride 3.13 ± 0.143 39.68 ± 3.253 110.1 ± 3.43
Group 3 deprivation (toxicant withdrawal) 3.09 ± 0.113 50.32 ± 1.542 105.3 ± 6.042
Group 4 deprivation + bread with Caliphene 4.87 ± 0.33c 92.48 ± 2.46c 138.3 ± 2.19b
Group 5 deprivation + bread with Diprim 4.90 ± 0.33c 91.75 ± 3.62c 137.3 ± 2.32c
Group 6 deprivation + bread with Eklikit 4.87 ± 0.20c 90.61 ± 2.59c 137.4 ± 1.53c
The differences are statistically significant at: 1 - P < 0.05; 2 - P < 0.01; 3 - P < 0.001 vs. control; a - P < 0.05; b - P < 0.01; c - P < 0.001 vs. group 3
radicals forming during carbon tetrachloride metabolism in the cytochrome P-450 system after intoxication [40].
The control group of mice with toxic hepatitis that did not receive bread displayed an imbalance and depletion of enzymatic antioxidant processes and antiradical protection. This was indicated by a decrease in antiradical activity to 6.65 ± 0.15 trolox units/mg protein, compared to the control group of intact mice (13.15 ± 0.21 trolox units/mg protein; P < 0.001). Moreover, the activity of superoxide dismutase, a key enzyme of the antioxidant defence system, was 2.5 times as low as in the control group (276.56 ± 8.64 units/mL of blood compared to 678.49 ± 6.47 units/mL of blood, respectively; P < 0.001), as shown in Table 3.
A study of the liver and blood antioxidant system revealed the following simultaneous changes in antioxidant indicators: a 21% decrease in glutathione peroxidase activity (GPO; P < 0.001), a 55% decrease in glutathione reductase (GR; P < 0.001), and a 33% decrease in reduced glutathione (P < 0.001), as shown in Table 4.
Such changes in the antioxidant protection system of carbon tetrachloride intoxicated mice can be regarded as evidence of its depletion.
A study of lipid metabolism in the liver of carbon tetrachloride intoxicated mice revealed a pronounced
metabolic disorder. This confirmed the efficacy of the experimental model and proved the development of toxic hepatitis in the experimental animals after the toxicant administration (Table 5).
Table 5 shows an increase in triacylglycerols (TAGs) by 17% (P < 0.001), cholesterol (CS) by 9% (P < 0.001), and free fatty acids (FFAs) by 14% (P < 0.001). These disorders were caused by a breakdown of triacylglycerols in adipose tissue (chemical stress), a release of fatty acids and glycerol into the blood, and their resynthesis as triacylglycerols in the liver. Due to impaired synthesis of phospholipids from triacylglycerols, fatty acids and TAGs accumulated in hepatocytes, leading to fatty infiltration of the liver. The increase in cholesterol was due to the inhibition of mitochondrial oxidation of acetyl CoA in the Krebs cycle, whose condensation lead to cholesterol production. A lower level of fatty acid esters (FAEs) (by 9%; P < 0.05) and cholesterol esters (CSEs) (by 22%; P < 0.001) indicated an impairment of cholesterol metabolism and esterifying function of the liver. This was due to the inhibition of the ACAT enzyme (acyl-CoA: cholesterol-acyltransferase) during carbon tetrachloride intoxication. This ratio of lipid fractions confirmed the fatty degeneration of the liver caused by the impairment of its esterifying function.
Table 5 Neutral lipid content in carbon tetrachloride intoxicated mice liver and their correction with extract-containing breads (M ± m)
Mice groups TAGs FFAs FAEs CS CSEs Residual
fraction
Group 1 control 21.91 ± 0.60 16.24 ± 0.24 15.49 ± 0.50 14.95 ± 0.19 16.97 ± 0.11 13.65 ± 0.15
Group 2 carbon tetrachloride 25.54 ± 0.423 18.47 ± 0.323 1 4.15 ± 0.271 16.15 ± 0.173 13.31 ± 0.173 12.37 ± 0.44 Group 3 deprivation 26.30 ± 0.353 17.27 ± 0.231 14.19 ± 0.241 15.69 ± 0.151 13.53 ± 0.233 13.51 ± 0.35
(toxicant withdrawal)
Group 4 deprivation + bread with 21.89 ± 0.31c 15.85 ± 0.28c 16.72 ± 0.23c 14.44 ± 0.26c 17.05 ± 0.19c 14.05 ± 0.25 Caliphene
Group 5 deprivation + bread with 21.70 ± 0.33c 15.63 ± 0.26c 16.75 ± 0.27c 14.44 ± 0.23c 17.25 ± 0.20c 14.23 ± 0.21 Diprim
Group 6 deprivation + bread with 21.80 ± 0.31c 16.30 ± 0.34a 16.74 ± 0.28c 14.80 ± 0.33a 15.34 ± 0.21c 15.02 ± 0.42 Eklikit_
The differences are statistically significant at: 1 - P < 0.05; 2 - P < 0.01; 3 - P < 0.001 vs. control; a - P < 0,05; b - P < 0,01; c - P < 0.001 vs. group 3. Abbreviations: TAGs - triacylglycerols, FFAs - free fatty acids, FAEs - fatty acids esters, CS - cholesterol, CSEs - cholesterol esters
The impaired metabolic reactions in the mice liver proved the efficacy of the experimental toxic hepatitis model through carbon tetrachloride intoxication.
Seven days after the toxicant withdrawal (deprivation period) did not normalize biochemical parameters in the liver of the mice with a bread-free diet. This indicated that their bodies' own defences were too weak to withstand pathologic development caused by intoxication with hepatotropic poison. The animals were lethargic; they had a poor appetite and dull hair.
The malondialdehyde content in the bread-free group remained 52% (P < 0.001) higher than in the control group, indicating a still high level of lipid peroxidation.
Further decrease in reduced glutathione to 3.09 ± 0.11 ^mol/g in group 3 confirmed the on-going destabilisation of liver cell membranes, causing their imbalance and depleting their antioxidant protection. The activity of superoxide dismutase was 57% (P < 0.001) higher than in group 2 (carbon tetrachloride), due to the toxicant withdrawal, but it was 36% (P < 0.001) lower than in the control group. Likewise, the antiradical activity in group 3 was 35% (P < 0.001) higher than in group 2 (carbon tetrachloride), but 32% (P < 0.001) lower than in the control. The results indicated the continuing free-radical processes.
Those indicators were associated with a high activity of alanine aminotransferase, a marker of liver damage, which was eight times as high as in the control group (13.38 ± 1.2 ^mol/mL/h; P < 0.001).
Among neutral lipids, the content of triacylglycerol, free fatty acids, and cholesterol remained significantly high, compared to the control groups. The blood serum of the mice with a bread-free diet showed an impaired cholesterol metabolism - a significantly low content of fatty acid and cholesterol esters - indicating a suppressed esterifying function of the liver. This was consistent with high ALT activity - 8 times as high as in the control group (13.38 ± 1.2 ^mol/mL/h; P < 0.001).
Thus, the lipid spectrum of the liver of mice that did not receive bread with extracts indicated a continuing deterioration of metabolic reactions even in the absence of the toxic agent (Tables 2-5).
Feeding the carbon tetrachloride intoxicated mice with bread containing plant extracts (groups 4-6) resulted in the correction and normalization of the parameters studied. The activity of ALT, an enzyme marker of toxic hepatitis, was not significantly different from the control values. This suggested that bread treated with plant extracts had membrane-stabilizing properties.
We found a complete normalization of the membrane lipid peroxidation parameters (in particular,
malonic dialdehyde) in groups 4-6 vs. the control. The antiradical and antioxidant protection systems also showed a full recovery.
The analysis of lipid metabolism in the liver of mice in groups 4-6 showed a marked decrease in triacylglycerols, free fatty acids, fatty acid esters, and cholesterol. At the same time, there was an increase in fatty acid esters and cholesterol, compared to group 3 (deprivation). This indicated the efficacy of Caliphene, Diprim, and Eklikit extracts in restoring the esterifying function of the liver and reversing fatty infiltration.
We believe that the biochemical mechanism of restoring the liver function with functional foods after carbon tetrachloride intoxication is based on the localization of polyphenols in the lipid bilayer of the hepatocyte plasma membrane, stabilizing its permeability [41, 42].
CONCLUSION
Thus, the experimental model of toxic hepatitis showed that breads enriched with the plant extracts of Caliphene, Diprim, and Eklikit had pronounced hepatoprotective and antioxidant properties.
Those properties were due to the effect of polyphenols contained in the extracts on the metabolism and function of the liver. In particular, polyphenols inhibited free radical reactions, increased the liver's antiradical and antioxidant activity, and reduced the amount of toxic lipid peroxidation products. They also stabilized hepatocyte membranes, normalized the liver's esterifying function, and restored the weight of the animals and their liver, as well as ALT activity [43-45]. Thus, breads enriched with water and ethanol extracts obtained from viburnum (Viburnum sargentii Koehne L.), magnolia-vine (Schisandra chinensis L.), and grapes (Vitis amurénsis L.) waste can be regarded as products with preventative properties and recommended as part of a hepatoprotective diet.
CONTRIBUTION
Authors are equally related to the writing of the manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The authors thank the Laboratory of Biochemistry and its Head, Prof. N.F. Kushnerova at V.I. Ilyichov Pacific Oceanological Institute, the Far Eastern Branch of the Russian Academy of Sciences.
REFERENCES
1. Postanovlenie Pravitel'stva RF ot 15 iyulya 2010 g. N 530 "Ob utverzhdenii Pravil ustanovleniya predel'no dopustimykh roznichnykh tsen na otdel'nye vidy sotsial'no znachimykh prodovol'stvennykh tovarov pervoy neobkhodimosti,
perechnya otdel'nykh vidov sotsial'no znachimykh prodovol'stvennykh tovarov pervoy neobkhodimosti, v otnoshenii kotorykh mogut ustanavlivat'sya predel'no dopustimye roznichnye tseny, i perechnya otdel'nykh vidov sotsial'no znachimykh prodovol'stvennykh tovarov, za priobretenie opredelennogo kolichestva kotorykh khozyaystvuyushchemu sub"ektu, osushchestvlyayushchemu torgovuyu deyatel'nost', ne dopuskaetsya vyplata voznagrazhdeniya" [The RF Government Decree No. 530 of July 15, 2010 "Approving the Rules for establishing maximum allowable retail prices for certain types of basic foodstuffs, the List of certain types of basic foodstuffs for which maximum allowable retail prices can be established, and the List of certain types of basic foodstuffs for the acquisition of a certain amount of which the retailer is not entitled to remuneration"].
2. Lobanov VG, Slepokurova YI, Zharkova IM, Koleva TN, Roslyakov YF, Krasteva AP. Economic effect of innovative flour-based functional foods production. Foods and Raw Materials. 2018;6(2):474-482. DOI: http://doi. org/10.21603/2308-4057-2018-2-474-482.
3. Vengerovskiy AI, Markova IV. Doklinicheskoe izuchenie gepatozashchitnykh sredstv [A preclinical study of hepatoprotective agents]. Vedomosti farmakologicheskogo komiteta [Vedomosti Farmakologicheskogo Komiteta]. 1999;(2):9-12. (In Russ.).
4. Sanz, MJ, Ferrandiz ML, Cejudo M, Terencio MC, Gil B, Bustos G, et al. Influence of a series of natural flavonoids on free-radical generating systems and oxidative stress. Xenobiotica. 1994;24(7):689-699. DOI: https://doi. org/10.3109/00498259409043270.
5. Es-Safi N-E, Ghidouche S, Ducrot PH. Flavonoids: hemisynthesis, reactivity, characterization and free radical scavenging activity. Molecules. 2007;12(9):2228-2258. DOI: https://doi.org/10.3390/12092228.
6. Terao J. Dietary flavonoids as antioxidants. ForumofNutrition. 2009;61:87-94. DOI: https://doi.org/10.1159/000212741.
7. De Souza RF, De Giovani WF. Antioxidant properties of complexes of flavonoids with metal ions. Redox Report. 2004;9(2):97-104. DOI: https://doi.org/10.1179/135100004225003897.
8. Pamplona R. Advanced lipoxidation end-products. Chemico-Biological Interactions. 2011;192(1-2):14-20. DOI: https://doi.org/10.1016/j.cbi.2011.01.007.
9. Momot TV Prirodnye rastitel'nye fenol'nye soedineniya v profilaktike stressovykh narusheniy metabolicheskikh reaktsiy organizma zhivotnykh i cheloveka [Natural plant phenolic compounds in preventing stress disorders of metabolic reactions in animals and humans: doctoral thesis in medical science]. Dr. med. sci. diss. Vladivostok: Pacific State Medical University; 2018. 347 p.
10. Olszewska MA, Michel P. Antioxidant activity of inflorescences, leaves and fruits of three Sorbus species in relation to their polyphenolic composition. Natural Product Research. 2009;23(16):1507-1521. DOI: https://doi. org/10.1080/14786410802636177.
11. Kharchenko YuA, Avdonina OO, Eremenko SV. Hepatoprotective properties of new drags. Modern problems of science and education. 2012;(1):215. (In Russ.).
12. Zain MZM, Baba AS, Shori AB. Effect of polyphenols enriched from green coffee bean on antioxidant activity and sensory evaluation of bread. Journal of King Saud University - Science. 2018;30(2):278-282. DOI: https://doi. org/10.1016/j.jksus.2017.12.003.
13. Sivam AS, Sun-Waterhouse D, Perera CO, Waterhouse GIN. Exploring the interactions between blackcurrant polyphenols, pectin and wheat biopolymers in model breads; a FTIR and HPLC investigation. Food Chemistry. 2012;131(3):802-810. DOI: https://doi.org/10.1016/j.foodchem.2011.09.047.
14. Sun-Waterhouse D, Sivam AS, Cooney J, Zhou J, Perera CO, Waterhouse GIN. Effects of added fruit polyphenols and pectin on the properties of finished breads revealed by HPLC/LC-MS and Size-Exclusion HPLC. Food Research International. 2011;44(9):3047-3056. DOI: https://doi.org/10.1016/j.foodres.2011.07.022.
15. Prokopov T, Chonova V, Slavov A, Dessev T, Dimitrov N, Petkova N. Effects on the quality and health-enhancing properties of industrial onion waste powder on bread. Journal of Food Science and Technology. 2018;55(12):5091-5097. DOI: https://doi.org/10.1007/s13197-018-3448-8.
16. Hidalgo A, Brandolini A, Canadanovic-Brunet J, Cetkovic G, Tumbas Saponjac V. Microencapsulates and extracts from red beetroot pomace modify antioxidant capacity, heat damage and colour of pseudocereals-enriched einkorn water biscuits. Food Chemistry. 2018;268:40-48. DOI: https://doi.org/10.1016/j.foodchem.2018.06.062.
17. Rico D, Martin-Diana AB, Lasa A, Aguirre, Milton-Laskibar I, de Luis DA, et al. Effect of wakame and carob pod snacks on non-alcoholic fatty liver disease. Nutrients. 2019;11(1). DOI: https://doi.org/10.3390/nu11010086.
18. Tang G-Y, Zhao C-N, Liu Q, Feng X-L, Xu X-Y, Cao S-Y, et al. Potential of grape wastes as a natural source of bioactive compounds. Molecules. 2018;23(10). DOI: https://doi.org/10.3390/molecules23102598.
19. Rico D, Alonso de Linaje A, Herrero A, Asensio-Vegas C, Miranda J, Martinez-Villaluenga C, et al. Carob by-products and seaweeds for the development of functional bread. Journal of Food Processing and Preservation. 2018;42(8). DOI: https://doi.org/10.1111/jfpp.13700.
20. Smertina ES, Fedyanina LN, Kalenik TK, Vigerina NS, Karaseva SV, Kushnerova NF, et al. Kompozitsiya dlya prigotovleniya testa dlya khlebobulochnykh izdeliy (varianty) [Bread dough composition (variants)]. Russian patent RU 2483548C2. 2013.
21. Petrov AN, Maslennikova GA. Physical and chemical aspects of vacuum drying of berry raw materials. Foods and Raw Materials. 2016;4(1):129-134. DOI: http://doi.org/10.21179/2308-4057-2016-1-129-134.
22. Parfenova TV. Obosnovanie primeneniya polifenol'nykh kompleksov iz dikorosov kak antioksidantov dlya proizvodstva marmelada funktsional'nogo naznacheniya i ego tovarovednaya kharakteristika [Substantiating the use of polyphenolic complexes from wild plants as antioxidants in producing functional fruit jelly and its product characteristics]. Cand. eng. sci. diss. Khabarovsk: Pacific National University; 2006. 26 p.
23. Fomenko SE, Kushnerova NF, Sprigin VG, Parfenova TV, Kushnerova TV. Application of plant polyphenols in the composition of functional foods. The bulletin of the Far Eastern Federal University. Economics and Management. 2009;49(1):62-69. (In Russ.).
24. Parfenova TV, Kushnerova NF, Korastyleva LA, Bystrova AM. Jelly fruit candy with biological active additives from wild plants. Storage and processing of farm products. 2007;(3):70-72. (In Russ.).
25. Vershinina AG, Kushnerova NF, Lentsova LV, Parfenova TV, Kalenik TK. Vozmozhnost' ispol'zovaniya novykh rastitel'nykh dobavok iz dikorosov ussuriyskoy taygi kak antioksidantov dlya ehmul'sionnoy pishchevoy produktsii dlitel'nogo khraneniya [The possibility of using new plant supplements from the Ussuri taiga as antioxidants for long shelf life emulsion food products]. News of institutes of higher education. Food Technology. 2004;278(1):62-64. (In Russ.).
26. Fedyanina LN, Kalenik TK, Smertina ES, Zvyagintseva TN, Imbs TI, Shevchenko NM, et al. To the question about security of ingredients using for the making bakery products for the purpose of medioprophylactic. The bulletin of the Far Eastern Federal University. Economics and Management. 2011;60(4):82-86. (In Russ.).
27. Smertina ES, Fedyanina LN, Lyakh VA. Seaweed BAA production waste usage as the functional ingredients in baking products contents. The bulletin of the Far Eastern Federal University. Economics and Management. 2012;64(4):94-102. (In Russ.).
28. Fedyanina LN, Smertina ES, Vigerina NS, Stepanov SV. On the efficiency of bakery products c bioactive food supplements adaptogennoe effect. Technology and merchandising of the innovative foodstuff. 2012;14(3):51-56. (In Russ.).
29. Sprygin VG, Kushnerova NF, Fomenko SE. Waste from processing the far-east wild-growing plants - perspective sources of nutritional antioxidants. Izvestia of Samara Scientific Center of the Russian Academy of Sciences. 2010;12(1-3):812-815. (In Russ.).
30. Momot TV, Kushnerova NF. Justification of the choice of raw sources from far east flora for receiving the pharmaceutical preparations. Izvestia of Samara Scientific Center of the Russian Academy of Sciences. 2016;18(2):146-149. (In Russ.).
31. Sbornik tekhnologicheskikh instruktsiy dlya proizvodstva khlebobulochnykh izdeliy [Collected technological instructions for bread production]. Moscow: Preyskurantizdat; 1989. 1080 p. (In Russ.).
32. Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-ciocalteu reagent. Methods in Enzymology. 1998;299:152-178. DOI: https://doi. org/10.1016/S0076-6879(99)99017-1.
33. Vegerovskiy AI, Markova IV, Saratikov AS. Metodicheskie ukazaniya po izucheniyu gepatozashchitnoy aktivnosti farmakologicheskikh veshchestv [Guidelines for studying hepatoprotective activity of pharmacological substances]. In: Khabriev RYu, editor. Rukovodstvo po ehksperimental'nomu (doklinicheskomu) izucheniyu novykh farmakologicheskikh veshchestv [Guidelines for an experimental (preclinical) study of new pharmacological substances]. Moscow: Meditsina; 1990. pp. 228-231. (In Russ.).
34. Khabriev RYu. Rukovodstvo po ehksperimental'nomu (doklinicheskomu) izucheniyu novykh farmakologicheskikh veshchestv [Guidelines for an experimental (preclinical) study of new pharmacological substances]. Moscow: Meditsina; 2005. 832 p. (In Russ.).
35. Paoletti F, Aldinucci D, Mocali A, Caparrini A. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Analytical Biochemistry. 1986;154(2):536-541. DOI: https://doi. org/10.1016/0003-2697(86)90026-6.
36. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS+ radical cation decolorization assay. Free Radical Biology and Medicine. 1999;26(9-10):1231-1237. DOI: https://doi.org/10.1016/S0891-5849(98)00315-3.
37. Novgorodtseva TP, Ehndakova EhA, Yan'kova VI. Rukovodstvo po metodam issledovaniya parametrov sistemy "perekisnoe okislenie lipidov-antioksidantnaya zashchita" v biologicheskikh zhidkostyakh [Guidelines for methods of studying the "lipid peroxidation - antioxidant protection" system parameters in biological fluids]. Vladivostok: Far Eastern Federal University; 2003. 80 p. (In Russ.).
38. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissue. The Journal of biological chemistry. 1957;226(1):497-509.
39. Amenta JS. A rapid chemical method for quantification of lipids separated by thin-layer chromatography. Journal of Lipid Research. 1964;5(2):270-272.
40. Brattin WJ, Glende AJ, Recknagel RO. Pathological mechanisms in carbon tetrachloride hepatotoxicity. Journal of Free Radicals in Biology and Medicine. 1985;1(1):27-38. DOI: https://doi.org/10.1016/0748-5514(85)90026-1.
41. Tsuchiya H. Effects of green tea catechins on membrane fluidity. Pharmacology. 1999;59(1):34-44. DOI: https://doi. org/10.1159/000028303.
42. Maffei Facino R, Carini M, Aldini G, Calloni MT, Bombardelli E, Morazzoni P. Sparing effect of procyanidins from Vitis vinifera on vitamin E: In vitro studies. Planta Medica. 1998;64(4):343-347. DOI: https://doi. org/10.1055/s-2006-957448.
43. Young IS, Woodside JV Antioxidants in health and disease. Journal of Clinical Pathology. 2001;54(3):176-186. DOI: https://doi.org/10.1136/jcp.54.3.176.
44. Sprygin VG, Kushnerova NF, Rakhmanin YuA. An antioxidant action produced by oligomeric anthocyanidins, isolated from viburnum, in lesions of the liver by carbon tetrachloride and prevention of its toxic effect. Hygiene and sanitation. 2003;(3):57-60. (In Russ.).
45. Kushnerova NF, Sprygin VG. Vliyanie kompleksnogo polifenol'nogo preparata "kalifen" na protsessy vosstanovleniya biokhimicheskikh pokazateley pecheni posle porazheniya ehtilovym spirtom [The effect of the Caliphene polyphenolic complex on the recovery of liver biochemical parameters after intoxication with ethyl alcohol]. Problems of Biological, Medical and Pharmaceutical Chemistry. 2002;(4):22-26. (In Russ.).
ORCID IDs
Elena S. Smertina https://orcid.org/0000-0002-2519-9170 Lyudmila N. Fedyanina https://orcid.org/0000-0002-9849-8358 Vladimir A. Lyakh https://orcid.org/0000-0001-6999-2210
