Antioxidant activity of some organosulfur compounds in vitro

2.1 General procedures and syntheses

Compounds 3–8 and all reagents were purchased from Sigma-Aldrich unless specified otherwise.

Compounds 1, 2 were synthesized starting from 2,6-di-tert-butylphenol according previously described method with slight modifications (Mueller et al., 1961).

2.2 DPPH radical scavenging activity

All experiments were performed with a 96-cell “Zenyth 200RT, Anthos” microplate spectrophotometer. The free radical scavenging activity was evaluated using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), according to the method described by Brand-Williams et al. (1995) with a slight modification. For each test compound different concentrations in MeOH were used (20, 40, 80, 120, 160 and 200 µM). The stock DPPH solution contained 0.2 mM of radical in MeOH. 0.1 mL of the test compound solution was added to 0.1 mL of DPPH solution (0.2 mM) in each cell so that the initial DPPH concentration in cells was 0.1 mM. The microplate was placed in a spectrophotometer and the decrease in the absorbance values of DPPH solution for 40 min at 20 °C was measured at λ_max = 517 nm. The results were expressed as scavenging activity, calculated as follows: $$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y},\%=[({\mathrm{A}}_{\mathrm{c}}-{\mathrm{A}}_{\mathrm{s}})/{\mathrm{A}}_{\mathrm{c}}]\times 100$$

The concentration of the compound needed to decrease 50% of the initial DPPH concentration (EC₅₀) was determined to evaluate the antioxidant effect. The EC₅₀ values were calculated graphically by plotting scavenging activity against compounds concentration.

2.3 Cupric reducing antioxidant capacity (CUPRAC assay)

Neocuproine (2,9-dimethyl-1,10-phenanthroline) and Trolox were used with no further purification. The method proposed by Apak et al. (2004) was used with slight modification. For these measurements, 0.05 mL of CuCl₂ solution (0.01 M), 0.05 mL of MeOH neocuproine solution (7.5 mM) and 0.05 mL of ammonium acetate buffer solution (1 M) were added to a test tube followed by mixing with the 0.05 mL tested compounds (0.5 mM). The mixtures were kept at room temperature for 30 min. All experiments were performed with a 96-cell “Zenyth 200RT, Anthos” microplate spectrophotometer, absorbance was measured at 450 nm against a reagent blank. The increase in absorbance of the reaction mixture in comparison with the control indicates the reduction capability of the test compound. The results were presented in Trolox equivalents (Trolox Equivalent Antioxidant Capacity, TEAC) obtained using absorbance data and the linear calibration curve plotted as absorbance vs. Trolox concentration.

2.4 Ferrous ions chelating activity (FIC)

The chelation of ferrous ions by compounds was estimated by method of Dinis et al. (1994). Briefly, 10 μl of 2 mM FeCl₂ was added to 20 μl of the investigated compound (5 mM) and 150 μl of EtOH. The reaction was initiated by the addition of 40 μl of 5 mM ferrozine solution. The mixture was left to stand at 35 °C for 10 min. The absorbance of the solution was thereafter measured at 562 nm.

The percentage inhibition of ferrozine–Fe²⁺ complex formation was calculated as: $$\mathrm{F}\mathrm{I}\mathrm{C},\%=[({\mathrm{A}}_{\mathrm{c}}-{\mathrm{A}}_{\mathrm{s}})/{\mathrm{A}}_{\mathrm{c}}]\times 100$$ where A_c was the absorbance of the control, and A_s was the absorbance of the sample. Na₂EDTA was used as positive control.

2.5 Determination of LOOH and TBARS concentrations in oleic acid

The determination of oleic acid oxidation level was performed by the kinetic measuring of the total concentration of the corresponding isomeric LOOH using iodometric titration (Rzhavskaya, 1976). The oxidation of constant volume of the acid (15 mL) was carried out in a thermostatic cell using an air flow at 65 °C during 3 h. The oxidation proceeds in the conditions, the oxidation rate is independent of air volume passing through the cell (Emanuel, 1974).

The concentrations of the additives were 1 mM compared with the initial concentration of LOOH in the reaction mixture. Oleic acid (1 mL), CHCl₃ (12 mL), glacial AcOH (18 mL), and freshly prepared cold-saturated KI solution (1 mL) were placed in a flask and shaken for 2 min; distilled water (100 mL) and a 1% starch solution (1 mL) were then added, and the resulting mixture was immediately titrated with Na₂S₂O₃ solution (0.01 N). Iodine released in an amount equivalent to that of LOOH was titrated with a standard thiosulfate solution. At the same time, a control test for reagents was carried out: all the reagents except for oleic acid were added to the flask.

The LOOH concentration was calculated according to the following formula: $$\left[\text{LOOH}\right]=\left[\left({\text{V}}_{\text{s}}-{\text{V}}_{\text{c}}\right)\times 0.001269\times \text{K}\times 100\right]/\text{m},$$ wherein V_s is the volume of 0.01 N Na₂S₂O₃ solution, consumed during the titration of working sample, mL; V_c is the volume of 0.01 N Na₂S₂O₃ solution, consumed during the titration of control, mL; K is the conversion factor to the exactly 0.01 N Na₂S₂O₃ solution; m is the mass of studied oleic acid; and 0.001269 is the amount of I₂ expressed in g, equivalent to 1 mL of 0.01 N Na₂S₂O₃ solution. The [LOOH, mmol/L] content equal to 1% corresponds to 78.7 mM of active O₂ per 1 L of lipids.

The accumulation rate for the final oxidation products (TBARS) was determined according to a modified standard method (Stroev et al., 2012). The studied compounds (1 mM) were added to oleic acid thermostated at the selected temperature. Samples (0.01 mL) of oleic acid were taken from the thermostat every 30 min. They were introduced into a test tube containing a mixture of Tris buffer (0.8 mL), distilled water (1.2 mL), and freshly prepared thiobarbituric acid solution (0.8%, 1 mL); the tube was placed for 10 min in a boiling water bath, and after cooling, the absorption of the samples was measured in comparison with that of control at λ = 532 nm. A similar mixture, but without added oleic acid, was used as the control.

The concentration of carbonyl compounds was calculated according to the formula: $$\left[TBARS\right]=\left(E\times 3\right)/0.156$$ where [TBARS] is the content of carbonyl compounds, nM; E is the extinction of a sample relative to the control; 3 is the sample volume, mL; and 0.156 is the extinction of malondialdehyde (1 nmol) dissolved in 1 mL at λ = 532 nm.

The kinetic curves of the oleic acid peroxidation in the presence of studied compounds follow the exponential law, the approximation coefficients of kinetic curves were in a range of 0.9031–0.9731. Kinetic investigation has shown the initials rate of the hydroperoxides LOOH accumulation to be pseudo-first-order in the air. The kinetic curves of content of secondary carbonyl products TBARS during the oleic acid oxidation in the presence of compounds follow the linear law, the approximation coefficients of kinetic curves were in a range of 0.9024–0.9922.

2.6 Determination of TBARS accumulation in the Russian sturgeon liver homogenate

The experiments in vitro were carried out using the liver of a Russian sturgeon raised in Unique aqua complex for the reproduction of valuable fish species of the Federal Research Center Southern Scientific Center of the Russian Academy of Sciences within the framework of State Assignment (Project No. 01201354245). All manipulations were conducted according to the international rules GLP (Good Laboratory Practice). The samples of fish liver were fixed in the cold.

The LPO intensity was estimated according to the accumulation of carbonyl products forming a colored complex with thiobarbituric acid (TBARS) (Stroev et al., 2012). A liver of Russian sturgeon (10 g) homogenized in the cold and studied compounds were added at the concentration of 0.1 mM in CHCl₃ to a solution of potassium chloride precooled to 0–4 °C. The absence of any influence of CHCl₃ on the LPO rate in the control was preliminarily established under these conditions. The resulting mixture was poured into flasks and incubated with the added studied compounds at the temperature of 5 °C for 48 h, sampling the mixture (2 mL) at a certain time interval into tubes for a subsequent centrifugation. Solutions of ascorbic acid (0.1 mL), Mohŕs salt (0.1 mL), and trichloroacetic acid (1 mL) were added to these tubes. The tubes were placed in a water bath at 37 °C for 10 min and then centrifuged for 10 min (3000 rpm). The supernatants (2 mL) were placed into clean tubes, thiobarbituric acid solutions (1 mL) were added, the samples were placed in a boiling water bath for 10 min, and then they were cooled in ice water down to room temperature (∼20 °C). After cooling, CHCl₃ (1.0 mL) was added to the samples to give a clear solution, and the resulting mixtures were centrifuged for 15 min (3000 rpm). The supernatant was sampled, and the extinction of sample was measured using a SF-103 spectrophotometer at λ = 532 nm relative to the control sample. The calculation was carried out according to the formula: $$\mathrm{X}=\left(E\times 3\times 3.2\right)/\left(0.156\times 2\right),$$

wherein X is the content of carbonyl products in the starting homogenate, nmol; E is the extinction of samples; 3 is the volume of samples, mL; 3.2 is the total volume of studied samples, mL; 0.156 is the extinction of malondialdehyde (1 nmol) dissolved in 1 mL at λ = 532 nm; 2 is the volume of supernatant used to determine carbonyl products, mL.

The efficiency of the antioxidant action (EAA, %) of the compounds 1–8 (Antonova et al., 2008) was calculated from the relation $$\mathrm{E}\mathrm{A}\mathrm{A}=\left[\left({\mathrm{C}}_{\mathrm{c}}-{\mathrm{C}}_{\mathrm{s}}\right)/{\mathrm{C}}_{\mathrm{c}}\right]\times 100\%,$$ where C_c and C_s are concentrations the carbonyl products forming a colored complex with thiobarbituric acid (TBARS) in the control and the sample of test liver homogenates Russian sturgeon, respectively. If the EAA is positive, the test compound shows an antioxidant action; a negative EAA value attests to a prooxidant action.

2.7 Statistical analysis

The acquired results were statistically processed using the Student t-test (implemented in Microsoft Excel software), and the average value and standard deviation were calculated. From five to ten repeats were carried out for the each experimental determination, and the nature of influence was estimated using the average values of activity, taking into account the experimental error (p < 0.01). Pearson’s correlation coefficient (r) was determined to establish the relationship between the values of the parameters of antioxidant activity on various model systems in vitro.

3.1 DPPH radical scavenging activity

The capacity to inhibit the development of radical chain reactions due to interaction with ROS and the formation of a corresponding highly stable radical is the main property of most known inhibitors of oxidative processes. One of the defining methods for revealing the antioxidant potential of compounds at the molecular level is the reaction with the stable 2,2-diphenyl-1-picrylhydrazyl radical DPPH (Gulcin et al., 2010).

The ability of compounds 1–8 to transfer a hydrogen atom to the stable DPPH radical with the formation of the corresponding radicals, which can then enter into reactions of recombination, dimerization and fragmentation, was investigated. Antiradical activity was measured by a colour change from violet to yellow and a decrease in absorbance at 517 nm. The amount of antioxidant required to reduce the initial DPPH concentration by 50% (half maximal inhibitory concentration (IC₅₀)) was calculated.

It is known that there are two main mechanisms of the antiradical action of antioxidants: hydrogen atom transfer (HAT) and single electron transfer (SET). Despite the difference in mechanisms the same end products are formed (Esfandi et al., 2019). These processes can proceed in parallel. Although, depending on the structure of the antioxidant and conditions, one of the mechanisms can dominate. According to the results obtained, it was found that only organosulfur compounds with phenolic and/or HS-groups 1 and 2 demonstrated a pronounced radical scavenging activity (IC₅₀ values were 68.01 ± 0.09 and 20.09 ± 0.02 μM, respectively) (Fig. 2), that indicates that these compounds act mainly through the HAT mechanism.

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Fig. 2

Dependence of the amount (%) of unreduced DPPH on the concentration of bis-(4-hydroxy-2,6-di-tert-butylphenyl) disulfide 2 in MeOH solution.

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This is consistent with literature data, according to which sterically hindered phenols are good antioxidants due to their ability to form stable aroxyl radicals (Leopoldini et al., 2004; Amić et al., 2020). The presence of two similar fragments in compound 2 promoted the increase in antioxidant properties in comparison with diphenyl disulfide and compound 1. Earlier, it was also found that sulfur-containing amino acids cysteine, methionine, taurine have different activities against DPPH (Kim et al., 2020), among them the highest one was explored for cysteine containing HS-group. On the contrary, the organic oligosulfides 3–8 do not exhibit antiradical activity in this reaction, but one must take into account that this method is not universal, since not all antioxidants can interact react quickly with active radicals like DPPH, and, therefore, it is necessary to study the antioxidant activity of such compounds by other test systems.

3.2 Cupric reducing antioxidant capacity (CUPRAC assay)

The CUPRAC assay is based on the ability of a potential antioxidant to act as an electron donor and reduce Cu²⁺ ion to Cu⁺ in combination with 2,9-dimethyl-1,10-phenanthroline (neocuproine). The absorbance of the solution compounds 1–8 were measured at 450 nm at room temperature. The calibration curves of each potential antioxidant were constructed as Trolox Equivalent Antioxidant Capacity (TEAC_CUPRAC), found as the ratio of the molar absorptivity of each compound to that of trolox were calculated (Dontha, 2016).

It was found that compound 2 in reducing ability was twice as active as trolox, and compound 4 exhibited activity similar to that of trolox despite the absence of a phenolic fragment in the structure, TEAC_CUPRAC = 1.06 (Table 1).

The rest of the organosulfur compounds did not show high reducibility in this test system. It is important to note that compound 1, despite the presence of HO- and HS-groups, also does not show a pronounced ability to act as an electron donor, reducing the copper ion in a complex with 2,9-dimethyl-1,10-phenanthroline.

3.3 Fe²⁺-chelating activity compounds (FIC)

An independent type of pharmacological and antioxidant activity of compounds is the ability of them to act as a chelator of transition metals that catalyze the decomposition reactions of LOOH with the formation of ROS, in particular, a hydroxyl radical HO^•. Antioxidants such as trolox, BHA and BHT can prevent the development of ferroptosis (Cao and Dixon, 2016). Absorption with food is considered among the main factors affecting the bioavailability of micronutrients for humans. Compounds with chelating activity enhance the absorption of calcium and iron ions in the digestive tract. It was shown that hydrolyzed proteins and peptides from food proteins can act as ligands of transition metals, inhibiting iron-dependent lipid peroxidation (Walters et al., 2018). Moreover, cysteine and methylcysteine can be used for decreasing of Fe(III) concentration in biological systems that helps to stop the development of oxidative stress (Tewari, 2002).

The Fe²⁺-chelating activity of compounds 1–8 was studied spectrophotometrically based on the ability to bind Fe²⁺ ions, reducing the content of the coloured Fe²⁺-ferrosine complex (λ = 562 nm). A pronounced Fe²⁺-chelating activity was demonstrated for di-tert-butyl disulfide (6) and methyl propyl trisulfide (8) (>95%) (Table 1).

For the rest of the compounds, the Fe²⁺-chelating activity is significantly lower than that of the standard EDTA, even in the case of compounds 1 and 2 with HO- and HS-groups in the structure. In this model system, the dependence of the Fe²⁺-chelating activity on the nature of the organic radical (aliphatic, aromatic) and the number of sulfur atoms in the structure of the compounds has not been established.

3.4 Influence of sulfur-containing compounds on the oxidation of cis-9-octadecenoic acid

Increased formation of primary and secondary products of lipid peroxidation (LPO) is a marker of the development of oxidative stress and is considered a universal pathogenetic factor that is responsible for the formation and development of a wide range of diseases and pathologies (Tsai and Huang, 2015; Krzystek-Korpacka et al., 2008). The oxidation of unsaturated fatty acids serves as a model reaction for the lipid peroxidation in cellular membranes bilayer. The oxidation of substrate (oleic acid) by molecular oxygen involves the generation of substituted radicals that interact with O₂ to produce peroxyl radicals LOO^•. The rate of LOO^• transformation to corresponding cis- and trans-isomeric hydroperoxides (LOOH) which are the main products of the initial reaction period (Porter et al., 1994) might be used as a criterion of the rate of the peroxidation (Frankel, 1980). The markers of carbonyl compounds formation following the LOOH decomposition are thiobarbituric acid reactive substances (TBARS), the accumulation of which in the presence of investigated compounds have been studied by UV–Vis spectrophotometry (λ_max = 532 nm).

On a model system for the oxidation of oleic acid with atmospheric oxygen at 65 °C for 3 h, the total antioxidant activity of organosulfur compounds was assessed by standard methods for the accumulation of LOOH and carbonyl products reacting with thiobarbituric acid.

The kinetic curves of LOOH accumulation in oleic acid are exponential and correspond to the equation C = a·e^кt + b with correlation coefficients close to 1, which indicates a pseudo-first order of reaction in the substrate, inherent in a radical process with degenerate chain branching (Figs. 3, 4).

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Fig. 3

Kinetic curves of LOOH formation in the presence of 1 mmol/L of different compounds at 65 °C: (control) oleic acid without additives; (1) 2,6-di-tert-butyl-4-mercaptophenol; (2) bis(3,5-di-tert-butyl-4-hydroxyphenyl) disulfide; (3) diphenyl disulfide; (4) dibenzyl disulfide.

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The effect of sulfur-containing compounds show that decrease the amount of oleic acid hydroperoxides formed, except for compound 5, the addition of which does not influence the level of oleic acid LOOH, the kinetic curves of oleic acid’s autooxidation and in the presence of compound 5 are identical (Fig. 4).

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Fig. 4

Kinetic curves of LOOH formation in the presence of 1 mmol/L additives at 65 °C: (control) oleic acid without additives; (5) dibutyl disulfide; (6) di-tert-butyl disulfide; (7) diallyl disulfide; (8) methyl propyl trisulfide.

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The total content of LOOH after 3 h in the presence of sulfur-containing compounds when compared with oleic acid without additives amount 64–87%. The influence of compound 2 is more significant and the total concentration of LOOH is 53% lower than in the oleic acid autooxidation. The relative rate constants of the LOOH formation are given in Table 1 and the comparison of the k₀/k₁ values for the LOOH accumulation in the presence of the compounds 1–8 only shows inhibitory effect.

The content of TBARS during the oleic acid oxidation is also appropriately increasing over time without and with additive the sulfur-containing compounds 1–8, the secondary carbonyl products accumulation curves are linear, corresponding to the equation [TBARS] = kt + b, the correlation coefficients are close to 1 (Figs. 5 and 6). However, the data confirm the inhibitory effect of sulfur-containing compounds, except for the compound 5 showing insignificant prooxidant activity (Fig. 6).

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Fig. 5

Kinetic curves for the accumulation of TBARS in oleic acid in the presence of 1 mmol/L additives at 65 °C: (control) oleic acid without additives; (1) 2,6-di-tert-butyl-4-mercaptophenol; (2) bis(3,5-di-tert-butyl-4-hydroxyphenyl) disulfide; (3) diphenyl disulfide; (4) dibenzyl disulfide.

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Fig. 6

Kinetic curves for the accumulation of TBARS in oleic acid in the presence of 1 mmol/L additives at 65 °C: (control) oleic acid without additives; (5) dibutyl disulfide; (6) di-tert-butyl disulfide; (7) diallyl disulfide; (8) methyl propyl trisulfide.

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The level of TBARS after 3 h decreases in the presence of added compounds 1, 3, 6, 7 and 8 by 11–32% relative to the control (Table 1). The level of LPO in oleic acid after 3 h of incubation with of compound 4 does not differ from the control. Compound 5 exhibits a prooxidant effect increasing the level of peroxidation by 9%. The addition of compound 2 leads to a greater decrease 73% in the level of secondary carbonyl compounds (Fig. 5, Table 1). Interestingly, dibutyl disulfide 5 exhibited insignificant promoting effect in contrast to di-tert-butyl disulfide 6.

It is known that organosulfur compounds exhibit antioxidant activity due to the ability to degrade LOOL and LOOH without the formation of active radicals that initiate chain radical processes leading to the development of oxidative stress (Chauvin et al., 2019). At the same time, organosulfur compounds can act as free radical scavenger, for example, as in the case of di-tert-butyl sulfide, the oxidation of which forms the corresponding sulfoxide. Chauvin et al. (2019) also found that the reactivity of polysulfides is preserved even at elevated temperatures, which allows them to surpass phenols and alkylated diphenylamines in antiradical activity, which are widely used in medicine as antioxidants.

Sinse compounds 1–4, 6–8 were active against both primary and secondary LPO products, these organosulfur compounds possess an intramolecular synergistic antioxidant effect. It was previously shown in various in vitro model systems that bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propyl)sulfide (thiophane) is an effective polyfunctional antioxidant due to synergistic combination of two phenol pendants and antiperoxidase bivalent sulfur activity (Prosenko et al., 2003).

We investigated also the impact of sulfur-containing compounds on oleic acid peroxidation and the inhibitory effect of compound 2 has been proven (Figs. 3 and 5) that is associated with the key role of 2,6-di-tert-butylphenol moieties and two atoms of sulfur in molecule. The data presented do not demonstration the significant influence of the organic group nature and amount of sulfur atoms upon the effectiveness of compounds 1–8.

3.5 Determination of TBARS level in the Russian sturgeon liver homogenate

The antioxidant potential of sulphur derivatives was studied using a model system of lipid peroxidation in sturgeon liver. The choice of the substrate is explained by the fact that liver cells are especially sensitive to any stressful conditions and can instantly respond, increasing the level of primary and secondary LPO products, as a result of which the process of apoptosis is activated, energy supply and detoxification mechanisms are disrupted. Oxidation of liver lipids which make up cell membranes directly causes damage to cells in vivo therefore this model system is similar to biological systems (Somerharju et al., 1999).

The inhibitory effect of organosulfur derivatives was studied under the conditions of a long-term LPO process, which made it possible to evaluate the effect of compounds under long-term oxidative stress, when the peroxidation process is enhanced, and the concentration of antioxidants is significantly consumed, and to reveal a possible inversion of anti-/prooxidant properties in vitro (Rao et al., 2011; Hořejší, 2005). The level of accumulation of secondary carbonyl products of LPO, forming colored complexes with thiobarbituric acid (TBARS), under autooxidation conditions and in the presence of the compounds under study is presented in Table 2. At subsequent stages of LPO, the activity of compound 1 is reduced to 15%, in contrast to compound 2, for which an increase in the effectiveness of the antioxidant action (EAA) is noted up to 64% (Fig. 7).

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Fig. 7

The efficiency of the antioxidant action (EAA, %) of the compounds 1–8 during long-term in vitro oxidation of Russian sturgeon liver lipids: (1) 2,6-di-tert-butyl-4-mercaptophenol; (2) bis(3,5-di-tert-butyl-4-hydroxyphenyl) disulfide; (3) diphenyl disulfide; (4) dibenzyl disulfide; (5) dibutyl disulfide; (6) di-tert-butyl disulfide; (7) diallyl disulfide; (8) methyl propyl trisulfide.

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Aliphatic symmetric disulfides 5–7 showed efficiency only at the initial (60 min) and middle (1441 min) stages of LPO, at the last stage (2880 min) a significant decrease in antioxidant activity was established, the level of TBARS is comparable to the control, but no inversion is observed.

It has previously been found that diallyl disulfide 7 can prevent acetaminophen-induced nephrotoxicity through its antioxidant properties (Ko et al., 2017).

It has also been shown in experimental rats with myocardial dysfunction that supplementation of garlic oil containing diallyl disulfide significantly reduced TBARS levels, cardiac lactate dehydrogenase activity, and increased endogenous antioxidant levels (Asdaq and Avula, 2015). At the same time, it was found that an essential oil extract from white cabbage containing dimethyltrisulfide and dimethyldisulfide are more potent inhibitors of LPO than diallyl disulfide, which explains its hepatoprotective effect (Morales-López et al., 2017).

Insignificant antioxidant activity is noted at all stages of oxidative destruction of sturgeon liver lipids dibenzyl disulfide 4 and methylpropyl trisulfide 8, and compound 3 exhibits prolonged prooxidant activity. Compound 2 exhibits a pronounced antioxidant effect at all stages, as in other model systems. The experimental results evidence that compound 2 acts as an effective inhibitor in this model system too, what is explained the hydrogen atom abstraction from the phenol fragments in compound 2 gives relatively stable radical.

3.6 Pearson’s correlation analysis

To assess the relationship between various indicators of the antioxidant activity of organosulfur compounds 1–8, Pearson's correlation analysis was carried out (Lesaffre et al., 2009) and the calculated coefficients are presented in Table 3.

Significant positive correlations were observed between the accumulation rate of LOOH and TBARS in oleic acid (r = 0.9179). A moderate positive correlation was found between the reducing activity of CUPRAC and LOOH and TBARS in oleic acid and EAA in sturgeon liver homogenate (r = 0.7636, 0.6807 and 0.8101, respectively), as well as between the effectiveness of the antioxidant action and the level of accumulation of primary and secondary products of oleic acid oxidation (r = 0.6701 and 0.7935).

The worst negative correlations were noted between iron chelating activity (FIC), reductive ability in CUPRAC and inhibition of the process of long-term LPO in Russian sturgeon liver (EAA,%) (r = −0.5746 and −0.4001, respectively).