COMPARATIVE EFFECTS OF HELMAR-1 AND HELMAR-2 POLYPHENOL EXTRACTS ON MITOCHONDRIAL ENERGY METABOLISM IN CCl₄-INDUCED EXPERIMENTAL HEPATITIS

ABSTRACT

Mitochondrial dysfunction plays a central role in liver injury, particularly in toxic hepatitis, leading to impaired respiration and reduced ATP production. This study aimed to evaluate the hepatoprotective effects of polyphenol extracts Helmar-1 and Helmar-2 on mitochondrial bioenergetics in a carbon tetrachloride (CCl₄)-induced hepatitis model in rats. Liver mitochondria were isolated, and respiration parameters were assessed. CCl₄ exposure significantly suppressed mitochondrial function, with a 34.7% decrease in State V₂ and a 28.6% decrease in State V₃ respiration, along with a 54.6% increase in State V₄, indicating uncoupling. RCR and ADP/O ratios decreased by 53.8% and 43.3%, respectively. Treatment improved mitochondrial parameters: Helmar-1 increased V₂ and V₃ by 15.8% and 29.9%, while Helmar-2 showed stronger effects (22.4% and 33.6%). RCR and ADP/O were also significantly restored.

These findings demonstrate that Helmar-2 exhibits pronounced hepatoprotective activity by improving mitochondrial function and energy metabolism.

АННОТАЦИЯ

Митохондриальная дисфункция играет ключевую роль в повреждении печени, особенно при токсическом гепатите, приводя к нарушению дыхания и снижению синтеза АТФ. Целью исследования было оценить гепатопротекторное действие полифенольных экстрактов Helmar-1 и Helmar-2 на митохондриальную биоэнергетику при гепатите, индуцированном тетрахлорметаном (CCl₄) у крыс.

Воздействие CCl₄ вызывало выраженное угнетение функции митохондрий: снижение дыхания в состоянии V₂ на 34,7% и V₃ на 28,6%, а также увеличение V₄ на 54,6%, что свидетельствует о разобщении окислительного фосфорилирования. Показатели RCR и ADP/O снижались на 53,8% и 43,3% соответственно. Введение экстрактов улучшало митохондриальные параметры: Helmar-1 увеличивал V₂ и V₃ на 15,8% и 29,9%, тогда как Helmar-2 проявлял более выраженный эффект (22,4% и 33,6%).

Полученные данные свидетельствуют о выраженном гепатопротекторном действии Helmar-2 за счет восстановления митохондриальной функции и энергетического обмена.

Keywords: Mitochondria, CCl₄, toxic hepatitis, liver, Helichrysum maracandicum, Helmar 1, Helmar 2, silymarin

Ключевые слова: митохондрии, CCl₄, токсический гепатит, печень, Helichrysum maracandicum, Helmar-1, Helmar-2, силимарин

INTRODUCTION

 The liver is a central metabolic organ responsible for detoxification, protein synthesis, bile production, and energy regulation, processes that require substantial ATP generation and rely heavily on mitochondrial function (Begriche et al. 2006; Grattagliano et al. 2012). Hepatocytes contain a high density of mitochondria, accounting for up to 18% of cellular volume, making hepatic function particularly sensitive to mitochondrial bioenergetic disturbances (Mansouri et al. 2018). Mitochondrial dysfunction has been recognized as a key pathogenic mechanism in a wide spectrum of liver diseases, including non-alcoholic fatty liver disease (NAFLD), drug-induced liver injury (DILI), and age-related hepatic impairment (Younossi et al. 2016; Fernandez-Checa et al. 2021).

Mitochondria generate cellular energy through oxidative phosphorylation (OXPHOS), which couples electron transport chain activity with ATP synthesis (Fromenty and Pessayre 1995). The respiratory chain consists of Complexes I–V, with Complex I playing a critical role in electron transfer from NADH (Ozawa 1997). In addition to energy production, mitochondria regulate fatty acid β-oxidation, reactive oxygen species (ROS) generation, calcium homeostasis, and apoptosis (Han et al. 2006a). Due to these functions, even minor impairments in mitochondrial activity can trigger significant pathological changes in liver tissue (Trauner et al. 2010; Genova et al. 1995). Previous studies have demonstrated that oxidative stress is closely associated with alterations in mitochondrial enzyme activity and cytochrome c release during lipid peroxidation processes (Ahmedova et al., 2020).

Mitochondrial ROS play a dual role in liver physiology and pathology. While low levels of ROS are involved in cellular signaling, excessive production leads to oxidative stress, damaging cellular macromolecules and disrupting metabolic homeostasis (Schieber and Chandel 2014; Cichoż-Lach and Michalak 2014). The liver is particularly vulnerable to oxidative damage due to its high metabolic rate and oxygen consumption, creating a feedback loop between mitochondrial dysfunction and oxidative stress (Serviddio et al. 2013). Complexes I and III are major sources of ROS, and their dysfunction enhances electron leakage and oxidative injury (Murphy 2009; Brand 2010). The glutathione system plays a crucial protective role in maintaining redox balance (Han et al. 2006b), while oxidative modifications of mitochondrial proteins further exacerbate mitochondrial damage (Aon et al. 2010).

Given the central role of mitochondrial dysfunction in liver pathology, therapeutic strategies aimed at restoring mitochondrial function are of considerable interest. These include stimulation of mitochondrial biogenesis via PGC-1α, use of mitochondria-targeted antioxidants, enhancement of mitophagy, and supplementation with metabolic cofactors (Scarpulla 2011; Smith and Murphy 2010). Lifestyle interventions such as physical activity have also been shown to improve mitochondrial function in liver diseases (Hallsworth et al. 2011).

Natural compounds, particularly plant-derived polyphenols, have attracted attention due to their antioxidant and hepatoprotective properties. Silymarin, derived from Silybum marianum, is widely used for its ability to stabilize mitochondrial membranes and reduce lipid peroxidation (Gillessen and Schmidt 2020). Carbon tetrachloride (CCl₄) remains a classical model of hepatotoxicity, inducing oxidative stress through formation of reactive radicals and subsequent mitochondrial damage (Recknagel et al. 1989). Recent studies have demonstrated that polyphenol-rich extracts can improve mitochondrial function, enhance antioxidant defenses, and restore energy metabolism in toxic liver injury models (Ahmed et al. 2018).

Despite increasing evidence, comparative evaluation of different polyphenol extracts on mitochondrial bioenergetics in toxic hepatitis remains limited. Therefore, the present study focuses on investigating the effects of Helmar-1 and Helmar-2 polyphenol extracts on mitochondrial respiration and oxidative phosphorylation in a CCl₄-induced experimental hepatitis model.

MATERIALS AND METHODS

Animal handling and experimental conditions

Male white outbred rats, ranging from 180 to 220 grams in weight, were kept in a controlled laboratory environment characterized by a temperature of 20 to 24 °C, humidity of 65%, and a natural light/dark cycle. They had unrestricted access to food and water at all times.

Ethical statement

The Local Ethics Committee of the Institute of Biophysics and Biochemistry, National University of Uzbekistan named after Mirzo Ulugbek, approved all experimental procedures in compliance with the Guide for the Care and Use of Laboratory Animals [Protocol No. BEC/IBB NUU/2019/02/22].

Acute carbon tetrachloride-induced model in animals

The rats were housed under standard laboratory conditions, with no more than five animals per cage, and were maintained on a standard laboratory diet. Food was withdrawn 24 hours prior to CCl₄ intoxication. An acute model of toxic liver injury was induced using carbon tetrachloride (CCl₄). To induce toxic hepatitis, CCl₄ was administered intraperitoneally twice a week at a dose of 1 ml/kg (in olive oil as the vehicle).

The toxin was mixed with sterile olive oil at a ratio of 1:1, resulting in a 50% CCl₄ solution in oil. This experimental model was performed according to the method described by Avtandilov (Avtandilov 1990), with minor modifications. The animals were divided into 5 groups: Group I control (n = 10); Group II CCl₄; Group III CCl₄ + Helmar-1; Group IV CCl₄ + Helmar 2; Group V CCl₄ + silymarin;

Mitochondria isolation

Mitochondria were extracted from rat liver tissue through differential centrifugation (Schneider and Hogeboom 1951). The procedure involved the use of a gentle ether anesthesia to immobilize the rats, followed by decapitation and opening of the abdominal cavity to retrieve the liver. The liver from a single rat was homogenized with a Teflon-glass homogenizer and then resuspended in an isolation buffer comprised of 250 mM sucrose, 1 mM sodium ethylenediaminetetraacetate (EDTA), and 10 mM Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) at pH 7.4. Nuclei and complete cells were removed by centrifugation at 1,500 × g for 7 minutes. Subsequently, mitochondria were pelleted from the supernatant through centrifugation at 6,000 × g for 15 minutes. The resultant mitochondrial pellet was suspended in a minimal volume of medium containing 250 mM sucrose and 10 mM Tris HCl, and was maintained on ice until utilized in experiments.

Evaluation of Lipid Peroxidation.

The degree of lipid peroxidation (LPO) in liver mitochondria was detected by measuring malondialdehyde (MDA) activity, which is associated with oxidative damage to mitochondrial membranes. Polyphenol extracts of the plant Helichrysum maracandicum were used to treat the oxidative stress in liver mitochondria caused by toxic hepatitis of carbon tetrachloride (CCl₄). According to the experimental protocol, these polyphenol extracts were given intraperitoneally at a dose of 20 mg/kg body weight for ten consecutive days following CCl₄ exposure. The antioxidant action of the polyphenol extracts was determined by comparing mitochondrial MDA levels, treated with treated liver, with untreated toxic hepatitis. Based on the obtained results, polyphenols from H. maracandicum were found to be effective in suppressing lipid peroxidation and preserving mitochondrial membranes. For LPO measured through rat liver mitochondrial membranes, an Fe²⁺/citrate-dependent system (in some experiments Fe²⁺/ascorbate) was adopted. Kinetic analysis of mitochondrial swelling by Fe²⁺/citrate (0.3 mg/mL) was performed through optical density monitoring at 540 nm in a cuvette open (3 mL volume) at 26 °C containing continually stirring solutions of mitochondrial suspension. Lipid peroxidation began with 50 µM FeSO₄ and 2 mM citrate as inducers (Almeida et al. 2006). The intensity of LPO in mitochondrial membranes was measured in the following incubation medium (mM): sucrose 125 mM, KCl 65 mM, Tris-HCl 10 mM, pH 7.2. To halt reaction, 0.220 mL of 70% trichloroacetic acid was added to the incubation medium before centrifugation at 4000 rpm for 15 minutes to separate components. The supernatant was then taken, which was combined with 1 mL 75% Thio barbituric acid in samples in which distilled water is a substitute for the supernatant (control samples, 2 mL). This mixture was incubated in a water bath for half an hour and cooled down for further analysis. The optical density was next read at 532 nm and malondialdehyde concentration determined by molar extinction coefficient (ε = 1.56×10⁵ M⁻¹·cm⁻¹) (Vladimirov and Archakov 1972) and thus the formula: nmol MDA/mg protein = D / (1.56 × 30).

Determination of mitochondrial respiration and oxidative phosphorylation
Respiration rates and oxidative phosphorylation parameters are determined by a polarographic method employing a rotating platinum electrode similar to that described earlier (Li and Graham 2012), with some modifications. Oxygen consumption rates from mitochondrial respiration and oxidative phosphorylation were analyzed to assess these. Specifically, the oxygen consumption rate of liver mitochondria from experimental rats was measured using a high-precision respiratory machine from Strathkelvin Instruments in a continuously stirred, thermostatically controlled closed chamber at 26°C, with a volume of 1 mg/ml in terms of protein, in the following incubation buffers (IB): 120 mM KCl, 1 mM KH₂PO₄ and Tris-HCl at 10 mM. Additionally, succinate was included as the respiratory substrate 5 mM, 1 mM MgCl2, 1 mM EGTA, 1 µM rotenone. The pH level was set to 7.2. The assessment of the mitochondrial respiratory chain was conducted based on the following parameters: V₂ (or Vsubstrate). This denotes the mitochondrial respiration rate when substrates are present, but ADP is absent. V₃ – This indicates the respiration rate during a metabolically active state after ADP is introduced, V₄ – Known as the resting (state 4) respiration rate, it reflects mitochondrial respiration with substrates present following the complete conversion of added ADP into ATP, VCCCP – maximal respiration in presence of 3 μM of CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, Sigma-Aldrich). This represents the maximum respiration rate observed in the presence of the uncoupler CCCP, indicating a total separation of oxidation from phosphorylation processes. The substrate oxidation rates in all metabolic states in O2 μg/mL are expressed compared to mitochondrial protein content. Peterson modified the Lowry method to determine protein concentration of mitochondria (Peterson 1977).

Statistical Analysis

The statistical processing of the data, such as the graphical visualization, was performed using OriginPro 8.6 software (OriginLab Corporation, USA). For example, mitochondrial swelling kinetics at each of the experiments was stated as a percentage of peak value and calculated on an arithmetic mean of 4 to 7 independent experiments. In order to examine differences among the control, experimental, and experimental + test compound groups, Student's t-test results are presented as M ± m. Significant findings are represented as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

RESULTS AND DISCUSSION

Carbon tetrachloride (CCl₄)-induced toxic hepatitis resulted in significant disturbances in mitochondrial respiration and oxidative phosphorylation in rat liver. In the pathological group (Group II), the mitochondrial respiration rate during succinate oxidation in State V₂ decreased by 34.7% (***p < 0.001) compared to the control group. Similarly, in the presence of 200 μM ADP, State V₃ respiration was significantly reduced by 28.6% (**p < 0.01), indicating impaired oxidative phosphorylation capacity. In contrast, State V₄ respiration increased by 54.6% reflecting mitochondrial uncoupling and enhanced proton leakage. Furthermore, the addition of the uncoupler CCCP led to decreased oxygen consumption compared to control values, confirming dysfunction of the respiratory chain.

These findings demonstrate that toxic hepatitis is associated with severe disturbances in mitochondrial bioenergetics, including increased membrane permeability to H⁺ ions and the development of a low-energy metabolic state due to the uncoupling of oxidative phosphorylation, primarily driven by excessive ROS generation.

Pharmacological intervention with polyphenol extracts significantly improved mitochondrial function. In rats with toxic hepatitis treated for 10 days, Helmar-1 (Group III), Helmar-2 (Group IV), and silymarin (Group V) all promoted recovery of mitochondrial respiration parameters.

Specifically, the V₂ respiration rate increased by 15.81% (*p < 0.05) in the Helmar-1 group, 22.4% (**p < 0.01) in the Helmar-2 group, and 27.5% (***p < 0.001) in the silymarin group compared to the pathological group. Similarly, State V₃ respiration increased by 29.9% (*p < 0.05), 33.6% (**p < 0.01), and 36.9% (***p < 0.001) in Helmar-1, Helmar-2, and silymarin-treated groups, respectively. These results indicate improved substrate oxidation and enhanced ATP synthesis capacity.

In addition, the elevated V₄ respiration observed under toxic hepatitis conditions was partially normalized following treatment, decreasing by 13.5% (*p < 0.05) (Helmar-1), 18.8% (**p < 0.01) (Helmar-2), and 25.2% (***p < 0.001) (silymarin). This suggests restoration of coupling between oxidation and phosphorylation processes.

The respiratory control ratio (RCR), which decreased by 53.8% in the pathological group, was significantly restored by 51.6% (*p < 0.05) in the Helmar-1 group and 65.5% (**p < 0.01) in the Helmar-2 group, while silymarin showed the highest recovery 82.7% (***p < 0.001). Likewise, the ADP/O ratio, reduced by 43.3% under toxic hepatitis, increased by 39.6% (*p < 0.05), 43.2% (**p < 0.01), and 51.35% (***p < 0.001) in Helmar-1, Helmar-2, and silymarin-treated groups, respectively (Table 1). The summarized results of mitochondrial respiration parameters are presented in Table 1.

Table 1.

 Effects of Helmar-1 and Helmar-2 on mitochondrial respiration and oxidative phosphorylation parameters in CCl₄-induced toxic hepatitis

Values are expressed as percentage of control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs CCl₄ group.

Importantly, comparative analysis demonstrates that Helmar-2 consistently exhibited stronger effects than Helmar-1 across all parameters, including mitochondrial respiration rates (V₂, V₃), coupling efficiency (RCR), and phosphorylation efficiency (ADP/O). Although silymarin showed the highest overall activity, Helmar-2 approached its efficacy, indicating a high therapeutic potential.

These findings confirm that polyphenol extracts from Helichrysum maracandicum effectively restore mitochondrial bioenergetics in toxic hepatitis. Previous studies have shown that polyphenolic compounds isolated from Helichrysum maracandicum exhibit strong antioxidant and antiradical activity (Ahmedova et al., 2022). The observed effects are likely mediated through reduction of oxidative stress, stabilization of mitochondrial membranes, and improvement of electron transport chain function, ultimately leading to enhanced ATP production.

Thus, Helmar-1 and Helmar-2 extracts significantly improve mitochondrial function under hepatotoxic conditions, with Helmar-2 demonstrating superior efficacy, suggesting its potential as a promising hepatoprotective agent.

Comparative Efficacy and Clinical Significance. The comparable activity of H. maracandicum extracts to silymarin is also noteworthy. Silymarin—a standardized extract of Silybum marianum (milk thistle), which includes a combination of flavonolignans including silybin, silydianin, and silychristin—has been well studied for hepatoprotection. Its mechanisms are antioxidant action, membrane stabilization, protein synthesis stimulation, and anti-inflammatory actions.

For translational reasons, there are three aspects to keep in mind. First, the classical utilization of Helichrysum species for folk medical purposes in liver ailments is proven by our findings.

The current results are in line with previous studies that were published in ANV publications and showed that herbal extracts significantly reduced liver damage caused by carbon tetrachloride by regulating oxidative stress and mitochondrial activity (Björnsson 2016). These investigations found improvements in cellular energy metabolism, preservation of mitochondrial membrane stability, and restoration of antioxidant enzyme activity, which is consistent with our findings and further supports the therapeutic value of polyphenol-based therapies in liver pathology.

CONCLUSION

Polyphenol extracts from Helichrysum maracandicum were shown to possess substantial hepatoprotective effects against the toxic hepatitis generated by CCl₄ through a dual mechanism involving inhibition of lipid peroxidation and enhancement of mitochondrial bioenergetic activity. The extracts considerably reduced oxidative damage markers (lipid peroxides and malondialdehyde), and increased mitochondrial respiratory performance, coupling, and ATP production capacities. The therapeutic potential observed is similar to that of silymarin; along with the conventional use and the availability of H. maracandicum, the results suggest that these extracts warrant further study as hepatoprotective agents. This study contributes to the growing body of evidence supporting the rational use of plant-derived polyphenols in management of liver disease, and reinforces the importance of focusing on mitochondrial bioenergetics for hepatoprotection.

ACKNOWLEDGMENTS: The authors express their heartfelt gratitude to the staff at the Laboratory of Experimental Technologies, Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, for their generous supply of the polyphenolic compounds utilized in this research.

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