Abdubokiev A, Khushmatov S, Komilov E, Ikramov S, Zhurakulov S. Analysis of Hepatoprotective Activity of Isoquinoline Alkaloids (F-5, F-24), and their Derivatives (KV-6, KV-8) in CCl₄-Induced Hepatotoxity in Mice. Biomed Pharmacol J 2025;18(4).

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Manuscript received on :10-10-2025
Manuscript accepted on :03-12-2025
Published online on: 22-12-2025

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Reviewed by: Dr. Subhasis Chakraborty
Second Review by: Dr. Hina Imran
Final Approval by: Dr. Patorn Piromchai

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Abduvali Abdubokiev^1*, Shunkor Khushmatov¹, Esohon Komilov¹, Sirojiddin Ikramov¹ and Sherzod Zhurakulov¹

¹Department of Physiology and Anatomy, Namangan State University, Namangan, Uzbekistan.

²Department for the Development of Scientific and Innovative Activities, Ministry of Higher Education, Science and Innovation of the Republic of Uzbekistan, Tashkent, Uzbekistan.

³Department of Molecular Biophysics, Institute of Biophysics and Biochemistry under the National University of Uzbekistan, Tashkent, Uzbekistan.

⁴Department of Medicine, University of Business and Science, Namangan, Uzbekistan.

⁵Department of Alkaloid Chemistry, Institute of the Chemistry of Plant Substances named after Academician S.Yu. Yunusov, Academy of Sciences of the Republic of Uzbekistan, Tashkent, Uzbekistan.

Corresponding Author’s E-mail: abduvaliabduboqiyev38@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/3332

Abstract

Carbon tetrachloride (CCl₄)-induced hepatotoxicity is characterized by profound oxidative stress, disruption of hepatocyte membrane integrity, and sharp elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In this study, the hepatoprotective potential of isoquinoline alkaloids (F-5, F-24) and their semi-synthetic derivatives (KV-6, KV-8) was evaluated using a CCl₄-induced toxic hepatitis model in mice. CCl₄ intoxication increased serum ALT and AST levels by up to 300% compared to the control, confirming severe hepatocellular injury. Administration of the tested compounds (25-100 mg/kg) produced a concentration-dependent reduction in ALT and AST activities. Among the alkaloids, F-5 (100 mg/kg) decreased ALT to 8.19±0.14 microkat/l in males and 7.14±0.25 microkat/l in females, while F-24 reduced these values to 9.43±0.18 and 8.45±0.26 μkat/l, respectively. The derivatives KV-6 and KV-8 demonstrated stronger hepatoprotective effects, with KV-6 (100 mg/kg) showing the greatest efficacy – reducing ALT to 5.10±0.17 and 4.04±0.23 microkat/l, and AST to 7.56±0.35 and 8.42±0.29 microkat/l in male and female mice, respectively. Calculation of the hepatoprotective action coefficient confirmed the effectiveness of the compounds in the order: F-24<F-5<KV-8<KV-6, with KV-6 exhibiting the highest protective activity (64-82% restoration relative to CCl₄ pathology). These findings indicate that isoquinoline alkaloids and their derivatives mitigate CCl₄-induced hepatocyte damage by normalizing aminotransferase activity, suggesting their promise as hepatoprotective agents. The results align with existing evidence on the protective effects of polyphenolic compounds in oxidative-stress–mediated liver injury.

Keywords

ALT; AST; CCl4-induced hepatotoxity; Hepatoprotective effect; Isoquinoline alkaloids

Introduction

According to World Health Organization statistics, approximately 3 million people will die from liver diseases by 2024, and this rapidly increasing rate represents a serious medical, socioeconomic, and global challenge. Therefore, developing new approaches to treating these disorders and creating effective next-generation pharmaceuticals is of critical importance.^1-7 Numerous studies have described in detail the origins of liver diseases, their pathogenesis, and the mechanisms of action of hepatoprotective agents. In particular, it has been noted that the therapeutic effects of broad-spectrum hepatoprotectors (including polyphenolic compounds and flavonoids) involve stimulation of the antioxidant system in hepatocytes, antiradical activity (suppression of ROS formation), and optimization of enzyme function, among other mechanisms.^8-10

Isoquinoline alkaloids and their derivatives represent one promising group of agents in this field. The biological activity of isoquinoline alkaloids – known for their wide range of pharmacological effects (anticancer, antibacterial, antiviral, antineurodegenerative, analgesic, antidepressant, cardioprotective, etc.) – has been extensively studied.^11-14 It has been demonstrated that isoquinoline alkaloids optimize lipid metabolism in hepatocytes by modulating the AMPK/SIRT1/PPAR-alfa signaling cascade, enhance the activity of antioxidant enzymes, and exert antioxidant and anti-apoptotic effects through modulation of the MAPK/NF-kappaB and ERK/JNK pathways.^15-18 It is also noteworthy that many hepatoprotective medications contain components with antioxidant, anti-inflammatory, detoxifying, and choleretic properties. A significant proportion of these components are polyphenolic compounds (such as flavonoids) that inhibit lipid peroxidation (LPO) and increase the stability of lipid biomembranes. In particular, the hepatoprotective activity of quercetin, closely related to its chemical structure, has been widely documented.^19-22

The aim of this study was to compare the hepatoprotective activity of several isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) by evaluating their effects on plasma transaminase enzymes – alanine aminotransferase (ALT) and aspartate aminotransferase (AST) – in a mouse model of CCl₄-induced toxic hepatitis.

Materials and Methods

The experiments were conducted in 2023-2024 in the Metabolomics laboratory of the Institute of Biophysics and Biochemistry of the National University of Uzbekistan named after Mirzo Ulugbek.

Experimental Animals

The objects of the study were white mice (male/female, m=20.5-26.2 g), which were fed standard food (water) under standard vivarium conditions (room temperature +20±5°C, relative air humidity 75±10%, light regime 12:12 hours).^23,24

Ethical Approval

When working with experimental animals in scientific research, the requirements of the rules developed by the International Council for International Organizations of Medical Sciences (1985), the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986), the Declaration developed by the European Union (86/609/EEC), and the Bioethical Statement of the Institute of Biophysics and Biochemistry of the National University of Uzbekistan (No. BRC/IBB; N44/2024/75-1) were observed.^25-28

Chemicals and Drugs

Isoquinoline alkaloids (F-5, F-24) and their derivatives with the flavonoid quercetin (KV-6, KV-8), provided by the staff of the S.Yu.Yunusov Institute of Plant Chemistry of the Academy of Sciences of the Republic of Uzbekistan (Table. 1) were used as objects of research.^29-33

Table 1: Chemical structure of isoquinoline alkaloids F-5, F-24, and their derivatives Click here to view Table

Experimental Design

The hepatoprotective activity of isoquinoline alkaloids and their derivatives was assessed using a standard method on a model of experimental toxic hepatitis in mice caused by CCl₄.^{34,35,36,37,38} To create an experimental model of toxic hepatitis, we used white outbred mice of both sexes (male/female, m=20.5-26.2 g) aged 2-3 months, kept in vivarium conditions on standard food and water. During the experiments, animals were administered intraperitoneal CCl₄ solution (diluted in olive oil) at a dose of 1 ml/kg for 14 days (on the ^1st, 5^th and 11^th days).^39-56

Biochemical Analysis of Enzymes

In biological research, spectrophotometric analysis of ALT, AST and other substances allows for the acquisition of highly accurate data.^57,58

The activity of ALT and AST in blood serum was analyzed using a standard method.^59-65

Aminotransferases or transaminases that use phosphopyridoxal and phosphopyridoxamine as coenzymes catalyze the reverse transfer of the amino group from an amino acid to an α-keto acid. Formed in the transamination reaction of an amino acid determination of the concentration of α-ketoacid forms the basis of the assessment of the activity of transaminases. Aspartic acid is converted to oxaloacetate by AST, and alanine is converted to pyruvic acid by ALT. During the enzymatic reaction, oxaloacetate is converted to pyruvate acid. Adding 2,4-dinitrophenylhydrazine to the medium halts the process, forming a brown pyruvate dinitrophenylhydrazone. The color intensity was used to estimate the concentration, which in turn, indicates the aminotransferase activity (expressed in micromol/h×mL), formed during 1 hour of incubation in 1 mL of the test serum at 37°C). The blood sample was centrifuged at 3000 rpm for 10 minutes, the serum was separated with a pipette into a test tube, dinitrophenylhydrazine (0.25 ml) was added to the test sample (0.05 ml), incubated for 60 minutes at t=+37±0.5°C, 0.4 M NaOH (2.5 ml) was added, after 10 minutes of incubation, the color intensity was assessed by optical density in the wavelength range 490-520 nm and the activity of ALT and AST was determined using a calibration graph. Based on the results of spectrophotometric analysis, a curve of the dependence of optical density and enzyme activity was constructed, where the optical density is plotted along the ordinate axis and the enzyme activity is plotted along the abscissa axis. The activity of ALT and AST (microkat/l) in the blood serum of mice was analyzed using a biochemical analysis reagent kit from Cupress Diagnostica (Belgium) and an Agilent Cary 60 Uv-Vis spectrophotometer (Agilent Technologies, USA).^66-80 A blood sample (1 ml) taken from a rat vein was centrifuged at 12,600 rpm for 10 minutes (t=+4±0.5°C), after which ALT and AST were analyzed spectrophotometrically.^81,82,83 In the spectrophotometric analysis of ALT activity, a method developed by Reitman S., Frenkel S. is used, based on the transfer of an amino group from the amino acid alanine to 2-oxoglutarate in the presence of ALT, the formation of pyruvate and glutamate, and in the next stage – the formation of lactate and NAD⁺ in the reaction of pyruvate (pyruvic acid) and NADH⁺ (H⁺) in the presence of lactate dehydrogenase and the assessment of the amount of ALT by the level of absorption of NADH⁺ (H⁺) in the range of 340 nm (Alanine + 2-oxoglutarate Pyruvate + Glutamate Pyruvate + NADH⁺ (H⁺) Lactate + NAD⁺).AST activity is analyzed by assessing the amount of oxaloacetate hydrazone formed from 2,4-dinitrophenylhydrazine under the action of oxaloacetate in the reaction of L-aspartate and 2-hydroxyglutarate with the participation of AST.^84-87

Statistical Analysis

Mathematical and statistical processing of the obtained experimental results was carried out by standard methods using special software packages “Microsoft Excel 2007” (Microsoft, USA), OriginPro v. 8.5 SR1 (EULA, USA). The results are presented as M±m, where M is the arithmetic mean and m is the standard error of the mean, calculated based on the results of experiments conducted with n=3-4 repetitions. Also, the level of statistical significance of the values between the experimental results and the control group was calculated based on Student’s t-test and was considered statistically significant at values of p<0.05 and p<0.01. The level of reliability of the difference between the values of the two experimental groups was calculated using the Student’s t-test.^{88,89,90,91,92} In the studies, the value of the hepatoprotective coefficient (HC) of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) was calculated using the following formula (1):

HC=[1-(E-C)/HC-C)]×100%                (1)

HC – is the hepatoprotective activity coefficient (%), E – is the experimental value (CCl₄+substance), C – is the control (healthy animal), and PC – is the pathological control value (CCl₄). The closer the HC – value is to 100%, the higher the level of hepatoprotective activity of the substance.

Results

In the pathogenesis of liver diseases under the influence of oxidative stress, a sharp change in enzyme activity is observed.^88,89

Aminotransferases (AST/ALT) – catalyze the reaction of amination of the aminogroup of an aminoacid to alfa-ketoglutaric acid (in the human body under physiological conditions, ALT is 0.68-1.08 microkat/l, AST – 0.67-0.83 microkat/l), and in experimental animals it has been confirmed that under conditions of hepatotoxic exposure to CCl4 their level increases up to 300% compared to the control.^90-94 Under the influence of CCl₄, the permeability of biological membranes in hepatocytes is disrupted, the activity of transaminases in the cytoplasm increases, the extracellular matrix is destroyed, and the amount of gamma-globulins decreases.⁹⁵ In turn, the next series of studies analyzed the effect of isoquinoline alkaloids and some of their derivatives on the ALT and AST enzymes in blood plasma in experimental toxic hepatitis caused by CCl₄. In the control group, ALT activity averaged 1.05±0.02 and 0.94±0.01 microkat/l in males and females, respectively, and with CCl₄ intoxication, these indicators increased to 12.23±0.19 and 10.53±0.16 microkat/l, respectively. Isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) were found to cause concentration-dependent (25-100 mg/kg) inhibition of ALT activity under CCl₄ intoxication conditions. It was established that the isoquinoline alkaloid F-5 at a maximum concentration of 100 mg/kg reduced ALT activity in male and female mice during CCl₄ intoxication to 8.19±0.14 and 7.14±0.25 microkat/l, respectively (Fig. 1A). It was also noted that the isoquinoline alkaloid F-24 at a maximum concentration of 100 mg/kg reduced ALT activity in experimental toxic hepatitis caused by CCl₄ to 9.43±0.18 and 8.45±0.26 microkat/l in male and female mice, respectively (Fig. 1B).

Figure 1: The effect of isoquinoline alkaloids – F-5 (A), F-24 (B) on the level of ALT in the blood serum in CCl4-induced hepatotoxity in mice. Click here to view Figure

It was found that the KV-6 at a maximum concentration of 100 mg/kg reduced the activity of ALT in experimental toxic hepatitis CCl₄ in male and female mice to 5.10±0.17 and 4.04±0.23 microkat/l, respectively, while the KV-8 (100 mg/kg) had this indicator of 7.13±0.28 and 6.05±0.15 microkat/l, respectively (Fig. 2A, B).

Figure 2: The effect of KV-6 (A), KV-8 (B) on the level of ALT in the blood serum in CCl4-induced hepatotoxity in mice. Click here to view Figure

It was also established that isoquinoline alkaloids (F-5, F-24), and their derivatives (KV-6, KV-8) inhibit AST activity depending on the concentration (25-100 mg/kg) under conditions of CCl4-intoxication. It was noted that the isoquinoline alkaloid F-5 at a maximum concentration of 100 mg/kg reduced AST activity in male and female mice under CCl4-intoxication to 10.35±0.27 and 12.64±0.32 microkat/l, respectively. For the isoquinoline alkaloid F-24 (100 mg/kg), a decrease in the value of this indicator was noted to 13.43±0.16 and 15.82±0.29 microkat/l, respectively (Fig. 3A, B).

Figure 3: The effect of isoquinoline alkaloids – F-5 (A), F-24 (A) on the AST level in the blood serum in CCl4-induced hepatotoxity in mice. Click here to view Figure

It was found that the KV-6 at a maximum concentration of 100 mg/kg reduced ALT activity in male and female mice poisoned with CCl₄ to 7.56±0.35 and 8.42±0.29 microkat/l, respectively, while the KV-8 (100 mg/kg) had this indicator down to 8.37±0.21 and 9.68±0.16 microkat/l, respectively (Fig. 4A, B).

Figure 4: The effect of isoquinoline alkaloids (F-5, F-24) – KV-6 (A), and their derivatives KV-8 (B) on the level of AST in the blood serum in CCl4-induced hepatotoxity in mice.   Click here to view Figure

The increase in ALT and AST levels during CCl₄-intoxication is explained by impaired liver function.^96-102

Under the influence of CCl₄, the generation of free radicals in hepatocytes, structural and functional disturbances of the biological membrane of the LPO process, and changes in the activity of enzyme complexes such as alkaline phosphatase, lactate dehydrogenase, ALT and AST are observed.¹⁰³ Also, the increase in ALT and AST during CCl₄ intoxication is explained by the release of these enzymes into the blood as a result of damage to mitochondrial membranes during hepatopathogenesis.^104-107 In this case, changes in serum activity of ALT, AST, total protein and other indicators against the background of CCl₄-toxic hepatitis are used as objective indicators.^108-111 The hepatoprotective activity of alkaloids and flavonoids, including a decrease in the level of ALT and AST in the blood serum, has been confirmed in studies. It has been established that flavonoids and other polyphenols optimize the levels of ALT, AST, and bilirubin in experimental animals under conditions of toxic hepatitis.¹¹² Under the influence of various stress factors, pathological changes occur in the liver of rats, in which the amount of ALT, AST and bilirubin in the blood serum increases, and quercetin optimizes these indicators.¹¹³ Studies have confirmed that quercetin (100-500 μg/kg) reduces the activity of ALT and AST in blood serum, decreases the amount of malondialdehyde in liver homogenate, optimizes the activity of glutathione peroxidase and superoxide dismutase, and has a therapeutic effect on the morphoanatomical and histopathological changes in hepatocytes under conditions of CCl₄-induced hepatotoxity in mice.¹¹⁴ Polyphenols have been shown to optimize the activity of ALT, AST, malondialdehyde, catalase and glutathione peroxidase, and also improve the cytological structure of hepatocytes in experimental animals with CCl₄-induced hepatotoxity.¹¹⁵ Flavonoids have been shown to exert hepatoprotective effects on hepatocytes by activating the PI₃K/Akt cascade, inhibiting ROS formation, activating the antioxidant cascade (activation of antioxidant enzymes) through modulation of MAPK/Nrf₂ signaling, reducing serum ALT and AST levels, regulating metabolism, inhibiting apoptosis through modulation of the MicroRNA-205/NF-κB cascade, optimizing lipogenesis, and improving mitochondrial function. Thus, under conditions of CCl₄-induced hepatotoxity, an increase in the formation of LPO and ROS in the liver of mice, a significant decrease in the activity of the antioxidant enzyme complex – superoxide dismutase, glutathione peroxidase, glutathione reductase, as well as an increase in the activity of iNOS, ALT and AST are observed. In liver diseases, structural and functional changes in hepatocytes, disturbances in carbohydrate, protein and lipid metabolism, and an increase in the number of enzymes (including aminotransferases) in the blood serum have been confirmed. At the next stage of the experiments, based on the data on the activity of ALT and AST, the values ​​of the coefficient of hepatoprotective action of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) were calculated (Table 2).

Table 2: The effect of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) on the activity of ALT and AST in blood serum in in CCl₄-induced hepatotoxity in mice (M±m)

Note: “kat” (katal) is the enzyme activity that converts 1 mol of substrate into product (P) in 1 second, and 1 microkat/L =  [(10^–6×(mol/sec)]/L (represents the activity that converts 10^–6 mol of substrate into product (P) in 1 second in 1 L of medium [Lothar, 1999; pp.771; Baltierra-Trejo et al., 2015; pp.126-131]. * – p<0.05 compared to control, ** – p<0.01 (n=3-4).

Based on the values of the analyzed parameters (ALT and AST activity in blood serum), the values of the coefficient of hepatoprotective action of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) were calculated (Table 3).

Table 3: alues ​​of the hepatoprotective coefficient of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) for ALT and AST activity

Note: Based on the change in ALT activity (microkat/l) in the blood of mice (male) under conditions of toxic hepatitis caused by CCl₄, for the isoquinoline alkaloid F-5 (100 mg/kg), the coefficient of hepatoprotective action is: HC_F-5=[(1-(8,19-1,05)/(12,23-1,05)]×100=36%.

Based on the analysis of the values of the analyzed parameters under control conditions, CCl₄-induced hepatotoxity and the action of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) at a concentration of 100 mg/kg, it was established that the coefficient of the hepatoprotective action of these substances has a characteristic increase in the series F-24<F-5<KV-8<KV-6. The maximum coefficient of hepatoprotective action was noted in the KV-6 (100 mg/kg), while it was established that the values of ALT (microkat/l), AST (microkat/l) in the blood serum of mice amounted to 64, 72, 82, 67, 73% of the values of CCl₄-induced hepatotoxity in males and females, respectively.

Mice are convenient subjects for various medical and physiological experimental studies, with the mean serum ALT level being 0.85 microkat/L and 0.82 microkat/L in male and female mice, respectively, and the mean AST level being 1.85 microkat/L and 1.95 microkat/L in male and female mice, respectively. Under conditions of CCl₄-induced hepatotoxity, ALT activity in the serum of mice increased from 0.51 microkat/l to 2.80 microkat/l in the control group, and AST activity increased from 0.84 microkat/l to 2.34 microkat/l in the control group. At the same time, gender differences in the values of the analyzed indicators in the blood of experimental animals were confirmed.

In the normal physiological norm in the human body, ALT in men is on average 41 units/l, in women 31 units/l, AST – 37 and 31 units/l, respectively, and an increase in its level (hepatopathologies are characterized by an increase in ALT, AST by 10-20 times compared to the control) indicates liver pathogenesis. In the CCl₄-induced hepatotoxity, changes (increases) in ALT and AST activities are used as sensitive objective indicators for assessing the functional state of the liver. CCl₄ exposure has been shown to induce fibrosis in hepatocytes, including TNF-α and transforming rowth factor (TGF), suggesting that plant extracts with anti-inflammatory activity may have beneficial effects.

Using an experimental model of acute hepatitis caused by CCl₄, it was confirmed that plant substances containing polyphenolic compounds and flavonoids (hepatoprotectors) significantly reduce the induction of lipid peroxidation, an increase in the concentration of toxic metabolites, necrosis and fibrosis in hepatocytes.

It is assumed that the hepatoprotective effect of Taraxacum officinale extract, consisting of polyphenolic compounds, in CCl₄-induced hepatotoxity is associated with the blockade of the KEAP1/JAK2 (Kelch-like ECH-related protein 1/Janus kinase 2) signaling pathway, which is involved in the activation of Nrf2 in the oxidative stress cascade in hepatocytes.

Discussion

Carbon tetrachloride (CCl₄)–induced hepatotoxicity is a well-established experimental model for evaluating liver injury and hepatoprotective agents, as it closely mimics oxidative stress–mediated liver damage observed in human hepatopathologies. In the present study, acute CCl₄ intoxication resulted in a pronounced elevation of serum aminotransferase activities (ALT and AST), reflecting severe hepatocellular injury and increased membrane permeability of hepatocytes. These findings are consistent with earlier reports demonstrating that CCl₄ exposure leads to excessive generation of reactive oxygen species (ROS), initiation of lipid peroxidation (LPO), and subsequent disruption of mitochondrial and cytoplasmic membranes, culminating in enzyme leakage into the bloodstream.

Under physiological conditions, ALT and AST are predominantly localized within hepatocytes and participate in amino acid metabolism via transamination reactions. Their sharp increase during CCl₄ intoxication – up to 10–20-fold compared with control values – confirms extensive hepatocyte damage and impaired liver function. The present data clearly demonstrate that CCl₄ intoxication caused a statistically significant rise in ALT and AST activities in both male and female mice, with a tendency toward higher AST elevation, indicating mitochondrial involvement, as AST is partially localized within mitochondria. Gender-related differences observed in enzyme activities may reflect sex-dependent variations in xenobiotic metabolism, antioxidant capacity, and hormonal regulation of hepatic enzymes.

Administration of isoquinoline alkaloids (F-5, F-24) and their derivatives (KV-6, KV-8) markedly attenuated the CCl₄-induced increase in serum ALT and AST activities in a dose-dependent manner. Among the tested compounds, KV-6 demonstrated the most pronounced hepatoprotective effect, followed by KV-8, F-5, and F-24. This ranking was consistently reflected in both absolute enzyme activity values and calculated hepatoprotective coefficients, indicating a clear structure–activity relationship within the isoquinoline alkaloid series.

The observed decrease in aminotransferase activity suggests that these compounds effectively stabilize hepatocyte membranes, limit enzyme leakage, and preserve cellular integrity under oxidative stress conditions. Such effects are likely mediated by the antioxidant properties of isoquinoline alkaloids, including their ability to scavenge free radicals, inhibit lipid peroxidation, and modulate redox-sensitive signaling pathways. Previous studies have shown that alkaloids and polyphenolic compounds can restore antioxidant enzyme activity (superoxide dismutase, glutathione peroxidase, glutathione reductase), suppress inducible nitric oxide synthase (iNOS), and reduce malondialdehyde accumulation in liver tissue during toxic hepatitis.

The superior efficacy of KV-6 may be attributed to enhanced bioavailability, stronger interaction with membrane lipids, or improved modulation of intracellular signaling pathways involved in oxidative stress responses. It is noteworthy that flavonoids and structurally related polyphenols exert hepatoprotective effects through activation of the PI3K/Akt pathway, induction of the Nrf2-dependent antioxidant response, inhibition of MAPK-mediated ROS generation, and suppression of apoptosis via regulation of NF-kappaB and microRNA-dependent mechanisms. Similar molecular mechanisms may underlie the protective effects observed for the tested isoquinoline alkaloids.

Furthermore, CCl₄-induced hepatotoxicity is associated not only with oxidative stress but also with inflammatory and fibrotic processes mediated by cytokines such as TNF-alfa and transforming growth factor-beta (TGF-beta). The reduction of ALT and AST activities observed in this study suggests that the tested compounds may also interfere with inflammatory signaling cascades and fibrogenic pathways, thereby limiting progression from acute hepatocellular damage to chronic fibrosis. This assumption is supported by literature data indicating that plant-derived hepatoprotectors reduce necrosis, fibrosis, and accumulation of toxic metabolites in hepatocytes.

The calculated hepatoprotective coefficients provide an integrated quantitative assessment of liver protection, allowing direct comparison between compounds. The increasing order of hepatoprotective efficacy (F-24 < F-5 < KV-8 < KV-6) highlights the importance of chemical modification of isoquinoline alkaloids for enhancing biological activity. Notably, KV-6 restored ALT and AST activities to levels approaching physiological norms, indicating substantial recovery of liver function even under severe toxic stress.

Conclusion

Thus, during the experiments it was established that the ALT activity in the control group of male and female mice was 1.05±0.02 and 0.94±0.01 microkat/l, respectively, and with CCl₄-intoxication it increased to 12.23±0.19 and 10.53±0.16 microkat/l. It was found that isoquinoline alkaloids F-5 and F-24 (25-100 mg/kg) significantly reduce the activity of ALT and AST during CCl₄-intoxication, with F-5 (100 mg/kg) reducing ALT activity to 8.19±0.14 microkat/l in males and 7.14±0.25 microkat/l in females, while for alkaloid F-24 these values are 9.43±0.18 and 8.45±0.26 microkat/l, respectively. It has been confirmed that derivatives of isoquinoline alkaloids F-5 and F-24 (KV-6, KV-8) have a more pronounced hepatoprotective effect than the alkaloids, with KV-6 reducing ALT activity in male mice by 5.10±0.17 and in females by 4.04±0.23 microkat/l, while for KV-8 these values are 7.13±0.28 and 6.05±0.15 microkat/l, respectively. Based on the analysis of the coefficient of hepatoprotective action, it was established that hepatoprotective activity increases in the series F-24<F-5<KV-8<KV-6. The obtained results can be used as a scientific basis for the development of hepatoprotective agents depending on the chemical structure based on isoquinoline alkaloids and their derivatives.

Acknowledgement

The author would like to thank Abdullaev Izzatullo and Gayibov Ulugbek, Ergashev Nurali for recommendations

Funding Source

This study was supported by the Project carriedout at the Institute of Biophysics and Biochemistry at the National Universityof Uzbekistan in 2021-2024 “Development of simulation models of pathological conditions and diseases of humans and animalsin vitro and in vivo” (F-OT-2021-465).

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article

Ethics statement

When working with experimental animals in scientific research, the requirements of the rules developed by the International Council for International Organizations of Medical Sciences (1985), the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986), the Declaration developed by the European Union (86/609/EEC), and the Bioethical Statement of the Institute of Biophysics and Biochemistry of the National University of Uzbekistan (No. BRC/IBB; N44/2024/75-1) were observed.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required

Clinical Trial Registration

This research does not involve any clinical trials

Permission to reproduce material from other sources

Not applicable

Author Contributions

• Abduvali Abdubokiev: Investigation, Methodology, Writing-review and editing.
• Khushmatov Shunkor: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing-review and editing.
• Esohon Komilov: Supervision, Writing-review and editing.
• Sirojiddin Ikromov: Data curation, Formal Analysis, Methodology, Writing-original draft.
• Sherzod Zhurakulov: Funding acquisition, Methodology, Project administration.

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