Evaluation of Hepatoprotective Activity of Silybum Marianum on Carbon Tetrachloride Induced Liver Damage in Rats

The liver is a vital organ responsible for numerous physiological functions, including metabolism of carbohydrates, proteins, and lipids, detoxification of xenobiotics, synthesis of plasma proteins, and regulation of biochemical homeostasis. Owing to its central role in the metabolism of endogenous and exogenous compounds, the liver is highly susceptible to injury caused by drugs, chemicals, alcohol, and environmental toxins [1, 2]. Liver diseases constitute a major global health problem, and the search for effective hepatoprotective agents remains an important area of biomedical research [3, 4].Carbon tetrachloride (CCl₄) is one of the most commonly used hepatotoxic agents for inducing experimental liver injury in laboratory animals. Following metabolic activation by the hepatic cytochrome P450 enzyme system, CCl₄ generates reactive free radicals, particularly trichloromethyl (CCl₃) and trichloromethyl peroxy (OOCCl₃) radicals. These radicals initiate lipid peroxidation, leading to oxidative stress, cellular membrane damage, inflammation, and hepatocellular necrosis [5,6]. Therefore, CCl₄-induced hepatotoxicity serves as a reliable experimental model for evaluating the efficacy of hepatoprotective compounds.

Oxidative stress is a key factor in the pathogenesis of liver injury. Excessive production of reactive oxygen species (ROS) overwhelms the body's antioxidant defense mechanisms, resulting in cellular damage and impaired liver function [7]. Consequently, natural antioxidants derived from medicinal plants have gained significant attention for their potential role in protecting the liver against oxidative damage [8].

Silybum marianum (Milk Thistle), a medicinal plant belonging to the family Asteraceae, has been traditionally used for the treatment of liver and biliary disorders for centuries[9, 10]. Its major bioactive constituent, silymarin, is a mixture of flavonolignans including silybin, silydianin, and silychristin. Silymarin possesses potent antioxidant, anti-inflammatory, membrane-stabilizing, and free radical scavenging properties, which contribute to its hepatoprotective effects [11,12,13]. Previous studies have demonstrated that silymarin can inhibit lipid peroxidation, enhance cellular antioxidant defenses, promote hepatocyte regeneration, and protect the liver against various toxic insults [14].

Therefore, the present study was undertaken to evaluate the hepatoprotective activity of Silybum marianum against carbon tetrachloride-induced liver damage in rats. The protective effect was assessed through biochemical and histopathological parameters to validate its potential as a natural hepatoprotective agent[15, 16].

MATERIALS AND METHODS:

Plant Material and Extraction

Dried seeds of Silybum marianum were procured from a reputed herbal supplier and authenticated by a qualified botanist. The seeds were cleaned, shade-dried, powdered, and subjected to Soxhlet extraction using 70% ethanol for 8 h. The extract was concentrated under reduced pressure using a rotary evaporator and stored at 4°C until use. Percentage yield was calculated based on the dry weight of the extract [17,18].

Phytochemical Screening

The ethanolic extract was screened qualitatively for major phytoconstituents such as flavonoids, alkaloids, tannins, phenolics, saponins, glycosides, and steroids using standard phytochemical tests described in the literature [19].

Experimental Animals and Ethical Approval

Healthy male Wistar rats (150–200 g) were obtained from a CPCSEA-approved animal facility and acclimatized for one week under standard laboratory conditions. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) and conducted according to CPCSEA guidelines [20,21].

Acute Oral Toxicity Study

Acute oral toxicity of the extract was evaluated according to OECD Guideline 423. Animals were observed for signs of toxicity and mortality for 14 days after administration. Based on the findings, doses of 100 and 200 mg/kg were selected for further studies [22].

Experimental Design

Table 1: Grouping of Animals for Antioxidant activity

Carbon tetrachloride (1:1 in olive oil) was administered intraperitoneally to induce hepatotoxicity, while treatments were given orally for 14 days [23].

Biochemical Analysis

Blood samples were collected at the end of the study, and serum was separated by centrifugation. Liver function markers including ALT, AST, ALP, total bilirubin, and total protein were estimated using standard diagnostic kits [24].

Estimation of Antioxidant Parameters

Liver tissues were homogenized in phosphate buffer (pH 7.4), centrifuged, and the supernatant was used for antioxidant assays [29].

Malondialdehyde (MDA)

MDA levels were determined using the TBARS method described by Ohkawa . Absorbance was measured at 532 nm, and results were expressed as nmol MDA/mg protein [30].

Superoxide Dismutase (SOD)

SOD activity was estimated by the Misra and Fridovich method based on inhibition of epinephrine auto-oxidation. Results were expressed as U/min/mg protein [31].

Catalase (CAT)

Catalase activity was measured according to Aebi’s method by monitoring the decomposition of hydrogen peroxide at 240 nm and expressed as μmol H₂O₂ decomposed/min/mg protein [32].

Histopathological Evaluation

Liver tissues were fixed in 10% neutral buffered formalin, processed routinely, and stained with hematoxylin and eosin (H&E). Histological examination was performed to assess necrosis, inflammation, fatty changes, and hepatocellular degeneration [33].

Statistical Analysis

Results were expressed as mean ± SEM. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s post hoc test. Values of p < 0.05 were considered statistically significant [34].

RESULT AND DISCUSSION

Plant Material and Extraction

The seeds of Silybum marianum were procured from a reputed herbal supplier and authenticated by a qualified taxonomist. A voucher specimen was deposited in the departmental herbarium for future reference. The seeds were cleaned, shade-dried, powdered, and extracted using 70% ethanol in a Soxhlet apparatus for 8 hours. The extract was concentrated under reduced pressure using a rotary evaporator (40–50°C) and stored in an amber-colored container at 4°C until further use. The extraction process yielded a thick brownish semi-solid ethanolic extract with a percentage yield of 13.5% w/w. The obtained yield indicates efficient extraction of bioactive constituents and is consistent with previously reported yields for Silybum marianum seeds extracted using hydroalcoholic solvents.

 

 

 

 

Table 4: Effect of Ethanolic Extract of Silybum marianum for Biochemical estimation

Group	ALT (U/L)	AST (U/L)	ALP (U/L)	Total Bilirubin (mg/dL)	Total Protein (g/dL)
I	41.25 ± 2.11	76.32 ± 2.45	96.42 ± 3.18	0.72 ± 0.04	6.72 ± 0.21
II	134.62 ± 4.58†	198.56 ± 5.67†	245.36 ± 6.01†	2.35 ± 0.09†	3.85 ± 0.17†
III	52.33 ± 3.04***	89.47 ± 3.01***	112.78 ± 3.92***	0.91 ± 0.05***	6.42 ± 0.18***
IV	66.72 ± 2.95**	102.36 ± 3.29**	132.64 ± 4.11**	1.12 ± 0.07**	5.92 ± 0.20**
V	49.56 ± 2.38***	84.25 ± 2.74***	108.25 ± 3.44***	0.89 ± 0.04***	6.55 ± 0.16***

 

Effect of Ethanolic Extract of Silybum marianum on ALT Levels in CCl₄-Induced Hepatotoxic Rats	Effect of Ethanolic Extract of Silybum marianum on AST Levels in CCl₄-Induced Hepatotoxic Rats
Effect of Ethanolic Extract of Silybum marianum on ALP Levels in CCl₄-Induced Hepatotoxic Rats	Effect of Ethanolic Extract of Silybum marianum on Total Bilirubin Levels in CCl₄-Induced Hepatotoxic Rats
Effect of Ethanolic Extract of Silybum marianum on Total Protein Levels in CCl₄-Induced Hepatotoxic Rats

Figure 1: Biochemical estimation of Silybum marianum for Antioxidant activity

Table 5: Effect of Ethanolic Extract of Silybum marianum for Antioxidant activity

Group	MDA (nmol/mg protein)	SOD (units/min/mg protein)	CAT (μmol H₂O₂ decomposed/min/mg protein)
I	1.28 ± 0.06	12.84 ± 0.45	48.75 ± 1.62
II	4.91 ± 0.12†	4.92 ± 0.22†	19.34 ± 0.95†
III	1.72 ± 0.09***	10.76 ± 0.41***	42.58 ± 1.33***
IV	2.14 ± 0.11**	9.14 ± 0.38**	36.47 ± 1.28**
V	1.54 ± 0.07***	11.42 ± 0.33***	45.12 ± 1.41***

 

Effect of Ethanolic Extract of Silybum marianum on MDA Levels in CCl₄-Induced Hepatotoxic Rats	Effect of Ethanolic Extract of Silybum marianum on SOD Levels in CCl₄-Induced Hepatotoxic Rats
Effect of Ethanolic Extract of Silybum marianum on CAT Levels in CCl₄-Induced Hepatotoxic Rats

Figure 2: Effect of Ethanolic Extract of Silybum marianum for Antioxidant activity

 

 

 

 

Figure 3: Histopathological Photomicrographs of Liver Tissues from Different Groups (Normal, CCl₄ Control, Standard, Extract 100 mg/kg, Extract 200 mg/kg)

The histopathological findings confirmed the hepatoprotective potential of *Silybum marianum* extract against CCl₄-induced liver injury. Severe hepatic damage observed in the CCl₄ control group was markedly reduced following treatment with the extract. The protective effect was dose-dependent, with the 200 mg/kg dose showing greater restoration of normal liver architecture, reduced necrosis, and decreased inflammatory changes. These effects may be attributed to the antioxidant and membrane-stabilizing properties of silymarin, the principal active constituent of *Silybum marianum*. The presence of regenerating hepatocytes further indicates the liver-restorative potential of the extract, with efficacy comparable to the standard silymarin-treated group.

CONCLUSION

The present study demonstrated that the ethanolic extract of Silybum marianum possesses significant hepatoprotective activity against CCl₄-induced liver damage in Wistar rats. Treatment with the extract effectively reduced elevated liver enzyme levels (ALT, AST, ALP, and bilirubin), restored antioxidant defenses (SOD and CAT), and decreased lipid peroxidation as indicated by reduced MDA levels. Histopathological findings further confirmed the protective effect of the extract, showing improved hepatic architecture, reduced necrosis and inflammation, and enhanced hepatocyte regeneration, particularly at the dose of 200 mg/kg. The hepatoprotective effect was dose-dependent and comparable to that of the standard drug silymarin. These findings suggest that the hepatoprotective activity of Silybum marianum may be attributed to its antioxidant and membrane-stabilizing properties. Therefore, Silybum marianum has potential as a natural therapeutic agent for the prevention and management of liver disorders.

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