This study evaluated the hepatoprotective and antioxidant activities (AA) of Pterocarpus santalinoides methanol leaf extract (PSMLE) on carbon tetrachloride (CCl₄)-induced hepatotoxicity in albino rats. Thirty male albino rats randomly assigned into 6 groups (A – F) of 5 rats each were used for the in vivo study. Hepatotoxicity was induced in groups A – E using CCl₄. Group A served as negative control. Groups B, C and D were treated with 50, 250 and 500 mg/kg PSMLE, respectively. Group E was treated with 100 mg/kg Silymarin, while Group F served as normal control. Treatment was given orally twice daily for 15 days, after which markers of hepatotoxicity and oxidative stress were evaluated. The in vitro AA of PSMLE was also evaluated using 1, 1-diphenyl 2-picryl hydrazyl. Results showed that treatment with PSMLE at 250 and 500 mg/kg led to significantly (p<0.05) lower serum alanine aminotransferase and malondialdehyde, significantly (p<0.05) higher superoxide dismutase and glutathione peroxidase levels, while 250 mg/kg dose further led to significantly (p<0.05) lower serum aspartate aminotransferase and serum total bilirubin levels, and significantly (p<0.05) higher serum total protein and serum globulin levels. 500 mg/kg dose treatment additionally led to significantly (p<0.05) lower serum total cholesterol. Treatment at all doses led to significantly (p<0.05) lower liver weight and relative liver weights and significantly (p<0.05) higher catalase and total glutathione levels. The PSMLE exhibited significantly (p<0.05) higher AA at concentrations ≥50 µg/ml in vitro. It was concluded that PSMLE was hepatoprotective and possesses significant antioxidant activity in vivo and in vitro.

Key words: Hepatotoxicity, oxidative stress, antioxidants, Pterocarpus santalinoides leaf extract, carbon tetrachloride.

INTRODUCTION

The liver is a vital organ for metabolism, excretion, clearance and transformation of chemicals in the body

(Singh et al., 2011). It is responsible for the detoxification of drugs and xenobiotics; thus, it is constantly and

variedly exposed to xenobiotics which may induce liver damage (Saukkonen et al., 2006). Most absorbed toxins and toxicants will first pass through liver, and the possible response elicited may range from inflammation to degeneration and/or neoplasia of the hepatocytes (Schiff and Schiff, 1987). Hepatotoxicity is a major health problem, and the manifestations vary from asymptomatic elevation of liver enzymes to fulminant liver failure  (Saukkonen et al., 2006). Toxic liver damage is commonly oxidative stress mediated, and constitutes a large proportion of liver disorders/diseases; its occurrence has been steadily increasing over the years (Suk and Kim, 2012, Rehm et al., 2013; Nwokediuko et al., 2013).

Carbon tetrachloride (CCl₄) is a commonly used model chemical for the experimental induction of hepatotoxicity (Kim et al., 2010). It is metabolized to trichloromethyl (CCl₃) free radical which induces hepatotoxicity by causing peroxidative degradation in the adipose tissue, resulting in fatty infiltration of the hepatocytes (Boll et al., 2001). Following administration, CCl₄ is activated by cytochrome CYP_2E1 and CYP_2B1 to form CCl₃ radical which binds to cellular molecules such as nucleic acids, proteins and lipids, thereby impairing crucial cellular processes like lipid metabolism, with the potential outcome of fatty degeneration (Boll et al., 2001). The CCl₃ radical reacts with oxygen to form highly reactive species, the trichloromethylperoxy (CCl₃OO) radical, which initiates the chain reaction of lipid peroxidation culminating in destruction of polyunsaturated fatty acids (Boll et al., 2001). This causes alteration in permeability of the mitochondria, endoplasmic reticulum, and plasma membranes, resulting in the loss of cellular calcium, disruption of calcium homeostasis and damage/death of hepatocytes (Weber et al., 2003).

Oxidative stress is a state in which oxidation and oxidants exceed the antioxidant systems in the body leading to imbalance between the generation of reactive oxygen species (ROS) and the level of antioxidants in the biological system (Yoshikawa and Naito, 2002). It occurs when free radicals which are not neutralized by antioxidants go on to create more volatile free radicals and damage cell membranes, vessels, proteins, fats and DNA. Biological free radicals are highly unstable reactive molecules that have electrons available to react with various organ substrates such as DNA, proteins and lipids. Oxidative stress is known to be involved in the pathogenesis of a variety of diseases including atherosclerosis, hypertension, diabetes mellitus, ischemic diseases, liver diseases and malignancies (Yoshikawa and Naito, 2002), or may exacerbate their symptoms (Halliwell and Gutteridge, 1989; Valko et al., 2007).

Antioxidants are compounds that inhibit the oxidation of other compounds and prevent chemical damage caused by free radicals (Sies, 1997). Oxidation reactions in living organisms produce free radicals which can initiate chain reactions that may cause damage or death to cells.

Antioxidants terminate these chain reactions by removing free radical intermediates and inhibiting other oxidation reactions (Sies, 1997; Valko et al., 2007). Insufficient levels of anti-oxidants or inhibition of the antioxidant enzymes in living organisms cause oxidative stress which may lead to injury and/or death of cells (Davies, 1995; Valko et al., 2007). Many of the natural antioxidants such as tannins, flavonoids and glycosides are very important in the prevention of diseases associated with oxidative stress (Yi-Fang et al., 2002; Aruoma, 2003).

Some plants such as Cussona barteri (leaves), Lannea vilutina (leaves), Sacoglotis gabonensis (stem bark), Trichilia roka (roots), Tinospora cordifolia (whole plant), Piptadeniastrum africanum (stem bark) and Gongronema latifolium (leaf) amongst others, have been reported to be rich sources of natural antioxidants that can protect against oxidative stress and thus play important role in the chemoprevention of diseases that have their etiology and pathophysiology in ROS (Ames et al., 1993; Atawodi, 2005; Karamalakova et al., 2018; Dlamini et al., 2019). There has been an increase in interest in the therapeutic potential of plants as antioxidants that may reduce free radical-induced tissue injury (Schuler, 1990; Karamalakova et al., 2018). A number of plants such as Ipomoea batatas (leaves), Allium cepa (leaves), Cnestus ferruginea (leaves stem and roots), Splenacentrum jollyanum (leaves and roots) and Voacanga africana (leaves) had been investigated in the search for novel antioxidants (Chu, 2000; Mantle et al., 2000; Koleva et al., 2002; Oke and Hamburger, 2002), while a lot more are still under investigation.