Plants serve various purposes in the world, one of which is treating various ailments. The neem tree (Azadirachta indica A. Juss) has been known as a wonder tree since time past in the Indian subcontinent. It is now known all over the world majorly because of its relevance in food production and its medicinal properties (Mugnai 2009). Neem plant is the most diverse and versatile tree with maximum useful non-wood products (leaves, barks, flowers, fruits, seeds, gum, oil and neem cake) (Uwimbabazi, Uwimana & Rutanga 2015). Neem plant popularly known as ‘village pharmacy’ contains limonoids in its seeds, bark and leaves which have been confirmed to exhibit antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer and antifungal properties (Nix 2007).

Since time past, herbal medicine has been known as the oldest form of medicine because it is believed to be safe, gentle, accessible and cost friendly (Abebe 2002). However, a standard dose for these herbal drugs is not established and may result in its minimised potential (lesser dosage) or toxicity effect (excess dosage). The physiological activities of neem (A. indica) leaf meal and the responses of rabbits at graded doses have been reported to have mild to moderately toxic effects (Ogbuewu 2008). A 24-hLD₅₀ of 14 mg/kg for acute toxicity in rats has been earlier reported for neem leaf meal. Gomase, Rangari and Verma (2011) reported that young stem bark extract of A. indica plant protected the liver of rats induced with carbon tetrachloride (CCL₄) using silymarin as a positive control. Doses of 200 mg/kg and 500 mg/kg of A. indica stem bark extract were reported to steady the functional status of liver cells. Also, neem leaf extract has been observed to provide hepatoprotective effects against paracetamol-induced hepatic cell damage in rats (Gupta, Khosla & Singh 2008). However, the present study aims at investigating the hepatoprotective activity of neem seed oil in rats induced with liver damage using CCL₄.

Neem seed oil at 0.25, 0.5 and 1.0 mL/kg significantly lowered the aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in a dose-dependent manner when compared with the control. AST, ALT and ALP levels of CCl₄-treated rats increased significantly (P < 0.05) when compared with the control. All doses of neem oil, positive control and negative control had no significant impact on the total protein (TP) and albumin (ALB) levels when compared with the control (Table 1). No significant difference (P > 0.05) was observed in all haematological parameters for animals treated with all doses of neem oil, positive and negative control when compared with the control (Table 2).

Gross examination of animal organs, livers, hearts, spleens, left kidneys, right kidneys and lungs, shows that there was no significant difference (P < 0.05) in all animal groups when compared with the control (Table 3). Also, the recorded body weight of animals at various days had no significant difference across the various groups (Table 4).

Fatty liver tissues are evident on CCl₄-treated animals only (Figure 1b), whereas moderate to mild fatty liver tissues were observed in silymarin (Figure 1c) and 0.25 mL/kg neem oil–treated rats (Figure 1d). Normal liver cells radially arranged around portal tracts and central veins were evident in 0.5 mL/kg and 1.0 mL/kg of neem oil–treated animals (Figure 1e and f), which are similar to control.

Carbon tetrachloride can cause liver damage at 0.5 mL/kg body weight (Hewawasam et al. 2004), which was less than the dose administered in this study. Some plants have been shown to have protective effects against organ damage, especially those caused by daily exposure to toxins (Amole & Ilori 2010). In the present study, the total bilirubin level increased significantly (P < 0.05) in serum of animals administered with CCl₄ and those co-administered with CCl₄ and silymarin as compared to the control. However, the lower the dose of the neem seed oil, the more the total bilirubin decreased significantly (P < 0.05) compared with the control. These observations are in line with the research findings of Prakash et al. (2008), suggesting that neem oil is more potent than the control drug in maintaining total bilirubin in animals. Conjugate bilirubin increased significantly (P < 0.05) in 1.0 mL/kg of neem oil treatment when compared with control animals. However, there was no significant difference (P > 0.05) in the conjugated bilirubin across the other groups. TP and ALB had no significant difference (P > 0.05) across the various groups, showing that CCl₄, silymarin and neem oil had no effect on the animals’ TP and ALB in this study. However, globulin of animals with CCl₄ treatment only and neem seed oil treatment (1.0 mL/kg) had no significant difference with that of the control group. Animals treated with 0.25 mL/kg and 0.5 mL/kg neem oil had a significantly different (P < 0.05) globulin level from that of the control.

AST (Range: 108–168), ALT (Range: 56–116) and ALP (Range: 45–105) values significantly increased (P < 0.05) in the CCl₄ group. This is in agreement with Kumar et al. (2009), Kaplowitz (2004), Kelly and Skidmore (2002), Basu (2003), Manibusan, Odin and Eastmond (2007) and Planaguma et al. (2005) who reported significant increase in the enzyme activity on administration of CCl₄. The reduction of the ALT, AST and ALP values decreased with increase in dose of neem oil when compared with the CCl₄ group only. More so, silymarin-treated groups had reduced ALT, AST and ALP values when compared with the control. This suggests that neem oil is more active at a higher dose (1.0 mL/kg) and more effective than silymarin (a known hepatoprotective drug).

There was no significant difference (P > 0.05) in the haematological parameters in all groups. This observation suggests that neem oil (dose: 0.25 mL/kg, 0.5 mL/kg, 1.0 mL/kg), CCl₄ (1 mL/kg) and silymarin (1 mg/kg) had no effect on the haematological parameters. The organ weight index and animal body weight were insignificant (P > 0.05) across the various groups in this study. This observation suggests that neem seed oil, CCl₄ and silymarin had no effect on organ weight and body weight.

Histological studies of liver tissues in each group are in agreement with the biochemical result in this study. High fat accumulation and massive necrosis were observed in the liver tissue of animal group administered with CCl₄ only. However, groups treated with silymarin and 0.25 mg/kg neem oil had a moderate fatty liver. In addition, liver tissues of control and 1.0 mg/kg of neem oil–treated group had a normal liver. This suggests that the neem oil dose 1.0 mL/kg is more effective than the lower doses in protecting the liver against CCl₄ damage. This might be because of the presence of some phytocompounds present in neem, which improve the ability of the liver to detoxify itself of chemical contamination (Ogbuewu et al. 2011).

This study reveals that neem oil is hepatoprotective (0.25 mL/kg, 0.5 mL/kg, 1.0 mL/kg) but might become toxic at a comparatively higher dose. Neem seed oil has been reported to show acute toxicity in rats and rabbits with LD50 of 14 mg/kg and 24 mg/kg, respectively, possibly targeting organs for toxic effects, especially the central nervous system and the lungs (Gandhi et al. 1988).

In conclusion, A. indica oil extract showed hepatoprotective activities. The activities were graded dose dependent. Furthermore, the hepatoprotective function of neem oil was comparable to the standard drug (silymarin). However, more work needs to be carried out on determining the toxicity profile (biosafety) of neem seed oil.