The stem bark of Spondias mombin was collected from Afféry (Region of Adzopé, south of Côte d’Ivoire). The plant was identified by her vernacular name and later authenticated by the National Floristic Center, University Felix Houphouët-Boigny, Côte d’Ivoire. The collected plant material was air dried in darkness at room temperature (20˚C). Stem barks were cut up and stored in tight-sealed dark containers until needed.

Carbon tetrachloride (CCl₄) and sylimarin were obtained, respectively, from Merck (Darmstadt, Germany) and Sigma Aldrich (St Louis, MO, USA).

One hundred grams (100 g) of washed, air-dried powdered stem of the plants were extracted with 1.5 L of methanol at room temperature for 48 hours with stirring at intervals. The methanolic solution obtained was filtered using a Buckner funnel and Whatman No 1. It was concentrated to dryness at 40˚C using a rotary evaporator under reduced pressure. The dried extracts were stored at 4˚C for subsequent analysis. The methanolic extract of the stem bark of Spondias mombin was named MESPM.

Albino rats (130 - 170 g) of both sexes were used for the experiment and resided in the animal rearing house of Ecole Normale Supérieure d’Abidjan (Côte d’Ivoire). They were provided with tap water and regular food with maintained 12 h day and night cycle, at a room temperature of 27˚C ± 1˚C. All experiments were performed in accordance with regulations on experimental animal administration issued by the Ethical Guidelines of the University Committee on Animal Resources (Côte d’Ivoire). Doses of the plant were selected based on our previous acute toxicity study [12] . Thirty animals were divided into 5 groups consisting of 6 animals each ( Figure 1 ). Group I served as a normal control group and received any treatment for fourteen (14) days. Group II served as a negative control group, receiving, intraperitoneally (i.p), only 2 mL/kg/day of 30% CCl₄ dissolved in liquid paraffin, on days 1, 2 and 3. Group III referred as a positive control group, was injected with CCl₄ (day 1, 2 and 3), and orally received silymarin (50 mg/kg/day) for eleven (11) consecutive days. The experimental groups IV and V, after intoxication with 30% CCl₄, the first 3 days, were given, orally, 200 mg/kg/day and 400 mg/kg/day of MESPM, respectively for 11 consecutive days.

Twenty-four hours after the last treatment i.e., on 15^th day, animals were anaesthetized using light ether anesthesia and blood was collected from the retro orbital plexus. The blood samples were allowed to clot and the serum was separated by centrifugation at 2500 rpm for 15 min at 37˚C and kept at –4^°C. Furthermore, the liver was rapidly excised, rinsed in ice-cold saline, and was fixed in 10% formalin solution for histopathological evaluation while a 10% w/v homogenate was prepared using 0.15M KCl, centrifuged at 3000 g for 20 min at 4˚C. This supernatant was ultimately used to assess parameters.

Three techniques were used to quantify the in vivo antioxidant activity in liver tissue and serum samples against CCl₄-induced oxidative stress in rats. TBARS (thiobarbituric acid reactive substances) level was assessed through the process of Ohkawa et al. [16] . The total antioxidant power was evaluated using FRAP (ferric reducing antioxidant power) assay developed by Benzie and Strain [17] . DPPH free radicals scavenging activity of serum samples was determined according to the protocol of Hasani et al. [18] .

To evaluate biochemical markers in liver injury, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein and total bilirubin were measured in plasma samples using an autoanalyzer (Roche/ INTEGRA) and commercial assay kits (COBAS INTEGRA). The manufacturer’s protocol was followed for all estimations.

The livers of rats were macroscopically examined for signs of damage, followed by histopathological analysis of liver samples fixed in 10% formalin solution. Afterward, livers were dissected into approximately 5 mm-thick pieces. These sections were dehydrated in gradual ethanol concentration, cleared in xylene and finally embedded in paraffin wax. The paraffin embedded tissues were sectioned (4 μm to 5 μm) serially using a rotary microtome. Tissue slices were then stained with eosin and hematoxylin for light microscopic observations.

All data are presented as a mean with standard error (SEM). Graphpad Prism version 5.0 (San Diego, United States of America) was used to analyze the data. To analyze data sets, one-way analysis of variance (ANOVA) was used, and statistical analysis was performed using Tukey’s multiple comparison. At p < 0.05, differences were considered statistically significant.

In this study, in vivo antioxidant activity of methanolic extract of S. mombin (MESPM) in serum and liver homogenate was assessed by measuring TBARS levels, FRAP values, and percentage inhibition of DPPH radicals. As shown in Table 1 , administration of CCl₄ in rats caused higher level (p < 0.001) of TBARS in liver tissue by 312.10% compared to normal group. Also, a significant decrease (p < 0.001) in the FRAP values and percentage inhibition of DPPH radicals in serum, respectively, by around 47.08% and 68.30%, was observed in CCl₄ intoxicated group (negative control) compared to normal control. However, post-treatment, both with MESPM, at 400 mg/kg of body weight and silymarin (50 mg/kg body weight), in CCl₄-intoxicated rats, reduced significantly (p < 0.001) hepatic concentration of TBARS, respectively by 43.20% and 39.90%, whereas exhibited a significant increase (p < 0.05) of percentage inhibition of DPPH radicals as well as total antioxidant power, compared to negative control group without bringing them back to normal values. After treatment with MESPM (400 mg/kg of body weight) and silymarin, serum DPPH radical scavenging activity returned to normal levels, except for TBARS concentrations.

The values of the parameters studied are expressed as mean ± standard deviation, n = 6. The symbols (*#) represent statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001 denotes a significant difference between the negative control group and the other experimental groups. #p < 0.05; ##p < 0.01; ###p < 0.001 denotes a significant difference between the normal control group and the treatment groups.

The effect of methanolic extract of stem barks of S. mombin (MESPM) on serum biochemical parameters in CCl₄-intoxicated rats is summarized in Table 2 . The ALT, AST, ALP, and total bilirubin levels in the CCl₄-intoxicated group (negative control) were significantly raised (p < 0.001) in serum, respectively by 132.65%, 126.98%, 164.83%, and 465.21%, whereas total proteins were decreased (27.61%) as compared to normal control. Serum marker enzyme activity and bilirubin level in rats treated after CCl₄ intoxication with 200 mg/kg of body weight (except for AST) and 400 mg/kg of body weight of MESPM, are significantly reduced, while total proteins concentration increased compared to negative control. Furthermore, only rats treated with MESPM, at 400 mg/kg of body weight and silymarin (50 mg/kg of body) reversed these biochemical parameters, bringing them closer to normal control values (p > 0.05).

The values of the parameters studied are expressed as mean ± standard deviation, n = 6. The symbols (*#) represent statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001 denotes a significant difference between the negative control group and the other experimental groups. #p < 0.05; ##p < 0.01; ###p < 0.001 denotes a significant difference between the normal control group and the treatment groups.

Gross necropsy and histopathological study were performed on the livers to observe any irregularities or anomalies on the structure. Figure 2 shows macroscopic observations. A normal control group liver displayed a reddish-brown color, a regular surface, and a normal anatomical structure. No lesions are visible to the naked eye ( Figure 2(a) ). According to Figure 2(b) , liver treated with only CCl₄ showed an unregular surface with a rounded shape, dented by large cysts, suggesting severe hepatocellular damage. Its surface is dotted with white spots which are signs of necrosis. Post treatment of intoxicated liver with either the high dose of MESPM (400 mg/kg of body weight) and silymarin presented a pale liver color with minimal white spots and micronodules ( Figures 2(d)-(e)) . No serious lesions such as macronodules or cysts are detectable, indicating a regression of lesions induced by CCl₄.

Histopathological examination of the liver section from the normal control group showed no abnormalities in hepatic cellular architecture ( Figure 3(a) , (Figure 3(b) ). Liver cells spaced by sinusoids were arranged radically from central vein. In the negative control group, a very significant dilatation of the centrilobular vein and numerous foci of inflammation around this vein is noted ( Figure 3(c) ). Liver tissue’s trabecular organization is completely unrecognizable due to lysis and loss of cellular cohesion. Furthermore, necrotic lesions are present throughout the entire photomicrograph and significant inflammation foci are identified. In addition, hypercolored nuclei of hepatocytes are a sign of pyknosis ( Figure 3(d) ). The photomicrograph of the liver of rats intoxicated with CCl₄ then treated with 400 mg/kg of MESPM or silymarin indicated ballooned hepatocytes and moderate dilatation of the centrilobular vein are noted as well as relative lesions of necrosis and inflammation infiltrates ( Figures 3(g)-(j) ). Post-treatment with these doses generally resulted in a strong regression of CCl₄-induced hepatocyte lesions in rats compared to rats in negative controls.