The chemicals used were NaAsO₂, ketamine hydrochloride (PVT Ltd., India,) rat loading activities of the antioxidant enzymes (CAT, SOD, GSH), concentration of MDA, and chemokines (TNF-alpha and IL-1ꞵ rat ELISA kit). The silymarin used in this study was silymarin capsules 70 mg/tablet, purchased from the Micro Labs Ltd, India. All other chemicals and reagents used in the studies were of analytical grade and obtained from standard commercial sources.

N. latifolia root extracts were collected from a community garden in Shika, Sabo, Kaduna State, Nigeria immediately after the flowering period. Characterization and authentication of the plant root was done by a Taxonomist of Ahmadu Bello University. The voucher specimen number for the plant is 3276, which is deposited in the herbarium of the department. Air-drying of N. latifolia roots was done at 25 °C until they reached a constant mass and then mechanically pounded using an automated mortar and pestle. Subsequently, each dry powder of the sample was dissolved in 600 ml of 95% ethanol and maintained at the same condition for 48 hours. The tested samples, weighing 500 g, were put into the mortar. The samples were filtered through Whatman filter paper no. 1 and then concentrated at 55 °C using rotary evaporator namely Rotavapor R-300, Buchi. The extract was stored on ice and at a temperature of 4 °C was used in this experiment. The rats under experimentation were administered with N. latifolia extract in a daily dose in distilled water.

The rats used in this experimental study were 25 healthy six-week-old Wistar albino rats with body weight of 140 to 160g. When housed in these chambers it is important to note that the rats were caged in clear plastic cages with a mesh roof and provisions for the feeding and watering of the animals. The rats were maintained under standard housing condition, temperature of 25 ± 2 °C and light-dark cycle of 12 hours in a vermiculated portion of the animal house of the faculty of pharmaceutical science, ABU, Zaria, Nigeria. The rats also received standard rodent diet and water ad libitum in good hygienic conditions as needed by any standard rodent. These rats were allowed to acclimatized for two weeks before the experiment. The follow-up of the study was enhanced with the permit of the ethic committee of the utilisation of animals in the process of ABU Zaria Animal Use and Care Committee (ABUCAUC/2023/033). All animal studies were carried out in compliance with the European Union guidelines for the use of animals for scientific research (Directive 2010/63/ EU).

Twenty-five (25) rats were randomly assigned into five groups of 5 rats each as follows:

Group 1 (normal control) included rats receiving distilled water (10 ml/kg b.w.);

Group 2 (positive control) treated with HFD/ NaAsO₂ rats);

Group 3 rats treated with HFD/NaAsO₂ + N. latifolia roots extract – NlREq (200 mg/kg b.w.);

Group 4 rats treated with HFD/NaAsO₂ + N. latifolia roots extract – NlREq (400 mg/kg b.w.);

Group 5 rats treated with HFD/NaAsO₂ + Silymarin (50 mg/kg b.w.);

The high-fat diet (consisting of 40 grams of animal fat and 100 grams of pelletised rat chow), and drinking water containing NaAsO₂ was supplied (prepared from sodium arenite solution in deionized water to a final concentration of 10 mg/L). The doses of both HFD and NaAsO₂ were selected based on previous studies, which demonstrated their ability to induce organ toxicity in Wistar rats [19, 20]. The selection of N. latifolia at doses of 200 mg/kg and 400 mg/kg was informed by previous studies demonstrating its therapeutic efficacy and favourable safety profile. These doses have been shown to exert significant biological activity without inducing adverse effects on hepatic or renal function, thus supporting their suitability for use in this study [21,22].

A blinded investigator assessed behavioural abnormalities. Neurobehavioral tests (using Elevated plus maze and T-maze test) were conducted in a well-lit testing room and recorded using a digital video recorder. The recordings were analyzed with ANY-maze video tracking software (Kim & Friends Inc., USA), and the data were exported to excel for further evaluation [23].

The EPM test is to evaluate anxiety-like behaviors in rats based on their preference for using an open field test [24,25]. The EPM for rats had two perpendicular open arms of size 21.5 × 7.5 cm and two closed ones 21.5 × 7.5 × 20 cm which were arranged from a central square platform of 7.5 × 7.5 cm. The platform, as well as the floored area of the maze, was made from wooden material; the lateral walls of the closed arms were made of wooden panels painted black. The whole maze was above the ground surface by a mean height of 38 cm [26].

In the course of each trial, one rat at a time was then put at the centre of the maze and the exploratory activities of the rat were tracked for five minutes. Some of the variables quantified included the totals of entries made into the open and closed arms as well as the amount of time spent in the open or closed arms. Data collected from rat activities was omitted for the time spent on the central platform. To avoid other scent clues affecting the subsequent experiments, the maze was washed with 70% ethanol and left to air dry in between trials.

This experiment employed a T-maze apparatus made from black plexiglass to minimise any prospective fresh stimuli during the experiment. This included a long start arm with a width of 16 cm and length of 50 cm and two choice arms each with width of 10 cm and length of 10 cm located perpendicularly on the upper end of the start arm. A partition projected 15 cm from the start arm and forced the rat into one of the left or right goal arm without its reach the junction. A rat was taken separately and placed in the start arm, facing the junction, and the rat was trained to choose the appropriate arm – left or right. A correct alternation was defined as a time when the rat switched between goal arms, an incorrect choice was considered an error. An arm choice was recorded only if all four legs of the rat was placed in it. Every rat was given seven trials with 30 second intertrial interval; the maximum switch between the left and right arms were six as recommended by Jagadeesan et al. [27].

Wistar rat brain tissues were removed and prepared according to procedures elaborated by Zatta et al. [28] and Habila et al. [29]. Following anaesthesia, the treated and control animals were sacrificed by decapitation immediately after sample collection. Then, the brain tissues were immediately dissected out and dropped on an inverted Petri dish which rested on ice. These tissues were stripped and washed in physiological saline and then homogenized in PBS solution 0.7, pH 7:4. In order to pellet the cellular debris from the homogenized samples, they were further centrifuged at 5,000 X g for 15 minutes, and the resultant supernatant was aliquoted and kept at -20 °C for subsequent antioxidant (SOD, CAT, GSH and MDA) and Inflammatory (TNF-α and IL1-β) analysis.

SOD activity was measured following the methodology outlined by Kakkar et al. [30]. The assay involved a reaction mixture containing 2 mL of 0.052 mM sodium pyrophosphate buffer (pH 8.0), 0.1 mL of 186 μM phenazine methosulfate, 0.3 mL of brain supernatant, and 0.1 mL of 300 μM nitro blue tetrazolium. The mixture was subsequently incubated at 37°C for 5 minutes, after which 0.2 mL of NADH (780 μM) was introduced into the solution. The reaction was then allowed to proceed for 2 minutes at 37°C and stopped by the addition of 1.0 mL of glacial acetic acid (17.4 mol/L). The enzymatic activity of the resulting-coloured product was determined at 560 nm against reagent blank using spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan),and reported as units (U) protein^-1.

CAT activity was assessed using a modified version of the Aebi, [31] method. The reaction mixture consisted of 2.5 mL of 50 mM phosphate buffer (pH 7.0), 0.4 mL of 5.9 mM hydrogen peroxide (H₂O₂), and 0.1 mL of enzyme extract. The activity was evaluated by monitoring the reduction in absorbance at 240 nM using spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan), with results expressed as units per milligram of protein.

Using Paglia and Valentine's [32] method, the activity of GSH was measured. The reaction mixture was prepared using 0.1 mL of 0.15 M reduced glutathione (GSH), 0.3 mL of 0.1 M sodium phosphate buffer (pH 7.4), 0.05 mL of 2.25 mM sodium azide, 0.2 mL of 0.85 mM NADPH, 0.05 mL of brain homogenate, and 0.05 mL of water. The mixture was placed on a vortex for 10 minutes Per sample. The experiment was kept at room temperature. To start the enzymatic reaction, 0.05 mL of hydrogen peroxide (H₂O₂, 0.0011 M) were added and the absorbance read at 340 nm for 3 minutesusing spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan). The data obtained for a particular enzyme was reported relative to milligrams of the extracted protein.

Oxidative stress was determined by estimating malondialdehyde (MDA) concentration, according to the method described by Ohkawa et al. [33]. Shortly, 1.5 mL acetic acid 20%, 0.2 mL sodium dodecyl sulfate 8.1%, 1.5 mL thiobarbituric acid 0.8% was mixed with 0.1 mL brain tissue. They added the mixture to heat at 100°C for one hour and finally cooled in running tap water. On cooling, distilled water 1.0 mL and 5.0 mL of n-butanol containing pyridine were also added. The mixture was centrifuged for 10 minutes at 4000 rpm and 4°C then the pink colored compound in the supernatant was carefully extracted for absorption measurement at 532 nm using spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan).

Concentrations of TNF-α and IL1-β (inflammatory cytokines) in the samples were estimated using ELISA kits from Ray Biotech, Inc. USA. In this study, we quantified the cytokines in the blood serum in pg/ml in this study.

The brain was dissected out, the prefrontal cortex, hippocampus cerebellum was carefully excised, tissue was post-fixed in Neutral buffer formalin for 24 hours then processed for histological staining with H&E to demonstrate cortical histoarchitectural changes [34].

The data obtained in these investigations was analyse with help of the SPSS software, version 20, produced by IBM, USA. The statistical analysis was done using a one-way ANOVA and then Tukey post hoc test to ascertain the difference in control group and experimental groups. The results obtained were presented as means ± standard error of the mean wherever applicable. The measure of significance was at p < 0.05.

Table 1, At the onset of the study, there was no statistically significant different (p > 0.05) in the weight of the Wistar rats across and within the experimental groups. At the end the experiment, HFD/NaAsO₂ exposure resulted in a substantial body weight loss (p < 0.05) in all rats treated with HFD/NaAsO₂ alone compared to rats in the treatment groups receiving 400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂. Altogether, body weight gain in HFD/NaAsO₂ treated rats was considerably (p < 0.05) less compared with control group and 400 mg/kg NlREq + HFD/NaAsO₂ treated rats. The change in body weight which was measured in percentage (%) was significantly low in the HFD/NaAsO₂ treated group compared to groups of rats that were treated with 400 mg/kg NlREq + HFD/NaAsO₂.

The data presented in Table 2 demonstrate significant differences across all groups regarding the number of closed and open arm entries by the rats. Similar patterns were observed in the percentage of entries into the closed and open arms and in the index of open arm avoidance. When compared to the control group, rats treated with HFD/NaAsO₂ exhibited reduced time spent in the open arms and increased time in the closed arms, accompanied by a significantly higher number of entries into the closed arms and fewer entries into the open arms.

Conversely, rats treated with 200/400 mg/kg NlREq + HFD/NaAsO₂ exhibited a significant, dose-dependent increase in the time spent in the open arms and a corresponding decrease in the time spent in the closed arms compared to the HFD/NaAsO₂-treated group. Furthermore, the index of open arm avoidance (%) was significantly reduced in HFD/NaAsO₂-treated rats compared to both the control group and the treatment groups (200/400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂).

The statistical analysis of the mean values of spontaneous alternation is presented in Fig. 1. Rats treated with HFD/NaAsO₂ exhibited a significant reduction (p < 0.05) in spontaneous alternation, with a mean value of 43.41 ± 1.39, compared to the control group, which had a mean value of 71.46 ± 2.61. Treatment with 200 mg/kg and 400 mg/kg NlREq + HFD/NaAsO₂ resulted in mean values of 61.35 ± 1.54 and 68.72 ± 1.56, respectively, showing a significant (p < 0.05) increase in the percentage of spontaneous alternation compared to the HFD/NaAsO₂-treated group. Similarly, the 50 mg/kg SLY + HFD/NaAsO₂ treatment group demonstrated a significant improvement in spontaneous alternation compared to the HFD/NaAsO₂-treated group.

The mean levels of oxidative stress biomarkers—SOD (4.10 ± 0.24), CAT (4.20 ± 0.22), and GSH (12.74 ± 1.18)—were significantly lower in the HFD/NaAsO₂-treated group compared to the control group, which showed mean levels of 15.38 ± 0.48 for SOD, 10.60 ± 0.58 for CAT, and 34.72 ± 1.73 for GSH, respectively. Rats treated with 400 mg/kg NlREq + HFD/NaAsO₂ showed a significant increase in SOD (9.42 ± 0.98), CAT (8.20 ± 0.26), and GSH (21.92 ± 1.46) levels compared to those treated only with HFD/NaAsO₂. Similarly, the 200/400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂-treated groups demonstrated significantly elevated CAT and GSH levels relative to the HFD/NaAsO₂ group. However, no significant differences in SOD levels were observed between the 200/400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂-treated groups when compared with the HFD/NaAsO₂ group.

Additionally, the MDA levels in the HFD/NaAsO₂-treated rats (196.20 ± 3.59) were significantly higher (p < 0.05) than those in the control group (148.60 ± 4.56). Treatment with 200 mg/kg and 400 mg/kg NlREq + HFD/NaAsO₂ resulted in a dose-dependent significant reduction in MDA levels, with values of 179.34 ± 2.85 and 156.80 ± 1.83, respectively, compared to the HFD/NaAsO₂-treated group. Similarly, rats treated with 50 mg/kg SLY + HFD/NaAsO₂ exhibited a significant decrease in MDA levels relative to the HFD/NaAsO₂-treated group (Fig. 2).

The statistical analysis of the mean values for oxidative stress biomarkers is presented in Fig. 3. Rats exposed to HFD/NaAsO₂ showed significant increases (p < 0.05) in TNF-α and IL-1β levels, with mean values of 91.21 ± 1.67 and 74.81 ± 2.46, respectively, compared to the control group, which had corresponding mean levels of 41.60 ± 2.06 and 40.73 ± 1.28. Treatment with 200 or 400 mg/kg NlREq in combination with HFD/NaAsO₂ significantly reduced TNF-α levels to 53.06 ± 4.00 and 51.64 ± 4.57, and IL-1β levels to 61.59 ± 5.19 and 62.23 ± 5.11, respectively, compared to the untreated HFD/NaAsO₂ group. Similarly, rats treated with 50 mg/kg SLY + HFD/NaAsO₂ showed a significant reduction in TNF-α and IL-1β levels, with values of 152.00 ± 2.66 and 182.80 ± 8.55, respectively, when compared to the HFD/NaAsO₂ group.

According to histological changes, there were noticeable tissue changes in all groups of the experiment. In the control group the areas of interest showed hippocampus had normal architecture with well developed granular and pyramidal neurons in CA1 and CA3 regions as depicted in the Figure 4A. In contrast, rats placed on HFD/NaAsO₂ developed significant neuronal pathology in terms of shrunken nuclei, vacuolated cytoplasm or pyknotic nuclei or karyolysis in the CA1 region of the hippocampus. Further, areas devoid of pyramidal neurons were noted; and apoptosis in of pyramidal cells of the CA3 region were detected (blue arrow), and clumped neuronal fibrils (arrow) were also noticeable (Fig. 4B). Treatments with 200 mg/kg NlREq + HFD/NaAsO₂ led to mild damage on the granular cells in the CA1 area and the pyramidal cells in the CA3 area with intracellular vacuoles with more intense staining as shown in Fig. 4C. However, the group given 400 mg/kg NlREq+ HFD/NaAsO₂ exhibited near-normal structure or architecture of the brain plus nearly normal histology or arrangement of cells; with only a few pyramidal as well as granule cells remained vacuolated in both the CA1 and CA3 areas of the hippocampus (Fig. 4D). Supplementation with 50 mg/kg SLY along with HFD/NaAsO₂ induced relatively mild neurotoxicity characterized by degeneration of the granular and pyramidal cells as well as vacuolation in the CA1 and CA3 regions of the hippocampus.

The control group exhibited intact neurons and a normal cytoarchitecture in the prefrontal cortex and cerebellum (Fig. 5A), including well-preserved molecular, granular, and Purkinje cell layers (Fig. 6A).

In contrast, the HFD/NaAsO₂ group had pronounced neuronal inflammation and loss of the neurons’cell, and most of the neurons were surrounded by widening of the perineuronal spaces (Fig. 5B), while for the cerebellum there is degeneration of the Purkinje cells (Fig. 6B). The rats, receiving 200/400 mg/kg NlREq, demonstrated neuronal preservation with multiple regenerated neurons and the decrease level of neuropil vacuolation(Fig. 5C&D), while for histology of the cerebellum revealed near normalorientation of the molecular layer, granular layer, and purkinje layer pyramidal and granule cells (Fig. 6C&D). The rats in the group receiving SLY of the 50 mg/kg backed up with HFD/ NaAsO₂ showed close to normal and crowded granular neuronal (Fig. 5E) cells and cerebellum densely populated granular layer, normal molecular layer and slight degeneration of the purkinje layer and cells (Fig 6E).