Effect of Diospyros mespiliformis seed extract on lead toxicity induced changes in prefrontal cortex antioxidants and inflammatory cytokines level in mice

Introduction

Heavy metals, naturally occurring elements with high atomic mass and density, are persistent environmental pollutants that bioaccumulate and resist biodegradation [1,2] . Human activities like mining, smelting, industrial processes, and agricultural use of metal-containing compounds drive environmental contamination and exposure [3] . Exposure occurs primarily through ingestion, inhalation, or dermal contact [4,5] . Non-essential heavy metals, such as cadmium, lead, and mercury, are systemic toxicants, inducing organ damage and generating reactive oxygen species [6,7] . These metals cause carcinogenic, mutagenic, neurotoxic, nephrotoxic, and immunotoxic effects, potentially leading to diseases like cancer or autoimmunity [3] . Lead, widely used in batteries, paints, ceramics, and other products, is a potent neurotoxicant, particularly affecting the developing brains of children [8] . It crosses the blood-brain barrier by mimicking calcium ions, disrupting cellular processes and causing intellectual and behavioral deficits [9] . Lead's systemic toxicity impacts multiple organs, notably the central nervous system, leading to functional impairments [8] . The brain, the central hub of the nervous system in vertebrates and most invertebrates, relies on the prefrontal cortex, located in the frontal lobe, for higher-order cognitive, emotional, and behavioral functions. Lead (Pb) exposure disrupts the blood-brain barrier, primarily damaging astrocytes and secondarily affecting endothelial microvasculature [10] . This leads to altered oxidative stress biomarkers, enzymes, and inflammatory markers, causing significant biochemical changes. Cytokines, small proteins critical for immune responses, coordinate immune cell actions, promote inflammation, and combat infections. Key cytokines include interleukins, interferons, tumor necrosis factors (TNF-α), and colony-stimulating factors. Lead-induced brain lesions primarily affect the prefrontal cortex, cerebellum, and hippocampus, triggering oxidative stress and inflammatory responses [10] . Medicinal plants, rich in phytochemicals, offer diverse biological activities, including antioxidant, antiinflammatory, antimicrobial, anticancer, and neuroprotective effects [11,12,13] . These plants produce secondary metabolites like alkaloids, phenolic acids, polyphenols, terpenoids, and saponins [14,15] . Diospyros mespiliformis, known as African ebony, belongs to the Ebenaceae family [16] . The plant's seed have been reported to offer nutraceutical benefits for cholesterol, diabetes and weight control [17,18] . D. mespiliformis leaves according to Von Maydell, and Ruffo et al. [19,20] treats fevers, leprosy and syphilis. However, there is dearth of information concerning the effect of D. mespiliformis on brain tissue of lead-induced mice. Hence, this research evaluates the changes in antioxidant and inflammatory cytokines in the prefrontal cortex of mice brain after exposure to lead and subsequent treatment with aqueous seed extracts of D. mespiliformis.

Materials and Methods

Collection of Plant (Diospyros mespiliformis) Seeds

The seeds of D. mespiliformis were purchased from a local market in Mararaban Guruku, Karu Local Government Area of Nasarawa State Nigeria. The seeds were identified at the Herbarium Unit of Ekiti State University, Ado-Ekiti Nigeria, as Diospyros mespiliformis (Ebenaceae) with voucher number UHAE 2025045.

Preparation of Plant (Diospyros mespiliformis) Extract

The Diospyros mespiliformis seeds were sun-dried to obtain a constant weight for two weeks, then grounded into powder using Warren blender. Twenty-five grams (25 g) of the grounded seeds was soaked in 100 ml distilled water boiled for 5 minutes and shaken for 10 minutes and allowed to cool then filtered. The extract was concentrated using a rotary evaporator at 40 to 50 o C under reduced pressure. The extracts were then stored frozen until required.

Ethical Approvals

The ethical approval was obtained from the Directorate of Research and Quality Assurance of the Federal University Otuoke, Bayelsa State, Nigeria. To ensure international conformity, the research protocol adhered strictly to the Animal Welfare Act of 1985 of the United States of America, as well as the guidelines of the Research and Institutional Animal Care and Use Committee (IACUC).

Experimental Design Toxicity Testing

Eighteen mice, divided into 6 groups were used for the determination of LD 50 of the extract (D. mespiliformis). The six groups were exposed to 10, 100, 1000, 1600, 2900 and 5000 mg/kg body weight of extract respectively. The LD 50 was subsequently determined by the method of Lorke [21] .

LD 50 = √𝑎 * 𝑏 Where; a = highest dose that gave no mortality, b = lowest dose that produced mortality

Treatment of Animals

Another set of twenty-five mice were divided into five groups and administered the following: Group A is control and were given distilled water. Group B mice received 50 mg/kg body weight of Lead (Pb) while animals in Group C were administered Pb and 200 mg/kg body weight of extract. Group D received Pb and 400 mg/kg body weight and Group E mice were administered Pb and vitamin E (100 mg/kg body weight). The mice were given this treatment daily for 28 days. At the end of the exposure time, mice were made to fast overnight, anaesthetized with chloroform and prefrontal cortex section of brain tissue was obtained.

Preparation of Brain Tissue Supernatant for Biochemical Assay

The mice were dissected, the brain tissue was quickly removed and the prefrontal cortex obtained. (0.025 g) of wet prefrontal cortex brain tissues was homogenized in 2.25 mL of the physiological solution (phosphate buffer, pH 7.4). The resulting homogenates were centrifuged at x5000 g for 20 minutes. The supernatants were decanted and used for further biochemical analysis.

Biochemical Analyses

The concentrations of total protein, protein thiol and tissue reduced glutathione were determined by the methods of Doumas et al. [22] , Sedlack & Lindsey [23] and Ellman [24] . The assays for the activities of superoxide dismutase, catalase and acetylcholinesterase were estimated in brain prefrontal cortex of mice employing the methods of Misra & Fridovich [26] , Kaplan & Groves [27] and Ellman et al. [25] respectively. The methods of Buege & Aust [28] and Green et al. [29] were used to evaluate malondialdehyde and nitric oxide concentrations. The levels of interleukin-6 and tumor necrosis factor-α was measured in prefrontal cortex of mice brain according to the methods of March et al. [30] and Engelmann et al. [31] . While the activity of myeloperoxidase activity in prefrontal tissue supernatant was assayed for, as described by Bradley et al. [32] .

Statistical analysis

The study data were analyzed using IBM SPSS Statistics (version 23.0). The data were presented as the means±standard deviations (SD) of the replicates in each group. The data from the groups were compared using a one-way analysis of variance (ANOVA). Tukey test was utilized as the descriptive statistical tool. The statistical significance level was set at p<0.05.

Results

According to the Globally Harmonized System (GHS) for classification of chemical substances, a substance with an LD 50 greater than 5000 mg/kg is considered to have low acute toxicity. Therefore, the LD 50 of D. mespiliformis is likely >5000 mg/kg, indicating that it is relatively non-toxic in acute exposure. However, we observed the presence of sublethal effects (reduced activity, fatigue, and writhing) at 2900 mg/kg and 5000 mg/kg indicating that the compound may cause physiological or neurological effects at higher doses. 2 showed that the levels of total protein in the prefrontal cortex of lead-induced mice were significantly higher (p<0.05) than the control (group A) but comparable (p>0.05) to that treated mice in groups C and D respectively. The administration of vitamin C reduced total protein concentration as compared with the lead-induced mice. The results also indicated lead exposure significantly reduced (p<0.05) protein thiol levels in group B mice as compared with group A (control). Treatment with D. mespiliformis aqueous seed extract reduces (p<0.05) protein thiol levels. Results in table 3 indicated that lead exposure causes an increase (p<0.05) in the levels of reduced glutathione (GSH) and malondialdehyde (MDA) in group B animals as compared with the control (group A) mice. Treatment with 200 mg/kg b. wt.; 400 mg/kg b.wt. of D. mesipiliformis extract and 100 mg/kg b.wt of vitamin E resulted in decreased (p<0.05) concentrations of GSH and MDA respectively. The activity of superoxide dismutase in prefrontal cortex of mice exposed to lead remain unaltered (p>0.05) while catalase activity was significantly reduced (p<0.05) as compared with the control animals (group A). Treatments with plant's extract and vitamin E increases (p<0.05) catalase activity but such increase in superoxide dismutase activity was only observed in mice treated with 200 mg/kg b.wt. of D. mespiliformis extract. It is observed from the results in table 4 that lead exposed mice had comparable (p>0.05) nitric oxide level with control mice. However, treatment with 400 mg/kg b. wt. of D. mesipiliformis extract reduces (p<0.05) the nitric oxide concentration. The results in table 4 also revealed that the activity of acetylcholinesterase was significantly (p<0.05) inhibited in lead induced mice (group B) as compared with the control mice. Treatment with D. mespiliformis extract at 200 mg/kg (C) and 400 mg/kg (D), as well as vitamin E (E), did not restore AChE activity. Lead exposure (Group B) significantly increased (p<0.05) all inflammatory markers compared to the control (Group A). Treatment with D. mespiliformis extract at 200 mg/kg (Group C) and 400 mg/kg (Group D), as well as vitamin E (Group E), significantly reduced TNF-α and IL-6 levels compared to lead alone, though IL-6 remained elevated relative to control. Myeloperoxidase activity was significantly higher in lead-exposed and extract-treated groups compared to control, except in the vitamin E group, which showed MPO levels similar to control.

Results in Table

Discussion

The acute toxicity study of D. mespiliformis aqueous seed extract revealed no mortality across all tested doses, including 5000 mg/kg, indicating an LD 50 >5000 mg/kg. According to the Globally Harmonized System (GHS), this classifies the extract as having low acute toxicity, suggesting a wide safety margin for acute exposure [33] . However, sublethal effects (e.g., reduced activity, fatigue, writhing) at 2900 mg/kg and 5000 mg/kg indicate potential physiological or neurological impacts at higher doses, necessitating further subacute and chronic toxicity studies. The low toxicity aligns with other medicinal plants, supporting its potential safety for traditional or clinical use, though the observed sublethal effects require comparison with similar plant extracts to elucidate mechanisms and organ-specific toxicity. According to the results, toxicity induction with Pb elicited significant perturbations in biomarkers of protein dysfunction, oxidative stress, neurotoxicity, and inflammation in the prefrontal cortex of mice and consistent with prior reports of Pb's ability to generate reactive oxygen species (ROS) and impair antioxidant defenses [34] . Treatment with aqueous seed extract of D. mespiliformis at 200 mg/kg (Group C) and 400 mg/kg (Group D), or vitamin E (100 mg/kg, Group E), demonstrated varying degrees of amelioration, highlighting the potential neuroprotective role of D. mespiliformis which may be attributable to its rich phytochemical profile including flavonoids and polyphenols with antioxidant and anti-inflammatory properties [35] . The total protein concentration in the prefrontal cortex reflects overall protein content that can be altered by lead-induced neurotoxicity or mitigated by treatments, while protein thiol levels indicate the availability of reduced sulfhydryl groups (-SH) essential for protein function and protection against oxidative stress, with decreases in thiol levels signaling oxidative damage caused by lead-induced ROS oxidation. Significant total protein observed in Pb-exposed mice may reflect compensatory protein synthesis or cellular hypertrophy in response to Pb-induced stress. . Concurrently, protein thiol levels, indicative of sulfhydryl (-SH) groups essential for protein stability and antioxidant function, were markedly reduced in lead induced and treated. This depletion may be caused by Pb's affinity for thiol groups, leading to protein oxidation and conformational changes that exacerbate neurotoxicity. Similar thiol reductions have been documented in Pbexposed models, where oxidative damage contributes to elevated protein carbonyls and impaired cellular redox status [36] . Elevated malondialdehyde (MDA), a lipid peroxidation byproduct observed in Group B mice could be related to Pb's induction of ROS-mediated membrane damage in the brain. D. mespiliformis treatment dose-dependently and vitamin E reduced MDA which suggest its antioxidant capacity to scavenge free radicals. Levels of reduced glutathione (GSH) increased in Group B compared to Group A, potentially as an adaptive response to initial oxidative insult, before depletion in chronic scenarios; this compensatory rise has been noted in early Pb exposure phases. The extract at 400 mg/kg and vitamin E normalized or lowered GSH (groups D & E), indicating reduced demand due to mitigated stress. Superoxide dismutase (SOD) activity remained unchanged in Group B but increased in group C mice, implying enhanced dismutation of superoxide radicals by the extract. Catalase (CAT) activity decreased in Pb-exposed mice relative to control, reflecting impaired hydrogen peroxide detoxification, a common Pb effect in cortical regions. Both doses of the extract restored CAT (groups C & D) maybe likely due to polyphenolic compounds present in the plant enhancing enzymatic antioxidants, as reported in studies on Diospyros species [37] . Acetylcholinesterase (AChE) is an enzyme essential for hydrolyzing acetylcholine in the cholinergic system, with its inhibition serving as a hallmark of neurotoxicity that can impair neurotransmission and cognitive function. At the same time, nitric oxide (NO), a signaling molecule involved in neuronal activity, can contribute to nitrosative stress and neurotoxicity when elevated, particularly during lead exposure. Acetylcholinesterase (AChE) activity was significantly inhibited in prefrontal cortex of lead exposed mice and this could be due to Pb's interference in neurotransmitter systems, leading to cognitive deficits in mice [38,39] . Treatment with plant' extract and vitamin E fail to restore the activity of acetylcholinesterase suggesting limited efficacy against Pb's direct enzyme binding. Nitric oxide (NO) levels did not show a significant increase following Pb and this could mean that NO-mediated nitrosative stress may not be a major pathway in the prefrontal cortex within this model. IL-6 and TNF-α are proinflammatory cytokines involved in neuroinflammatory responses, with elevated levels of both being associated with neuronal damage, cognitive impairment, and neuronal dysfunction, whereas MPO is an enzyme associated with neutrophil activity and oxidative stress, serving as a marker of inflammatory and oxidative damage in tissues. Inflammatory markers (Table 5) demonstrated Pb's proinflammatory action, with significant elevations in IL-6 & TNF in Pb-induced mice as compared with the control, partially attenuated. Treatment with plant's extract reduces the concentrations of these inflammatory markers. Myeloperoxidase (MPO) activity, a neutrophil infiltration marker, increased in Group B. Administration of D. mespiliformis did not appreciably reduced the activity of MPO. However, vitamin E, a potent antioxidant significantly decreases MPO activity.as against that of group B mice.

Conclusion

The acute toxicity study of D. mespiliformis aqueous seed extract showed no mortality up to 5000 mg/kg, indicating an LD50 >5000 mg/kg and low acute toxicity per GHS standards, suggesting a wide safety margin. However, sublethal effects at higher doses (2900 and 5000 mg/kg) necessitate further subacute and chronic toxicity studies. Lead (Pb) exposure (50 mg/kg, 28 days) caused neurotoxicity in mice prefrontal cortex, increasing total protein and malondialdehyde (MDA), reducing protein thiols and catalase (CAT) activity, and elevating IL-6, TNFα, and myeloperoxidase (MPO), indicating oxidative stress and inflammation. Glutathione (GSH) unexpectedly increased, possibly as a compensatory response, while superoxide dismutase (SOD) remained stable. Acetylcholinesterase (AChE) inhibition confirmed cholinergic disruption. D. mespiliformis extract (200 and 400 mg/kg) and vitamin E (100 mg/kg) mitigated these effects. The 400 mg/kg extract dose-dependently reduced MDA, normalized TNF-α, IL-6, and CAT, often surpassing vitamin E, likely due to its antioxidant flavonoids and polyphenols. Neither fully restored AChE or thiol levels, suggesting limited counteraction of Pb's direct binding.

Vitamin E better reduced MPO, indicating broader antioxidant effects. D. mespiliformis shows promise as a neuroprotective agent against Pb-induced neurotoxicity, with low acute toxicity and efficacy rivaling vitamin E. Further studies are needed to clarify its active compounds' mechanisms, long-term safety, and optimization for thiol and cholinergic pathways.

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