Lead and Cadmium-Induced Testicular Toxicity: Protective Role of Soursop (Annona muricata) Phytowaste Extract via Antioxidant and Anti-Inflammatory Pathways

275 Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 p ISSN: 2635-3342; e ISSN: 2635-3350 Original Research Article Lead and Cadmium-Induced Testicular Toxicity: Protective Role of Soursop (Annona muricata) Phytowaste Extract via Antioxidant and Anti-Inflammatory Pathways *1,3Ekhator, O.C., 1Enaibre, F.O., 1Ahonle, P., 2Egbe, J.U. and 3Imoobe, T.O.T. 1Faculty of Science Laboratory Technology, University of Benin, Nigeria. 2Department of Life Sciences, Manchester Metropolitan University, United Kingdom. 3Department of Animal and Environmental Biology, University of Benin, Nigeria. *[email protected] http://doi.org/10.5281/zenodo.18060863 ARTICLE INFORMATION ABSTRACT Article history: Received 25 Jun. 2025 Revised 17 Sep. 2025 Accepted 06 Oct. 2025 Available online 30 Dec. 2025 Lead (Pb) and cadmium (Cd) exposure induces oxidative stress, inflammation, and testicular dysfunction. This study investigated the protective effects of Hydro-Ethanolic Extract of Annona muricata phytowaste (HEEAM) in male Wistar rats exposed to Pb (200 mg/kg) and Cd (100 mg/kg) in drinking water for 90 days. Treatment groups received HEEAM (250 or 500 mg/kg) or zinc-selenium (Zn-Se) supplements. Pb-Cd exposure significantly increased malondialdehyde (MDA: 19.58 ± 1.05 nmol/mL), testicular Pb (2.30 ± 0.06 mg/kg), Cd (1.62 ± 0.01 mg/kg), sperm abnormalities (51.20%), interleukin 6 (IL-6 (5.76 ± 1.35 pg/mL), and tumor necrosis factor alpha (TNF-α (23.18 ± 0.69 pg/mL), while reducing super oxide dismutase (SOD (4.72 ± 0.63 U/mL), glutathione (GSH (3.97 ± 0.01 nmol/mL), and testosterone (0.48 ± 0.02 ng/mL) (P < 0.05). HEEAM at 500 mg/kg significantly reduced MDA (10.21 ± 0.83 nmol/mL), Pb (0.92 ± 0.04 mg/kg), and Cd (0.65 ± 0.02 mg/kg), improved SOD (9.03 ± 0.52 U/mL), GSH (6.02 ± 0.11 nmol/mL), testosterone (1.85 ± 0.04 ng/mL), and lowered IL-6 (2.10 ± 0.36 pg/mL) and TNF-α (12.12 ± 0.48 pg/mL) (P < 0.05). Sperm count, motility, and morphology were also restored. HEEAM outperformed Zn-Se in reducing oxidative stress and metal burden. Phytochemical screening revealed flavonoids, phenolics, and alkaloids as likely active constituents. These findings highlight Annona muricata phytowaste as a promising, sustainable intervention against heavy metal-induced reproductive toxicity. © 2025 RJEES. All rights reserved. Keywords: Anti-inflammation Lead Cadmium Antioxidant Toxicity 1. INTRODUCTION Contamination by heavy metals, particularly lead (Pb) and cadmium (Cd), has become a critical public health concern due to their ability to induce oxidative stress, inflammation, and tissue damage (Peana et al., 2022). These metals are persistent environmental pollutants that bioaccumulate in living 276 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 organisms, leading to chronic and prolonged toxic effects. Notably, Pb and Cd are known to disrupt the body’s antioxidant defense systems, resulting in excessive generation of reactive oxygen species (ROS) (Goyal et al., 2021). This imbalance promotes oxidative damage to lipids, proteins, and DNA, thereby triggering cellular dysfunction, inflammation, and injury to vital organs such as the liver, kidneys, brain, and reproductive organs (Rajput et al., 2015; Carpena et al., 2022). Among the affected systems, the male reproductive system is particularly vulnerable. Testicular toxicity induced by environmental toxicants like Pb and Cd is associated with impaired spermatogenesis, hormonal imbalances, and histopathological damage. These adverse effects are largely mediated by oxidative stress mechanisms, including lipid peroxidation, mitochondrial dysfunction, and apoptosis of testicular cells, which compromise male fertility (Rajput et al., 2015). Parallel to concerns about heavy metal toxicity is the growing issue of agro-industrial waste, especially in the context of a rapidly increasing global population and the need for greater food production. During processing, parts of fruits and vegetables such as peels, seeds, and skinsare often rich in bioactive compounds which are typically discarded as phytowaste (Carpena et al., 2022). Improper disposal of this organic waste contributes to environmental pollution, including greenhouse gas emissions, water system contamination, and excessive landfill use (Kumar et al., 2017). In response, there is increasing interest in sustainable waste management practices within the agro-food sector. Driven by regulatory pressures, health and environmental concerns, and a growing appreciation for the pharmacological potential of bioactive-rich waste materials, industries are exploring strategies for repurposing phytowaste (Carpena et al., 2022). Utilizing phytowaste as a source of natural antioxidants presents a cost-effective and eco-friendly alternative, with potential applications in the development of novel therapeutics and nutraceuticals (Dike et al., 2021). One promising candidate is Annona muricata, commonly known as soursop, a tropical fruit belonging to the Annonaceae family. Widely distributed in tropical and subtropical regions, including parts of South America, the Caribbean, Southeast Asia, and Africa, Annona muricata has gained attention for its medicinal properties. Various parts of the plant, particularly its fruit, leaves, and seeds, are rich in bioactive compounds such as acetogenins, alkaloids, flavonoids, and polyphenols. These compounds possess potent antioxidant and anti-inflammatory activities, making the plant a viable natural agent against oxidative stress-related pathologies (Moghadamtousi et al., 2015; Santos et al., 2023). Given the increasing prevalence of testicular toxicity due to environmental exposure to Pb and Cd and the potential health benefits of underutilized plant waste, this study aims to investigate the testicular protective effects of Annona muricata phytowaste in Wistar rats exposed to Pb and Cd through drinking water. 2. MATERIALS AND METHODS 2.1. Preparation of the Ethanolic Extract of Annona Muricata Phytowaste Fresh Soursop fruit was bought from New Benin market, Benin City, Edo State. It was identified and authenticated by a plant taxonomist in the Department of Plant Biology and Botany, Faculty of Life Sciences, University of Benin, Benin City, Nigeria. Following a thorough washing of the soursop, the peels, fibers and seeds were taken out and cut into small pieces). The pieces were placed in a dehydrator to dry for 7 days. The fibers, peels, and seeds were separately ground into fine powder using a mechanical blender. In a plastic container, 150 g of the seed, 100 g of the fiber, and 250 g of the peel powder were soaked in a solution of 1000 ml of absolute ethanol and 1000 ml of distilled water, sealed with foil paper to prevent the evaporation of the ethanol and macerated for 72 hours. After 72 hours, the extract was sieved using a sieve cloth and Whatman paper, afterward ensuring a homogenous solution was obtained. The filtrate was poured into stainless containers and was concentrated in a water bath into a paste. The paste form of extract was stored in small containers, properly sealed, labeled, and kept in the refrigerator at 4 °C for preservation prior to usage (Owolabi et al., 2008). 277 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 2.2. Qualitative and Quantitative Phytochemical Screening of Hydro-Ethanol Extracts of Annona muricata Phytowaste Phytochemical screens were performed on the extracts using standard procedures by Trease and Evans (2002) and Lawal et al. (2014). 2.3. Animal Model A total of thirty (30) healthy male Wistar rats with weights ranging from 105.4 g to 154.4 g were purchased from the University of Ibadan animal house in Oyo State and housed in adequately ventilated plastic cages in the Department of Science Laboratory Technology (SLT), University of Benin, Benin City, Nigeria. The rats were acclimatized for two weeks prior to the commencement of the experiments. All rats received food and water ad libitum and exposed to 12 hours’ light and darkness. The rats were weighed once every week for 90 days. They were handled in accordance with laboratory animal standard protocols (National Institute of health USA public Health service policy on humane care and use of laboratory animals, 2002) and the guidelines of the Institutional Animal Ethics Committee of the Department of Science Laboratory Technology, University of Benin. 2.4. Experimental Design After 14 days of acclimatizing to laboratory conditions, the Wistar rats were grouped into five (5) groups of six (6) rats in each group. Group I (control) was administered 0.5 ml of distilled water via oral route, Group 2 was exposed to lead (200 mg/kg) + cadmium (100 mg/kg) in 300ml of drinking water, Group 3 was administered zinc (Zn (1 mg/kg) + selenium (Se (1.5 mg/kg) via oral route and exposed to lead (200 mg/kg) + cadmium (100 mg/kg) in 300ml of drinking water, Group 4 and 5 were administered soursop extract (250 and 500 mg/kg) via oral route and exposed to lead (200 mg/kg) + cadmium (100 mg/kg) in 300ml of drinking water respectively. Orogastric tube was used for the administration. 2.5. Blood Sample Collection After 90 days of administration, the animals were sedated using chloroform-induced anesthesia and humanely sacrificed to expose the abdominal aorta and organs. Blood was collected from the abdominal aorta (4 ml total) and then separated into three parts: 1 ml was placed in a plain bottle for heavy metals analysis, while 3 ml was placed in a plain bottle as whole blood (WB), which was subsequently centrifuged to isolate serum for biochemical and anti-inflammatory analysis. The WB samples were labeled accurately and sent to the laboratory for analysis. The testes were subsequently harvested using dissecting kits, trimmed of all fat and stored in a universal container, it was labeled appropriately. The epididymis was carefully cut for semen analysis. 2.6. Determination of Catalase Activity Catalase activity was measured by adding 0.05 ml of sample or distilled water (blank) into test tubes, followed by 500 µL of 30 M hydrogen peroxide. Immediately before reading, 100 µL of 6M H₂SO₄ and 700 µL of 0.01 M KMnO₄ were added. Absorbance was recorded within 30–60 seconds using a uv/vis spectrophotometer (UV-1100) zeroed with distilled water. A standard was prepared using phosphate buffer, sulfuric acid, and potassium permanganate. 2.7. Determination of Malondialdehyde (MDA) Lipid peroxidation was assessed using the TBARS method. A reagent mix of 15% TCA, 0.25 M HCl, and 0.875% TBA was heated to homogeneity. 200 µL of serum was added to 1.5 ml of this reagent. After cooling, samples were centrifuged at 10,000 rpm for 10 minutes. The absorbance of the supernatant was measured at 534 nm. 278 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 2.8. Determination of Superoxide Dismutase (SOD) SOD activity was evaluated by mixing 1000 µL carbonate buffer with 80 µL of serum and 100 µL of epinephrine. The absorbance was measured every 30 seconds up to 120 seconds at 480 nm. A reference standard was prepared with SOD reagent and adrenaline. 2.9. Determination of Glutathione (GSH) Trichloroacetic acid (TCA) solution (5%, 0.5 µL) was combined with 1.5 µL of blood sample, forming a precipitate. After centrifugation (10 min), 0.5 µL Ellman's reagent and 1.5 µL phosphate buffer were added to fresh tubes, followed by 2.5 µL of the centrifuged sample. Absorbance was read at 416 nm against a blank (without sample). 2.10. Heavy Metals Analysis Blood and testes were homogenized and 0.5 ml was digested with a mixed acid solution (HNO₃:HClO₄:H₂SO₄ in 5:2:1 ratio) until clear. After cooling, the mixture was diluted with deionized water, filtered, and made up to 100 ml. Heavy metal concentrations were measured using Atomic Absorption Spectrophotometry (BK-AA320N) 2.11. Anti-Inflammatory, Pro-Inflammatory, and Apoptotic Analysis Serum was collected after clotting and centrifugation. 100 µL of standards, blanks, and samples were added to ELISA plate wells. After incubation and sequential washing, biotinylated detection antibody, HRPconjugate, substrate, and stop solutions were added per kit instructions (Elabscience). Absorbance was measured at 450 nm using a microplate reader (BK-EL10A). 2.12. Sperm Cell Morphology and Count Sperm was extracted from the vas deferens into a petri dish with pre-warmed saline. A drop was placed on a slide and covered for analysis. 2.13. Morphology Smears were stained with Eosin and Leishman’s stain for 15 minutes, rinsed, dried, and viewed under x100 magnification. Sperm morphology was assessed and quantified by percentage. 2.14. Motility A drop of sperm was placed on a slide and observed under x20 and x40 magnification. Motility was scored based on progressive movement. 2.15. Testosterone Assay Reagents, calibrators, and samples were equilibrated to room temperature. Samples and controls were pipetted into wells with enzyme reagent and incubated for 60 minutes. After washing, a substrate was added, followed by a stop solution. Absorbance was recorded at 450 nm using a microplate reader (BK-EL10A). 2.16. Histopathological Examination The testis specimen was dehydrated in alcohol, processed in xylene and then embedded in paraffin wax before sectioning with a microtome. Sections (5 um thick) of the tissue were strained with hematoxylin and eosin. All sections were viewed under a Leica light microscope (model DM500) at x400 magnification by a histopathologist who was unaware of the experimental grouping and protocols. 279 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 2.17. Statistical Analysis The data was presented as a mean + standard error of mean. Significant differences in the values between the treatment and control groups were assessed using one-way analysis of variance (ANOVA) after confirming the homogeneity of variance among groups. Significance was determined at P<0.05. The analysis was conducted using Graph Pad Prism 9.0. 3. RESULTS AND DISCUSSION The qualitative phytochemical screening of Annona muricata revealed the following phytochemicals as presented in Table 1. The result obtained from the qualitative phytochemical screening of Annona muricata revealed the presence of key bioactive compounds, including glycosides, saponins, alkaloids, phenolics, eugenols, terpenoids, flavonoids, and tannins, with reducing sugars being highly abundant (++) and absent of steroids. These findings align with previous research (Agu et al., 2017; Anaya-Esparza and Montalvo-González, 2020), confirming the plant's rich phytochemical profile and its potential pharmacological benefits, such as antioxidant, anti-inflammatory, and nephroprotective properties. The absence of steroids in this study is consistent with earlier reports (Agu et al., 2017), reinforcing the reliability of the findings. Table 1: Qualitative phytochemical screening of Annona muricata phytowaste extract Phytochemical compound Annona muricata phytowaste. Glycoside + Saponins + Alkaloids + Phenolics + Eugenols + Steroids - Terpenoids + Flavonoids + Tannins + Reducing sugar ++ Key: + = present; ++ = highly present; -= not detected The quantifiable phytochemical content of alkaloid, saponins, phenolics, flavonoids and Tannins in Annona muricata phytowaste extract are presented in Table 2. The quantitative phytochemical estimation of Annona muricata phytowaste extract. Annona muricata contains significantly higher alkaloids (18.32%). The high alkaloid content in Annona muricata confirms their therapeutic potency, particularly in neuroprotection, anticancer activity and anti-inflammatory. This supports the findings of (Agu et al., 2019), where ethanol extracts of Annona muricata was effective in protecting against cycad-induced oxidative stress in wistar rats. High alkaloid content aligns with their ability to chelate heavy metals like Pb and Cd, reducing bioavailability and toxicity (Oladele et al., 2019). Tannin content in Annona muricata was (25.41 mg/kg). Tannins are polyphenolic chemicals with significant metal-chelating capabilities, meaning they bind to heavy metals and limit their absorption and accumulation in tissues (Zhang et al., 2023). The significant presence of tannins Annona muricata waste suggests a protective role in preventing Pb and Cd accumulation. Pb and Cd primarily accumulate in the liver and kidneys, causing chronic nephrotoxicity and hepatotoxicity (Satarug et al., 2020). Tannins Annona muricata can reduce metal absorption in the intestines, minimizing their toxic effects on the body. This supports previous findings that tannins prevent heavy metal-induced liver damage and renal dysfunction by forming insoluble metal complexes that are excreted from the body (Matović et al., 2015). The results in Table 3 indicate levels of Pb and Cd present in the blood of experimental animals before acclimatization, extracts of Annona muricata, administered, feed and water given to Wistar rats ad libitum. All parameters show minute levels of Pb and Cd exposure. 280 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 Table 2: Quantifiable phytochemical content of Annona muricata phytowaste extract. Phytochemicals Annona muricata phytowaste. Alkaloid [%] 18.32 ± 0.52 Saponins [mg/kg] 0.93 ± 0.00 Phenolics [mg/kg] 1.45 ± 0.01 Flavonoids [mg/kg] 1.68 ± 0.07 Tannins [mg/kg] 25.41 ± 1.09 Table 3: Baseline study for heavy metals accumulation in Pb and Cd induced Wistar rats Parameters Lead (200 mg/kg) Cadmium (100 mg/kg) Blood (Control) 0.004 0.002 Extract 0.005 0.003 Water 0.007 0.003 Feed 0.007 0.004 From Table 4, the control group showed a significant increase in the weight at day 30 and day 60 when compared to Pb and Cd exposed group and the combined treatment with Zn and Se (P<0.05). The control group also exhibited very significant increase in the weight at day 90 (243.30 ±1.89) when compared to Pb and Cd exposed group (189.14 ± 0.07) and the combined treatment with Zn and Se (P<0.01). Heavy metal exposure is known to impair growth and metabolic processes, as evidenced by stunted weight gain in Pb and Cd-exposed subjects (Bersenyi et al., 2003; Sarkar et al., 2013). The partial weight recovery observed with combined Zn and Se treatment suggests their supportive role in mitigating toxicity, though their efficacy was surpassed by Annona muricata extract (HEEAM), particularly at the 250 mg dose. This aligns with studies highlighting the role of polyphenols in enhancing nutrient uptake and protecting gastrointestinal integrity (Oladele et al., 2019; Zubaidi et al., 2023). The reduced efficacy of the higher dose (500 mg) may indicate a biphasic response, warranting further investigation into optimal dosing. Table 4: The Effect of HEEAM on the weight of Wistar rats exposed to Pb and Cd in drinking water from day one and an interval of 30 days till the final day of treatment Group Day 1 (g) Day 30 (g) Day 60 (g) Day 90 (g) Control 121.82 ± 0.56 204.14 ± 0.39 195.44 ± 0.56 ab 243.30 ± 1.89 ab Pb (200 mg/kg) and Cd (100 mg/kg) 137.26 ± 0.06 181.36 ± 1.10 168.90 ± 0.30 a 189.14 ± 0.07a Zn (1 mg/kg) and Se (1.5 mg/kg) 139.48 ± 0.89 169.35 ± 0.47 164.85 ± 0.85 a 191.00 ± 0.47a HEEAM (250 mg/kg) 119.20 ± 1.38 183.65 ± 0.83 170.70 ± 0.78 a 209.03 ± 0.96c HEEAM (500 mg/kg) 122.43 ± 0.45 165.80 ± 0.07 165.77 ± 0.4 a 202.70 ± 0.92c Values were expressed as Mean ± SD, n=5. Data with same alphabet in a column demonstrate no significant difference (P0.05) different from each other while data with different alphabet in a column are significantly different from each other. The accumulation of Pb and Cd in blood across groups is presented in Figure 1. The control group had the lowest levels of Cd and Pb, representing baseline levels. The Pb and Cd exposed group showed significant increase in both Cd and Pb levels compared to control. Zn and Se treatment resulted in significant decrease in Cd and Pb levels compared to Pb and Cd exposed group. HEEAM 250mg showed significant decrease in Cd and Pb levels compared to Pb and Cd exposed group, while HEEAM 500mg demonstrated a more significant decrease in Cd and Pb levels when compared to the 250mg/kg group, proving more effective than Zn and Se treatment. The blood analysis demonstrated significant heavy metal accumulation in Pb and Cdexposed subjects, consistent with their known toxicity even at low concentrations (Papanikolaou et al., 2005; 281 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 Jaishankar et al., 2014). The protective effects of Zn and Se were evident in their ability to reduce metal levels, likely through antioxidant and chelation mechanisms (El-Boshy et al., 2015; Kim et al., 2016). However, comparatively HEEAM, especially at 500 mg, exhibited superior efficacy, suggesting a dosedependent enhancement of its metal-chelating and antioxidant properties (Oladele et al., 2019; Mutakin et al., 2022). Figure 1: The effect of HEEAM on Pb and Cd accumulation in the blood of Wistar rats exposed Pb and Cd in drinking water Testicular Pb and Cd levels is presented in Figure 2. The Pb and Cd exposed group exhibited significantly elevated testicular levels of both metals compared to control. The Zn and Se group showed reduced metal levels compared to Pb and Cd group. HEEAM at both 250mg and 500mg demonstrated dose-dependent reductions in metal accumulation, with the 500mg dose achieving Cd and Pb reduction levels comparable to or lower than control levels. Reproductive toxicity, a critical consequence of heavy metal exposure, was evident in elevated testicular metal levels and associated oxidative damage (Järup, 2003; Wirth and Mijal, 2010). The Zn and Se combination mitigated some of these effects, likely through zinc's displacement of Cd and selenium's enhancement of glutathione peroxidase activity (Yamaguchi et al., 2009; Al-Ani et al., 2015). HEEAM, particularly at 500 mg, achieved near-normal metal reduction levels, underscoring the potential of plant-derived antioxidants like flavonoids and acetogenins in restoring testicular function (El-Monem et al., 2020; Mobasher et al., 2024). Assessed oxidative stress markers including SOD, CAT, and GSH levels are presented in Figure 3. In the Pb and Cd exposed group, SOD and CAT were significantly decreased (P<0.05), and GSH was reduced compared to control. Zn and Se treatment restored SOD and CAT activity, with GSH levels showing similarities to control group. HEEAM 250mg showed SOD lower than control but higher than Pb and Cd group, while CAT and GSH were significantly increased (P<0.05) compared to control. HEEAM 500mg demonstrated CAT and GSH significantly increased (P<0.05) compared to control, and SOD significantly increased (P<0.01). Oxidative stress occurs when harmful molecules (free radicals) injure cells more rapidly than the body can heal them. Exposure to lead and cadmium enhances oxidative stress by increasing harmful compounds like MDA and reducing protective enzymes like SOD and CAT. In the Pb and Cd exposed group, SOD and CAT, were significantly decreased (P<0.05), reflecting impaired enzymatic defense. GSH level was reduced compared to the control group resulting in a disrupted cellular redox balance as Cd directly conjugates with GSH to generate inert complexes (Sarkar et al., 2013). 0 1 2 3 4 Control Pb (200 mg/kg) & Cd (100 mg/kg) Zn (1 mg/kg) & Se (1.5 mg/kg) HEEAM (250 mg/kg) HEEAM (500 mg/kg) Concentration (mg/kg) Groups Cd conc (mg/kg) Pb conc (mg/kg) 282 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 Figure 2: The effect of HEEAM on Pb and Cd accumulation in the Testes of Wistar rats exposed Pb and Cd in drinking water In the combined treatment with Zn and Se, the SOD and CAT activity were restored, indicating enhanced antioxidant defenses, which is in line with their roles in mitigating heavy metal toxicity by activating antioxidant enzymes (El-Boshy et al., 2015; Rehman et al., 2018; Das and Al-Naemi, 2019). GSH levels were similar when compared to the control group. The SOD level in the HEEAM 250 mg was lower compared to the control group but higher than the Pb and Cd exposed group which indicates partial restoration of SOD activity. This may be due to the presence of flavonoids, and this aligns with previous research that suggests that flavonoids present in Annona muricata may stabilize catalase structure or directly scavenge hydrogen peroxide (Oladele et al., 2019). Figure 3: The Effect of HEEAM on anti-oxidant markers in the serum of Wistar rats exposed to Pb and Cd in their drinking water CAT and GSH showed a significant increase (P<0.05) compared to the control group. This suggests that Annona muricata effectively restores GSH (Agu et al., 2019). The HEEAM 500mg group levels of CAT and GSH showed significant increases (P<0.05) when compared to the control group, indicating a potent antioxidant effect probably as a result of the induction of Nrf2-mediated antioxidant genes. 0.0 0.5 1.0 1.5 2.0 2.5 Control Pb (200 mg/kg) & Cd (100 mg/kg) Zn (1 mg/kg) & Se (1.5 mg/kg) HEEAM (250 mg/kg) HEEAM (500 mg/kg) Concentration (mg/kg) Groups Cd conc (mg/kg) Pb conc (mg/kg) 0 50 100 150 200 250 300 Control Pb (200 mg/kg) & Cd (100 mg/kg) Zn (1 mg/kg) & Se (1.5 mg/kg) HEEAM (250 mg/kg) HEEAM (500 mg/kg) Concentration (units/ml) Groups SOD (units/ml) CAT (units/ml) GSH (nmol/ml) 283 O.C. Ekhator et al. / Nigerian Research Journal of Engineering and Environmental Sciences 10(2) 2025 pp. 275-289 This aligns with previous research that linked Annona muricata’s protective effects to its potent antioxidant contents (Agu et al., 2019). The significant SOD increase (P<0.01) in HEEAM 500 mg may be due to dose-dependent antioxidant enzymes stimulation, though the high variability points to individual variations in metabolic response to phytochemicals like annonacin and quercetin. The result of MDA levels as a lipid peroxidation marker is presented in Figure 4. Malondialdehyde (MDA) is a lipid peroxidation marker that confirms oxidative membrane damage. In the Pb and Cd exposed group, MDA levels were elevated indicative of severe oxidative stress, lipid peroxidation, and cell membrane damage (Ara and Usmani, 2015; Matović et al., 2015; Dobrakowski et al., 2017; Genchi et al., 2020). In the combination treatment with Zn and Se, MDA reduced compared to the lead and cadmium group but was still significantly increased (P<0.05) compared to control suggesting that less damage occurred. The MDA level in the HEEAM 250 mg was significantly increased (P<0.05) compared to the control, combined treatment of Zn and Se, and HEEAM 500mg group but is lesser than the Pb and Cd exposed group. The MDA levels in the HEEAM 500 mg were lesser compared to the Pb and Cd exposed group but were slightly higher than the control group indicating less oxidative damage. These results support earlier studies that indicated that Annona muricata can assist in preventing organ damage and reducing oxidative stress in cases of heavy metal poisoning (Oladele et al., 2019; Agu et al., 2019). The natural antioxidants found in abundance in the fruit protect the body from harmful toxins (Sears, 2013). Figure 4: The effect of HEEAM on the oxidative stress marker MDA, in the serum of Wistar rats exposed to Pb and Cd in their drinking water Examined inflammatory and apoptotic markers results are presented in Table 5. The control group exhibited low IL-6 levels (1.01 ± 0.58 pg/ml), high IL-10 levels (7.21 ± 0.61 pg/ml), low Caspase-3 levels (0.63 ± 0.11 ng/ml), and low TNF-α levels (6.72 ± 1.50 pg/ml). The Pb and Cd exposed group showed highly significant increase (P<0.001) in Caspase-3 compared to control, significant increase (P<0.01) in TNF-α compared to control, increased IL-6 levels, and reduced IL-10 levels. Zn and Se treatment resulted in increased IL-10 levels compared to control, TNF-α elevated but lower than Pb and Cd group, and Caspase-3 marginally higher than control but reduced compared to Pb and Cd group. HEEAM groups showed IL-6 reduced compared to control in both 250mg and 500mg doses. For TNF-α, the 250mg dose showed significant decrease (P<0.01) compared to Pb and Cd group, while 500mg showed reduced levels but slightly higher than 250mg. IL-10 levels in 250mg were slightly increased compared to Pb and Cd but reduced compared to control, while 500mg showed higher levels than 250mg dose. 0 4 8 12 16 20 Control Pb (200 mg/kg) & Cd (100 mg/kg) Zn (1 mg/kg) & Se (1.5 mg/kg) HEEAM (250 mg/kg) HEEAM (500 mg/kg) MDA (nmol/ml) Groups