The chemical and reagents used included acetylcholine iodide, gallic acid and Folin‐Ciocalteu reagent obtained from Sigma‐Aldrich (St. Louis, MO, United States). β‐CBP (purity C98.5%), metronidazole, Tween 80 and Durcupan™ ACM Fulka embedding mixture were from Sigma‐Aldrich, United States. Thiobarbituric acid (TBA), trichloroacetic acid (TCA), quercetin, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2‐deoxyribose were procured from Sigma‐Aldrich Chemie (United States). Sodium carbonate, ferric chloride, aluminium chloride, potassium acetate, potassium ferricyanide, tris salt, ferric sulphate and other reagents used were of analytical grade; glass‐distilled water was also used.

The radical scavenging ability of the sample against DPPH free radical was evaluated as described by Gyamfil et al. [10]. An appropriate sample dilution was mixed with 1 mL of 0.4 mM methanolic solution containing DPPH radical. The mixture was left in the dark for 30 min, and the absorbance was measured at 516 nm. The percentage of DPPH radical scavenging ability was subsequently calculated as a percentage of the control.

The method of Puntel et al. was used to determine the iron chelating ability of the samples [11]. A mixture containing 150 μL of freshly prepared 500 μM FeSO₄, 168 μL of 0.1 M Tris–HCL (pH 7.4) buffer, 218 μL of saline solution and an appropriate concentration of the samples was prepared. After incubation for 5 min, 13 μL of 0.25% 1,10‐phenanthroline (w/v) was added. Absorbance was measured at 510 nm, and the iron chelating ability was subsequently calculated and expressed as a percentage.

The reducing property of the samples was determined by assessing the ability of the samples to minimise FeCl₃ solution as described by Oyiazu [12]. A 2.5‐mL aliquot of each sample was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. Following incubation at 50 °C for 20 min, 2.5 mL of 10% trichloroacetic acid (TCA) was added. After centrifugation, 5 mL of the supernatant was mixed with an equal volume of water and 1 mL of 0.1% ferric chloride. Absorbance was measured at 700 nm.

The total antioxidant capacity of the sample (ABTS) was determined against 2,2′‐azino‐bis (ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) radical according to the method described by Re et al. [13]. The ABTS radical was generated by reacting 7 mM ABTS aqueous solution with K₂S₂O₈ (2.45 mM) for 16 h. The absorbance was adjusted at 734 nm, and Trolox equivalent antioxidant capacity (TEAC) was calculated using Trolox as the standard.

The method of Halliwell and Gutteridge was used to determine the ability of the samples to prevent Fe²⁺/H₂O₂ induced decomposition of deoxyribose [14]. Samples were added to a reaction mixture containing 120 μL of 20 mM deoxyribose, 400 μL of 0.1 M phosphate buffer and 40 μL of 500 mM FeSO₄. After incubation at 37 °C for 30 min, the reaction was stopped, and absorbance was measured at 532 nm using a spectrophotometer.

In this study, male and female Wistar rats, each weighing between 200 and 220 g, were utilised. These animals, aged between 9 and 10 weeks, had reached sexual maturity and were sourced from the Federal University of Technology, Akure, Nigeria. All procedures involving the animals adhered to the guidelines set by the National Council for Animal Experiments Control (CONCEA) and the International Guiding Principles for Biomedical Research Involving Animals. Throughout the study, both male and female rats were provided with standard feed.

Following a 2‐week acclimatisation period, the rats were randomly assigned into nine groups, each consisting of six animals (one female in each group), and were fed for 14 days with unrestricted access to food and water. Animal handling strictly followed international standards. The dosage of β‐CBP was based on prior research, with an initial dose of 10 mg/kg/day [15, 16]. However, this study also included a higher dose of 20 mg/kg/day to evaluate a broader dose–response relationship, as the 20 mg/kg dose demonstrated significant effects on erectile function and oxidative stress markers. The inclusion of both dosing regimens aimed to explore the dose‐dependent efficacy of β‐CBP further. Paroxetine (20 mg/kg) and sildenafil citrate (20 mg/kg) were also administered daily, based on previous studies with slight modifications [17, 18].

Group I: normal control rats which were administered the vehicle (1% ethanol)

All groups received treatments with 1% ethanol as the vehicle. On the 14th day, male rats were paired with females for sexual behavioural assays following the protocol described by Thawatchai et al. and modifications informed by Adefegha et al.

The sexual behaviour assay was conducted based on a modified protocol from Thawatchai et al., with additional methodological support from Adefegha et al. This modification involved the use of higher doses of hormones compared to the original study. Specifically, oestradiol benzoate was administered at a dose of 10 μg/mg body weight (five times higher than in Thawatchai et al.), and progesterone was given at 1 mg/kg body weight (double the dose) through subcutaneous and intraperitoneal routes, respectively. While there is a previous study that utilised the modified dosing in the evaluation context of this study [19], the design was informed by existing functional data from Adefegha et al.

Male rats were trained prior to the experiment to ensure that sexual behaviours could be reliably measured. The training involved exposure to receptive females to allow the males to become familiar with the testing environment and reduce variability in sexual behaviour performance. Female rats were ovariectomised to ensure controlled induction of oestrus using the hormone regimen. Only females confirmed to be in oestrus through vaginal smear cytology were included in the assay, following a previously described methodology [20]. Each oestrus female was paired with a single male from each group. After the male test was completed, the female rats were euthanised in accordance with ethical guidelines through an overdose of pentobarbital administered intraperitoneally. This method ensures a humane and rapid process, minimising any animal distress in line with CONCEA guidelines. The same female was not used for multiple males to avoid potential confounding effects of fatigue or prior mating.

Sexual behaviours were observed using a clear rectangular plastic chamber (60 × 40 × 30 cm) in a designated room. A digital video recorder was positioned to capture all activities without interfering with the behaviour of the animals. The recorded videos were analysed to assess key sexual parameters including mounting number, mounting latency, intromission number and intromission latency. Although we did not assess female sexual behaviours directly, care was taken to ensure that all females were proceptive to the males regardless of the treatment group. This was confirmed by the consistent occurrence of proceptive behaviours, such as solicitation and lordosis, prior to pairing with the males.

To evaluate the involvement of CB2 receptors in the effect of β‐CBP, a separate group of animals (Group X) was included, which received a selective CB2 receptor antagonist, AM630, administered at a dose of 3 mg/kg intraperitoneally alongside β‐CBP (20 mg/kg). The CB2 receptor antagonist AM630 was administered 30 min prior to each β‐CBP administration to ensure effective CB2 receptor blockade throughout β‐CBP's action. The 3‐mg/kg dose of AM630, primarily used in mouse models, was selected based on modifications from prior studies, such as García‐Gutiérrez et al., which demonstrated its efficacy and receptor‐binding affinity in Swiss ICR mice [21]. We aligned this protocol with previous approaches, such as those outlined by Munawar et al., in which antagonists were pre‐administered to investigate receptor‐specific effects [22].

Penile tissue was collected from all groups, including the CB2 receptor antagonist group, for Western blot analysis. CB2 receptor expression was quantified using a primary antibody specific for CB2 receptors (Santa Cruz Biotechnology, United States; antibody number sc‐293188). The procedure involved the following steps:

Sample preparation: Homogenised penile tissue samples were lysed in RIPA buffer with protease inhibitors.

Superoxide dismutase (SOD) activity was determined by the method of Mistra and Fridovich [24]. Penile tissue (0.1 mL) was diluted in 0.9 mL of water to make one in 10 dilutions. An aliquot of the diluted homogenate was added to carbonate buffer (0.05 M, pH 10.2) and adrenaline substrate (0.3 M). Absorbance at 480 nm was monitored every 30 s for 150 s.

This was carried out according to the modified method of Clairborne [25]. Test samples (50 μL) were added to a reacting mixture containing H₂O₂ and phosphate buffer (pH 7.0). Absorbance at 570 nm was monitored for 3 min at intervals of 150 s.

Reduced GSH level was determined by the method of Jollow et al. [26]. The supernatant (1 mL) was treated with Elman's reagent and phosphate buffer, and absorbance was read at 412 nm.

This was carried out according to Mannervik and Guthenberg's [27] method. Test samples (30 μL) were added to a reacting mixture containing CDNB and phosphate buffer (pH 6.5). Absorbance at 340 nm was monitored for 3 min at 15 s intervals.

The results were expressed as the means ± SD. Statistical analysis was determined using JMP Pro (Version 16, JMP, SAS Institute Inc.).

The ferric reducing antioxidant properties (FRAP) analysis of β‐CBP yielded results indicating comparatively lower reducing potentials in contrast to sildenafil citrate, as depicted in Figure 1a. Likewise, the ABTS radical scavenging capacity of β‐CBP is illustrated as Trolox equivalent antioxidant capacity in Figure 1b, revealing the highest ABTS radical scavenging ability. The OH radical scavenging capability of β‐CBP, presented in Figure 1c along with associated IC50 values in Table 1, shows a marked increase in the ‘Sick’ group (18.00 μg/mL), indicating a diminished antioxidant capacity relative to the ‘Control’ group (8.50 μg/mL). This supports the hypothesis that oxidative stress contributes to the altered antioxidant defence in the diseased state.

Furthermore, lipid peroxidation, as displayed in Figure 1d, exhibited a significant increase in the percentage of Thiobarbituric acid reactive substances (TBARS) produced upon the addition of pro‐oxidants. Conversely, a concentration‐dependent reduction in the % TBARS produced was observed upon the inclusion of β‐CBP. Figure 1e showcases the DPPH radical scavenging capacity of β‐CBP, while Table 1 details the corresponding IC₅₀ values. This presentation highlights the concentration‐dependent scavenging of DPPH radicals. In addition, Figure 1f illustrates the iron chelating ability of β‐CBP, where results indicate a concentration‐dependent chelation of Fe²⁺. The IC₅₀ values in Table 1 provide insight into the chelating potential of β‐CBP.

Figure 2a illustrates the activity of SOD within liver tissue. The results highlight a significant variance (p < 0.05) between the PIED and control groups. Additionally, all other groups exhibited increased SOD activity, significantly differing (p < 0.05) from both the control and Paroxetine groups. Notably, the β‐CBP groups displayed a further increase compared to the control and PIED groups. Figure 2b presents the impact of β‐CBP on catalase activity in liver tissue. The results reveal a significant rise in catalase activity in the PIED group compared to the control group (p < 0.05). All remaining groups also showed marked differences (p < 0.05) when compared to the control and PIED groups. Moving on to Figure 2c, the influence of β‐CBP on GST activity in liver tissue is depicted. The data illustrates an increase in GST activity in the PIED group, signifying a significant distinction (p < 0.05) from the control group. Furthermore, all groups displayed notable variances (p < 0.05) compared to the normal control and PIED groups.

Figure 2d demonstrates the effect of β‐CBP on glutathione peroxidase (GPx) in liver tissue. The results exhibit a significant elevation in GPx activity in the PIED group (p < 0.05), and all other groups displayed marked differences (p < 0.05) in comparison to the normal control and PIED groups. Figure 2e portrays the effects of β‐CBP on lipid peroxidation within liver homogenate. β‐CBP effectively reduced lipid peroxidation activity in comparison to the PD group. These findings indicate significant distinctions (p < 0.05) among all groups, with the PIED groups treated with β‐CBP displaying a noteworthy difference (p < 0.05) compared to the control groups.

Figure 3 presents the relative expression levels of CB2 receptors across different experimental groups. The CB2 receptor expression was significantly increased in the groups treated with β‐CBP compared to the normal control group. Notably, the β‐CBP (20 mg/kg) group exhibited the highest expression levels among all the groups, including the combination treatment with sildenafil citrate. The PIED rat group showed reduced CB2 receptor expression, which was partially restored by β‐CBP treatment in a dose‐dependent manner. Interestingly, the co‐administration of β‐CBP with the CB2 antagonist AM630 resulted in a notable decrease in CB2 receptor expression, underscoring the role of CB2 receptor activation in mediating the observed effects.