Phenylephrine (PHE), noradrenaline (NA), carbachol (CcH), methacholine (McH), n-hexane, CDCl₃, KCl, and Cell Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich (St. Louis, MO, United States). 5-Ethynyl-2ʹ-deoxyuridine (EdU) solution, the EdU-Click 555 Cell Proliferation Assay Kit (Baseclick, Munich, Germany), 4ʹ,6-diamidino-2-phenylindole (DAPI), 96-well plates, and 16-well chambered coverslips were obtained from Thermo Fisher Scientific (Munich, Germany).

Leaves and roots of Acridocarpus smeathmannii were collected in 2023 from a farmland secondary settlement forest in Akinmorin village, Oyo State, Nigeria (7°51′9.25″N, 3°55′52.5″E; 298 m above sea level). The collection was processed out by Dr. Odewo A. Samuel, a botanist at the Forest Research Institute of Nigeria (FRIN), Ibadan. The plant voucher was deposited in the FRIN herbarium (Voucher No. FHI: 113685) and at the University of Lagos Herbarium (LUH 6638) by Dr. O. O. Oyebanji. Research involving the plant was approved by the Health Research and Ethics Committee, College of Medicine, the University of Lagos (CMUL/HREC/09/18/424). In addition, a phytosanitary certificate (No. 0124876) was issued by the Nigeria Agricultural Quarantine Service. The plant material was dried at approximately 23°C and pulverized using a Christy and Norris Lab Mill (No. 50158, England) at the Department of Pharmacognosy, Olabisi Onabanjo University, Nigeria.

A Soxhlet extractor thimble was loaded with 25 g of A. smeathmannii root powder and extracted with n-hexane (EMPLURA^®, Merck KGaA, Germany) (Kale et al., 2025).

Gas chromatography–mass spectrometry (GC-MS) analysis was performed using a Shimadzu QP2010 Plus instrument with a fused silica capillary column (Equity TM-5; 30 m × 0.25 mm, 0.25 µm film thickness; Supelco Merck KGaA, Darmstadt, Germany) (Kale et al., 2025).

Nuclear magnetic resonance (NMR) spectroscopy was performed using 5 mg of A. smeathmannii extract dissolved in 600 μL of CDCl₃ (Kale et al., 2025).

This study was in line with the Declaration of Helsinki and was approved by the Ethics Committee of Ludwig-Maximilians University (LMU), Munich, Germany. Informed consent was obtained from all patients. Prostate (periurethral zone) and bladder (lateral wall) tissues were collected from patients undergoing radical prostatectomy or cystectomy, respectively. Only tissues free from macroscopic tumor infiltration were used. The urothelial layer was removed from bladder samples to isolate the detrusor muscle. Tissues were transported in Custodiol^® solution (Köhler, Bensheim, Germany), and experiments were initiated within 3 h of collection. Tissue strips (6 × 3 × 3 mm) were mounted in 10 mL tissue baths (Danish Myotechnology, Denmark) containing Krebs–Henseleit solution (37°C, pH 7.4) and aerated with 95% O₂ and 5% CO₂. After equilibration at 4.9 mN for 45 min, maximum contraction was induced with 80 mM KCl. Tissues were rinsed and treated with either A. smeathmannii extract (0.05–0.5 mg/mL final concentration) or ethanol control (1%). Cumulative concentration–response curves for NA and PHE in prostate tissues, as well as for CcH and McH in bladder tissues, were generated 30 min after treatment. Each sample was divided into control and A. smeathmannii-treated groups for intra-patient comparisons. Contractile responses were normalized to the KCl-induced maximum (100%). Dose–response curves were fitted using GraphPad Prism (GraphPad Software Inc., San Diego, CA, United States) to calculate maximum agonist-induced contraction (EC₅₀), maximum possible contraction (E_max), and maximum EFS-induced contraction (Ef₅₀) values. EC₅₀ values were also expressed as pEC₅₀ (-log molar concentration) (Kale et al., 2025; Keller et al., 2025).

Porcine hearts and kidneys were obtained from a local abattoir (Metzgerei Brehm, Planegg, Germany), in compliance with LMU ethics approval (LMU/MH060922). Organs were kept at 4°C (Custodiol^® solution) and processed within 2 h. Segments of coronary and interlobar arteries were dissected, cleared of connective and adipose tissue, and cut into rings (3–4 mm). Arterial rings were mounted in tissue baths under a resting tension of 9.8 mN (interlobar) or 19.8 mN (coronary) (Huang et al., 2022), and adjusted during a 45-min equilibration period. Functional studies with A. smeathmannii and controls were conducted similarly to prostate and bladder tissue protocols.

The anticontractile effects of increasing concentrations of A. smeathmannii extract (0.05, 0.1, 0.25, and 0.5 mg/mL) on prostate and bladder tissues were assessed by comparing to contractions induced with ethanol (1%) as a vehicle control. Contractions were elicited using NA (nonselective) and PHE (α₁-selective) agonists (0.1–100 µM) in prostate tissues and using CcH and McH (0.1–1,000 µM) in bladder tissues. Anticontractile effects (A. smeathmannii extract or vehicle) were expressed as a positive percentage relative to the contraction induced by the agonist (Kale et al., 2025; Keller et al., 2025).

The anticontractile effects of increasing concentrations of A. smeathmannii on porcine coronary and interlobar arteries were assessed using a similar protocol. Contractions were induced using CcH (0.1–1,000 µM) in coronary arteries and using NA (0.1–100 µM) in interlobar arteries. A reduction in agonist-induced tone by A. smeathmannii compared to the corresponding vehicle control was considered an anticontractile effect and expressed as a positive percentage relative to the control response (Huang et al., 2022).

Electrical field stimulation (EFS) was used to generate frequency–response curves for contractions mediated by neurogenic activation, 30 min after the addition of A. smeathmannii extract or vehicle (ethanol). EFS evokes action potentials that cause the release of endogenous neurotransmitters, including noradrenaline and acetylcholine. Tissue strips were mounted between two parallel platinum electrodes connected to a CS4 stimulator (Danish Myotechnology Aarhus, Denmark). Square-wave pulses (positive monophasic) of 1 m duration and 20 V amplitude were delivered at frequencies of 2, 4, 8, 16, and 32 Hz, with 60-s intervals between stimulations. Only one frequency–response curve was recorded for each sample. EFS-induced contraction amplitudes were expressed as a percentage of the maximal response to 80 mM KCl. E_max and the frequency producing 50% of maximal EFS-induced contraction (Ef₅₀) were calculated using curve fitting in GraphPad Prism.

WPMY-1 cells were cultured in a medium containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere and seeded at a density of 50,000 cells/well on 16-well chambered coverslips. Cells were treated with 10 µL of A. smeathmannii extract at concentrations of 0.05, 0.10, and 0.25 mg/mL or with vehicle control and incubated for 12 h. Parallel experiments were performed with 24- and 48-h incubation periods. Following treatment, the medium was replaced with 10 mM EdU in an FCS-free medium containing the respective treatments, and cells were fixed with 3.7% formaldehyde. EdU incorporation into DNA was detected using a fluorescent 5-carboxy tetramethylrhodamine probe. Nuclear counterstaining was performed using DAPI. Fluorescence microscopy was used for analysis (excitation: 546 nm; emission: 479 nm) (Tamalunas et al., 2022).

The cell viability in response to A. smeathmannii extract was assessed in 96-well plates seeded with 20,000 cells/well and incubated for 24 h. Cells were treated for 12, 24, or 48 h, after which 10 μL of WST-8 reagent from the CCK-8 was added to each well. Absorbance at 450 nm was measured after 2 h of incubation at 37°C (Wang H. et al., 2020).

The cryo-EM structure of the alpha1A-adrenergic receptor (α1AAR) bound to tamsulosin (complexed with Nb6) is available from the Protein Data Bank (PDB ID: 7YMJ). Protein structures were visualized and analyzed using Swiss-PdbViewer 4.1.0 and Biovia Discovery Studio 2024, respectively. Chemical structures of GC-MS-identified compounds from the hexane root extract of A. smeathmannii were retrieved from ChemAxon and PubChem and converted to a PDB format using Open Babel. These PDB files, with Gasteiger charges, were converted to PDBQT format for protein–ligand docking using AutoDock Vina v4.2.6. Docking results were visualized using PyMOL 2.6.0, and interaction analyses were performed in Biovia Discovery Studio to generate 2D diagrams and 3D visualizations. Pharmacokinetic, physicochemical, and medicinal chemistry properties were evaluated using the SwissADME and pkCSM web servers.

Data from concentration–response and frequency–response curves are expressed as mean ± standard deviation (SD). Post hoc analyses of multiple comparisons at different agonist concentrations or stimulation frequencies were conducted using two-way ANOVA with multiple comparisons. Cell culture results were analyzed using repeated-measures one-way ANOVA and column statistics. All data are presented as scatterplots, showing individual values from each independent experiment (Michel et al., 2020). Each series of organ bath and cell culture experiments consisted of n = 5 independent replicates with paired samples. Statistical analyses were performed using GraphPad Prism 9.5.0 (GraphPad Software Inc., San Diego, CA, United States). E_max, pEC₅₀, and Ef₅₀ values were compared using paired t-tests. Changes in responses are expressed as percentage differences relative to the control [mean difference (MD) with 95% confidence intervals], normalized to the KCl-induced maximal contraction.

Figure 1 shows contractions of human prostate tissue induced by the nonselective adrenergic agonist NA. Following incubation with A. smeathmannii (0.05–0.50 mg/mL) or ethanol (control), the NA response (0.1–100 µM) was concentration dependent but attenuated by the extract compared to controls (Figures 1A–D; Table 1). At 0.05 mg/mL A. smeathmannii, NA-induced contractions were largely unaffected at most concentrations, with only a small reduction [up to 12% at 100 μM NA; MD 12.98 (−25.44 to 55.41) % of KCl]. However, higher A. smeathmannii concentrations produced sustained inhibition. In particular, 0.10 mg/mL A. smeathmannii reduced contractions by 52%–54% at 10, 30, and 100 µM NA [e.g., MD 66.8 (16.1–117.2) %, p = 0.005] (Figure 1B). At 0.25 mg/mL, contraction at 10 µM NA decreased by 51% [MD 50.3 (2.8–97.7) %] (Figure 1C). At 0.50 mg/mL, NA-induced contractions were reduced by 47%–57% across 3–30 µM [e.g., 57.3 (16.5–98.1) %, p = 0.003 at 3 µM] (Figure 1D). Curve-fitting revealed that A. smeathmannii (0.10 and 0.50 mg/mL) lowered E_max values, whereas EC₅₀ shifts were minor and not statistically significant (Figures 1A–H, J–L).

PHE (0.1–100 µM), an α₁-selective agonist, also induced concentration-dependent prostate contractions, which A. smeathmannii reduced in a dose-dependent manner (Figures 1E–H; Table 1). At 0.05 mg/mL A. smeathmannii, contraction at 100 µM PHE decreased by 17% [MD 30.7 (−18.6 to 80.0) %, p = 0.45] (Figure 1E). At 0.10 mg/mL A. smeathmannii, the reduction reached 36.8% [MD 67.0 (−6.7 to 140.7])%, p = 0.09] at the same PHE level (Figure 1F). At 0.25 mg/mL, reductions reached 52.9%–54% at 30 and 100 µM PHE (p = 0.02) (Figure 1G); similar effects occurred at 0.50 mg/mL (44.8%–51.2%; p = 0.02) (Figure 1H). Acridocarpus smeathmannii reduced E_max compared to ethanol controls.

Electric field stimulation (2–32 Hz) to produce neurogenic contraction showed that A. smeathmannii inhibited prostate responses (Figures 1I–L; Table 3). At 0.05 mg/mL A. smeathmannii, inhibitory effects reached 45% at 32 Hz [MD 14.5 (−1.7 to 31.0) %]. At 0.10 mg/mL, inhibition was 54.1% at 16 Hz [MD 65.0 (−1.9 to 131.9), p = 0.05] and 63.9% at 32 Hz [MD 94.7 (27.8–161.5)]. At 0.25 mg/mL, reductions were 50% at both 16 Hz [MD 22.0 (9.7–34.3), p = 0.003] and 32 Hz [MD 30.6 (18.3–42.9), p < 0.0001]. At 0.50 mg/mL, inhibition reached 68% at 32 Hz [MD 176.0 (−5.0 to 357.0), p = 0.05]. Acridocarpus smeathmannii decreased E_max values for EFS-induced contractions; Ef₅₀ was unaffected except at 0.50 mg/mL.

Carbachol-induced (0.1–1,000 µM) contractions of detrusor smooth muscle were attenuated by A. smeathmannii (0.05–0.50 mg/mL) (Figures 2A–D; Table 1). At 0.05 mg/mL, inhibition reached 39.1% at 100 µM [MD 41.5 (11.1–72.0) %, p = 0.003]. At 0.10 mg/mL, inhibition was 47.2% at 3 µM [MD 43.4 (3.3–83.4), p = 0.03] and 35% at 10 µM [MD 42.0 (2.0–82.0), p = 0.03]. At 0.25 mg/mL, inhibition ranged between 57% and 72% across 1–30 µM (p < 0.01–0.03). At 0.50 mg/mL, inhibition reached 59%–62% at 3–30 µM (all p < 0.01–0.02). E_max values were reduced at 0.10 and 0.50 mg/mL, whereas EC₅₀ remained unchanged.

Methacholine-induced (0.1–1,000 µM) bladder contractions were similarly inhibited by A. smeathmannii (Figures 2E–H). At 0.05 mg/mL, inhibition ranged from 33% to 59% across 3–1,000 µM (p < 0.01–0.032). At 0.10 mg/mL, inhibition was 48% (3 µM), 40% (10 µM), and 42% (30 µM) (all p < 0.001–0.004). Both 0.25 and 0.50 mg/mL produced 44% and 65% inhibition at 1 µM. E_max values were reduced by A. smeathmannii at 0.05–0.25 mg/mL.

Acridocarpus smeathmannii inhibited EFS-induced bladder contractions (2–32 Hz) (Figures 2I–L; Table 3). At 0.05 mg/mL, inhibition reached 30.7% at 16 Hz and 15.8% at 32 Hz (p > 0.05). At 0.10 mg/mL, contraction was reduced by 52%–58% across 8–32 Hz (p < 0.001). At 0.25 mg/mL, inhibition ranged from 48% to 52% at 16–32 Hz (p < 0.001–0.003). At 0.50 mg/mL, inhibition reached 69%–86% across 16–32 Hz (p < 0.001–0.009). Acridocarpus smeathmannii lowered E_max without significantly altering Ef₅₀.

In porcine coronary artery, A. smeathmannii (less than 0.50 mg/mL) produced only mild, insignificant inhibition of carbachol-induced contraction (less than 14%) (Figures 3A–D; Table 2). EFS at 32 Hz was inhibited by 76% at 0.10 mg/mL (p = 0.01) and 46% at 0.25 mg/mL (p = 0.06); a concentration of 0.50 mg/mL had no significant effect (Figures 3E–H; Table 3).

In porcine interlobar artery, NA-induced contractions were inconsistently inhibited at less than 0.25 mg/mL, but at 0.50 mg/mL, 100 µM NA-induced contraction decreased by 38.1% (p = 0.03) (Figures 4A–D). For EFS, 0.05 mg/mL A. smeathmannii reduced contraction by 78.7% at 32 Hz (p = 0.004); a concentration of 0.25 mg/mL showed a 35% decrease at 32 Hz (p = 0.03); at 0.50 mg/mL, inhibition at 16 and 32 Hz reached 47%–60% (p < 0.001). E_max and Ef₅₀ remained unchanged.

CCK-8 assays demonstrated a profound, dose-dependent reduction in WPMY-1 cell viability. At 12 h, viability decreased by 98.2% (0.05 mg/mL), 94.5% (0.10 mg/mL), and 83.8% (0.25 mg/mL) compared to controls (Figure 5Ai). After 24 h, colony formation decreased by 94.2% and 50.6% at 0.05 and 0.10 mg/mL, respectively (Figure 5Aii), and by 90.1% and 73.2% at 0.05 and 0.10 mg/mL, respectively, after 48 h (Figure 5Aiii).

EdU assays revealed dose-dependent inhibition of proliferation, with an IC₅₀ of 0.0518 μg/mL at 12 h. Compared to controls, proliferation decreased by 37%, 72%, and 91% at 0.05, 0.10, and 0.25 mg/mL, respectively (Figure 5Bi). Cell counts per field decreased by 65% (0.10 mg/mL) and 78% (0.25 mg/mL) (Figure 5Bii).

Acridocarpus smeathmannii reduced contractility elicited by exogenous (NA and CcH) and endogenous (EFS) stimuli in prostate and bladder tissues, with no significant differences pre- versus post-washout curves (Figure 6).

GC-MS of the hexane extract identified the following major compounds: glutaric acid di(2-methoxybenzyl) ester (10.35%), p-cymene (5.64%), α-terpinene (4.53%), benzyl benzoate (4.44%), tau-cadinol (4.42%), amorpha-4,7(11)-diene-2-α-acetoxy (4.69%), cis-mentha-1(7),8-dien-2-ol (7.40%), pinostrobin chalcone (2.94%), stigmasterol (2.05%), γ-sitosterol (1.41%), α-patchoulene (1.95%), and 9-octadecenoic acid (5.15%), among others (Supplementary Table S1).

The ¹H NMR spectrum of the hexane extract downfield-shifted singlet signals >11.5 ppm suggested the presence of carboxylic acid moieties (R–COOH) and hydrogen-bonded OH protons (R–OH) (e.g., phenolic moieties); strong signals between 7.0 and 8.0 ppm reflected the presence of several aromatic systems, possibly from molecules containing multiple benzene or heteroaromatic rings. Signals in the 3.5–4.0 ppm region suggested alcohol (HO–CH_x–), ether (–O–CH_x–), or ester functional groups, and signals below 3.0 ppm indicated aliphatic chains indicative of branched methylene and methyl groups (Figure 7).