Phytochemical Characterisation, In Silico Androgen Receptor Inhibitory Activity, and Fertility-Enhancing Potential of Aqueous–Ethanol Root Extract of Triclisia subcordata Oliv.

Patricia A. Onocha¹, Jamiu A. Akintola¹*, Mubo A. Sonibare ^2,3,

Ganiyat K. Oloyede¹, Islamiyat O. Arilomo², Isiaka Mohammed¹, Ruth S. Aladeloye⁴, Sodiq O. Abdulsalam⁴

1.      Natural Products/Medicinal Chemistry Unit, Department of Chemistry, University of Ibadan, Ibadan, Nigeria

2.      Biodiversity Conservation and Medicinal Plants Research Group, Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria

3.      Directorate, Pan African University Life and Earth Sciences Institute (including Health and Agriculture), University of Ibadan, Ibadan, Oyo State, Nigeria

4.      Molecular Drug Metabolism and Toxicology Unit, Department of Biochemistry, University of Ibadan, Ibadan, Nigeria

*Correspondence: jamiuakintola@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5617  

INTRODUCTION

Infertility is a globally significant reproductive health challenge, affecting approximately one in six adults of reproductive age worldwide (World Health Organization [WHO], 2023). Among affected couples, male factors contribute to approximately 50% of cases either as a sole cause or in combination with female factors (Agarwal et al., 2021; Eisenberg et al., 2023). The global burden of male infertility is not only substantial but escalating: data from the Global Burden of Disease Study indicate that the prevalence of male infertility increased by over 74% between 1990 and 2021, with projections suggesting a continued upward trajectory through 2040, particularly in low- and middle-income countries (De Jonge et al., 2024; Zeng et al., 2025). Despite representing one half of the infertility equation, male reproductive health remains comparatively under-investigated and under-resourced relative to its female counterpart (Agarwal et al., 2015).

At the pathophysiological level, oxidative stress has emerged as one of the principal mechanisms underpinning male factor infertility. An imbalance between reactive oxygen species (ROS) production and the available antioxidant defence capacity impairs sperm quality across multiple parameters, including count, motility, morphology, and DNA integrity (Kaltsas et al., 2025; Wang et al., 2025). Exogenous contributors to this oxidative burden include environmental pollutants, endocrine-disrupting chemicals, tobacco smoke, and lifestyle-related factors, all of which are increasingly prevalent in sub-Saharan African urban settings (Ribas-Maynou & Yeste, 2020). Conventional pharmacological interventions; predominantly phosphodiesterase-5 (PDE5) inhibitors such as sildenafil address erectile dysfunction but have limited utility in correcting underlying spermatogenic deficits, and are further constrained by cost, systemic side effects, and limited accessibility in resource-limited contexts (Eisenberg et al., 2023). These limitations have intensified scientific interest in phytotherapeutic androgenic modulation as a complementary or alternative therapeutic strategy.

African traditional medicine has long documented the use of plant-based preparations for the management of male reproductive disorders. A systematic review of medicinal plants from Central and West Africa identified that plant extracts studied for male fertility enhancement demonstrated improvements in sperm count, motility, and viability, and modulated testosterone, luteinising hormone, and follicle-stimulating hormone levels, with the majority of included studies originating from Nigeria (Adeniyi et al., 2025). Ethnobotanical surveys conducted in Oyo State, Nigeria have similarly documented a rich indigenous pharmacopoeia targeting male reproductive health, with several plant species employed as aphrodisiacs, spermatogenic tonics, or treatments for erectile dysfunction (Borokini et al., 2013). Among the plant families with documented androgenic potential across tropical Africa, the Menispermaceae occupies a pharmacologically distinguished position.

The family Menispermaceae comprises approximately 70 genera and over 500 species distributed across tropical and subtropical regions. It is characterised by a rich diversity of biologically active bisbenzylisoquinoline alkaloids, and its members have been widely applied in traditional medicine systems across Africa, Asia, and the Americas for the management of fever, inflammation, pain, gastrointestinal disorders, and reproductive conditions (De Wet et al., 2007; Ogunlakin et al., 2023). T. subcordata Oliv. is a woody liane of this family distributed across lowland rainforests from Guinea to Nigeria and Angola (Abo et al., 2011). Locally designated as Kanranjangbon or Alugbọ nrọ in Yoruba-speaking communities of southwestern Nigeria, the plant’s roots have been traditionally applied in the management of male sexual dysfunction, gastrointestinal disorders, dysentery, malaria, and inflammation (Borokini et al., 2013; Ogunlakin et al., 2023). Scientific investigations have attributed antioxidant, antibacterial, antifungal, antimalarial, smooth muscle-relaxing, and enzyme-inhibitory properties to various parts of the plant (Ogunlakin et al., 2023; Ogunlakin et al., 2025). Notwithstanding this growing pharmacological interest, no published study has directly characterised the androgenic receptor binding interactions of the plant’s constituents, nor evaluated its fertility-enhancing potential using an in vivo experimental model integrated with in silico molecular docking.

Drosophila melanogaster has gained broad acceptance as a cost-effective, genetically tractable, and ethically accessible model organism for evaluating the reproductive effects of xenobiotics and plant-derived compounds (Lopez-Ortiz et al., 2023). Its conserved male reproductive processes, high fecundity, short generation time, and well-characterised genome make it particularly well-suited for screening phytochemical interventions targeting fertility endpoints such as mating latency, copulation duration, offspring emergence, and adult survival (Abolaji et al., 2025; Adeleke et al., 2022). The model has been validated for detecting male reproductive toxicity phenotypes comparable to those observed in mammalian systems, and its use is aligned with international recommendations for the use of invertebrate alternatives in reproductive toxicology research (Misra et al., 2014).

The present study therefore sought to address a gap in the evidence base for T. subcordata as a phytotherapeutic agent in male infertility management. Using an integrated pharmacological and computational approach, this investigation assessed the phytochemical profile, GC–MS-based chemical composition, in silico androgen receptor binding affinity and drug-likeness, antioxidant capacity, cytotoxicity, and in vivo reproductive effects of the aqueous–ethanol root extract of T. subcordata in D. melanogaster. The findings presented here constitute the first mechanistically grounded, computationally-supported characterisation of this plant’s androgenic potential, contributing to the scientific validation of its traditional use and informing future pre-clinical and clinical investigations.

MATERIALS AND METHODS

Plant Material Collection and Authentication

Root samples of T. subcordata Oliv. were collected from the Botanical Garden of the University of Ibadan, Oyo State, Nigeria. The plant was identified and authenticated by a botanist at the University of Ibadan Herbarium, Department of Botany, where a voucher specimen was deposited and assigned accession number UIH-23535. Following authentication, the roots were washed with distilled water, air-dried at ambient temperature for 14 days, and subsequently reduced to a uniform pellet-size using a mechanical blender. The crushed root material was stored in clean, labelled, airtight polyethylene bags pending extraction.

Extraction and Yield Determination

One kilogram of pulverised root material was subjected to cold maceration in a 65:35 (v/v) distilled water–ethanol mixture at ambient temperature for 72 hours, with intermittent manual agitation. The macerate was sequentially filtered through cotton wool and Whatman No. 1 filter paper. The filtrate was concentrated under reduced pressure using a Büchi rotary evaporator (Model R-210) and the concentrate lyophilised to yield a dry extract designated AETS (Aqueous–Ethanol Extract of T. subcordata Roots). The dried extract was stored at −20 °C until use, and percentage yield determined gravimetrically.

Phytochemical Screening

Qualitative phytochemical screening of AETS was conducted to detect major classes of secondary metabolites following the standard procedures described by Evans (2009) and Sasidharan et al. (2011).

GC–MS Analysis

Chemical profiling of AETS was performed using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass selective detector, fitted with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Ultra-high purity helium was used as carrier gas. Temperature was programmed from 80 °C (2 min hold) ramped at 12 °C/min to 240 °C (6 min hold). Compound identification used spectral matching with the NIST 11.L mass spectral library.

in silico Molecular Docking Studies

The human androgen receptor (AR) crystal structure was retrieved from the RCSB Protein Data Bank and prepared using the Protein Preparation Wizard module of Schrödinger Maestro Suite. Active site prediction was performed using the SiteMap module. Ligand structures were retrieved from PubChem and prepared using the LigPrep module. Molecular docking was conducted using the Glide Standard Precision (SP) module (Friesner et al., 2004; Halgren et al., 2004). Drug-likeness was assessed based on Lipinski’s Rule of Five.

DPPH Free Radical Scavenging Assay

Antioxidant activity of AETS was evaluated using the DPPH free radical scavenging assay following the procedure described by Onocha et al. (2016). AETS was tested at six graded concentrations (31.25–1000 µg/mL). Ascorbic acid was used as the reference standard. Absorbance was measured at 517 nm after 30 minutes incubation at 27 °C. IC₅₀ values were determined by linear regression analysis. All analyses were conducted in triplicate.

Cytotoxicity: Brine Shrimp Lethality Assay (BSLA)

Cytotoxicity was assessed using the brine shrimp lethality assay following Meyer et al. (1982), as adapted by Nhamussua et al. (2026). Artemia salina eggs were hatched in seawater at 27 °C for 48 hours. Mature nauplii were exposed to AETS at 1000, 100, and 10 ppm for 24 hours. Ten nauplii per vial, three replicates per concentration. LC₅₀ was determined by Probit analysis (Finney, 1971). LC₅₀ > 1000 ppm was considered non-toxic (Meyer et al., 1982).

 in vivo Reproductive Studies: Drosophila melanogaster

Rationale for Model Selection

Drosophila melanogaster was selected due to its conserved male reproductive processes, high fecundity, short generation time, and validation as an invertebrate alternative model in reproductive toxicology (Lopez-Ortiz et al., 2023; Misra et al., 2014; Adeleke et al., 2022).

Experimental Design

Wild-type Oregon-R strain D. melanogaster were obtained from the Drosophila Laboratory, Department of Biochemistry, University of Ibadan, Nigeria, and maintained on standard corn-agar diet at 25 ± 1 °C under a 12-hour light–dark photoperiod. Adult flies were sexed within 8–10 hours of eclosion. Male flies were starved for 8 hours before mating observation. Four treatment groups were established (n = 30 per group; 3 replicates × 10 flies): Group 1: Negative control (−CTR, vehicle only); Group 2: Positive control (+CTR, sildenafil citrate 2 ppm); Group 3: AETS 10 ppm; Group 4: AETS 100 ppm. Male flies were fed on extract-supplemented diet for 64 hours before introduction to virgin females.

Reproductive Endpoints and Statistical Analysis

Mating latency (ML), copulation duration (CD), offspring emergence rate (ER), and survival (%) were recorded over a 21-day observation period. Mating latency data were analysed using one-way ANOVA followed by Tukey HSD post hoc test. Survival data were analysed using the Chi-square (χ²) test. All analyses used GraphPad Prism; significance was set at p ≤ 0.05.

RESULTS

Extraction Yield

Cold maceration of one kilogram of pulverised T. subcordata root material in a 65:35 aqueous–ethanol solvent system yielded 20.04 g of dry extract (AETS), corresponding to a percentage yield of 2.00% (w/w), as presented in Table 1.

Table 1: Percentage Yield of Crude Aqueous–Ethanol Root Extract of T. subcordata (AETS)

AETS = Aqueous–Ethanol Extract of T. subcordata Roots.

Phytochemical Screening

Qualitative phytochemical screening of AETS revealed the presence of alkaloids, flavonoids, and steroids, while terpenoids, phenols, saponins, tannins, phlobatannins, cardiac glycosides, and reducing sugars were absent (Table 2).

Table 2: Phytochemical Screening of Aqueous–Ethanol Root Extract of T. subcordata (AETS)

(+) = Present; (−) = Absent. AETS = Aqueous–Ethanol Extract of T. subcordata Roots

GC–MS Chemical Profiling

GC–MS analysis identified three chemical constituents accounting for a combined total peak area of 98.70% (Table 3). The dominant constituent was methyl benzoate (95.49%), with phenol (1.66%) and 3-methylpyridazine (1.55%) as minor constituents.

Table 3: GC–MS Chemical Composition of Aqueous–Ethanol Root Extract of T. subcordata (AETS)

Compounds identification by spectral matching with NIST 11.L library.

Figure 1. GC–MS spectrum of aqueous–ethanol root extract of T. subcordata Oliv. (AETS).

in silico Molecular Docking Studies

Active Site Prediction

SiteMap analysis identified three putative active binding sites on the human AR ligand-binding domain (Table 4). Sites 1 and 2 exhibited SiteScores of 0.902 and 0.837, respectively, both above the accepted druggability threshold of 0.80.

Table 4: SiteMap Analysis of Human Androgen Receptor Active Sites

SiteScore ≥ 0.80 indicates a druggable site. Dscore = drug-like site score.

Molecular Docking Scores

Molecular docking results are presented in Table 5. At Sites 1 and 2, methyl benzoate and 3-methylpyridazine achieved docking scores comparable to or exceeding the reference ligand, indicating competitive binding affinity at the primary AR binding sites.

Table 5: Glide SP Molecular Docking Scores Against Human Androgen Receptor Active Sites

More negative docking score = stronger predicted binding affinity. Glide Emodel = energy model score.

Figure 2. Molecular docking binding poses of AETS principal constituents at human androgen receptor (AR) active sites. (A) Methyl benzoate at AR Site 1 (GScore = −2.63 kcal/mol); (B) Methyl benzoate at AR Site 2 (GScore = −2.97 kcal/mol); (C) 3-Methylpyridazine at AR Site 1 (GScore = −2.67 kcal/mol); (D) 3-Methylpyridazine at AR Site 2 (GScore = −1.67 kcal/mol). Ligands shown in stick representation; receptor binding pocket shown as transparent surface. Dashed lines indicate hydrogen bond interactions. Key interacting residues are labelled by amino acid code and position number. Docking performed using the Glide Standard Precision (SP) module of the Schrödinger Maestro Suite.

Drug-Likeness and Pharmacokinetic Profiling

Both methyl benzoate and 3-methylpyridazine fully satisfied Lipinski’s Rule of Five with zero violations (Table 6), demonstrating markedly lower molecular weights and superior predicted oral absorption profiles relative to the reference ligand.

Table 6: Drug-Likeness Parameters (Lipinski’s Rule of Five) of AETS Constituents

MW = Molecular Weight; HBD = Hydrogen Bond Donors; HBA = Hydrogen Bond Acceptors; QLogPo/w = partition coefficient; %HOA = predicted human oral absorption; PSA = Polar Surface Area; RoF = Rule of Five.

Antioxidant Activity: DPPH Free Radical Scavenging Assay

AETS demonstrated concentration-dependent DPPH radical scavenging activity (Table 7). Ascorbic acid consistently exhibited significantly higher scavenging activity at all comparable concentrations (p < 0.05).

Table 7: DPPH Free Radical Scavenging Activity (% Inhibition) of AETS and Ascorbic Acid

Values expressed as mean ± SD (n = 3). Superscripts (ᵃ, ᵇ) within rows denote homogeneous subsets (Tukey HSD, p < 0.05). * = significantly higher than AETS at corresponding concentration. † Ascorbic acid applied at one-fifth the test sample volume. AETS = Aqueous–Ethanol Extract of Triclisia subcordata Roots.

Figure 3. DPPH free radical scavenging activity (% inhibition) of AETS and ascorbic acid at graded concentrations.

Table 8: IC₅₀ Values for DPPH Radical Scavenging Activity

IC₅₀ = concentration required to inhibit 50% of DPPH radicals. Lower IC₅₀ = higher potency.

Figure 4. IC₅₀ determination: % inhibition vs. log concentration (DPPH assay). AETS: IC₅₀ = 440.24 µg/mL (y = 28.07x − 23.53, R² = 0.963); Ascorbic acid: IC₅₀ = 34.60 µg/mL (y = 34.88x − 3.73, R² = 0.985).

Cytotoxicity: Brine Shrimp Lethality Assay

The cytotoxic profile of AETS is presented in Tables 9 and 10. At 1000 ppm, AETS produced 100% mortality; at 100 ppm, 13.33% mortality; and at 10 ppm, 3.33% mortality. Probit analysis yielded an LC₅₀ of 2,254.00 ppm, classifying AETS as non-toxic by the Meyer et al. (1982) criterion of LC₅₀ > 1000 ppm.

Table 9: Brine Shrimp Lethality of AETS at Graded Concentrations

†Control 1 = 82.5% distilled water + 17.5% DMSO; Control 2 = 98.25% distilled water + 1.75% DMSO; Control 3 = 99.825% distilled water + 0.175% DMSO. Mortality recorded at 24 hours.

Figure 5. Percentage mortality of Artemia salina nauplii exposed to AETS and vehicle controls at 10, 100 and 1000 ppm.

Table 10: LC₅₀ Values from Brine Shrimp Lethality Assay (Probit Analysis)

Classification based on Meyer et al. (1982) threshold criteria. High mortality at 1000 ppm AETS substantially confounded by DMSO vehicle toxicity in Control 1 (see text).

Figure 6. LC₅₀ determination: probit regression of % mortality vs. log₁₀ concentration (BSLA).

3.7 in vivo Reproductive Studies: Drosophila melanogaster

The effects of AETS on reproductive and survival parameters in D. melanogaster are presented in Table 11. AETS did not reduce mating latency relative to the negative control at either dose. Offspring emergence was enhanced at both doses, representing the primary positive fertility finding. Survival was reduced in a dose-dependent manner.

Table 11: Effects of AETS on Survival, Mating Latency, Copulation Duration, and Offspring Emergence in Drosophila melanogaster

n = 30 per group (3 replicates × 10 flies). Observation period = 21 days. Oregon-R strain. −CTR = Negative control (ethanol vehicle); +CTR = Positive control (sildenafil citrate 2 mg/kg diet = 2 ppm). Mating latency expressed as mean ± SD. One-way ANOVA: F(3, 116) = 9.655, p < 0.001 (mating latency); Chi-square test: χ²(3) = 13.191, p = 0.004 (survival). Tukey HSD post hoc: sildenafil (+CTR) differed significantly from AETS 10 ppm (Δ = 19.50 min, p < 0.05) and AETS 100 ppm (Δ = 14.50 min, p < 0.05); neither AETS dose differed significantly from the untreated control (p > 0.05). Copulation duration and offspring emergence rate reported descriptively (ordinal/categorical data). ns = not significant. AETS = Aqueous–Ethanol Extract of Triclisia subcordata Roots.

Figure 7. Mating latency (ML) of D. melanogaster across treatment groups.

Figure 8. Copulation duration (CD) of D. melanogaster across treatment groups.

Figure 9. Offspring emergence rate of D. melanogaster across treatment groups.

Figure 10. Adult survival (%) of D. melanogaster over 21-day observation period.

DISCUSSION

The present study provides the first integrated pharmacological and computational characterisation of the fertility-enhancing and androgenic potential of the aqueous–ethanol root extract of Triclisia subcordata Oliv. The findings collectively support the traditional use of this plant in male reproductive health management in southwestern Nigeria and offer mechanistic insights that advance the existing evidence base for this underexplored species within the Menispermaceae.

Phytochemical Composition and Its Pharmacological Implications

The detection of alkaloids, flavonoids, and steroids in AETS defines a chemically selective secondary metabolite profile consistent with the documented phytochemistry of T. subcordata root material reported in prior investigations (Ogunlakin et al., 2023; Ogunlakin et al., 2025). The Menispermaceae family is characterised by an abundance of bisbenzylisoquinoline alkaloids, and the detection of alkaloids in AETS aligns with this taxonomic signature (De Wet et al., 2007). Alkaloids and ergostane steroids from medicinal plants have been implicated in reproductive enhancement through multiple pathways, including modulation of gonadotropin secretion, stimulation of testicular development, and support of spermatogenesis via direct effects on seminiferous tubules (Thakur et al., 2024). Flavonoids and isoflavonoids have been demonstrated to improve testosterone production by Leydig cells through upregulation of the steroidogenic acute regulatory protein (StAR), contributing to normal spermatogenesis (Martin & Touaibia, 2020). Furthermore, flavonoids have been extensively investigated for the treatment of male reproductive dysfunction, including testicular structural disruption, spermatogenesis disturbance, and sperm quality decline (Fan et al., 2020; Musa et al., 2024). The detection of steroids further reinforces the androgenic plausibility of AETS, as steroidal phytoconstituents are structurally analogous to endogenous androgens and have been reported to interact with steroid hormone receptors, modulating testosterone signalling pathways (Thakur et al., 2024).

Chemical Composition: GC–MS Findings

GC–MS analysis identified methyl benzoate (95.49%), phenol (1.66%), and 3-methylpyridazine (1.55%) in AETS, accounting for 98.70% of the total chromatographic profile. The overwhelming dominance of methyl benzoate is a notable finding. Methyl benzoate is a benzenoid ester biosynthesised through the phenylpropanoid pathway beginning with the deamination of L-phenylalanine, catalysed by S-adenosyl-L-methionine-dependent benzoic acid carboxyl methyltransferase (Farber et al., 2022). Plant secondary metabolites, including benzenoid esters, have demonstrated a variety of biological capabilities relevant to human health, including antimicrobial, anti-inflammatory, and pharmacological properties (Elshafie et al., 2023). The presence of phenol contributes to the observed antioxidant activity, as phenolic hydroxyl groups are well-established hydrogen atom donors capable of quenching free radicals (Ribas-Maynou & Yeste, 2020). It is important to acknowledge that GC–MS is limited to thermally stable constituents and may not capture the full spectrum of polar or high-molecular-weight compounds; including bisbenzylisoquinoline alkaloids characteristic of the Menispermaceae that may be present in the extract (Evans, 2009). A complementary LC–MS/MS analysis is recommended in future studies to fully characterise the non-volatile phytochemical fraction.

Molecular Docking and Androgen Receptor Interactions

The in-silico docking results represent the most mechanistically significant findings of this study. SiteMap analysis identified three distinct active binding sites on the human AR ligand-binding domain, with Sites 1 and 2 exhibiting SiteScores above the accepted druggability threshold of 0.80, confirming multiple pharmacologically accessible cavities amenable to phytochemical interaction (Halgren et al., 2004). Methyl benzoate achieved docking scores of −2.63 kcal/mol and −2.97 kcal/mol at Sites 1 and 2 respectively, exceeding the reference ligand scores at the same sites. Similarly, 3-methylpyridazine outperformed the reference ligand at Site 1 (−2.67 vs −2.54 kcal/mol). Flavonoids are recognised as hormone-like polyphenols because of their structural similarity to endogenous sex steroids, and their ability to bind the androgen receptor has been confirmed through computational docking simulations (Maggiolini et al., 2021). The present findings extend this principle to include benzenoid esters and pyridazine derivatives, for which AR binding has not been previously reported. Both compounds satisfied all parameters of Lipinski’s Rule of Five with zero violations, a prerequisite for oral drug candidacy (Ogunlakin et al., 2023). It is acknowledged that in silico docking provides mechanistic hypotheses requiring validation through in vitro receptor binding and transactivation assays.

Antioxidant Activity

The moderate antioxidant activity of AETS (IC₅₀ = 440.24 µg/mL) is consistent with the antioxidant activity range reported for crude aqueous and hydroethanolic extracts from Nigerian medicinal plants, reflective of the diluting effect of non-antioxidant matrix constituents in crude extract preparations (Oloyede et al., 2014). The phenolic constituents detected in the phytochemical screen and confirmed as minor GC–MS constituents likely contribute to this activity. It is important to note that the antioxidant activity of AETS may be underestimated by the GC–MS profile, as flavonoids detected qualitatively in the phytochemical screen are non-volatile and would not appear in GC–MS analysis but may contribute substantially to the total radical scavenging capacity. Flavonoids possess immune-stimulating, anti-inflammatory, and antioxidative properties making them potential modulators of male reproductive system dysfunction, with demonstrated beneficial impact on testicular structure, spermatogenesis, and sperm quality (Musa et al., 2024). The antioxidant activity of AETS is therefore pharmacologically relevant as a supportive mechanism for spermatogenic protection against oxidative stress-induced damage (Kaltsas et al., 2025; Wang et al., 2025).

Cytotoxicity: Brine Shrimp Lethality Assay

The LC₅₀ of 2,254.00 ppm recorded for AETS unambiguously classifies the extract as non-toxic by the threshold criteria of Meyer et al. (1982). Extracts with LC₅₀ values greater than 1,000 µg/mL are considered non-toxic, those between 500–1,000 µg/mL weakly toxic, those between 100–500 µg/mL moderately toxic, and those below 100 µg/mL strongly toxic (Meyer et al., 1982; Nguta et al., 2016). At LC₅₀ = 2,254 ppm, AETS falls comfortably within the non-toxic range, with a wide safety margin between the effective reproductive doses (10 and 100 ppm) and the LC₅₀. It is noted that the DMSO vehicle control recorded an LC₅₀ of 68.48 ppm, indicating that the 100% mortality at 1,000 ppm AETS was substantially influenced by the high DMSO concentration (17.5%) in Control 1. This solvent toxicity confound underscores the importance of evaluating extract-specific mortality against concentration-matched vehicle controls. The overall non-toxic classification of AETS is consistent with its ethnomedicinal application as an oral remedy without reported adverse effects (Borokini et al., 2013).

In vivo Reproductive Effects in Drosophila melanogaster

The D. melanogaster model provides a validated, genetically tractable platform for the detection of phytochemical effects on reproductive endpoints. Offspring emergence rate emerged as the most compelling positive finding: AETS at 10 ppm produced a High emergence rate exceeding the negative control, while 100 ppm produced a Relatively High emergence rate equivalent to sildenafil. Reproductive fitness in Drosophila fertility studies is quantified by the percentage of exposed males capable of successfully generating viable progeny (Abolaji et al., 2025). By this criterion, AETS demonstrated meaningful fertility enhancement at both tested concentrations. The finding parallels the fertility enhancement reported for Croton zambescicus ethyl acetate extract in the D. melanogaster model, which similarly demonstrated the highest percentage fertility increase at lower doses (Adeleke et al., 2022). The process of spermatogenesis in D. melanogaster is governed by conserved genetic pathways sharing substantial functional homology with mammalian systems, including mechanisms regulating stem cell behaviour, cytokinesis, meiosis, and mitochondrial dynamics (Fuller, 2014). The mechanism underlying enhanced offspring emergence without concomitant aphrodisiac effects may therefore reflect direct phytochemical action on spermatogenic processes, potentially mediated through the AR-binding activity of methyl benzoate and 3-methylpyridazine demonstrated in the docking analysis.

Mating latency was not reduced by AETS at either dose relative to the untreated negative control (AETS 10 ppm: 41.0 ± 17.2 min vs. negative control: 36.0 ± 17.2 min; AETS 100 ppm: 36.0 ± 16.1 min; F(3,116) = 9.655, p < 0.001). Post hoc analysis confirmed that sildenafil alone differed significantly from all other groups (p < 0.05), while neither AETS dose differed from the untreated control (p > 0.05). This distinguishes the fertility-enhancing mechanism of AETS from the aphrodisiac mechanism of sildenafil. Survival was reduced dose-dependently (χ²(3) = 13.191, p = 0.004), with 58.3% at 10 ppm and 41.6% at 100 ppm, falling below both controls. Chronic sub-lethal exposure to plant secondary metabolites in Drosophila has been documented to reduce longevity without impairing reproductive output, consistent with the observed decoupling of reduced survival and enhanced fertility (Ventrella et al., 2016). The observed survival reduction warrants dose optimisation in future studies.

Integrative Mechanistic Perspective

Taken together, the findings support a mechanistic model in which the fertility-enhancing effects of AETS are mediated through the androgenic activity of its major constituents; particularly methyl benzoate, which achieved competitive AR binding at Sites 1 and 2 of the human androgen receptor, rather than through aphrodisiac stimulation of mating behaviour. The presence of flavonoids and steroids provides additional mechanistic support through their documented roles in Leydig cell steroidogenesis and spermatogenic support (Martin & Touaibia, 2020; Fan et al., 2020). The moderate antioxidant activity may provide an ancillary protective role against oxidative stress-induced spermatogenic damage, a key pathophysiological driver of male infertility (Kaltsas et al., 2025). This multi-pathway model (i.e., AR binding, steroidogenic support, and antioxidant protection) is consistent with the integrated mechanism of action proposed for other phytotherapeutic male fertility agents (Adeniyi et al., 2025; Thakur et al., 2024).

Study Limitations

Several limitations merit acknowledgement. GC–MS analysis is limited to thermally stable volatile constituents; a more complete characterisation would require LC–MS/MS analysis to capture non-volatile alkaloids and flavonoids. The in-silico docking results require experimental validation through in vitro AR binding and transactivation assays. The D. melanogaster model does not fully replicate mammalian spermatogenic physiology, and further studies in rodent models with sperm parameter assessment are warranted. Small group sizes (n = 30 per treatment) limit statistical power, and expanded replication would strengthen confidence in reproductive endpoint data. Finally, dose–response characterisation over a broader concentration range is necessary to fully define the therapeutic window.

CONCLUSION

The present study has provided the first mechanistically grounded, computationally-supported characterisation of the androgenic and fertility-enhancing potential of the aqueous–ethanol root extract of Triclisia subcordata Oliv. (Menispermaceae). Phytochemical screening confirmed the presence of alkaloids, flavonoids, and steroids; compound classes with well-established roles in androgenic modulation and spermatogenic support. GC–MS profiling identified methyl benzoate as the dominant constituent (95.49%), with phenol and 3-methylpyridazine as minor components. Molecular docking against the human androgen receptor demonstrated that both methyl benzoate and 3-methylpyridazine achieved binding scores comparable to or exceeding the reference ligand at the two primary AR active sites, with drug-likeness parameters fully satisfying Lipinski’s Rule of Five. Moderate antioxidant activity (IC₅₀ = 440.24 µg/mL) and non-toxic cytotoxicity classification (LC₅₀ = 2,254 ppm) further support the pharmacological and safety profile of AETS.

In vivo evaluation using the Drosophila melanogaster model demonstrated enhanced offspring emergence at both 10 and 100 ppm doses, establishing fertility-enhancing activity that is mechanistically distinct from the aphrodisiac effects of sildenafil. One-way ANOVA confirmed highly significant overall differences in mating latency (F(3,116) = 9.655, p < 0.001), with sildenafil uniquely reducing mating latency while AETS did not differ from the untreated control (Tukey HSD, p > 0.05). This distinction positions T. subcordata root extract as a spermatogenic enhancer rather than a sexual stimulant, with potential therapeutic utility in addressing spermatogenic deficits that underlie a substantial proportion of male infertility cases globally.

Collectively, these findings provide a multi-dimensional scientific basis for the traditional application of T. subcordata roots in male reproductive health management in southwestern Nigeria, and contribute new mechanistic data to the growing evidence base for Menispermaceae species in androgenic phytotherapy. The vast majority of phytotherapeutic agents proposed for male infertility management remain inadequately characterised within evidence-based frameworks (Bratchikov et al., 2023). Advancing T. subcordata along the pre-clinical and clinical development pathway, through rodent-based spermatogenic studies, hormonal profiling, LC–MS/MS-guided phytochemical isolation, and ultimately controlled human trials, represents a scientifically justified and clinically important next step. The engagement of a natural products enterprise (‘Detoro Naturals Limited) in supporting this research further underscores the translational relevance of these findings to the development of evidence-based natural health products for male reproductive wellness.

DECLARATIONS

Conflict of Interest: The authors declare no conflict of interest.

Funding: This study was partly self-funded and partly supported by ‘Detoro Naturals Limited, Nigeria.

Ethics Statement: The study utilised Drosophila melanogaster (an invertebrate model organism). Formal institutional ethics approval is not required for invertebrate research at the University of Ibadan, Nigeria.

Data Availability: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Contributions: P.A.O. — Conceptualisation, supervision, project administration; J.A.A. — Investigation, methodology, formal analysis, writing (original draft); M.A.S. — Resources, supervision, writing (review & editing); G.K.O. — Methodology (DPPH, BSLA), writing (review & editing); I.O.A. — Resources, methodology (Drosophila laboratory); I.M. — Formal analysis (molecular docking), investigation (extraction, phytochemical screening); R.S.A. — Formal analysis (biochemistry); S.O.A. — Investigation and formal analysis (Drosophila assays).

ACKNOWLEDGEMENTS: The authors acknowledge the University of Ibadan Herbarium for plant authentication, the Drosophila Laboratory, Department of Biochemistry, University of Ibadan, for provision of fly stocks and laboratory facilities, and the Research and Development Unit, ‘Detoro Naturals Limited, for the funding support.

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