Phytochemical Profiling, Antioxidant Capacity, Cytotoxicity, and In silico Androgen Receptor Binding Interactions of the Aqueous–Ethanol Root Extract of Cnestis ferruginea Vahl ex DC. (Connaraceae)

Male infertility affects an estimated 55 million men worldwide, with prevalence rising across sub-Saharan Africa where affordable interventions remain limited. Oxidative stress is a key driver of male reproductive dysfunction, disrupting spermatogenesis via lipid peroxidation, DNA damage, and impaired androgen receptor (AR) signalling. Medicinal plants with antioxidant and AR-interacting phytoconstituents offer an underexplored strategy for fertility-modulating lead discovery. Cnestis ferruginea Vahl ex DC. (Connaraceae), a West African shrub used ethnomedicinally for male sexual dysfunction, lacks molecular-level pharmacological evidence. This study reports the phytochemical profile, GC–MS composition, DPPH antioxidant activity, brine shrimp lethality, and in silico AR binding of C. ferruginea aqueous–ethanol root extract (AECF). Cold maceration (65:35 v/v ethanol–water) yielded 4.70% (w/w) extract containing flavonoids, phenols, terpenoids, steroids, saponins, tannins, phlobatannins, and reducing sugars. GC–MS identified 17 compounds (89.27% of the profile), dominated by methyl benzoate (67.12%). Docking against the AR ligand-binding domain (PDB: 2AM9) showed methyl benzoate scored −2.632 and −2.921 kcal/mol at AR Sites 1 and 2, comparable to testosterone (−2.539 and −2.926 kcal/mol), with slightly superior binding. All key constituents satisfied Lipinski’s Rule of Five, supporting oral bioavailability. DPPH assay gave an IC50 of 97.94 µg/mL (vs 34.60 µg/mL for ascorbic acid), indicating moderate antioxidant activity; brine shrimp lethality gave an LC50 of 347.80 ppm, indicating moderate cytotoxicity. These findings provide the first computational evidence for AR-mediated androgenic potential of C. ferruginea, supporting its traditional use in male reproductive health and identifying methyl benzoate as a priority candidate for isolation and in vivo validation.

Cnestis ferruginea; androgen receptor; methyl benzoate; antioxidant; brine shrimp lethality; GC–MS; male reproductive health

1. Liang, Y.; Huang, J.; Zhao, Q.; Mo, H.; Su, Z.; Feng, S.; Li, S.; Ruan, X. Global, Regional, and National Prevalence and Trends of Infertility among Individuals of Reproductive Age (15–49 Years) from 1990 to 2021, with Projections to 2040. Hum. Reprod. 2025, 40 (3), 529–544. https://doi.org/10.1093/humrep/deae292. [Google Scholar] [Crossref]

2. Zeng, G.; Liu, L.; Wang, Y.; Yu, J.; Wang, H.; Li, F. Global, Regional, and National Burden and Trends of Reproductive-Aged Male and Female Infertility from 1990–2021. Front. Endocrinol. 2025, 16, 1506229. https://doi.org/10.3389/fendo.2025.1506229. [Google Scholar] [Crossref]

3. Kaltsas, A. Oxidative Stress and Male Infertility: The Protective Role of Antioxidants. Medicina 2023, 59 (10), 1769. https://doi.org/10.3390/medicina59101769. [Google Scholar] [Crossref]

4. Panner Selvam, M. K.; Agarwal, A. Oxidative Stress-Associated Male Infertility: Current Diagnostic and Therapeutic Approaches. Medicina 2024, 60 (6), 1008. https://doi.org/10.3390/medicina60061008. [Google Scholar] [Crossref]

5. Ankomah, A. D.; Boakye, Y. D.; Agana, T. A.; Boamah, V. E.; Ossei, P. P. S.; Adu, F.; Agyare, C. Evaluation of Dermal Toxicity and Wound Healing Activity of Cnestis ferruginea Vahl ex DC. Evid.-Based Complementary Altern. Med. 2022, 5268613. https://doi.org/10.1155/2022/5268613. [Google Scholar] [Crossref]

6. Barbagallo, F.; D’Andrea, S. Editorial: Androgen Receptors in Male and Female Reproduction. Front. Endocrinol. 2025, 16, 1638220. https://doi.org/10.3389/fendo.2025.1638220. [Google Scholar] [Crossref]

7. Akharaiyi, F. C.; Boboye, B.; Adetuyi, F. C. Antibacterial, Phytochemical and Antioxidant Properties of Cnestis ferruginea DC (Connaraceae) Extracts. J. Microbiol. Biotechnol. Food Sci. 2012, 2 (1), 592–609. [Google Scholar] [Crossref]

8. World Health Organization. WHO Global Report on Traditional and Complementary Medicine 2019; WHO Press: Geneva, Switzerland, 2019. [Google Scholar] [Crossref]

9. Ojetayo, T. A.; Durojaiye, A. F.; Bamidele, N. A.; Ibitunde, O. R.; Sofowote, O. A. Phytochemical Constituents of Cnestis ferruginea and Its Toxicity in Fish. Jurnal Perikanan Universitas Gadjah Mada 2022, 24 (1), 47–54. https://doi.org/10.22146/jfs.69651. [Google Scholar] [Crossref]

10. Ishola, I. O.; Akindele, A. J.; Adeyemi, O. O. Analgesic and Anti-Inflammatory Activities of Cnestis ferruginea Vahl ex DC (Connaraceae) Methanolic Root Extract. J. Ethnopharmacol. 2011, 135 (1), 55–62. https://doi.org/10.1016/j.jep.2011.02.028. [Google Scholar] [Crossref]

11. Evans, W. C. Trease and Evans’ Pharmacognosy, 16th ed.; Saunders/Elsevier: Edinburgh, U.K., 2009; ISBN 978-0-7020-2933-2. [Google Scholar] [Crossref]

12. Sasidharan, S.; Chen, Y.; Saravanan, D.; Sundram, K. M.; Yoga Latha, L. Extraction, Isolation and Characterisation of Bioactive Compounds from Plants’ Extracts. Afr. J. Tradit., Complementary Altern. Med. 2011, 8 (1), 1–10. https://doi.org/10.4314/ajtcam.v8i1.60483. [Google Scholar] [Crossref]

13. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem. 2004, 47 (7), 1739–1749. https://doi.org/10.1021/jm0306430. [Google Scholar] [Crossref]

14. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. J. Med. Chem. 2004, 47 (7), 1750–1759. https://doi.org/10.1021/jm030644s. [Google Scholar] [Crossref]

15. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Delivery Rev. 2001, 46 (1–3), 3–26. https://doi.org/10.1016/S0169-409X(00)00129-0. [Google Scholar] [Crossref]

16. Onocha, P. A.; Oloyede, G. K.; Akintola, J. A. Chemical Composition, Free Radical Scavenging and Antimicrobial Activities of Essential Oil of Mariscus alternifolius Vahl. Open Conf. Proc. J. 2016, 7, 160–167. [Google Scholar] [Crossref]

17. Aiyedun, P. O.; Sonibare, M. A.; Gueye, B.; Adedoyin, R. A. Antidiabetic and Antioxidant Profiling of 67 African Trifoliate Yam Accessions. Fitoterapia 2025, 180, 106299. https://doi.org/10.1016/j.fitote.2024.106299. [Google Scholar] [Crossref]

18. Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin, J. L. Brine Shrimp: A Convenient General Bioassay for Active Plant Constituents. Planta Med. 1982, 45 (5), 31–34. https://doi.org/10.1055/s-2007-971236. [Google Scholar] [Crossref]

19. Vanhaecke, P.; Persoone, G.; Claus, C.; Sorgeloos, P. Proposal for a Short-Term Toxicity Test with Artemia Nauplii. Ecotoxicol. Environ. Saf. 1981, 5 (3), 382–387. [Google Scholar] [Crossref]

20. Sleet, R. B.; Brendel, K. Homogeneous Populations of Artemia Nauplii and Their Potential Use for In Vitro Testing in Developmental Toxicology. Teratog., Carcinog., Mutagen. 1985, 5 (1), 41–54. [Google Scholar] [Crossref]

21. Finney, D. J. Probit Analysis, 3rd ed.; Cambridge University Press: Cambridge, U.K., 1971. [Google Scholar] [Crossref]

22. Hamdi, M.; Teixeira da Silva, J. A. Plants are Capable of Synthesizing Animal Steroid Hormones. Molecules 2019, 24 (14), 2585. https://doi.org/10.3390/molecules24142585. [Google Scholar] [Crossref]

23. Effmert, U.; Große, J.; Rose, U. S. R.; Ehrig, F.; Kägi, R.; Piechulla, B. Volatile Composition, Emission Pattern, and Localization of Floral Scent Emission in Mirabilis jalapa (Nyctaginaceae). Am. J. Bot. 2005, 92 (1), 2–12. [Google Scholar] [Crossref]

24. Korany, A. M.; Aboelhadid, S. M. Practical Antimicrobial Efficacy of Methyl Benzoate against Staphylococcus aureus Biofilm. J. Appl. Biol. Biotechnol. 2025. https://doi.org/10.7324/JABB.2025.189991. [Google Scholar] [Crossref]

25. Mostafiz, M. M.; Jhan, P. K.; Shim, J.-K.; Hwang, Y.-S.; Lee, K.-Y. Methyl Benzoate Exhibits Insecticidal and Repellent Activities against Bemisia tabaci. PLoS One 2018, 13 (11), e0208552. https://doi.org/10.1371/journal.pone.0208552. [Google Scholar] [Crossref]

26. Alemdar, S.; Kalkan, A.; Çelik, M. Systematically Investigation on the Spectral, Antioxidant and Antibacterial Properties of Fragrant Methyl Benzoate Esters. J. Mol. Struct. 2023, 1291, 136085. https://doi.org/10.1016/j.molstruc.2023.136085. [Google Scholar] [Crossref]

27. Panda, S. K.; Reynders, M.; Kipanga, P. N.; Luyten, W. The Anti-Staphylococcal Activity of Cnestis ferruginea Is Due to Benzoquinone, the Oxidation Product of Hydroquinone. Front. Cell. Infect. Microbiol. 2025, 14, 1494589. https://doi.org/10.3389/fcimb.2024.1494589. [Google Scholar] [Crossref]

28. Pereira de Jésus-Tran, K.; Côté, P. L.; Cantin, L.; Blanchet, J.; Labrie, F.; Breton, R. Comparison of Crystal Structures of Human Androgen Receptor Ligand-Binding Domain Complexed with Various Agonists. Protein Sci. 2006, 15 (5), 987–999. https://doi.org/10.1110/ps.051905906. [Google Scholar] [Crossref]

29. Wu, S.; Oceguera Nava, E. I.; Ashong, D.; Chen, G.; Chen, Q.-H. Natural Products Targeting the Androgen Receptor Signaling Pathway. Curr. Issues Mol. Biol. 2025, 47 (9), 780. https://doi.org/10.3390/cimb47090780. [Google Scholar] [Crossref]

30. Abisola, A.-A. M.; Ogunlakin, A. D.; Sonibare, M. A. Evaluation of Antioxidant and Membrane Stabilizing Activities of Croton lobatus L. and Croton zambesicus Mull. Arg. Leaves. J. Phytother. 2025, 24 (1). https://doi.org/10.4314/jopat.v24i1.19. [Google Scholar] [Crossref]

31. Kouakou, K.; Boyvin, L.; M’boh, G. M.; Méité, S.; Djaman, A. J.; N’guessan, J. D. Isolation of Antimicrobial Compounds from Cnestis ferruginea Vahl ex DC (Connaraceae) Leaves through Bioassay-Guided Fractionation. Microorganisms 2019, 7 (4), 96. https://doi.org/10.3390/microorganisms7040096. [Google Scholar] [Crossref]

32. Abbott, W. S. A Method of Computing the Effectiveness of an Insecticide. J. Econ. Entomol. 1925, 18 (2), 265–267. https://doi.org/10.1093/jee/18.2.265a. [Google Scholar] [Crossref]

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