Gazi University Journal of Science 2147-1762 Gazi University 10.35378/gujs.1727978 Natural Products and Bioactive Compounds Doğal Ürünler ve Biyoaktif Bileşikler Bioguided isolation of 8-epideoxyloganic acid from Nepeta italica: In vitro and in silico assessments https://orcid.org/0000-0002-1041-267X İpek Yaşar ÇANKIRI KARATEKİN ÜNİVERSİTESİ https://orcid.org/0000-0002-1979-5427 Yenigün Semiha ONDOKUZ MAYIS ÜNİVERSİTESİ https://orcid.org/0000-0002-7785-3242 Başar Yunus IĞDIR ÜNİVERSİTESİ https://orcid.org/0000-0001-8946-647X Demirtas İbrahim ONDOKUZ MAYIS ÜNİVERSİTESİ https://orcid.org/0000-0003-0133-5630 Ozen Tevfik ONDOKUZ MAYIS ÜNİVERSİTESİ https://orcid.org/0000-0001-8334-7816 Behçet Lütfi BİNGÖL ÜNİVERSİTESİ Advanced Online Publication 06 26 2025 02 05 2026 Copyright © 1988, Gazi University Journal of Science 1988 Gazi University Journal of Science

In this study, 8‑epideoxyloganic acid (8‑ELA) was isolated from the methanol fraction of Nepeta italica subsp. italica using a bioactivity‑guided approach. The active NIMFr3 fraction was purified, and the structure of 8‑ELA was elucidated by NMR and MS analyses. The compound exhibited significant inhibitory activity against butyrylcholinesterase (BChE) and α‑glucosidase, with IC50 values of 7.768 ± 0.000 and 3.985 ± 0.066 µg/mL, respectively. Enzyme kinetics revealed a non‑competitive inhibition profile for most enzymes, except lipase. Molecular docking and 100 ns molecular dynamics simulations confirmed stable binding orientations, supported by MM‑PBSA free energy calculations (ΔGbind = −1.94 kcal/mol). Density functional theory (DFT) analysis indicated a HOMO–LUMO energy gap of 5.660 eV, while PASS prediction suggested potential anti‑inflammatory, antifungal, and metabolic activities. Overall, this work provides complementary in vitro and in silico insights into 8‑ELA, supporting its potential as a candidate molecule for future pharmacological research.

 Nepeta italica 8-epideoxyloganic acid In vitro bioactivity In silico studies TÜBİTAK TÜBİTAK 119Z442 [1] Bhat, S. G., “Medicinal plants and its pharmacological values”, Natural Medicinal Plants, 12:217-228, (2021). https://doi.org/10.5772/intechopen.99848. [2] Rodrigues, F., and Delerue-Matos, C., “Plant extracts: Chemical composition, bioactivity and potential applications”, (2022). https://doi.org/10.3390/books978-3-0365-2876-2. [3] Bassani, D., and Moro, S., “Past, present, and future perspectives on computer-aided drug design methodologies”, Molecules, 28:3906, (2023). https://doi.org/10.3390/molecules28093906. [4] Gozalbes, R., and Pineda-Lucena, A., “Small molecule databases and chemical descriptors useful in chemoinformatics: an overview”, Combinatorial Chemistry & High Throughput Screening, 14:548-558, (2011). https://doi.org/10.2174/138620711795767857. [5] Schneider, P., Walters, W. P., Plowright, A. T., Sieroka, N., Listgarten, J., Goodnow Jr, R. A., Fisher, J., Jansen, J. M., Duca, J. S., and Rush, T. S., “Rethinking drug design in the artificial intelligence era”, Nature Reviews Drug Discovery, 19:353-364, (2020). https://doi.org/10.1038/s41573-019-0050-3. [6] Akpinar, N., Akpinar, M., Gorgun, S., Dirmenci, T., and Aktumsek, A., “Fatty acid composition of the seeds of five Nepeta species from Turkey”, Chemistry of Natural Compounds, 44:90-92, (2008). https://doi.org/10.1007/s10600-008-0025-9. [7] Joshi, R. K., Satyal, P., and Setzer, W. N., “Himalayan aromatic medicinal plants: a review of their ethnopharmacology, volatile phytochemistry, and biological activities”, Medicines, 3:6, (2016). https://doi.org/10.3390/medicines3010006. [8] Eser, F., Altun, M., Demirtas, I., Behcet, L., and Aktas, E., “Three new iridoids from the aerial parts of Nepeta teucriifolia Willd. and antiproliferative activities of extracts”, Natural Product Research, 1-10, (2022). https://doi.org/10.1080/14786419.2022.2036148. [9] Başar, Y., Yenigün, S., İpek, Y., Behçet, L., Gül, F., Özen, T., and Demirtaş, İ., “DNA protection, molecular docking, enzyme inhibition and enzyme kinetic studies of 1,5,9-epideoxyloganic acid isolated from Nepeta aristata with bio-guided fractionation”, Journal of Biomolecular Structure and Dynamics, 42:9235-9248, (2024). https://doi.org/10.1080/07391102.2023.2250461. [10] Yenigün, S., Başar, Y., İpek, Y., Behçet, L., Gül, F., Özen, T., and Demirtaş, İ., “Determination of antioxidant, DNA protection, enzyme inhibition potential and molecular docking studies of a biomarker ursolic acid in Nepeta species”, Journal of Biomolecular Structure and Dynamics, 42:5799-5816, (2024). https://doi.org/10.1080/07391102.2023.2229440. [11] Wang, C., Gong, X., Bo, A., Zhang, L., Zhang, M., Zang, E., Zhang, C., and Li, M., “Iridoids: research advances in their phytochemistry, biological activities, and pharmacokinetics”, Molecules, 25:287, (2020). https://doi.org/10.3390/molecules25020287. [12] Saha, S., Prinsa, V. J., Mahato, A. K., Srivastava, S., Dobhal, K., and Kawsar, S. M., “In Silico Assessment of the Role of Iridoid in the Treatment of Zika and Influenza Virus Infection”, Philippine Journal of Science, 152:1953-1988, (2023). https://doi.org/10.56899/152.05.35. [13] Başar, Y., Demirtaş, İ., Yenigün, S., İpek, Y., Özen, T., and Behçet, L., “Molecular docking, molecular dynamics, MM/PBSA approaches and bioactivity studies of nepetanudoside B isolated from endemic Nepeta aristata”, Journal of Biomolecular Structure and Dynamics, 42:1-14, (2024). https://doi.org/10.1080/07391102.2024.2309641. [14] Basar, Y., Yenigün, S., Behçet, L., Özen, T., and Demirtaş, İ., “Antibacterial and Antioxidant Molecule Isolated from Nepeta aristata Boiss Et Kotschy Ex Boiss plant: 1,5,9-Epideoxyloganic Acid”, International Journal of Chemistry and Technology, 8:27-31, (2024). https://doi.org/10.32571/ijct.1381998. [15] Greig, N. H., Utsuki, T., Ingram, D. K., Wang, Y., Pepeu, G., Scali, C., Yu, Q., Mamczarz, J., Holloway, H. W., Giordano, T., Chen, D., Furukawa, K., Sambamurti, K., Brossi, A., and Lahiri, D. K. “Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer β-amyloid peptide in rodent”. Proceedings of the National Academy of Sciences, 102(47), 17213-17218, (2005). https://doi.org/10.1073/pnas.0508575102. [16] Akmal, M., Patel, P., and Wadhwa, R. “Alpha Glucosidase Inhibitors”. In: StatPearls. StatPearls Publishing, Treasure Island (FL), (2024), 32496728. [17] Kim, G. N., Shin, M.R., Shin, S. H., Lee, A. R., Lee, J. Y., Seo, B. I., Kim, M. Y., Kim, T. H., Noh, J. S., Rhee, M. H., and Roh, S. S. “Study of antiobesity effect through inhibition of pancreatic lipase activity of Diospyros kaki fruit and Citrus unshiu peel”. BioMed Research International, 2016(1), 1723042. https://doi.org/10.1155/2016/1723042. [18] Aslam, S., and Gupta, V. “Carbonic Anhydrase Inhibitors”, In: StatPearls. StatPearls Publishing, Treasure Island (FL), (2020), 32491668. [19] Yenigun, S., Başar, Y., İpek, Y., Gok, M., Behçet, L., Özen, T., and Demirtaş, İ., “A potential DNA protector, enzyme inhibitor and in silico studies of daucosterol isolated from six Nepeta species”, Process Biochemistry, 143:234-247, (2024). https://doi.org/10.1016/j.procbio.2024.04.039. [20] Ellman, G. L., Courtney, K. D., Andres Jr, V., and Featherstone, R. M., “A new and rapid colorimetric determination of acetylcholinesterase activity”, Biochemical Pharmacology, 7:88-95, (1961). https://doi.org/10.1016/0006-2952(61)90145-9. [21] Mayur, B., Sandesh, S., Shruti, S., and Sung-Yum, S., “Antioxidant and α-glucosidase inhibitory properties of Carpesium abrotanoides L.”, Journal of Medicinal Plants Research, 4:1547-1553, (2010). https://doi.org/10.5897/JMPR.9000218. [22] Chanda, J., Mukherjee, P. K., Biswas, R., Biswas, S., Tiwari, A. K., and Pargaonkar, A., “UPLC‐QTOF‐MS analysis of a carbonic anhydrase‐inhibiting extract and fractions of Luffa acutangula (L.) Roxb (ridge gourd)”, Phytochemical Analysis, 30:148-155, (2019). https://doi.org/10.1002/pca.2800. [23] Trentin, R., Custódio, L., Rodrigues, M. J., Moschin, E., Sciuto, K., da Silva, J. P., and Moro, I., “Exploring Ulva australis Areschoug for possible biotechnological applications: In vitro antioxidant and enzymatic inhibitory properties, and fatty acids contents”, Algal Research, 50:101980, (2020). https://doi.org/10.1016/j.algal.2020.101980. [24] Yenigun, S., Ipek, Y., Marah, S., Demirtas, I., and Ozen, T., “DNA protection, molecular docking, antioxidant, antibacterial, enzyme inhibition, and enzyme kinetic studies for parietin, isolated from Xanthoria parietina (L.) Th. Fr”, Journal of Biomolecular Structure and Dynamics, 42:848-862, (2024). https://doi.org/10.1080/07391102.2023.2196693. [25] Abraham, M. J., Murtola, T., Schulz, R., Páll, S., Smith, J. C., Hess, B. and Lindahl, E., “GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers”, SoftwareX, 1:19-25, (2015). https://doi.org/10.1016/j.softx.2015.06.001. [26] Bjelkmar, P., Larsson, P., Cuendet, M. A., Hess, B., and Lindahl, E., “Implementation of the CHARMM force field in GROMACS: analysis of protein stability effects from correction maps, virtual interaction sites, and water models”, Journal of Chemical Theory and Computation, 6:459-466, (2010). https://doi.org/10.1021/ct900549r. [27] Akkoc, S., Karatas, H., Muhammed, M. T., Kökbudak, Z., Ceylan, A., Almalki, F., Laaroussi, H., and Ben Hadda, T., “Drug design of new therapeutic agents: Molecular docking, molecular dynamics simulation, DFT and POM analyses of new Schiff base ligands and impact of substituents on bioactivity of their potential antifungal pharmacophore site”, Journal of Biomolecular Structure and Dynamics, 41:6695-6708, (2023). https://doi.org/10.1080/07391102.2022.2111360. [28] Valdés-Tresanco, M. S., Valdés-Tresanco, M. E., Valiente, P. A., and Moreno, E., “gmx_MMPBSA: a new tool to perform end-state free energy calculations with GROMACS”, Journal of Chemical Theory and Computation, 17:6281-6291, (2021). https://doi.org/10.1021/acs.jctc.1c00645. [29] Frisch, M., Trucks, G., Schlegel, H., Scuseria, G., Robb, M., Cheeseman, J., Scalmani, G., Barone, V., Mennucci, B., and Petersson, G., “Gaussian 09, revision C”. 01, Gaussian Inc., Wallingford, 2009, Received: August 28 (2013). https://doi.org/10.1039/C4DT02443E. [30] Lee, C., Yang, W., and Parr, R. G., “Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density”, Physical Review B, 37:785, (1988). https://doi.org/10.1103/PhysRevB.37.785. [31] Becke, A. D., “Density-functional exchange-energy approximation with correct asymptotic behavior”, Physical Review A, 38:3098, (1988). https://doi.org/10.1103/PhysRevA.38.3098. [32] Reed, A. E., and Curtiss, L. A., F. “Weinhold, Intermolecular interactions from a natural bond orbital, donor-acceptor viewpoint”, Chemical Reviews, 88:899-926, (1988). https://doi.org/10.1021/cr00088a005. [33] Gökçimen, S. Ş., İpek, Y., Behçet, L., Demirtaş, İ., and Özen, T., “Isolation, characterization and evaluation of oxypeucedanin and osthol from local endemic Prangos aricakensis Behçet and Yapar root as antioxidant, enzyme inhibitory, antibacterial and DNA protection: molecular docking and DFT approaches”, Journal of Biomolecular Structure and Dynamics, Published online: 12 Jan 2024, 1-18, (2024). https://doi.org/10.1080/07391102.2024.2303387. [34] Lagunin, A., Stepanchikova, A., Filimonov, D., and Poroikov, V., “PASS: prediction of activity spectra for biologically active substances”, Bioinformatics, 16:747-748, (2000). https://doi.org/10.1093/bioinformatics/16.8.747. [35] Yenigun, S., Basar, Y., Gül, F., Ipek, Y., Gok, M., Behcet, L., Ozen, T., and Demirtas, I., “In vitro bioactivities and in silico ADME profile for potential biological target prediction of selected phytochemicals using six Nepeta species”, Chemistry Select, 9:e202400919, (2024). https://doi.org/10.1002/slct.202400919. [36] Li, Z., Ryenchindorj, L., Liu, B., Shi, J., Zhang, C., Hua, Y., Liu, P., Shan, G., and Jia, T., “Chemical profiles and metabolite study of raw and processed Cistanche deserticola in rats by UPLC-Q-TOF-MS(E)”, Chinese Medicine, 16:95, (2021). https://doi.org/10.1186/s13020-021-00508-0. [37] Acquaviva, A., Di Simone, S. C., Nilofar, Bouyahya, A., Zengin, G., Recinella, L., Leone, S., Brunetti, L., Uba, A. I., Guler, O., Balos, M., Cakilcioğlu, U., Menghini, L., Ferrante, C., Orlando, G., Libero, M. L., and Chiavaroli, A. “Screening for chemical characterization and pharmacological properties of different extracts from Nepeta italica”. Plants, 12(15), 2785, (2023). https://doi.org/10.3390/plants12152785. [38] Liu, X., Shi, D., Zhou, S., Liu, H., Liu, H., and Yao, X., “Molecular dynamics simulations and novel drug discovery”, Expert Opinion on Drug Discovery, 13:23-37, (2018). https://doi.org/10.1080/17460441.2018.1403419. [39] Majewski, M., Ruiz-Carmona, S., and Barril, X., “An investigation of structural stability in protein-ligand complexes reveals the balance between order and disorder”, Communications Chemistry, 2: 110, (2019). https://doi.org/10.1038/s42004-019-0205-5. [40] Gautam, V., Nimmanpipug, P., Zain, S. M., Rahman, N. A., and Lee, V. S., “Molecular dynamics simulations in designing DARPins as phosphorylation-specific protein binders of ERK2”, Molecules, 26:4540, (2021). https://doi.org/10.3390/molecules26154540.