SavedAdd your journal

Journals / Adıyaman Üniversitesi Fen Bilimleri Dergisi / 2026 / Cilt: 16 Sayı: 1

The Phytochemical Content and DPPH Activity of Trifolium repens L. Methanol Extract, and In Silico Studies

Trifolium repens L. Metanol Ekstresinin Fitokimyasal İçeriği ve DPPH Aktivitesi ve İn Silico Çalışmaları

Yunus Başar(IGDIR UNIVERSITY)0000-0002-7785-3242
İlyas Yıldız(TOKAT GAZİOSMANPAŞA ÜNİVERSİTESİ)0000-0003-1254-1069
İbrahim Hosaflıoğlu(IĞDIR ÜNİVERSİTESİ, IĞDIR MESLEK YÜKSEKOKULU)0000-0002-0455-0515
Belkıs Muca(IĞDIR ÜNİVERSİTESİ)0000-0002-3966-4774
Ramazan Erenler(TOKAT GAZİOSMANPAŞA ÜNİVERSİTESİ)0000-0002-0505-3190
Pages
61–76
Publication Date
2026-07-01
🏛️Bibliographic metadata and full text access for this article are provided via DergiPark.
DergiPark Record ↗
✨

AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

In conclusion, the bioactive compounds of T. repens appear to be responsible for its biological activities.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:Trifolium repens L., commonly known as white clover, is an herbaceous perennial plant belonging to the Fabaceae family.
📊
Key Findings & Data:Furthermore, molecular docking was used to theoretically determine the interactions between the dominant compounds and the enzymes responsible for antioxidant activity: thioredoxin reductase (TrxR) and heme oxygenase-1 (HO-1).
💡
Conclusion & Impact:In conclusion, the bioactive compounds of T. repens appear to be responsible for its biological activities.
🔬Design:Araştırma Makalesi
📍Scope:Comprehensive Sample
⚙️Variables:Ak üçgül · biyosensör · antioksidan

Abstract

Trifolium repens L., commonly known as white clover, is an herbaceous perennial plant belonging to the Fabaceae family. It is widely used in traditional medicine to treat diarrhoea, menstrual irregularities, skin damage, coughs, rubella, and stress, irritability and insomnia. In our study, we investigated the phenolic profile of the methanol extract of T. repens by LC-MS/MS and determined the DPPH and total phenolic content (TPC) for the first time using potentiometric biosensor methods. Furthermore, molecular docking was used to theoretically determine the interactions between the dominant compounds and the enzymes responsible for antioxidant activity: thioredoxin reductase (TrxR) and heme oxygenase-1 (HO-1). LC-MS/MS analysis revealed the main components of T. repens to be coumarin (29,673.13 µg/mg extract) and isoquercitrin (21,608.99 µg/mg extract). The extract exhibited lower antioxidant activity than the standard extract, as determined by DPPH (27.43%) and FCR (179.6257 GA/plant extract) analyses. Additionally, isoquercitrin (-7.6, 8.9 kcal/mol) and the proposed coumarin-isoquercitrin complex (-9.2, -9.5 kcal/mol) exhibited significant binding affinity to HO-1 and TrxR enzymes, respectively. In conclusion, the bioactive compounds of T. repens appear to be responsible for its biological activities. Therefore, this application could be widespread in fields such as food, pharmacology and cosmetics.

Özet

Beyaz yonca olarak da bilinen Trifolium repens L., Fabaceae familyasına ait otsu çok yıllık bir bitkidir. Geleneksel tıpta ishal, adet düzensizlikleri, cilt hasarı, öksürük, kızamıkçık, stres, sinirlilik ve uykusuzluğun tedavisinde yaygın olarak kullanılır. Çalışmamızda, T. repens'in metanol özütünün fenolik profilini LC-MS/MS ile, DPPH ve toplam fenolik içeriğini (TPC) ise potansiyometrik biyosensör yöntemleri ile araştırdık. Ayrıca, baskın bileşikler ile antioksidan aktiviteden sorumlu enzimler olan tiyoredoksin redüktaz (TrxR) ve hem oksijenaz-1 (HO-1) arasındaki etkileşimleri teorik olarak belirlemek için moleküler yerleştirme kullanıldı. LC-MS/MS analizi, T. repens'in ana bileşenlerinin kumarin (29.673,13 µg/mg özüt) ve izokuersitrin (21.608,99 µg/mg özüt) olduğunu ortaya koydu. DPPH (%27,43) ve FCR (179,6257 GA/bitki özütü) analizlerine göre, özüt standart özütten daha düşük antioksidan aktivite göstermiştir. Ayrıca, izokuersitrin (-7,6, 8,9 kcal/mol) ve önerilen kumarin-izokuersitrin kompleksi (-9,2, -9,5 kcal/mol), sırasıyla HO-1 ve TrxR enzimlerine önemli bağlanma afinitesi göstermiştir. Sonuç olarak, T. repens'in biyoaktif bileşikleri, biyolojik aktivitelerinden sorumlu görünmektedir. Bu nedenle, uygulamaları gıda, farmakoloji ve kozmetik gibi alanlarda yaygınlaşabilir.

🔖

Cite This Article

Başar, Y., Yıldız, İ., Hosaflıoğlu, İ., Muca, B., Erenler, R. (2026). The Phytochemical Content and DPPH Activity of Trifolium repens L. Methanol Extract, and In Silico Studies. Adıyaman Üniversitesi Fen Bilimleri Dergisi, Cilt: 16 Sayı: 1, 61–76. https://doi.org/10.37094/adyujsci.1813336

📄 Full Text PDF Viewer

📄

PDF görüntüleyici hazırlanıyor...

References (43)

  1. Petrovska, B.B., Historical Review of Medicinal Plants' Usage, Pharmacognosy Reviews, 6(11), 1-5, 2012.
  2. Sun, W., Shahrajabian, M.H., Therapeutic Potential of Phenolic Compounds in Medicinal Plants Natural Health Products for Human Health, Molecules, 28(4), 1845, 2023.
  3. Dias, M.C., Pinto, D.C.G.A., Silva, A.M.S., Plant Flavonoids: Chemical Characteristics and Biological Activity, Molecules, 26(17), 5377, 2021.
  4. Ayad, R., Akkal, S., Chapter 12 - Phytochemistry and Biological Activities of Algerian Centaurea and Related Genera, in: Atta ur, R. (Ed.), Studies in Natural Products Chemistry, Elsevierpp. 357-414, 2019.
  5. Rigobello, M.P., Bindoli, A., Chapter Seven - Mitochondrial Thioredoxin Reductase: Purification, Inhibitor Studies, and Role in Cell Signaling, in: Cadenas, E., Packer, L. (Eds.), Methods in Enzymology, Academic Presspp. 109-122, 2010.
  6. Arnér, E.S.J., Zhong, L., Holmgren, A., Preparation and Assay of Mammalian Thioredoxin and Thioredoxin Reductase, Methods in Enzymology, Academic Presspp. 226-239, 1999.
  7. Tamura, T., Stadtman, T.C., [27] - Mammalian Thioredoxin Reductases, in: Sies, H., Packer, L. (Eds.), Methods in Enzymology, Academic Presspp. 297-306, 2002.
  8. Biaglow, J.E., Miller, R.A., The Thioredoxin Reductase/thioredoxin System: Novel Redox Targets for Cancer Therapy, Cancer Biology & Therapy, 4(1), 13-20, 2005.
  9. Watanabe, R., Nakamura, H., Masutani, H., Yodoi, J., Anti-oxidative, Anti-cancer and Anti-inflammatory Actions by Thioredoxin 1 and Thioredoxin-Binding protein-2, Pharmacology & Therapeutics, 127(3), 261-270, 2010.
  10. Fernandes, A.P., Holmgren, A., Glutaredoxins: Glutathione-Dependent Redox Enzymes with Functions far Beyond a Simple Thioredoxin Backup System, Antioxidants & Redox Signaling, 6(1), 63-74, 2004.
  11. Wood, Z.A., Schröder, E., Robin Harris, J., Poole, L.B., Structure, Mechanism and Regulation of Peroxiredoxins, Trends in Biochemical Sciences, 28(1), 32-40, 2003.
  12. Lu, J., Holmgren, A., The Thioredoxin Antioxidant System, Free Radical Biology and Medicine, 66, 75-87, 2014.
  13. Lee, S.E., Park, Y.S., The Emerging Roles of Antioxidant Enzymes by Dietary Phytochemicals in Vascular Diseases, Life (Basel), 11(3), 2021.
  14. Consoli, V., Sorrenti, V., Grosso, S., Vanella, L., Heme Oxygenase-1 Signaling and Redox Homeostasis in Physiopathological Conditions, Biomolecules, 11(4), 589, 2021.
  15. Schipper, H.M., Heme oxygenase expression in human central nervous system disorders, Free Radic Biol Med, 37(12), 1995-2011, 2004.
  16. Zoric, L., Merkulov, L., Lukovic, J., Boza, P., Comparative Analysis of Qualitative Anatomical Characters of Trifolium L. (Fabaceae) and Their Taxonomic Implications: Preliminary Results, Plant Systematics and Evolution, 298(1), 205-219, 2012.
  17. Rahman, I.U., Afzal, A., Iqbal, Z., Hart, R., Abd_Allah, E.F., Hashem, A., Alsayed, M.F., Ijaz, F., Ali, N., Shah, M., Bussmann, R.W., Calixto, E.S., Herbal Teas and Drinks: Folk Medicine of the Manoor Valley, Lesser Himalaya, Pakistan, Plants, 8(12), 581, 2019.
  18. Ahmad, S., Zeb, A., Phytochemical Profile and Pharmacological Properties of Trifolium repens, Journal of Basic and Clinical Physiology and Pharmacology, 32(1), 2021.
  19. Engelmann, N.J., Reppert, A., Yousef, G., Rogers, R.B., Lila, M.A., In Vitro Production of Radiolabeled Red Clover (Trifolium pratense) Isoflavones, Plant Cell, Tissue and Organ Culture (PCTOC), 98(2), 147-156, 2009.
  20. Leporatti, M.L., Ivancheva, S., Preliminary Comparative Analysis of Medicinal Plants Used in theTraditional Medicine of Bulgaria and Italy, Journal of Ethnopharmacology, 87(2), 123-142, 2003.
  21. Yildiz, İ., Başar, Y., Erenler, R., Alma, M.H., Calimli, M.H., A phytochemical content analysis, and antioxidant activity evaluation using a novel method on Melilotus officinalis flower, South African Journal of Botany, 174, 686-693, 2024.
  22. Basar, Y., Yenigun, S., Yılmaz, S., Kocaman, A.Y., Demirtas, I., Alma, M.H., Temel, S., Ozen, T., Urease Inhibitory Potency of Chenopodium Quinoa Seed Extract: LC–ESI–MS/MS, GC–MS/MS Analysis, Biological Evaluations, Molecular Docking, ADMET Property, DFT Calculation and PASS Prediction, Journal of Food Measurement and Characterization, 19(8), 5311-5332, 2025.
  23. Isildak, Ö., Yildiz, I., Genc, N., A new Potentiometric PVC Membrane Sensor for the Determination of DPPH Radical Scavenging Activity of Plant Extracts, Food Chemistry, 373, 131420, 2022.
  24. Işıldak, Ö., Yıldız, İ., New Potentiometric Sensor for Total Phenolic Assay of Plant extracts, Bulletin of the Chemical Society of Japan, 97(5), 2024.
  25. Başar, Y., Yıldız, I., Hosaflioglu, I., Erenler, R., Alma, M.H., Phytochemical Study of Astragalus onobrychis: Bioactive Compounds, Antioxidant Activity, in silico Molecular Docking, PASS and DFT Study, Brazilian Archives of Biology and Technology, 68, e25240574, 2025.
  26. Meng, X.-Y., Zhang, H.-X., Mezei, M., Cui, M., Molecular Docking: a Powerful Approach for Structure-Based Drug Discovery, Current computer-aided drug design, 7(2), 146-157, 2011.
  27. Chi, G.F., Khan, S., Başar, Y., Kuete, J.R.N., Matieta, V.Y., Kuete, J.B.T., Megaptche, J.F., Chongong, M.A., Yenigün, S., Ayimele, G.A., Mbaveng, A.T., Kuete, V., Shaheen, F., Antibacterial Flavonoids from Tetrapleura tetraptera (Fabaceae) Fruit Pulp, In Silico Studies, South African Journal of Botany, 180, 96-106, 2025.
  28. Gok, M., Basar, Y., Yenigun, S., Ipek, Y., Behcet, L., Ozen, T., Demirtas, I., Bioactive Apigenin‐7‐O‐β‐Glucoside and Rosmarinic Acid Molecules From Two Nepeta Species: Bioactivity‐Guided Isolation, In Vitro Evaluations, Pharmacokinetic and In Silico Approaches as Metabolic Enzyme Inhibition Agents, Phytochemical Analysis, 36(2), 1677-1694, 2025.
  29. Basar, Y., Yenigun, S., Ozen, T., Demirtas, I., Balpınar, O., Behcet, L., Nepetanudoside B, a potential new food additive isolated from Nepeta aristata: antioxidant, antibacterial, DFT, ADME/T, PASS prediction and MTT analysis studies, European Food Research and Technology, 251, 3123–3139, 2025.
  30. Nurgün, K., Kadioğlu, O., Adigüzel, N., Tamer, U., Güvenç, A., BANİ, B., Determination of Isoflavone Content By Hplc-Uv Method and In Vitro Antioxidant Activity of Red Clover (Trıfolıum Pratense L.), Turkish Journal of Pharmaceutical Sciences, 10(3), 463-472, 2013.
  31. Halliwell, B., The Wanderings of a Free Radical, Free Radical Biology and Medicine, 46(5), 531-542, 2009.
  32. Lushchak, V.I., Free Radicals, Reactive Oxygen Species, Oxidative Stress and Its Classification, Chemico-Biological Interactions, 224, 164-175, 2014.
  33. Erenler, R., Adak, T., Karan, T., Elmastas, M., Yildiz, İ., Aksit, H., Topcu, G., Sanda, M.A., The Eurasia Proceedings of Science Technology Engineering and Mathematics, (1), 139-145, 2017.
  34. Erenler, R., Yıldız, İ., Geçer, E.N., Hosaflıoğlu, İ., Alma, M.H., Işıldak, Ö., Determination of antioxidant activity of Medicago sativa L. flowers by biosensor and quantitative analysis of bioactive compounds by LC-MS/MS, Turkish Journal of Sensors and Biosensors, 2(1), 9-17, 2025.
  35. McCord, J.M., The Evolution of Free Radicals and Oxidative Stress, The American Journal of Medicine, 108(8), 652-659, 2000.
  36. Gulcin, İ., Antioxidants and Antioxidant Methods: an Updated Overview, Archives of Toxicology, 94(3), 651-715, 2020.
  37. Zhang, D., Liu, Q., Biosensors and Bioelectronics on Smartphone for Portable Biochemical Detection, Biosensors and Bioelectronics, 75, 273-284, 2016.
  38. Ibrahim, H., Issa, Y.M., Abu-Shawish, H.M., Chemically Modified Carbon Paste Electrode for the Potentiometric Determination of Dicylomine Hydrochloride in Batch and in FIA Conditions, Analytical Sciences, 20(6), 911-916, 2004.
  39. Başar, Y., Yenigün, S., Alma, M.H., Demirtaş, İ., & Özen, T., Fruit extracts of Rosa canina L. and Rosa pimpinellifolia L.: Phytochemical profiles, in vitro antioxidant, anti-inflammatory, xanthine oxidase inhibitory effects, and in silico molecular dynamics studies. KSU J. Agric Nat 28 (3), 636-649, 2025.
  40. Tundis, R., Marrelli, M., Conforti, F., Tenuta, M.C., Bonesi, M., Menichini, F., Loizzo, M.R., Trifolium Pratense and T. repens (Leguminosae): Edible Flower Extracts as Functional Ingredients, Foods, 4(3), 338-348, 2015.
  41. Ahmad, S., Zeb, A., Phytochemical Profile and Pharmacological Properties of Trifolium repens, Journal of Basic and Clinical Physiology and Pharmacology, 32(1), 20200015, 2021.
  42. Ahmad, S., Zeb, A., Ayaz, M., Murkovic, M., Characterization of Phenolic Compounds Using UPLC–HRMS and HPLC–DAD and Anti-cholinesterase and Anti-oxidant Activities of Trifolium repens L. Leaves, European Food Research and Technology, 246(3), 485-496, 2020.
  43. Bitencourt-Ferreira, G., Veit-Acosta, M., de Azevedo, W.F., Hydrogen Bonds in Protein-Ligand Complexes, in: de Azevedo Jr, W. F. (Ed.), Docking Screens for Drug Discovery, Springer New York, New York, NY, pp. 93-107, 2019.