Environmental impact on Ononis natrix: Phytochemical profiling, heavy metal bioaccumulation, and multifunctional bioactivities

A. Ghellab; F. Menaceur; Y. Gouasmia

doi:10.15421/0225200

A. Ghellab Echahid Cheikh Larbi Tebessi University
F. Menaceur Higher School of Food Science and Agri-Food Industry
Y. Gouasmia Biotechnology Research Center

Keywords: environmental pollution, heavy metal bioaccumulation, UPLC-ESI-MS/MS, phytochemical composition.

Abstract

Ononis natrix L., a Mediterranean medicinal species traditionally used for its diuretic and anti-inflammatory prope r ties, has recently attracted global scientific interest for its rich profile of bioactive metabolites with potential pharma cological and ecological significance. This study aimed to evaluate the comparative impact of environmental contamin a tion on phytochemical composition, biological activities and heavy metal bioaccumulation in Ononis natrix L. Samples were collected from a polluted site and a non-polluted site in Berezguel (Tebessa, Algeria). The aerial parts were analyzed for heavy metal content: lead (Pb), cadmium (Cd), zinc (Zn), and copper (Cu). Methanolic extracts were prepared from the same plant parts and were analyzed for total phenolic (TPC), flavonoid (TFC), and condensed tannin (TTC) contents. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), phenanthroline, and reducing power. In addition, enzyme inhibitory potential was examined against α-amylase, acetylcholinesterase, and butyrylcholinesterase, while antimicrobial activity was also assessed. Plants from the polluted site bioaccumulated higher levels of Pb, Cd, Zn and Cu. Phytochemical content was also higher in these samples with significantly elevated TPC and TFC. Plants collected from the polluted site exhibited superior antioxidant activity across all assays, with lower IC50 values for ABTS (15.49 ± 1.89 μg/mL) and DPPH (26. 1 ± 0.9 μg/mL) compared to those from the non-polluted site (23. 8 ± 1. 9 and 33. 7 ± 1.9 μg/mL, respectively). Extracts from the polluted site also showed stronger enzyme inhibition, particularly against α-amylase (IC 50 : 268.1 ± 9.6 μg/mL) compared to acarbose (IC 50 : 3177.3 ± 11. 7 μg/mL). Both extracts exhibited high antibacterial activity with the polluted-site extract producing larger inhibition zones for Gram-positive and Gram-negative bacteria. These findings suggest that pollution induced enviro n mental stress enhanced bioactive compound production and therapeutic potential in O. natrix , though metal accumulation in the raw plant raises safety concerns, underscoring the need for standardization and sustainable sourcing strategies.

References

Aithani, D., & Kushawaha, J. (2024). Heavy metals contamination in environment. In: Selvasembian, R., Thokchom, B., Singh, P., Jawad, A. H., Gwenzi, W. (Eds.). Remediation of heavy metals: Sustainable technologies and recent advances. John Wiley & Sons, Ltd., Hoboken. Pp. 15–30.

Altaf, M. M., Ahmad Khan, M. S., & Ahmad, I. (2019). Diversity of bioactive compounds and their therapeutic potential. In: Ahmad Khan, M. S., Ahmad, I., & Chattopadhyay, D. (Eds.). New look to phytomedicine. Academic Press. Pp. 15–34.

Awasthi, A. (2023). Bioaccumulation of Cd, Cr, Cu, Ni, and Pb in a wild grass, Parthenium hysterophorus L. (Asteraceae), growing naturally on barren land and evaluation of phyto-extraction potential of the plant for studied metals. Asian Journal of Research in Chemistry, 16, 277–284.

Bhairam, M., Roy, A., Bahadur, S., Banafar, A., & Turkane, D. (2013). Standardization of herbal medicines – an overview. Journal of Applied Pharmaceutical Research, 1(1), 14–21.

Bhiri, N., Masquelez, N., Nasri, M., Nasri, R., Hajji, M., & Li, S. (2025). Synthesis, characterization, and stability study of selenium nanoparticles coated with purified polysaccharides from Ononis natrix. Nanomaterials, 15(6), 435.

Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181(4617), 1199–1200.

Cichon, N., Grabowska, W., Gorniak, L., Stela, M., Harmata, P., Ceremuga, M., & Bijak, M. (2025). Mechanistic and therapeutic insights into flavornoid-based inhibition of acetylcholinesterase: Implications for neurodegenerative diseases. Nutrients, 17(1), 78.

Cîrţînă, D., Chirigiu, L. M. E., & Nănescu, V. (2022). Considerations regarding the presence of heavy metals in medicinal plants and their health risks. Journal of Research and Innovation for Sustainable Society, 4(1), 18–23.

Dahiru, M. M., Badgal, E. B., & Musa, N. (2022). Phytochemistry, GS-MS analysis, and heavy metals composition of aqueous and ethanol stem bark extracts of Ximenia americana. GSC Biological and Pharmaceutical Sciences, 21(3), 145–156.

Devesa Alcaraz, J. A., & López González, G. (1997). Taxonomic and nomenclatural notes on the genus Ononis L. (Leguminosae) in the Iberian Peninsula and Balearic Islands. Anales del Jardín Botánico de Madrid, 55(2), 245–260.

Dias, M. C., Mariz-Ponte, N., & Santos, C. (2019). Lead induces oxidative stress in Pisum sativum plants and changes the levels of phytohormones with antioxidant role. Plant Physiology and Biochemistry, 137, 121–129.

Donald, A. N., Raphael, P. B., Olumide, O. J., & Amarachukwu, O. F. (2022). The synopsis of environmental heavy metal pollution. American Journal of Environmental Sciences, 18, 125–134.

Ejaz, U., Khan, S. M., Khalid, N., Ahmad, Z., Jehangir, S., Fatima Rizvi, Z., Lho, L. H., Han, H., & Raposo, A. (2023). Detoxifying the heavy metals: A multipronged study of tolerance strategies against heavy metals toxicity in plants. Frontiers in Plant Science, 14, 1154571.

Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88–95.

Ertaş, E., Aygan, A., Sarikurkcu, C., İstifli, E. S., & Tepe, B. (2025). Investigation of the chemical composition, antioxidant, and enzyme inhibitory activities of Ononis natrix subsp. natrix, performance of in silico analyses of its major compounds. Journal of Molecular Structure, 1319, 139428.

Ertürk, A. G., Ertürk, Ö., Ayvaz, M. Ç., & Ertürk, E. Y. (2018). Screening of phytochemical, antimicrobial and antioxidant activities in extracts of some fruits and vegetables consumed in Turkey. Celal Bayar University Journal of Science, 14(1), 81–92.

Ewubare, P. O., Aliyu, S. O., Michael, I., Ejakhaegbe, J. O., Obomejero, J., Okoro, S. O., & Olukayode, O. J. (2024). An academic review on heavy metals in the environment: Effects on soil, plants, human health, and possible solutions. American Journal of Environmental Economics, 3(1), 71–81.

Goncharuk, E. A., & Zagoskina, N. V. (2023). Heavy metals, their phytotoxicity, and the role of phenolic antioxidants in plant stress responses with focus on cadmium: Review. Molecules, 28(9), 3921.

Gorelova, V., Ambach, L., Rébeillé, F., Stove, C., & Van Der Straeten, D. (2017). Folates in plants: Research advances and progress in crop biofortification. Frontiers in Chemistry, 5, 21.

Hagerman, A. E. (2002). The tannin handbook. Miami University, Oxford.

Hegstad, K., Giske, C. G., Haldorsen, B., Matuschek, E., Schønning, K., Leegaard, T. M., Kahlmeter, G., & Sundsfjord, A. (2014). Performance of the EUCAST disk diffusion method, the CLSI agar screen method, and the Vitek 2 automated antimicrobial susceptibility testing system for detection of clinical isolates of enterococci with low- and medium-level VanB-type vancomycin resistance: A multicenter study. Journal of Clinical Microbiology, 52(5), 1582–1589.

Ibrahim, M. M., Zaki, E. R., & Rady, M. R. (2024). Alpha-amylase inhibitory activity and in silico studies of in vitro sweet basil plantlets treated with chitosan and ZnO NPs. In Vitro Cellular and Developmental Biology – Plant, 60(2), 147–160.

Jacobo-Velázquez, D. A., & Cisneros-Zevallos, L. (2012). An alternative use of horticultural crops: Stressed plants as biofactories of bioactive phenolic compounds. Agriculture, 2(3), 259–271.

Kanso, M., Hijazi, M., Aboul Ela, M., & El-Lakany, A. (2022). Medicinal plants’ stress factors: Effects on metabolites and novel perspectives for tolerance. BAU Journal – Health and Well-Being, 5(1), 6.

Karunakaran, K. B., Thiyagaraj, A., & Santhakumar, K. (2022). Novel insights on acetylcholinesterase inhibition by Convolvulus pluricaulis, scopolamine and their combination in zebrafish. Natural Products and Bioprospecting, 12(1), 6.

Kaur, H., & Goyal, N. (2022). Biochemical adaptations in plants under heavy metal stress: A revisit to antioxidant defense network. In: Aftab, T., & Hakeem, K. (Eds.). Metals metalloids soil plant water systems. Phytophysiology and Remediation Techniques. Academic Press, New York, Boston, San Diego, Oxford, London. Pp. 51–90.

Khare, S., Singh, N. B., Singh, A., Hussain, I., Niharika, K., Yadav, V., Bano, C., Yadav, R. K., & Amist, N. (2020). Plant secondary metabolites synthesis and their regulations under biotic and abiotic constraints. Journal of Plant Biology, 63(3), 203–216.

Kováčik, J., & Klejdus, B. (2008). Dynamics of phenolic acids and lignin accumulation in metal-treated Matricaria chamomilla roots. Plant Cell Reports, 27, 605–615.

Kowalczyk, A., Przychodna, M., Sopata, S., Bodalska, A., & Fecka, I. (2020). Thymol and thyme essential oil – New insights into selected therapeutic applications. Molecules, 25(18), 4125.

Kumar, K., Debnath, P., Singh, S., & Kumar, N. (2023). An overview of plant phenolics and their involvement in abiotic stress tolerance. Stresses, 3(3), 570–585.

Mhamdi, B., Abbassi, F., & Abdelly, C. (2015). Chemical composition, antioxidant and antimicrobial activities of the edible medicinal Ononis natrix growing wild in Tunisia. Natural Product Research, 29(12), 1157–1160.

Molnar, M., Jakovljević Kovač, M., & Pavić, V. (2024). A comprehensive analysis of diversity, structure, biosynthesis and extraction of biologically active tannins from various plant-based materials using deep eutectic solvents. Molecules, 29(11), 2615.

Müller, L., Gnoyke, S., Popken, A. M., & Böhm, V. (2010). Antioxidant capacity and related parameters of different fruit formulations. LWT – Food Science and Technology, 43(6), 992–999.

Oyaizu, M. (1986). Studies on products of browning reaction. Antioxidative activities of products of browning reaction prepared from glucosamine. The Japanese Journal of Nutrition and Dietetics, 44(6), 307–315.

Pandey, N., & Singh, G. K. (2012). Studies on antioxidative enzymes induced by cadmium in pea plants (Pisum sativum). Journal of Environmental Biology, 33(2), 201–206.

Pradhan, J., Pramanik, K., Jaiswal, A., Kumari, G., Prasad, K., Jena, C., & Srivastava, A. K. (2024). Biosynthesis of secondary metabolites in aromatic and medicinal plants in response to abiotic stresses: A review. Journal of Experimental Biology and Agricultural Sciences, 12(3), 318–334.

Rao, M. J., & Zheng, B. (2025). The role of polyphenols in abiotic stress tolerance and their antioxidant properties to scavenge reactive oxygen species and free radicals. Antioxidants, 14(1), 74.

Raza, A., Habib, M., Kakavand, S. N., Zahid, Z., Zahra, N., Sharif, R., & Hasanuzzaman, M. (2020). Phytoremediation of cadmium: Physiological, biochemical, and molecular mechanisms. Biology, 9(7), 177.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9–10), 1231–1237.

Sanal, F. (2019). The effects of heavy metal stress in wheat plant on certain phenolic compounds. Journal of BioScience and Biotechnology, 8(1), 45–50.

Sayari, N., Saidi, M. N., Sila, A., Ellouz-Chaabouni, S., & Bougatef, A. (2016). Chemical composition, angiotensin I-converting enzyme (ACE) inhibitory, antioxidant and antimicrobial activities of Ononis natrix leaves extracts. Free Radicals and Antioxidants, 6(1), 23–33.

Sharma, A., Shahzad, B., Rehman, A., Bhardwaj, R., Landi, M., & Zheng, B. (2019). Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules, 24(13), 2452.

Sharma, P., Gautam, A., Kumar, V., Khosla, R., & Guleria, P. (2021). Naringenin reduces Cd-induced toxicity in Vigna radiata (mungbean). Plant Stress, 1, 100005.

Shri, R., & Bansal, G. (2020). Does abiotic stresses enhance the production of secondary metabolites? A review. The Pharma Innovation Journal, 9(1), 412–422.

Son, Y.-J., Park, J.-E., Lee, N., Ju, Y.-W., Pyo, S.-H., Oh, C., Yoo, G., & Nho, C. W. (2024). Copper- or zinc-fortified nutrient solution in vertical farming system enriches copper or zinc and elevates phenolic acid and flavonoid contents in Artemisia annua L. Agronomy, 14(1), 135.

Szydlowska-Czerniak, A., Dianoczki, C., Recseg, K., Karlovits, G., & Szlyk, E. (2008). Determination of antioxidant capacities of vegetable oils by ferric-ion spectrophotometric methods. Talanta, 76(4), 899–905.

Tian, L., Wu, M., Guo, W., Li, H., Gai, Z., & Gong, G. (2021). Evaluation of the membrane damage mechanism of chlorogenic acid against Yersinia enterocolitica and Enterobacter sakazakii and its application in the preservation of raw pork and skim milk. Molecules, 26(21), 6748.

Topçu, G., Ay, M., Bilici, A., Sarıkürkcü, C., Öztürk, M., & Ulubelen, A. (2007). A new flavone from antioxidant extracts of Pistacia terebinthus. Food Chemistry, 103(3), 816–822.

Tripathi, P., & Tripathi, R. D. (2019). Metabolome modulation during arsenic stress in plants. In: Srivastava, S., Srivastava, A. K., & Suprasanna, P. (Eds.). Plant-metal interactions. Springer International Publishing, Cham. Pp. 119–140.

World Health Organization (2007). WHO guidelines for assessing quality of herbal medicines with reference to contaminants and residues. World Health Organization, Geneva.

Yu, X., Li, Y., Yang, X., He, J., Tang, W., Chai, Y., Duan, Z., Li, W., Zhao, D., Wang, X., Huang, A., Li, H., & Shi, Y. (2024). Chlorogenic acid: A promising strategy for milk preservation by inhibiting Staphylococcus aureus growth and biofilm formation. Foods, 13(24), 4104.

Zengin, G., Sarikurkcu, C., Aktumsek, A., & Ceylan, R. (2014). Sideritis galatica Bornm.: A source of multifunctional agents for the management of oxidative damage, Alzheimer’s and diabetes mellitus. Journal of Functional Foods, 11, 538–547.