Research article    |    Open Access
Acta Natura et Scientia 2026, Vol. 7(1) 74-84

LC-MS/MS-Based Phenolic Profiling of Flaxseed (Linum usitatissimum L.) and Safflower (Carthamus tinctorius L.) Seed Extracts and Their Antibacterial Activity Against Selected Fish Pathogenic Bacteria

Mabrokah Adrees Rafallah Saed, Mustafa Karga, Osman Nezih Kenanoğlu, Soner Bilen

pp. 74 - 84   |  DOI: https://doi.org/10.61326/actanatsci.v7i1.491

Publish Date: June 24, 2026  |   Single/Total View: 0/0   |   Single/Total Download: 0/0


Abstract

The growing demand for sustainable disease-control strategies in aquaculture has increased interest in plant-derived compounds with potential antibacterial activity against fish pathogens. This study investigated the LC-MS/MS-based phenolic profile and in vitro antibacterial activity of aqueous methanolic flaxseed (Linum usitatissimum L.) and safflower (Carthamus tinctorius L.) seed extracts against selected fish pathogenic bacteria. The extracts were obtained using 40% methanol, concentrated, and prepared as aqueous stock solutions. The stock concentrations were 0.148 g mL-1 for flaxseed and 0.101 g mL-1 for safflower. The phenolic profiles of the final aqueous stock solutions were then determined by LC-MS/MS and expressed as µg L-1 (ppb). LC-MS/MS analysis showed that the flaxseed extract was mainly characterized by a tannic acid-rich profile, together with trans-ferulic acid, caffeic acid, gallic acid, quercetin, rutin trihydrate, cinnamic acid, and 2,5-dihydroxybenzoic acid. In contrast, the safflower seed extract contained trans-ferulic acid, cinnamic acid, 2,5-dihydroxybenzoic acid, ellagic acid, caffeic acid, and quercetin as the detected phenolic constituents. Antibacterial activity was tested by the broth microdilution method against Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas putida, Yersinia ruckeri, and Vibrio anguillarum. The strongest antibacterial activity was observed for flaxseed extract against A. hydrophila and A. salmonicida, with MIC values of 25 and 50 µg mL-1, respectively. Safflower seed extract showed its highest inhibitory activity against A. salmonicida, with an MIC value of 50 µg mL-1, while higher MIC values were recorded against A. hydrophila, Y. ruckeri, and V. anguillarum. No inhibitory activity was detected against P. putida for either extract within the tested concentration range. These results indicate that aqueous methanolic flaxseed and safflower seed extracts, particularly flaxseed, have species-dependent antibacterial activity against fish pathogens. Although these findings suggest that the tested extracts may be considered as preliminary natural antibacterial candidates for aquaculture-related research, further studies are needed to evaluate their safety, stability, and in vivo applicability.

Keywords: Antibacterial activity, Aquaculture, Broth microdilution, Fish pathogens, LC-MS/MS, MIC


How to Cite this Article?

APA 7th edition
Saed, M.A.R., Karga, M., Kenanoglu, O.N., & Bilen, S. (2026). LC-MS/MS-Based Phenolic Profiling of Flaxseed (Linum usitatissimum L.) and Safflower (Carthamus tinctorius L.) Seed Extracts and Their Antibacterial Activity Against Selected Fish Pathogenic Bacteria  . Acta Natura et Scientia, 7(1), 74-84. https://doi.org/10.61326/actanatsci.v7i1.491

Harvard
Saed, M., Karga, M., Kenanoglu, O. and Bilen, S. (2026). LC-MS/MS-Based Phenolic Profiling of Flaxseed (Linum usitatissimum L.) and Safflower (Carthamus tinctorius L.) Seed Extracts and Their Antibacterial Activity Against Selected Fish Pathogenic Bacteria  . Acta Natura et Scientia, 7(1), pp. 74-84.

Chicago 16th edition
Saed, Mabrokah Adrees Rafallah, Mustafa Karga, Osman Nezih Kenanoglu and Soner Bilen (2026). "LC-MS/MS-Based Phenolic Profiling of Flaxseed (Linum usitatissimum L.) and Safflower (Carthamus tinctorius L.) Seed Extracts and Their Antibacterial Activity Against Selected Fish Pathogenic Bacteria  ". Acta Natura et Scientia 7 (1):74-84. https://doi.org/10.61326/actanatsci.v7i1.491

References
  1. Bilen, S., Ünal, S., & Güvensoy, H. (2016). Effects of oyster mushroom (Pleurotus ostreatus) and nettle (Urtica dioica) methanolic extracts on immune responses and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss). Aquaculture, 454, 90–94. https://doi.org/10.1016/j.aquaculture.2015.12.010 [Google Scholar] [Crossref] 
  2. Bouarab-Chibane, L., Forquet, V., Lantéri, P., Clément, Y., Léonard-Akkari, L., Oulahal, N., Degraeve, P., & Bordes, C. (2019). Antibacterial properties of polyphenols: Characterization and QSAR (Quantitative structure–activity relationship) models. Frontiers in Microbiology, 10, 829. https://doi.org/10.3389/fmicb.2019.00829 [Google Scholar] [Crossref] 
  3. CLSI. (2018). Clinical and Laboratory Standards Institute, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 11th edition. Standard M07. Clinical and Laboratory Standards Institute (CLSI). [Google Scholar]
  4. Cushnie, T. T., & Lamb, A. J. (2005). Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents, 26(5), 343-356. https://doi.org/10.1016/j.ijantimicag.2005.09.002 [Google Scholar] [Crossref] 
  5. Dallaire-Dufresne, S., Tanaka, K. H., Trudel, M. V., Lafaille, A., & Charette, S. J. (2014). Virulence, genomic features, and plasticity of Aeromonas salmonicida subsp. salmonicida, the causative agent of fish furunculosis. Veterinary Microbiology, 169(1-2), 1-7. https://doi.org/10.1016/j.vetmic.2013.06.025 [Google Scholar] [Crossref] 
  6. Farha, A. K., Yang, Q. Q., Kim, G., Li, H. B., Zhu, F., Liu, H. Y., Gan, R. Y., & Corke, H. (2020). Tannins as an alternative to antibiotics. Food Bioscience, 38, 100751. https://doi.org/10.1016/j.fbio.2020.100751 [Google Scholar] [Crossref] 
  7. Frans, I., Michiels, C. W., Bossier, P., Willems, K. A., Lievens, B., & Rediers, H. (2011). Vibrio anguillarum as a fish pathogen: Virulence factors, diagnosis and prevention. Journal of Fish Diseases, 34(9), 643–661. https://doi.org/10.1111/j.1365-2761.2011.01279.x [Google Scholar] [Crossref] 
  8. Hajji, M., Jarraya, R., Lassoued, I., Masmoudi, O., Damak, M., & Nasri, M. (2010). GC/MS and LC/MS analysis, and antioxidant and antimicrobial activities of various solvent extracts from Mirabilis jalapa tubers. Process Biochemistry, 45(9), 1486–1493. https://doi.org/10.1016/j.procbio.2010.05.027 [Google Scholar] [Crossref] 
  9. Karga, M., Kenanoğlu, O. N., & Bilen, S. (2020). Investigation of antibacterial activity of two different medicinal plants extracts against fish pathogens. Journal of Agricultural Production, 1(1), 5–7. [Google Scholar]
  10. Katsuda, S. I., Suzuki, K., Koyama, N., Takahashi, M., Miyake, M., Hazama, A., & Takazawa, K. (2009). Safflower seed polyphenols (N-(p-coumaroyl) serotonin and N-feruloylserotonin) ameliorate atherosclerosis and distensibility of the aortic wall in Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. Hypertension Research, 32(11), 944-949. https://doi.org/10.1038/hr.2009.144 [Google Scholar] [Crossref] 
  11. Kim, E. O., Oh, J. H., Lee, S. K., Lee, J. Y., & Choi, S. W. (2007). Antioxidant properties and quantification of phenolic compounds from safflower (Carthamus tinctorius L.) seeds. Food Science and Biotechnology, 16(1), 71-77. [Google Scholar]
  12. Koçak, M. Z. (2024). Phenolic compounds, fatty acid composition, and antioxidant activities of some flaxseed (Linum usitatissimum L.) varieties: A comprehensive analysis. Processes, 12(4), 689. https://doi.org/10.3390/pr12040689 [Google Scholar] [Crossref] 
  13. Li, M., Wei, D., Huang, S., Huang, L., Xu, F., Yu, Q., Liu, M., & Li, P. (2022). Medicinal herbs and phytochemicals to combat pathogens in aquaculture. Aquaculture International, 30(3), 1239-1259. https://doi.org/10.1007/s10499-022-00841-7 [Google Scholar] [Crossref] 
  14. Liu, R. (2003). Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. The American Journal of Clinical Nutrition, 78(3), 517S–520S. https://doi.org/10.1093/ajcn/78.3.517S [Google Scholar] [Crossref] 
  15. Metin, S., Kara, N., Didinen, B. I., & Kubilay, A. (2021). Antibacterial activity of essential oils and extracts of some medicinal plants against bacterial fish pathogens. The Israeli Journal of Aquaculture – Bamidgeh, 73, 1305530. https://doi.org/10.46989/001c.21456 [Google Scholar] [Crossref] 
  16. Oomah, B. D., Kenaschuk, E. O., & Mazza, G. (1995). Phenolic acids in flaxseed. Journal of Agricultural and Food Chemistry, 43(8), 2016–2019. https://doi.org/10.1021/jf00056a011 [Google Scholar] [Crossref] 
  17. Öztürk, R. Ç., & Altınok, I. (2014). Bacterial and viral fish diseases in Turkey. Turkish Journal of Fisheries and Aquatic Sciences, 14(1), 275–297. https://doi.org/10.4194/1303-2712-v14_1_30 [Google Scholar] [Crossref] 
  18. Pang, Z., Raudonis, R., Glick, B. R., Lin, T.-J., & Cheng, Z. (2019). Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and alternative therapeutic strategies. Biotechnology Advances, 37(1), 177–192. https://doi.org/10.1016/j.biotechadv.2018.11.013 [Google Scholar] [Crossref] 
  19. Pires, L. d. C., Rodrigues, P., Garlet, Q. I., Barbosa, L. B., da Silveira, B. P., Bandeira Junior, G., Silva, L. d. L., Gindri, A., Coldebella, R., Pedrazzi, C., de Vargas, A. P. C., Baldisserotto, B., & Heinzmann, B. M. (2021). Maclura tinctoria extracts: In vitro antibacterial activity against Aeromonas hydrophila and sedative effect in Rhamdia quelen. Fishes, 6(3), 25. https://doi.org/10.3390/fishes6030025 [Google Scholar] [Crossref] 
  20. Ramesh, D., & Souissi, S. (2018). Antibiotic resistance and virulence traits of bacterial pathogens from infected freshwater fish, Labeo rohita. Microbial Pathogenesis, 116, 113–119. https://doi.org/10.1016/j.micpath.2018.01.019 [Google Scholar] [Crossref] 
  21. Semwal, A., Kumar, A., & Kumar, N. (2023). A review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture. Heliyon, 9(3), e14088. https://doi.org/10.1016/j.heliyon.2023.e14088 [Google Scholar] [Crossref] 
  22. Tadese, D. A., Song, C., Sun, C., Liu, B., Liu, B., Zhou, Q., Xu, P., Ge, X., Liu, M., Xu, X., Tamiru, M., Zhou, Z., Lakew, A., & Kevin, N. T. (2022). The role of currently used medicinal plants in aquaculture and their action mechanisms: A review. Reviews in Aquaculture, 14(2), 816–847. https://doi.org/10.1111/raq.12626 [Google Scholar] [Crossref] 
  23. Terzi, E., & Isler, H. (2019). Antibiotic resistance genes of Escherichia coli in coastal marine environment of Eastern Black Sea, Turkey. Fresenius Environmental Bulletin, 28(2A), 1594–1601. [Google Scholar]
  24. Terzi, E., Tahiluddin, A. B., & Kadak, A. E. (2023). Evaluation of the antibacterial activity of cultivated Caucasian whortleberry (Vaccinium arctostaphylos L.) against fish pathogens. Fisheries & Aquatic Life, 31(2), 79–86. https://doi.org/10.2478/aopf-2023-0009 [Google Scholar] [Crossref] 
  25. Tobback, E., Decostere, A., Hermans, K., Haesebrouck, F., & Chiers, K. (2007). Yersinia ruckeri infections in salmonid fish. Journal of Fish Diseases, 30(5), 257–268. https://doi.org/10.1111/j.1365-2761.2007.00816.x [Google Scholar] [Crossref] 
  26. Van Hai, N. (2015). The use of medicinal plants as immunostimulants in aquaculture: A review. Aquaculture, 446, 88–96. https://doi.org/10.1016/j.aquaculture.2015.03.014 [Google Scholar] [Crossref] 
  27. Yu, S.-Y., Lee, Y.-J., Kim, J.-D., Kang, S.-N., Lee, S.-K., Jang, J.-Y., Lee, H.-K., Lim, J.-H., & Lee, O.-H. (2013). Phenolic composition, antioxidant activity and anti-adipogenic effect of hot water extract from safflower (Carthamus tinctorius L.) seed. Nutrients, 5(12), 4894–4907. https://doi.org/10.3390/nu5124894 [Google Scholar] [Crossref]