Evaluation of oxidative stress biomarkers in equine erythrocytes after in vitro treatment with leaf extract of thymus pulegioides l. (lamiaceae)

DOI: 10.32900/2312-8402-2021-125-47-59

Tkachenko H.,
Doctor of Biological Sciences,
Kurhaluk N.,
Doctor of Biological Sciences,
Institute of Biology and Earth Sciences, Pomeranian University in Słupsk, Poland,
Honcharenko V.,
Ph.D. https://orcid.org/0000-0001-6888-2124,
Nachychko V.,
Prokopiv A.,
Ivan Franko National University of Lviv, Ukraine,
Aksonov Ie.,
The Institute of Animal Science NAAS, Kharkiv, Ukraine

Keywords: Thymus pulegioides L., leaf extract, equine erythrocytes, lipid peroxidation, oxidatively modified proteins, total antioxidant capacity, hemolysis


In line with our previous study, we continue to evaluate the antioxidant potential of four species and one interspecific hybrid of the Thymus genus sampled in the Western part of Ukraine on the equine erythrocyte model. Therefore, in the present study, the oxidative stress biomarkers [2-thiobarbituric acid reactive substances (TBARS), carbonyl derivatives content of protein oxidative modification, total antioxidant capacity (TAC)], as well as HCl-induced hemolysis in the equine erythrocytes, was used for assessing the antioxidant activity of extract obtained from the leaves of Thymus pulegioides L. in dose 5 mg/mL. Leaves of Th. pulegioides were collected among grass nearby land parcels (Syvky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°02´02,8´´, E 26°14´13,9´´, 306 m a.s.l.). Equine erythrocyte aliquots were used in the study. For positive control (blank), phosphate buffer was used. After incubation of the mixture at 37°C for 60 mins with continuous stirring, samples were used for the biochemical assays. Results of the current study revealed that the extract obtained from leaves of Th. pulegioides (5 mg/mL) has a mild cytotoxic activity on the equine erythrocytes increasing the level of lipid peroxidation biomarker and hemolysis rate. The investigation also revealed that this extract exhibited hemolytic activity. These findings suggest the use of Th. pulegioides extract in dose 5 mg/mL as a source of prooxidant compounds and warrant further studies to evaluate their therapeutic potential. The aldehydic and ketonic derivatives level, as well as total antioxidant capacity, was non-significantly altered after in vitro incubation with an extract obtained from leaves of Th. pulegioides. Screening of Thymus species for other biological activities including antioxidant activities is essential and may be effective for searching the preventive agents in the pathogenesis of some diseases as well as prevention and treatment of some disorders in medicine and veterinary.


  1. Afonso, A. F., Pereira, O. R., & Cardoso, S. M. (2020). Health-Promoting Effects of Thymus Phenolic-Rich Extracts: Antioxidant, Anti-Inflammatory and Antitumoral Properties. Antioxidants (Basel), 9 (9), 814. https://doi.org/10.3390/antiox9090814.
  2. Afonso, A. F., Pereira, O. R., Neto, R. T., Silva, A. M., & Cardoso, S. M. (2017). Health-Promoting Effects of Thymus herba-barona, Thymus pseudolanuginosus, and Thymus caespititius Int. J. Mol. Sci., 18 (9), 1879. https://doi.org/10.3390/ijms18091879.
  3. Butt, A. S., Nisar, N., Mughal, T. A., Ghani, N., & Altaf, (2019). Anti-oxidative and anti-proliferative activities of extracted phytochemical compound thymoquinone. J. Pak. Med. Assoc., 69 (10), 1479–1485.
  4. Costa, P., Gonçalves, S., Valentão, P., Andrade, P. B., Coelho, N., & Romano, A. (2012). Thymus lotocephalus wild plants and in vitro cultures produce different profiles of phenolic compounds with antioxidant activity. Food Chem., 135 (3), 1253–1260. https://doi.org/10.1016/j.foodchem.2012.05.072.
  5. Dubinina, E. E., Burmistrov, S. O., Khodov, D. A., & Porotov, I. G. (1995). Okislitel’naia modifikatsiia belkov syvorotki krovi cheloveka, metod ee opredeleniia [Oxidative modification of human serum proteins. A method of determining it]. Med. Khim., 41 (1), 24–26. [in Russian].
  6. Farag, M. R., Alagawany, M. (2018). Erythrocytes as a biological model for screening of xenobiotics toxicity. Biol. Interact., 279, 73–83. https://doi.org/10.1016/j.cbi.2017.11.007.
  7. Galaktionova, L. P., Molchanov, A. V., El’chaninova, S. A., & Varshavskiĭ, B. Ia. (1998). Sostoianie perekisnogo okisleniia u bol’nykh s iazvennoĭ bolezn’iu zheludka i dvenadtsatiperstnoĭ kishki [Lipid peroxidation in patients with gastric and duodenal peptic ulcers]. Lab. Diagn., (6), 10–14. [in Russian].
  8. Honcharenko, V., Tkachenko, H., Nachychko, V., Prokopiv, A., & Osadowski, Z. (2018). Oxidative stress biomarkers in the equine erythrocyte suspension after in vitro incubation with leaf extract obtained from Thymus serpyllum emend. Mill. (Lamiaceae). Agrobiodiversity for Improving Nutrition, Health and Life Quality, (2): 201–211. https://doi.org/10.15414/agrobiodiversity.2018.2585-8246.201-211.
  9. Honcharenko, V., Tkachenko, H., Nachychko, V., Prokopiv, A., & Osadowski, Z. (2018). The antibacterial activities of some Thymus (Lamiaceae) representatives against Salmonella enteriditis strain locally isolated. Agrobiodiversity for Improving Nutrition, Health, and Life Quality, (2), 212–222. https://doi.org/10.15414/agrobiodiversity.2018.2585-8246.212-222
  10. Honcharenko, V., Tkachenko, H., Osadowski, Z., Nachychko, V., & Prokopiv, A. (2018). The antibacterial activities of ethanolic extracts obtained from leaves of some Thymus (Lamiaceae) representatives against β-lactamase producing Pseudomonas aeruginosa Słupskie Prace Biologiczne, 15, 59–78.
  11. Honcharenko, V., Tkachenko, H., Prokopiv, A., Nachychko, V., Kurhaluk, N., & Osadowski, Z. (2019). Oxidative stress biomarkers in the equine erythrocytes after In Vitro treated with leaf extract obtained from Thymus × porcii Borbás (Lamiaceae). Agrobiodiversity for Improving Nutrition, Health, and Life Quality, (3), 382–393. https://doi.org/10.15414/agrobiodiversity.2019.2585-8246.382-393.
  12. Kamyshnikov, V.S. (2004). A reference book on the clinic and biochemical researches and laboratory diagnostics. MEDpress-inform, Moscow.
  13. Kindl, M., Blažeković, B., Bucar, F., & Vladimir-Knežević, S. (2015). Antioxidant and Anticholinesterase Potential of Six Thymus Evid. Based Complement. Alternat. Med., 2015, 403950. https://doi.org/10.1155/2015/403950.
  14. 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. https://doi.org/10.3390/molecules25184125.
  15. Levine, R. L., Garland, D., Oliver, C. N., Amici, A., Climent, I., Lenz, A. G., Ahn, B. W., Shaltiel, S., & Stadtman, E. R. (1990). Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol., 186, 464–478. https://doi.org/10.1016/0076-6879(90)86141-h.
  16. Li, X., He, T., Wang, X., Shen, M., Yan, X., Fan, S., Wang, L., Wang, X., Xu, X., Sui, H., & She, G. (2019). Traditional Uses, Chemical Constituents and Biological Activities of Plants from the Genus Thymus. Biodivers., 16 (9), e1900254. https://doi.org/10.1002/cbdv.201900254.
  17. Meeran, M. F., Prince, P. S. (2012). Protective effects of thymol on altered plasma lipid peroxidation and nonenzymic antioxidants in isoproterenol-induced myocardial infarcted rats. Biochem. Mol. Toxicol., 26(9), 368–373. https://doi.org/10.1002/jbt.21431.
  18. Nachychko, V. (2014). The genus Thymus (Labiatae Juss.) in the Ukrainian Carpathians’ flora: systematics and taxonomic problems. Visnyk of Lviv University. Biological Series, 64, 159–169 [in Ukrainian].
  19. Nachychko, V. O. (2015). Diagnostic features of representatives of Thymus Serpyllum and T. sect. Marginati (Lamiaceae) and guidance for their herborization. The Journal of V. N. Karazin Kharkiv National University. Series: Biology, 25, 77–89. [In Ukrainian].
  20. Nachychko, V. O., Honcharenko, V. I. (2016). Hybrids of Thymus (Lamiaceae) genus in flora of the western regions of Ukraine: taxonomic composition and distribution. Studia Biologica, 10 (1), 163–186. [in Ukrainian].
  21. Nagoor Meeran, M. F., Jagadeesh, G. S., Selvaraj, P. (2015). Thymol attenuates inflammation in isoproterenol induced myocardial infarcted rats by inhibiting the release of lysosomal enzymes and downregulating the expressions of proinflammatory cytokines. J. Pharmacol., 754, 153–161. https://doi.org/10.1016/j.ejphar.2015.02.028.
  22. Nagoor Meeran, M. F., Javed, H., Al Taee, H., Azimullah, S., & Ojha, S. K. (2017). Pharmacological Properties and Molecular Mechanisms of Thymol: Prospects for Its Therapeutic Potential and Pharmaceutical Development. Pharmacol., 8, 380. https://doi.org/10.3389/fphar.2017.00380.
  23. Pagano, M., Faggio, C. (2015). The use of erythrocyte fragility to assess xenobiotic cytotoxicity. Cell Biochem. Funct., 33(6), 351–355. https://doi.org/10.1002/cbf.3135.
  24. Saidi, M., Sadeghifard, N., Kazemian, H., Sekawi, Z., Badakhsh, B., Friadian, S., & Ghafourian, S. (2016). Ex Vivo Evaluation of Thymus daenensis as an Antioxidant and Antibacterial Medicinal Herb. Drug Res. (Stuttg.), 66 (12), 657–659. https://doi.org/10.1055/s-0042-113457.
  25. Salehi, B., Mishra, A. P., Shukla, I., Sharifi-Rad, M., Contreras, M. D. M., Segura-Carretero, A., Fathi, H., Nasrabadi, N. N., Kobarfard, F., & Sharifi-Rad, J. (2018). Thymol, thyme, and other plant sources: Health and potential uses. Res., 32(9), 1688–1706. https://doi.org/10.1002/ptr.6109.
  26. Taghouti, M., Martins-Gomes, C., Félix, L. M., Schäfer, J., Santos, J. A., Bunzel, M., Nunes, F. M., & Silva, A. M. (2020). Polyphenol composition and biological activity of Thymus citriodorus and Thymus vulgaris: Comparison with endemic Iberian Thymus Food Chem., 331, 127362. https://doi.org/10.1016/j.foodchem.2020.127362.
  27. Taghouti, M., Martins-Gomes, C., Schäfer, J., Félix, L. M., Santos, J. A., Bunzel, M., Nunes, F. M., & Silva, A. M. (2018). Thymus pulegioides as a rich source of antioxidant, anti-proliferative and neuroprotective phenolic compounds. Food Funct., 9(7), 3617–3629. https://doi.org/10.1039/c8fo00456k.
  28. Terskov, I. A., Gitelson, I. I. (1957). Method of chemical (acid) erythrograms. Biofizika, 2, 259–266.
  29. Webster, N. R., Toothill, C. (1987). Inorganic phosphate transport across the red blood cell membrane: the effect of exposure to hyperoxia. Chim. Acta, 167(3), 259–265. https://doi.org/10.1016/0009-8981(87)90345-7.
  30. Youdim, K. A., Deans, S. G. (2000). Effect of thyme oil and thymol dietary supplementation on the antioxidant status and fatty acid composition of the ageing rat brain. J. Nutr., 83 (1), 87–93.
  31. Zar, J. H. (1999). Biostatistical Analysis. 4th, Prentice Hall Inc., New Jersey.