Oxidatively modified proteins level in the muscle tissue of the rainbow trout (oncorhynchus mykiss walbaum) as a biomarker of antioxidant properties of leaf extracts of camellia japonica l. Cultivars (theaceae d. Don)

DOI: 10.32900/2312-8402-2019-122-4-17

Buyun L.,
Doctor of Biological Sciences,
Kharchenko I.,
Maryniuk M.,
Post-graduate student,
M. M.Gryshko National Botanic Garden, National Academy of Science of Ukraine, Kyiv, Ukraine,
Tkachenko H.,
Doctor of Biological Sciences,
Osadowski Z.,
Doctor of Biological Sciences,
Institute of Biology and Environmental Protection, Pomeranian University in Słupsk, Poland

Keywords: rainbow trout, Oncorhynchus mykiss Walbaum, muscle tissue, Camellia japonica L., cultivars, oxidatively modified proteins


In this study, we investigated the antioxidative activity of plant extracts by the measurement of aldehydic and ketonic derivatives of oxidatively modified proteins. In our study, Camellia japonica L. ‘Kramer’s Supreme’, Camellia japonica ‘C. M. Wilson’, Camellia japonica ‘La Pace’, Camellia japonica ‘Benikarako’, Camellia japonica ‘Fanny Bolis’ were protectively effective extracts caused the decrease of protein oxidative damage in the muscle tissue of rainbow trout. The leaves of various Camellia japonica cultivars, cultivated under glasshouse conditions, were sampled at M. M. Gryshko National Botanical Garden (NBG), National Academy of Sciences of Ukraine. Freshly collected leaves were washed, weighed, crushed, and homogenized in 100 mM phosphate buffer (pH 7.2) (in proportion 1:19, w/w) at room temperature. The extracts were then filtered and investigated. Clinically healthy rainbow trout with a mean body mass of 80-120 g were used in the experiments. The muscle tissue samples were homogenized in ice-cold buffer (100 mM Tris-HCl, pH 7.2) using a glass homogenizer immersed in ice water bath. Homogenates were centrifuged at 3,000 g for 15 min at 4°C. The supernatant of the muscle tissue was used to incubate with extracts of various cultivars of C. japonica (in a ratio 19:1) at room temperature. The positive control group (trout muscle tissue) was incubated with 100 mM Tris-HCl buffer (pH 7.2) (in a ratio 19:1). The incubation time was 2 hours. When muscle tissue was incubated with leaf extracts of various C. japonica cultivars, the aldehydic derivatives level was ranged to the value of the control group. Among the six plant extracts, C. japonica ‘La Pace’ exhibited the highest inhibitory effect (the decrease of ketonic derivatives was 28.2%, p<0.05 compared to the control group). The level of ketonic derivatives of oxidatively modified proteins in the sample with C. japonica ‘Mrs. Lyman Clarke’ was increased by 2.8% (p>0.05) compared to controls. The least level of ketonic derivatives is attributed to C. japonica ‘La Pace’ extract. Therefore, Therefore, Camellia japonica may be used as an antioxidant agent in aquaculture as it can be easily obtained and is not expensive. Furthermore, the use of such plant products as antioxidants and immunostimulants in aquaculture systems may also have environmental value because of their biodegradability.


  1. Akihisa, T., Yasukawa, K., Kimura, Y., Takase, S., Yamanouchi, S., Tamura, T. (1997). Triterpene alcohols from camellia and sasanqua oils and their anti-inflammatory effects. Pharm. Bull. (Tokyo), 45(12), 2016-2023. DOI:10.1248/cpb.45.2016.
  2. Amar, E. C., Kiron, V., Satoh, S., Watanabe, T. (2004). Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products. Fish Shellfish Immunol., 16(4), 527-537. DOI: 10.1016/j.fsi.2003.09.004.
  3. Bashir, S., Khan, B. M., Babar, M., Andleeb, S., Hafeez, M., Ali, S., Khan, M. F. (2014). Assessment of Bioautography and Spot Screening of TLC of Green Tea (Camellia) Plant Extracts as Antibacterial and Antioxidant Agents. Indian J. Sci., 76(4), 364-370. PMCID: PMC4171874.
  4. Berlett, B. , Stadtman, E. R. (1997). Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry, 272, 20313-20316. DOI:10.1074/jbc.272.33.20313.
  5. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Biochem., 72, 248-254. DOI: 10.1006/abio.1976.9999.
  6. Chacko, S. , Thambi, P. T., Kuttan, R., Nishigaki, I. 2010. Beneficial effects of green tea: a literature review. Chin. Med., 5, 13. DOI: 10.1186/1749-8546-5-13.
  7. Dalle-Donne, I., Giustarini, D., Colombo, R., Rossi, R., Milzani, A. (2003). Protein carbonylation in human diseases. Trends in Molecular Medicine, 9(4), 169-176. DOI: 10.1111/j.1582-4934.2006.tb00407.x.
  8. Dubinina, E. , Burmistrov, S. O., Khodov, D. A., Porotov, I. G. (1995). Oxidative modification of human serum proteins. A method of determining it. Voprosy Meditsinskoĭ Khimii, 41, 24-26 (Article in Russian, Abstract in English).
  9. Facciola, S. (1990). Cornucopia. A source book of edible plants. Vista: Kampong Publishing, 677 p.
  10. Fassina, G., Buffa, A., Benelli, R., Varnier, O. E., Noonan, D. M., Albini, A. (2002). Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent. AIDS, 16(6), 939-941. DOI: 10.1097/00002030-200204120-00020.
  11. Hasanpour, S., Salati, A. P., Falahatkar, B., Azarm, H. M. (2017). Effects of dietary green tea (Camellia sinensis) supplementation on growth performance, lipid metabolism, and antioxidant status in a sturgeon hybrid of Sterlet (Huso huso ♂ × Acipenser ruthenus ♀) fed oxidized fish oil. Fish Physiol. Biochem., 43(5), 1315-1323. DOI: 10.1007/s10695-017-0374-z.
  12. Jeong, C. H., Kim, J. H., Choi, G. N., Kwak, J. H., Kim, D. O., Heo, H. J. (2010). Protective effects of extract with phenolics from camellia (Camellia japonica) leaf against oxidative stress-induced neurotoxicity. Food Sci. Biotechnol., 19(5): 1347-1353. DOI: 10.1007/s10068-010-0192-x.
  13. Kanth, B. K., Lee, K. Y., Lee, G. J. (2014). Antioxidant and radical-scavenging activities of petal extracts of Camellia japonica Hortic. Environ. Biotechnol., 55: 335-341. DOI: 10.1007/s13580-014-0024-7.
  14. Kharchenko, I., Maryniuk, M., Buyun, L., Tkachenko, H., Pażontka-Lipiński, P., Witaszek, M., Osadowski, Z. (2017). Lipid peroxidation level in the muscle tissue of the rainbow trout (Oncorhynchus mykiss Walbaum) under in vitro incubation with extracts from leaves of various cultivars of Camellia japonica (Theaceae). Науково-технічний бюлетень Інституту тваринництва, Нац. акад. аграр. наук України. Харків, N 118, 2017. – C. 3-13. [The Scientific and Technical Bulletin of the Institute of Animal Science NAAS of Ukraine, National Academy of Agrarian Sciences of Ukraine, Kharkiv, N 118, 2017.]
  15. Kharchenko, I., Maryniuk, M., Tkachenko, H., Buyun, L., Pażontka-Lipiński, P., Witaszek, M., Osadowski, Z. (2018). Superoxide dismutase activity level in the muscle tissue of the rainbow trout (Oncorhynchus mykiss Walbaum) as a biomarker of antioxidant properties of leaf extracts of Camellia japonica cultivars (Theaceae). Рыбохозяйственные водоёмы России: фундаментальные и прикладные исследования. Материалы II Всероссийской научной конференции с международным участием, ФГБНУ «Государственный научно-исследовательский институт озерного и речного рыбного хозяйства им. Л.С. Берга» (ФГБНУ «ГосНИОРХ»), Санкт-Петербург, 2-4 апреля 2018 г. [Fishery reservoirs of Russia: fundamental and applied research. Proceedings of the II All-Russian Scientific Conference with International Participation, FGBIU “L.S. Berg State Research Institute of the Lake and River Fisheries» (FGBNU «GosNIORH»), St. Petersburg, April 2-4, 2018.] – P. 528-535.
  16. Kharchenko, I., Maryniuk, M., Tkachenko, H., Buyun, L., Pażontka-Lipiński, P., Witaszek, M., Osadowski, Z. (2017)b. Total antioxidant activity of the muscle tissue of the rainbow trout (Oncorhynchus mykiss Walbaum) under incubation with extracts from leaves of various cultivars of Camellia japonica Scientific Journal of DALRYBVTUZ, 43(4): 18-26. (Харченко И. И., Маринюк М. М., Ткаченко Г. М., Буюн Л. И., Пажонтка-Липинский П., Виташек М., Осадовский З. 2017. Общая антиоксидантная активность мышечной ткани радужной форели (Oncorhynchus mykiss Walbaum) в исследованиях in vitro с экстрактами из листьев различных сортов камелии японской (Camellia japonica L.). Научные труды Дальрыбвтуза, 43(4), 18-26.)
  17. Kunkel, G. (1984). Plants for human consumption. An annotated checklist of the edible phanerogams and ferns. Koenigstein: Koeltz Scientific Books, 393.
  18. Landolt, M.L. (1989). The relationship between diet and the immune response. Aquaculture, 79: 193-206. DOI: 10.1016/0044-8486(89)90461-4.
  19. Lee, H. H., Cho, J. Y., Moon, J. H., Park, K. H. 2011. Isolation and identification of antioxidative phenolic acids and flavonoid glycosides from Camellia japonica Hortic. Environ. Biotechnol., 52(3): 270-277. DOI: 10.1007/s13580-011-0157-x.
  20. Lee, J. H., Kim, J. W., Ko, N. Y., Mun, S. H., Kim, D. K., Kim, J. D., Kim, H. S., Lee, K. R., Kim, Y. K., Radinger, M., Her, E., Choi, W.S. (2008). Camellia japonica suppresses immunoglobulin E-mediated allergic response by the inhibition of Syk kinase activation in mast cells. Exp. Allergy, 38(5), 794-804. DOI:10.1111/j.1365-2222.2008.02936.x.
  21. Levine, R. L. (2002). Carbonyl modified proteins in cellular regulation, aging, and disease. Free Radic. Biol. Med., 32(9), 790-796. DOI: 10.1016/s0891-5849(02)00765-7.
  22. Levine, R. L., Garland, D., Oliver, C. N., Amic, A., Climent,, Lenz, A. G., Ahn, B. W., Shaltiel, S., Stadtman, E. R. (1990). Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology, 186, 464-478. DOI:10.1016/0076-6879(90)86141-h.
  23. Long, M., Lin, W., Hou, J., Guo, H., Li, L., Li, D., Tang, R., Yang, F. 2017. Dietary supplementation with selenium yeast and tea polyphenols improve growth performance and nitrite tolerance of Wuchang bream (Megalobrama amblycephala). Fish Shellfish Immunol., 68, 74-83. DOI: 10.1016/j.fsi.2017.07.017.
  24. Martínez, A., Portero-Otin, M., Pamplona, R., Ferrer, I. (2010). Protein targets of oxidative damage in human neurodegenerative diseases with abnormal protein aggregates. Brain Pathology, 20(2), 281-297. DOI: 10.1111/j.1750-3639.2009.00326.x.
  25. Mizutani, T., Masaki, H. (2014). Anti-photoaging capability of antioxidant extract from Camellia japonica Exp. Dermatol., Suppl. 1, 23-26. DOI:10.1111/exd.12395.
  26. Nakamura, S., Fujimoto, K., Nakashima, S., Matsumoto, T., Miura, T., Uno,, Matsuda, H., Yoshikawa, M. (2012). Medicinal flowers. XXXVI. Acylated oleanane-type triterpene saponins with inhibitory effects on melanogenesis from the flower buds of Chinese Camellia japonica. Chem. Pharm. Bull., 60(6), 752758. DOI: 10.1248/cpb.60.752.
  27. Nootash, S., Sheikhzadeh, N., Baradaran, B., Oushani, A. K., Maleki Moghadam, M. R., Nofouzi, K., Monfaredan, A., Aghebati, L., Zare, F., Shabanzadeh, S. (2013). Green tea (Camellia sinensis) administration induces expression of immune relevant genes and biochemical parameters in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol., 35(6), 1916-1923. DOI: 10.1016/j.fsi.2013.09.030.
  28. Oh, G. S., Kang, S. S., Chung, M. G. (1996). Temporal genetic structure in Camellia japonica (Theaceae). Genes Genet. Syst., 71, 9-13. DOI: 10.1266/ggs.71.9.
  29. Onodera, K., Tsuha, K., Yasumoto-Hirose, M., Tsuha, K., Hanashiro, K., Naoki, H., Yasumoto, T. (2010). Okicamelliaside, an extraordinarily potent anti-degranulation glucoside isolated from leaves of Camellia japonica. Biotechnol. Biochem., 74(12), 2532-2534. DOI: 10.1271/bbb.100630.
  30. Salinero, C., Feás, X., Mansilla, J. P., Seijas, J. A., Vázquez-Tato, M. P., Vela, P., Sainz, M. J. (2012). ¹H-nuclear magnetic resonance analysis of the triacylglyceride composition of cold-pressed oil from Camellia japonica. Molecules, 17(6), 6716-6727. DOI: 10.3390/molecules17066716.
  31. Sayed, A.E.H., Soliman, H.A.M. (2018). Modulatory effects of green tea extract against the hepatotoxic effects of 4-nonylphenol in catfish (Clarias gariepinus). Environ. Saf., 149, 159-165. DOI: 10.1016/j.ecoenv.2017.11.007.
  32. Sheikhzadeh, N., Nofouzi, K., Delazar, A., Oushani, A. K. (2011). Immunomodulatory effects of decaffeinated green tea (Camellia sinensis) on the immune system of rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol., 31(6): 1268-1269. DOI: 10.1016/j.fsi.2011.09.010.
  33. Sousa, C., Quintelas, C., Augusto, C., Ferreira, E.C., Páscoa, R. N. M. J. (2019). Discrimination of Camellia japonica cultivars and chemometric models: An interlaboratory study. Computers and Electronics in Agriculture, 159, 28-33. DOI:1016/j.compag.2019.02.025.
  34. Stadtman, E. R., Levine, R. L. (2000). Protein oxidation. N Y Acad. Sci., 899, 191-208. DOI: 10.1111/j.1749-6632.2001.tb05632.x.
  35. Stuart, R. G. A. (1979). Chinese Materia Medica: vegetable kingdom. Southern Materials Centre Inc., Taipei.
  36. Tang, G. Y., Zhao, C. N., Xu, X. Y., Gan, R. Y., Cao, S. Y., Liu, Q., Shang, A., Mao, Q. Q., Li, H. B. (2019). Phytochemical Composition and Antioxidant Capacity of 30 Chinese Teas. Antioxidants (Basel), 8(6). DOI: 10.3390/antiox8060180.
  37. Thawonsuwan, J., Kiron, V., Satoh, S., Panigrahi, A., Verlhac, V. (2010). Epigallocatechin-3-gallate (EGCG) affects the antioxidant and immune defense of the rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem., 36(3), 687-697. DOI:1007/s10695-009-9344-4.
  38. Usher, G. (1974). A dictionary of plants used by man. London: Constable, 619 p.
  39. Vela, P., Salinero, C., Sainz, M. 2013. Phenological growth stages of Camellia japonica. Ann. Appl. Biol., 162, 82-190. DOI: 10.1111/aab.12010.
  40. Wang, C.-C., Ho, C.-T., Lee, S.-C.,Way, T.-D. (2016). Isolation of eugenyl b-primeveroside from Camellia sasanqua and its anticancer activity in PC3 prostate cancer cells. Food Drug Analysis, 24, 105-111. DOI: 10.1016/j.jfda.2015.06.005.
  41. Yoshikawa, M., Morikawa, T., Asao, Y., Fujiwara, E., Nakamura, S., Matsuda, H. (2007). Medicinal flowers. XV. The structures of noroleanane- and oleanane-type triterpene oligoglycosides with gastroprotective and platelet aggregation activities from flower buds of Camellia japonica. Pharm. Bull., 55(4), 606-612. DOI: 10.1248/cpb.55.606.
  42. Zar, J. H. (1999). Biostatistical Analysis, 4th Prentice Hall Inc., New Jersey, 663 p.
  43. Zhao, C. N., Tang, G. Y., Cao, S. Y., Xu, X. Y., Gan, R. Y., Liu, Q., Mao, Q. Q., Shang, A., Li, H. B. (2019). Phenolic Profiles and Antioxidant Activities of 30 Tea Infusions from Green, Black, Oolong, White, Yellow and Dark Teas. Antioxidants (Basel), 8(7). DOI: 10.3390/antiox8070215.