Effects of Four-Week Intake of Blackthorn Flower Extract on Mice Tissue Antioxidant Status and Phenolic Content
Vedran Balta 1  
,   Domagoj Đikić 2, 1  
,   Irena Crnić 3  
,   Dyna Odeh 1  
,   Nada Orsolic 1  
,   Ivana Kmetič 4  
,   Teuta Murati 4  
,   Verica Dragović Uzelac 5  
,   Irena Landeka Jurčević 3  
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Biology Department, Faculty of Science, University of Zagreb, Croatia
Department of Animal Physiology, Faculty of Science Univeristy of Zagreb, Croatia
Department of Food Quality Control Laboratory for Food Chemistry and Biochemistry, Faculty of Food Technology and Biotechnology University of Zagreb, Croatia
Department of food toxicology, Faculty of Food Technology and Biotechnology University of Zagreb, Croatia
Department of Food Engineering Laboratory for Technology of Fruits and Vegetables Preservation and Processing, Faculty of Food Technology and Biotechnology University of Zagreb, Croatia
Domagoj Đikić   

Department of Animal Physiology, Faculty of Science Univeristy of Zagreb, Rooseveltov trg 6, 10000, Zagreb, Croatia
Submission date: 2020-04-30
Final revision date: 2020-09-29
Acceptance date: 2020-10-05
The study examined the antioxidative physiological effects of phenolics from an ethanol-water extract of blackthorn flowers orally administrated to C57/BL6 mice for 28 days in daily doses of 25 mg of total phenolics/kg body weight. Contents of phenolics in the intestine, liver, and kidneys collected after 1, 7, 14, 21, and 28 days of extract administration were analyzed by UPLC-MS/MS method. In the same tissues, the antioxidative properties were determined as ferric reducing antioxidant power (FRAP), ABTS•+ scavenging activity, content of reduced glutathione (GSH), and activity of superoxide dismutase (SOD) and catalase (CAT). The lipid peroxidation in tissues was also evaluated by thiobarbituric acid reactive substances (TBARS) assay. The exposed mice (compared to the control ones) had a lower content of TBARS in all tissues mostly on the third/fourth week of daily consumption. SOD activity and GSH content increased on the 28th day in tissues. CAT activity was higher only in the liver after one week of consumption but remained unchanged in other organs throughout the experiment. Phenolic profiles were different in individual tissues. The most prominent increases compared to the control were determined for contents of 3-O-feruloylquinic acid, 4-O-p-coumaroylqiunic acid, kaempferol pentoside, and quercetin rhamnoside in the intestine; for ferulic acid and quercetin 3-O-rutinoside in the liver; and for quercetin 3-O-rutinoside, ferulic acid, and 4-O-p-coumaroylquinic acid in the kidneys. The screened phenolics with different distribution in tissues could be responsible for slight differences in the recorded antioxidative effects.
We are grateful to the staff of the Animal Breeding Facility of the Department of Animal Physiology Faculty of Science, University of Zagreb, for their help during conduction of the in vivo experiment and to Dr. Zoran Zoric and Dr. Sandra Pedisic for their great effort during the preparation of UPLC-MS/MS data and results.
This work was supported by the project “Bioactive molecules of medical plants as natural antioxidants, microbicides, and preservatives” (KK., co-financed by the Croatian Government and the European Union through the European Regional Development Fund-Operational Programme Competitiveness and Cohesion (KK.
Aebi, H. (1984). Catalase in vitro. Methods in Enzymology, 105, 121–126.
Alarcόn, R., Pardo-de-Santayana, M., Priestley, C., Morales, R., Heinrich, M. (2015). Medicinal and local food plants in the south of Alava (Basque Country, Spain). Journal of Ethnopharmacology, 176, 207-224.
Bao, Y., Qu, Y., Li, J., Li, Y., Ren, X., Maffucci, K.G., Li, R., Wang, Z., Zeng, R. (2018). In vitro and in vivo antioxidant activities of the flowers and leaves from Paeonia rockii and identification of their antioxidant constituents by UHPLC-ESI-HRMSn via pre-column DPPH• reaction. Molecules, 23(2), art, no. 392.
Benzie, I.F., Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Analytical Biochemistry, 239(1),70-76.
Bioethic approval of the experimental procedure within the project, (2014). Document No. 251-58-10617-14-37, Faculty of Science, University of Zagreb, Croatia. (in Croatian).
Broncel, M., Kozirog, M., Duchnowicz, P., Koter-Michalak, M., Sikora, J., Chojnowska-Jezierska, J. (2010). Aronia melanocarpa extract reduces blood pressure, serum endothelin, lipid, and oxidative stress marker levels in patients with metabolic syndrome. Medical Science Monitor, 16(1), 28-34.
Đikić, D., Balta, V., Kmetić, I., Murati, T., Orsolić, N., Dragovic Uzelac, V., Landeka Jurčević, I. (2018). UPLC/MS analysis of plasma bioavailability of 32 polyphenols in C57BL/6 mice treated with single acute dose (24 h) of flower extract of the blackthorn Prunus spinosa L. Molecular and Experimental Biology in Medicine, 2, 23-31.
Dominko, K., Đikić, D. (2018). Glutathionylation: a regulatory role of glutathione in physiological processes. Arhiv za Higijenu Rada i Toksikologiju, 69(1), 1-24.
Dominko, K., Đikić, D., Hecimovic, S. (2020). Enhanced activity of superoxide dismutase is a common response to dietary and genetically induced increased cholesterol levels. Nutritional Neuroscience, 23(5), 398-410.
Elez-Garofulić, I., Zorić, Z., Pedisić, S., Brnčić, M., Dragović-Uzelac, V. (2018). UPLC-MS2 Profiling of blackthorn flower polyphenols isolated by ultrasound-assisted extraction. Journal of Food Science, 83(11), 2782-2789.
Flohé, L., Ötting, F. (1984). Superoxide dismutase assays. Methods in Enzymology, 105, 93-104.
Ganguly, S.G., Mantha, S., Panda, K. (2016). Simultaneous determination of black tea-derived catechins and theaflavins in tissues of tea consuming animals using Ultra-Performance Liquid-Chromatography Tandem Mass Spectrometry. PLoS One, 11(10), art. no. e0163498.
Gonzales, G.B., Smagghe, G., Grootaert, C., Zotti, M., Raes, K., Van Camp, J. (2015). Flavonoid interactions during digestion, absorption, distribution and metabolism: a sequential structure-activity/property relationship-based approach in the study of bioavailability and bioactivity. Drug Metabolism Reviews, 47(2), 175-90.
Guide for the Care and Use of Laboratory Animals (2011). Washington DC, USA, National Academies Press. 2011, 86-123.
Harwood, M., Danielewska-Nikiel, B., Borzelleca, J.F., Flamm, G.W., Williams, G.M., Lines, T.C.A. (2007). Critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties. Food and Chemical Toxicology, 45(11), 2179-2205.
Jin, S.L., Yin, Y.G. (2012). In vivo antioxidant activity of total flavonoids from indocalamus leaves in aging mice caused by D-galactose. Food and Chemical Toxicology, 50(10), 3814-3818.
Katalinic, V., Modun, D., Music, I., Boban, M. (2005). Gender differences in antioxidant capacity of rat tissues determined by 2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays. Comparative Biochemistry and Physiology C. Toxicology & Pharmacology, 140(1), 47-52.
Kelly, E., Vyas, P., Weber, J.T. (2017). Biochemical properties and neuroprotective effects of compounds in various species of berries. Molecules, 23(1), art. no. 26.
Landeka Jurčević, I., Dora, M., Guberović, I., Petras, M., Rimac Brnčić, S., Đikić, D. (2017). Polyphenols from wine lees as a novel functional bioactive compound in the protection against oxidative stress and hyperlipidaemia. Food Technology and Biotechnology, 55(1), 109-116.
Lovrić, V., Putnik, P., Kovačević, D.B., Jukić, M., Dragović-Uzelac, V. (2017). Effect of microwave-assisted extraction on the phenolic compounds and antioxidant capacity of blackthorn flowers. Food Technology and Biotechnology, 55(2), 243-250.
Lowry, D.H., Rosebrough, N.J., Farr, A.L. (1951). Protein measurement with the Folin–phenol reagent. The Journal of Biological Chemistry, 193(1), 265-275.
Marchelak, A., Owczarek, A., Matczak, M., Pawlak, A., Kolodziejczyk-Czepas, J., Nowak, P., Olszewska, M.A. (2017). Bioactivity potential of Prunus spinosa L. flower extracts: phytochemical profiling, cellular safety, pro-inflammatory enzymes inhibition and protective effects against oxidative stress in vitro. Frontiers in Pharmacology, 8, art. no. 680.
Menendez-Baceta, G., Aceituno-Mata, L., Tardío, J., Reyes-García, V., Pardo de Santayana, M. (2012). Wild edible plants traditionally gathered in Gorbeialdea (Biscay, Basque Country). Genetic Resources and Crop Evolution, 59(7), 1329-1347.
Meschini, S., Pellegrini, E., Condello, M., Occhionero, G., Delfine, S., Condello, G., Mastrodonato, F. (2017). Cytotoxic and apoptotic activities of Prunus spinosa Trigno ecotype extract on human cancer cells. Molecules, 22(9), art. no. 1578.
Mikulic-Petkovsek, M., Stampar, F., Veberic, R., Sircelj, H. (2016). Wild Prunus fruit species as a rich source of bioactive compounds. Journal of Food Science, 81(8), C1928-C1937.
Murati, T., Miletić, M., Kolarić, J., Lovrić, V., Kovačević, D.B., Putnik, P., Jurčević, I.L., Đikić, D., Dragović-Uzelac, V., Kmetič, I. (2019). Toxic activity of Prunus spinosa L. flower extract in hepatocarcinoma cells. Arhiv za higijenu rada i toksikologiju, 70(4), 303–309.
Nakhaee, A., Bokaeian, M., Saravani, M., Farhangi, A., Akbarzadeh, A. (2009). Attenuation of oxidative stress in streptozotocin-induced diabetic rats by Eucalyptus globulus. Indian Journal of Clinical Biochemistry, 24(4), 419–425.
Nardi, G.M., Farias Januario, A.G., Freire, C.G., Megiolaro, F., Schneider, K., Perazzoli, M.R., Do Nascimento, S.R., Gon, A.C., Mariano, L.N., Wagner, G., Niero, R., Locatelli, C. (2016). Anti-inflammatory activity of berry fruits in mice model of inflammation is based on oxidative stress modulation. Pharmacognosy Research, 8(1), 42-49.
NN 55/2013. Act on Animal Welfare, Croatia (2013). Official Gazette of the Republic of Croatia, 1129 (in Croatian).
Ohkawa, H., Ohishi, N., Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95(2), 351-358.
Olszewska, M., Głowacki, R., Wolbís, M., Bald, E. (2001). Quantitative determination of flavonoids in the flowers and leaves of Prunus spinosa L. Acta Poloniae Pharmaceutical, 58(3), 199-203. PMID: 11712737.
Olszewska, M., Wolbiś, M. (2001). Flavonoids from the flowers of Prunus spinosa L. Acta Poloniae Pharmaceutical, 58(5), 367-372.
Olszewska, M., Wolbiś, M. (2002). Further flavonoids from the flowers of Prunus spinosa L. Acta Poloniae Pharmaceutical, 59(2), 133-137. PMID: 12365605.
Olthof, M.R., Hollman, P.C., Vree, T.B., Katan, M.B. (2000). Bioavailabilities of quercetin-3-glucoside and quercetin-4'-glucoside do not differ in humans. The Journal of Nutrition, 130(5), 1200-1203.
Peng, K.Z., Zhang, S.Y., Zhou, H.L. (2016). Toxicological evaluation of the flavonoid-rich extract from Maydis stigma: Subchronic toxicity and genotoxicity studies in mice. Journal of Ethnopharmacology, 192,161-169.
Pilaczynska-Szczesniak, L., Skarpanska-Steinborn, A., Deskur, E., Basta, P., Horoszkiewicz-Hassan, M. (2005). The influence of chokeberry juice supplementation on the reduction of oxidative stress resulting from an incremental rowing ergometer exercise. International Journal of Sport Nutrition and Exercise Metabolism, 15(1), 48-58.
Pinacho, R., Cavero, R., Astiasarán, I., Ansorena, D., Calvo, M. (2015). Phenolic compounds of blackthorn (Prunus spinosa L.) and influence of in vitro digestion on their antioxidant capacity. Journal of Functional Foods, 19, 49-62.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS•+ radical cation decolourization assay. Free Radical Biology & Medicine, 26(9-10), 1231–1237.
Renfrew, J.M. (1973). Palaeoethnobotany: The Prehistoric Food Plants of the Near East and Europe. Methuen, London, UK.
Salahshoor, M.R., Mohammadi, M.M., Roshankhah, S., Najari, N., Jalili, C. (2019). Effect of Falcaria vulgaris on oxidative damage of liver in diabetic rats. Journal of Diabetes and Metabolic Disorders, 18(1), 15-23.
SPSS version 17.0 (SPSS Inc, Chicago, IL). Available at: [https://www.hks.harvard.edu/.....].
Squillaro, T., Cimini, A., Peluso, G., Giordano, A., Melone, M.A.B. (2018). Nano-delivery systems for encapsulation of dietary polyphenols: An experimental approach for neurodegenerative diseases and brain tumors. Biochemical Pharmacology, 154, 303-317.
Teng, H., Chen, L. (2019). Polyphenols and bioavailability: an update. Critical Reviews in Food Science and Nutrition, 59(13), 2040-2051.
Tietze, F. (1969). Enzyme method for quantitative determination of nanogram amounts of total and oxidized glutathione. Analytical Biochemistry, 27, 502–522.
Wang, W., Sun, C., Mao, L, Ma, P., Liu, F., Yang, J., Gao, Y. (2016). The biological activities, chemical stability, metabolism and delivery systems of quercetin: a review. Trends in Food Science & Technology, 56, 21–38.
Yuksel, A.K. (2015). The effects of blackthorn (Prunus spinosa L.) addition on certain quality characteristics of ice cream. Journal of Food Quality, 38(6), 413-421.