Numerous in vitro assays are used to characterize the antioxidant properties of natural-based matrices. However, many of them generate contradictory and non-compliant results. In our study, we focused on the characterization of traditionally used biochemical (2,2'-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS), Oxygen Radical Absorption Capacity (ORAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and cellular (CAA) antioxidant tests on a broad set of milk thistle dietary supplements containing silymarin. In addition to 26 commercially available preparations, also the natural silymarin extract available from Sigma Aldrich, St. Louis, MI, USA, and a model mixture of pure flavonoid/flavonolignans mimicking the silymarin composition were investigated as control samples. Significant differences in the antioxidant capacity of the supplements were observed. Unlike the DPPH, the results of the ABTS and ORAC methods correlated with the silymarin components determined by U-HPLC-HRMS/MS. The responses in CAA were considerably lower than in other assays. Silymarin exhibited a significantly higher antioxidant capacity than the artificially prepared flavonoid/flavonolignans mixture in all tests, indicating possible presence of other antioxidants of natural origin. The follow-up U-HPLC-HRMS/MS screening revealed the presence of tens of non-silymarin compounds with reported antioxidant activity (not only in the silymarin extract, but also in the milk thistle preparations). The sum of the total phenolics and the sum of the simple phenolics correlated with CAA results more than silymarin.

4th Department of Internal Medicine and Institute of Medical Biochemistry and Laboratory Diagnostics 1st Faculty of Medicine Charles University Katerinska 32 12000 Prague Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Food Analysis and Nutrition University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republice

Laboratory of Biotransformation Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republice

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Moon J.-K., Shibamoto T. Antioxidant assays for plant and food components. J. Agric. Food Chem. 2009;57:1655–1666. doi: 10.1021/jf803537k. PubMed DOI

Alves C.Q., David J.M., David J.P., Bahia M.V., Aguiar R.M. Methods for determination of in vitro antioxidant activity for extracts and organic compounds. J. Food Sci. Tech. Mysore. 2010;33:2202–2210. doi: 10.1590/S0100-40422010001000033. DOI

Kim N.-C., Graf T.N., Sparacino C.M., Wani M.C., Wall M.E. Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum) Org. Biomol. Chem. 2003;1:1684–1689. doi: 10.1039/b300099k. PubMed DOI

Skottová N., Vecera R., Urbanek K., Vana P., Walterová D., Cvak L. Effects of polyphenolic fraction of silymarin on lipoprotein profile in rats fed cholesterol-rich diets. Pharmacol. Res. 2003;47:17–26. doi: 10.1016/S1043-6618(02)00252-9. PubMed DOI

Fried M.W., Navarro V.J., Afdhal N., Belle S.H., Wahed A.S., Hawke R.L., Doo E., Meyers C.M., Reddy K.R. Effect of silymarin (milk thistle) on liver disease in patients with chronic hepatitis c unsuccessfully treated with interferon therapy: A randomized controlled trial. JAMA. 2012;308:274–282. doi: 10.1001/jama.2012.8265. PubMed DOI PMC

Abenavoli L.M., Capasso R., Milic N., Capasso F. Milk thistle in liver diseases: Past, present, future. Phytother. Res. 2010;24:1423–1432. doi: 10.1002/ptr.3207. PubMed DOI

Seeff L.B., Curto T.M., Szabo G., Everson G.T., Bonkovsky H.L., Dienstag J.L., Shiffman M.L., Lindsay K.L., Lok A.S., Di Bisceglie A.M., et al. Herbal product use by persons enrolled in the hepatitis c antiviral long-term treatment against cirrhosis (halt-c) trial. Hepatology. 2008;47:605–612. doi: 10.1002/hep.22044. PubMed DOI

Féher J., Lengyel G. Silymarin in the prevention and treatment of liver diseases and primary liver cancer. Curr. Pharm. Biotechnol. 2012;13:210–217. doi: 10.2174/138920112798868818. PubMed DOI

Zhong S., Fan Y., Yan Q., Fan X., Wu B., Han Y., Zhang Y., Chen Y., Zhang H., Niu J. The therapeutic effect of silymarin in the treatment of nonalcoholic fatty disease: A meta-analysis (prisma) of randomized control trials. Medicine. 2017;96:e9061. doi: 10.1097/MD.0000000000009061. PubMed DOI PMC

Ferenci P., Scherzer T.M., Kerschner H., Rutter K., Beinhardt S., Hofer H., Schöniger–Hekele M., Holzmann H., Steindl–Munda P. Silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy. Gastroenterology. 2008;135:1561–1567. doi: 10.1053/j.gastro.2008.07.072. PubMed DOI

De Avelar C.R., Pereira E.M., Costa P.R., De Jesus R.P., De Oliveira L.P.M. Effect of silymarin on biochemical indicators in patients with liver disease: Systematic review with meta-analysis. World J. Gastroenterol. 2017;23:5004–5017. doi: 10.3748/wjg.v23.i27.5004. PubMed DOI PMC

Yang Z., Zhuang L., Lu Y., Xu Q., Chen X. Effects and tolerance of silymarin (milk thistle) in chronic hepatitis C virus infection patients: a meta-analysis of randomized controlled trials. BioMed. Res. Int. 2014;2014:9. doi: 10.1155/2014/941085. PubMed DOI PMC

Lee V., Goyal A., Hsu C.C., Jacobson J.S., Rodriguez R.D., Siegel A.B. Dietary supplement use among patients with hepatocellular carcinoma. Integr. Cancer Ther. 2015;14:35–41. doi: 10.1177/1534735414550038. PubMed DOI

Simanek V., Křen V., Ulrichová J., Vicar J., Cvak L. Silymarin: What is in the name…? An appeal for a change of editorial policy. Hepatology. 2000;32:442–444. doi: 10.1053/jhep.2000.9770. PubMed DOI

Fenclova M., Novakova A., Viktorova J., Jonatova P., Dzuman Z., Ruml T., Kren V., Hajslova J., Vitek L., Stranska-Zachariasova M. Poor chemical and microbiological quality of the commercial milk thistle-based dietary supplements may account for their reported unsatisfactory and non-reproducible clinical outcomes. Sci. Rep. 2019;9 doi: 10.1038/s41598-019-47250-0. PubMed DOI PMC

Zheleva-Dimitrova D., Nedialkov P., Kitanov G. Radical scavenging and antioxidant activities of methanolic extracts from Hypericum species growing in Bulgaria. Pharmacogn. Mag. 2010;6:74–78. doi: 10.4103/0973-1296.62889. PubMed DOI PMC

Zulueta A., Esteve M.J., Frígola A. ORAC and TEAC assays comparison to measure the antioxidant capacity of food products. Food Chem. 2009;114:310–316. doi: 10.1016/j.foodchem.2008.09.033. DOI

Pyrzynska K., Pękal A. Application of free radical diphenylpicrylhydrazyl (DPPH) to estimate the antioxidant capacity of food samples. Anal. Methods. 2013;5:4288–4295. doi: 10.1039/c3ay40367j. DOI

Liu S., Huang H. Assessments of antioxidant effect of black tea extract and its rationals by erythrocyte haemolysis assay, plasma oxidation assay and cellular antioxidant activity (CAA) assay. J. Funct. Foods. 2015;18:1095–1105. doi: 10.1016/j.jff.2014.08.023. DOI

Dixit N., Baboota S., Kohli K., Ahmad S., Ali J. Silymarin: A review of pharmacological aspects and bioavailability enhancement approaches. Indian J. Pharmacol. 2007;39:172–179. doi: 10.4103/0253-7613.36534. DOI

Anthony K.P., Saleh M.A. Free radical scavenging and antioxidant activities of silymarin components. Antioxidants. 2013;2:398–407. doi: 10.3390/antiox2040398. PubMed DOI PMC

Barnes P.M., Bloom B., Nahin R.L. Complementary and alternative medicine use among adults and children: United States, 2007. Natl. Health Stat. Rep. 2008;12:1–23. PubMed

Smith T., Kawa K., Eckl V., Morton C., Stredney R. Herbal supplement sales in us increase 7.7% in 2016 consumer preferences shifting toward ingredients with general wellness benefits, driving growth of adaptogens and digestive health products. Am. Bot. C. 2017;115:56–65.

Chambers C.S., Holečková V., Petrásková L., Biedermann D., Valentová K., Buchta M., Křen V. The silymarin composition… and why does it matter??? Food Res. Int. 2017;100:339–353. doi: 10.1016/j.foodres.2017.07.017. PubMed DOI

Anthony K., Saleh M.A. Chemical profiling and antioxidant activity of commercial milk thistle food supplements. J. Chem. Pharm. Res. 2012;4:4440–4450.

Fibigr J., Šatínský D., Solich P. A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract using UHPLC core-shell column with F5 stationary phase. J. Pharm. Biomed. Anal. 2017;134:203–213. doi: 10.1016/j.jpba.2016.11.042. PubMed DOI

Filippopoulou K., Papaevgeniou N., Lefaki M., Paraskevopoulou A., Biedermann D., Křen V., Chondrogianni N. 2,3-Dehydrosilybin A/B as a pro-longevity and anti-aggregation compound. Free. Radic. Biol. Med. 2017;103:256–267. doi: 10.1016/j.freeradbiomed.2016.12.042. PubMed DOI

Šuk J., Jašprová J., Biedermann D., Petrásková L., Valentová K., Křen V., Muchová L., Vítek L. Isolated silymarin flavonoids increase systemic and hepatic bilirubin concentrations and lower lipoperoxidation in mice. Oxid. Med. Cell. Longev. 2019;2019:12. doi: 10.1155/2019/6026902. PubMed DOI PMC

Biedermann D., Buchta M., Holečková V., Sedlák D., Valentová K., Cvačka J., Bednárová L., Křenková A., Kuzma M., Škuta C., et al. Silychristin: Skeletal alterations and biological activities. J. Nat. Prod. 2016;79:3086–3092. doi: 10.1021/acs.jnatprod.6b00750. PubMed DOI

Hammouda F.M., Ismail S.I., Hassan N.M., Zaki A.K., Kamel A., Rimpler H. Evaluation of the silymarin content in Silybum marianum (L.) Gaertn. Cultivated under different agricultural conditions. Phytother. Res. 1993;7:90–91. doi: 10.1002/ptr.2650070122. DOI

Lobo V., Patil A., Phatak A., Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn. Rev. 2010;4:118–126. doi: 10.4103/0973-7847.70902. PubMed DOI PMC

Amorati R., Valgimigli L. Advantages and limitations of common testing methods for antioxidants. Free. Radic. Res. 2015;49:633–649. doi: 10.3109/10715762.2014.996146. PubMed DOI

Miguel-Chávez R.S. Phenolic antioxidant capacity: A review of the state of the art. In: Soto-Hernandez M., Palma-Tenango M., Garcia-Mateos M.R., editors. Phenolic Compounds—Biological Activity. IntechOpen Limited; London, UK: 2017.

Köksal E., Gulcin I., Beyza S., Sarikaya Ö., Bursal E. In vitro antioxidant activity of silymarin. J. Enzym. Inhib. Med. Chem. 2009;24:395–405. doi: 10.1080/14756360802188081. PubMed DOI

Sharifi R., Pasalar P., Kamalinejad M., Dehpour A.R., Tavangar S.M., Paknejad M., Mehrabani Natanzi M., Nourbakhsh M., Ahmadi Ashtiani H.R., Akbari M., et al. The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model. Pharm. Biol. 2013;51:298–303. doi: 10.3109/13880209.2012.721789. PubMed DOI

Thaipong K., Boonprakob U., Crosby K., Cisneros-Zevallos L., Byrne D.H. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food Compos. Anal. 2006;19:669–675. doi: 10.1016/j.jfca.2006.01.003. DOI

Lucas-Abellán C., Mercader-Ros M.T., Zafrilla M.P., Gabaldon J.A., Núñez-Delicado E. Comparative study of different methods to measure antioxidant activity of resveratrol in the presence of cyclodextrins. Food Chem. Toxicol. 2011;49:1255–1260. doi: 10.1016/j.fct.2011.03.004. PubMed DOI

Blasa M., Angelino D., Gennari L., Ninfali P. The cellular antioxidant activity in red blood cells (CAA-RBC): A new approach to bioavailability and synergy of phytochemicals and botanical extracts. Food Chem. 2011;125:685–691. doi: 10.1016/j.foodchem.2010.09.065. DOI

Panche A.N., Diwan A.D., Chandra S.R. Flavonoids: An overview. J. Nutr. Sci. 2016;5:e47. doi: 10.1017/jns.2016.41. PubMed DOI PMC

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