Calibration curves for Folin-Ciocalteu assay for reliable quantification of total polyphenols.

Five different protocols (P1-P5) for determining total polyphenol content in samples were investigated and compared. Detailed information on the protocols are provided in:

P1 Singleton, V.L.; Rossi, J.A. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic. 1965, 16, 144–158, doi:10.5344/ajev.1965.16.3.144.

P2  Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. [14] Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin-Ciocalteu Reagent. In Methods in Enzymology; Oxidants and Antioxidants Part A; Academic Press, 1999; Vol. 299, pp. 152–178.

P3  Cicco, N.; Lanorte, M.T.; Paraggio, M.; Viggiano, M.; Lattanzio, V. A Reproducible, Rapid and Inexpensive Folin–Ciocalteu Micro-Method in Determining Phenolics of Plant Methanol Extracts. Microchemical Journal 2009, 91, 107–110, doi:10.1016/j.microc.2008.08.011.

P4. Sotenko, M.; Coles, S.; Barker, G.; Song, L.; Jiang, Y.; Longhurst, P.; Romanova, T.; Shuvaeva, O.; Kirwan, K. Phytoremediation-Biorefinery Tandem for Effective Clean-up of Metal Contaminated Soil and Biomass Valorisation. International Journal of Phytoremediation 2017, 19, 965–975, doi:10.1080/15226514.2016.1267705.

P5         Tsibranska, I.; Tylkowski, B.; Kochanov, R.; Alipieva, K. Extraction of Biologically Active Compounds from Sideritis Ssp. L. Food and Bioproducts Processing 2011, 89, 273–280, doi:10.1016/j.fbp.2010.10.004.

Total polyphenol content was determined spectrophotometrically using different variation of Folin–Ciocalteu’s method by means of UV-VIS UV-1800 Shimadzu spectrophotometer (Tokio, Japan). The Folin-Ciocalteu reagent, a mixture of phosphomolybdic and phosphotungstic acids reacts with phenolic compounds and is reduced, forming a blue chromophore detectable spectrophotometrically. Seven concentration levels of gallic acid standard were used to construct the calibration curves: L1 - 0.6 g/L, L2 - 0.4 g/L, L3 - 0.2 g/L, L4 - 0.1 g/L, L5 - 0.05 g/L, L6 - 0.025 g/L and L7 - 0.0125 g/L. A 1 g/L gallic acid stock solution was prepared in Milli-Q water. Each concentration level was measured in triplicate using all the investigated polyphenol determination methods. Once the calibration curves for each method were prepared, two independent standard solutions were used for methods validation. The first one was gallic acid at a concentration of 0.2 g/L prepared in Milli-Q water. The second standard was a commercial polyphenol extract Vitaflavan at a concentration of 0.2 g/L which contains 80% of total polyphenols. Each standard solution was measured in triplicate using all the methods investigated.