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Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent


Non-structural phenolic compounds perform a variety of functions in plants, including acting as antioxidants. We describe a microplate-adapted colorimetric total phenolics assay that utilizes Folin–Ciocalteu (F–C) reagent. The F–C assay relies on the transfer of electrons in alkaline medium from phenolic compounds to phosphomolybdic/phosphotungstic acid complexes, which are determined spectroscopically at 765 nm. Although the electron transfer reaction is not specific for phenolic compounds, the extraction procedure eliminates approximately 85% of ascorbic acid and other potentially interfering compounds. This assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. At least 64 samples can be processed in 1 d.

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Figure 1: Example of a gallic acid standard curve.

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  1. Croteau, R., Kutchan, T.M. & Lewis, N.G. Natural products (secondary metabolites). In Biochemistry & Molecular Biology of Plants (eds. Buchanan, B., Gruissem, W. & Jones, R.L.) 1250–1318 (American Society of Plant Physiologists, Beltsville, MD, USA, 2000).

    Google Scholar 

  2. Grace, S.C. & Logan, B.A. Energy dissipation and radical scavenging by the plant phenylpropanoid pathway. Phil. Trans. R. Soc. Lond. B 355, 1499–1510 (2000).

    Article  CAS  Google Scholar 

  3. Grace, S.C. Phenolics as antioxidants. In Antioxidants and Reactive Oxygen Species in Plants (ed. Smirnoff, N.) 141–168 (Blackwell Publishing, Oxford, UK, 2005).

    Google Scholar 

  4. Bors, W., Heller, W., Michel, C. & Saran, M. Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods Enzymol. 186, 343–355 (1990).

    Article  CAS  Google Scholar 

  5. Bors, W., Michel, C. & Saran, M. Flavonoid antioxidants: rate constants for reactions with oxygen radicals. Methods Enzymol. 234, 420–429 (1994).

    Article  CAS  Google Scholar 

  6. Dixon, R.A. & Paiva, N.L. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085–1097 (1995).

    Article  CAS  PubMed  Google Scholar 

  7. Pasqualini, S. et al. Ozone-induced cell death in tobacco cultivar Bel W3 plants. The role of programmed cell death in lesion formation. Plant Physiol. 133, 1122–1134 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Robards, K. & Antolovich, M. Analytical chemistry of fruit bioflavonoids a review. Analyst 122, 11R–34R (1997).

    Article  CAS  Google Scholar 

  9. Magalhães, L.M., Segundo, M.A., Reis, S., Lima, J.L. & Rangel, A.O. Automatic method for the determination of Folin–Ciocalteu reducing capacity in food products. J. Agric. Food Chem. 54, 5241–5246 (2006).

    Article  Google Scholar 

  10. Folin, O. & Ciocalteu, V. On tyrosine and tryptophane determinations in proteins. J. Biol. Chem. 73, 627–650 (1927).

    CAS  Google Scholar 

  11. Singleton, V.L. & Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158 (1965).

    CAS  Google Scholar 

  12. Prior, R.L., Wu, X. & Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53, 4290–4302 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Singleton, V.L., Orthofer, R. & Lamuela-Raventós,, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 299, 152–178 (1999).

    Article  CAS  Google Scholar 

  14. Huang, D., Ou, B. & Prior, R.L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 53, 1841–1856 (2005).

    Article  CAS  Google Scholar 

  15. Booker, F.L. & Miller, J.E. Phenylpropanoid metabolism and phenolic composition of soybean [Glycine max (L.) Merr.] leaves following exposure to ozone. J. Exp. Bot. 49, 1191–1202 (1998).

    Article  CAS  Google Scholar 

  16. Gillespie, K.M., Chae, J.M. & Ainsworth, E.A. Rapid measurement of total antioxidant capacity in plants. Nat. Protoc. 2, 867–870 (2007).

    Article  CAS  Google Scholar 

  17. Gillespie, K.M. & Ainsworth, E.A. Measurement of reduced, oxidized and total ascorbate content in plants. Nat. Protoc. 2, 871–874 (2007).

    Article  CAS  PubMed  Google Scholar 

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This research was supported by the Office of Science (BER), U.S. Department of Energy, Grant no. DE-FG02-04ER63849.

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Correspondence to Elizabeth A Ainsworth.

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Ainsworth, E., Gillespie, K. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc 2, 875–877 (2007).

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