Chemical determination of phenolic compounds

Protocol

Authors

Hendrik Poorter, Yvonne de Jong-Van Berkel

Author Affiliations

Hendrik Poorter IBG-2, Forschungszentrum Jülich and Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University

Overview

This protocol follows the Folin Ciocalteu method.

Different phenolic compounds occur in plant material. Extraction of these compounds can be done with different methods. The phenolic acids and aldehydes with low molecular weight are soluble in water. Other phenolic compounds are conjugated in the cell wall, i.e., as esters or polysaccharides. To extract these compounds the pH must be increased to pH 13. Furthermore, there is a group of phenolic compounds called phenolic glycosides, as well as condensed and hydrolysable tannins. They can be extracted using organic solvents. To reduce the chance that some phenolic compounds are oxidized, the extractions must take place in a nitrogen-gas saturated environment.

Soluble phenolic compounds can be determined in extracts using Folin Ciocalteu reagent. This reagent reacts with the benzene ring of the phenolic compounds and gives a specific colour. The reagent is a mixture of phosphomolybdic acid and phosphotungstic acid. p-Coumaric acid is used as a standard.

Background

Note: The methods described here are for ‘bulk estimation’, and are mainly focussed on getting a simple estimate of the proximate chemical composition of plant material with just a small number of different groups of compounds (cf. Poorter & Villar 1997). Thus, they are not intended to be specific for one particular type of compound, such as a specific phenolic constituent. If you are a specialist and interested in the fine detail, there are much better methods, using HPLC, GC or MS. However, if you can accept some roughness, the methods described here are generally faster, cheaper and more easy to apply.

Materials/Equipment

Reagents

  • Folin-Ciocalteu reagent 2 M
  • p-Coumaric acid
  • Deionized water
  • Sodium carbonate
  • Ethanol 80% and 96%
  • Chloroform

Equipment

  • Water bath
  • Table centrifuge
  • Test tubes
  • Spectrophotometer

Procedure

Preparation of reagent A

  • 75 ml Folin-Ciocalteu reagent is made up to 750 ml with deionized water

Preparation of reagent B

  • 57.5 g of sodium carbonate is dissolved in deionized water and filled up to 500 ml with deionized water

Standard solution

  • 0.050 g of p-Coumaric acid is dissolved in 2 ml 96 % ethanol and filled up to 50 ml with deionized water
  • A standard range is diluted: 0; 0.1; 0.2; 0.4; 0.6; 0.8 and 1.0 mg/ml

Extraction

  • 50 to 100 mg of dry plant material is extracted with 5 ml 80% ethanol in a centrifugation tube.
  • Or 500 mg of fresh plant material is extracted with 4 ml 80% ethanol in a centrifugation tube.
  • Put the tubes in a water bath for 30 minutes at 30∘C.
  • Remove the supernatant after centrifugation at 4500 rpm (2649 g) for 10 minutes.
  • Extract the pellet again with 2.5 ml 80 % ethanol, put the tubes in a water bath for 30 minutes at 30∘C.
  • After centrifugation the supernatant is added to the other supernatant.
  • To remove other compounds, such as chlorophyll and lipids, that can disturb the colour reaction, extract the solution with 5 ml chloroform and 2.5 ml deionized water.
  • Remove the chloroform by centrifugation for 10 minutes at 4500 rpm (2649 g).
  • The ethanol-water fraction is ready to be used for the determination. The residue can be used for extraction of insoluble sugars (fructans and starch).

Determination

  • Transfer 0.1 ml of sample or standard solution to a test tube
  • Add 5 ml of reagent A
  • Mix well
  • After 0.5 to 8 min 3.5 ml of reagent B is added
  • Mix well
  • Incubate the tubes either 2 h at 20∘C or 1 h at 40∘C in a water bath
  • Measure the absorption at 765 nm with a spectrophotometer. The colour is stable for several hours.

Notes and troubleshooting tips

  • Be sure that the phenol content in the samples is less than 1.0 mg/ml.
  • Glucose and fructose may interfere with the absorption measurement.
  • The colour is stable for several hours.
  • A large quantity of protein in the extracts can also interfere with the determination. Protein can be removed with TCA. After a few hours of precipitation, the proteins can be removed by centrifugation.

Reference values

In leaves of herbaceous species: 2 – 130 mg g-1, generally less in stems and roots.

In leaves of woody species: 25 – 150 mg g-1 (Poorter & Villar 1997)

Kuiters, L 1987, Phenolic acids and plant growth in forest ecosystems, PhD Thesis, Vrije Universiteit, Amsterdam. pp 19-32.

Poorter, H & Villar, R. 1997. The fate of acquired carbon in plants: chemical composition and construction costs. In: Plant Resource Allocation. Academic Press, pp 30-72.

Singleton, V,L 1988. Wine phenols. In: Modern Methods of Plant Analysis, Eds: Linskens, H,F, & Jackson, Z,J,F, pp: 201-202,

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