Some of the commonly used methods to determine the content of phenolics are approximately 100 years old, and were initially developed by Folin and colleagues at Harvard Medical School to study the metabolism of proteins in humans. Folin and Denis (1912b) reported on a colorimetric method to detect the amino acid tyrosine (3.28) in protein hydrolysates. This method relied the reduction of a mixture of phosphotungstic (WO42-)-phosphomolybdic (MoO42-) reagent by the phenolic hydroxyl group of tyrosine, resulting in the formation of a blue product. The color intensity could be quantified based on absorbance readings using an early version of the spectrophotometer. The Folin-Denis reagent is prepared by mixing sodium tungstate and (phospho)molybdic acid in phosphoric acid, boiling it for 2 hours, followed by cooling, diluting and filtering it (Folin and Denis, 1912a). This method was subsequently applied to the determination of phenolics in urine (Folin and Denis, 1915). A modification of this method was reported by Folin and Ciocalteu (1927). The modification consisted of the addition of lithium sulfate and bromine to the phosphotungstic-phosphomolybdic reagent at the end of the boiling period, followed by cooling and dilution. The addition of the lithium prevents the formation of a precipitate that would interfere with the quantification of the color intensity. The resulting reagent, referred to as the Folin-Ciocalteu reagent, was used to determine the content of tyrosine and tryptophan in protein hydrolysates, but can be used to determine the content of phenolics from a wide range of sources. Below follows a protocol for this method.
1. Dilute an aliquot of the sample 10:1 with water (9 parts water to 1 part sample). This is not necessary if the phenol content is low.
2. Add 2 ml of freshly prepared 2% (w/v) sodium carbonate (anhydrous) to 0.1 ml of the sample extract (diluted if necessary).
3. Mix vigorously on a Vortex mixer.
5. While mixing on a Vortex add 0.1 ml of a 1:1 dilution of Folin-Ciocalteu reagent. This reagent can be purchased from chemical supply companies, such as Merck. If the reagent has a green color, it is no longer good and should be replaced.
6. Allow the sample to stand for a minimum of 30 minutes, but not more than one hour.
7. Read the absorbance in a spectrophotometer at 750 nm.
For blanks one can use ethanol, water or methanol, whichever the tissue extract was dissolved in last. Since this is a spectrophotometric assay, it is important to have a standard curve to relate the absorbance value to a concentration. Common compounds used to generate a standard curve are chlorogenic acid (1.18) or gallic acid (1.5). The concentration of phenolic compounds is then reported as chlorogenic acid or gallic acid equivalents, respectively.
Scalbert et al. (1989) used a slight modification of this method, whereby a 2.5 mL aliquot of the Folin-Ciocalteu reagent (diluted 10 times in water) and 2 mL of a 75g/L solution of sodium carbonate are added to 0.5 mL of the extract (diluted in methanol), followed by a 5 min. incubation in a 50°C waterbath. A potential complication of this method is the deglycosylation of phenolic compounds due to the heating.
The colorimetric assay based on the protocol developed by Folin and Ciocalteu (1927) can be used to determine the concentration of soluble phenolics, such as anthocyanins in the example above, as well as complex phenolics such as hydrolysable and condensed tannins. Swain and Hillis (1959) pointed out that variation in phenolic composition (e.g. tannins versus flavonoids) can influence the efficiency of the reduction of the Folin-Ciocalteu reagent, so that comparisons between samples may not always be appropriate, depending on what the origin of the samples is. Apple et al. (2001) investigated the appropriateness of the Folin-Ciocalteu reagent in comparisons of leaf samples obtained from different tree species. They argued that the use of this reagent offers an efficient method to estimate the reducing capacity of the sample, but due to variation in phenolic content and composition between samples, comparing phenolic contents across samples may not be meaningful. To demonstrate this, Apple et al. (2001) determined the tannin content of a set of leaves from sixteen tree species, a subset collected at different times of the year, and mixtures of commercially available tannins, using the Folin-Ciocalteu reagent as a measure of total tannin content, the butanol-HCl assay (Bate-Smith, 1977) to determine the content of condensed tannins (see Section 184.108.40.206), and the potassium iodate method (Haslam, 1965; Bate-Smith, 1977; Schultz and Baldwin, 1982; see Section 220.127.116.11) to determine the content of hydrolysable tannins. Since the tannins were extracted from the leaves, they could perform the reactions with the same amount of starting material for each of the samples, and evaluate to what extent the composition of the sample impacted the reducing capacity. The total phenolic content obtained with the Folin-Ciocalteu reagent was primarily correlated with either the content of hydrolysable tannins, or the content of condensed tannins, or the combination, depending on the actual sample, confirming that the phenolic composition affected the reducing potential of the sample. They concluded that the use of the Folin-Ciocalteu assay resulted in over- or underestimation of the phenolic content values obtained with other methods.
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