Folin-Ciocalteu phenol reagent

The color produced in the Lowry method results from the biuret reaction plus the reduction of the phosphomolybdate-phosphotungstate reagent (Folin CiOcalteu phenol reagent) by tyrosine residues. The Lowry method is suitable for solutions containing 20 to 400 /rg protein/ml. 

In a study of the structure and functions of feruloylated pectins in primary cell walls in spinach, about one feruloyl group was found per sixty sugar residues ( ). Ferulic acid was determined after alkaline hydrolysis by the Folin-Ciocalteu phenol reagent. 

A. Lowry assay (Lowry et al., 1951) Folin-Ciocalteu phenol reagent (Merck). 

Reagent D (Folin-Ciocalteu phenol reagent) Dilute with an equal volume of water just prior to use. 

SINGLETON v L, ORTHOFER R and LAMUELA-RAVENTOS R M (1999) Analysis of total phenolics and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Meth Enzymol, 299, 152-78. 

Singleton, V. L., Orthofer, R., and Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. In "Methods in Enzymology, Oxidant and Antioxidants (Part A)", (L. Packer, Ed.), vol. 299 pp. 152-178. Academic Press, San Diego, CA. 

Materials required Sample solution in methanol, distilled water, 20% Na2C03, Folin-Ciocalteu s phenol reagent, water bath, graduated tube, cuvette, micro pipette (0.1 ml), pipette of 1,2 and 10 ml, test tubes of 20 ml, pure ferulic acid (Serva, Germany), Shimadzu UV 160 spectrophotometer. 

Procedure A 0.002 ml aliquot of the sample solution in methanol was taken and 7 ml distilled water plus 0.1 ml Folin-Ciocalteu s phenol reagent was added and after 3 min 0.2 ml of 20% Na2C03 was included. After boiling at 90 °C (exactly 5 min) samples were cooled at room temperature and were diluted with HzO to 10 ml volume. Only distilled water and reagents were used as a blank. The absorbance of total phenolics was measured at 660 nm spectrophotometrically (a Shimadzu UV 160 spectrophotometer) as per Feldman and Hanks (1968), with a sensitivity of 0.05 pig/g d.w. A standard curve was constructed with different concentrations of ferulic acid (Serva, Germany). Concentrations of ferulic acid varied from 0.33-80jig/ml (Table 1). 

The Folin-Ciocalteu assay is the most widely used method to determine the total content of food phenolics (Fleck and others 2008). Folin-Ciocalteu reagent is not specific and detects all phenolic groups found in extracts, including those found in extractable proteins. A disadvantage of this assay is the interference of reducing substances, such as ascorbic acid (Singleton and others 1999). The content of phenolics is expressed as gallic acid or catechin equivalents. 

Total Phenols Assay (Folin-Ciocalteu Reagent)... 

Methods used for the detection of PAs in cmde or partially purified extracts can also be adapted for post-column analysis after fractionation (see below). Direct quantitative analysis of PAs in crude grape phenolic extracts is often impossible due to the complex sample matrix. Thus, fractionation or purification is often necessary before analysis. The Folin-Ciocalteu and Pmssian Blue assays are widely used for the quantification of total polyphenols in plants [27,28]. These methods are not specific for PAs due to the reaction of other phenolic compounds with these reagents. 

G Mix just before use Soln. C, D, E, and F in a ratio of 1 1 28 10 H Folin-Ciocalteu s phenol reagent (stock), diluted 1+3 with ddH20... 

The Falin-Ciocalteu reagent (FCR) is a complex formed in a reaction between sodium tungstate and sodium molybdenate in hydrochloric add and phosphoric acid, which turns yellow after lithium sulphate is added. The reagent reads in an alkaline environment with reducing compounds. Such a reaction gives a blue chromophore which is observed by colorimetry. The Folin-Ciocalteu method is highly sensitive - both to phenolic and non-phenolic compounds, e.g. proteins, vitamin C, vitamin Bj, folic acid, Cu(I). The method is applied most frequently to determine the total content of phenolic compounds [34,35]. If that is the case, a sample for determination should be prepared in a proper manner to minimise the effect of non-phenolic... 

Total phenolic content the Folin-Ciocalteu reagent... 

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. 

A third method relies on the precipitation of proanthocyanidins with formaldehyde. First, the total phenolic content is measured using the Folin-Ciocalteu reagent as described before. A 0.5 mole equivalent of phloroglucinol (1.3) is added for every gallic acid equivalent in the extract. To 2 mL of this plant extract and phloroglucinol is added 1 mL of a 2 5 HC1 /H20 solution and 1 mL of an aqueous solution of formaldehyde (13 mL of 37% formaldehyde diluted to 100 mL in water). After an overnight incubation at room temperature, the unprecipitated phenols are estimated in the supematent by the Folin-Ciocalteu method. The precipitate contains the proanthocyanidins and the known amount of phloroglucinol, which is always quantitatively precipitated. 

The concentrations of phenolic compounds were determinedby HPLC (Waters) using a method described previously by Nilvebrant et al. (21). The total concentration of phenols was also estimated by a spectrophotometric method (23) based on the Folin and Ciocalteu s reagent (Sigma). 

Fumed silica A-200 (Pilot plant at the Institute of Surface Chemistry, Kalush, Ukraine specific surface area Ascorbic acid (vitamin C) and all-rac-a-Tocopheryl acetate (vitamin E acetate) were used as adsorbates. Folin-Ciocalteu s phenol reagent (Merck) was used to measure the total polyphenolic index. Silica samples with different degree of surface silylation were obtained using gas-phase chemical modification of highly disperse silica (A-200) surface by trimethylchlorosilane.6... 

Antioxidant activity of silica nanocomposites with immobilized vitamin C was tested using the polyphenolic activity index.8 After adsorption of ascorbic acid on the silica surface and centrifugation, the excess solution was removed to obtain the suspension of a fixed volume (2 ml). Distilled water, sodium carbonate solution, and Folin-Ciocalteu s phenol reagent were subsequently added to suspensions and to the reference Vitamin C solution. The suspensions were then stored for 30 min, and the optical density of supernatant was measured at X = 750 nm. The reference solution of ascorbic acid was used to compare antioxidant activity of vitamin-containing nanocomposites with the activity of dissolved vitamin C. 

Indications. A red or orange-red colour indicates the presence of a phenolic substance. Primary aryl amines also react. Some basic compounds which contain a phenolic group do not react to this test a combination of this test with the Folin-Ciocalteu Reagent is therefore advised for phenolic compounds. 

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