FOL-01_FolinReviewIJFSND

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International Journal of Food Science, Nutrition and Dietetics, 2014 © 2
Gabriel A Agbor (2014) Folin-Ciocalteau Reagent for Polyphenolic Assay 3:801
Summary of Single reagent Procedure
To 10-100 µL of sample, add to a total volume of 1000 µL 10-
fold diluted F-C reagent (Sigma) and read 10-20 min later at 750
nm vs. a reagent blank and standards.
Specific antioxidant classes and compounds detected by the
F-C reagent
There are over 4000 citations of Folin in Chemical Abstracts and
over 500 in PubMed as of December, 2008. Both the dual and
the single Folin methods are good for the detection of a wide
range of antioxidant compounds in a large variety of plants and
plant-derived foods and beverages. The single reagent has been
used for phenolic antioxidants from fruits , vegetable
cereals , fruit juices caffeinated beverages al-
coholic beverages chocolate , herbs and spices
and plant extracts by our group and other investigators. The
major classification of antioxidant compounds: flavonols, fla-
vones, flavanones, flavanols, proanthocyanidins, isoflavones, an-
thocyanins, phenolic acids are detected by the Folin methods.
Procedure for the dual and single reagent assay
methods for comparison
Antioxidant compounds representing the different classes of
polyphenols listed above were analyzed using both the dual and
single reagent methods.
Dual reagent assay
The Singleton and Rossi original method has been modified
to suit different laboratory needs. The procedure was applied in
our laboratory.
Standard Preparation
Preparation of catechin standard: Dissolve 2.9 mg of catechin
powder in 10ml of methanol, therefore resulting in a 1000μM
solution. Keep in refrigerator when not in use. Prepare a new
solution monthly.
Standard Catechin Sample Preparation and Analysis
Standard concentrations of catechin of 05, 0μM1, 00μM20, 0μM,
400μM and 800μM, were prepared from the (1000μM) standard.
Prepare standard curve by taking 40 μl of catechin standard solu-
tion in to 6 different 10 ml screw cap tubes, the first tube (blank)
40μl of nanopure water.
Add 800 μl of a 10 fold diluted Folin-Ciocalteau reagent in to
each tube and mix well.
Allow the tubes to stand for 5 minutes.
Then add 800 μl of 7% w/v sodium carbonate aqueous solution
to each tube and mix well.
Make up volume in each tube with nanopure water (360 μl) to
2mL, mix well, and allowed the tubes to stand for 2hr.
Read absorbance at 760 nm against the blank using a UV-Visible
spectrophotometer.
Standard Curve Determination
The absorbances of the standards were plotted against the stand-
ard concentrations. The regression line to this graph (Figure 1)
was used to determine the catechin equivalent of the polyphenol
concentration in the various samples analyzed.
Folin assay of polyphenolic compounds (see Table 1)
Polyphenolic compounds (1mM) were prepared in methanol.
40 μl of sample was transferred in to a 10 ml screw cap tube.
Add 800 μl of a 10-fold diluted Folin-Ciocalteau reagent in to the
tube and mix well.
Allow the tube to stand for 5 minutes.
Then add 800 μl of 7% w/v sodium carbonate aqueous solution
to the tube and mix well.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 50 100 150
Average absorbance against concentration
Concentration of catechin statndard (uM)
Absorbance (700 nm)
Figure. 1. Standard curve: Concentration of catechin against absorbance for the dual reagent method
y=0.0013x-0.0069
R2=0.9814