This document describes a method for estimating total phenols using the Folin-Ciocalteu reagent. Phenolic compounds are important plant antioxidants. The method involves preparing plant extracts in methanol and mixing them with the Folin-Ciocalteu reagent and sodium carbonate. The absorbance is then measured to determine the total phenol content based on a gallic acid standard curve. The total phenolic content measurement provides information about a plant's antioxidant properties and potential health benefits.

1. Estimation of total phenols by
Folin –ciocalteau reagent
Jasmine Juliet .R
Teaching Assistant
Biotechnology Dept
AC&RI, Madurai.

2. Estimation of total phenols by
Folin –ciocalteau reagent

3. Phenols - Introduction
• Polyphenols are a group of secondary metabolites involved in the
hydrogen peroxide scavenging in plant cells.
• Polyphenolic compounds have high antioxidant potency.
• Phenolic antioxidants, play the very important role of protecting
organisms against harmful effects of oxygen radicals and other highly
reactive oxygen species.
• Phenolic compounds have multiple biological effects.
• Flavonoids and other phenolics have been suggested to play a preventive
role in the development of cancer and heart disease.

6. Estimation of total phenols - Aim
• The assessment of antioxidant activity indicates that edible plants are
significant source of natural antioxidants.
• The selected plants with high antioxidant activity might be proposed for
the treatment of coronary diseases.
• The investigation of isolation and identification of responsible antioxidant
components is necessary to better understand their ability to control
diseases.
• Phenolic compounds are important plant constituents with redox
properties responsible for antioxidant activity.

7. Estimation of total phenols - Principle
• The Folin–Ciocalteu reagent also called the gallic acid equivalence method , is a
mixture of phosphomolybdate and phosphotungstate used for the
colorimetric invitro assay of polyphenolic antioxidants.
• The F–C reaction is an antioxidant assay(quantification of total phenolics) based
on electron transfer, which measures the reductive capacity of an antioxidant.
• In this method, protein is pretreated with copper(II) in a modified biuret reagent
(alkaline copper solution stabilized with sodium potassium tartrate).
• Addition of the phenol reagent generates chromogens that give increasing
absorbance between 550-750nm.

8. Estimation of total phenols –
Materials and methods
• Standards of phenolic acids (gallic acid).
• The FolinCiocalteu’s phenol reagent.
• 7% Na2CO3 solution.
• Test tubes.
• Volumetric flask.
• Pipette.
• Incubator.
• UV – Spectrophotometer.
• Methanol.

10. Estimation of total phenols – Procedure
Unknown solution (Preparation of plant extracts)
• Plant extracts were prepared according to a standard protocol.
• Prepared plant material (10 g) was transferred to dark-coloured flasks and
mixed with 200 ml of solvents (methanol, acetone, ) and stored at room
temperature.
• After 24 h, infusions were filtered through Whatman No. 1 filter paper and
residue was re-extracted with equal volume of solvents.
• After 48 h, the process was repeated.
• Combined supernatants were evaporated to dryness under vacuum at 40 º C
using Rotary evaporator.
• The obtained extracts were kept in sterile sample tubes and stored in a
refrigerator at 4ºC.

11. Estimation of total phenols - Procedure
• Methanolic solution of extract in concentration of 1 mg/ml was used in analysis.
• The reaction mixture was prepared by mixing 0.5 ml of methanolic solution of
extract, 2.5 ml of 10% Folin-Ciocalteu’s reagent dissolved in water and 2.5 ml
7.5% Na2CO3.
• Blank was prepared, containing 0.5 ml methanol, 2.5 ml 10% Folin-Ciocalteu’s
reagent dissolved in water and 2.5 ml of 7.5% of NaHCO3.
• The samples were incubated in a thermostat at 45ºC for 45 min.
• The absorbance was determined using spectrophotometer at λmax = 765 nm.
• The same procedure was repeated for the standard solution of gallic acid and the
calibration line was constructed.
• The content of phenolics in extracts was expressed in mg of GA/g of extract.

14. Conclusion
• The total phenolic content will be helpful for standardizing the drug.
• The presence of a high total phenolic content shows that antioxidant
properties, which could lead to a new field of research in the future.
• It measures anti-oxidant capacity in vitro, the reagent has been used to
assay foods and supplements in food science.
• The oxygen radical absorbance capacity (ORAC) used to be the industry
standard for antioxidant strength of whole foods, juices and food
additives.