Antioxidants
An antioxidant is defined as “any substance that when present at low concentrations, compared
with those of the oxidizable substrate, significantly delays or inhibits oxidation of that substrate”.
Oxidative metabolism is essential for the survival of cells. However, it results in free radicals and
reactive oxygen species (ROS) that causes oxidative damage. Excess of free radicals overwhelm
protective enzymes such as catalases, peroxidases which is essential for cellular respiration.
However, ROS influences in cell signaling pathways.
Antioxidants can be classified in many ways. It can be classified as enzymatic antioxidants and
non-enzymatic antioxidants. Enzymatic antioxidants such as catalase and superoxide dismutase
directly or indirectly involve in the defense against reactive oxygen species (ROS).
Antioxidants are classified into 3 groups known as primary/natural, secondary/synthetic and
tertiary. Primary antioxidants involve in chain breaking process. Chain breaking process is
achieved either by delaying or inhibiting the initiation step by reacting with a lipid radical or
inhibiting the propagation step by reacting with peroxyl or alkoxyl radicals. The antioxidant free
radical further interferes with chain propagation by forming peroxy antioxidant compounds.
Secondary antioxidants capture free radicals and stop the chain reaction.
Imbalance between ROS and cellular antioxidant capacity leads to oxidative stress. If this
condition is not properly regulated by the body it will results many diseases such as cancer, stroke
and heart disease.
Natural antioxidants could be more effective in reducing ROS level compared to synthetic single
dietary antioxidants due to synergistic action of many compounds. Number of compounds present
in a system give pronounced effect than single compound present in a system is known as
synergism. Vitamin C, vitamin E, carotenoids, phenolic compounds are few natural antioxidants.
Phenols and flavonoids act as natural antioxidants. Presence of these two compounds can
be confirmed by conducting total Phenolic content (TPC) assay and total Flavonoid content
(TFC) assay.
1|P ag e
, Total Phenolic content/ Folin- Ciocalteu assay (F-C assay)
Phenolic compounds include phenols, flavonoids, stilbenes, tannins and lignins. They are
secondary metabolites which act as antioxidants, protective agents against UV light, contributors
to plant pigmentation etc. Hydroxyl groups of phenols confer scavenging activity. Researchers
have been found that plant phenols are capable of inhibiting superoxide anion radical. Reference
substance is used to measure concentration of all phenolic hydroxyl groups.
Phenolic compounds act as reducing agents, metal chelating agents as well as antioxidants.
Multiple hydroxyl groups in polyphenols make them ideal for radical scavenging reaction and
metal chelating reaction. The arrangement of hydroxyl groups around the phenolic molecule is
also important in antioxidant ability. The method is based on electron transfer which measures the
reductive capacity of an antioxidant. The assay measures all phenolic compounds. However, all
heterogenous phenolic compounds do not react similar manner.
Total phenolic content is determined using F-C method. The sample changes its color from yellow
to green after addition of F-C reagent. F-C reagent reacts easily with polyphenols and gives blue
chromophore as most phenolic compounds are in its dissociated form at the working pH of the
assay. Though, maximum absorption depends on alkaline condition this reagent is not stable in
alkaline medium. Therefore, more reagents should be used which results high turbidity. Turbidity
is avoided by Lithium salt in the reagent.
Phenolic compounds are oxidized in basic medium resulting superoxide ion. It reacts with
molybdate to form molybdenum oxide (MoO4+). It has an intense blue color. This blue
chromophore is Phosphotungstic- Phosphomolybdenum complex. Its maximum absorption
depends on alkaline solution and the concentration of polyphenols.
Pyrogallol was taken as the reference substance and results are expressed as milligrams of
pyrogallol equivalent (PGE) per gram of dried sample. It has the largest number of hydroxyl groups
propotional to its molar mass. Pyrogallol has one ring with no substitute groups forming three-
dimensional structure with hydroxyl groups. The structure is less influenced by electronic
interactions such as steric or resonance effects. Hence, it has the highest specific absorptivity
indicating that, it is the best reference substance to determine total phenols. Pyrogallol, indirectly
2|P ag e
An antioxidant is defined as “any substance that when present at low concentrations, compared
with those of the oxidizable substrate, significantly delays or inhibits oxidation of that substrate”.
Oxidative metabolism is essential for the survival of cells. However, it results in free radicals and
reactive oxygen species (ROS) that causes oxidative damage. Excess of free radicals overwhelm
protective enzymes such as catalases, peroxidases which is essential for cellular respiration.
However, ROS influences in cell signaling pathways.
Antioxidants can be classified in many ways. It can be classified as enzymatic antioxidants and
non-enzymatic antioxidants. Enzymatic antioxidants such as catalase and superoxide dismutase
directly or indirectly involve in the defense against reactive oxygen species (ROS).
Antioxidants are classified into 3 groups known as primary/natural, secondary/synthetic and
tertiary. Primary antioxidants involve in chain breaking process. Chain breaking process is
achieved either by delaying or inhibiting the initiation step by reacting with a lipid radical or
inhibiting the propagation step by reacting with peroxyl or alkoxyl radicals. The antioxidant free
radical further interferes with chain propagation by forming peroxy antioxidant compounds.
Secondary antioxidants capture free radicals and stop the chain reaction.
Imbalance between ROS and cellular antioxidant capacity leads to oxidative stress. If this
condition is not properly regulated by the body it will results many diseases such as cancer, stroke
and heart disease.
Natural antioxidants could be more effective in reducing ROS level compared to synthetic single
dietary antioxidants due to synergistic action of many compounds. Number of compounds present
in a system give pronounced effect than single compound present in a system is known as
synergism. Vitamin C, vitamin E, carotenoids, phenolic compounds are few natural antioxidants.
Phenols and flavonoids act as natural antioxidants. Presence of these two compounds can
be confirmed by conducting total Phenolic content (TPC) assay and total Flavonoid content
(TFC) assay.
1|P ag e
, Total Phenolic content/ Folin- Ciocalteu assay (F-C assay)
Phenolic compounds include phenols, flavonoids, stilbenes, tannins and lignins. They are
secondary metabolites which act as antioxidants, protective agents against UV light, contributors
to plant pigmentation etc. Hydroxyl groups of phenols confer scavenging activity. Researchers
have been found that plant phenols are capable of inhibiting superoxide anion radical. Reference
substance is used to measure concentration of all phenolic hydroxyl groups.
Phenolic compounds act as reducing agents, metal chelating agents as well as antioxidants.
Multiple hydroxyl groups in polyphenols make them ideal for radical scavenging reaction and
metal chelating reaction. The arrangement of hydroxyl groups around the phenolic molecule is
also important in antioxidant ability. The method is based on electron transfer which measures the
reductive capacity of an antioxidant. The assay measures all phenolic compounds. However, all
heterogenous phenolic compounds do not react similar manner.
Total phenolic content is determined using F-C method. The sample changes its color from yellow
to green after addition of F-C reagent. F-C reagent reacts easily with polyphenols and gives blue
chromophore as most phenolic compounds are in its dissociated form at the working pH of the
assay. Though, maximum absorption depends on alkaline condition this reagent is not stable in
alkaline medium. Therefore, more reagents should be used which results high turbidity. Turbidity
is avoided by Lithium salt in the reagent.
Phenolic compounds are oxidized in basic medium resulting superoxide ion. It reacts with
molybdate to form molybdenum oxide (MoO4+). It has an intense blue color. This blue
chromophore is Phosphotungstic- Phosphomolybdenum complex. Its maximum absorption
depends on alkaline solution and the concentration of polyphenols.
Pyrogallol was taken as the reference substance and results are expressed as milligrams of
pyrogallol equivalent (PGE) per gram of dried sample. It has the largest number of hydroxyl groups
propotional to its molar mass. Pyrogallol has one ring with no substitute groups forming three-
dimensional structure with hydroxyl groups. The structure is less influenced by electronic
interactions such as steric or resonance effects. Hence, it has the highest specific absorptivity
indicating that, it is the best reference substance to determine total phenols. Pyrogallol, indirectly
2|P ag e
