ANTIOXIDANT CAPACITY: DEVELOPMENT OF
METHODS BASED ON FREE RADICALS
M. Cortina-Puig, Y. Wang, B. Liu, C. Calas-Blanchard and J.L. Marty
INTRODUCTION
FREE RADICALS
 Highly unstable molecules with available electrons.
 Generated in vivo during metabolic processes.
 Reactive oxygen species (ROS).
•-
 Superoxide
Radical
(O2 humans
)
 In order to protect
against
them,
have evolved with
 Hydrogen
antioxidant (AOx)
systems.Peroxide (H2O2)
 Hydroxyl Radical (OH•)
 Singlet oxygen (1O2)
Equilibrium
 Hypochlorous
Acid (HOCl)
AOx = Free Radicals
 Alkoxyl Radicals (RO•)
 Peroxyl Radicals (RO2•)
AOx
Free
Radicals
INTRODUCTION
FREE RADICALS
pollution
FREE
RADICAL
EXCESS
Stressors
(environmental
or behavioural)
sunlight
exposure
excessive
alcohol
consumption
OXIDATIVE
STRESS
cigarette
smoking
Antioxidant
Oxidative
Stress
production
Excess
Free Radicals
malfunction
AOx
Free
Radicals
INTRODUCTION
FREE RADICALS
Normal Lipids
Lipid Peroxyl &
Alkoxyl Radicals
Normal DNA
Altered DNA
Loss of Temporal
Control of Gene
Functions
Excess of
Specific
Proteins
Normal RNA
Normal
Proteins and
Enzymes
Altered RNA
Altered
Proteins and
Enzymes
Impairment of
Essential Cellular &
Tissue Functions
Immunological
Response to
Altered Proteins
Absence of
Specific
Proteins
HUMAN DISEASES (CANCER, ALZHEIMER) & AGING PROCESS
INTRODUCTION
ANTIOXIDANTS
 Substances which counteract free radicals and prevent the
damage caused by them.
 Reduction of the adverse damage due to oxidants through
different protective mechanisms:
• crumbling ROS before they react with biological targets
• preventing chain reactions
• preventing the activation of O2 to highly reactive products
INTRODUCTION
ANTIOXIDANTS
Antioxidants
Enzymatic AOx
Non-enzymatic AOx
Primary Enzymes
Minerals
Vitamins
SOD, catalase, glutathione,
peroxidase
Zinc, Selenium
Vit A, Vit C, Vit E, ViitK
Secondary Enzymes
b-carotene, lycopene, lutein, zeaxanthin
Carotenoids
glutathione reductase,
glucose 6-phosphate
dehydrogenase
Organosulfur compounds
allium, allyl sulfide, indoles
Low molecular weight AOx
AOx cofactors
glutathione, uric acid
Coenzyme Q10
Polyphenols
Flavonoid
s
Phenolic acids
Flavonols
Isoflavonoids
Flavanols
Flavanones
Flavones
Quercetin,
kaempferol
genistein
Catechin, EGCG
hesperitin
chrysin
Hydroxycinnamic
acids
ferulic acid, p-Coumaric
Hydroxybenzoic
acids
gallic acid, ellagic acid
INTRODUCTION
ANTIOXIDANT CAPACITY (AOC) DETERMINATION
Hydrogen Atom Transfer (HAT)
Measure the classical ability of an antioxidant to quench free radicals
by hydrogen donation (AH = any H donor)
X•+ AH
XH + A•
Single Electron Transfer (SET)
Detect the ability of a potential antioxidant to transfer one electron to
reduce any compound, including metals, carbonyls and radicals
X•+ AH
X- + AH•+
AH•+
X-+ H3O+
M(III) + AH
A • + H 3 O+
XH + H2O
AH+ + M(II)
INTRODUCTION
ANTIOXIDANT CAPACITY METHODS
HAT:
Oxygen Radical Absorbance Capacity (ORAC)
Total Radical-trapping Antioxidant Parameter (TRAP)
SET:
Ferric Reducing Antioxidant Power (FRAP)
HAT/SET:
Trolox Equivalent Antioxidant Capacity (TEAC)
2,2-Diphenyl-1-picrylhydrazyl (DPPH assay)
OBJECTIVES
Original biosensors using ROS
Free radical scavenging capacity
“total antioxidant capacity”
 Development of a cytochrome c (cyt c)-based biosensor
for the quantification of the antioxidant capacity against O2•-.
 Development of a simple and sensitive electrochemical
method for the determination of antioxidant capacity based
on the photogenerated •OH radicals.
CYTOCRHOME C-BASED BIOSENSOR
Gold electrode
DETECTION PRINCIPLE OF CYT C-BASED BIOSENSORS
S
HX: Hypoxanthine
XOD: Xanthine oxidase
S
S
COO-
S
Cyt c
Heme (Fe3+)
COOH
S
O2
COO-
S
e-
COOH
COO-
Cyt c
Heme (Fe2+)
COOH
E = 150 mV vs Ag/AgCl
XOD
catalase
O2 + H2O
I
[O2•-]
HX
O2•-
I
H2O2
O2
[O2•-]
Antioxidant Capacity
uric
acid
CYTOCRHOME C-BASED BIOSENSOR
MEASUREMENT OF THE O2•- SCAVENGING CAPACITY
IHX (1)
IHX (2)
+ HX (100 mM)
+ HX (100 mM)
+ Antioxidant
Iantioxidant
Signal inhibition (%) =
Antioxidant
Capacity
buffer +
catalase
(10 U mL-1)
buffer +
catalase
(10 U mL-1)
IHX(1) – IHX(2)
IHX(1)
x 100
IC50
AC
IC50
CYTOCHROME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX
Ascorbic acid
 Hydrophilic AOx
 Standards for other
antioxidative substances
Trolox
(6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
CYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX
Hill equation
y=
B•x
C+x
y: % signal inhibition
x: [AOx]
B,C: constant values
IC50 =
50 • C
B - 50
IC50
IC50 = 6.0 ± 0.9 mg mL-1
Ascorbic acid
CYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX
IC50
IC50 (Trolox) = 40.8 ± 0.7 mg mL-1
Trolox
IC50 (Ascorbic acid) = 6.0 ± 0.9 mg mL-1
IC50
AC
CYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES
AOx: flavonoids, carotenoids and vitamin C
+ Orange juice
Intensity
Antioxidant
capacity
CYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES
Other AOx:
 flavonoids (hesperetin and
naringenin)
 carotenoids (xanthophylls,
cryptoxanthins, carotenes)
IC50 / mg mL-1
66% AC
Vitamin C
Ascorbic acid
6.0 ± 0.9
Brand 1
3.4 ± 0.6
Brand 2
3.5 ± 0.5
Brand 3
5.2 ± 0.6
Brand 4
3.7 ± 0.8
Natural orange juice
5.0 ± 0.4
CYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES
Cyt c-based biosensor
NBT method
IC50 / mg mL-1
AEAC
IC50 / mg mL-1
AEAC
Ascorbic acid
6.0 ± 0.9
1.00
6.7 ± 0.8
1.00
Brand 1
3.4 ± 0.6
1.76
3.4 ± 0.7
1.97
Brand 2
3.5 ± 0.5
1.71
3.5 ± 0.7
1.91
Brand 3
5.2 ± 0.6
1.15
5.9 ± 0.8
1.14
Brand 4
3.7 ± 0.8
1.62
4.0 ± 0.5
1.68
Natural orange juice
2.1 ± 0.4
2.86
2.3 ± 0.6
2.91
AEAC 
1 IC50 sample
IC ascorbic acid
 50
1 IC50 ascorbic acid
IC50 sample
OBJECTIVES
Original biosensors using ROS
Free radical scavenging capacity
“total antioxidant capacity”
 Development of a cytochrome c (cyt c)-based biosensor
for the quantification of the antioxidant capacity against O2•-.
 Development of a simple and sensitive electrochemical
method for the determination of antioxidant capacity based
on the photogenerated •OH radicals.
SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH
DETECTION PRINCIPLE
•OH
generation: photocatalytic oxidation of water by TiO2
nanoparticles
•OH
trapping agent: 4-hydroxybenzoic acid
4-hydroxybenzoic acid
3,4-dihydroxybenzoic acid
SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH
DETECTION PRINCIPLE
3,4-DHBA
4-HBA
Blank solution
Square Wave Voltammetry (SWV) of 3,4-DHBA
Quantification of •OH
SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH
DETECTION PRINCIPLE
Without antioxidant compounds
Maximum 3,4-DHBA peak current
With antioxidant compounds
Competition AOx / 4-HBA for the elimination of •OH
Decrease of 3,4-DHBA peak current
SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH
DETERMINATION OF THE ANTIOXIDANT CAPACITY
Antioxidant
Capacity
IC50
Hill equation
y=
B•x
C+x
y: % signal inhibition
x: [AOx]
B,C: constant values
IC50
AC
Lipoic acid > Caffeic acid > Glutathione > Trolox > Ascorbic acid
SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH
DETERMINATION OF THE ANTIOXIDANT CAPACITY
Electrochemical method
Fluorimetric method
IC50 / mM
TEAC
IC50 / mM
TEAC
Trolox
22.15
1.00
34.67
1.00
Lipoic acid
1.75
12.66
6.75
5.14
Caffeic acid
2.72
8.14
11.23
3.09
Glutathione
13.52
1.64
17.90
1.94
Ascorbic acid
60.55
0.37
140.70
0.25
TEAC =
1/ IC50
Sample
1/ IC50 Trolox
IC50 Trolox
=
IC50 Sample
CONCLUSIONS
 An amperometric cyt-c based biosensor for the quantification of
the scavenging capacity of AOx has been developed.
 A MUA/MU-modified gold electrode with immobilized cyt c and
XOD has been characterized and applied to the AOx analysis.
 The applicability of this method has been shown by analyzing
the antioxidant capacity of ascorbic acid, Trolox and 5 orange
juices.
 The antioxidant capacity have been also determined by using a
simple electrochemical method.
 Based on the photogenerated •OH radicals, 4-HBA was
hydroxylated and the product 3,4-DHBA was measured by SWV.
 A good correlation between a fluorimetric method and the
proposed electrochemical method was obtained.
THANK YOU FOR YOUR ATTENTION
ANTIOXIDANT CAPACITY: DEVELOPMENT OF
METHODS BASED ON FREE RADICALS
M. Cortina-Puig, Y. Wang, B. Liu, C. Calas-Blanchard and J.L. Marty