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ANTIOXIDANT CAPACITY: DEVELOPMENT OF METHODS BASED ON FREE RADICALS. M. Cortina-Puig, Y. Wang, B. Liu, C. Calas-Blanchard and J.L. Marty. Equilibrium AOx = Free Radicals. AOx. Free Radicals. INTRODUCTION. FREE RADICALS. Highly unstable molecules with available electrons. - PowerPoint PPT Presentation
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ANTIOXIDANT CAPACITY: DEVELOPMENT OF METHODS BASED ON FREE RADICALS
M. Cortina-Puig, Y. Wang, B. Liu, C. Calas-Blanchard and J.L. Marty
FREE RADICALS
Highly unstable molecules with available electrons.
Generated in vivo during metabolic processes.
In order to protect against them, humans have evolved with antioxidant (AOx) systems.
AOx FreeRadicals
EquilibriumAOx = Free Radicals
Reactive oxygen species (ROS). Superoxide Radical (O2
•-) Hydrogen Peroxide (H2O2) Hydroxyl Radical (OH•) Singlet oxygen (1O2) Hypochlorous Acid (HOCl) Alkoxyl Radicals (RO•) Peroxyl Radicals (RO2
•)
INTRODUCTIONINTRODUCTION
FREE RADICALS
Stressors(environmental or behavioural)
pollution
sunlight exposure
cigarette smoking
excessive alcohol
consumption
Antioxidant production malfunctionAOx
FreeRadicals
Oxidative StressExcess Free Radicals
FREE RADICAL EXCESS
OXIDATIVE STRESS
INTRODUCTIONINTRODUCTION
FREE RADICALS
Normal Lipids Normal DNA Normal RNANormal
Proteins and Enzymes
Lipid Peroxyl &Alkoxyl Radicals Altered DNA Altered RNA
Altered Proteins and
Enzymes
Loss of Temporal Control of Gene
Functions
Impairment of Essential Cellular &
Tissue Functions
Immunological Response to
Altered Proteins
Excess of Specific Proteins
Absence of Specific Proteins
HUMAN DISEASES (CANCER, ALZHEIMER) & AGING PROCESS
INTRODUCTIONINTRODUCTION
ANTIOXIDANTS
Substances which counteract free radicals and prevent the damage caused by them.
• crumbling ROS before they react with biological targets• preventing chain reactions• preventing the activation of O2 to highly reactive products
Reduction of the adverse damage due to oxidants through different protective mechanisms:
INTRODUCTIONINTRODUCTION
INTRODUCTIONINTRODUCTIONANTIOXIDANTS
Antioxidants
Non-enzymatic AOx
MineralsZinc, Selenium
VitaminsVit A, Vit C, Vit E, ViitK
Carotenoids-carotene, lycopene, lutein, zeaxanthin
Organosulfur compoundsallium, allyl sulfide, indoles
Low molecular weight AOxglutathione, uric acid
AOx cofactorsCoenzyme Q10
Polyphenols
Flavonoids Phenolic acids
Hydroxycinnamic acids
ferulic acid, -Coumaric
Hydroxybenzoic acids
gallic acid, ellagic acid
Flavoneschrysin
Flavanoneshesperitin
FlavanolsCatechin, EGCG
Isoflavonoidsgenistein
FlavonolsQuercetin, kaempferol
Enzymatic AOx
Primary EnzymesSOD, catalase, glutathione,
peroxidase
Secondary Enzymesglutathione reductase, glucose 6-phosphate
dehydrogenase
ANTIOXIDANT CAPACITY (AOC) DETERMINATION
Hydrogen Atom Transfer (HAT)
X•+ AH XH + A•
Measure the classical ability of an antioxidant to quench free radicals by hydrogen donation (AH = any H donor)
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•+ A• + H3O+
X-+ H3O+ XH + H2O
M(III) + AH AH+ + M(II)
INTRODUCTIONINTRODUCTION
ANTIOXIDANT CAPACITY METHODS
Oxygen Radical Absorbance Capacity (ORAC)Total Radical-trapping Antioxidant Parameter (TRAP)
HAT:
Ferric Reducing Antioxidant Power (FRAP)SET:
Trolox Equivalent Antioxidant Capacity (TEAC)2,2-Diphenyl-1-picrylhydrazyl (DPPH assay)
HAT/SET:
INTRODUCTIONINTRODUCTION
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.
OBJECTIVESOBJECTIVES
DETECTION PRINCIPLE OF CYT C-BASED BIOSENSORS
Cyt cHeme (Fe3+)
Gol
d el
ectr
ode
COOH
COOH
S
S
COO-
SS
COO-S
COO-
S
COOH Cyt cHeme (Fe2+)
XOD
HX
uricacid
H2O2O2 + H2Ocatalase
O2
e-
O2
O2•-
E = 150 mV vs Ag/AgCl
[O2•-]I
[O2•-] I Antioxidant Capacity
HX: HypoxanthineXOD: Xanthine oxidase
CYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
MEASUREMENT OF THE O2•- SCAVENGING CAPACITY
IHX (1)
+ HX (100 M)
+ AntioxidantIantioxidant
IHX (2)
buffer + catalase
(10 U mL-1)
+ HX (100 M)
buffer + catalase
(10 U mL-1)
Signal inhibition (%) =IHX(1) – IHX(2)
IHX(1)x 100 IC50
Antioxidant Capacity
IC50 AC
CYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX
Ascorbic acid
Trolox(6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
Hydrophilic AOx Standards for other antioxidative substances
CYTOCHROME C-BASED BIOSENSORCYTOCHROME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX
Ascorbic acid
Hill equation
y =B • xC + x
y: % signal inhibitionx: [AOx]B,C: constant values
IC50 =50 • CB - 50
IC50 = 6.0 ± 0.9 g mL-1
IC50
CYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX
IC50 (Trolox) = 40.8 ± 0.7 g mL-1
IC50
Trolox IC50 (Ascorbic acid) = 6.0 ± 0.9 g mL-1
IC50 AC
CYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES
AOx: flavonoids, carotenoids and vitamin C
+ Orange juice Intensity Antioxidant capacity
CYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
IC50 / g mL-1
Ascorbic acid 6.0 ± 0.9Brand 1 3.4 ± 0.6Brand 2 3.5 ± 0.5Brand 3 5.2 ± 0.6Brand 4 3.7 ± 0.8
Natural orange juice 5.0 ± 0.4
ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES
Other AOx: flavonoids (hesperetin and naringenin) carotenoids (xanthophylls, cryptoxanthins, carotenes)
66% AC
Vitamin C
CYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
ANTIOXIDATIVE PROPERTIES OF ORANGE JUICESCYTOCRHOME C-BASED BIOSENSORCYTOCRHOME C-BASED BIOSENSOR
Cyt c-based biosensor NBT methodIC50 / g mL-1 AEAC IC50 / g 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
sampleIC
acidascorbicICacidascorbicIC
sampleICAEAC
50
50
50
50
11
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.
OBJECTIVESOBJECTIVES
DETECTION PRINCIPLESIMPLE ELECTROCHEMICAL METHOD BASED ON SIMPLE ELECTROCHEMICAL METHOD BASED ON ••OHOH
•OH generation: photocatalytic oxidation of water by TiO2 nanoparticles
•OH trapping agent: 4-hydroxybenzoic acid
4-hydroxybenzoic acid 3,4-dihydroxybenzoic acid
DETECTION PRINCIPLESIMPLE ELECTROCHEMICAL METHOD BASED ON SIMPLE ELECTROCHEMICAL METHOD BASED ON ••OHOH
3,4-DHBA 4-HBA
Blank solution
Square Wave Voltammetry (SWV) of 3,4-DHBA Quantification of •OH
DETECTION PRINCIPLESIMPLE ELECTROCHEMICAL METHOD BASED ON SIMPLE ELECTROCHEMICAL METHOD BASED ON ••OHOH
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
DETERMINATION OF THE ANTIOXIDANT CAPACITYSIMPLE ELECTROCHEMICAL METHOD BASED ON SIMPLE ELECTROCHEMICAL METHOD BASED ON ••OHOH
IC50
Antioxidant Capacity
IC50 AC
Hill equation
y =B • xC + x
y: % signal inhibitionx: [AOx]B,C: constant values
Lipoic acid > Caffeic acid > Glutathione > Trolox > Ascorbic acid
SIMPLE ELECTROCHEMICAL METHOD BASED ON SIMPLE ELECTROCHEMICAL METHOD BASED ON ••OHOHDETERMINATION OF THE ANTIOXIDANT CAPACITY
Electrochemical method Fluorimetric methodIC50 / M TEAC IC50 / M 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
50 Sample 50 Trolox
50 Trolox 50 Sample
1/ = =
1/ IC IC
TEACIC IC
CONCLUSIONSCONCLUSIONS
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
