ANTIOXIDANT CAPACITY: DEVELOPMENT OF METHODS BASED ON FREE RADICALS

1. ANTIOXIDANT CAPACITY: DEVELOPMENT OF METHODS BASED ON FREE RADICALS M. Cortina-Puig, Y. Wang, B. Liu, C. Calas-Blanchard and J.L. Marty

2. Equilibrium AOx = Free Radicals AOx Free Radicals INTRODUCTION FREE RADICALS • Highly unstable molecules with available electrons. • Generated in vivo during metabolic processes. • 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•) • In order to protect against them, humans have evolved with antioxidant (AOx) systems.

3. Stressors (environmental or behavioural) excessive alcohol consumption pollution sunlight exposure cigarette smoking FREE RADICAL EXCESS OXIDATIVE STRESS AOx Oxidative Stress Excess Free Radicals Free Radicals INTRODUCTION FREE RADICALS Antioxidant production malfunction

4. 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 INTRODUCTION FREE RADICALS Normal Proteins and Enzymes Normal Lipids Normal DNA Normal RNA

5. 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

6. Enzymatic AOx Non-enzymatic AOx Primary Enzymes SOD, catalase, glutathione, peroxidase Minerals Zinc, Selenium Vitamins Vit A, Vit C, Vit E, ViitK Carotenoids b-carotene, lycopene, lutein, zeaxanthin Organosulfur compounds allium, allyl sulfide, indoles Secondary Enzymes glutathione reductase, glucose 6-phosphate dehydrogenase Low molecular weight AOx glutathione, uric acid AOx cofactors Coenzyme Q10 Polyphenols Flavonoids Phenolic acids Hydroxycinnamic acids ferulic acid, p-Coumaric Flavonols Quercetin, kaempferol Isoflavonoids genistein Flavanols Catechin, EGCG Flavanones hesperitin Flavones chrysin Hydroxybenzoic acids gallic acid, ellagic acid INTRODUCTION ANTIOXIDANTS Antioxidants

7. X•+ AH XH + A• X•+ AH X- + AH•+ AH•+ A• + H3O+ X-+ H3O+ XH + H2O M(III) + AH AH+ + M(II) 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) • Single Electron Transfer (SET) Detect the ability of a potential antioxidant to transfer one electron to reduce any compound, including metals, carbonyls and radicals

8. 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)

9. 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.

10. Gold electrode S ANTIOXIDANT S S COO- O2 S COOH S COO- S e- COOH Cyt c Heme (Fe2+) COO- COOH HX O2•- catalase O2 + H2O H2O2 uric acid O2 [O2•-] I I Antioxidant Capacity [O2•-] CYTOCRHOME C-BASED BIOSENSOR DETECTION PRINCIPLE OF CYT C-BASED BIOSENSORS HX: Hypoxanthine XOD: Xanthine oxidase Cyt c Heme (Fe3+) E = 150 mV vs Ag/AgCl XOD

11. IHX (1) IHX (2) + HX (100 mM) + HX (100 mM) Antioxidant Capacity + Antioxidant Iantioxidant buffer + catalase (10 U mL-1) IHX(1) – IHX(2) IC50 AC x 100 Signal inhibition (%) = IHX(1) CYTOCRHOME C-BASED BIOSENSOR MEASUREMENT OF THE O2•- SCAVENGING CAPACITY buffer + catalase (10 U mL-1) IC50

12. 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)

13. B• x y = C + x 50 •C IC50 = B - 50 Ascorbic acid CYTOCRHOME C-BASED BIOSENSOR ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX Hill equation y: % signal inhibition x: [AOx] B,C: constant values IC50 IC50 = 6.0 ± 0.9 mg mL-1

14. IC50 AC Trolox CYTOCRHOME C-BASED BIOSENSOR ANTIOXIDATIVE PROPERTIES OF ASCORBIC ACID AND TROLOX IC50 IC50 (Trolox) = 40.8 ± 0.7 mg mL-1 IC50 (Ascorbic acid) = 6.0 ± 0.9 mg mL-1

15. Antioxidant capacity Intensity + Orange juice CYTOCRHOME C-BASED BIOSENSOR ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES AOx: flavonoids, carotenoids and vitamin C

16. 66% AC Vitamin C CYTOCRHOME C-BASED BIOSENSOR ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES • Other AOx: • flavonoids (hesperetin and naringenin) • carotenoids (xanthophylls, cryptoxanthins, carotenes)

17. CYTOCRHOME C-BASED BIOSENSOR ANTIOXIDATIVE PROPERTIES OF ORANGE JUICES

18. 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.

19. 4-hydroxybenzoic acid 3,4-dihydroxybenzoic acid SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH DETECTION PRINCIPLE •OH generation: photocatalytic oxidation of water by TiO2 nanoparticles •OH trapping agent: 4-hydroxybenzoic acid

20. 4-HBA 3,4-DHBA Blank solution Square Wave Voltammetry (SWV) of 3,4-DHBA Quantification of •OH SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH DETECTION PRINCIPLE

21. Without antioxidant compounds Maximum 3,4-DHBA peak current SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH DETECTION PRINCIPLE With antioxidant compounds Competition AOx / 4-HBA for the elimination of •OH Decrease of 3,4-DHBA peak current

22. B• x y = C + x Antioxidant Capacity IC50 AC SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH DETERMINATION OF THE ANTIOXIDANT CAPACITY IC50 Hill equation y: % signal inhibition x: [AOx] B,C: constant values Lipoic acid > Caffeic acid > Glutathione > Trolox > Ascorbic acid

23. SIMPLE ELECTROCHEMICAL METHOD BASED ON •OH DETERMINATION OF THE ANTIOXIDANT CAPACITY

24. 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.

25. 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