Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

Isabelle Yang

Antioxidant Assay Selection

What is oxidation

•Generally: Oxidation is loss of electrons

Oxidation in Food

Different category

Oxidation in aqueous phase; eg. Protein oxidation

Oxidation in lipid phase; eg. Lipid oxidation

Enzymatic reaction; eg. Browning in apple slides

Non-enzymatic reaction; eg. Browning in whit wine

Reactive oxygen species; (ROS)

Reactive nitrogen species; (RNS)

Free radical

Non free radical

Oxidation type

• ROS

– Triplet oxygen (Ground state Oxygen)

- Singlet oxygen (Strong electron robber)

- Superoxide (O2.-)

- Peroxyl (ROO`)

-Alkoxyl (RO`)

-Hydroxyl (HO`)

•RNS : `NO, ONOO-

Non-radicals

Free radical

Oxidation type• Free radical oxidation

Free radicals are Any molecular species capable of independent existence, which contains one or more unpaired valence electrons not contributing to intramolecular bonding

Food system

• Structure: - cells (compounds transfer, interface with ROS, RNS)

- Phase: water/lipid; polar/non-polar

- pH, temperature, metal ions

•Oxidation order:

-how vulnerable the compounds are;

-which one is going to die first; (polyphenol, linoeicacid; carotenoids.)

Antioxidant

•Antioxidant is

- In food: protectors against oxidative stress

- Dietary antioxidant: substance in foods that significantly decreases the adverse effects of reactive species, ROS/RNS, on normal physiological function in humans. (Institute of Medicine)

Antioxidant• Physical barriers

•Metal chelators: siderphores, proteins

•Work on enzymes

- Inhibit oxidative enzymes through pH, temperature: lipidoxidase, Polyphenol oxidase

- Antioxidant enzyme cofactors (Q10, Selenium)

•Reducing (reductive) compounds

Antioxidant – reductive compounds

• Free radical scavenger

• Inhibit the reactive oxidants

•Reducing oxidation number : ferric, cupper

•Different antioxidants could be mixed together (antagonistic response; synergic response)

Antioxidant capacity

•Relative antioxidant efficiency (RAE)

RAE=Scd

Sbc

Antioxidant capacity assays• Simulate the target food system in order to

determine the Relative antioxidant efficiency

•HAT : Hydrogen atom transfer

- Simulate free radical in food, measure antioxidants’ capacity of competing free radicals, and terminate the radical chain.

• SET: Single electron transfer

- Measure antioxidants’ reducing capacity

HAT & SET

HAT Assay

• Test radical chain breaking capacity

Free radical generator / Initiator (AAPH; AMVN)

Oxidizable molecular probe (UV ; fluorescence; BODIPY)

antioxidant

Similar food system: lipid phase (cholesterol, carotenoids, linoleic acid)

• TRAP : Total radical trapping antioxidant parameter

•ORAC : Oxygen radical absorbance capacity

•Crocin bleaching assay ( natural carotenoid)

HAT Assay

HAT Assay

• The more vulnerable, the more competitive in sacrificial and terminate the radical chain by dying together.

HAT Assay

ORAC : Oxygen radical absorbance capacity

HAT Assay• TRAP : Total radical trapping antioxidant

parameter

HAT Assay

•Crocin Bleaching Assay

-10

0

10

20

30

40

50

60

10 20 30 40 50 60 70 80 90 100

Quercetin (ppm)

Rat

e of

Inh

ibiti

on

Quercetin Only

Quercetin + 5 mM Ascorbic acid

Quercetin + 2.5 mM Caffeic acid

SET Assay• Test reducing capacity. More general

antioxidant capacity than HAT

• Probe: Oxidant with metal ion, measure reducing capacity by different color of same metal ion with different oxidation number

•Metal ion: Molybdenum, Ferric, Copper

SET Assay

• Folin-Ciocalteu assay – total phenols

• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

• Ferric Ion Reducing Antioxidant Power Assay

• Total Antioxidant Potential Assay (Cu II)

• 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity Assay (DPPH)

SET Assay• FCR

-sodium tungstate (Na2WO4.2H2O, 100 g), sodium molybdate (Na2MoO4.2H2O, 25 g)

-Total phenolics

- pH ~10

SET Assay

SET Assay

• Ferric Ion Reducing Antioxidant Power Assay

SET Assay

• Total Antioxidant Potential Assay (Cu II)

SET Assay

• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

SET Assay

• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

SET Assay

• 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity Assay (DPPH)

•Radical scavenging

•HAT or SET

SET Assay

Antioxidant capacity application

Antioxidant capacity application •Antioxidant for homemade

guacamole

- Lemon juice; ascorbic acid, low pH to inhibit Polyphenol oxidase

- Package: physical barrier

- Metal chelalor: EDTA, calcium disodium EDTA