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Isabelle Yang Antioxidant Assay Selection

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

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Page 1: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

Isabelle Yang

Antioxidant Assay Selection

Page 2: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

What is oxidation

•Generally: Oxidation is loss of electrons

Page 3: Isabelle Yang Antioxidant Assay Selection. 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

Page 4: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 5: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 6: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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.)

Page 7: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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)

Page 8: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 9: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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)

Page 10: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

Antioxidant capacity

•Relative antioxidant efficiency (RAE)

RAE=Scd

Sbc

Page 11: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 12: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

HAT & SET

Page 13: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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)

Page 14: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

HAT Assay

Page 15: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

HAT Assay

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

Page 16: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

HAT Assay

ORAC : Oxygen radical absorbance capacity

Page 17: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

HAT Assay• TRAP : Total radical trapping antioxidant

parameter

Page 18: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 19: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 20: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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)

Page 21: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay• FCR

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

-Total phenolics

- pH ~10

Page 22: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

Page 23: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

• Ferric Ion Reducing Antioxidant Power Assay

Page 24: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

• Total Antioxidant Potential Assay (Cu II)

Page 25: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

Page 26: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

Page 27: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

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

•Radical scavenging

•HAT or SET

Page 28: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

SET Assay

Page 29: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

Antioxidant capacity application

Page 30: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons

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

Page 31: Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons