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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