Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons.
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Transcript of Isabelle Yang Antioxidant Assay Selection. Generally: Oxidation is loss of electrons.
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Isabelle Yang
Antioxidant Assay Selection
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What is oxidation
•Generally: Oxidation is loss of electrons
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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
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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
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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
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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.)
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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)
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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
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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)
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Antioxidant capacity
•Relative antioxidant efficiency (RAE)
RAE=Scd
Sbc
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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
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HAT & SET
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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)
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HAT Assay
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HAT Assay
• The more vulnerable, the more competitive in sacrificial and terminate the radical chain by dying together.
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HAT Assay
ORAC : Oxygen radical absorbance capacity
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HAT Assay• TRAP : Total radical trapping antioxidant
parameter
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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
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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
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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)
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SET Assay• FCR
-sodium tungstate (Na2WO4.2H2O, 100 g), sodium molybdate (Na2MoO4.2H2O, 25 g)
-Total phenolics
- pH ~10
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SET Assay
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SET Assay
• Ferric Ion Reducing Antioxidant Power Assay
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SET Assay
• Total Antioxidant Potential Assay (Cu II)
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SET Assay
• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)
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SET Assay
• Trolox Equivalent Antioxidant Capacity Assay ( ABTS)
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SET Assay
• 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity Assay (DPPH)
•Radical scavenging
•HAT or SET
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SET Assay
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Antioxidant capacity application
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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
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