Transcript How to Select an Antioxidant Assay

Antioxidant Assay
Selection
Isabelle Yang
• Generally: Oxidation is loss of electrons
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
•
ROS
Non-radicals
– Triplet oxygen (Ground state Oxygen)
- Singlet oxygen (Strong electron robber)
- Superoxide (O2.-)
- Peroxyl (ROO`)
Free radical
-Alkoxyl (RO`)
-Hydroxyl (HO`)
• RNS : `NO, ONOO-
• 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
• 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 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)
• 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
• Free radical scavenger
• Inhibit the reactive oxidants
• Reducing oxidation number : ferric, cupper
• Different antioxidants could be mixed together
(antagonistic response; synergic response)
• Relative antioxidant efficiency (RAE)
RAE=
Scd
Sbc
• 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
• 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)
• The more vulnerable, the
more competitive in
sacrificial and terminate
the radical chain by dying
together.
• TRAP : Total radical trapping antioxidant
parameter
• Crocin Bleaching Assay
Rate of Inhibition
60
Quercetin Only
50
Quercetin + 5 mM Ascorbic acid
40
Quercetin + 2.5 mM Caffeic acid
30
20
10
0
-10
10
20
30
40
50
60
70
Quercetin (ppm)
80
90 100
• 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
• 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)
• FCR
-sodium tungstate (Na2WO4.2H2O, 100 g), sodium
molybdate (Na2MoO4.2H2O, 25 g)
-Total phenolics
- pH ~10
• Ferric Ion Reducing Antioxidant Power Assay
• Total Antioxidant Potential Assay (Cu II)
• Trolox Equivalent Antioxidant Capacity Assay
ABTS)
(
• Trolox Equivalent Antioxidant Capacity Assay
( ABTS)
• 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging
Capacity Assay (DPPH)
• Radical scavenging
• HAT or SET
• Antioxidant for homemade guacamole
- Lemon juice; ascorbic acid, low pH
to inhibit Polyphenol oxidase
- Package: physical barrier
- Metal chelalor: EDTA, calcium
disodium EDTA