Antioxidant Capacity (AOC)

One typical analysis for characterizing medicinal plant extracts is determining their antioxidant activity. Free radicals formed during the metabolism are: O₂^.- (superoxide radical), O₂^2- (peroxide) and HO^. (hydroxyl radical) aka ROS (Reactive Oxygen Species) as well as NO (nitric oxide), ONOO (peroxy nitrate) and NO₂ (nitrogen dioxide) aka RNS (Reactive Nitrogen Species). As radicals are molecules with an unpaired free electron in their outer valance they are highly reactive and can damage several biomolecules as DNA, proteins and lipids (membranes). This is a special problem for mitochondria as they produce in the respiratory chain easily ROS. Now, proteins of the Krebs cycles (final oxidation of substrates to produce NADH) and of the respiratory chain (NADH reoxidation to generate Proton Motive Force, PMF) can be damaged, the membrane becomes leaky due to lipid-peroxidation (PMF dissipate) thus not enough ATP can be regenerated. If additionally (mt)DNA is damaged, protein damages can not be corrected by resynthesizing the proteins. This situation is described as oxidative stress and can cause serious to lethal cell damage - many disease like Alzheimer, Parkinson, artherosclerosis, cancer or immunological disorders are related to oxidative stress. Now, antioxidants are substances, which can eleminate the unpaired free electron and thus "detoxifying" radicals into a normal (i.e. not highly reactive) molecules.

For determining the total antioxidant capacity (AOC) of an extract several asepects have to be considered:
1) Chemical properties:
a) hydrophilic (e.g. vitamine C, which protect the cytosol) and
b) lipophilic antioxidants (e.g. vitamine E, which protect the membrane).
2) Mode of action:
a) single electron transfer (SET) or radical reduction and
b) H-Atom-Transfer (HAT) or radical quenching.
3) Test method:
a) in-vitro using chemical reactions for quantifying antioxidant compounds and
b) in-vivo using cells thus considering biological factors like uptake or distribution.

Of course, exist many different methods, each one with its advantages (and disadvantages), biological relevance and degree of difficulty.

Controls

Solvent: Liquid in which the substances (test system, pos. control, sample) are disssolved.
Test system: Principle of the test, mostly a dye which changes its absorbance
Sample: The substance which want to be analysed.

Blank:
Solvent of the system, mostly water sometimes MeOH. Used for autozero for photometer.

Mock Control:
Mixture of solvent (for the sample) and test system to measure the influence of the solvent on the test system; measured values have to be corrected for this.
Increasing absorbance (test system has low absorbance, which increases by the sample): Sample - Mock-control
Diminishing absorbance (test system has high absorbance, which reduces by the sample): Mock-control - Sample

Negative Control:
Mixture of sample and solvent to measure the influence of the sample on the test system (e.g. auto-absorbance of sample at wavelength of test system); measured values have to be corrected for this.

Positive Control:
To compare the antioxidant effect of the test substance it should be compared to a substance with a well known antioxidant effect (positive control). Typical positive controls are:
SET (very fast reaction: seconds to few minutes)
- Ascorbate (vitamine C):
- Gallic acid
Mixed (initially delayed: 30-120 min)
- Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid): better water soluble than vitamine E
HAT (slow reaction: hours)
- GSH
The best way to address the problem of different reaction kinetics (depending on the unknown antioxidant principal(s) in the sample) is a continious measurement of the absorbance, which result in a reaction curve.

Calculate:
Increasing Abs.: Standard curve: A_Positive - A_Mock; Sample: A_Sample - A_Mock - A_Negative
Diminishing Abs.: Standard curve: A_Mock - A_Positive; Sample: A_Mock - A_Sample - A_Negative

Results can be compared to standard antioxidants, typically 1 mM Trolox (TEAC = Trolox Equivalent Antioxidant Capacity):
TEAC = ΔA_sample / ΔA_{1 mM Trolox}

under construciton

1) DPPH (2,2-DiPhenyl-1-PicrylHydrazyl)

DPPH is a stable radical, which has a violet colour (abs. max. 515 nm) in methanolic solution; it can be used in ranges of 0-60% water and the rest MeOH; a higher water content interfere with test system (critical for very hydrophilic antioxidants). In contact with another radical (an antioxidant) it gets reduced, it lose its properties as a free radical and thus changes its colour to light yellow.
Advantages: The system is sentitive for radical reaction/H-transfer (HAT) or reduction/single e^--transfer (SET).
Disadvantages: DPPH is not soluble in water, thus the test component has to be disolved in MeOH. DPPH is not a physiological radical (i.e. is not similar to peroxyl-radicals). Light, O₂, pH have influence on DPPH absorbance (variance between laboratories). Test compounds with absorption at 515 nm (e.g. carotenoids) interfere with the absorption maximum of DPPH. Small molecules react more easy with DPPH than polymers (thus polymeric antioxidants in plant extracts are underestimated).

Protocol:

Solutions:
Degas solvents before use (e.g. 30 min sonication)
DPPH-stock : 19.7 mg DPPH in 100 ml MeOH (500 µM)
storage: -20 °C, dark. DPPH is not water soluble.
working DPPH: 11 ml DPPH-stock + 39 ml MeOH (110 µM)
=> absorbance of ~1.38 @ 515 nm

Procedure:
- 100 µl sample, pos. (Trolox) or Mock (MeOH) control
(unknown sample: 1, 10 & 100 µl and dilute w/ MeOH)
- 900 µl DPPH (in MeOH)
- mix, incubate 24 h at 37 °C, dark (100 µM DPPH)
(usually 1 h is enough, but in the presence
of polymeric antioxidants 24 h are recommended)
- measure absorbance @ 515 nm
=> absorbance of Mock control: ~1.25
- Standard: 0-50-100-200-300-400-500 µM Trolox

% Inhibition = 100 * (A_Mock - A_Sample - A_Neg.) / A_Mock
with different sample concentrations EC₅₀ can be calculated (Dose-Response curve).

Troubleshooting:
- If the sample absorption falls below 0.2 than dilute sample

original DPPH paper

DPPH pitfalls

2) ABTS (2,2'-Azinobis(3-ethyl-BenzoThiazoline-6-Sulfonic acid))

ABTS gets oxidysed by Na-persulfate to a stable, blue-green (Abs. 734 nm) cation-radical; which is more stable at pH 4.5 than at pH 7.4. This ABTS^•+ gets reduced by several antioxidants forming the colourless ABTS again.
Advantages: ABTS is soluble in water and organic solvents. ABTS^•+ has 4 absorption maxima (415, 645, 734 and 815 nm) for the case that test compounds interfere with the standard wavelength (734 nm).
Disadvantages: ABTS is a non-physiological radical (i.e. is not similar to peroxyl-radicals). (Poly)phenols are sterically hindered thus the reaction is slow, means the test is not very sentitive for radical reaction/H-transfer (HAT). Light and pH have influence on ABTS reduction, acid pH facilitates SET (and thus reaction kinetic).

Protocol:

Solutions:
Acetate-buffer: 581 mg NaAc + 738 µl HAc in 1 l A. dest. (20 mM, pH 4.5)
ABTS-stock: 22.6 mg ABTS in 10 ml acetate-buffer (4.125 mM)
storage: -20 °C, dark?
K-persulfate: 3.9 mg in 10 ml acetate-buffer (1.44 mM)
ABTS: 10 ml ABTS-stock + 10 ml K₂S₂O₈ (2.06 mM + 0.72 mM)
=> incubate 12-16 h, RT, dark
=> dilute 2 ml ABTS in 50 ml acetate-buffer or solvent (ABTS^•+: 82.5 µM)
=> absorbance of ~1.24 @ 734 nm

Procedure:
- 100 µl sample, pos. (Trolox) or Mock (acetate-buffer) control
(unknown sample: 1, 10 & 100 µl and dilute w/ buffer)
- 900 µl ABTS^•+
- mix, incubate 4 h at 37 °C, dark (75 µM ABTS^•+)
- measure absorbance @ 734 nm
=> absorbance of Mock control: ~1.13
- Standard: 0-50-100-200-300-400-500 µM Trolox

% Inhibition = 100 * (A_Mock - A_Sample - A_Neg.) / A_Mock
with different sample concentrations EC₅₀ can be calculated (Dose-Response curve).

Troubleshooting:
- If the sample absorption falls below 0.3 than dilute sample

ABTS

3) FRAP (Ferric Reducing Antioxidant Power)

Reduction of blue ferric (Fe³⁺)-trispyridyltriazine to ferrous (Fe²⁺)-trispyridyltriazine.
Advantages: simple, cheap, robust (absorption increase).
Disadvantages: The power to reduce ferric (Fe³⁺) is not biologically relevant. This test does not detect radical quencher (HAT) containing -SH groups like GSH and reaction time (kinetic) can vary strongly, specially polyphenols react slowly. Substances like biliverdin have a similar abs.max.

Protocol:

Solutions:
Acetate-buffer: 1.59 g NaAc + 16 ml HAc in 1 l A. dest. (300 mM, pH 3.6)
HCl: 83 ml A. dest. + 500 µl 25% HCl (40 mM)
TPTZ (2,4,6-Tris(2-Pyridyl-s-TriaZine): 31.2 mg TPTZ in 10 ml 40 mM HCl (10 mM)
FeCl₃: 54 mg FeCl₃ * 6 H₂O in 10 ml A. dest. (20 mM)
FRAP: 25 ml acetate-buffer, 2.5 ml TPTZ (0.83 mM), 2.5 ml FeCl₃ (1.67 mM)

Procedure:
- 100 µl sample, pos. (Trolox) or Mock (acetate-buffer) control
(unknown sample: 1, 10 & 100 µl and dilute w/ buffer)
- 900 µl FRAP
- mix, incubate 2 h at 37 °C, dark? (0.75 mM TPTZ, 1.5 mM FeCl₃)
- measure absorbance @ 593 nm (formed Fe^II(TPTZ)₂)
- Standard: 0-50-100-200-300-400-500 µM Trolox

Antioxidant Power = A_Sample - A_Mock - A_Neg.
with different sample concentrations EC₅₀ can be calculated (Dose-Response curve).

Troubleshooting:
- Preparation of FeCl₃-soln

original FRAP paper

Last modified: 28.04.2015