氧族自由基清除体外抗氧化方法(ORAC)-康奈尔大学刘瑞海实验室实验方法

Oxygen Radical Absorbance Capacity Assay Description:
The ORAC assay measures a compounds ability to scavenge a free radical from ABAP compared to that Trolox. (Huang, et al. 2002)
Materials:
●Fluoroskan Ascent FL
●15 mL, 50mL conical /ˈkɑ:nɪkl tube
● 1.8 mL Eppendorf tubes
●V olumetric flasks
●96-well culture cluster, flat bottom, black plates
●Potassium phosphate dibasic二盐
●Potassium phosphate monobasic
●β-cyclodextrin
●Fluorescein disodium salt
●Trolox(6-hydroxy-2,5,7,8-tetramethylchronman（色烷）-2-carboxylic acid) 水溶
性Ve
●ABAP(2,2’-azobis(2-amidinopropane)dihydrochloride (2,2'-偶氮二异丁基脒二盐
酸盐)
●Gallic acid(F.W=170.12) (没食子酸)
●Deonized water
Solution Preparation:
Phosphate Buffers
75 mM potassium phosphate monobasic(A):
1. Add 10.21 g potassium phosphate monobasic to 1000 mL volumetric flask
2. Add 700 mL deionized water and magnetic stir bar. Stir until dissolved.
3. Remove stir bar and deionized water to 1000 mL.
75 mM potassium phosphate dibasic(B):
1. Add 13.06 g potassium phosphate diasic to 1000 mL volumetric flask
2. Add 700 mL deionized water and magnetic stir bar. Stir until dissolved.
3. Remove stir bar and deionized water to 1000 mL.
Phosphate buffer working solution:
1. Add 800 ml B to 1000 mL beaker and begin reading pH meter.
2. Add 200 mL A to B and monitor pH.
3. Add additional A to adjust final pH to 7.
4.
4. Store phosphate buffer working solution in amber glass bottle.
For lipophilic antioxidants, replace phosphate buffer working solution with 0.8% W/V β- cyclodextrin in water.
Trolox Standards
Trolox stock solution
1.dissolve 25 mg Trolox in 100 mL phosphate buffer working solution to make 1
mM Trolox solution.
2.Dilute to 500 μM with phosphate buffer working solution.
3.Aliquot 1.5 mL of stock solution into eppendorf tubes and store at -70 ℃until
use.
Trolox standard solution:
1.Thaw （溶化）an aliquot of 500μM Trolox stock solution.
2.Add 1 mL 500μM Trolox stock solution to 9 mL phosphate buffer working
solution (50μM final concentration).
3.make serial dilutions of 50μM Trolox solution to make standard solutions of 6.25,
12.5, and 25μM Trolox standards.
4.Keep remaining Trolox solution at 4 ℃.
Fluoroscein Solutions
Fluoroscein stock solution A
1.Dissolve 2
2.5 mg fluoroscein stock solution A in 50 mL phosphate buffer working
solution and mix well.
Fluoroscein stock solution B
1.Dissolve 50 μl fluoroscein stock solution A in 10 mL phosphate buffer working
solution and votex.
2.Aliquot fluoroscein stock solution B into Eppendorf tubes and store at -20 ℃ until
use.
Fluoroscein working solution (no more than 8 h before using)
1.Add 800 μl fluoroscein stock solution B to 50 mL phosphate buffer working
solution in a 50 mL conical.
2.Incubate solution at 37℃. Solution can be kept in water bath for a long time. ABAP Solution- Solution is made daily
1.Incubate phosphate buffer working solution at 25 ℃.
2.Dissolve 162 mg ABAP into 5 mL phosphate buffer working solution immediately
before the start of the assay(119.4 μmol/mL or 2.4 μmol/20 μL aliquot).
You must keep the ABAP at 4 ℃before adding to warm buffer. ABAP reaction is temperature dependent.
Sample Preparation
1.Based on the total phenolic content of the sample, dissolve the sample to less than
5-10 μM gallic acid with phosphate buffer working solution.
2.Record the dilution factor.
Gallic Acid Preparation
1.Dissolve 29.8 mg gallic acid in 250 mL phosphate buffer working solution to
make 700 μM stock solution.
2.add 125 μL to 4875 μL phosphate buffer working solution to make 17.5 μM gallic
acid.
3.aliquot in Eppendorf tubes and store at -80 ℃ away from light.
Gallic acid is used to measure the precision of the assay. The precision of the assay will be determined by the inter-run mean and the standard deviation of the 17.5 μM gallic acid.
Method of the Analysis:
1.Pipette 20 μL sample, blank, or Trolox standard or 200 μL fluoroscein working
solution (the F well, also 40 μL phosphate buffer) to the appropriate well according to Figure 1 and incubate for 10 min.
2.Set the microplate well reader according to parameters in Figure 2(if necessary).
3.Add 200 μL fluoroscein working solution to each well(except the F well).
4.Cover microplate and incubate at 37 ℃ for at least 20 min.
5.Verify that plate reader is warmed up to 37 ℃ before reading samples.
6.Add 20 μL AAPH working solution to each well.
7.Read plate immediately after adding AAPH.
The relative area under the curve is calculate as:
AUC = (0.5* f1/f1 + f2/f1+f3/f1+ … + f i/f1+… f34/f1 + 0.5 * f35/f1) × CT
Where f1 is the first fluorescence reading, f i is the reading at the cycle i and the CT is cycle time in min.
Subtract the AUC from the blank to get the net AUC. The final ORAC value is calculate by linear regression between Trolox concentration (μM) and the net AUC. The linear regression is used in the range of 6.25-50 μM Trolox. Data is expressed as μmol Trolox equivalents(TE) per 100 g sample.
ORAC is calculated using the linear equation y = ax + b ( y, Net AUC; x, Trolox concentration; a, slope; b, y-intercept)
Figure 1-Microplate layout:
F = 200 μL fluoroscein working solution + 40 μL phosphate buffer, B = blank, T = Trolox, S = sample
A
B
C
D
E
F
G
H
Figure 2–Fluoroskan Ascent Fluorescent 96-well Plate Reader Basic Parameters: Positioning Delay 0.3s
Numbe i=of kinetic windows 1
Number of cycles 35
Measurement starting time 0s
Number of flashing per cycle 15
Cycle time Depends on the layout
Filters and integration Fluorescence intensity
Number of multichromatics 1
Gain Depends on gain adjustment
Pause before cycle 0
Excitation filter 485 nm
Emission filter 538 nm
Calculation range Start 1 stopn 35
V olume and pump speed 200 μL and 420 μL/s
V olume 2 and pump speed 200 μL and 420 μL/s
Skaking mode Orbital
Shaking width 4 mm
Additional shaking Before each cycle
Shaking time 8 s
Figure 4 – Calculation of ORAC
Figure 5 – Example of Standard Cure
References:
DEJIAN HUANG, BOXIN OU, MAUREEN HAMPSCH-WOODILL, et al. (2002). High-Throughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. J. Agric. Food Chem. 50(16):4437-4444.。