Peeling Away Problems

The Antioxidating, Antimicrobial, and Antimutagenic Effects of Tree Bark

FRAP Assay

(See Reference 1 for more details)

Using iron (II) sulphate (FeSO₄7H₂O) as a standard:

1.      Make a 10mmol/L stock solution of FeSO₄7H₂O by weighing 27.8mg of FeSO₄7H₂O and dissolving it in 10ml of water.

2.      Add 0, 10, 20, 50, 80, and 100µL of FeSO₄7H₂O stock solution to a tube and then add 1000, 990, 980, 950, 920, and 900 µL of water respectively (so that the total solution is 1000µL) to make 0, 0.1, 0.2, 0.5, 0.8, and 1 mmol/L FeSO₄7H₂O working solution.

3.      Add 30µL of each FeSO₄7H₂O working solution and 5µL (1µL in case of White Spruce extract) of each tree bark sample (since the bark extracts are more concentrated) into separate tubes.

4.      Add 900µL of freshly prepared FRAP reagent (see below).

5.      Add 90µL (for FeSO₄7H₂O samples) or 65µL (for bark extract samples) of H₂O.

6.      Mix, pipette into separate cuvettes and put into a spectrophotometer.

7.      Read the absorbance of each of the samples at A₅₉₃ (Absorbance at 593 nm wavelength) against 0 mmol/L of FeSO₄7H₂O.

FRAP reagent  (prepare freshly):

       25   mL     acetate buffer, 300mmol/L, pH3.6  

       2.5  mL     TPTZ solution (10 mmol/L 2, 4, 6-tripyridyl-1, 3, 5-triazine or C₃N₃(C₅H₄N)₃ in 40 mmol/L HCl)

       2.5  mL     FeCl₃ · 6H₂O solution, 20 mmol/L in distilled water

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