Antioxidant Analysis on Roselle using Ferric Reducing Antioxidant Power (FRAP) Assay

Materials and Method

  1. The total antioxidant activity was determined by using FRAP assay as adapted from Benzie and Strain (1996).
  2. The samples were first extracted with three different solvents: water, ethanol and methanol.
  3. Then, the samples were analyzed in the concentration range of 0.2 to 1.0 mM.
  4. The FRAP reagent was prepared by mixing 25 ml of 300 mM acetate buffer with pH 3.6, 2.5 ml of 10 mM 2,4,acetate 6-tri-(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl and 2.5 ml of 20 mM FeCl3.6H2
  5. After the 300 µl of the freshly prepared FRAP reagent was warmed to 37 ºC in water bath (Memmert/WNB 14, Germany), a reagent blank reading was taken at 593 nm by using UV spectrophotometer (Shimadzu/UV 1240, North America). Then, 10 µl of sample and 30 µl of distilled water were added.
  6. The absorbance reading was taken and the difference between the final reading of FRAP reagent with sample and the reagent blank reading was calculated and recorded.
  7. Standards of FeSO4.7H2O were run in triplicate with different concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 mM and a standard curve will be prepared by plotting the mean value for each concentration by using Microsoft Excel.
  8. The FRAP values of samples were determined using the standard curve.
  9. The total antioxidant activity of roselle was expressed as mM Fe(II)/g dry weight.

Results

Standard Curve

Concentration (mM) 1st reading 2nd reading 3rd reading Mean
0.2 0.157 0.148 0.124 0.143
0.4 0.348 0.418 0.395 0.387
0.6 0.508 0.509 0.544 0.520
0.8 0.823 0.818 0.778 0.806
1.0 1.051 1.133 1.035 1.073

Extraction Solvent: Water

Concentration (mM) 1st reading 2nd reading 3rd reading Mean
0.2 0.123 0.131 0.109 0.121
0.4 0.223 0.238 0.202 0.221
0.6 0.348 0.302 0.331 0.327
0.8 0.468 0.493 0.475 0.479
1.0 0.530 0.542 0.548 0.540

Extraction Solvent: Ethanol

Concentration (mM) 1st reading 2nd reading 3rd reading Mean
0.2 0.186 0.168 0.183 0.179
0.4 0.292 0.297 0.268 0.286
0.6 0.438 0.446 0.422 0.435
0.8 0.488 0.505 0.505 0.499
1.0 0.649 0.640 0.679 0.656

Extraction Solvent: Methanol

Concentration (mM) 1st reading 2nd reading 3rd reading Mean
0.2 0.264 0.250 0.243 0.252
0.4 0.354 0.378 0.363 0.365
0.6 0.510 0.507 0.532 0.516
0.8 0.616 0.608 0.636 0.620
1.0 0.773 0.762 0.775 0.770

Total Antioxidant Activity (mM Fe(II)/g dry weight)

Solvent

Concentration

 Water Ethanol Methanol
0.2 29.8 50.3 72.9
0.4 54.5 80.3 105.6
0.6 80.5 122.3 149.4
0.8 104.3 140.3 179.4
1.0 132.8 184.6 222.9

 

 

Discussion

From the experiment, the sample extracted with methanol showed the highest antioxidant activity, followed by ethanol and then distilled water. According to Zhou and Yu (2004), the use of methanol for extraction can obtain higher antioxidant reading compared to ethanol. This suggested that using methanol might extract higher amount of antioxidants. Besides, methanol is more polar than ethanol and this might be the answer why the extract using methanol has higher antioxidant activity compared to ethanol. However, both ethanol and methanol showed effectiveness in extracting phenolic compounds (Murugan and Parimelazhagan, 2014). In this experiment, we did not identify the antioxidant component present in roselle. Hence we cannot conclude the polarity of antioxidant as there may be more than one type of antioxidants present in roselle. Based on the result, extraction using methanol showed higher antioxidant activity. This can indicated that the antioxidant present in roselle may be slightly polar. As bioactive component mostly made up of non-polar molecule, we expected that the antioxidant activity will be less if we extracted roselle using distilled water.

Reference

Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239, 70-76.

Murugan, R., & Parimelazhagan, T. (2014). Comparative evaluation of different extraction methods for antioxidant and anti-inflammatory properties from Osbeckia parvifolia Arn.–An in vitro approach. Journal of King Saud University-Science, 26(4), 267-275.

Zhou, K., & Yu, L. (2004). Effects of extraction solvent on wheat bran antioxidant activity estimation. LWT-Food Science and Technology, 37(7), 717-721.