**2. Identification of variability factors on the measurement of antioxidant activity**

The scientific community is facing an accumulation of data on antioxidant activity. These data indicated a great variability depending on the method used, which does not render their comparison easy. To understand such variability, we analyzed several cases to highlight the factors that could be at the origin of the variability observed.

**Case 1**: Investigation of the antioxidant activity of pure components

The antioxidant activity obtained by different methods (ABTS, DPPH and ORAC) of 25 phenols was compared. The results obtained are summarized in table 1.


With: AA (ascorbic acid); C (cyanidin); C-G (cyanidin-3-O-glucoside); C-Ga (cyanidin-3-O-galactoside); C-R (cyani‐ din-3-O-rutinoside); Ca (catechin); CA (chlorogenic acid) ; D (delphinidin); D-G (delphinidin-3-O-glucoside); EA (ella‐ gic acid); ECa (epicatechin); EGCa (epigallocatechin); EGCaG (epigallocatechin gallate); GA (gallic acid); GCa (gallocatechin); Gl (reduced glutathione); HE (hesperidin); K (kaempferol); K-G(kaempferol-3-O-glucoside); M (myrice‐ tin); M-R (myricetin-3-rhamnoside); NA (naringenin); Q (quercetin); Q-R (rutin); T (Trolox).

**Table 1.** Antioxidant values of phenolic compounds measured by ABTS, DPPH and ORAC methods [2]

It appears that the values obtained of antioxidant activities are not of the same order of magnitude; the ORAC method gives the highest values followed by the ABTS and the DPPH method. Moreover, phenolic compounds having a high antioxidant activity with a given method may have low antioxidant activity with another method. For example, kaempferol 3 glucoside and quercetin have respectively an antioxidant value of 6.6 and 4.2 µmol Trolox with the ORAC method and only 0.2 and 1.8 with the ABTS method. The variability observed is due only to the method used. So, it seems that it is difficult to compare the numerical values of antioxidant activity provided by different methods of determination.

To analyse the effects of the extraction step and the variety of food, the antioxidant activities of extracts resulting from different raw materials such as onions and apples were investigated.

**Case 2**: Antioxidant activity of extracts from the food matrix

**2. Identification of variability factors on the measurement of antioxidant**

The scientific community is facing an accumulation of data on antioxidant activity. These data indicated a great variability depending on the method used, which does not render their comparison easy. To understand such variability, we analyzed several cases to highlight the

The antioxidant activity obtained by different methods (ABTS, DPPH and ORAC) of 25

1.8 0.5 1.5 0.6 0.2 6.6

2.0 2.3 1.9 1.7 2.3 3.5

0.0

1.1 1.3 1.2 4.9 2.0

1.3 0.7

tin); M-R (myricetin-3-rhamnoside); NA (naringenin); Q (quercetin); Q-R (rutin); T (Trolox).

**Table 1.** Antioxidant values of phenolic compounds measured by ABTS, DPPH and ORAC methods [2]

With: AA (ascorbic acid); C (cyanidin); C-G (cyanidin-3-O-glucoside); C-Ga (cyanidin-3-O-galactoside); C-R (cyani‐ din-3-O-rutinoside); Ca (catechin); CA (chlorogenic acid) ; D (delphinidin); D-G (delphinidin-3-O-glucoside); EA (ella‐ gic acid); ECa (epicatechin); EGCa (epigallocatechin); EGCaG (epigallocatechin gallate); GA (gallic acid); GCa (gallocatechin); Gl (reduced glutathione); HE (hesperidin); K (kaempferol); K-G(kaempferol-3-O-glucoside); M (myrice‐

**ABTS DPPH ORAC**

0.9 0.8 1.8 1.0 0.0 2.0

0.5 1.5 0.6 0.8 0.5 1.4

0.0 0.0

0.8 1.0 1.5 8.5 3.7

0.9 0.9 0.8 4.2 6.2 3.6 4.6 6.6 6.0

4.4 3.8 7.3 5.5 5.8 5.9

4.5 5.6

7.9 5.1 3.1 8.3 3.4

1.0 5.3 2.9

factors that could be at the origin of the variability observed.

K M Q-R K-G M-R

D C-G C-R C-GA D-G

Flavanones HE NA 0.5

ECa EGCa GCa EGCaG

Phenolic acids GA CA EA 2.1

Flavonols Q

Anthocyanins C

Flavan-3-ols CA

**Case 1**: Investigation of the antioxidant activity of pure components

phenols was compared. The results obtained are summarized in table 1.

**activity**

78 Biotechnology

To highlight on the effect of the origin and the variety of food raw materials, [3] evaluated the total antioxidant capacity (TAC) by three methods (ABTS, FRAP and DPPH) using four varieties of onions with a similar procedure of extraction (methanol:water (70:30 v/v)). The activities obtained are summarized in table 2.


**Table 2.** Antioxidant activity of different onion varieties (Allium cepa).obtained with ABTS, FRAP and DPPH methods

These results indicate significant variations of the antioxidant activity depending on the variety tested. Antioxidant activity of the red onion is higher than that of white one. The magnitude of the variation is different according to the method used. For example, there is a factor of 2.4 between the red and white onion with ABTS, whereas this factor is only of 1.3 for FRAP and 1.7 for DPPH. For a given variety, the ABTS values are two times higher than FRAP or DPPH values, while values obtained by FRAP and DPPH are closer and sometimes do not indicate any difference between varieties. This behavior could be attributed to the fact that the active compounds in the origin of the antioxidant activity are not similar for the four varieties tested. This fact will have to be taken into account to compare the antioxidant activity obtained by different methods of determination.

To analyze the effect of the step of extraction, we gathered several results on the antioxidant activity of the golden delicious apple using different methods of determination (ABTS, FRAP and ORAC). The results obtained are given in table 3.


**Table 3.** Total antioxidant capacity of golden delicious apples (in µmol TE/ g FW) according to [4-7].

These results indicates that for a given method, the extraction procedure has a great impact on antioxidant activity values. For example with the ABTS method, using an extraction with methanol as solvent and assisted by ultrasound, this leads to 6.7 µmol TE/g FW; while the extraction with a mixture of methanol and water (80%v/v) or with acetone furnishes only 0.94 µmol TE/g FW. The measurement of bioavailability directly in plasma gives a value of 8.3 µmol TE/g FW. Different values are also obtained depending on the method of the extraction used with FRAP or ORAC protocols.

The analysis of the results of antioxidant activity of pure components and extracts from the food matrix indicates a broad variability in antioxidant values whatever the method used. This variability is also observed for a given method with the variety or the degree of maturation of the food raw material. This variability of the antioxidant activity determination can be attributed to three sources (i) factors related to food products such as the variety, and the growth method. (ii) Factors related to the extraction method such as pH, temperature, solvent, presence of an accelerator and (iii) factors related to the method used for the antioxidant activity determination.
