J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 7, September 2019, pages 36-42

Revisiting DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as a useful tool in antioxidant evaluation: a new IC100 concept to address its limitations

Figure 1. The changes of chemical structure (a), color (a), and the absorption spectrum of DPPH radical after reduction by an antioxidant.

Figure 3. Determination of IC₁₀₀ by using two equations having a different pattern in the measurement of DPPH radical scavenging ability.

Figure 4. The IC₁₀₀ of new DPPH method in the presence or absence of pigments.

**Table 1.** DPPH radical scavenging ability of representative antioxidants using the original DPPH assay
	Concentration (mM)	Ratio (DPPH/Antioxidant)	Scavenged DPPH (%)	Stoichiometric factor (n, DPPH/Antioxidant)
α-Tocopherol	1.000	2/1	65.7 ± 0.5	1.3
0.500	4/1	51.8 ± 1.1	2.1
0.250	8/1	27.5 ± 1.3	2.2
0.125	16/1	17.1 ± 0.8	2.7
0.063	32/1	10.1 ± 0.5	3.2
Ascorbyl-6-palmitate	1.000	2/1	31.1 ± 2.1	0.6
0.500	4/1	17.5 ± 0.6	0.7
0.250	8/1	10.4 ± 1.5	0.8
0.125	16/1	6.7 ± 0.8	1.1
0.063	32/1	4.1 ± 0.1	1.3
BHT	1.000	2/1	62.7 ± 0.6	1.3
0.500	4/1	46.9 ± 1.4	1.9
0.250	8/1	30.1 ± 0.3	2.4
0.125	16/1	17.7 ± 0.1	2.8
0.063	32/1	11.4 ± 0.3	3.6
Catechin	1.000	2/1	67.9 ± 2.1	1.4
0.500	4/1	68.1 ± 1.7	2.7
0.250	8/1	61.2 ± 1.5	4.9
0.125	16/1	41.6 ± 1.0	6.7
0.063	32/1	26.9 ± 0.3	8.6

Table 1. DPPH radical scavenging ability of representative antioxidants using the original DPPH assay

Concentration (mM)

Ratio (DPPH/Antioxidant)

Scavenged DPPH (%)

Stoichiometric factor (n, DPPH/Antioxidant)

α-Tocopherol

1.000

2/1

65.7 ± 0.5

1.3

0.500

4/1

51.8 ± 1.1

2.1

0.250

8/1

27.5 ± 1.3

2.2

0.125

16/1

17.1 ± 0.8

2.7

0.063

32/1

10.1 ± 0.5

3.2

Ascorbyl-6-palmitate

1.000

2/1

31.1 ± 2.1

0.6

0.500

4/1

17.5 ± 0.6

0.7

0.250

8/1

10.4 ± 1.5

0.8

0.125

16/1

6.7 ± 0.8

1.1

0.063

32/1

4.1 ± 0.1

1.3

BHT

1.000

2/1

62.7 ± 0.6

1.3

0.500

4/1

46.9 ± 1.4

1.9

0.250

8/1

30.1 ± 0.3

2.4

0.125

16/1

17.7 ± 0.1

2.8

0.063

32/1

11.4 ± 0.3

3.6

Catechin

1.000

2/1

67.9 ± 2.1

1.4

0.500

4/1

68.1 ± 1.7

2.7

0.250

8/1

61.2 ± 1.5

4.9

0.125

16/1

41.6 ± 1.0

6.7

0.063

32/1

26.9 ± 0.3

8.6

**Table 2.** The IC₁₀₀ and stoichiometric factors of representative antioxidants using the modified DPPH method
	Concentration (mM)	IC₁₀₀ (mM)	Stoichiometric factor (n, DPPH/Antioxidant)
α-Tocopherol	1.00	0.124	2.488
0.50	0.060	2.389
0.25	0.025	1.985
Ascorbyl 6-palmitate	1.00	0.133	2.655
0.50	0.060	2.396
0.25	ND	ND
BHT	1.00	0.011	0.221
0.50	0.007	0.265
0.25	ND	ND
Catechin	1.00	0.198	3.963
0.50	0.103	4.106
0.25	0.045	3.637

Table 2. The IC₁₀₀ and stoichiometric factors of representative antioxidants using the modified DPPH method

Concentration (mM)

IC₁₀₀ (mM)

Stoichiometric factor (n, DPPH/Antioxidant)

α-Tocopherol

1.00

0.124

2.488

0.50

0.060

2.389

0.25

0.025

1.985

Ascorbyl 6-palmitate

1.00

0.133

2.655

0.50

0.060

2.396

0.25

BHT

1.00

0.011

0.221

0.50

0.007

0.265

0.25

Catechin

1.00

0.198

3.963

0.50

0.103

4.106

0.25

0.045

3.637

**Table 3.** The calculation of IC₁₀₀ and scavenged DPPH radical by different food extracts
	Concentration(mg/mL)	IC₁₀₀ (mM)	Scavenged DPPH mg/g of DW
Bell pepper	100	0.296	1.164
50	0.156	1.229
25	0.072	1.134
Correlation coefficient		0.998
Blackberry	100	0.521	2.052
50	0.242	1.908
25	0.132	2.081
Correlation coefficient		0.997
Raspberry	100	0.345	1.360
50	0.170	1.338
25	0.084	1.324
Correlation coefficient		1.000
Beet	200	0.072	0.141
100	0.034	0.133
50	0.021	0.162
Correlation coefficient		0.993

Table 3. The calculation of IC₁₀₀ and scavenged DPPH radical by different food extracts

Concentration(mg/mL)

IC₁₀₀ (mM)

Scavenged DPPH mg/g of DW

Bell pepper

100

0.296

1.164

0.156

1.229

0.072

1.134

Correlation coefficient

0.998

Blackberry

100

0.521

2.052

0.242

1.908

0.132

2.081

Correlation coefficient

0.997

Raspberry

100

0.345

1.360

0.170

1.338

0.084

1.324

Correlation coefficient

1.000

Beet

200

0.072

0.141

100

0.034

0.133

0.021

0.162

Correlation coefficient

0.993