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 63-72

Optimization of extraction of antioxidants from aromatic herbs and their synergistic effects in a lipid model system

Figure 1. Effect of ethanol percentage and temperature on total phenolic content in (a) rosemary; (b) thyme; (c) sage; (d) oregano extracts. TPC, total phenolic contents; GAE, gallic acid equivalent.

Figure 2. (a) Hydroperoxide content (HC); (b) UV absorbance at 232 nm of RBD soybean oil in oven test (60 °C) on day 8 and C: Induction period (IP) of RBD soybean oil in Rancimat test. The hydroperoxide content (mmol LOOH/L oil) was calculated using a standard curve of cumene hydroperoxide. The UV absorption at 232 nm indicates the generation of primary oxidation products (e.g. conjugated dienes). The induction period was expressed in hours.

Figure 3. Inhibition percentage of phenolics from herb extracts against (a) hydroperoxide and (b) conjugated diene accumulation and (c) oxidation in Rancimat test. R is rosemary, T is Thyme, and O is oregano.

**Table 1.** Total phenolic content of extracts of selected herbs
Run	Independent variables	TPC (mg GAE/g)*
*Values expressed as means of triplicate determinations. GAE: gallic acid equivalents, TPC: total phenolic content. Runs 9–12, the central point (n = 4), allows to estimate the variation in the responses at the central point and provides a basis for the lack-of-fit test.
1	15	35	43.65	80.58	28.09	46.53
2	85	35	33.77	64.88	13.83	33.77
3	15	55	42.42	94.28	33.76	54.95
4	85	55	30.83	62.09	27.09	35.94
5	0	45	40.15	64.36	27.52	42.27
6	99	45	13.31	21.49	12.69	20.07
7	50	30	36.98	90.73	35.11	45.50
8	50	60	52.25	93.28	53.91	64.55
9	50	45	43.60	91.65	46.20	66.59
10	50	45	56.18	92.36	41.66	59.26
11	50	45	57.93	98.82	46.39	63.32
12	50	45	58.92	94.77	41.80	67.11

Table 1. Total phenolic content of extracts of selected herbs

Run

Independent variables

TPC (mg GAE/g)*

% EtOH

T (°C)

Rosemary

Oregano

Sage

Thyme

*Values expressed as means of triplicate determinations. GAE: gallic acid equivalents, TPC: total phenolic content. Runs 9–12, the central point (n = 4), allows to estimate the variation in the responses at the central point and provides a basis for the lack-of-fit test.

43.65

80.58

28.09

46.53

33.77

64.88

13.83

33.77

42.42

94.28

33.76

54.95

30.83

62.09

27.09

35.94

40.15

64.36

27.52

42.27

13.31

21.49

12.69

20.07

36.98

90.73

35.11

45.50

52.25

93.28

53.91

64.55

43.60

91.65

46.20

66.59

56.18

92.36

41.66

59.26

57.93

98.82

46.39

63.32

58.92

94.77

41.80

67.11

**Table 2.** Variance analysis of regression models fitted to experimental designs of TPC of selected herbal extracts
Source of variation (sum of squares)	Responses
*If F calculated is three-fold higher than F tabulated, the model may be used for prediction purposes.
Regression	1,947.536	4,973.575	1,664.749	2,555.898
Residual	131.066	253.406	140.428	82.775
Pure error	3.850	31.397	20.867	39.291
Total	2,078.602	5,226.981	1,805.77	2,638.673
F_calc*	22.289	88.322	31.612	53.953
F_tab	4.53	4.26	4.07	4.12
R²	0.9307	0.9515	0.9222	0.9671

Table 2. Variance analysis of regression models fitted to experimental designs of TPC of selected herbal extracts

Source of variation (sum of squares)

Responses

Rosemary

Oregano

Sage

Thyme

*If F calculated is three-fold higher than F tabulated, the model may be used for prediction purposes.

Regression

1,947.536

4,973.575

1,664.749

2,555.898

Residual

131.066

253.406

140.428

82.775

Pure error

3.850

31.397

20.867

39.291

Total

2,078.602

5,226.981

1,805.77

2,638.673

F_calc*

22.289

88.322

31.612

53.953

F_tab

4.53

4.26

4.07

4.12

R²

0.9307

0.9515

0.9222

0.9671

**Table 3.** Antiradical activities and corresponding Pearson’s correlation with total phenolic contents (TPC) of selected extracts of herbs
Sample	Antiradical activity (μmol TE/g dry weight)	Pearson’s correlation
¹Data represent the means of triplicate analysis for each sample ± standard deviations; TE is trolox equivalent. ²means with the same letters within a column are not significantly different according to Tukey’s multiple test (p > 0.05); **significant for Pearson’s correlation (p < 0.01).
Oregano	295.6 ± 16.4a²	520.8 ± 7.86a	0.9962**	0.9957**
Thyme	181.9 ± 2.48b	263.4 ± 3.39b	0.9861**	0.9683**
Rosemary	178.0 ± 7.90b	239.0 ± 2.57c	0.9984**	0.9955**

Table 3. Antiradical activities and corresponding Pearson’s correlation with total phenolic contents (TPC) of selected extracts of herbs

Sample

Antiradical activity (μmol TE/g dry weight)

Pearson’s correlation

DPPH

ABTS

TPC × DPPH

TPC × ABTS

¹Data represent the means of triplicate analysis for each sample ± standard deviations; TE is trolox equivalent. ²means with the same letters within a column are not significantly different according to Tukey’s multiple test (p > 0.05); **significant for Pearson’s correlation (p < 0.01).

Oregano

295.6 ± 16.4a²

520.8 ± 7.86a

0.9962**

0.9957**

Thyme

181.9 ± 2.48b

263.4 ± 3.39b

0.9861**

0.9683**

Rosemary

178.0 ± 7.90b

239.0 ± 2.57c

0.9984**

0.9955**

**Table 4.** Hydroperoxide content (mmol LOOH/L oil)¹, absorbance at 232 nm, and induction period of soybean oil with added herbal extracts subjected to thermal oxidation under Schaal oven test condition (60 °C) and Rancimat testing at 110 °C
Run	Mixture²	Relative concentration	Hydroperoxide content (mmol LOOH/L oil)³	Absorbance at 232 nm³	Induction Period⁴
¹values are expressed as means of triplicate determinations; ²Total concentration of phenolics for each run was 100 mg gallic acid equivalent/kg oil and the control was free from any herbal extracts or synthetic antioxidants. ³Results of oven test. ⁴Results of Rancimat test.
		(x₁, x₂, x₃)	0	4	6	8	0	4	6	8
1	Thyme (x₁)	(1,0,0)	0.76	2.44	6.59	17.5	5.40	5.52	6.08	8.26	8.26
2	Oregano (x₂)	(0,1,0)	1.01	4.70	19.8	29.0	5.40	5.76	7.20	10.2	8.03
3	Rosemary (x₃)	(0,0,1)	0.87	2.43	3.93	5.12	5.34	5.49	5.71	6.21	10.8
4	x₁ + x₂	(0.5, 0.5, 0)	0.55	2.70	14.2	20.7	5.37	5.64	7.35	8.77	8.37
5	x₁ + x₃	(0.5, 0, 0.5)	0.37	2.92	4.92	6.37	5.40	5.69	5.86	6.48	9.27
6	x₂ + x₃	(0, 0.5, 0.5)	0.92	2.97	5.06	6.38	5.41	6.15	5.95	6.61	9.03
7	x₁ + x₂ + x₃	(0.33, 0.33, 0.33)	0.40	2.88	4.56	5.69	5.21	5.51	5.59	6.07	8.94
8	Control	(0,0,0)	0.45	2.41	21.1	32.6	5.42	5.78	7.33	10.8	7.82

Table 4. Hydroperoxide content (mmol LOOH/L oil)¹, absorbance at 232 nm, and induction period of soybean oil with added herbal extracts subjected to thermal oxidation under Schaal oven test condition (60 °C) and Rancimat testing at 110 °C

Run

Mixture²

Relative concentration

Hydroperoxide content (mmol LOOH/L oil)³

Absorbance at 232 nm³

Induction Period⁴

Time (days)

(h)

¹values are expressed as means of triplicate determinations; ²Total concentration of phenolics for each run was 100 mg gallic acid equivalent/kg oil and the control was free from any herbal extracts or synthetic antioxidants. ³Results of oven test. ⁴Results of Rancimat test.

(x₁, x₂, x₃)

Thyme (x₁)

(1,0,0)

0.76

2.44

6.59

17.5

5.40

5.52

6.08

8.26

Oregano (x₂)

(0,1,0)

1.01

4.70

19.8

29.0

5.40

5.76

7.20

10.2

8.03

Rosemary (x₃)

(0,0,1)

0.87

2.43

3.93

5.12

5.34

5.49

5.71

6.21

10.8

x₁ + x₂

(0.5, 0.5, 0)

0.55

2.70

14.2

20.7

5.37

5.64

7.35

8.77

8.37

x₁ + x₃

(0.5, 0, 0.5)

0.37

2.92

4.92

6.37

5.40

5.69

5.86

6.48

9.27

x₂ + x₃

(0, 0.5, 0.5)

0.92

2.97

5.06

6.38

5.41

6.15

5.95

6.61

9.03

x₁ + x₂ + x₃

(0.33, 0.33, 0.33)

0.40

2.88

4.56

5.69

5.21

5.51

5.59

6.07

8.94

Control

(0,0,0)

0.45

2.41

21.1

32.6

5.42

5.78

7.33

10.8

7.82

**Table 5.** Variance analysis of regression models fitted to experimental designs of hydroperoxide content and absorbance at 232 nm
Source of variation (sum of squares)	Responses
*If F calculated is three-fold higher than F tabulated, the model may be used for prediction purposes.
Regression	1,617.548	45.59242	9.98347
Residual	144.284	4.40783	0.07705
Total	1,761.32	50.00025	10.06052
F_calc	26.15872	24.13484	151.1666
F_tab	2.85	2.85	3.87
R²	0.9181	0.9118	0.9923

Table 5. Variance analysis of regression models fitted to experimental designs of hydroperoxide content and absorbance at 232 nm

Source of variation (sum of squares)

Responses

Hydroperoxide content

Absorbance (232 nm)

Rancimat test

*If F calculated is three-fold higher than F tabulated, the model may be used for prediction purposes.

Regression

1,617.548

45.59242

9.98347

Residual

144.284

4.40783

0.07705

Total

1,761.32

50.00025

10.06052

F_calc

26.15872

24.13484

151.1666

F_tab

2.85

3.87

R²

0.9181

0.9118

0.9923

**Table 6.** Special cubic polynomial models of the synergistic effects of mixtures containing phenolic extracts of thyme, oregano and rosemary
Hydroperoxide content = 17.48 × Thyme + 29.02 × Oregano + 5.12 × Rosemary − 10.30 × Thyme × Oregano − 19.70 × Thyme × Rosemary − 42.76 × Oregano × Rosemary − 92.54 × Thyme × Oregano × Rosemary
Absorbance at 232 nm = 8.26 × Thyme + 10.23 × Oregano + 6.20 × Rosemary − 1.89 × Thyme × Oregano − 3.01 × Oregano × Rosemary − 6.45 × Oregano × Rosemary − 24.16 × Thyme × Oregano × Rosemary
Induction Period = 8.26 × Thyme + 8.03 × Oregano + 10.75 × Rosemary + 0.89 × Thyme × Orenago − 0.94 × Thyme × Rosemary − 1.44 × Oregano × Rosemary + 2.46 × Thyme × Oregano × Rosemary

Table 6. Special cubic polynomial models of the synergistic effects of mixtures containing phenolic extracts of thyme, oregano and rosemary

Hydroperoxide content = 17.48 × Thyme + 29.02 × Oregano + 5.12 × Rosemary − 10.30 × Thyme × Oregano − 19.70 × Thyme × Rosemary − 42.76 × Oregano × Rosemary − 92.54 × Thyme × Oregano × Rosemary

Absorbance at 232 nm = 8.26 × Thyme + 10.23 × Oregano + 6.20 × Rosemary − 1.89 × Thyme × Oregano − 3.01 × Oregano × Rosemary − 6.45 × Oregano × Rosemary − 24.16 × Thyme × Oregano × Rosemary

Induction Period = 8.26 × Thyme + 8.03 × Oregano + 10.75 × Rosemary + 0.89 × Thyme × Orenago − 0.94 × Thyme × Rosemary − 1.44 × Oregano × Rosemary + 2.46 × Thyme × Oregano × Rosemary