Optimizing the Extraction of Catechin from Peanut Red Skin Using Response Surface Methodology and its Antioxidant Activity

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ScienceDirect IERI Procedia 5 (2013) 312 – 320

2013 International Conference on Agricultural and Natural Resources Engineering

Optimizing the Extraction of Catechin from Peanut Red Skin Using Response Surface Methodology and its Antioxidant Activity Haiyue Zhang a, *, Man Liua, Shengting Hana, Ying Weia a

College of Chemical and Life Science, Changchun University of Technology, Changchun 130012, PR China.

Abstract Catechin was rich in the peanut red skin. In this paper, response surface methodology was used to optimize the catechin from peanut red skin extraction conditions. The results showed that the optimal conditions were the ethanol concentration 78%, the ratio of material to liquid 1:17, extraction temperature43 and extraction time 1.5h. The ultraviolet spectrum and infrared spectrum analysis was used to determine that there was catechin in the extract. And the superoxide anion (O2 .) inhibition rate of the catechin was 42.50% through the pyrogallol autoxidation - spectrophotometric method which indicated that the catechin had certain antioxidant ability.

© 2013The Published by Elsevier B.V. B.V. © 2013 Authors. Published by Elsevier Selection and review under responsibility of Engineering Information Engineering Selection and peerpeer review under responsibility of Information Research Institute Research Institute Key word: Peanut red skin, Catechin, Response surface methodology, Antioxidant activity

1. Introduction Peanut red skins are the seed coat of peanut. Peanut resources are extremely rich in China which is the country of the highest peanut production and the largest export volume, accounting for 1/3 of the world's total [1]. Thus the use of peanut red skins which are the by-products of peanut protein and peanut butter manufacturing to extract the natural active ingredients have good economic returns and far-reaching * Corresponding author. Tel.: +86-156-3055-8239; fax:+86-431-8593-6362 E-mail address: [email protected]

2212-6678 © 2013 The Authors. Published by Elsevier B.V. Selection and peer review under responsibility of Information Engineering Research Institute doi:10.1016/j.ieri.2013.11.109

Haiyue Zhang et al. / IERI Procedia 5 (2013) 312 – 320

significance. Peanut red skins contain high concentrations of natural antioxidants which can be extracted and potentially utilized in a variety of food and pharmaceutical applications [2-4]. Catechin is alkanols derivative. It is colorless crystalline solid and could be dissolved in water. Its aqueous solution could polymerizate into amorphous tannin easily under the conditions of heating and the presence of inorganic acids [5-7]. It can be very important in improving the body’s immune function, clearing the body excess free radicals and strong antioxidant capacity. High catechin concentration would evidently injure red cell membrane at the present of iron. Catechin possesses a powerful ability to chelat with iron [8]. Response surface methodology (RSM) is a kind of experimental design and optimization methods. RSM can be effectively used to find the combination of factor levels that produce an optimum response [9]. In the paper, response surface methodology was used to handle the data and it provided the theoretical basis for extracting catechin from peanut skins. The catechin extraction rate was used to choose the optimal parameters. 2. Materials and Methods 2.1. Materials and Instruments Peanut red skins were obtained from Hongbaolai Company (Siping, China). The skins were stored in a freezer at -4 °C in sealed plastic bags until analysis. Electronic balance (Sartorius, Beijing, China); SHA-C water bath oscillator (Hengfeng, Jiangsu, China); Blast Oven (Jinghong, Shanghai, China); UV-9l00 spectrophotometer (Ruili, Beijing, China). Ethanol, citric acid and sodium dihydrogen phosphate are AR. The purity of cathchin standard (Beiyanxinlv, Beijing, China) is more than 98%. 2.2. Methods 2.2.1 Extract process Peanut red skin Ethanol reflux extract Filter Concentrated under vacuum Drying Crude extracts 2.2.2 The determination of maximum absorption wavelength Five g peanut red skins were weighed and were put into Erlenmeyer flask. Some 70% ethanol was added into it which was in constant temperature water bath at 50 . After a certain time, filtered and collected the filtrate. 200nm-800nm wavelength scanning was carried out with citric acid-disodium hydrogen phosphate (pH3.0) as a buffer blank [10]. 2.2.3 The drawing of catechin standard curve Two mg catechin standard product was weighed accurately and was placed in a 50 mL volumetric flask. Anhydrous ethanol was added to volume to get the catechin standard solution of 0.004mg/mL. 0mL, 2mL, 4mL, 6mL, 8mL and 10mL that standard solution was removed respectively with a pipette into 10mL volumetric flask and then anhydrous ethanol was added to volume to obtain a certain concentration gradient solution. The above solution was placed in UV-Vis spectrophotometer to measure the absorbance at the maximum wavelength. The data was recorded and the standard curve was drawn with software (origin7.5). 2.2.4 Choice of the extraction solvent Each part 10 g peanut skins were weighed accurately and were extracted of 100mL distilled water, anhydrous ethanol, acetone, ethyl acetate and chloroform for 10h at room temperature. The filter was placed in UV-Vis spectrophotometer to measure the absorbance at the maximum wavelength.

313

314

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Extraction rate=

X V m

(1)

X-The concentration of catechin in filter V-The volume of filter m-The weight of the peanut red skin 2.2.5 Single factor test The ethanol concentration, solid-liquid ratio, extraction temperature and extraction time was determined. The ethanol concentration was 50%, 60%, 70%, 80% and 90%. The solid-liquid ratio was 1:10, 1:15, 1:20, 1:25 and 1:30. The extraction temperature was 30 , 40 , 50 , 60 and 70 . The extraction time was 0.5h, 1.0h, 1.5h, 2.0h and 2.5h. 2.2.6 Response surface methodology design Table 1 Levels of factors in response surface analysis

Levels

Factors A (%)

B(mL/g)

C( )

D (h)

-1

60

1:15

40

1.0

0

70

1:20

50

1.5

1

80

1:25

60

2.0

Box-Benhnken of Design-Expert 8.0 was used to analyze the data. The analysis was designed that the ethanol concentration (A), liquid- solid ratio (B), extraction temperature (C) and extraction time (D) as independent variables and catechin extraction rate as response value combining the results of single factor test. The levels of factors were shown in Table 1. 2.2.7 Infrared spectrum One mg extracts mixing with a little KBr was pressed and was carried scanning in the range of 4000cm-1400cm-1. 2.2.8 The determination of the antioxidant ability Fifty mmol/L Tris-HCl buffer solution (pH8.2) 4.5 mL mixing with distilled water 4.2 mL was put in 25 water bath for 20 min. Removed and joined with pyrogallol solution 0.3 mL which had been preheated in 25 water bath. Absorbance was measured every 30s under 320nm. The steps above were followed. Just 0.1mL catethin extract was put before the pyrogallol. Inhibition rate (%) = ( A1/ t -

A2/ t)/ A1/ t*100%

A1/ t—the reaction rate of the pyrogallol autoxidation A2/ t—the reaction rate of the pyrogallol autoxidation after the extract was put

(2)

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3. Results and Discussion 3.1. The determination of maximum absorption wavelength 5

1.2 4

Y = 167.26X + 0.0261 2 R = 0.9954

1.0 281 3

0.8 0.6

2

0.4 1

0.2 0

200

0.0 300

400

500

600

700

800

Fig.1 Ultraviolet spectrogram of peanut red skin extract

0.000

0.001

0.002

0.003

0.004

0.005

0.006

Fig.2 Catechin standard curve

Fig.1 showed that the peanut red skin extract had a maximum absorption peak at near 280nm. The previous literature suggested that the catechin maximum absorbance was 280nm [11, 12]. The result was compliant with it. 3.2. Catechin standard curve The standard curve was drawn according to the data at the maximum absorption wavelength of different concentrations of standard catechin solutions. The relational expression of the concentration and absorbance was Y =167.26X+0.0261, R2=0.9954 which was showed in Fig.2. The extraction rate of catechin was calculated according to the curve. 3.3. Choice of the extraction solvents The Table 2 showed that the extraction rate with ethanol was the optimal, so ethanol was chose as extraction solvent. Table2. Different extraction rates of different extraction solvents Extraction solvents

Extraction rate (%)

Distilled water

1.02

Anhydrous ethanol

1.61

Acetone

0.99

Ethyl acetate

0.37

Chloroform

0.72

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3.4. The results of single factor tests 1.70

1.74 1.80

1.72

1.72

1.78

1.68

1.70

1.76

1.70

1.74

1.66

1.68

1.72

1.68

1.70

1.64

1.66

1.68

1.66

1.66 1.64

1.62

1.64

1.64

1.62 1.60

1.62 50

60

70

80

90

1.60

1.62

30

40

50

60

70

1:10

1:15

1:20

1:25

1:30

0.5

1.0

1.5

2.0

2.5

Fig. 3 (1) Effects of ethanol concentration on extraction rate (2) Effects of temperature on extraction rate (3) Effects of solid-liquid ratio on extraction rate (4) Effects of extraction time on extraction rate

These figures showed obviously that the optimal ethanol concentration was 70%, the optimal temperature was 50 , the optimal solid-liquid ratio was 1:20 and the optimal extraction time was 1.5h. These results provided the data infrastructure for the response surface methodology design. 3.5. The determination of optimal parameters with the response surface methodology 3.5.1 Response surface methodology design and results The analysis was designed that the ethanol concentration (A), liquid-solid ratio (B), extraction temperature (C) and extraction time (D) as independent variables and catechin extraction rate as response value combining the results of single factor test. The result was as table 3. Table3. Results of response surface design No.

A (%)

B (mL/g)

C( )

D (h)

Extraction rate (%)

1 2 3

70 70 70

25 25 15

60 50 50

1.5 2 2

1.89 1.87 1.79

4

70

20

40

1

1.71

5

70

25

40

1.5

1.83

6 7 8 9 10

80 60 70 70 70

20 20 20 15 20

40 50 50 60 50

1.5 2 1.5 1.5 1.5

1.83 1.85 2.21 1.82 2.21

11

70

20

50

1.5

2.20

12 13 14

60 80 60

25 20 15

60 50 50

1.5 1 1.5

1.82 1.85 1.69

15 16 17 18

70 60 70 70

15 20 20 20

40 40 50 40

1.5 1.5 1.5 2

1.71 1.68 2.21 1.83

317

Haiyue Zhang et al. / IERI Procedia 5 (2013) 312 – 320 19 20

70 70

20 15

50 50

1.5 1

2.21 1.72

21

60

20

60

1.5

1.84

22 23 24

80 70 70

15 20 20

50 60 60

1.5 2 1

1.82 1.92 1.83

25

80

20

60

1.5

1.91

26 27

60 70

20 25

50 50

1 1

1.73 1.79

28

80

25

50

1.5

1.92

29

80

20

50

2

1.85

The data in the Table 3 was analyzed by using the Design Expert software. After fitting, the quadratic regression equation of A, B, C, D was as follows: Catechin extraction rate = +2.21+0.048*A+0.047*B+0.052*C+0.040*D-7.500E-0.003*A*B-0.020*A*C-0.030*A*D0.013*B*C+2.500E-003*B*D-7.500E-003*C*D-0.19*A2-0.21*B2-0.19*C2-0.20*D2 The quadratic terms A2, B2, C2 and D2of that model was extremely significant. The interaction term AD was significant. The F value was 142.86. All these could be shown obviously in Table 4. The significant level p of the model was less than 0.0001 which indicated that the model was significant. The R2 and R2 (Adj) were 0.9930 and 0.9861 respectively which showed that the fitting degree of regression equation was well. So the regression equation was used for analysis instead of the real test point. The response surface map was drawn according to the mathematical model which was showed in Fig.4. Table4. Variance analysis of regression model

a

Source

Sum of Squares

df

Mean Square

F Value

P Value

Significance

Model A-Ethanol concentration

0.76 0.027

14 1

0.054 0.027

142.86 71.23

<0.0001 <0.0001

** **

B-Liquid-solid rate

0.027

1

0.027

71.23

<0.0001

**

C- Extraction temperature

0.032

1

0.032

84.27

<0.0001

**

D- Extraction time

0.019

1

0.019

50.51

<0.0001

**

AB AC AD BC

2.250E-004 1.600E-003 3.600E-003 6.250E-004

1 1 1 1

2.250E-004 1.600E-003 3.600E-003 6.250E-004

0.59 4.21 9.47 1.64

0.4545 0.0594 0.0082 0.2206

BD

2.500E-005

1

2.500E-005

0.066

0.8013

CD A2 B2

2.250E-004 0.24 0.28

1 1 1

2.250E-004 0.24 0.28

0.59 633.98 736.50

0.4545 <0.0001 <0.0001

** **

C2 D2

0.24 0.26

1 1

0.24 0.26

625.79 675.77

<0.0001 <0.0001

** **

Residual

5.322E-003

14

3.801E-004

Lack of Fit Pure Error Cor Total

5.242E-003 8.000E-005 0.77

10 4 28

5.242E-004 2.000E-005

26.21

0.0033

*

Notes: “*” represented p<0.05, “**” represented p<0.0001.

*

Haiyue Zhang et al. / IERI Procedia 5 (2013) 312 – 320

2.23

2.23

2.0925

2.095

1.96

1.825

1.69

25.00

1.955

1.8175

1.68

60.00

80.00 22.50 65.00 15.00

2.23

2.00

40.00

A

60.00

1.825

1.69

60.00

1.71

2.00

D

B

15.00

A

60.00

1.97

1.84

1.71

2.00

25.00 1.75

60.00 1.75

22.50 1.50

17.50 40.00

1.00

2.1

20.00 45.00

C

65.00

2.23

1.84

22.50 50.00

70.00 1.25

D

1.97

25.00 55.00

75.00 1.50

Extraction rate

1.96

80.00 1.75

2.1

Extraction rate

Extraction rate

65.00

2.23

2.095

1.69

70.00 45.00

C

A

60.00

1.96

1.825

75.00 50.00

70.00 17.50

B

80.00 55.00

75.00 20.00

Extraction rate

2.23

2.095

Extraction rate

Extraction rate

318

17.50 1.00

15.00

55.00 1.50

20.00 1.25

D

B

50.00 1.25

45.00 1.00

C

40.00

Fig. 4 Response surface plot and contour diagram for alternative effects of various factors on catechin extraction from peanut red skin

The response surface slope reflected the interaction of the various factors. The gentle and steepness of the response surface slope directly reflected the response sensitivity level the catechin extraction rate when the extraction conditions were changed. The response was slow when the slope was gentle, so did the opposite. Fig.4 indicated that the interactive effect of the ethanol concentration and extraction time influenced the catechin extraction rate significantly. 3.5.2 The optimization of conditions The boundary values and extreme values were analyzed. The optimal extraction conditions were A=77.62(%), B=16.67(mL/g), C=43.38( ), D=1.49(h). The extraction conditions were amended to the condition that was A=78(%), B=17(mL/g), C=43( ), D=1.5(h). The verification test was conducted as this condition for 3 times. The result indicated the theoretical optimal value was higher than the actual optimal value. 3.6. Infrared spectrum results 88

912

2.0

86

1.8

84

1.6

1417

82

1.2

78

Abs( A)

Transmittance( %

)

1.4

80

1264 1114

76 74

4000

0.8 0.6

1617

0.4

72 70

1.0

0.2

3400 3500

3000

2500

2000

1500

1000

-1 Wavenumber( cm )

Fig. 5 Infrared spectra of peanut red skin extract

500

0

0.0 -20

0

20

40

60

80

100

120

140

160

180

200

Time( S)

Fig. 6 Catechin antioxidant function diagram

Fig.5 showed that at near 1600-1450 cm-1 there were two absorption bands. This group of bands and the absorption band of aromatic ring Ar-H stretching vibration provided an important basis for judging the

Haiyue Zhang et al. / IERI Procedia 5 (2013) 312 – 320

compounds had aromatic ring or not [13]. In the range of 3250-3500 cm-1 were vibration absorption peaks of –OH and at near 1230 cm-1 indicated C=O [14]. The extract had aromatic ring, -OH and C=O through infrared spectrum analysis. These were consistent with the catechin structure. 3.7. Antioxidant experiments results The scavenging results of the catechin extract to superoxide anion radical (O 2 - .) was showed in Fig. 6. It displayed the changes in absorbance with the change of time. The absorbance was becoming smaller obviously when joined with the extract. The inhibition of catechin extract to O 2 - . was 42.50% through calculating. The results indicated it had certain antioxidant ability. 4. Conclusions According to the central composite design principle, the response surface analysis was used to get the optimal extraction. The results were the ethanol concentration 78%, the ratio of material to liquid 1:17, extraction temperature 43 and extraction time 1.5h. The inhibition of catechin extract to superoxide anion radical (O2·-) was 42.50%. Because the certain content of catechin has the promotion to iron supplementation, this experiment provides the basis to following work for developing the enrich blood oral of peanut red skin. As it is biological active substance, we should also study its stability. Acknowledgements This work was financially supported by the Jilin Province pharmaceutical industry development foundation (YYZX201130). References [1] J. Chen, H. L. Xu, Z. W. Fang. Peanut skins research and development applications, J. Agricultural Sciences of Guangzhou. 2007; 8 80-81. [2] M. S. Li, K. Yao, D. Y. Jia. Peanut functional component and comprehensive utilization, J. China oil and fats. 2004; 9: 13-14. [3] Q. L. Wang, J. Dong, H. Ji. Research progress of peanut skins proanthocyanidins, J. Food research and development. 2011; 4: 184-186. [4] S. Yi. Peanut skin physiological activity, J. Food and Ferment. 2001; 4: 44-45. [5] S. L. Liu, Z. W. Liu, P. Q. Lu, Y. Zhang, J. D. Zhang, D. H. Jia, Y. O. Yao, Z. B. Cao. Protective effects of catechin on cerebral ischemia-reperfusion injury in rats and its mechanism, J. Chinese Pharmacological Bulletin. 2010; 2: 121-123. [6] X. J. He, Z. W. Yi. Effect of catechin on serum free radical and endogenous antioxidease in nephritic rats, J. Chinese Journal of Nephrology, Dialysis & Transplantation. 2002; 4: 47-51. [7] H. X. Li. Study on the Extraction and Purification of Catechins and the Separation and Preparation of Epigallocatechin Gallate by Resins, J. Hefei University of Technology. 2006; 8. [8] S. R. Shen, Y. F. Zhao, B. L. Zhao. Inhibiting effect of catechins to free radicals at the present iron, J. Tea Science. 1997; 17: 119-123. [9] T. S. Ballard, P. Mallikarjunan, K. Zhou, S. f. O, Keefe. Optimizing the extraction of phenolic antioxidants from peanut skins using response surface methodology, J. Journal of agriculture and food chemistry. 2009; 57:

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