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BY 4.0 license Open Access Published by De Gruyter Open Access May 8, 2023

Optimization of ultra-high pressure-assisted extraction of total phenols from Eucommia ulmoides leaves by response surface methodology

  • Xiao-yan Ren EMAIL logo , Xue-yuan Jin and Wei Zong
From the journal Open Chemistry

Abstract

The ultra-high pressure-assisted extraction (UHPE) of total phenols from Eucommia ulmoides leaves (TPEU) was optimized and its antioxidant activity on Eucommia ulmoides seed oil was studied. The effects of UHPE pressure, UHPE time, and ethanol concentration on the extraction yield of TPEU were examined by response surface methodology. In addition, TPEU was added to Eucommia ulmoides seed oil, and the effects of TPEU on the antioxidant activity (acid value, peroxide value) of Eucommia ulmoides seed oil during storage were studied. The optimal UHPE conditions were as follows: UHPE pressure, 406 MPa; UHPE time, 8.3 min; and ethanol concentration, 60.2%. Under these UHPE conditions, the yield of TPEU was 7.58%. TPEU had a good antioxidant effect when the TPEU content was 0.06%. The antioxidant effect of TPEU was lower than that of BHA. Both TPEU and BHA have a synergistic effect.

1 Introduction

Eucommia ulmoides (Eucommia ulmoides Oliver), also known as Bakelite, comes from the Eucommia family plant [1,2]. Eucommia ulmoides leaves (EUL) are the dry leaves of Eucommia ulmoides. In China, EUL are distributed in Shanxi, Gansu, Zhejiang, Henan, Hubei, Sichuan, Guizhou, Yunnan, and other places [3,4]. Modern pharmaceutical research found that the active components of EUL have the functions of neuroprotective effect [5], lipid-lowering activity [6], aldose reductase inhibitory activity [7], antimicrobial activity [8,9], and anti-gastric ulcer activity [10]. The chemical composition of EUL contains phenolic compounds [11], flavonoids [12], and polysaccharides [13]. The phenols of EUL (TPEU) are one of its main active ingredients [14]. At present, many extraction methods have been applied in the extraction of plant extracts [15,16,17], such as ionic liquids’ extraction [18] and refluxing extraction [19]. Among these extraction methods, refluxing extraction is the main extraction method of total phenols for its simplicity; however, the refluxing extraction takes a long time and it can destroy TPEU. Ultra-high pressure-assisted extraction (UHPE) has been used in the extraction of active compounds from natural resources [20]. It is faster and has little damage to the active ingredient.

Eucommia ulmoides seed oil is extracted from Eucommia ulmoides seed; the unsaturated fat content in Eucommia ulmoides seed oil was 91.18% [21]. Because of its unique lipid-lowering and anti-aging effect on the human body, it has aroused extensive attention. However, as it oxidizes easily, it is necessary to add 2-tert-butyl-4-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT), and other antioxidants [22]. However, in recent years, the use of natural antioxidants in oil is of more concern [23]; polyphenols are natural compounds with strong antioxidant properties and are used in food antioxidant applications, etc. [24,25]. Therefore, the extraction of TPEU from EUL and its application in the anti-oxidation of oil are of great significance.

In this study, the UHPE conditions of TPEU from EUL were optimized using response surface methodology (RSM) and used in the anti-oxidation of Eucommia ulmoides seed oil.

2 Experimental section

2.1 Materials

EUL harvested from Zhengzhou of China in September 2022 were used. Samples were dried at 60°C using a vacuum dryer (DHG9030A; Yiheng Lab Co, China) until the moisture was 5%. Dried samples were crushed into 80 mesh pieces. The powder sample was stored in sealed jars at −18°C. Eucommia ulmoides seed oil (EUSO) was prepared by a press device (ZY28; Daxiang Co, China) and was provided by the Peron Company (Zibo, China). Folin–Ciocalteu reagent was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). BHA, BHT, gallic acid, potassium iodide, sodium thiosulfate, and ethanol were of analytical purity, and purchased from the National Pharmaceutical Group Co Ltd (Shanghai, China).

2.2 UHPE process

An ultra-high pressure treatment device (2 L; Kefa Ltd., Baotou, China) (Figure 1) was used for the UHPE process. EUL samples (20 g each) were weighed, mixed with 500 mL of ethanol solution, and then placed in the high-pressure chamber of the device for extraction. The UHPE parameters were controlled through the control panel. The high pressure (100–600 MPa) is produced to the high-pressure oil by the high-pressure pump and is conducted to the high-pressure chamber through the high-pressure oil pipeline. The UHPE process was conducted at different UHPE pressures, different UHPE times, and different ethanol concentrations. After extraction, the solution was centrifuged at 5,000 rpm for 20 min using a centrifugal machine (H1850; Hunan Xiangyi Ltd., Changsha, China). The supernatant was collected and concentrated in a vacuum concentrator (R5003KE; Taikang Ltd., Xian, China) to obtain the extract. The total phenols in the extract were determined according to the Folin–Ciocalteu method as described by Mechikova et al. [26] with slight modification. The yield of TPEU was expressed as follows:

Y ( % ) = m W × 100 % ,

where W is the weight (mg) of EUL and m is the weight (mg) of TPEU (mg).

Figure 1 
                  Device of ultra-high-pressure treatment.
Figure 1

Device of ultra-high-pressure treatment.

2.3 Signal test

2.3.1 Effect of UHPE pressure on the yield of TPEU

The yield of TPEU was taken as an index; the ethanol concentration was 60%, the UHPE time was 8 min, and the pressure was changed from 100 to 600 MPa. The effect of UHPE pressure on the yield of TPEU was studied.

2.3.2 Effect of UHPE time on the yield of TPEU

The yield of TPEU was taken as an index; the ethanol concentration was 60%, the UHPE pressure was 400 MPa, and the UHPE time was changed from 0 to 10 min. The effect of UHPE time on the yield of TPEU was studied.

2.3.3 Effect of ethanol concentration on the yield of TPEU

The yield of TPEU was taken as an index; the UHPE pressure was 400 MPa, the UHPE time was 8 min, and the ethanol concentration was changed from 40 to 65%. The effect of ethanol concentration on the yield of TPEU was studied.

2.4 RSM test

On the basis of a single-factor test, the UHPE pressure, UHPE time, and ethanol concentration were selected as the variables, and the TPEU yield was taken as the response value. The TPEU yield was optimized using the Box–Behnken design. The levels and codes are shown in Table 1.

Table 1

Levels and code of extraction variables used in the Box–Behnken design

Variable Symbols Coded levels
Coded −1 0 1
UHPE pressure (MPa) A 380 400 420
UHPE time (min) B 7 8 9
Ethanol concentration (%) C 58 60 62

2.5 Refluxing extraction

EUL samples (20 g each) were weighed, mixed with 500 mL ethanol solution, and then were placed in a 2,000 mL flask for refluxing extraction at 80°C for 2 h. After extraction, the solution was centrifuged at 5,000 rpm for 20 min using a centrifugal machine (H1850; Hunan Xiangyi Ltd., Changsha, China). The supernatant was collected and concentrated in a vacuum concentrator (R5003KE; Taikang Ltd., Xian, China) to obtain the extract. The total phenols were determined according to the Folin–Ciocalteu method and the yield of TPEU was determined.

2.6 Effect of TPEU on the oxidative stability of EUSO

TPEU (0.03, 0.06, and 0.09%) samples were fully dissolved in cones containing 100 g of EUSO in bottles and placed in an oven set to (60 ± 2)°C. To the blank EUSO and samples, 0.06% BHA was added as a control; the peroxide value and acid value of EUSO were measured every 3 days, and the oxidative deterioration of EUSO was observed within 25 days.

2.7 Determination of the peroxide value

The peroxide value was determined according to the AOAC official method 965.33 peroxide value of oils and fats.

2.8 Determination of the acid value

The acid value was determined according to the AOAC official method cd3d-63 acid value.

2.9 Statistical analysis

All data in the test were expressed as mean ± standard deviation (n = 3). P < 0.05 was considered a significant difference. RSM was performed using the software Design-Expert 8.0.6.

3 Results and discussion

3.1 Effect of UHPE pressure on yield of TPEU

The effect of UHPE pressure on the yield of TPEU is shown in Figure 2.

Figure 2 
                  Effect of UHPE pressure on the yield of TPEU (n = 3).
Figure 2

Effect of UHPE pressure on the yield of TPEU (n = 3).

As can be seen from Figure 2, when the UHPE pressure increased from 100 to 600 MPa, the yield of TPEU increased first and then decreased, and reached the maximum at a UHPE pressure of 400 MPa. In the UHPE process, the extraction process can be divided into three stages: pressure increase, pressure holding, and pressure unloading. During the pressure increase stage, the pressure inside the cell is much lower than that outside the cell. The higher the pressure, the higher the rate of solvent permeation. In the pressure holding stage, the extracellular solvent continues to enter the cell at a high rate of penetration, and TPEU were dissolved in the solvent. In the pressure unloading stage, the pressure inside the cell is much higher than that outside the cell. The cell breaks down and the TPEU are released. The higher the pressure, the greater the degree of cell rupture. But high pressure also leads to the release of impurities into the cell, affecting the release of TPEU. He et al. [27] used UHPE for extracting total phenolics from deodeok (Codonopsis lanceolata). They found that a pressure of 385 MPa gives the highest total phenol yield. It is similar to the optimal pressure in this study. Therefore, 400 MPa is chosen as the UHPE pressure factor (code level 0) of the RSM test.

3.2 Effect of UHPE time on the yield of TPEU

The effect of UHPE time on the yield of TPEU is shown in Figure 3.

Figure 3 
                  Effect of UHPE time on the yield of TPEU (n = 3).
Figure 3

Effect of UHPE time on the yield of TPEU (n = 3).

As can be seen from Figure 3, when the UHPE time increased from 0 to 10 min, the yield of TPEU increased with an increase in time. At a UHPE time of 8 min, TPEU reached the maximum. But after the UHPE time of 8 min, with the UHPE time prolonged, the TPEU increase was not significant (P > 0.05). This is because, in the pressure holding stage, it takes a certain time for the solvent to diffuse into the cell and dissolve the TPEU. If the UHPE time is too short, it is difficult for the solvent to diffuse into the cell and dissolve the TPEU completely. Alexandre et al. [28] used UHPE for extracting total phenols from pomegranate peels. They also observed that the higher the pressure time, the more solvent can enter the cell and more compounds can permeate the cell membrane. Therefore, 8 min was chosen as the UHPE time factor (code level 0) of the RSM test.

3.3 Effect of ethanol concentration on the yield of TPEU

The effect of ethanol concentration on the yield of TPEU is shown in Figure 4.

Figure 4 
                  Effect of ethanol concentration on the yield of TPEU (n = 3).
Figure 4

Effect of ethanol concentration on the yield of TPEU (n = 3).

As can be seen from Figure 4, when the ethanol concentration increased from 40 to 65%, the yield of TPEU increased first and then decreased, and reached a maximum at an ethanol concentration of 60%. The reason is that the polarity of 60% ethanol is similar to that of TPEU, and the yield of TPEU is the highest under the condition of similar solubility. With the increase in the concentration of ethanol, the polarity of the solution decreased and the solubility of TPEU decreased. Li et al. [29] extracted TPEU from EUL. They also found that the ethanol concentration had a significant effect on the TPEU yield. Therefore, 60% is chosen as the ethanol concentration factor (code level 0) of the RSM test.

3.4 RSM test

On the basis of the single-factor test, the RSM test was carried out according to the factors and levels given in Table 1. The results are shown in Table 2.

Table 2

RSM test plan and results

Test number Coded variable levels Y TPEU (%)
A: UHPE pressure (MPa) B: UHPE time (min) C: ethanol concentration (%)
1 −1 0 1 5.31
2 −1 0 −1 5.43
3 1 1 0 6.48
4 −1 1 0 6.36
5 0 −1 −1 6.51
6 1 −1 0 6.81
7 0 1 −1 6.43
8 0 0 0 7.68
9 0 0 0 7.52
10 0 0 0 7.26
11 1 0 1 6.98
12 0 1 1 6.41
13 0 0 0 7.70
14 −1 −1 0 6.47
15 0 −1 1 6.90
16 1 0 −1 6.62
17 0 0 0 7.38

The response factors in Table 2 were fitted by multiple regression, and the quadratic regression equation is obtained as follows:

Y TPEU = 7.51 + 0.42 A 0.13 B + 0.076 C 0.055 A B + 0.12 A C 0.10 B C 0.73 A 2 0.256 B 2 0.70 C 2

Here, Y is the yield of TPEU (%), and A, B, and C are the coded values of the UHPE pressure, UHPE time, and ethanol concentration, respectively.

ANOVA for the RSM quadratic model of TPEU is shown in Table 3. The model F-value of 5.92 implies that the model is significant. There is only a 1.43% chance that such a large “model F-value” could occur due to noise. Values of “Prob > F” < 0.0500 indicate model terms are significant. In this case, A, A 2, and C 2 are significant model terms. For “lack of fit,” values of “Prob > F” > 0.0500 indicate that model terms are not significant. A negative “Pred R-Squared” implies that the overall mean is a better predictor of response than the current model. “Adeq Precision” measures the signal-to-noise ratio, and a ratio greater than 4 is desirable. A ratio equal to 6.956 indicates an adequate signal. This model can be used to navigate the design space. From the F value, the order of the influencing factors was water UHPE pressure > UHPE time > ethanol concentration.

Table 3

ANOVA for the response surface quadratic model for the yield of polysaccharides

Source SS Df MS F value P value (Prob > F)
Model 6.64 9 0.74 5.92 0.0143
A 1.38 1 1.38 11.05 0.0127
B 0.13 1 0.13 1.02 0.3455
C 0.047 1 0.047 0.37 0.5606
AB 0.012 1 0.012 0.097 0.7645
AC 0.058 1 0.058 0.46 0.5185
BC 0.042 1 0.042 0.34 0.5797
A 2 2.24 1 2.24 17.94 0.0039
B 2 0.27 1 0.27 2.13 0.1876
C 2 2.04 1 2.04 16.37 0.0049
Residual 0.87 7 0.12
Lack of fit 0.72 3 0.24 6.51 0.015
Pure error 0.15 4 0.037
Total variation 7.51 16
R 2 0.8839
Adj. R 2 0.7345
Pre. R 2 −0.5732
Adeq. precision 6.956
CV (%) 5.25

Note: P < 0.05 was considered significant.

According to Figure 5a and b, the yield of TPEU increased first and then decreased with the increase of the UHPE pressure and UHPE time, indicating that the interaction between the UHPE pressure and UHPE time is significant. According to Figure 5c and d, the yield of TPEU increased first and then decreased with the increase of UHPE time and ethanol concentration, indicating that the interaction between UHPE time and ethanol concentration is significant. According to Figure 5e and f, the yield of TPEU increased first and then decreased with the increase of UHPE pressure and ethanol concentration, indicating that the interaction between the UHPE pressure and ethanol concentration is significant.

Figure 5 
                  Response surface and contour map of the effects of factor interaction on the yield of TPEU. (a) Response surface map of A to B, (b) countour map of A to B, (c) response surface map of C to B, (d) countour map of C to B, (e) response surface map of C to A, (f) countour map of C to A.
Figure 5

Response surface and contour map of the effects of factor interaction on the yield of TPEU. (a) Response surface map of A to B, (b) countour map of A to B, (c) response surface map of C to B, (d) countour map of C to B, (e) response surface map of C to A, (f) countour map of C to A.

The optimum extraction conditions of TPEU when the response value was 7.60%, obtained by RSM, were as follows: UHPE pressure, 406.1 MPa; UHPE time, 8.29 min; and ethanol concentration, 60.21%. In order to facilitate the operation of the process in practical application, the UHPE pressure, the UHPE time, and ethanol concentration were adjusted to 406 MPa, 8.3 min, and 60.2%, respectively. On this basis, the yield of TPEU was 7.58% in three parallel and repeated experiments. It was shown that the model could effectively predict the yield of TPEU.

3.5 Comparison of different extraction methods

The EUL were extracted under the optimum conditions of UHPE and compared with the refluxing extraction method. The results are shown in Table 4.

Table 4

Comparison of different extraction methods

Method Y TPEU (%) Time (min)
UHPE 7.58 8.3
Refluxing extraction 6.82 120

The results show that UHPE has a shorter time and higher extraction yield compared with the refluxing extraction method, so it is a suitable method for extracting TPEU.

3.6 Effect of TPEU on the oxidative stability of EUSO

The effects of TPEU on the peroxide value and acid value of EUSO are shown in Figure 6.

Figure 6 
                  Effects of TPEU on the peroxide value and acid value of EUSO (different letters represent significant differences at the same time [P < 0.05]) (a: peroxide, b: acid value).
Figure 6

Effects of TPEU on the peroxide value and acid value of EUSO (different letters represent significant differences at the same time [P < 0.05]) (a: peroxide, b: acid value).

From Figure 6a and b, compared with the blank EUSO, the peroxide value and the acid value of EUSO were significantly decreased by the addition of 0.03 and 0.06% TPEU, which indicated that the addition of 0.03 and 0.06% TPEU could significantly decrease the peroxide value and acid value of EUSO; the oxidation rancidity rate of EUSO was significantly reduced by 0.03 and 0.06% TPEU (P < 0.05). But, with the increase of TPEU, the addition of 0.09% TPEU accelerated the oxidation rancidity rate of EUSO, which could be because the excessive TPEU leaves have the function of supporting oxidation. Shang et al. [30] studied the oxidative stability of Camellia seed oil by the addition of 0.04% of olive polyphenols (OP); they, however, added 0.06% of OP to control the oxidation of Camellia seed oil and found that the antioxidant activity of TPEU was similar to that of OP.

Compared with EUSO, the addition of 0.06% TPEU and 0.06% BHA significantly decreased the peroxide value and acid value of the seed oil (P < 0.05); the peroxide value and the acid value of 0.06% BHA were lower than those of TPEU, which indicated that the antioxidant effect of BHA was better than that of TPEU. However, the peroxide value of 0.03% BHA + 0.03% TPEU was higher than that of 0.06% TPEU and 0.06% BHA (P < 0.05), which indicated that TPEU had a synergistic effect with BHA. Shang et al. [30] also studied the oxidative stability of Camellia seed oil with the addition of 0.02% OP + 0.02% BHA to control the oxidation of Camellia seed oil. They found that BHA and OP had a synergistic effect. The antioxidant activity of TPEU and BHA on EUSO was similar to this. In recent years, increasing attention has been paid to the safety of antioxidants. The use of natural antioxidants is favored by consumers. As TPEU is a good antioxidant, it is expected to be widely used in food anti-oxidation.

4 Conclusion

RSM was used to optimize the extraction conditions of TPEU with UHPE. The optimum conditions were as follows: UHPE pressure, 406 MPa; UHPE time, 8.3 min; and ethanol concentration, 60.2%. Under these conditions, the yield of TPEU was 7.58%. The effect of TPEU on the oxidative stability of EUSO was studied. The results showed that TPEU had a good antioxidant effect on EUSO when the TPEU content was 0.06%, and the antioxidant effect of TPEU was lower than that of BHA. TPEU and BHA have a synergistic effect.

  1. Funding information: This study was supported by the natural science project of universities in Anhui province, China (KJ2021A1312) and the vocational education innovation development pilot zone project in Anhui province, China (WJ-RCPY-007).

  2. Author contributions: Xiao-yan Ren: conceptualization, data curation, funding acquisition, project administration, resources, writing – original draft; Xue-yuan Jin: supervision, validation; Wei Zong: software, writing - review and editing.

  3. Conflict of interest: The authors state that there was no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2023-02-02
Revised: 2023-03-13
Accepted: 2023-03-14
Published Online: 2023-05-08

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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