Chemical compositions, extraction technology, and antioxidant activity of petroleum ether extract from Abutilon theophrasti Medic. leaves

ABSTRACT

Petroleum ether extract from Abutilon theophrasti Medic. leaves was optimized by response surface methodology, and the optimal extraction conditions were as follows: ratio of solvent to material (20.12 mL/g), extraction time (5.45 h), and Soxhlet extraction temperature (61.32°C). And the yield of petroleum ether extract collected in August, September, and October was (2.05 ± 0.02)%, (2.39 ± 0.01)%, and (2.32 ± 0.02)%, respectively. The September and October extracts exhibited a better antioxidant activity, which was proved by DPPH·scavenging ability (IC₅₀ value of 327.5 and 331.5 μg/mL), ABTS^·+ scavenging ability (IC₅₀ value of 170.1 and 182.1 μg/mL), and reducing power (0.31 and 0.28 mmol Fe²⁺/100 μg/mL). Meanwhile, the gas chromatograph-mass spectrometry analysis revealed that the main antioxidant components contained 9, 12, 15-octadecatrienoic acid and 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z) in three petroleum ether extracts. Therefore, petroleum ether extract from Abutilon theophrasti Medic. leaves can be a potential resource of natural antioxidants in pharmaceutical, medicine, food, and chemical industries.

KEYWORDS:

• Abutilon theophrasti Medic
• Antioxidant activity
• Extraction technology
• Gas chromatography-mass spectrometry
• Response surface methodology

Introduction

As an annual herb plant in the Malvaceae family, Abutilon theophrasti Medic. (A. theophrasti) is widely distributed all over tropics and sub-tropics.^[1] The different parts of A. theophrasti, including roots, stems, leaves, flowers, seeds, and episperms, have been used for treating many illnesses, such as swell, ulcer, and inflammation in folk.^[2,3] In recent years, many bioactive compounds, including tannin, phenols, and flavonoids, have been detected and isolated from A. theophrasti leaves,^[4–6] and these compounds exhibited important pharmacological effects, such as antibacterial, antioxidant, anti-inflammatory, hepatoprotective, and anticancer activities.^[7–12]

It is well known that low-polarity constituents are not only a major energy source but also containing many biological activities such as antioxidant, anti-inflammatory, and anti-cancer activities.^[13–16] Liu et al.^[3] reported that fatty acids from A. theophrasti seeds can be used as antidiuretic, antidote, and antipyretic. However, low-polarity compounds from A. theophrasti leaves have not been well studied.

Response surface methodology (RSM) is a powerful tool for evaluating the effects of each factor and their interactions in the extraction process.^[17,18] Zhang et al.^[19] employed RSM to optimize the extraction conditions of polysaccharides from lotus (Nelumbo nucifera) leaves, such as liquid/solid ratio, processing pressure, processed times, extraction temperature, and extraction time. RSM was also used by Singh and Pradhan^[20] for modelling a second-order response surface to estimate the optimum machining condition.

Now there are more and more assay methods for evaluating the antioxidant activity of low-polarity extract, such as superoxide radical, hydroxyl radical, oxygen radical absorbance capacity, β-carotene/linoleic acid, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, 2, 2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS) radical scavenging assay, and ferric-ion reducing antioxidant power (FRAP) methods. As reported by Villa-Rodríguez,^[12] there was a positive correlation between DPPH· and ABTS^·+ radical scavenging ability and the content of some unsaturated fatty acids. Aktumsek et al.^[7] focused on the assessment of antioxidant property and fatty acid composition of four Centaurea species, and evaluated their antioxidant activity with phosphomolybedum assay, DPPH·assay, β-carotene/linoleic acid, and ferric and cupric reducing power. Pumpkin seed oil exhibited stronger antioxidant activities with the IC₅₀ values 123.93 and 152.84 mg/mL for DPPH·radical scavenging assay and β-carotene/linoleic acid bleaching test.^[21] The above two methods were also adopted for evaluating the antioxidant activities of Isatis indigotica seeds oil with IC₅₀ values 9.58 and 15.03 mg/mL, respectively.^[13]

In this work, we firstly optimized the Soxhlet extraction parameters for petroleum ether extract (PEE) of A. theophrasti leaves by a 17-run Box–Behnken design (BBD) with a quadratic regression model set up by RSM. Moreover, the chemical components and antioxidant activities (DPPH·and ABTS^·+ radical scavenging ability, FRAP) of PEE obtained in August, September, and October were compared and studied.

Materials and methods

Plant material

A. theophrasti leaves were gathered from Jilin province of China in August, September, and October 2014, and authenticated by professor Shaofan Du of Shenyang Agricultural University according to Compendium of Materia Medica. The voucher specimens KT/JL/CH/AT/08/14, KT/JL/CH/AT/09/14, and KT/JL/CH/AT/10/14 were deposited at college of Animal Science and Veterinary Medicine, Shenyang Agricultural University for future reference. Plant materials were washed and dried at room temperature.

Apparatus and reagents

The antioxidant assay was determined by Tecan Infinite M200 Pro NanoQuant (Tecan Inc., Switzerland). DPPH, ABTS, and 2, 4, 6-tri (2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). All other chemicals and reagents used in this study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Preparation of petroleum ether extract

Three grams of A. theophrasti leaves was extracted with petroleum ether (bp, 60–90°C). The Soxhlet extraction conditions were as follows: ratio of solvent to material 20.12 mL/g, extraction time 5.45 h, and extraction temperature 61.32°C. Then, PEE was concentrated by rotary evaporator, and stored in the dark glass bottle at 4°C.

Experimental design

In the present study, three major influence factors, including ratio of solvent to material, extraction time, and extraction temperature, were investigated for response surface experiments with the yield of PEE as index. The range of levels for the three factors was as follows: ratio of solvent to material 12, 14, 16, 18, 20, 22 mL/g; extraction time 2, 4, 6, 8, 10 h; and extraction temperature 40°C, 45°C, 50°C, 55°C, 60°C, 65°C. When evaluating one factor, the levels of the other two were set in the middle.

According to the results of the single factor experiments, three levels of each factor were selected. In the response surface experiment, BBD (Design-Expert software, Trial Version 8.0.6, State, Inc., Minneapolis, USA) with three variables (X₁, ratio of solvent to material; X₂, extraction time; X₃, extraction temperature) and three levels was introduced to optimize the extraction process of PEE with the yield of PEE as dependent variable. The BBD consisted of 12 factorial points and five replicates of central points (Table 1). Each experimental run was assayed in triplicate except the five centre points, and the averages of yield of PEE were taken as response.

Table 1. Box–Behnken experimental design and the results for extraction yield of PEE from A. theophrasti leaves (n = 3).

No.	The ratio of solvent to material (X1)/(mL/g)	Extraction time (X2)/(h)	Extraction temperature (X3)/(°C)	Yield of PEE (%)^a
1	20	6	60	2.38
2	22	6	55	1.68
3	20	6	60	2.37
4	18	6	55	1.56
5	18	4	60	2.01
6	20	4	55	1.81
7	22	6	65	1.93
8	20	6	60	2.36
9	20	6	60	2.37
10	22	4	60	2.17
11	20	8	65	2.15
12	18	8	60	2.06
13	20	8	55	1.87
14	20	4	65	2.21
15	18	6	65	1.96
16	22	8	60	2.04
17	20	6	60	2.38

^aEach value is the mean of triplicate measurements.

Antioxidant activity assay

Measurement of DPPH radical scavenging activity

DPPH radical scavenging assay was performed according to the method of Brand-Williams et al.^[22] with a few modifications. One hundred microlitres of different concentrations of PEE was mixed with 900 μL of 0.1 mmol/L DPPH·solution, and incubated for 30 min at 37°C in the dark. Then the absorbance was measured at 517 nm, and IC₅₀ value was determined for evaluating the antioxidant activity. Each assay was measured in triplicate, and the percentage of inhibition effect was calculated as follows: Inhibition (%) = (1–OD_test/OD_control) × 100.

Measurement of ABTS^·+ radical scavenging activity

The ABTS^·+ radical scavenging ability of PEE was assayed as the method of Ali^[23] with minor modifications. The ABTS cation radical was prepared with 2.45 mmol/L potassium persulphate and 7 mmol/L ABTS, and incubated at room temperature in the dark for 12–16 h, and then diluted by phosphate buffer solution to obtain an absorbance of 0.700 ± 0.02 at 734 nm. One hundred microlitres of PEE of A. theophrasti leaves was mixed with 900 μL of ABTS^·+ solution. After 30 min incubation at room temperature in the dark, the absorbance of the resulting solution was measured at 734 nm. The values of IC₅₀ were determined as reported above.

Measurement of ferric-ion reducing antioxidant power

According to the method reported by Benzie^[24] with some modifications, FRAP reagent was prepared with 100 mL of 10 mmol/L TPTZ in 40 mmol/L HCl, 10 mL of 20 mmol/L FeCl₃, and 10 mL acetate buffer (pH 3.6). One hundred microlitres of PEE was added to 400 μL of FRAP reagent, and the absorbance was measured at 593 nm after 30 min of incubation at 37°C. FRAP was expressed as mmol of Fe²⁺ per 100 μg/mL PEE.

Constituent analysis of PEE

Derivatization reaction

0.1 g of PEE was mixed with 5 mL of petroleum-anhydrous ether (4:3, v/v), 4 mL of 0.5 mol/L potassium hydroxide methanol solution was added, and then mixed for 1 min. After 15 min, the mixture was added 1 mL of distilled water, and the liquid supernatant was analysed.

Conditions of gas chromatography-mass spectrometry

The prepared samples were analysed on Agilent 6890-5973N gas chromatograph-mass spectrometry (GC-MS) by using hydrogen as the carrier gas and a TR-FAME column (30.0 m × 0.25 mm ID × 0.25 μm film thickness, Thermo Fisher, USA). The peaks were identified by using a Database Nist 98.l, and then each constituent was expressed as a percentage of total compounds quantified. The initial temperature of the column was set at 80°C for 1 min, and then raised at 4°C/min to 220°C for 1 min. The temperatures of MS Source and Quad were 230°C and 150°C, respectively. The range of MS scan was m/z 35–520.

Statistical analysis

All determinations were performed in triplicate and processed using Design Expert 8.0.6 software and SPSS 17.0 (SPSS 17.0 for WINDOWS; SPSS Inc., Chicago, IL) with p-values < 0.05 as statistically significant and p-values < 0.01 as very significance.

Results and discussion

Optimization of extraction conditions

Results of single factor experiments

Three factors (ratio of solvent to material, extraction time, and Soxhlet extraction temperature) were evaluated in the single factor experiments (Fig. 1). As shown X₁ line in Fig. 1, the ratio of solvent to material was set at 12, 14, 16, 18, 20, and 22 mL/g with the extraction temperature of 50°C and the extraction time of 6 h. X₁ line indicated that the yield of PEE increased with increasing ratio of solvent to material, and the yield increased more significantly when the ratio of solvent to material increased from 18 to 20 mL/g, and reached the peak value (1.98%). The positive effect of ratio of solvent to material could be explained by the higher volume of solvent, the higher dissolution rate of PEE in the solvent. However, the yield of PEE started to decrease when the ratio of solvent to material continued to rise; further, it may be that loss of solvent evaporation increased with the increase of solvent volume.

Figure 1. Effect of ratio of solvent to material (X₁), extraction time (X₂), and extraction temperature (X₃) on the extraction yield of PEE.

In order to evaluate the influence of Soxhlet extraction time on the yield of PEE, extraction time was set at 2, 4, 6, 8, and 10 h with extraction temperature of 50°C and ratio of solvent to material of 16 mL/g. According to X₂ line in Fig. 1, the yield of PEE increased rapidly with the increase of extraction time from 2 to 4 h, and increased slowly from 4 to 10 h. The results indicated that the extraction time would not increase dramatically yield of PEE.

X₃ line in Fig. 1 represented the effect of Soxhlet extraction temperature on extraction yield of PEE. Temperatures of 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C were investigated with ratio of solvent to material of 16 mL/g and extraction time of 6 h. X₃ line described that the yield increased rapidly when the temperature increased from 40°C to 45°C, while yield increased slowly from 45°C to 50°C, and reached the peak value at 60°C. The boiling range of petroleum ether was 60°C to 90°C, so the higher extraction efficiency was achieved at 60°C. However, the possibility of petroleum ether evaporation increased when the temperature was higher than 60°C, and the extraction efficiency decreased.

According to the results of single factor experiments, three factors with three levels were adopted for the RSM experiments, and experiment parameters were as follows: ratio of solvent to material (18, 20, 22 mL/g), extraction time (4, 6, 8 h), and Soxhelt extraction temperature (55°C, 60°C, 65°C).

RSM experiments

Model fitting and statistical analysis

Table 1 presents 17 experiments in the design matrix, and the yield of PEE ranged from 1.56% to 2.38%. The experimental data were adopted to calculate the coefficients of the quadratic equation, and the following final equation in terms of coded factors was set up by multiple regression analysis of Design Expert software (version 8.0.6):

$\begin{array}{rl}& Y=2.37+0.029X_1-0.01X_2+0.17X_3-0.045X_1{X}_2-0.038X_1{X}_3-0.030X_2{X}_3-0.26{X_1}^2\\ & -0.037{X_2}^2-0.32{X_3}^2\end{array}$

In statistical analysis, a larger regression coefficient and a smaller p-value would indicate a more significant effect on the respective response variables.^[18] The determined coefficient (R² = 0.9986), which was presented by the quadratic regression model, indicated that only 0.14% of total variants cannot be explained by the model. A larger value of R² does not always imply the adequacy of the model, so it is appropriate to adopt an adjusted R² of over 90% to evaluate the model adequacy.^[18] In this research, the adjusted R² value at 0.9967 suggested a better model adequacy. Meanwhile, the lower p-value (p＜0.0001) and coefficient of the variation (C.V., 0.71) all indicated that the model can significantly represent the actual relationship between parameters and response with higher precision, accuracy, reliability, and reproducibility. The results suggest that the model used in this study was able to identify the optimum extraction condition of PEE from A. theophrasti leaves.

ANOVA was applied to determine the statistical significance of regression coefficients in equation, and the significance of each coefficient was evaluated by p-value. The linear coefficients and quadratic coefficients of ratio of solvent to material (X₁) and Soxhlet extraction temperature (X₃) were very significant (p < 0.01). However, no significant linear and quadratic effect was observed for extraction time (X₂, p > 0.05). Meanwhile, a very significant interaction effect was obtained among all interaction factors (p < 0.01). The p-value indicated that Soxhlet extraction temperature (X₃) was the most significant factor influenced on PEE, followed by ratio of solvent to material (X₁), while extraction time (X₂) exhibited insignificant effect.

Response surface analysis of the content of PEE (CPEE)

The graphical interpretation of regression equation was provided by the 3D response surface curves (Fig. 2) to illustrate the relationship between dependent variable and experimental levels of each independent variable, and the interaction of any two variables was evaluated with the third factor at zero level. Intensive contour indicated much higher influences of parameters on CPEE, and thin contour indicated weak influences.

Figure 2. (a–c) Response surface plots of ratio of solvent to material (X₁, mL/g), extraction time (X₂, h), and extraction temperature (X₃, ^oC) on the yield of PEE.

The interactions among ratio of solvent to material (X₁), extraction time (X₂), and Soxhlet extraction temperature (X₃) on the CPEE were described in Fig. 2. Intensive contour and steeper surface indicated that Soxhlet extraction temperature exhibited a higher influence on CPEE than ratio of solvent to material and extraction time. The CPEE improved remarkably with increasing Soxhlet extraction temperature with the ratio of solvent to material at zero level. However, CPEE began to decrease when extraction temperature beyond 61.3°C. This result may be the volatilization loss of extraction solvent under high extraction temperature. CPEE increased slowly with increasing ratio of solvent to material from 18 to 20 mL/g with extraction temperature at zero level, and decreased over 20 mL/g. However, there was no significant effect between extraction time and CPEE. These results suggested that there were quadratic effects between extraction temperature, ratio of solvent to material, and CPEE, and that there was no significant effect for extraction time.

Graphical interpretation and optimization of procedure

The PEE final equation obtained in this study was applied for response surface optimization by Design-Expert software, and the optimum conditions were ratio of solvent to material (20.12 mL/g), extraction time (5.45 h), and Soxhlet extraction temperature (61.32°C). The experimental values (2.39 ± 0.01)% were in agreement with the predicted value (2.40%).

Content of PEE in 3-month samples

With the optimal extraction condition, the yields of PEE are (2.05 ± 0.02)%, (2.39 ± 0.01)%, and (2.32 ± 0.02)% for the three samples collected in August, September, and October 2014, respectively (Table 2). The results indicated that CPEE varied with the collection month, and the content was highest in September, then October, and last in August.

Table 2. Yield of PEE, IC₅₀ values for evaluated antioxidant assays and EC₅₀ values reducing power of PEE from A. theophrasti leaves.

No.	Yield of extracts (%)	IC50 DPPH (μg/mL)	IC50 ABTS (μg/mL)	FRAP (mmol Fe^2+/100 μg/mL)
August	2.05 ± 0.02	831.5	278.0	0.15
September	2.39 ± 0.01	327.5	170.1	0.31
October	2.32 ± 0.02	331.5	182.1	0.28
BHT		9.02	21.16	0.33

IC₅₀, inhibition concentration 50%; sample 1, 2, and 3 were collected in August, September, and October 2014.

Antioxidant activity

Table 2 lists the scavenging DPPH radical, ABTS^·+ radical effect, and reducing power of PEE from A. theophrasti leaves collected in August, September, and October. As presented in Table 2, the DPPH radical-scavenging capacity of September and October extracts was stronger 2.5 times than August extract with IC₅₀ 327.5 and 331.5 μg/mL, respectively.

As described in Table 2, three extracts all presented potential ability to scavenge the ABTS^·+ radical, and the order of antioxidant activity was August＜October＜September. The PEE of September and October had a higher antioxidant capacity with IC₅₀ of 170.1 and 182.1 μg/mL compared to August (IC₅₀ 278.0 μg/mL).

Contrary to scavenging DPPH radical and ABTS^·+ radical assay, the FRAP value is proportional to antioxidant activity. The results of FRAP assay for three extracts were listed in Table 2. The September extract exhibited a higher FRAP with 0.31 mmol Fe²⁺/100 μg/mL, followed by October extract (0.28 mmol Fe²⁺/100μg/mL). The FRAP of August PEE was 50% lower than other two extracts. The results of the above three antioxidant experiments were consistent, and the antioxidant activity of September and October extracts was similar, and better than August extract. Moreover, the FRAP of September and October extracts was almost the same with BHT.

Chemical component of PEE

The compositions of oils and/or the ratio of saturated-unsaturated fatty acid will affect directly the medicinal and nutritional value.^[25] The compositions profiles of PEE from A. theophrasti leaves were analysed by GC-MS, and the results were summarized in Table 3. Unsaturated fatty acid (C18:3) 9, 12, 15-octadecatrienoic acid was the most predominant constituent in the PEE collected in August, September, and October, and its content was 43.36%, 50.42%, and 53.14%, respectively. Hexadecanoic acid was the most abundant saturated fatty acid (C16:0) with the content of 21.07%, 17.41%, and 16.39%. 9, 12-Octadecadienoic acid (C18:2) and 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z) (C18:3) were two unsaturated fatty acids with relatively high content. In addition, other 21 ingredients were the mainly active constituents with the content lower than 3%. The PEE from A. theophrasti leaves was rich in 9, 12, 15-octadecatrienoic acid, and hence may be viewed as a healthy, nutritive, and cheap alternative to other vegetable oils.

Table 3. Chemical composition of PEE from A. theophrasti leaves.

Peak no.	Retention time (min)	Compounds	RA^a (%)
			August^b	September	October
1	11.648	Undecanoic acid, 10-methyl-	0.22	0.29	–
2	15.748	Methyl tetradecanoate	1.20	1.34	1.98
3	16.609	Tetradecanoic acid, ethyl ester	–	–	0.27
4	17.721	Pentadecanoic acid	0.17	0.28	0.17
5	19.087	Phenol, 2, 4-bis(1, 1-dimethylethyl)	–	0.62	0.47
6	19.632	Hexadecanoic acid	21.07	17.41	16.39
7	20.282	7-Hexadecenoic acid	1.35	0.77	0.61
8	20.398	Hexadecanoic acid, ethyl ester	1.82	2.59	2.22
9	21.448	Hexadecanoic acid, 14-methyl-	0.19	–	0.20
10	23.215	Octadecanoic acid	2.93	1.60	1.26
11	23.671	9-Octadecenoic acid	1.37	0.57	0.81
12	24.515	8, 11-Octadecadienoic acid	0.25	–	–
13	24.637	9, 12-Octadecadienoic acid	11.36	9.08	8.28
14	25.243	Linoleic acid ethyl ester	0.80	1.02	0.93
15	25.710	Phytol	1.46	–	–
16	25.849	9, 12, 15-Octadecatrienoic acid	43.36	50.42	53.14
17	26.387	9, 12, 15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)	3.81	7.13	7.50
18	26.521	Eicosanoic acid	0.22	0.21	–
19	27.315	Nonadecane	0.36	–	–
20	27.326	Docosane	–	–	0.46
21	29.460	Folic acid	0.31	–	–
22	29.587	Docosanoic acid	0.51	0.54	0.63
23	30.321	Heneicosane	–	0.71	–
24	30.337	Tetratetracontane	–	–	0.54
25	30.476	Dibutyl phthalate	2.80	0.65	1.17
Total amount			95.56	95.23	97.03

^a Relative area; ^bcollecting season; ^cundetected (<0.1%).

Pearson correlation analysis

Correlations of DPPH·radical, ABTS^·+ radical scavenging activity, and FRAP activity with CPEE

In this study, DPPH·radical, ABTS^·+ radical scavenging activity, and FRAP activity assay were used to evaluate antioxidant activity of PEE from A. theophrasti leaves. The correlations parameters were calculated by Pearson correlation analysis test, and the correlation coefficients were listed in Table 4. The results indicated that the highest correlation coefficient was found between the CPEE and FRAP activity (1.000, p＜0.05), then CPEE and ABTS^·+ radical scavenging activity (−0.996), and the last one between the CPEE and DPPH·radical scavenging activity (−0.982). The high correlation between CPEE and DPPH·radical, ABTS^·+ radical scavenging activity and FRAP activity assay, suggests that PEE may play an important role in antioxidant activity aspects of A. theophrasti leaves.

Table 4. Correlation coefficients between antioxidant activities and yield of extracts.

	DPPH	ABTS	FRAP	Yield of extracts (%)
DPPH	1	0.996	−0.986	−0.982
ABTS	–	1	−0.997*	−0.996
FRAP	–	–	1	1.000*
Yield of extracts (%)	–	–	–	1

**significant at p＜0.01; *significant at p＜0.05.

Correlations between methods used to evaluate antioxidant activity of PEE

As presented in Table 4, the significant correlation (p＜0.05) was found between ABTS^·+ radical scavenging activity and FRAP activity (−0.997), followed by ABTS^·+ and DPPH· (0.996), and then by FRAP and DPPH· (−0.986). In brief, three antioxidant methods used in this research all indicated better correlation, which was the basis of further study antioxidant activity of PEE from A. theophrasti leaves.

Correlations between antioxidant activities and main chemical component of PEE

Table 5 presents the correlations between antioxidant activities and main chemical component of PEE, including 9, 12, 15-octadecatrienoic acid, hexadecanoic acid, 9, 12-octadecadienoic acid, and 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z). As shown in Table 5, the contents of 9, 12, 15-octadecatrienoic acid and 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z) were negatively correlated with DPPH· (−0.961 and −0.995) and ABTS^·+ (−0.931 and −0.981) values, and positively correlated with FRAP (0.900 and 0.964) value. However, the contents of hexadecanoic acid and 9, 12-octadecadienoic acid were positively correlated with DPPH· (0.977 and 0.966) and ABTS^·+ (0.952 and 0.938) values, and negatively correlated with FRAP (−0.926 and −0.909) value. The finding was in accordance with the result of antioxidant activities of the PEE collected in different months. Meanwhile, the highest correlation coefficient was found between the content of 9, 12, 15-octadecatrienoic acid and 9, 12-octadecadienoic acid (−1.000, p＜0.05), followed by 9, 12-octadecadienoic acid and hexadecanoic acid (0.999, p＜0.05), and then by 9, 12, 15-octadecatrienoic acid and hexadecanoic acid(−0.998, p＜0.05). Furthermore, the correlation coefficient was much better among the content of 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z) and 9, 12, 15-octadecatrienoic acid, hexadecanoic acid, 9, 12-octadecadienoic acid (0.984, −0.993, and −0.987). This further validated the correlations between antioxidant activities and main chemical component of PEE.

Table 5. Correlation coefficients between antioxidant activities and content of four main components.

	DPPH	ABTS	FRAP	9, 12, 15-Octade-catrienoic acid	Hexadec-anoic acid	9, 12-Octade-cadienoic acid	9, 12, 15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)
DPPH	1	0.996	−0.986	−0.961	0.977	0.966	−0.995
ABTS	–	1	−0.997*	−0.931	0.952	0.938	−0.981
FRAP	–	–	1	0.900	−0.926	−0.909	0.964
9, 12, 15-Octadecatrienoic acid	–	–	–	1	−0.998*	−1.000*	0.984
Hexadecanoic acid	–	–	–	–	1	0.999*	−0.993
9, 12-Octadecadienoic acid	–	–	–	–	–	1	−0.987
9, 12, 15-Octadecatrienoic acid,ethyl ester, (Z,Z,Z)	–	–	–	–	–	–	1

**significant at p＜0.01; *significant at p＜0.05.

Conclusion

This study is the first report on the extraction of PEE from A. theophrasti leaves collected in August, September, and October, and optimization of their extraction conditions with Soxhlet extraction method by RSM. The optimized extraction parameters were as follows: ratio of solvent to material (20.12 mL/g), extraction time (5.45 h), and Soxhlet extraction temperature (61.32°C). Also, the yield of PEE was 2.39%. In addition, September and October extracts exhibited stronger antioxidant activity than August extract, which were demonstrated by DPPH·radical, ABTS^·+ radical scavenging assay, and FRAP assay. The analysis of chemical component of three PEEs indicated that 9, 12, 15-octadecatrienoic acid and 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z) were the major constituents, followed by hexadecanoic acid and 9, 12-octadecadienoic acid. It revealed that the material base of the antioxidant activity of PEE may be 9, 12, 15-octadecatrienoic acid and 9, 12, 15-octadecatrienoic acid, ethyl ester (Z,Z,Z), and PEE from A. theophrasti leaves might be explored as a new resource of natural antioxidants and applied in pharmaceutical, medicine, food, and chemical industries.

Additional information

Funding

This project was supported by National Natural Science Foundation of China [Grant No.31402252].