Optimization of ultrasonic-assisted extraction of wedelolactone and antioxidant polyphenols from Eclipta prostrate L using response surface methodology

Separation and Purification Technology 138 (2014) 55–64

Contents lists available at ScienceDirect

Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur

Optimization of ultrasonic-assisted extraction of wedelolactone and antioxidant polyphenols from Eclipta prostrate L using response surface methodology Xinsheng Fang a,b, Jianhua Wang a,b,⇑, Yingzi Wang c, Xueke Li a,b, Hongying Zhou a,b, Lixiang Zhu a,b a b c

State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Taian 271018, China Shandong Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Taian 271018, China School of Chinese Materia Medica, Beijing University of Traditional Chinese Medicine, Beijing 100102, China

a r t i c l e

i n f o

Article history: Received 14 August 2014 Received in revised form 2 October 2014 Accepted 11 October 2014 Available online 18 October 2014 Keywords: Ultrasonic-assisted extraction Wedelolactone Antioxidant Central composite design Eclipta prostrate L

a b s t r a c t An efficient ultrasonic-assisted extraction using ultrasonic probe system (UPAE) was developed for the extraction of wedelolactone (WEL) and antioxidant polyphenols from Eclipta prostrate. A central composite design and response surface methodology (RSM) were used to optimize the UPAE. The key parameters considered for the UPAE included solvent type, temperature, ultrasonic power, solvent to material ratio and extraction time. The dependent variables included the yield of WEL, total phenolic content (TPC) and scavenging activity of DPPH radical. For simultaneously maximizing the three responses, the optimal conditions were 48% ethanol–water mixture as solvent, temperature of 40 °C, ultrasonic power of 90 W, solvent to material ratio of 50 mL/g and extraction time of 11 min. The predicted values from the developed quadratic polynomial equations were in close agreement with the actual experimental values. The TPC and %DPPH of the optimized UPAE were significantly higher than those of ultrasonic-assisted extraction using ultrasonic bath, heat reflux extraction and Soxhlet extraction. Compared to conventional extraction methods, UPAE provided higher extraction efficiency and offered many advantages such as reduction of time and energy for extraction. The developed UPAE appears to be a green method and have great potential for the extraction of active components and antioxidants from natural products. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Eclipta prostrata Linn (Mo han lian in Chinese, abbreviated as MHL) is an annual herb in China and many other countries in the world. The overground part of the herb is a famous traditional medicine and is also an edible natural product. It is used for the treatment of hemorrhages, hepatic disease, renal injuries, hair loss, tooth mobility, and viper bites in traditional Chinese medication [1]. Modern pharmacological research has demonstrated that MHL exhibits various bioactivities including anti-tumor, antisnake venom, anti-inflammatory, anti-oxidation, anti-HIV-1 integrase, reduction of blood lipids, and prevention of CCl4-induced liver damage [2–5]. Phenolic compounds have demonstrated their remarkable activities in cardiovascular and cerebrovascular diseases, liver disease, antioxidation, anti-bacterium, anti-HIV and ⇑ Corresponding author at: State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Taian 271018, China. Tel./fax: +86 538 8242226. E-mail address: [email protected] (J. Wang). http://dx.doi.org/10.1016/j.seppur.2014.10.007 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.

Alzheimer’s disease. So, these metabolites may be the great important bioactive components in MHL. Wedelolactone (WEL) is one of the important active components that have been isolated from E. prostrata. Various bioactivities of WEL have been demonstrated in previous publications. WEL can significantly inhibit the protein expression levels of iNOS and COX-2 in LPS-stimulated cells, as well as the downstream products, including NO, PGE2 and TNF-alpha [6]. Xia et al. demonstrated the anti-fibrotic effects of wedelolactone on activated human hepatic stellate cell (HSC) line LX-2 [7]. Chen et al. discovered that WEL increased IFN-c signaling by inhibiting STAT1 dephosphorylation and prolonging STAT1 activation through specific inhibition of T-cell protein tyrosine phosphatase (TCPTP) [8]. Sarveswaran et al. found that WEL selectively induced caspasedependent apoptosis in prostate cancer cells, which suggested that WEL may be as a novel therapeutic agent against clinical prostate cancer in human [9]. Lee et al. found that WEL exert anti-invasive growth effect on breast cancer cells [10]. Benes et al. found that WEL can act as growth suppressor independently of NFjB and androgen receptors [11]. Patil et al. studied the extraction of WEL

56

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64

using supercritical carbon dioxide extraction [12]. The chemical structure of WEL is shown in Fig. 1. Extraction is one of the key steps in the investigation and utilization of phenolic components from various materials. Ultrasoundassisted extraction (UAE) attracts much more attention in extraction of bioactive components from natural products. The extraction mechanisms of UAE mainly include cavitation effect, mechanical effect, and thermology effect. The acoustic cavitation is the primary mechanism for the high extraction efficiency of ultrasonic treatment [13]. Ultrasonic cavitation creates shear forces that break cell walls mechanically and improve material transfer. UAE has been used in the extraction of phenolic compounds [14,15], essential oils [16], polysaccharides [17], and etc. [18,19]. However, the mechanism of solute extraction from plant material by UAE is still under study [20]. Two different types of ultrasound equipments are commonly used in laboratory. The first one is the ultrasonic cleaning bath and the second one is the ultrasonic probe or horn system. The ultrasound-assisted extraction using ultrasonic bath (UBAE) is easy to handle and economically advantageous. But the delivered intensity is low and is highly attenuated by the water in the bath and the walls of the glassware used for the experiment [21]. However, the ultrasound-assisted extraction using ultrasonic probe (UPAE) is much more powerful because the ultrasonic intensity is delivered on a small surface (only the tip of the probe) compared to the ultrasonic bath. In UPAE, the probe is directly immersed into the reaction flask, so less attenuation can happen [19,21]. Up to now, no paper has been published to study the UAE of WEL and polyphenols from MHL. The objective of this study was to optimize the UPAE of WEL and polyphenols using response surface methodology (RSM) and evaluate the antioxidant activity of polyphenols. The key parameters of UPAE procedure such as solvent, ultrasonic power, temperature, solvent to material ratio and extraction time were optimized. The results would provide information for understanding the mechanism of UPAE. The efficiency of the developed UPAE was also compared with those of UBAE and other conventional methods.

2. Materials and methods 2.1. Plant materials, chemicals and reagents Herba Ecliptae, the overground part of E. prostrata L, was chipped to small segment. All the segments were dried in the drying room with active ventilation at room temperature (about 25 °C) until constant weight. The standard substance of wedelolactone and gallic acid was obtained from the National Institute for the Control of Pharmaceuticals and Biological Products (Beijing, China). The purity of the reference standards was determined to be more than 98%. Formic acid, acetonitrile (HPLC grade), absolute ethanol (analytical grade) were purchased from Tianjin Kemiou Chemical Reagent Company (Tianjin, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and Folin-Phenol reagent were purchased from Sigma–Aldrich. Double-distilled water was made in our laboratory.

OH

OH

O

2.2. Extraction method The sonochemical reactor used in the present work consisted of an ultrasonic probe (Dakshin India Ltd., Mumbai) operating at a frequency of 20 kHz with rated power of 150 W. Ultrasonic probe was fitted with PZT transducer with tip diameter of 0.6 cm. The segments of MHL were crushed by a pulverizer and sieved by standard sieve (80–100 mesh). An amount of 2 g MHL powder was added into a 100 mL round-bottomed glass vessel and soaked with solvent for 3 min. The sample was extracted using the designed conditions. The ultrasound was introduced using the ultrasonic probe operating in 50% duty cycle (5 s ON and 5 s OFF). About 1.0 cm length of the ultrasonic probe tip was dipped into the solvent from its surface. After extraction, the weight loss was complemented with the solvent. 2.3. Central composite design and optimization A four-factor (X1, X2, X3 and X4) and five-level (1.682, 1, 0 and +1, +1.682) central composite design was used to optimize the extraction process [22]. The independent variables were ethanol percentage (X1, %), ultrasonic power (X2, W), solvent to material ratio (X3, mL/g), and extraction time (X4, min). The coded and uncoded levels of the independent variables are shown in Table 1. The dependent variable included the yield of WEL, total phenolic content (TPC) and scavenging activity of DPPH radical. The experiment design was carried out by Design-Expert 7.1.6 software. The generalized second-order polynomial model proposed for the response surface analysis was as follows:

Y ¼ b0 þ

k X

bi X i þ

i¼1

k k1 X k X X bii X 2i þ bij X i X j i¼1

i

where b0, bi, bii, bij are regression coefficients for intercept, linear, quadratic and interaction terms, respectively. Xi and Xj are coded value of the independent variables while k equals to the number of the tested factors (k = 4). All experiments were performed randomly in triplicate. Statistical analysis was performed using Design-Expert 7.1.6 software and fitted to a second-order polynominal regression model containing the coefficient of linear, quadratic and interaction terms. An analysis of variance (ANOVA) with 95% confidence level was then carried out for each response variable in order to test the model significance and suitability. In order to find the conditions that give the maximum of the three responses simultaneously, the desirability function approach of Derringer [22] was used to optimize all the three responses. The Derringer desirability function was carried out with the software of Design-Expert 7.1.6. Varying the degrees of importance assigned to the responses (1–5) the objective function is given as follows: ri

D ¼ ðd1 

r d22

  

r dnn Þ

P1

ri

¼

O

Fig. 1. Chemical structure of wedelolactone.

r di i

!P1

ri

ð2Þ

where di is the partial desirability function of each response obtained from the transformation of the individual response of each

Table 1 Coded and uncoded levels of the independent variables. Factors

O

n Y i¼1

OH CH3O

ð1Þ

j

X1/Ethanol concentration (%) X2/Ultrasonic power (W) X3/Solvent to material ratio (mL/g) X4/Extraction time (min)

Levels 1.682

1

0

+1

+1.682

30 40 30 1

40 50 35 3.8

55 65 42.5 8

70 80 50 12.2

80 90 55 15

57

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64

experiment, n is the number of responses in the measure and ri reflects the importance of each response. 2.4. HPLC analysis The quantitative analysis of WEL was carried out by HPLC–DAD. A Waters UPLC system equipped with Quaternary Solvent Manager, Sample Manager, PDA detector and Empower Chromatography Data Software (Waters Technologies, USA) was used for the analysis. A Thermo ODS2-HypersilÒ column (250 mm  4.6 mm, 5 lm, Thermo, USA) was used as the analysis column. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid aqueous solution (B) using the following elution program for separation: 0–7 min, 33% A, and then increased to 100% A at 8 min (hold 3 min) to clean up the hydrophobic residues on the column. The system was subsequently returned to the initial condition and equilibrium for 5 min before the next injection. The flow rate was 1 mL/min and the injection volume was 10 lL. UV wavelength was 350 nm. Quantitative determination of WEL was performed using external standards by means of a six points calibration curve. The HPLC chromatograms of the standard and one sample are shown in Fig. 2. 2.5. Determination of total polyphenols Total phenolic contents (TPC) of the MHL extracts were determined using Folin–Ciocalteu (FC) reagent assay described by Singleton et al. [23]. Methanolic gallic acid solutions were used as standards. One hundred microlitres of properly diluted extract solution were mixed with 1.5 mL of FC reagent. The reagent was pre-diluted to 6 mL with distilled water and then 3 mL of (10.0% w/v) sodium carbonate solution were added. Finally the solution was diluted to 25 mL with distilled water. The mixed solution was allowed to stand for 1 h at room temperature. The absorbance was measured at 756 nm using a UV–visible spectrophotometer (UV 2450 Shimadzu, Japan). A calibration graph [A = 0.0057 C + 0.0071, r2 = 0.9992] was constructed by plotting absorbance difference against the gallic acid concentration at seven concentration levels. The results of total phenolic content were expressed as mg gallic acid equivalents per g dry weight of MHL.

2.6. Determination of antioxidant capacity of polyphenols DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical-scavenging activity was used to evaluate the antioxidant capacity of the total polyphenols. The antioxidant activity was determined according to the method reported by Brand-Williams et al. [24] with some modification. The test sample solutions (2 mL) at different concentrations were mixed with 2 mL DPPH (0.15 mM). The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The UV absorbance of the reaction mixtures was measured at 517 nm using a UV–Visible spectrophotometer (UV 2450 Shimadzu, Japan). Percentage of inhibition of the DPPH radical was calculated according to the following equation:

Inhibition of DPPH ð%Þ ¼ ðAC  AS Þ=AC  100

where AC is the absorbance of DPPH solution without extracts, AS is the absorbance of DPPH solution with extracts. 3. Results and discussion 3.1. Effect of solvent The correct choice of solvent is fundamental for obtaining an optimal extraction process. Ethanol (or methanol) and water are the conventional solvents for extraction. A suitable mixed solvent system of methanol/water or ethanol/water could improve the efficiency of UAE and economize the use of energy and organic solvents. Therefore, the effects of the mixed solvent systems of methanol/water and ethanol/water were firstly investigated. Two gram of powder (80–100 mesh) was extracted with 50 mL different mixed solvent for 5 min. The ultrasonic power was 50 W. The results are shown in Fig. 3. The yields of WEL (Fig. 3A) increased significantly (p < 0.05) when the solvent changed from 30% to 70% aqueous ethanol (or methanol) and reached the maximum when the solvent was 70% ethanol. The yield decreased significantly when the solvent was 90% ethanol (or methanol). The yields of WEL of 50–70% ethanol were significantly higher than those of 50–70% methanol. But the yield of WEL using 90% methanol was significantly higher than that of 90% ethanol. The variation trends of TPC and inhibition of DPPH using different solvent were similar

0.15

AU

A 0.10

0.05

0.00 0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

min

0.25

AU

0.20

B

0.15 0.10 0.05 0.00 0.00

1.00

2.00

3.00

ð3Þ

4.00

5.00

6.00

7.00

min Fig. 2. The HPLC chromatograms of the standard (A) and one extract of MHL (B).

8.00

58

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64 3.5

A

3

Yield (mg/g)

2.5 2 1.5 1 0.5 0 30%EtOH

50%EtOH

70%EtOH

90%EtOH

30%MeOH

50%MeOH

70%MeOH

90%MeOH

50%MeOH

70%MeOH

90%MeOH

Solvent 60

B

TPC (mg/g)

%DPPH

50 40 30 20 10 0

30%EtOH

50%EtOH

70%EtOH

90%EtOH

30%MeOH

Solvent Fig. 3. Effect of different solvent on the extraction yield of WEL (A), TPC and %DPPH (B). Solvent to material ratio: 25 mL/g; ultrasonic power: 50 W; extraction time: 5 min.

TPC (mg/g)

70

%DPPH

WEL

4.5 4

60

3.5 3 40

2.5

30

2

Yield (mg/g)

50

1.5 20 1 10

0.5

0

0 20

30

40

50

60

Temperature (°C) Fig. 4. Effect of temperature on the extraction yield of WEL, TPC and %DPPH. The values of TPC (mg/g) and %DPPH are shown on the y-axis on the left. The value of the yield of WEL is shown on the secondary y-axis on the right. Solvent to material ratio: 25 mL/g; ultrasonic power: 50 W, extraction time: 5 min.

with that of WEL (Fig. 3B). The ethanol concentration of 50% was most suitable for the extraction of total polyphenols. In conventional extraction methods, the solubility of the target components in solvent was an important factor. But in UAE, except for the solubility of the components in solvent, the cavitation effect is the primary extraction mechanism [13,21]. Polyphenols usually have higher polarity and WEL is a low polarity compound. In conventional extraction methods, pure methanol or ethanol was more suitable for the extraction of WEL and lower concentration of ethanol (or methanol) was suitable for the extraction of polyphenols. In the present study, the yield of WEL with 70% ethanol was significantly higher than that of with 90% ethanol. The TPC with 50% ethanol was significantly higher than that of 30% ethanol. The results indicated that the cavitation effect of UAE had great influence on the extraction efficiency of WEL and total phenols. The highest yield of WEL was obtained with 70% ethanol and the highest yield of TP was obtained with 50% ethanol, which was the result of com-

bination of ultrasonic effect and solubility of the target components in solvent. 3.2. Effect of temperature In the pre-experiment, temperature showed significant effect on the extraction efficiency and antioxidant activity. Therefore, a suitable temperature should be chosen for the following experiments. An amount of 2 g powder (80–100 mesh) was extracted with 50 mL 70% ethanol for 5 min and the ultrasonic input power was 50 W. The extraction yields at different temperature (20, 30, 40, 50 and 60 °C) are shown in Fig. 4. The yield of WEL, TPC and inhibition of DPPH increased significantly from the temperature of 20–30 °C. From 30 to 40 °C TPC and %DPPH increased significantly but the yield of WEL increased slightly. For the yield of WEL, TPC and %DPPH there was not significant difference between 40 and 50 °C, as well as 50 and 60 °C. In UAE, the extraction yield

59

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64 Table 2 Central composite design of four variables with their observed responses.

a

No.

X1/Ethanol concentration (%)

X2/Ultrasonic power (W)

X3/Solvent to material ratio (mL/g)

X4/Extraction time (min)

Yield of WEL (mg/g)a

TPC (mg GAE/g DW)

%DPPH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

80.0 30.0 55.0 40.0 55.0 70.0 70.0 40.0 40.0 55.0 40.0 55.0 55.0 55.0 70.0 55.0 70.0 55.0 55.0 55.0 55.0

65.0 65.0 65.0 80.0 65.0 50.0 50.0 50.0 50.0 40.0 80.0 65.0 65.0 65.0 80.0 65.0 80.0 90.0 65.0 65.0 65.0

42.5 42.5 42.5 50.0 42.5 50.0 35.0 50.0 35.0 42.5 35.0 42.5 42.5 30.0 50.0 42.5 35.0 42.5 42.5 55.0 42.5

8.0 8.0 8.0 12.2 8.0 12.2 12.2 3.8 3.8 8.0 12.2 15.0 8.0 8.0 3.8 1.0 3.8 8.0 8.0 8.0 8.0

3.36 2.32 3.78 3.56 3.73 3.70 3.71 3.19 3.05 3.83 3.27 3.92 3.79 3.66 3.59 3.40 3.43 3.71 3.76 3.90 3.73

14.35 20.86 20.77 22.50 20.99 16.54 17.49 20.62 18.73 21.25 20.62 21.66 21.69 19.78 18.41 19.73 16.94 21.83 21.84 20.47 21.93

35.06 37.91 51.95 51.80 48.44 42.09 47.79 45.61 44.05 53.74 51.50 51.41 52.36 51.82 47.25 45.49 46.71 51.91 51.50 52.69 49.50

Yield of WEL (mg/g) = (quantity of WEL in extract/quantity of raw material).

could be affected by the combined action of acoustic cavitation and thermal effect. High temperature leads to the decrease of cavitation effect because of the increase of vapor pressure and decrease of viscosity and surface tension of solvent [25]. However, higher temperature increased solubility of the components and reduced the solvent viscosity, which resulted in the increase of mass transfer. The influence of cavitation effect and thermal effect on the yield would arrive at equilibrium state at certain temperature. For the yield of WEL there was not significant difference between 40 and 60 °C. But the TPC at 60 °C was significantly higher that of at 40 °C. The reason was due to that 70% ethanol was most suitable for the extraction of WEL but not for total polyphenols. Therefore, the temperature showed significant effect on the extraction of TP. Considering the saving of energy and easy operation, 40 °C was used as the temperature in the following optimized experiments. 3.3. Optimization of UPAE using RSM

Y 1 ðyield of WELÞ ¼ 0:316 þ 0:1918X 1  0:0486X 2  0:0172X 3  0:0285X 4 þ 1:36  104 X 1 X 2  3:1  104 X 1 X 3  9:7  104 X 1 X 4 þ 3:42  104 X 2 X 3 þ 2:279  103 X 2 X 4  5:1  105 X 3 X 4  1:44  103 X 21 þ 4:66  105 X 22 þ 2:6  104 X 23  1:71  103 X 24

Table 3 Analysis of the variance of the regression coefficients of the fitted polynomial quadratic equation for the yield of WEL (mg/g), TPC (mg GAE/g DW) and %DPPH. Coefficient

3.3.1. Modeling of the responses of the yield of WEL, TPC and %DPPH The responses (yield of WEL, total phenolic content and scavenging activity of DPPH radical) of each run of the experimental design were presented in Table 2. The yields of WEL varied from 2.32 to 3.92 mg/g. Total phenolic content of MHL extract varied from 14.35 to 22.50 mg gallic acid/g dry weight. The inhibition of DPPH varied from 35.06% to 53.74%. ANOVA was used to estimate the statistical significance of the factors and interactions between them. Regression coefficient and analysis of variance of the second-order polynomial models for the yield of WEL, TPC and %DPPH are summarized in Table 3. The ANOVA analysis showed that the models were significant (p < 0.0001, p < 0.001 and p < 0.01 for the yield of WEL, TPC and %DPPH, respectively) and the lack of fits were not significant at 95% confidence, which indicated that the fitted models were considered adequate. The fit value, termed R2 (determinant coefficient) of the polynomial models ranged from 0.97 to 0.998 and the adjusted determination coefficient (R2Adj) ranged from 0.9 to 0.994, which further indicated that the models adequately represent the experimental results and a high degree of correlation between the observed and predicted values. The three models for the yield of WEL, TPC and %DPPH are represented as follows:

ð4Þ

b0 b1 b2 b3 b4 b11 b22 b33 b44 b1 b2 b1 b3 b1 b4 b2 b3 b2 b4 b3 b4 Model Lack of fit R2 R2adj ns

Response Yield of WEL

TPC

%DPPH

0.316 0.192d 0.0486a 0.0172d 0.0285d 1.44  103d 4.66  105ns 2.6  104ns 1.71  103a 1.36  104ns 3.1  104a 9.7  104b 3.42  104b 2.279  103d 5.1  105ns p < 0.0001 0.545 0.998 0.994

21.865 0.730c 0.0981ns 1.015a 1.611ns 6.56  103d 2.6  104ns 0.0101b 0.0205a 1.198  103ns 3.67  103ns 7.51  103ns 2.731  103ns 4.81  103ns 9.85  103ns p < 0.001 0.350 0.979 0.931

6.112 2.878ns 1.011ns 0.849ns 4.412a 0.0219d 4.279  103ns 0.0135ns 0.0348ns 2.553  103ns 7.957  103ns 0.0446a 5.635  103ns 4.751  103ns 0.0303ns p < 0.01 0.576 0.970 0.900

Not significant (p > 0.05). Significant at p < 0.05. b Significant at p < 0.01. c Significant at p < 0.001. d Significant at p < 0.0001. a

60

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64

3.48

3.52

Yield (mg/g)

4.00

Yield (mg/g)

3.90

3.05 2.63

A

2.20

B

55.00

52.50

Power (W)

48.75

30.00

65.00

42.50

77.50

67.50 55.00

36.25

67.50 80.00

80.00

42.50

S/L (mL/g)

55.00

90.00

42.50

30.00

Ethanol concentration (%)

4.00

30.00

Ethanol concentration (%)

4.00

3.45

3.88

2.90

Yield (mg/g)

Yield (mg/g)

2.58 2.10

40.00

2.35

C

1.80

3.77 3.65

D

3.53

15.00

55.00

11.50 67.50 30.00

3.95

3.90

Yield (mg/g)

4.09

3.60 3.25

E 90.00

11.50

3.51

F

15.00

65.00 4.50

Time (min)

40.00

55.00 48.75

8.00

52.50 1.00

Power (W)

11.50

77.50

8.00

40.00

3.71

3.32

15.00

52.50 30.00

Ethanol concentration (%)

4.30

2.90

65.00 36.25

S/L (mL/g)

42.50 1.00

77.50

42.50

55.00

4.50

Time (min)

90.00

48.75

80.00

8.00

Yield (mg/g)

3.05

Power (W)

Time (min)

42.50 4.50

36.25 1.00

30.00

S/L (mL/g)

Fig. 5. Responses surface plots for the effect of (A) EtOH/power, (B) EtOH/solvent ratio, (C) EtOH/time, (D) power/solvent ratio, (E) power/time, and (F) solvent ratio/time on the extraction yield of WEL.

Y 2 ðTPCÞ ¼ 21:865 þ 0:73X 1  0:0981X 2 þ 1:015X 3

Y 3 ð% DPPHÞ ¼ 6:112 þ 2:878X 1  1:011X 2  0:849X 3

3

þ 1:611X 4 þ 1:198  10 X 1 X 2  3:67

þ 4:412X 4 þ 2:553  103 X 1 X 2  7:957

 103 X 1 X 3  7:51  103 X 1 X 4 þ 2:731

 103 X 1 X 3  0:0446X 1 X 4 þ 5:635

3

 10 X 2 X 3 þ 4:81  10 X 2 X 4  9:85

 103 X 2 X 3 þ 4:751  103 X 2 X 4

 103 X 3 X 4  6:56  103 X 21  2:6  104 X 22

 0:0303X 3 X 4  0:0219X 21 þ 4:279  103 X 22

 0:0101X 23  0:0205X 24

3

ð5Þ

þ 0:0135X 23  0:0348X 24

ð6Þ

61

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64

UAE, appropriate ultrasonic power and solvent would improve the extraction efficiency. However, in some circumstances, with the increase of acoustic intensity, more bubbles were formed which hampers the propagation of shock waves and the bubbles may coalesce to form bigger ones and implode weakly. Hence the extraction efficiency would decrease [19,26]. In addition, powerful cavitation effect would lead to the degradation of components [27]. Ethanol concentration and solvent to material ratio showed significant interaction effect on the yield (p < 0.05) (Fig. 5B). The yield increased with the increasing of solvent to material ratio when

22.3

23

19.95

20.25

17.6 15.25

A

12.9 90.00

Total phenols (mg/g)

Total phenols (mg/g)

3.3.2. Response surface analysis for the yield of WEL The three-dimensional response surface plots of the model for the yield of WEL are shown in Fig. 5. All the four factors had a significant (p < 0.05) effect on the yield of WEL. The yields of WEL were at higher level when the ethanol concentration changed from 55% to 70% but showed decreasing trend when the ethanol concentration increased to 80% (Fig. 5A). When the ethanol concentration was less than 55% the yield decreased with the increasing of ultrasonic power. When the ethanol concentration was over than 70% the yield showed slight increase with the increasing of power. In

77.50

30.00 55.00

Ethanol concentration (%)

80.00

B

12 55.00 48.75 42.50

42.50

Power (W)

52.50

67.50

14.75

30.00

65.00

42.50

17.5

55.00

36.25

67.50

40.00

Ethanol concentration (%)

80.00

S/L (mL/g)

30.00

22.10 23

Total phenols (mg/g)

20.58

Total phenols (mg/g)

20.25 17.5 14.75

C

12 15.00

19.05 17.52

D 16.00

55.00

11.50 30.00 55.00

4.50

67.50 80.00

Time (min)

S/L (mL/g)

20.58

20.58

19.05 17.52

E

16.00

15.00

90.00

11.50

77.50

8.00 1.00

40.00

Power (W)

40.00

19.05 17.52

F

16.00

15.00

55.00

11.50

48.75

8.00

65.00 52.50

52.50 30.00

22.10

4.50

65.00 36.25

1.00

22.10

Time (min)

77.50

42.50

Total phenols (mg/g)

Total phenols (mg/g)

Ethanol concentration (%)

90.00

48.75

8.00

42.50

Power (W)

Time (min)

42.50 4.50

36.25 1.00

30.00

S/L (mL/g)

Fig. 6. Responses surface plots for the effect of (A) EtOH/power, (B) EtOH/solvent ratio, (C) EtOH/time, (D) power/solvent ratio, (E) power/time, and (F) solvent ratio/time on the TPC.

62

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64

Ultrasonic power and solvent to material ratio also showed significant interaction (p < 0.01) on the yield of WEL (Fig. 5D). The yield decreased significantly with the increase of ultrasonic power when the solvent to material ratio was lower than 40 mL/g. The reason may be due to the degradation of WEL because of the powerful cavitation effect when the solvent to material ratio was in lower level [25]. But the yield showed slightly increase with the increase of power when solvent to material ratio was over than 45 mL/g. The yield increased with the increase of solvent to material ratio when the power was between 55 and 90 W. Ultrasonic power and extraction time also showed significant interaction (p < 0.001) on the yield of WEL (Fig. 5E). The changes of the yield

55.00

55.00

48.75

48.75

42.50

42.50

% DPPH

% DPPH

the ethanol concentration was less than 60%. But when the ethanol concentration was over than 70% the yield showed slight decrease with the increasing of solvent to material ratio. In UAE the increase of solvent to material ratio may have negative effects on the extraction [27]. Ethanol concentration and extraction time showed significant interaction (p < 0.01) on the yield of WEL (Fig. 5C). The yield increased significantly with the prolonging of time when the ethanol concentration was lower than 50%. When the ethanol concentration was over than 60% the yield increased slightly with the increase of time. The highest yields were obtained at ethanol concentration between 60% and 70% and extraction time between 12 and 15 min.

36.25

A

30.00

55.00

77.50

Power (W)

48.75

80.00

65.00 55.00

52.50

S/L (mL/g)

42.50 30.00

30.00

58.30

46.25

53.72

37.50

49.15

% DPPH

55.00

C

20.00

67.50 55.00

36.25

Ethanol concentration (%)

28.75

80.00

42.50

67.50 40.00

% DPPH

B

30.00

90.00

42.50 30.00

Ethanol concentration (%)

44.58

D

40.00

55.00

15.00

48.75

11.50

80.00

8.00

Time (min)

S/L (mL/g)

55.00

4.50

77.50 65.00

36.25

42.50 30.00

90.00

42.50

67.50 1.00

30.00

52.50 40.00

Power (W)

Ethanol concentration (%)

58.30

58.30

53.72

% DPPH

53.72

% DPPH

36.25

49.15 44.58 40.00

E

44.58

F

40.00

15.00

15.00

11.50

11.50

90.00

8.00

Time (min)

49.15

77.50 65.00

4.50

52.50 1.00

40.00

Power (W)

55.00

8.00

Time (min)

48.75 42.50

4.50 1.00

36.25 30.00

S/L (mL/g)

Fig. 7. Responses surface plots for the effect of (A) EtOH/power, (B) EtOH/solvent ratio, (C) EtOH/time, (D) power/solvent ratio, (E) power/time, and (F) solvent ratio/time on%DPPH.

63

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64 Table 4 Comparison of the optimized UPAE with other three extraction methods. Extraction methods

Solvent

Ultrasonic power (W)

Solvent to material ratio (mL/g)

Extraction time (min)

Yields of WEL (mg/g)

TPC (mg/g)

%DPPH

UPAE-1 UPAE-2 UBAE-1 UBAE-2 HRE SE

60% Ethanol 48% Ethanol 48% Ethanol 70% Ethanol 70% Ethanol Methanol

75 90 200 200 – –

40 50 50 40 40 100

12 11 11 30 90 180

3.97 ± 0.12 3.90 ± 0.10 2.88 ± 0.07 3.94 ± 0.16 3.89 ± 0.11 4.01 ± 0.08

18.33 ± 0.65 22.57 ± 0.90 20.85 ± 0.77 20.49 ± 0.98 18.20 ± 0.51 13.79 ± 0.40

48.9 ± 2.0 53.6 ± 2.4 49.2 ± 1.9 50.4 ± 2.5 47.3 ± 1.8 37.5 ± 1.2

UPAE and UBAE are the abbreviations of ultrasonic-assisted extraction using ultrasonic probe system and ultrasonic bath system, respectively.

were not obvious when the power was between 40 and 50 W. When the power was between 55 and 70 W the yield increased with the increase of time from 1 to 10 min. But when the power was between 75 and 90 W the yield increased significantly with the prolonging of time. Solvent to material ratio and extraction time showed not significant interaction on the yield of WEL (Fig. 5F). From 1 to 10 min the yield increased obviously with the increase of time. The yield increased obviously with the increase of solvent to material ratio when the time was between 1 and 5 min. 3.3.3. Response surface analysis for TPC and %DPPH The three-dimensional response surface plots of the model of TPC are shown in Fig. 6. Ethanol concentration and solvent to material ratio showed significant (p < 0.05) effect on the total phenol content. The TPC increased significantly when the ethanol concentration changed from 30% to 50% but decreased significantly when ethanol concentration increased from 55% to 80% (Fig. 6A– C). In general, the polarity of ethanol–water mixture would increase continuously with the addition of water to ethanol. More polar phenolic compounds could be extracted according to ‘‘like dissolves like’’ principle. The results indicated that 40–55% ethanol was suitable for the extraction of total polyphenols. In Fig. 6E the TPC increased with the increase of power when the extraction time was at low level. But when the extraction time was over 12 min the TPC showed decreased trend with the increase of power. The reason may be the degradation of polyphenols with powerful cavitation effect for a long time. From Fig. 6F one can see that the higher TPC could be obtained at the conditions of solvent to material 45–50 mL/g and extraction time 10–12 min. The three-dimensional response surface plots of the model of %DPPH are shown in Fig. 7. Extraction time showed significant (p < 0.05) effect on %DPPH. The interaction between ethanol concentration and extraction time was statistically significant for DPPH scavenging activity. The higher antioxidant activity could be obtained at ethanol concentration of 45–65% (Fig. 7A–C). In Fig. 7D the high %DPPH was observed when solvent to material ratio and ultrasonic power were simultaneously at low level or high level. From Fig. 7F one can see that when the solvent to material ratio was at low level the extraction time showed more obvious effect on %DPPH. In Fig. 7A, D and E, the %DPPH decreased firstly and then increased when the ultrasonic power changed from 40 to 90 W. The results would be the combined contribution of the changes of WEL yield and TPC, which can be seen from Figs. 5A, D and E and 6A, D and E. 3.3.4. Optimization of UPAE and comparison with other methods The RSM guided optimization demonstrated that the optimum treatment conditions for maximizing the yield of WEL were: ethanol concentration 60%, ultrasonic power 75 W, solvent to material ratio 40 mL/g and extraction time 12 min. The optimal conditions for TPC and %DPPH were: 47% (ethanol concentration), 85 W (power), 48 mL/g (S/L), 7.5 min (time) and 43% (ethanol concentration),

56 W (power), 36 mL/g (S/L), 12.9 min (time), respectively. Furthermore, the desirability function approach of Derringer was used to search the experimental conditions that optimize all the responses simultaneously. When the three responses were given the same importance degree the predicted optimal extraction conditions were: ethanol concentration 48%, ultrasonic power 90 W, solvent to material ratio 50 mL/g and extraction time 11 min, which predicted the yield of WEL, TPC and %DPPH value as 3.92 mg/g, 22.40 mg GAE/g DW, and 55%, respectively. Based on the results of the optimization, the predicted optimum condition was verified by three parallel experiments, respectively. The results were presented in Table 4. Heat reflux extraction (HRE), ultrasonic-assisted extraction using ultrasonic bath (UBAE), and Soxhlet extraction (SE) were also applied for the extraction of WEL and total polyphenols from MHL powder (80–100 mesh). The results are shown in Table 4. UPAE-1 was the optimized method for the extraction of WEL. UPAE-2 was the optimized method for the desirability function of the three responses. The yields of WEL showed not significant difference (p > 0.05) between UPAE-1 and UPAE-2. But the TPC and %DPPH of UPAE-2 were higher (p < 0.05) than those of UPAE-1. When using 48% ethanol as solvent the yield of WEL and TPC of UPAE were higher than those of UBAE. The high yield of WEL could be obtained when using UBAE with 70% ethanol as solvent in 30 min. SE was suitable for the extraction of WEL with the highest yield of 4.01 mg/g in 180 min using methanol as solvent. HRE using 70% ethanol was suitable for the extraction of WEL. Comparison with other three methods the optimized UPAE could simultaneously obtain the highest yield of WEL, TPC and antioxidant activity in a short time and consumed a lower quantity of energy.

4. Conclusion Ultrasound-assisted extraction is a potential green method in the extraction of active components from natural products. Ultrasound-assisted extraction using ultrasonic probe is much more powerful and energy-saving than using ultrasonic bath. In the present study, an efficient ultrasound-assisted extraction using ultrasonic probe system was developed for the extraction of wedelolactone and antioxidant polyphenols from Eclipta prostrate L. The response surface methodology was successfully employed to optimize the UAE conditions including four independent variables such as ethanol concentration, ultrasonic power, solvent to material ratio and extraction time. The developed prediction models showed good correlation with the experimental data at a 95% confidence level. The maximum of the yield of WEL, TPC and %DPPH could be simultaneously obtained using the optimized UPAE. The developed UPAE process was validated to be fast, economical and reliable with highest yield compared with other three conventional methods. The developed method appears to be a green method and have great potential for the extraction of active components and antioxidants from natural products.

64

X. Fang et al. / Separation and Purification Technology 138 (2014) 55–64

Acknowledgments We greatly acknowledge the financial supports from the National Natural Science Foundation of China (No. 81274012) and State Key Laboratory of Crop Biology.

References [1] National Commission of Chinese Pharmacopoeia, Pharmacopoeia of Peoples Republic of China, Chemical Industry Press, Beijing, 2010, p. 351. [2] P. Pithayanukul, S. Laovachirasuwan, R. Bavovada, N. Pakmanee, R. Suttisri, Anti-venom potential of butanolic extract of Eclipta prostrate against Malayan pit viper venom, J. Ethnopharmacol. 90 (2004) 347–352. [3] K.C. Santhosh, S. Govindasamy, E. Sukumar, Lipid lowering activity of Eclipta prostrata in experimental hyperlipidemia, J. Ethnopharmacol. 105 (2006) 332– 335. [4] M.K. Lee, N.R. Ha, H. Yang, Antiproliferative activity of triterpenoids from Eclipta prostrata on hepatic stellate cells, Phytomedicine 15 (2008) 775–780. [5] S. Tewtrakul, S. Subhadhirasakul, S. Cheenpracha, C. Karalai, HIV-1 protease and HIV-1 integrase inhibitory substances from Eclipta prostrate, Phytother. Res. 21 (2007) 1092–1095. [6] F. Yuan, J. Chen, P.P. Sun, S. Guan, J. Xu, Wedelolactone inhibits LPS-induced pro-inflammation via NF-kappaB Pathway in RAW 264.7 cells, J. Biomed. Sci. 20 (2013) 84–90. [7] Y. Xia, J. Chen, Y. Cao, C. Xu, R. Li, Y. Pan, X. Chen, Wedelolactone exhibits antifibrotic effects on human hepatic stellate cell line LX-2, Eur. J. Pharmacol. 714 (2013) 105–111. [8] Z. Chen, X. Sun, S. Shen, H. Zhang, X. Ma, J. Liu, S. Kuang, Q. Yu, Wedelolactone, a naturally occurring coumestan, enhances interferon-c signaling through inhibiting STAT1 protein dephosphorylation, J. Biol. Chem. 288 (2013) 1417– 1427. [9] S. Sarveswaran, S.C. Gautam, J. Ghosh, Wedelolactone, a medicinal plantderived coumestan, induces caspase-dependent apoptosis in prostate cancer cells via downregulation of PKCe without inhibiting Akt, Int. J. Oncol. 41 (2012) 2191–2199. [10] Y.J. Lee, W.L. Lin, N.F. Chen, S.K. Chuang, T.H. Tseng, Demethylwedelolactone derivatives inhibit invasive growth in vitro and lung metastasis of MDA-MB231 breast cancer cells in nude mice, Eur. J. Med. Chem. 56 (2012) 361–367. [11] P. Benes, L. Knopfova, F. Trcka, A. Nemajerova, D. Pinheiro, K. Soucek, M. Fojta, J. Smarda, Inhibition of topoisomerase IIa: novel function of wedelolactone, Cancer Lett. 303 (2011) 29–38.

[12] A.A. Patil, B.S. Sachin, D.B. Shinde, P.S. Wakte, Optimization of sample preparation variables for wedelolactone from Eclipta alba using Box-Behnken experimental design followed by HPLC identification, Ann. Pharm. Fr. 71 (2013) 249–259. [13] M. Vinatoru, An overview of the ultrasonically assisted extraction of bioactive principles from herbs, Ultrason. Sonochem. 8 (2001) 303–313. [14] L. Wang, Z.Y. Wang, X.Y. Li, Optimization of ultrasonic-assisted extraction of phenolic antioxidants from Malus baccata (Linn.) Borkh. using response surface methodology, J. Sep. Sci. 36 (2013) 1652–1658. [15] R. Tabaraki, E. Heidarizadi, A. Benvidi, Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) peel antioxidants by response surface methodology, Sep. Purif. Technol. 98 (2012) 16–23. [16] M.B. Hossain, N.P. Brunton, A. Patras, B. Tiwari, C.P. O’Donnell, A.B. MartinDiana, C. Barry-Ryan, Optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (Origanum majorana L.) using response surface methodology, Ultrason. Sonochem. 19 (2012) 582–590. [17] Z. Ying, X. Han, J. Li, Ultrasound-assisted extraction of polysaccharides from mulberry leaves, Food Chem. 127 (2011) 1273–1279. [18] X.G. Chen, Y.J. Luo, B.K. Qi, Y.H. Wan, Simultaneous extraction of oil and soy isoflavones from soy sauce residue using ultrasonic-assisted two-phase solvent extraction technology, Sep. Purif. Technol. 128 (2014) 72–79. [19] S. Dey, V.K. Rathod, Ultrasound assisted extraction of b-carotene from Spirulina platensis, Ultrason. Sonochem. 20 (2013) 271–276. [20] V. Kotovicz, E.F. Zanoelo, Pulsed hydrostatic pressure and ultrasound assisted extraction of soluble matter from mate leaves (Ilex paraguariensis): experiments and modeling, Sep. Purif. Technol. 132 (2014) 1–9. [21] F. Chemat, Z. Huma, M.K. Khan, Applications of ultrasound in food technology: processing, preservation and extraction, Ultrason. Sonochem. 18 (2011) 813– 835. [22] G.A. Lewis, D. Mathieu, R. Phan-Tan-Luu, Pharmaceutical Experimental Design, Marcel Dekkler, New York, 1999. [23] V.L. Singelton, R. Orthofer, R.R. Lamuela-Raventos, Methods Enzymol. 299 (2009) 152–178. [24] W. Brand-Williams, M.E. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, LWT – Food Sci. Technol. 28 (1995) 25–30. [25] Y. Xu, S.Y. Pan, Effects of various factors of ultrasonic treatment on the extraction yield of all-trans-lycopene from red grapefruit (Citrus paradise Macf.), Ultrason. Sonochem. 20 (2013) 1026–1032. [26] J. Raso, P. Mañas, R. Pagán, F.J. Sala, Influence of different factors on the output power transferred into solvent by ultrasound, Ultrason. Sonochem. 5 (1999) 157–162. [27] X.S. Wang, Y.F. Wu, G.Y. Chen, W. Yue, Q.L. Lianga, Q.A. Wu, Optimisation of ultrasound assisted extraction of phenolic compounds from Sparganii rhizoma with response surface methodology, Ultrason. Sonochem. 20 (2013) 846–854.