Food Chemistry 141 (2013) 1361–1368

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Food Chemistry

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Analytical Methods

Optimisation of ultrasonic-assisted extraction of antioxidant compoundsfrom Artemisia absinthium using response surface methodology

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.04.003

⇑ Corresponding author. Tel.: +90 224 2941747; fax: +90 224 2941899.E-mail address: salihabilgi@uludag.edu.tr (S. S�ahin).

Saliha S�ahin ⇑, Önder Aybastıer, Esra Is�ıkUniversity of Uludag, Faculty of Science and Arts, Department of Chemistry, 16059 Bursa, Turkey

a r t i c l e i n f o

Article history:Received 27 March 2012Received in revised form 20 March 2013Accepted 2 April 2013Available online 10 April 2013

Keywords:Ultrasonic-assisted extractionArtemisia absinthiumResponse surface methodologyAntioxidant capacityPhenolic compounds

a b s t r a c t

Response surface methodology was used to optimise experimental conditions for ultrasonic-assistedextraction of phenolic compounds from Artemisia absinthium. The central composite design wasemployed, the extracts were characterised by the determination of total phenolic content and antioxidantcapacity. The total phenolic contents of extracts were determined by Folin method and also total antiox-idant capacities of extracts were determined by ABTS and CUPRAC methods. The phenolic compounds ofA. absinthium at optimum extraction conditions were determined by HPLC-DAD. The optimum conditionswere determined as HCl concentration between 0.41 and 0.44 mol/L, methanol volume between 55% and59% (v/v), extraction temperature between 64 and 70 �C, extraction time between 101 and 107 min. Theexperimental values agreed with those predicted within a 95% confidence level, thus indicating the suit-ability of response surface methodology in optimising the ultrasound-assisted extraction of phenoliccompounds from A. absinthium.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Artemisia absinthium (wormwood) distributed in Europe andAsia (Tan, Zheng, & Tang, 1998) is commonly used in food industryin the preparation of aperitives, bitters and spirits (Mishra, Naik,Srivastava, & Prasad, 1999). In the late nineteenth century, A. absin-thium, in the meantime called ‘‘green fairy’’ was the most popularspirit drink in Europe (Lachenmeier, Walch, Padosch, & Kröner,2006). Phytochemical studies have revealed that A. absinthium con-tains essential oils, sesquiterpenoid lactones, flavonoids, phenolicacids and lignans (Wichtl, 1994). Essential oils (0.2–1.5%), whichhave a colour ranging from dark-green or brown to blue, give anastringent bitter taste and smell strongly of A. absinthium. Severalflavonol-3-glycosides have been isolated from leaves of A. absin-thium such as quercetin, isorhamnetin, patuletin and spinacetinderivates (Hoffmann & Herrmann, 1982). A. absinthium extractshave been demonstrated to possess strong antioxidant and anti-parasitic activity (Canadanovic-Brunet, Djilas, Cetkovic, & Tumbas,2005). And, the aqueous methanolic extract has been reported toprotect the liver against chemical toxins such as acetaminophenand CCl4 (Gilani & Janbaz, 1995).

The phenolic compounds have been shown to have anticarcino-genic, antimicrobial, antiviral, antimutagenic and antioxidant prop-erties (Lampe, 2003; Sarıburun, S�ahin, Demir, Türkben, & Uylas�er,2010; Srinivasan, 2005; S�ahin, Demir, & Malyer, 2011). This diverse

range of biological properties makes spice phenolic compounds aninteresting target for optimising extraction from natural source(Hossain, Barry-Ryan, Martin-Diana, & Brunton, 2011). Because ofdifference in phenolic compounds with respect to polarity, acidity,number of hydroxyl groups and aromatic rings, concentration, andmatrix complexity, specific extractions techniques must be de-signed and optimised for each phenolic. Many factors contributeto the efficiency of extraction such as the type and concentrationof the solvent, pH, temperature and time, pressure and the particlesize (Juntachote, Berghofer, Siebenhandl, & Bauer, 2006). Therefore,it is appropriate to choose optimal pre-treatment according to thechemical structure and properties of target compounds.

Conventional extractions such as heating, boiling, or reflux canbe used to extract phenolic compounds. However, disadvantagesinclude the loss of phenolic compounds due to oxidation, ionisa-tion and hydrolysis during extraction as well as the long extrac-tions times (Li, Chen, & Yao, 2005). Recently, new extractiontechniques have been developed for the extraction of targetcompounds from different materials including ultrasound- andmicrowave-assisted, supercritical fluid and accelerated solventextraction (Wang & Weller, 2006). Among these, ultrasound-as-sisted extraction is an inexpensive, simple and efficient alternativeto conventional extraction techniques (Wang, Sun, Cao, Tian, & Li,2008). The mechanism of action for ultrasound-assisted extractionare attributed to cavitation, mechanical forces and thermal impact,which result in disruption of cells walls, reduce particle size, andenhance mass transfer across cell membranes (Pan, Qu, Ma, Atung-ulu, & McHugh, 2011).

1362 S. S�ahin et al. / Food Chemistry 141 (2013) 1361–1368

Plant material extracts are a mixture of different classes ofphenolics that are soluble in a variety of solvents. The most com-mon solvents are aqueous mixtures with methanol, ethanol andacetone (Borges, Vieira, Copetti, Gonzaga, & Fett, 2011; Ma, Chen,Liu, & Ye, 2009; Rodrigues, Pinto, & Fernandes, 2008). Various chro-matographic techniques have been employed for separation andquantification of phenolic compounds. High performance liquidchromatography (HPLC) has been most widely used for both sepa-ration and quantification of phenolic compounds (Türkben,Sarıburun, Demir, & Uylas�er, 2010).

Response surface methods have been used widely for the pro-duction and optimisation of different industrially important bio-technological and biochemical products (Aybastıer & Demir,2010; Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). A centralcomposite design (CCD) is a suitable for response surface methodsbecause optimisation with CCD not only allows rapid screening of awide range conditions but also indicates the role of each factor(Aybastıer, S�ahin, Is�ık, & Demir, 2011). CCD is used to describethe individual and cumulative effect of the parameters on theresponse. CCD has been the most successful factorial design forthe optimisation of parameters with a limited number of experi-ments (Aybastıer & Demir, 2010; Bezerra et al., 2008).

The aim of this work was to employ response surface method-ology to optimise the extraction parameters for A. absinthiumbased on phenolic content and antioxidant capacity. Optimisationof temperature, time, solvent and acid concentration for extractionof antioxidant compounds from A. absinthium was carried outusing ultrasound-assisted extraction. A five level, four-variablecentral composite design was employed to maximize simulta-neously the total phenolic content and total antioxidant capacity.

Table 1Central composite design of factors with coded values.

Treatment Factors

x1 x2 x3 x4

HClconcentration

Methanolvolume (%, v/v)

Temperature(�C)

Time(min)

2. Materials and methods

2.1. Plant material

Dried A. absinthium was obtained from Akıllıoglu Akdem, Tur-key. The purchased samples were milled into uniform dry powderby rondo and stored at 4 �C.

(mol/L)

1 �1 �1 �1 12 1 �1 �1 13 �1 1 �1 14 1 1 �1 15 �1 �1 1 16 1 �1 1 17 �1 1 1 18 1 1 1 19 �1 �1 �1 �110 1 �1 �1 �111 �1 1 �1 �112 1 1 �1 �113 �1 �1 1 �114 1 �1 1 �115 �1 1 1 �116 1 1 1 �1

2.2. Chemicals and reagents

2,20-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS),Folin–Ciocalteu reagent, trolox [(±)-6-hydroxy-2,5,7,8-tetrame-thylchroman-2-carboxylic acid] were purchased from Sigma.Methanol and acetonitrile and formic acid were obtained fromMerck (Merck, Darmstadt, Germany). Protocatechuic acid(P97.0% HPLC grade), chlorogenic acid were purchased from Fluka(Switzerland). Ferulic acid (P99.0% HPLC grade) and rosmarinicacid (P96.0% HPLC grade) were supplied by Aldrich (Germany).Also caffeic acid (P98.0% HPLC grade) was supplied by Sigma(Germany). All standard solutions were prepared in methanol.

17 �2 0 0 018 2 0 0 019 0 �2 0 020 0 2 0 021 0 0 �2 022 0 0 2 023 0 0 0 �224 0 0 0 225 0 0 0 026 0 0 0 027 0 0 0 028 0 0 0 029 0 0 0 030 0 0 0 0

2.3. Ultrasound-assisted extraction

Ultrasound-assisted extraction was performed in a temperaturecontrolled ultrasonic cleaner. Furthermore, temperature was alsomonitored with thermometer. The A. absinthium powder (0.5 g)was put in a glass vial (45 mL) and acidic methanol solvent(30 mL) added before the vial was placed in the ultrasonic cleaningbath (United) at 40 kHz. The acidic methanol solution was pre-pared across the range of concentrations of HCl (0.4–1.6 mol/L)and methanol volume (20–80%, v/v). The extract was filtered andanalysed.

2.4. Experimental design

The response surface methodology (RSM) is an empirical statis-tical technique employed for multiple regression analysis usingquantitative data. It uses multivariable data obtained from care-fully designed experiments to resolve multivariable scenarios,simultaneously (Tan, Ahmad, & Hameed, 2008). The total numberof experiments (N) in a central composite design (CCD) can be cal-culated using the following Eq. (1):

N ¼ 2k þ 2kþ x0 ð1Þ

where k is the number of variables and x0 is the number of centralpoints. Thus a five-level-four-factor CCD was employed in this study,requiring 30 (k = 4; x0 = 6) experiments for the optimisation ofextraction parameters. Among them, six replications were at centerpoints, while the axial points were determined to be

p4 = 2 (Singh,

Gupta, Singh, & Sinha, 2011). Thirty experiments were performedaccording to Table 1 to optimise the parameters (Aybastıer & Demir,2010). Twenty-four experiments were augmented with six replica-tions at the center points to evaluate the pure error (Yuan et al., 2008).

The parameters and their levels were HCl concentration (0.4–1.6 mol/L), methanol volume (20–80%, v/v), temperature (30–70 �C) and time (20–120 min) (Table 2).

A second-order polynomial Eq. (2), which includes all terms,was used to calculate the predicted response:

y ¼ b0 þX4

i¼1

bixi þX4

i¼1

biix2i þ

X3

i¼1

X4

j¼iþ1

bijxixj ð2Þ

where y is response; b0 is the offset term, bi is the linear effect, bii isthe squared effect, bij is the interaction effect and xi and xj are inde-pendent variables.

Table 2Range of coded and actual values for central composite design.

Factor Level

�2 �1 0 1 2

HCl concentration (M) 0.4 0.7 1 1.3 1.6Methanol concentration (%, v/v) 20 35 50 65 80Extraction temperature (�C) 30 40 50 60 70Extraction time (min) 20 45 70 95 120

S. S�ahin et al. / Food Chemistry 141 (2013) 1361–1368 1363

The experimental results of the CCD were fitted with a second-order polynomial equation by a multiple regression technique. Thedata were analysed using Design Expert program (7.0.0 version)and the coefficients were interpreted using F test. Three main ana-lytical steps: analysis of variance (ANOVA), regression analysis andplotting of response surface plot were performed to establish opti-mum conditions for antioxidant capacities and total phenoliccontent.

2.5. HPLC-DAD analysis

An Agilent 1200 HPLC system (Waldbronn, Germany), consist-ing of a vacuum degasser, binary pump, autosampler and adiode-array detector, was used for determination of phenolic com-pounds in fractions. Chromatographic separations were carried outusing an XBridge C18 (4.6 � 250 mm, 3.5 lm) column fromWaters. The mobile phase consisted of 1% formic acid in water (sol-vent A) and acetonitrile (solvent B). Gradient conditions were asfollows; 0–10 min 13% B, 10–20 min 41.5% B, 20–25 min 70% B,25–35 min 10% B, total run time is 35 min. The column was equil-ibrated for 10 min prior to each analysis at 25 �C. Flow rate was0.5 mL/min and injection volume was 10 lL. A Chemstation forLC (Agilent) was used for data acquisition and pre-processing.The monitoring wavelengths of interest were 280 nm for proto-catechuic acid and 360 nm for chlorogenic acid, caffeic acid, ferulicacid, rosmarinic acid. Peaks were identified on the basis of compar-ison of retention times and UV spectra with standards of proto-catechuic acid, chlorogenic acid, caffeic acid, ferulic acid androsmarinic acid.

2.6. Folin–Ciocalteu method

The total phenolic content by Folin–Ciocalteu reagent was car-ried out according to the procedure reported in the literature(Apak, Güçlü, Özyürek, & Çelik, 2008). The solution used in the Fo-lin assay of phenolics were prepared as follows: Lowry A: 2% aque-ous Na2CO3 in 0.1 M NaOH; Lowry B: 0.5% CuSO4 aqueous solutionin 1% NaKC4H4O6 solution; Lowry C: prepared freshly as mixture50 mL Lowry A and 1 mL Lowry B; Folin–Ciocalteu reagent was di-luted with H2O at a volume ratio 1:3 prior to use. The sample(0.1 mL), 1.9 mL of H2O and 2.5 mL of Lowry C solution were mixedand the mixture was let to stand for 10 min. At the end of this per-iod, 0.25 mL of Folin reagent was added, and 30 min was allowedfor stabilisation of the blue colour. The absorbance was measuredby spectrophotometry (Varian Cary 50, Australia) at 750 nm. Totalphenols were expressed as mg of gallic acid equivalent (GAE) per gof dried weight.

2.7. ABTS method

The total antioxidant capacity of extract was determined withABTS method, as described in the literature (Apak et al., 2008).ABTS�+ was produced by reacting 20 mM ABTS solution with2.45 mM potassium persulphate solution and allowing the mixtureto stand in dark at room temperature for 12–16 h before use. Theprocedure for A. absinthium was performed by adding 0.25 mL

extract, 3.75 mL of ethanol and 1 mL of the ABTS�+ radical cationsolution which was diluted with ethanol at a ratio of 1:10 andthe absorbance was recorded at 734 nm against blank after6 min. The results were expressed as mg of trolox equivalent (TE)per g dried weight.

2.8. CUPRAC method

The cupric ion reducing antioxidant capacity of A. absinthiumwas determined according to the method of (Apak et al., 2008).To a test tube, 1 mL each of CuCl2 solution (1.0 � 10–2 mol/L), neo-cuproine alcoholic solution (7.5 � 10–3 mol/L), and NH4Ac (1 mol/L,pH 7.0) buffer solution and 1 mL of water and 0.1 mL of extract wasadded to the initial mixture so as to make the final volume 4.1 mL.After 30 min, the absorbance was recorded at 450 nm against thereagent blank. The results were expressed as mg of trolox equiva-lent (TE) per g dried weight.

3. Results and discussion

3.1. Fitting the models

It was necessary to investigate the extraction variables in orderto determine the best combination of variables for the total pheno-lic content and antioxidant capacity from A. absinthium. Prelimin-ary trials enabled the range of HCl concentration (0.4–1.6 mol/L),methanol volume (20–80%, v/v), temperature (30–70 �C) and time(20–120 min) to be fixed. The experimental and predicted data interms of total phenolic content and antioxidant capacity are shownin Table 3.

Among the 30 experiments including 6 replicates (Table 3),experiment 18 (HCl concentration 1.6 mol/L, methanol volume50% (v/v), temperature 50 �C and time 70 min) provided the high-est total phenolic content (55.87 mg GAE/g dried plant) and exper-iment 19 (HCl concentration 1.0 mol/L, methanol volume 20% (v/v),temperature 50 �C and time 70 min) produced the least phenolics(41.6 mg GAE/g dried plant). Experiment 11 for ABTS (HCl concen-tration 0.7 mol/L, methanol volume 65% (v/v), temperature 40 �Cand time 95 min) and experiment 22 for CUPRAC (HCl concentra-tion 1.0 mol/L, methanol volume 50% (v/v), temperature 70 �Cand time 70 min) offered the highest total antioxidant capacity(57.70 mg TE/g dried plant for ABTS and 270.17 mg TE/g driedplant for CUPRAC). Experiment 26 for ABTS (HCl concentration1.0 mol/L, methanol volume 50% (v/v), temperature 50 �C and time70 min) and experiment 19 for CUPRAC (HCl concentration1.0 mol/L, methanol volume 20% (v/v), temperature 50 �C and time70 min) had the lowest antioxidant capacity (41.53 and164.18 mg TE/g dried plant obtained by ABTS and CUPRAC meth-ods, respectively).

The effects of each factor and their interaction were calculatedusing a Design Expert program (version 7.0.0). Fitting the data withvarious models and, subsequently, the analysis of variance (ANO-VA) showed that total phenolic content and total antioxidantcapacity were best described with quadratic polynomial model.The quadratic polynomial model was highly significant and suffi-cient to represent the actual relationship between the responseand significant parameters with very low p-value (<0.0001 for Fo-lin and CUPRAC, 0.0001 for ABTS) from the ANOVA (Table 4). Themodel F-value (14.98, 8.09 and 10.43 for Folin, ABTS and CUPRACmethods, respectively) implies it was significant at 95% confidencelevel. The model also showed statistically insignificant lack of fit, asis evident from the computed F-values of 0.1783, 0.0506 and0.0663 at 95% confidence level for Folin, ABTS and CUPRAC meth-ods, respectively. Furthermore the value of pure error was low,which indicates good reproducibility of the data obtained with a

Table 3Central composite design of factors with experimental and predicted values.

Treatment Total phenolic content (mg GAE/g dried plant) Total antioxidant capacity (mg TE/g dried plant)

ABTS CUPRAC

Experimental Predicted Experimental Predicted Experimental Predicted

1 42.59 42.8 46.33 47.44 219.96 205.522 47.48 47.17 46.81 44.8 176.47 167.813 45.67 44.71 55.3 52.88 230.75 233.974 48.73 49.73 47.59 48.28 214.33 213.075 48.01 47.46 49.65 49.32 240.13 247.546 49.84 49.58 48.08 46.68 192.87 187.387 49.9 48.74 52.52 50.62 266.14 253.468 52.5 51.51 44.69 46.03 203.9 210.119 45.06 44.68 51.06 49.68 208.04 201.110 45.43 47.29 45.88 45.2 203.03 210.0311 48.99 49.95 57.7 56.53 225.41 225.2112 54.02 53.19 49.8 50.1 259.11 250.9613 50.07 49.77 57.32 54.06 247.15 242.7214 50.53 50.12 47.21 49.6 233.16 229.2115 55.47 54.4 54.81 56.78 236.38 244.3116 54.9 55.39 54.05 50.36 238.86 247.6117 48.61 49.89 55.23 57.61 240.61 247.4618 55.87 55.26 48.32 48.55 213.49 213.0519 41.6 41.33 41.59 43.06 164.18 175.7220 47.58 48.52 48.12 49.26 227.69 222.5721 48.57 47.46 50.94 52.41 214.91 226.4122 52.54 54.32 53.42 54.55 270.17 265.0823 44.18 45.35 42.43 43.59 191.19 200.8224 51.62 51.12 48.7 50.15 237.11 233.8925 50.32 51.2 42.71 42.95 213.01 214.6326 51.25 51.2 41.53 42.95 220.36 214.6327 49.98 51.2 42.51 42.95 205.3 214.6328 51.32 51.2 43.73 42.95 219.89 214.6329 52.51 51.2 45.02 42.95 211.54 214.6330 51.81 51.2 42.19 42.95 217.66 214.63

Table 4Analysis of variance (ANOVA) for the fitted quadratic polynomial model for optimization of extraction parameters.

Source Folin (R2 = 0.9332) ABTS (R2 = 0.8831) CUPRAC (R2 = 0.9068)

DF SS MS F value p Value DF SS MS F value p Value DF SS MS F value p Value

Model 14 348.71 24.91 14.98 <0.0001 14 608.94 43.50 8.09 0.0001 14 15130 1080.72 10.43 <0.0001Lack of fit 10 20.58 2.06 2.36 0.1783 10 72.86 7.29 4.71 0.0506 10 1386.06 138.61 4.10 0.0663Pure error 5 4.37 0.87 5 7.74 1.55 5 168.90 33.78

DF, degree of freedom; SS, sum of squares; MS, mean square.

Table 5Second order polynomial equations and regression coefficients of the response variables (the HCl concentration; x1, the methanol concentration; x2, the extraction temperature;x3, the extraction time; x4).

Responses Second order polynomial equations

Total phenol content (mg GAE/g dried plant) y = 51.20 + 1.34x1 + 1.80x2 + 1.72x3 + 1.44x4 + 0.84x2x4 � 1.57x22–0.74x2

4

ABTS (mg TE/g dried plant) y = 42.95 � 2.27x1 + 1.55x2 + 0.53x3 + 1.64x4 + 2.53x21 + 2.63x2

3 + 0.98x24

CUPRAC (mg TE/g dried plant) y = 214.63 � 8.60x1 + 11.71x2 + 9.67x3 + 8.27x4 � 5.61x1x3 + 11.66x1x4 � 5.63x2x3 + 7.78x23

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small p-value from the ANOVA and a satisfactory coefficient ofdetermination (Table 4). The coefficient of determination also re-vealed that there are excellent correlations between the indepen-dent variables.

3.2. Response surface analysis of total phenolic content

The effects of extraction parameters such as HCl concentration,methanol volume, temperature and time were investigated on theultrasonic-assisted extraction of antioxidant compounds from A.absinthium. The significance of each coefficient was determinedby F-values and p-values, which are listed in Table 5. Response

surface analysis of the data in Table 5 demonstrated the relation-ship between the total phenolic content and extraction parameterswas quadratic with good regression coefficient (R2 = 0.9332). Thelarger the magnitude of the F-value and smaller the p-value, themore significant the corresponding coefficient. x1 (HCl concentra-tion), x2 (methanol volume), x3 (temperature), x4 (time), x2x4, x2

2

and x24 were the most significant parameters (p-value less than

0.05). However, x1x2, x1x3, x1x4, x2x3, x3x4, x21 and x2

3 had less effect(p-value more than 0.05) on the ultrasonic-assisted extraction fortotal phenolic content values.

The relationship between extraction parameters and total phe-nolic content were investigated by response surface plots. Fig. 1

Fig. 1. Response surface plots of Artemisia absinthium showing the effect of (A) HCl concentration and temperature, (B) HCl concentration and time, (C) methanol volume andtime on total phenolic content.

S. S�ahin et al. / Food Chemistry 141 (2013) 1361–1368 1365

shows the effect of HCl concentration, methanol volume, temper-ature, time and their mutual interaction on the total phenoliccontent. The highest total phenolic content was observed at lowerHCl concentrations and higher temperature (Fig. 1A). However,the increase in temperature at a fixed HCl concentration led toan increase in the total phenolic content, and reached a maxi-mum at the highest temperature tested (Fig. 1A). High tempera-tures might have increased the diffusion and solubility rate ofthe many compounds resulting in antioxidant compounds beingextracted at a higher rate. However, elevated temperatures couldalso affect the activity of the extracts due to the degradation andloss of the phenolic compounds (Dorta, Lobo, & Gonzalez, 2012;Yap et al., 2009) or phenolic compounds reacting with other com-ponents of the plant material. This behavior is similar to the find-ings in the literature describing extraction of banana peel(Gonzalez-Montelongo, Lobo, & Gonzalez, 2010). However, the ef-fect of changing extraction temperature was significant forextracting phenolic compounds in A. absinthium. It can be seenfrom Table 2 (experiment 21 and 22) that raising the extractiontemperature from 30 to 70 �C yielded a higher content of phenoliccompounds and antioxidant capacity in extracts obtained from A.absinthium.

The effect of HCl concentration and time shown in Fig. 1B dem-onstrated that the total phenolic content increased with increasingHCl concentration over a shorter time. The total phenol content in-creased with increasing time, at higher and lower HCl concentra-tions. Fig. 1C shows the effect of time and methanol volume onthe ultrasonic-assisted extraction of antioxidant compounds fromA. absinthium. Methanol had a positive linear impact on the totalphenolic content, and was a controlling factor in total phenolicrecovery. The total phenolic content started to decrease above65% (v/v). According to the literature (Escribano-Bailon & Santos-Buelga, 2003), a maximum recovery of phenolic compounds frommost plants is achieved with 50% of methanol. We found similarresults for the extraction of antioxidant compounds from A.absinthium.

3.3. Response surface analysis of total antioxidant capacity

The response surface analysis in Table 4 also shows goodregression values (R2 = 0.8831 for ABTS and 0.9068 for CUPRAC)and the relationship between total antioxidant capacity andextraction parameters such as HCl concentration, methanol vol-ume, temperature and time. The quadratic polynomial equations

Fig. 2. Response surface plots of Artemisia absinthium showing the effect of (A) HCl concentration and methanol volume, (B) methanol volume and temperature, (C)temperature and time on ABTS values.

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with significance for antioxidant activity (ABTS and CUPRAC) fromA. absinthium are given in Table 5. x1, x2, x3, x4, x2

1, x23, x2

4 for ABTSand x1, x2, x3, x4, x1x3, x1x4, x2x3, x2

3 for CUPRAC were the mostsignificant parameters on the ultrasonic-assisted extraction ofantioxidant compounds A. absinthium x1x2, x1x3, x1x4, x2x3, x2x4,x3x4, x2

2 for ABTS and x1x2, x2x4, x3x4, x21, x2

2, x24 for CUPRAC had less

effect on total antioxidant capacity values from ultrasonic-assistedextraction.

The relationship between extraction parameters and antioxi-dant capacity were investigated by response surface plots in theFigs. 2 and 3. Fig. 2A shows the effect of HCl concentration, meth-anol volume and their mutual interaction on the antioxidantcapacity as determined using the ABTS method. An increase inantioxidant capacity was observed with increasing methanol vol-ume. A decrease in the antioxidant capacity was observed withincreasing HCl concentration at first, but the trend was reservedwhen the HCl concentration reached to 1.1 mol/L and antioxidantcapacity increased thereafter. Fig. 2B represents the effect of vary-ing methanol volume, temperature and their mutual interaction onthe antioxidant capacity as determined using the ABTS method.Antioxidant capacity decreased with increasing methanol volumeat the high temperature (80 �C) and it increased with decreasingmethanol volume at the low temperature (20 �C). The effect of

time, temperature and their mutual interaction on the antioxidantcapacity as determined using the ABTS method was illustrated inFig. 2C. Generally, antioxidant capacity increased with increasingtime. Antioxidant capacity decreased with increasing temperatureinitially but increased above 50 �C. Highest antioxidant capacitywas observed at higher temperatures (80 �C) and longer times(120 min).

Fig. 3A represents the effect of varying HCl concentration,extraction temperature their mutual interaction on the antioxidantcapacity using the CUPRAC method. Antioxidant capacity increasedwith increasing temperature at the low HCl concentration. Highestantioxidant capacity was observed at lower HCl concentration(0.4 mol/L) and higher temperature (70 �C). Fig. 3B shows the effectof HCl concentration, time and their mutual interaction on theantioxidant capacity using the CUPRAC method. A decrease in anti-oxidant capacity was observed with increasing of HCl concentra-tion at shorter times (20 min). The effect of methanol volume,temperature and their mutual interaction on the antioxidantcapacity using the CUPRAC method is illustrated in Fig. 3C. An in-crease in the antioxidant capacity was observed with increasingmethanol volume up to 50%, but decreased with further additionof methanol. Antioxidant capacity increased with increasing meth-anol volume at lower temperatures (30 �C).

Fig. 3. Response surface plots of Artemisia absinthium showing the effect of (A) HCl concentration and temperature, (B) HCl concentration and time, (C) methanol volume andtemperature on CUPRAC values.

Table 7The amounts of phenolic compounds (milligrams per gram dried plant) extracted from Artemisia absinthium L. by using optimum ultrasonic-assisted extraction.

Optimum conditions Protocatechuic acid Chlorogenic acid Caffeic acid Ferulic acid Rosmarinic acid

Folin 0.09 ± 0.01 4.64 ± 0.04 1.99 ± 0.03 17.30 ± 0.52 7.82 ± 0.01ABTS 0.08 ± 0.01 4.50 ± 0.11 1.95 ± 0.02 16.30 ± 0.54 7.59 ± 0.12CUPRAC 0.10 ± 0.01 5.72 ± 0.11 2.07 ± 0.01 19.57 ± 0.44 4.57 ± 0.10

Table 6Optimum conditions, predicted and experimental values of responses.

Responses Optimum ultrasonic-assisted extraction conditions Maximum values

HCl concentration(M)

Methanol concentration(%, v/v)

Extraction temperature(�C)

Extraction time(min)

Predicted Experimental

Total phenolic content (mg GAE/g driedplant)

0.44 59 70 101 57.83 60.58 ± 1.03

ABTS (mg TE/g dried plant) 0.43 58 64 107 70.29 68.96 ± 2.80CUPRAC (mg TE/g dried plant) 0.41 55 70 105 296.05 294.27 ± 5.82

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1368 S. S�ahin et al. / Food Chemistry 141 (2013) 1361–1368

3.4. Optimisation of extraction parameters and validation of the model

The optimum ultrasonic-assisted extraction conditions for thetotal phenolic content and the total antioxidant activities from A.absinthium are given in Table 6. HCl concentrations in the range0.41–0.44 mol/L, methanol volumes in the range 55–59% (v/v),ultrasonic-assisted extraction temperature of 64–70 �C and timein the range 101–107 min produced the optimal total phenoliccontent (57.83 mg GAE/g dried plant) and total antioxidant activi-ties (70.29 mg TE/g dried plant for ABTS and 296.05 mg TE/g driedplant for CUPRAC) from A. absinthium. The predicted resultsmatched well with experimental results obtained using optimumextraction conditions, which were confirmed with good correlation(R2 > 0.95) As a result, the model from central composite designwas considered to be accurate and reliable for predicting the totalphenolic content and the total antioxidant activities of extracts ob-tained from A. absinthium for ultrasonic assisted extraction.

3.5. HPLC analysis

Phenolic compounds [rosmarinic acid, quercetin, rutin, chloro-genic acid, caffeic acid, ferulic acid, gallic acid, protocatechuic acid,p-hydroxybenzoic acid, ellagic acid, kaempferol 3-b-D-glucopyran-oside, myricetin, 2-hydroxybenzoic acid, protocatechuic acid, vani-lic acid, p-coumaric acid, luteolin, kaempferol, trans-cinnamic acid,(+)-catechin and (�)-epicatechin)], which it was thought might bein A. absinthium, were investigated. Protocatechuic acid, chlorogen-ic acid, caffeic acid, ferulic acid and rosmarinic acid were deter-mined in extract of A. absinthium using the optimum extractionconditions. The amounts of phenolic compounds of extract in A.absinthium determined by HPLC-DAD are shown in Table 7. Theamount of individual phenolic compounds (expressed as mg/gdried plant) were as follows: 0.10 ± 0.01 protocatechuic acid,5.72 ± 0.11 chlorogenic acid, 2.07 ± 0.01 caffeic acid, 19.57 ± 0.44ferulic acid, 7.82 ± 0.01 rosmarinic acid in A. absinthium. Theamounts of ferulic acid, caffeic acid and chlorogenic acid deter-mined were higher than previously reported by Craciunescu et al.,2012. On the other hand, there is no information in literature aboutprotocatechuic acid and rosmarinic acid contents in A. absinthium.

4. Conclusions

Response surface methodology was successfully employed tooptimise extraction from A. absinthium using ultrasonic-assistedextraction. Optimised conditions for maximum extraction totalphenolics and total antioxidant capacity were determined includ-ing HCl concentration, methanol concentration, extraction temper-ature and extraction time, which were identified as controllingfactors. Protocatechuic, chlorogenic, caffeic, ferulic and rosmarinicacids were determined by HPLC-DAD in ultrasonic-assisted ex-tracts from A. absinthium. This study indicates that A. absinthiumcan be considered a good source of naturally-occuring antioxidantcompounds, which have an application in food industry.

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