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Extraction of Polyphenolic Antioxidants from Green Tea by

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Extraction of Polyphenolic Antioxidants from Green Tea by

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Extraction of Polyphenolic Antioxidants from Green Tea by

    Ultrahigh Pressure Technique

    XI Jun

    5 (School of Chemical Engineering, Sichuan University, ChengDu 610065)

    Abstract: A new extraction technique, ultrahigh pressure extraction, was used to obtain antioxidants from green tea. The response surface methodology was employed to optimize the extraction process. The effects of ethanol concentration (33.2-66.8 %), pressure (281.8-618.2 MPa) and liquid/solid ratio (11.6-28.4 mL/g) on the total phenolic content and DPPH free radical scavenging activity were

    10 investigated. ANOVA showed that second order polynomial models produced a satisfactory fitting of the experimental data with regard to total phenolic content (R2 = 0.9996, P<0.0001) and antioxidant capacity (R2 = 0.9986, P<0.0001). The optimal condition determined was as follows: ethanol concentration 50%, pressure 490 MPa and liquid/solid ratio 20 mL/g. Under this condition, the maximum total phenolic content and antioxidant activity of 583.8?0.9 mg/g DW and 85.6?0.7% could

    15 be achieved, respectively, which were well matched with the predicted value.

    Keywords: ultrahigh pressure extraction; total phenolic content; antioxidant activity; response surface methodology; green tea

    0 Introduction

    20 Green tea, a water extract of the non-fermented leaves of Camellia sinensis L., is a very

    popular drink in East Asian countries and is becoming increasingly popular worldwide, partly

    [1]. because of more documented evidence about its beneficial health propertiesSome studies have

    suggested that these properties are related to the antioxidant activity coming from polyphenols. Tea polyphenols account for 30-42% of the dry weight of green tea leaves due to climate, season

    [2] 25 or variety. The main polyphenols in green tea are epicatechin, epicatechin-3-gallate,epigallocatechin and epigallocatechin-3-gallate, with the latter playing the most important role in the total antioxidant capacity of green tea. The tea polyphenols are free radical scavengers, metal

    [3]chelators, inhibitors of transcription factors and enzymes. Therefore green tea extracts have been

    used as natural antioxidants, antibacterial and antiviral agents. Also, it has been reported that tea

    [4] 30 polyphenols has anticarcinogenic and antimutagenic activity.

    Extraction is the initial and the most important step in the recovery and purification of bioactive compounds from plant materials. In general, the conventional techniques for green tea extraction are heating, boiling, Soxhlet extraction and cold extraction, which are all limited by

    [5]long extraction periods and low extraction efficiency. Recently, a new extraction technique,

    35 ultrahigh pressure extraction (UPE), has been widely employed in the extraction of target compounds from different plant materials. It yields some advantages, such as short extraction time, mild extraction condition, high extraction yield, less impurity, high reproducibility at shorter times,

    [6-8]simplified manipulation, and lowered energy input, as well as solvent consumption. Above all,

    this extraction technique could be operated at ambient temperature, so the structural change and

    40 degradation of green tea ingredients can be avoided. Thus, UPE may be an effective and advisable technique for the extraction of green tea.

    In order to maximize the polyphenolic content and antioxidant activity, it is a prerequisite to design an optimal extraction conditions. Response surface methodology (RSM) has been used

     increasingly to optimize processing parameters. RSM explores the relationship between several

Foundations: The Specialized Research Fund for the Doctoral Program of Higher Education (No.

    20100181120076).

    Brief author introduction:XI Jun, (1973-), Male, Lecturer, Separation and purification engineering. E-mail: xijun@scu.edu.cn

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    45 explanatory variables and one or more response variables by means of a mathematical model,

    which is able to properly predict the values of the response variables. In this methodology,

    multivariate experiments are designed to reduce the number of assays necessary to optimize the

    process and to gather results more precise than those obtainable by traditional full factorial

    [9]designs.

    50 To our knowledge, there is little information on the optimization of extraction of

    polyphenolic antioxidants from green tea using ultrahigh pressure extraction. Hence, the

    objectives of this study were to explore the potential of ultrahigh pressure extraction of green tea

    in producing polyphenolic antioxidants and to optimize extraction parameters (ethanol

    concentration, pressure and liquid/solid ratio) using RSM approach to obtain the highest 55 polyphenolic content and antioxidant capacity. The correlation between the antioxidant activity of

    green tea extracts and their total polyphenol content was also determined.

    1 Materials and methods

    1.1 Plant materials and chemicals

    The fresh green tea leaves (Thea sinensis L.) (Place of origin: Hangzhou, China) were

    60 purchased from a local market. The sample was dried for 24h using a hot air oven at 50?, and

    then ground into powder using a milling machine and the material that passed through a 60 mesh

    sieve was kept in sealed polyethylene bags at -20? until use.

    Ethanol used in the experimental work was analytical reagent grade chemicals (Beijing

    Chemical Reagents Company, Beijing, China). Deionized water was prepared using a Milli-Q Plus 65 system (Millipore, USA). α, α-diphenyl-b-picrylhydrazyl (DPPH) was purchased from

    Sigma-Aldrich Chemical Co. (Sigma, USA). Folin-Ciocalteau reagent and other chemicals for

    analysis of tea polyphenols were also from Beijing Chemical Reagents Company (analytical grade,

    Beijing, China). Gallic acid, pharmaceutical grade standard, was purchased from the National

    Institute for Control of Pharmaceuticals and Biological Products (China). Other reagents were of 70 analytical grade and purchased from Chengdu Chemical Industry (Chengdu, China). The

    spectrophotometer (751-GW) was from Shanghai Analytical Instrument Overall Factory

    (Shanghai, China).

    The ultrahigh pressure apparatus was purchased from Shanghai Dalong Super-high Pressure

    Machine Co. Ltd. (Shanghai, China). Effective volume of vessel: 5 L; maximal working pressure: 75 700 MPa; inner diameter: 200 mm; pressure transmitting media: water and glycol (20/80, v/v).

    1.2 Ultrahigh pressure extraction

    The dried green tea powder (10 g) was mixed with aqueous ethanol at desired concentrations,

    and then placed into a sterile polyethylene bag. The bag was sealed after eliminating air from the

    inside and placed into the pressure vessel. The extraction was carried out at ambient temperature 80 for 15 min using an ultrahigh pressure apparatus at specified pressure and liquid/solid ratio as

    dictated by the experimental design. The optimization procedure was designed based on a central

    composite design consisting of ethanol concentration (33.2-66.8%), pressure (281.8-618.2 MPa)

    and liquid/solid ratio (11.6-28.4 mL/g), using five levels of each variable as shown in Table 1.

    After extraction, the mixture was filtered through a filter paper. The extracts were centrifuged at 85 4000×g for 10 min, and the supernatants were pooled. The supernatants obtained were combined

    and concentrated in a rotary evaporator under reduced pressure at 40? and then the supernatant

    [8]was lyophilized. In this manner, the green tea extracts (GTE) by UPE were prepared.

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     1.3 Conventional extraction

     [10] Conventional extraction (CE) was performed as described by Jin et al.with some

    modifications. Sample was weighed exactly (10 g) in a three-neck flask, and 200 ml 50% ethanol 90 extraction solvent was added. A stirring apparatus and a reflux condenser were fixed. Extraction

     was carried out at boiling state (about 85?) for 4 h. Then the extracts were prepared as UPE

    procedure.

     1.4 Determination of total polyphenol content of green tea extracts

    The amount of polyphenol was reference measured by a photometric Folin-Ciocalteu assay 95

     [11]according to a proposed international standard method. The method was based on the reduction of phosphotungstic acid (HP[WO]) in alkaline solution to phosphotungstic blue. The 33104 matic phenolic absorbance of formed phosphotungstic blue was proportional to the number of aro groups and was used for their quantification with gallic acid as the standard. Briefly, a calibration

    curve of gallic acid (ranging from 0.005 to 0.05 mg/ml) was prepared and the results, determined 100

     by regression equation of the calibration curve, were expressed as mg gallic acid equivalents per gramme of the sample. In this method, 1 ml of tea extract diluted 10-75 times with deionized

     water (to obtain absorbance in the range of the prepared calibration curve) was mixed with 1 ml of 3-fold-diluted Folin-Ciocalteu phenol reagent. Two milliliter of 35% sodium carbonate solution is

    added to the mixture, which was then shaken thoroughly and diluted to 6 ml by adding 2 ml of 105

     water. The mixture was allowed to stand for 30 min and blue color formed was measured at 700 nm using a spectrophotometer. The total phenolic contents were determined using the standard

     gallic acid calibration curve. 1.5 Determination of free radical scavenging activity by DPPH assay

    The free radical scavenging activities of green tea extracts were analyzed by the method of 110 [12] Sheng et al.with some modifications. Initially, 2 ml aliquot of each solution of 50 μg/ml was -4 added to 2 ml of 2×10mol/L ethanolic DPPH solution in a cuvette. The mixture was shaken vigorously. The reaction mixture was incubated at 28?in a dark room for 30 min. The control

     contained all reagents except the extract sample while ethanol was used as blank. The scavenging activity against DPPH radicals was determined by measuring the absorbance at 517 nm with a 115 spectrophotometer. The inhibition of DPPH radicals by the test samples was calculated as

     scavenging activity (%) = (1?absorbance of sample/absorbance of control)×100. Measurements were performed in triplicate. A higher value indicates a higher antioxidant activity. 1.6 Experimental design The extraction parameters were optimized using RSM. A central composite design (CCD) 120

     was used to identify the relationship between the response functions and the independent variables, as well as to determine those conditions that optimized the extraction process of total phenolic

     content and antioxidant capacity of green tea extracts. Ethanol concentration ( x, 33.2-66.8 %), 1 pressure ( x, 281.8-618.2 MPa) and liquid/solid ratio ( x, 11.6-28.4 mL/g) were chosen for 2 3 independent variables. The range and center point values of three independent variables presented 125

    in Table 1 were based on the results of preliminary experiments. Each variable to be optimized

    was coded at five levels (-α, -1, 0, +1, +α). Star points were carried out using α of 1.682. Twenty

    randomized experiments including six replicates as the centre points were assigned based on CCD.

    The total phenolic content ( y) and DPPH free radical scavenging activity ( y) were selected as 1 2

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    the responses for the combination of the independent variables given in Table 2. Three 130

     experiments of each condition were carried out and the mean values were stated as measured responses. Unless otherwise stated, the data were expressed as mean?standard deviation (SD). Experimental runs were randomized to minimize the effects of unexpected variability in the

    observed responses.

    135

     Tab. 1 The coded values and corresponding actual values of the optimization parameters. Coded levels IndependentUnits Symbol variable -1 0 1 -1.682 (-α) +1.682 (+α) Ethanol40 50 60 33.2 66.8 % x1 concentration

    Pressure MPa 350 450 550 281.8 618.2 x 2

    Liquid/solid ratio mL/g x15 20 25 11.6 28.4 3

     Tab. 2 The central composite design with experimental and predicted values of the investigated responses.

     Response value x: y: y: 1 1 2 x: x: 3 2 Ethanol DPPH free radical Total phenolic contentRun Pressure Liquid/solid aconcentrationscavenging activity (%) (mg/g DW)(MPa)ratio (mL/g) (%) Experimental Predicted Experimental Predicted

    value value value value 1 60 350 25 452.2?1.4 450.6 63.5?0.3 64.2 2 50 450 20 579.4?1.0 578.7 84.1?0.2 84.8 3 66.8 450 20 532.1?0.6 534.7 75.1?0.6 75.5 4 50 450 28.4 482.2?1.2 480.7 70.3?0.5 69.5 5 50 450 11.6 445.6?0.5 446.3 65.5?0.4 65.9 6 50 450 20 576.9?0.5 577.9 85.2?0.2 85.5 7 50 450 20 579.5?0.6 579.5 85.3?0.4 85.1 8 60 350 15 438.2?0.9 436.4 63.6?0.5 62.5 9 50 450 20 578.6?0.6 579.9 85.8?0.4 85.3 10 50 618.2 20 513.2?1.1 512.8 73.5?0.3 73.1 11 60 550 15 508.4?1.0 507.5 73.4?0.6 73.1 12 33.2 450 20 527.2?0.8 524.3 74.2?0.6 73.9 13 40 350 25 454.3?0.7 455.1 65.4?0.5 64.8 14 60 550 25 529.5?0.5 527.9 75.4?0.3 74.7 15 40 550 25 516.4?0.8 517.3 73.1?0.4 73.8 16 50 450 20 578.5?1.3 579.6 85.9?0.6 85.2 17 50 281.8 20 402.6?0.9 400.0 57.6?0.5 57.1 18 40 550 15 488.5?0.8 490.6 71.5?0.4 70.9 19 40 350 15 432.6?0.9 434.7 61.9?0.4 62.2 20 50 450 20 577.3?0.9 578.4 84.3?0.6 84.7 a DW: dry weight. 140

    Based on the experimental data, regression analysis was performed and was fitted into the

    following second order polynomial model:

     3 3 2 32 y = A(1) + Ax+ Ax+ Axx 0? ? ? ? i i ii iij i ji =1 i =1 i =1 j =i +1 x are the independent variables (i ? j ), Where y is the response variable, j xandA, i 0

    145 A,AandAare the regression coefficients for intercept, linear, quadratic and interaction ij ij i terms, respectively. The predicted values of total phenolic content and antioxidant capacity were obtained

     according to the recommended optimum conditions. The total phenolic content and antioxidant capacity were determined after ultrahigh pressure extraction of polyphenolic antioxidants under

    150 optimal conditions. The predicted and experimental values were compared in order to determine

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     the validity of the model. 1.7 Statistical analysis All the data are shown as the mean of three replicate determinations within significance P<0.05 after subjecting to an analysis of variance (ANOVA) and processed with SAS (Version

    155 8.02; SAS). Stat-Ease Design Expert 8.0.6.1 (Trial version, Stat-Ease Inc., Minneapolis, MN,

     USA) was used for the experimental design and regression analysis of the experimental data. The Students t-test permitted the checking of the statistical significance of the regression coefficient, and the Fischers F-test determined the second order model equation at a probability (P) of 0.001, 0.01 or 0.05. The adequacy of the model was determined by evaluating the lack of fit, the 2160 coefficient of determination (R) and the F-test value obtained from the analysis of variance (ANOVA) that was generated. The relationship between the independent variables and the response variables was demonstrated by the 3D response surface plots. The Pearson's correlation coefficients between the total phenolic content and antioxidant capacity were demonstrated by employing SAS (Version 8.02; SAS).

    2 Results and discussion 165 The ultrahigh pressure extraction of polyphenolic antioxidants from green tea was optimized through RSM approach. In order to obtain the highest not only total phenolic content but also

     antioxidant activity, the central composite design with 20 experiments was employed to optimize parameters including ethanol concentration, pressure and liquid/solid ratio (Table 2). These

    parameters were chosen during the preliminary study which gave the higher total phenolic content 170

    and with desired antioxidant activity. The results of 20 runs using CCD were given in Table 2 that

    includes the design, the experimental and the predicted values. Results showed that the total

     phenolic content ranged from 402.6?0.9 to 579.5?0.6 mg/g DW. The maximum value (579.5?0.6

    mg/g DW) was found under the experimental conditions of x= 50%,x= 450 MPa and 1 2

    175 x= 20 mL/g. A wide range of antioxidant activity (61.9?0.485.9?0.6%) was found, and the 3

    maximum point (85.9?0.6%) was also found in conditions of x= 50%,x= 450 MPa and 1 2 x= 20 mL/g. By comparison of the experimental conditions of the maximum values, similar 3 experimental conditions were found. This could be explained that the polyphenols in green tea [13]extracts are largely responsible for the antioxidant activities. In order to obtain the highest

    polyphenolic content and antioxidant capacity, the optimum process condition should be 180

     investigated. 2.1 Model fitting

     By applying multiple regression analysis on the experimental data, the design expert software was generated the following second order polynomial equations to demonstrate the relationship

    between three factors and the predicted responses: 185

    y= 578.43 + 3.12 x+ 33.31 x+ 10.23 x+ 3.81 xx 1 12 3 1 2 2 2 2(2) -1.56 xx+ 1.56 xx-17.29 x- 42.99 x- 40.62 x 1 3 2 3 1 2 3

     y= 85.15 + 0.34 x+4.89 x+ 1.07 x+ 0.5 xx- 0.25 xx 2 1 2 3 1 2 1 3 2 2 2 - 3.75 x- 7.02 x- 6.14 x(3)1 2 3

    190 Where, yand yare the total phenolic content and antioxidant activity, respectively. x, 1 2 1

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    xandxare the coded values of the ethanol concentration, pressure and liquid/solid ratio, 2 3 respectively.

     Table 3 presented the ANOVA for response surface quadratic polynomial models. The statistical significance of regression equation was checked by F-test. The models had a very high

    195 F-value (2907.53 for total phenolic content and 771.66 for antioxidant activity) and a very low

     P-value (P<0.0001 for total phenolic content and P<0.0001 for antioxidant activity), which 2 ) for each indicated that the models were highly significant. The coefficients of determination (R

     predicted model were 0.9996 for total phenolic content and 0.9986 for antioxidant activity, while 2the adjusted determination coefficient (Adj.R) values were 0.9993 and 0.9973 for total phenolic

    content and antioxidant activity, respectively, which indicated a high degree of correlation 200

     between the experimental and predicted values. The fitness of the model was studied through the lack of fit test. The F-value of 4.70 and P-value of 0.0573 for total phenolic content, as well as

     4.50 and 0.0622 for antioxidant activity, which indicated the suitability of models to accurately predict the variations. All these results suggested that the models were adequate for predicting

    within the range of the variables employed. 205

     In term of total phenolic content, it could be observed that the independent variables 2 2 2 ( x , x , x ), the quadratic terms ( x, x, x) and the interaction term ( x x , xx, xx) 1 2 2 3 1 2 3 1 2 3 1 3 2were all significant (P<0.05). The linear and quadratic terms of ethanol concentration ( x, x), 1 1 2 2gave the largest effect pressure ( x, x), liquid/solid ratio ( x, x) and interaction term ofxx 2 2 3 3 1 2

    210 (P < 0.0001) followed by interaction term of and xxxx. 1 32 3

    Tab. 3 ANOVA for response surface models: estimated regression model of relationship between response

    variables (the total phenolic content and antioxidant capacity) and independent variables (ethanol concentration,

    pressure and liquid/solid ratio).

     Degrees of MeanSun of F-value P-valueTerm freedom square squares

    Total phenolic content

    Model 64130.45 9 7125.61 2907.53 < 0.0001

    132.83 1 132.83 54.20 < 0.0001 x- Ethanol concentration 1

    x- Pressure 15149.18 1 15149.18 6181.47 < 0.0001 2

    x- Liquid/solid ratio 1428.64 1 1428.64 582.94 < 0.0001 3

    116.28 1 116.28 47.45 < 0.0001 xx 1 2 xx 19.53 1 19.53 7.97 0.0181 1 3 19.53 1 19.53 7.97 0.0181 xx 2 3 2 x 1 4307.66 1 4307.66 1757.70 < 0.0001 2 x 2 26636.93 1 26636.93 10868.93 < 0.0001 2 x 3 23782.49 1 23782.49 9704.20 < 0.0001

    Residual 24.51 10 2.45

    Lack of Fit 20.21 5 4.04 4.70 0.0573

    Pure Error 4.30 5 0.86 Cor Total 64154.96 192 R0.99962 Adj.R0.9993

    DPPH radical scavenging activity

    Model 1584.79 9 176.09 771.66 < 0.0001

    1.60 1 1.60 7.03 0.0242x- Ethanol concentration 1

    326.25 1 326.25 1429.69 < 0.0001x- Pressure 2

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    - Liquid/solid ratio 15.54 1 15.54 68.10 < 0.0001x 3

    2.00 1 2.00 8.76 0.0143 xx 1 2 xx 0.50 1 0.50 2.19 0.1696 1 3 0.000 1 0.000 0.000 1.0000 xx 2 3 2 x 1 203.11 1 203.11 890.08 < 0.0001 2 x 2 711.12 1 711.12 3116.28 < 0.0001 2 x 3 543.42 1 543.42 2381.39 < 0.0001 Residual 2.28 10 0.23 Lack of Fit 1.87 5 0.37 4.50 0.0622 Pure Error 0.42 5 0.083 Cor Total 1587.07 19 2 R0.9986 2 Adj.R0.9973

    215

    As to antioxidant activity, it could be observed that the independent variables ( x, x, x), 1 2 3 2 2 2 the quadratic terms ( x,x,x) and the interaction term ( x x,) were significant (P<0.05).1 2 3 1 2

    However, the interaction term of and were not significant (P > 0.05). The variables xxxx 1 32 3

     with the largest effect on scavenging activity were the linear and quadratic terms of pressure 2 2 2220 x, x), liquid/solid ratio ( x, x) and quadratic term of ethanol concentration ( x), followed( 2 2 3 3 1

     by the linear of ethanol concentration ( x) and interaction term of xx. 1 1 2 2.2 Optimization of the procedure The 3D response surface plots simulated by Design-Expert software were the graphical representations of regression equation, which illustrated the relationship between independent and

    225 response variables. Two variables within the experimental range were depicted in three

     dimensional surface plots when the third variable is kept constant at zero level. As shown in Fig. 1, three independent variables (ethanol concentration, pressure and liquid/solid ratio) all have a

     positive impact on the total phenolic content and antioxidant activity of green tea extracts. Three dimensional plots for the response variables (total phenolic content and antioxidant

    activity) as functions of pressure and ethanol concentration at a fixed liquid/solid ratio were given 230

     in Fig. 1 (a, b). It could be observed that the pressure level demonstrated a strongly positive influence on the response variables, whereas ethanol concentration had a slight impact. The plots

     also showed that the influence of ethanol concentration almost disappeared at relatively higher pressure ( ? 400 MPa). The combination of pressure and liquid/solid ratio also has a similar effect

    on the response variables when the ethanol concentration was fixed (Fig. 1 c, d). As shown in 235

     Figure 1 (e, f), increased liquid/solid ratio would increase the response variables at the low and moderate ethanol concentration (33.2-55%). With further increase in ethanol concentration

     (55-66.8%), a decrease in the response variables was observed. This effect may be attributed to the [2]change of solvent polarity with change in ethanol concentration. Our results were in good [14]agreement with Spigno et al., who reported higher phenolic content from grape seeds were 240

    obtained when 50% ethanol was used.

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     (a) (b)

     245 (c) (d) (e) (f) centration (a, b), pressure and Fig.1 Response surface plots of the combined effect of pressure and ethanol con liquid/solid ratio (c, d), liquid/solid ratio and ethanol concentration (e, f) on the total phenolic content and

    antioxidant capacity of green tea extracts, respectively. 250 The aforementioned studies suggested that the pressure was the most significant extraction factor affecting response variables (total phenolic content and antioxidant activity). This is likely due to the destructive effects of pressure on the structures of the tea leaves tissue, cell wall,

    255 membrane and organelles (especially vacuole), which enhanced the mass transfer of the solvents

    [8][15] . Butz et al.also into the leaves materials and the soluble constituents into the solvents reported that 100 MPa pressure were enough to cause rupture of intracellular vacuoles and plant

     cell walls in onions. During this rupture process, the chemical substances within the cell are rapidly released into the surrounding extraction solvents. Thereby the compounds are more [16]260 accessible to extraction up to equilibrium.

     In order to obtain the highest polyphenolic content and antioxidant capacity, the optimum levels of the independent variables and their combination were generated by analyzing the

     quadratic polynomial regression equations using Design Expert software. The optimal extraction condition that provided a maximum total phenolic content of 585.9 mg/g DW was predicted as

    follows: 51.3% for ethanol concentration, 489.6 MPa for pressure and 20.7 mL/g for liquid/solid 265

    ratio. The optimal value of the tested variables for obtaining a maximum antioxidant activity of

    86.1% could be predicted as: 50.7% for ethanol concentration, 485.0 MPa for pressure and 20.4

    mL/g for liquid/solid ratio.

    The polyphenols have been reported to be responsible for the antioxidant activities of

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270 botanical extracts. The Pearson's correlation coefficients between the total phenolic content and

     2 antioxidant capacity were demonstrated (R=0.99). Correlations indicated the strong association between phenolic content and the antioxidant activities of green tea extracts, which was in

     agreement with the conclusion that the polyphenols in green tea contributed most to its antioxidant [17][18] behavior. Tabart et al.reported that the similar high correlation between antioxidant activity

    and total phenolic content of bud and leaf extracts of black currant was obtained. Thus, the total 275

     phenolic content in this study could be used as an indicator of antioxidant capacity. Through comparison of the optimal extraction conditions of total phenolic content and antioxidant activity,

     similar parameter groups for total phenolic content and antioxidant activity were found. Thus, we were able to use the same predicted optimal condition to obtain the highest polyphenolic content

    and antioxidant capacity of green tea extracts. Considering the operability in actual production, the 280

     optimal condition could be modified as follows: 50% for ethanol concentration, 490 MPa for pressure and 20 mL/g for liquid/solid ratio.

     2.3 Verification of predictive models Based on the above findings, a verification experiment was performed to evaluate the optimal

    operating conditions mentioned above for the ultrahigh pressure extraction with highest 285

     polyphenolic content and antioxidant capacity. Under this condition, the experimental values for total phenolic content and antioxidant activity were 583.8?0.9 mg/g DW and 85.6?0.7% (n = 3), respectively, which were well matched with the predicted value (Table 4). Only small deviations were found between the experimental and predicted values. Thus, the model could be used to

     optimize the process of ultrahigh pressure extraction of polyphenolic antioxidants from green tea. 290

     Tab. 4 Predicted and experimental values at optimum conditions. Ethanol ExtractionPressureTimeLiquid/solidy (mg/g 1Temperature concentration y(%) 2 method (MPa) (min) ratio (mL/g) DW)(%) UPE490 15 25? 50 20 585.9 86.1 (predicted) UPE 490 15 25 50 20 583.8?0.9 85.6?0.7? (experimental)

     CE 4h 85? 50 20 440.2?0.6 58.6?0.4

    y: Total phenolic content (mg/g DW),y: DPPH free radical scavenging activity (%). DW: dry weight. UPE: 1 2 ultrahigh pressure extraction. CE: conventional extraction. 295

     3 Conclusion In the paper, response surface methodology was successfully implemented for optimization

     of ultrahigh pressure extraction of polyphenolic antioxidants from green tea. A central composite design was used to determine the optimum process parameters that could give the highest

    polyphenolic content and antioxidant capacity. ANOVA showed that the effects of the variables 300

     (ethanol concentration, pressure and liquid/solid ratio) were all significant and the second order polynomial models for predicting responses were obtained. The 3D response surfaces were plotted

     from the mathematical model. The optimal condition determined was as follows: 50% for ethanol concentration, 490 MPa for pressure and 20 mL/g for liquid/solid ratio. Under the optimal

    condition, the maximum total phenolic content and antioxidant activity of 583.8?0.9 mg/g DW 305

    and 85.6?0.7% could be achieved, respectively, which were well matched with the predicted

    value.

    Compared to the conventional extraction techniques, UPE requires less extraction time, lower

    temperature and provides higher extraction efficiency. Thus, this study indicated that this new

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310 technology can benefit the food and pharmaceutical industries.

     Acknowledgements This work was financially supported by the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20100181120076).

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