Extraction of Polyphenolic Antioxidants from Green Tea by

By Ronnie Olson,2014-09-09 11:27
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Extraction of Polyphenolic Antioxidants from Green Tea by


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.


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

    - 1 -


    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


    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.

    - 2 -


     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


     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

    - 3 -


    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.


     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 (