Journal of Applied Botany and Food Quality 91, 163 - 170 (2018), DOI:10.5073/JABFQ.2018.091.022 1Tea Research Institute, Guangdong Academy of Agricultural Sciences, Guangdong Provincial Key Laboratory of Tea Plant Resources Innovation & Utilization, Guangzhou, China 2College of Horticulture, South China Agricultural University, Tianhe District, Guangzhou, China Optimization of brewing conditions in epigallocatechin-3-gallate (EGCG) extraction from Jinxuan summer green tea by response surface methodology Limin Xiang1,#, Shunshun Pan1,2,#, Xingfei Lai1, Lingli Sun1, Zhigang Li1, Qiuhua Li1,*, Yahui Huang2,*, Shili Sun1,* (Submitted: December 11, 2017; Accepted: June 12, 2018) * Corresponding author # These authors contributed equally to this work Summary The extraction conditions of epigallocatechin-3-gallate (EGCG) from Jinxuan summer green tea and antitumor activity against human gastric cancer SGC-7901 cells of the green tea extracts were inves- tigated. On the basis of a single factor experiment, Box-Behnken design and response surface methodology were employed to opti- mize the hot water extraction conditions. The optimal extraction conditions for EGCG were determined as: extraction temperature of 85 °C, extraction time of 34 min, water-tea ratio of 41 mL/g, a solu- tion of pH 6, and extraction twice. Under these conditions, the experi- mental extraction yield value of EGCG was 33.82 mg/g, which was not significantly different in comparison to predicted values. The re- sults indicated that the regression models were suitable for the EGCG extraction from Jinxuan summer green tea. The summer green tea extract prepared under the optimal conditions had a higher antitumor activity against human gastric cancer SGC-7901 cells than the green tea extract made with traditional tea brewing method. Keywords: Response surface methodology, Summer green tea, EGCG, Gastric cancer Introduction Camellia sinensis tea is the second most widely consumed non- alcoholic beverage in the world (Dalar and KonczaK, 2013). Tea is usually classified into unfermented (green tea), slight-fermented (white tea), semi-fermented (oolong tea), fermented (black tea), and post-fermented (dark green tea) forms (zhao et al., 2014). Among them, green tea is one of the most popular beverages consumed worldwide. And its consumption is increasing as a result of accumu- lated scientific evidence on its beneficial effects for human health. The epidemiological studies have suggested that daily consumption of green tea will help to prevent cancer, cardiovascular diseases, dental decay, obesity, diabetes, and improve the immune system (Kao et al., 2006; Khan and MuKhtar, 2007; Vuong et al., 2013; Wheeler and Wheeler, 2004). The aforementioned health benefits of green tea have been closely associated with tea catechins, purine alkaloids and theanine. Tea catechins which usually account for 30% of the dry weight of the green tea are associated with various health benefits, including antioxidant activity, antitumor activity, and anti- aging activity (Khan and MuKhtar, 2007; SaVic et al., 2016). Epigallocatechin-3-gallate (EGCG) has been considered as the most abundant and active constituent in green tea and may account for up to 50% of the catechins. It is usually used as a quality indicator (gaDKari et al., 2015; Khan and MuKhtar, 2007; Perva-UzUnalić et al., 2006). A number of studies have reported that EGCG has vari- ous biological, physiological, and pharmaceutical activities, such as prevention of obesity, hyperglycemia, insulin resistance, hypercho- lesterolemia, and hepatic steatosis (chen et al., 2011; hininger- FaVier et al., 2009; liu et al., 2012; Sae-tan et al., 2011). Additio- nally, it has been reported to have excellent anticancer and antioxi- dant activities (garbiSa et al., 2001; Singh et al., 2011; Xu et al., 2010). Hence, EGCG may be extensively applied in the fields of medication, cosmetics, beverages, and foodstuffs (PaVloVic et al., 2015). Owing to its great potential as a health promoter, the extraction of EGCG from green tea is considered to be an important research work for its wide use in a variety of health and food products (zhang et al., 2012). The extraction efficiency of the tea constituents and quality of obtained extracts have been directly influenced by several extraction conditions, including brewing temperature, extraction time, the ratio of solvent to tea, tea particle size, the pH of the brewing solution and the number of times the same sample is extracted (caStiglioni et al., 2015; hajiaghaaliPour et al., 2016; SaKlar et al., 2015). Thus, the extraction efficiency can be improved by optimizing each of these factors during the extraction process. The conventional single factor experiment for performing optimization is very time- consuming and the interactions among multiple parameters are not considered in this method (D’archiVio et al., 2016). Response sur- face methodology (RSM) is a helpful statistical technique which is useful for developing, improving and optimizing processes. It can minimize the number of experiments and provide sufficient informa- tion for a statistically acceptable result. RSM has become more and more attractive in process optimization, which has been successfully applied in the extraction of biologically active constituents from plant sources (Kong et al., 2010; SaVic et al., 2015; Savić et al., 2013). Therefore, the aim of the present study was to extract EGCG using the RSM with a Box-Behnken design (BBD) to optimize the extrac- tion conditions and obtain maximum EGCG from summer green tea (the lower grade green tea). According to the results of a single factor experiment, extraction temperature, extraction time and water-to-tea ratio were selected as independent variables for a Box-Behnken de- sign. Meanwhile, the number of times the tea is extracted and the pH of the brewing solution were set as fixed values. The chemical compositions of the lower grade green tea extract was analyzed, iden- tified and quantified using an HPLC (high performance liquid chro- matography) gradient type elution system and authentic standards. Additionally, the lower grade green tea extract was tested in vitro for its antitumor activity against human gastric cancer cells. Materials and methods Materials The summer green tea of the Jinxuan variety (Camellia sinensis), harvested in June 2013, was kindly provided by Guangdong Min- ghuang Tea Co., Ltd. (Guangdong, China). The dried green tea was ground by a disintegrator of FW100 (Taisite Co., Ltd, Tianjin, China) and then passed through a 40-mesh sieve. The tea power was kept in airtight bags at -20 °C until use. The conventional chemical composi- 164 L. Xiang, S. Pan, X. Lai, L. Sun, Z. Li, Q. Li, Y. Huang, S. Sun tions of green tea used in brewing trails were determined according to the GB/T of Chinese standard (GB/T 8313-2008 and GB/T 8314- 2013). The data analysis for the green tea used in brewing trails is shown in Tab. 1. Deionized water was prepared using a Millipore ZMQS5001 system (Millipore, Bedford, MA, USA). Standards of C (catechin), CG (catechin gallate), EC (epicatechin), ECG (epicatechin gallate), EGC (epigallocatechin), EGCG (epigallocatechin-3-gallate), GC (gallocatechin), GCG (gallocatechin gallate) and caffeine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solvents used for HPLC analysis were HPLC grade and were purchased from Thermo Scientific (Waltham, MA, USA). All other reagents were of analytical grade. (1) where Y represents the predicted extraction yield of EGCG, Xi and Xj represent the independent variables. β0, βi, βii and βij represent the regression coefficient for intercept, linearity, quadratic and interac- tive terms, respectively. The data were analyzed using Design Expert program (7.0.0 version, Stat-Ease Inc., Minneapolis, Minnesota, USA). Additional confirma- tion experiments were subsequently conducted to verify the validity of the statistical experimental design. Tab. 1: The composition of green tea leaves (g/100 g dry matter). Green tea g/100 g dry matter* Tea polyphenols 27.33 ± 0.25 Amino acids 4.58 ± 0.05 Caffeine 4.74 ± 0.07 Soluble sugars 3.42 ± 0.04 Soluble protein 0.64 ± 0.01 * Values are mean ± standard deviation of three measurements EGCG extraction Three grams of pretreated green tea powder was extracted with dis- tilled water using a designed temperature, time, brewing solution pH, water-tea ratio and the extraction times in a HHS thermostat water- bath (Aohua Co., Ltd, Changzhou, China). Following extraction, the mixtures were centrifuged at 8000 rpm for 10 min by the centri- fuge (HC-3018, Anhui USTC Zonkia Scientific Instruments Co., Ltd, Anhui, China). The supernatant was collected and transferred into a 250 mL measuring flask for determination of EGCG content. Each extraction was performed in three replicates. Samples (the extracts) were stored at -20 °C until HPLC analysis. Single-factor design for EGCG extraction The extraction yield of EGCG depends on the factors, including tem- perature, extraction time, brewing solution pH, water-tea ratio and the extraction times. The single-factor design was used to determine the preliminary range of extraction factors. Single factor experiment was used to study the effect of different factors on the extraction yield of EGCG. For single factor experiment, one factor was changed in a certain range while all other factors were kept constant. The extraction parameters were temperature (50, 60, 70, 80 and 90 °C), time (10, 20, 30, 40 and 50 min), water-tea ratio (10, 20, 30, 40 and 50 mL/g), times (once, twice, thrice, and four times), and brewing solution pH (4, 5, 6, 7 and 8), of which the single factor experiment was investigated in this paper, respectively. Response surface optimization designs According to the results of single-factor experiment, three major factors and their appropriate ranges were finally determined for a three-level factorial Box-Behnken design (BBD) in order to inves- tigate the relationship between process variables and the extraction yield of EGCG as the response value (Tab. 2). The whole design comprising 17 experimental runs were carried out in a randomized order as shown in Tab. 3. The following second-order polynomial equation was used to explain the behavior of the system: Tab. 2: The uncoded and coded levels of independent variables used in the RSM design. Levels Independent variable Symbol Low (-1) Middle (0) High (+1) Temperature (°C) X1 50 70 90 Time (min) X2 20 40 60 Water-tea ratio (mL/g) X3 10 30 50 Determination of tea catechins content The tea catechins in extract were quantified by HPLC using an Agi- lent 1200 series HPLC system equipped with a UV-detector and an analytical workstation (Agilent Co., Ltd, Santa Clara, CA, USA). The separation was performed on an Agilent ZORBAX EclipseXDB-C18 column (4.6 mm × 150 mm, 5 μm; Agilent Technologies, Wilming- ton, DE, USA). The solvents used were: (A) acetonitrile/acetic acid/ methanol/water (1:0.5:2:96.5, v/v/v/v), and (B) acetonitrile/acetic acid/methanol/water (10:0.5:20:69.5, v/v/v/v). The following linear gradient elution was used: 0-30 min, A from 72.5% to 20%; 30- 35 min, A from 20% to 72.5%. The column temperature was 28 °C. The flow rate was 1 mL/min. The detection wavelength was 280 nm and the injection volume was 10 μL. Quantification was carried out with the external standard method. Calibration plot was constructed by tea catechins concentrations versus each plotting peak areas. The concentrations of tea catechins in extracted samples were obtained by linear calibration curve. Determination of antitumor activity of green tea extracts Preparation of green tea extracts for cell viability assay In order to evaluate the antitumor activity of green tea infusions brewed with different methods, traditional tea brewing method (TTBM) and improved tea brewing method (ITBM) were chose to prepare tea infusion. Different brewing methods to prepare tea infu- sion are mentioned below: (1) Traditional tea brewing method (TTBM): tea infusions were pre- pared by placing 1 g of tea in 50 mL of distilled water at boiling temperature (100 °C) twice, and 3 min each time. (2) Improved tea brewing method (ITBM): tea infusions were pre- pared by placing 1 g of tea in 41 mL of distilled water (pH 6) at 85 °C twice, and 34 min each time. The tea infusions were freeze-dried. The concentrated green tea ex- tracts were stored at -20 °C for assay. Cell viability assay Human gastric cancer cell SGC-7901 was obtained from Shanghai Institute of Cell Biology (Shanghai, China) and maintained in an atmosphere of 5% CO2 and 95% air at 37 °C in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco/BRL, Gaithersburg, MD, USA). Cell viability assay was determined by EGCG enrich brewing method of green tea 165 MTT assay as described with some modification. Briefly, SGC-7901 was seeded at a density of 2 × 104 cells per well in 96-well plate, grown for 24 h, and then treated with different concentration of green tea extracts for 24 h. After incubation, 20 μL MTT (5 mg/mL in PBS) was added to each well and the cells were incubated at 37 °C for 4 h. The supernatants were discarded and 150 μL DMSO was added to each well to dissolve the precipitate and the absorbance of the well was measured at 570 nm with a microplate reader (Thermo Scientific Multiskan GO, Vantaa, Finland). Results were expressed as percent- age of cell viability (%), assuming control cells as 100%. Statistical analysis All experiments were performed in triplicate and centered. Analysis of variance (ANOVA) was performed by ANOVA procedure. P < 0.05 and P < 0.01 were regarded as significant and very significant, respectively. The optimal extraction conditions were established based on regression analysis and plotting of three-dimensional (3D) response surface plots. Results and discussion Single-factor experiment of EGCG extraction Effect of temperature on the content of EGCG extraction The effect of temperature on the EGCG extraction yield using an extraction time of 30 min, a solution pH of 6, water-tea ratio of 30 mL/g and extraction once, is shown in Fig. 1a. It demonstrates that the extraction temperature significantly affected the EGCG ex- traction yield. The EGCG yield increased significantly as the tem- perature increased from 50 to 70 °C. This could be explained by the fact that the rise in temperature can decrease solvent viscosity and surface tension, increase the solubility and diffusion rate of EGCG (Kong et al., 2010; Vuong et al., 2010; Vuong et al., 2011). How- ever, beyond 70 °C, the EGCG yield decreased, suggesting that the higher extraction temperatures induced the concurrent decomposi- tion of the compounds. This result was in agreement with several previous studies (Kong et al., 2010; Vuong et al., 2011). Therefore, a temperature of 70 °C was chosen as the central point for the RSM. Tab. 3: Box-Behnken design of factors with experimental and predicted values. EGCG content (mg/g) Run X1Temperature (°C) X2Time (min) X3Water-tea ratio(mL/g) Experimental Predicted 1 70 (0) 40 (0) 30 (0) 32.5 31.0 2 70 (0) 60 (+1) 10 (-1) 10.6 10.8 3 50 (-1) 20 (-1) 30 (0) 21.8 24.4 4 50 (-1) 60 (+1) 30 (0) 23.4 24.4 5 90 (+1) 40 (0) 50 (+1) 31.5 32.7 6 70 (0) 40 (0) 30 (0) 32.3 31.0 7 90 (+1) 60 (+1) 30 (0) 30.2 27.1 8 50 (-1) 40 (0) 10 (-1) 13.8 12.1 9 70 (0) 40 (0) 30 (0) 28 31.0 10 70 (0) 40 (0) 30 (0) 32.6 31.0 11 70 (0) 60 (-1) 10 (-1) 19.5 10.8 12 90 (+1) 40 (0) 10 (-1) 13.4 15.4 13 50 (-1) 40 (0) 50 (+1) 28 25.6 14 70 (0) 40 (0) 30 (0) 30.7 31.0 15 70 (0) 20 (-1) 50 (+1) 29.4 28.7 16 70 (0) 60 (+1) 50 (+1) 30.2 31.1 17 90 (+1) 20 (-1) 30 (0) 33.7 32.2 Fig. 1: Effect of extraction temperature (a), time (b), water-tea ratio (c), brewing solution pH (d), and extraction times (e) on the extraction yield of EGCG 166 L. Xiang, S. Pan, X. Lai, L. Sun, Z. Li, Q. Li, Y. Huang, S. Sun Effect of time on the content of EGCG extraction The EGCG was extracted at extraction time varying from 10 to 50 min and a temperature of 70 °C, a solution pH of 6, water-tea ratio of 30 mL/g and extraction once. As shown in Fig. 1b, a positive effect was caused by a longer extraction time. When extraction time changed from 10 min to 30 min, the EGCG yield increased from 20.91 ± 1.30 to 23.42 ± 0.74 mg/g due to the increase in reactive sites (chen et al., 2017). However, beyond 30 min, the EGCG yield decreased, suggesting that prolonging extraction time may expose the EGCG to oxidative degradation (Kong et al., 2010). Thus, an optimal extraction time of 30 min was chosen as the central point for the RSM. Effect of water-tea ratio on the content of EGCG extraction The effect of water-tea ratio on EGCG yield was investigated un- der fixed conditions (a temperature of 70 °C, an extraction time of 30 min, a solution pH of 6 and extraction once). Fig. 1c exhibits that the yield of EGCG significantly increased from 14.57 ± 0.71 to 23.17 ± 0.88 mg/g when the ratio rose from 10:1 to 30:1 mL/g because of the increasing contact surface area between tea powders and water, and the increase of the solid-liquid concentration gradient as well as the diffusion rate in the extraction (Yang et al., 2017). However, further increase in the ratio did not significantly improve the extrac- tion yield (p > 0.05), which is in agreement with several previous studies (FelKai-haDDache et al., 2016). Thus, a water-tea ratio of 30:1 mL/g was selected as the central point for the RSM. Effect of pH on the content of EGCG extraction The EGCG was extracted at solution pH varying from 4 to 8 and a time of 30 min, a temperature of 70 °C, water-tea ratio of 30 mL/g and extraction once. As shown in Fig. 1d, pH level showed a posi- tive effect on the EGCG extraction ratio, which reached at 24.04 ± 0.71 mg/g at pH = 6 and then decreased rapidly. This is due to the fact that EGCG was quite stable in acidic solutions but its stability decreased when the pH of the extraction solution rose from pH 4 to 8 (Vuong et al., 2013). Consequently, a solution pH of 6 was selected as the central point for the RSM. Effect of extraction times on the content of EGCG extraction The effect of the extraction times on EGCG yield was investigated under fixed conditions (a temperature of 70 °C, an extraction time of 30 min, a solution pH of 6 and water-tea ratio of 30 mL/g). Fig. 1e showed that the extraction ratio increased significantly with the in- creasing number of extraction times at first. However, the extraction ratio increased slightly with the increasing number of extraction times after extraction twice. This could be explained by the fact that most of EGCG had been dissolved out in the first two extraction cy- cle. And this tendency was also in accordance with previous studies (Yang et al., 2017). Thus, the number of extraction times was fixed as twice in the RSM. Optimization of EGCG extraction by response surface designs According to the above results of single factor experiment, the three major parameters (extraction temperature, time and water-tea ratio) and their ranges for EGCG extraction were determined and adopted for Box-Behnken design with response surface methodology with the consideration of cost, energy and solvent consumption. Model fitting Tab. 3 showed the design matrix and the experimental and predicted values of the response. According to the multiple regression analysis, the fitted second-order polynomial model for EGCG in coded vari- ables was shown in the following Eq. (2). Y = -35.2775 + 1.0123X1 + 0.2983X2 + 1.0528X3 - 0.0032X1X2 + 0.0024X1X3 + 0.0061X2 X3 - 0.0059X12- 0.004X22- 0.018X32 (2) where Y represents the EGCG extraction ratio, X1, X2 and X3 re- present the experimental value of extraction temperature, time, and water-tea ratio, respectively. In order to determine whether the quadratic model is significant, it is necessary to run ANOVA analysis. The significance of each coef- ficient was measured using F-values and p-values. The smaller the p-value and the greater the F-value, the more significant the corres- ponding coefficient (gan and latiFF, 2011; hu et al., 2016). The regression coefficients of the linear, quadratic and interactive para- meters are shown in Tab. 4. Lack of fit was also given in Tab. 4 in order to confirm model fit. The p-value of the model was less than 0.001, which indicated that the fitting model is adequate to describe the experimental data. F-value for the lack of fit was insignificant (p > 0.05) imply that the model is valid. Among all variables, the linear of X1 and X3, and the quadratic of X32 exhibited significant effects (p < 0.05), whereas the others had negligible effects on the EGCG extraction ratio (p > 0.05). The linear variable of water-tea ratio had the highest influence on the EGCG content to its p-value below 0.001, followed by the linear effect of extraction temperature. However, extraction time had no remarkable effect (p > 0.05). Besides, the quadratic effect of water-tea ratio has the significant influence on the content of EGCG (p < 0.001). It could be concluded that water-tea ratio played the most important role in the EGCG extraction among three independent variables, followed by extraction temperature and extraction time. Tab. 4: ANOVA of the regression model for the prediction of MLP ex- tractions. Source Sum DF Mean F value p-value of squares square Prob > F Model 851.82 9 94.65 12.85 p < 0.001 X1 59.41 1 59.41 8.07 p < 0.05 X2 12.5 1 12.5 1.7 p > 0.05 X3 477.4 1 477.4 64.82 p < 0.001 X1X2 6.5 1 6.5 0.88 p > 0.05 X1X3 3.8 1 3.8 0.52 p > 0.05 X2 X3 23.52 1 23.52 3.19 p > 0.05 X12 23.2 1 23.2 3.15 p > 0.05 X22 10.75 1 10.75 1.46 p > 0.05 X32 218.12 1 218.12 29.62 p < 0.001 Residual 51.55 7 7.36 - - Lack of fit 36.21 3 12.07 3.15 p > 0.05 Pure error 15.35 4 3.84 - - Total 903.37 16 - - - Analysis of response surface plot and contour plot The three-dimensional response surface plots and two-dimensional contour plots were applied to visualize the interactive effects of the independent variables on the EGCG yield (Fig. 2). The surface re- sponse plots of the model were created by changing two variables within their test range and keeping the other variable at their central level, which is the best way of showing the effect of the independent variable on the content of EGCG (FelKai-haDDache et al., 2016; guo et al., 2016). The 3D response surface and 2D contour plots are the graphical representations of regression equation. Through these plots, it is easy to judge interactions between two variables and deter- EGCG enrich brewing method of green tea 167 mine optimum ranges for independent variables (DahMoune et al., 2014; zhang et al., 2011). The variables of temperature/time, temperature/water-tea ratio, and time/water-tea ratio collaboratively worked in a nonlinear synergistic manner to the content of EGCG (Fig. 2A, 2B, and 2C). As it can be found from Fig. 2A and Fig. 2C, the response curve of extraction time was smooth. It indicated that the extraction time is less signifi- cant than other two effects. In Fig. 2B and Fig. 2C, water-tea ratio has the huge influence on the content of EGCG. With the increase of water-tea ratio from 30 to 41 mL/g, the content of EGCG increased to a definite value and subsequently maintained stable or decrease. The content of EGCG was not found to be significantly affected by an enhancement of the extraction temperature at a fixed water-tea ratio and extraction time, shown in Fig. 2A and Fig. 2B. This suggested that the change in extraction temperature had an alternative influence on the content of EGCG. Model verification By applying the regression analysis to Eqs. (1) and (2), the predic- ted optimized conditions were concluded as follows: X1= 85.42 °C, X2 = 34.22 min, and X3 = 40.79 mL/g. Based on Eq. (2), the predicted Fig. 2: Three-dimensional response surfaces and contour plots for the effect of variables on the extraction yield of EGCG maximum extraction yield of EGCG was 34.54 mg/g. For convenient operation in actual experiments, the verification experiment was per- formed under predicted optimal conditions with slightly modifica- tion as follows: temperature of 85 °C, time of 34 min, water-tea ratio of 41 mL/g, a solution pH of 6 and extraction twice. Under these conditions, the experimental extraction yield value of EGCG was 33.82 mg/g, which was no significant difference to predicted value. Therefore, the results revealed the accuracy and adequacy of the response model in the optimization of extraction process of EGCG from the lower grade green tea (liM et al., 2017; Yang et al., 2017). Effect of brewing conditions on the content of individual cate- chins The concentrations of eight main tea catechins in green tea extract made with two brewing methods were quantified by HPLC (Tab. 5). The HPLC profile is shown in Supplementary materials 1. Compared with the TTBM group, the contents of all kinds of tested catechins in ITBM group were much higher, especially for the contents of C, CG, GCG, EGCG. Additionally, the contents of gallated catechins and total catechins in ITBM group were significantly higher than TTBM group (p < 0.001 and p < 0.05, respectively). 168 L. Xiang, S. Pan, X. Lai, L. Sun, Z. Li, Q. Li, Y. Huang, S. Sun Antitumor activity Gastric cancer is one of the most serious malignant tumors, which is the leading cause of cancer-related death in most countries. Case- control studies have suggested that green tea consumption has pro- tective effect of against gastric cancer (Xu et al., 2010; zhu et al., 2007). It is reported that EGCG suppresses tumor growth by inhi- biting proliferation and inducing apoptosis through a variety of me- chanisms. In this study, we investigated cell growth inhibition effect of above green tea extract made with two different brewing methods on human gastric cancer SGC-7901 cells. Cells were treated with 1.5, 2.5 and 3.5 mg/mL of green tea extract for 24 h and cell viability was determined using the MTT assay. As shown in Fig. 3, green tea extract had significant growth inhibition effect on gastric cancer cells Tab. 5: Catechin contents of green tea extract brewing with TTBM and ITBM (mg/L). Catechins TTBM ITBM GC 163.34±0.54 164.43±1.69 EGC 256.06±6.05 256.31±4.81 C 28.58±0.83 36.11±0.76** EC 68.8±5.28 72.5±2.15 CG 4.15±0.67 13.68±0.79** ECG 1.59±0.16 3.14±0.12 GCG 41.93±3.46 52.66±2.87* EGCG 281.98±16.80 340.07±13.77* Gallated catechins 329.65±21.56 409.55±17.1** Total catechins 846.43±25.63 938.92±25.95* TTBM, traditional tea brewing method. ITBM, improved tea brewing method. Values are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01 versus TTBM group. in a dose-dependent manner with a maximum loss at concentration of 3.5 mg/mL. Moreover, the decrease of the cell viability exposed to green tea extract made with ITBM was more significant than that of TTBM, which was consistent with the results of HPLC analysis. This indicated that the antitumor activity of green tea extract was due to the cumulative effects of tea catechins (cooPer et al., 2005). Conclusions In the present study, Box-Behnken design based on the response sur- face methodology was successfully employed to optimize the EGCG extraction from summer green tea. The optimal extraction conditions for EGCG were: temperature of 85 °C, time of 34 min, water-tea ratio of 41 mL/g, a solution pH of 6, and extraction twice. Water-tea ratio was also found to have the major influence on the yield of EGCG. Under these conditions, the experimental extraction yield value of EGCG was 33.82 mg/g. Furthermore, the summer green tea extract prepared under the optimal conditions had the higher antitumor ac- tivity against human gastric cancer SGC-7901 cells. The results in- dicated that the rich and valuable resource of summer green tea can be utilized much better by using the optimal extraction conditions (zhang et al., 2012). It is worth mentioning, however, that from the point of view of industrial application, future work need to be done to establish the pilot-plant trials of EGCG extraction and to claim the mechanism of the healthy benefits of summer green tea benifits in human body or food industry (DahMoune et al., 2014). Acknowledgements This work was financially supported by the Fund for Guangdong Pro- vincial Key Laboratory of Tea Plant Resources Innovation and Uti- lization (No. 2008A060301004), the construction of industrial tech- nology innovation platform for black tea of Guangdong big leaves species, the earmarked fund for Modern Agro-industry Technology Research System (No. 2016LM2151), the Science and Technology Planning Project of Guangdong Province (No. 2016B090918118), and the President Foundation of Guangdong Academy of Agricul- tural Sciences (Grant No. 201534 and 201720). References caStiglioni, S., DaMiani, e., aStolFi, P., carloni, P., 2015: Influence of steeping conditions (time, temperature, and particle size) on antioxidant properties and sensory attributes of some white and green teas. Int. J. Food Sci. Nutr. 66, 491-497. 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