Optimization of polyphenol extraction from green tea leaves (Camellia sinensis) using aqueous ethanol

Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 299 OPTIMIZATION OF POLYPHENOL EXTRACTION FROM GREEN TEA LEAVES (CAMELLIA SINENSIS) USING AQUEOUS ETHANOL 2,**Hoang Van Thanh; 1Tran Tu Ngan; 1Ngo Thi Thuy Vy; 1,*Dong Thi Anh Dao 1Hochiminh city University of Technology - VNU-HCM 2Hochiminh city University of Food Industry Email:*dtanhdao@hcmut.edu.vn;** thanhhv@cntp.edu.vn ABSTRACT Extraction of polyphenol from green tea leaves (Camellia sinensis) using aqueous ethanol was optimized for yield. The response surface methodology (RSM) was used to optimize the extraction conditions of polyphenol. Four factors that effect to the extraction process are the concentration of ethanol, the solvent-material ratio, the temperature and time of extraction. The results of single-factor showed that all four factors were the main influencing variables. The RMS showed that the optimal extraction conditions of polyphenol from green tea leaves was were the ethanol concentration of 62%, the solvent-material ratio of 9.9 (v/w), the temperature of 78oC and the time of 75 minutes; the polyphenol content achieved up to 276.18 mg/g (dry basis). The result of HPLC analysis showed that at the optimal extraction conditions, the EGCG content was 127.28 mg/g (dry basis). A second degree polynomial equation describes the effects on the total polyphenol of four factors content fitted well to experimental values (R2 = 0.994). This study is an evidence in order to extract the highest polyphenol from green tea leaves in aqueous ethanol conditions. Keywords: green tea leaves, Camellia sinensis, polyphenol, optimization, response surface methodology. INTRODUCTION Tea has been recognized as the most consumed beverage in over the world, which has been drunk for over 4000 years in Asia [HYPERLINK \l "Sus161" 1 ]. Futhermore, tea is also investigated and applied in pharmaceutical and industrial field because of the special bioactivity and nutrient functions of tea 2]. It has been known that there are serveral phenolic compounds composed of flavanoids in tea leaves, in which flavan-3-ols (catechins) is the major component. Catechins make up to 30% of its dry weight, including epigallocatechingallate (EGCG), epigallocatechin (EGC), epicatechingallate (ECG), epicatechin (EC), catechin (C), and gallocatechin (GC) [HYPERLINK \l "Chi081" 2]. Because catechins are able to donate hydrogens from the hydroxyl groups in their Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 300 structure, they have well antioxidant activities. Morever, their free-radical scavenging ability is more powerful than that of vitamin C, vitamin E, or β-carotene 3]. Considering the healthy effects of the green tea, catechins have been found to have ability of antioxidant, anticancer, antimicrobial, reducing cardiovascular diseases, anti-inflammatory [HYPERLINK \l "Chi081" 2]. Polyphenols are a common ingredient of plant-based foods, most commonly in our diets and are common in fruits, vegetables, grains, olive oil, dried beans, chocolate and beverages like tea, coffee and wine. Despite its presence in many foods, the effects of polyphenols have only been considered in recent years by nutritionists because polyphenols are highly diverse and chemically complex. Polyphenols can protect the antioxidant cellular components, thus limiting the risk of various degenerative diseases [4]. The market demand for products of tea that having several polyphenol compound increased significantly since its healthy benefit. However, the process of producing polyphenol from green tea with higher polyphenol amount is limited if extraction is in liquid CITATION Wen13 \l 1033 [5]. Thus, extraction by aqueous ethanol is the better way in order to boost extraction efficiency. MATERIALS AND METHODS Materials Sample Fresh green tea leaves were purchased in a local market, Bao Loc District, Lam Dong Province, Vietnam, located at altitude of 800-1000 meters above the sea level and an average temperature ranging from 18-25oC. Green tea leaves which were stored in a cool place (15-20oC) for three days were analyzed after one day of harvest. Chemicals Folin-Ciocalteu reagent were purchased from EMP Millipore Corporation (Germany), gallic acid was purchased from Merck Millipore Corporation (Germany), and sodium carbonate, ethanol were from China. Methods Selection of relevant variables and experimental ranges Fresh tea leaves were cleaned of impurities, blanched at 90oC for 30 seconds, drained off water quickly, and then blended by the blender (Philips Blender, HR2096/00, Netherlands) into the size of 0.4 ÷ 0.5cm. For the extraction experiment of Fresh tea leaves of 20g was placed into 1000 mL beacher, infused in aqueous ethanol, after which extraction was carried out in a water bath under the certain conditions. The detacted variables conducted of survey were the concentration of ethanol, the solvent-material ratio, the temperature and time of extraction. The experimental values of the independent variables were described in Table.1. If a variable isn’t studied, it is kept at constant value. The constant values for the concentration of ethanol, the solvent-material ratio, Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 301 the extraction temperature and the time of extraction were 50% v/v, 5 mL/g, 50oC and 30 minutes, respectively. On top of that, the extracts were centrifugated at 4oC, 2000rpm for 15 minutes (Centrifuge, Hermle, Z 326 K, Germany). All experiments were performed in triplicate. Table SEQ Table \* ARABIC 1: Experimental values of the independent variables for the single-factor experiment No. Ce (%V/V) rSTM (mL/g) T (oC) t (min) 1 30 5 30 30 2 40 6 40 45 3 50 7 50 60 4 60 8 60 75 5 70 9 70 90 6 80 10 80 Ce: ethanol concentration; rSTM: solvent-to-material ratio; T: extraction temperature; t: extraction time; The experiment was carried out under static conditions in a becher covered with a film to prevent loss from solvent, so the volume of the extract is not given. Design of statistical experiments for polyphenol optimization by RSM The effects on total polyphenol content of four factors (include ethanol concentration, solvent- material ratio, extraction temperature and time) were investigated by a single-factor method. The major influencing ranges of factors were identified from the results, and then using the response surface methodology in order to design the experiment project with four factors with three levels and seven replicated experiments at the center point. The Modde 5.0 software was utilized to establish a mathematical model (regression analysis) and perform the optimum conditions of extraction. The variables were coded according to the below equation: xi = (Xi – X0)/∆X, i = 1, 2, 3,... Where: xi is the (dimensionless) coded value of the variable Xi. X0 is the value of Xi at the center point. ΔX is the step change. Table.2 shows the actual design of the experiment. The following second degree polynomial equation describes the interrrelationship effects of four factors on the total polyphenol content: Y = QUOTE Where: Y is the response. A0 is the constant coefficient. Ai, Aii, Aij are the linear, quadratic and interactional coefficients, respectively. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 302 Determination of the optimal conditions Modde 5.0 software was set to search for the optimum desirability of the response variables – the maximum polyphenol content. Total polyphenol determination The polyphenol in the extraction were determined by colorimetry using Folin-Ciocalteu’s reagent with standard agent is gallic acid, according to the described method by Briefly, 1.0 mL of diluted sample of extract was transferred into the test tubes containing 5.0 mL of a 10-1 dilution of Folin-Ciocalteu’s reagent in water and then mixing gather. After 3 minutes, added 4.0 mL of sodium carbonate solution 7.5% (w/v) into each tube and mixed. The tubes were kept at room temperature for 60 minutes, then they were measured the absorbance by using UV-Vis spectrophotometer (PhotoLab 6100 Vis, WTW, Germany) at the wavelength of 760 nm. The total polyphenol content was expressed as mg gallic acid equivalents/g material of dry basis. Analysis of EGCG content by HPLC The EGCG content in tea leaves was determined by HPLC with the conditions: HPLC system of Agilent 1260, USA, and using DAD probe. Using the Agilent column of LiChrospher 100 RP-18 (5μm-250x3.0 mm), the measured wavelength of the UV-VIS detector was 274 nm, the sample load was 20 μl, the flow rate was 1 ml / min. For the equilibrium, the mobile phase was solvent acetonitrile-water: 15/85 (v/v). The EGCG standard purchased from Sigma (USA) with a minimum content of 98% is used to bundle the standard solutions. Statistical analysis All experiments were performed in triplicate and these values were presented as mean ± standard deviation (SD). Statistical analyses were performed by IBM SPSS Statistics 20 software. One- way analysis of variance (ANOVA) was also performed by using IBM SPSS Statistics software. Comparisons between samples were calculated by using Ducan’s test for independent observations. The differences were considered significantly at P<0,05. RESULTS AND DISCUSSIONs Effect of ethanol concentration on the polyphenol content Fig.1 showed that there is a significant effect of ethanol concentration in water in extraction of polyphenol in green tea leaves when solvent-material ratio, extraction temperature and time were 5mL/g, 50oC and 30 minutes, respectively. In the beginning, the amount of polyphenol extraction grew gradually from roughly 40 to 45mg GAE/g (dry basis), before rising significantly and reaching a peak of approximately 60mg GAE/g (dry basis). However, when the amounts of ethanol concentration were 70 and 80% (v/v), the total polyphenol content declined rapidly. Consequently, 60% (v/v) ethanol concentration in water was used in the following experiments. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 303 Figure 1: Influences of ethanol concentration Effect of solvent-material ratio on the polyphenol content Solvent – material ratio effect to the extraction of polyphenol in green tea leaves was given in Fig.2 when ethanol concentration, extraction temperature and time were 60% v/v, 50oC and 30 minutes, respectively. The amount of polyphenol extraction increased corresponding the increase of liquid/solid ratio. Starting at roughly 60mg GAE/g (dry basis), total polyphenol content increased gradually to about 80mg GAE/g (dry basis), after which it rised doubly and get a record high at about 120 mg GAE/g (dry basis) with solvent-material ratio is 9:1. Nevertheless, there was a leveling out in the polyphenol extraction when the liquid/solid ratio still increased. Thus, the liquid/solid ratio of 9:1 (mL/g) was sufficient to reach the high extraction, and it was used afterwards. Figure 2: Influences of solvent-material ratio Effect of extraction temperature on the polyphenol content Fig.3 described the effect of extraction temperature on polyphenol extraction process in green tea leaves when the ethanol concentration, the solvent-material ratio and extraction time 50% (v/v), 9mL/g and 30 minutes, respectively. The polyphenol content started at approximately 75mg GAE/g (dry basis) at 30oC, followed by significantly increases by 50 mg GAE/g (dry basis) from 30oC to 50oC. Moreover, total polyohenol content grew doubly in temperature from 50oC to Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 304 60oC, hittting a record high of around 200mg GAE/g (dry basis) at extraction temperature 70oC, before reducing at 80oC. Thus, 70oC was used in the following experiments. Figure 3: Influences of extraction temperature Effect of time on the polyphenol content The influence of time on the extraction of polyphenol was illustrated in Fig. 4 when the concentration of ethanol, the solvent-material ratio, the extraction temperature were 60% v/v, 9 mL/g and 70oC, respectively. It was showed clearly from the graph that, the extraction of polyphenol increased when the increasing of extraction time. Commencing at 200 mg GAE/g (dry basis), the polyphenol content rise considerably, after reaching a high point in 60 minutes of 275 mg GAE/g (dry basis). And then if extraction time was longer than, the extraction of polyphenol decreased gradually during the increasing of the time. The extraction time of 60 minutes was sufficient to reach the high extraction. Figure 4: Influences of extraction time Mathematical model and optimization of extraction conditions The appropriate condition of ethanol concentration (30, 40, 50, 60, 70, 80% v/v), solvent – material ratio (5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1 mL/g), extraction temperature (30, 40, 50, 60, 70, Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 305 80oC), extraction time (30, 45, 60, 75, 90 minutes) were determined in single-factor experiments. From these results, the extraction parameters were investigated for the optimum of extraction conditions by using Box-Hunter. The experimental values and coded levels of the four independent variables were given in Table 2. Value of the independent process variables considered (X1, X2, X3 and X4). Table 2: Actual values and coded levels of the independent variables xi X1 (% V/V) X2 (mL/g) X3 (oC) X4 (min) -2 40 7 50 30 -1 50 8 60 45 0 60 9 70 60 1 70 10 80 75 2 80 11 90 90 X1: ethanol concentration, X2: solvent-material ratio, X3: extraction temperature, X4: extraction time Figure 5: Effect of each factor on the Total polyphenol content Plan and results for response surface methodology of total polyphenol content. It’s clear that extraction temperature and extraction time affected considerably in the polyphenol extraction by using aqueous ethanol, followed by solvent-material ratio and finally, ethanol concentration. Consequently, temperature and time are the vital conditions need to be considerable in extraction process. The experimental data apply the multiple regression analysis and regression variables as well as test variables well fit to the second-order polynomial equation shown below: 2 1 2 3 4 1 2 2 2 2 3 4 1 4 3 4 Y 274.644 3.068X 16.016X 18.759X 7.699X 16.586X 13.629X 15.837X 12.054X 2.760X X 2.769X X = + + + + − − − − − + + Where X1: ethanol concentration; X2: solvent-material ratio; X3: extraction temperature; X4: extraction time. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 306 Figure 6: The effect of co-variance in: (a): (X1)/(X2); (b): (X1)/(X3); (X1)/(X4); (d): (X3)/(X2); (e): (X4)/(X2); (f): (X4)/(X3) on the TPC The obtained equation well fit to the experimental values with R2 = 0,825. This equation can be used to predict the polyphenol content value with the various extraction condition. Modde 5.0 software was set to search for the optimum desirability of the response variables – the maximum polyphenol content. The optimal conditions for polyphenol extraction were ethanol concentration of 62% (v/v), solvent-material ratio of 9.9 (mL/g), temperature of 78oC and time of 75 minutes; the polyphenol content achieved up to 276.18 mg/g (dry basis). The study of P. A. Nimal Punyasiri (2015) showed that the polyphenol content in green tea leaves is 266.79 ± 2.79 mg/g (dry basis) CITATION PAN15\l 1033 [HYPERLINK \l "PAN15" 6]. Compare to this research, when extracting at the above extraction conditions, the polyphenol content is higher. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 307 Analysis of EGCG content by HPLC method Figure 7: The HPLC chromatograms of EGCG extracted from green tea leaves The results showed that the amount of EGCG extracted from tea leaves was 11,394% (dry basis). Compared with the content of EGCG in tea leaves published from 8 to 12% [7] [8]. The extracted amount from the tea here is very significant and so can make use of this material for the item targeted exploitation of functional food production in food applications. CONCLUSIONS Conditions for extraction of polyphenol from green tea leaves have been studied. Extraction by ethanol 62% (v/v) as well as liquid/solid ratio of 9.9:1 (mL/g) has been shown to be considerable elements for extraction of polyphenol in green tea leaves. On the other hand, appropriate extraction temperature and time are also vital factors. Base on the priority result, temperature of 78oC and time of 75 minutes will help to extract high polyphenol content. This conditions provide polyphenol content is higher and acceptable color characteristic when extracted product becomes powder by spray drying. REFERENCES [1] Susantikarn, P. and Donlao, N, "Optimization of green tea extracts spray drying as affected by temperature and maltodextrin content," International Food Research Journal, vol. 23, no. 3, pp. 1327-1331, 2016. [2] C. T. Ho, Tea and Tea Products: Chemistry and Health-Promoting Properties, CRC Press, 2008. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 308 [3] Yang CS, Maliakal P, Meng X., "Inhibition of carcinogenesis by tea," Annu Rev Pharmacol Toxicol, 2002. [4] M. D Archivio, C. Filesi, R. Di Benedetto, R. Gargiulo, C. Giovannini, and R. Masella,, ""Polyphenols, dietary sources and bioavailability," Annali-Istituto Superiore di Sanita.," vol. 43, p. 348, 2007. [5] Wen-Ying Huang, Yu-Ru Lin, Ruei-Fen Ho, Ho-Yen Liu, Yung-Sheng Lin, "Effects of Water Solutions on Extracting Green Tea Leaves," The Scientific World Journal, vol. 2013, 2013. [6] P. A. Nimal Punyasiri, Brasathe Jeganathan, Jeevan Dananjaya Kottawa-Arachchi, Mahasen A. B. Ranatunga, I. Sarath B. Abeysinghe, M. T. Kumudini Gunasekare, B. M. Rathnayake Bandara, "New Sample Preparation Method for Quantification of Phenolic Compounds of Tea (Camellia sinensis L. Kuntze): A Polyphenol Rich Plant," Journal of Analytical Methods in Chemistry, 2015. [7] Pokorny Jan, Antioxidants in Food, Woodhead Publishing Limited, Cambridge, 2001. [8] Yukihiko Hara, Green tea: Health benefits and applications, Marcel Dekker Incorporated, 2001. [9] "ISO 14502-1:2005, Determination of substances characteristic of green and black tea — Part 1: Content of total polyphenols in tea — Colorimetric method using Folin Ciocalteu reagent," 2005. TỐI ƯU HÓA HIỆU SUẤT TRÍCH LY POLYPHENOL TỪ LÁ TRÀ XANH (CAMELLINA SINENSIS) BẰNG DUNG MÔI ETHANOL TÓM TẮT Hiệu suất trích ly polyphenol từ lá trà xanh (Camellia sinensis) bằng dụng môi ethanol - nước được tối ưu hóa trong nghiên cứu này. Phương pháp bề mặt đáp ứng (RSM) được sử dụng để tối ưu hóa các điều kiện trích ly polyphenol. Bốn yếu tố ảnh hưởng đến quá trình trích ly polyphenol là nồng độ ethanol, tỷ lệ dung môi-nguyên liệu, nhiệt độ và thời gian. Kết quả khảo sát từng yếu tố cho thấy rằng tất cả bốn yếu tố là các biến ảnh hưởng chính. RMS cho thấy điều kiện tách chiết tối ưu của polyphenol từ lá trà xanh là nồng độ ethanol 62%, tỷ lệ dung môi- nguyên liệu là 9.9 (v/w), nhiệt độ 78oC và thời gian 75 phút; hàm lượng polyphenol đạt tới 276.18mg/g (chất khô). Kết quả phân tích HPLC cho thấy ở điều kiện trích ly tối ưu, hàm lượng EGCG là 127.28 mg/g (chất khô). Một phương trình hồi quy đa biến bậc 2 đã mô tả ảnh hưởng trên tổng polyphenol từ giá trị tối ưu của bốn yếu tố phù hợp với các giá trị thực nghiệm (R2 = 0,994). Nghiên cứu này là một minh chứng cho quá trình trích ly polyphenol hiệu quả từ lá trà xanh bởi dung môi ethanol nước.