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KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 127 OPTIMIZATION OF EXTRACTION CONDITIONS FOR PHENOLIC COMPOUNDS FROM LEAVES OF CAMELLIA DALATENSIS LUONG, TRAN & HAKODA Huynh Dinh[.]
KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 OPTIMIZATION OF EXTRACTION CONDITIONS FOR PHENOLIC COMPOUNDS FROM LEAVES OF CAMELLIA DALATENSIS LUONG, TRAN & HAKODA Huynh Dinh Dunga, Lu Hoang Truc Linhb, Luong Van Dungb, Nguyen Thi To Uyena, Trinh Thi Diepa* a Faculty of Chemistry, Dalat University, Lamdong Province, Vietnam b Faculty of Biology, Dalat University, Lamdong Province, Vietnam * Corresponding author: Email: dieptt@dlu.edu.vn Abstract The extraction conditions of polyphenols from Camellia dalatensis leaves were optimized by experiment design of five variables using Design-Expert.V11.1.0.1 software Through the response surface method optimization, the optimal extraction conditions for the extraction of polyphenols were an ethanol concentration of 49.29% , an extraction temperature of 60°C, a sonication time of 40 min, a material size of 0.5mm, and a solvent/material ratio of 5.47 Keywords: Camellia dalatensis; Optimization of extraction; Polyphenol extraction; Response surface methodology 127 KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 TỐI ƯU HÓA ĐIỀU KIỆN CHIẾT XUẤT HỢP CHẤT PHENOL TỪ LÁ TRÀ ĐÀ LẠT CAMELLIA DALATENSIS LUONG, TRAN & HAKODA Huỳnh Đình Dũnga, Lữ Hồng Trúc Linhb, Lương Văn Dũngb, Nguyễn Thị Tố Uyêna, Trịnh Thị Điệpa,* a b Khoa Hóa học, Trường Đại học Đà Lạt, Lâm Đồng, Việt Nam Khoa Sinh học, Trường Đại học Đà Lạt, Lâm Đồng, Việt Nam * Tác giả liên hệ: Email: dieptt@dlu.edu.vn Tóm tắt Các điều kiện chiết xuất polyphenol từ trà mi Đà Lạt (C dalatensis) tối ưu hóa phương pháp quy hoạch thực nghiệm, sử dụng phần mềm Design-Expert.V11.1.0.1 Qua phương pháp tối ưu hóa đáp ứng bề mặt, điều kiện chiết xuất polyphenol tối ưu xác định là: dung môi chiết cồn 49,29%, nhiệt độ chiết 60oC, thời gian siêu âm 40 phút, kích thước nguyên liệu 0,5mm tỷ lệ dung mơi/ngun liệu 5,47 Từ khóa: Camellia dalatensis; Optimization of extraction; Polyphenol extraction; Response surface methodology 128 KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 INTRODUCTION Polyphenolic compounds are a group of biologically active molecules and one of the most important classes of secondary plant metabolites Plant polyphenols play important roles in the prevention of chronic diseases, such as cardiovascular diseases, neurodegenerative disorders, cancer, type II diabetes and osteoporosis (Scalbert et al., 2005) One of the rich sources of polyphenols is green tea (Camellia sinensis), a type of drink that has been used for thousands of years Recent studies on green tea show that tea polyphenols have many beneficial effects on human health such as antioxidant, cholesterol-lowering, anti-inflammatory, antibacterial, antiviral, anti-cancer, antidiabetic effects (Higdon et al., 2003; Maron et al., 2003; Fu et al., 2017; Rafieianet al., 2017) The predominant source of tea polyphenols are catechins such as epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate EGCG) (Higdon et al., 2003; Maron et al., 2003; Kanwar et al., 2012) Dalat tea (Camellia dalatensis Luong, Tran & Hakoda) is an endemic tea species of Dalat, recently discovered and named by Luong Van Dung, Tran Ninh and Hakoda in 2012 Through a preliminary investigation of chemical composition, we found that Dalat tea leaves contain relatively high level of total polyphenols (Tran et al., 2017) Therefore, the present study was undertaken to optimize the extraction of polyphenols from Dalat tea leaves by using response surface methodology (RSM) to provide scientific reference for development of a healthy product from this local source of polyphenols MATERIALS AND METHODS 2.1 Plant materials and chemicals The leaves of C dalatensis were collected in Tram Hanh, Dalat city in January 2018 and identified by Biologist Luong Van Dung, Department of Biology, Dalat University After collecting, the leaves were packed in sealed plastic bags, stored in the refrigerator at 5oC, and then ground to the desired sizes A voucher specimen has been deposited at the Natural Product Lab, Department of Chemistry, Dalat University 2.2 Methods 2.2.1 Polyphenol extraction Four grams of samples were placed in a capped triangular flask (100 mL) and mixed with ethanol-water The extraction process was conducted in an ultrasonic bath (Elma – Xtra 30 H Elmasonic, 35kHz, 400W) at a certain temperature After ultrasonic extraction, the mixture was filtered (Whatman No.1 paper) Then the filtrate was collected in volumetric flask and used for the determination of the total polyphenol content 129 KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 2.2.2 Determination of total polyphenol content Total polyphenol content (TPC) in the extracts was determined by colorimetric method according to TCVN 9745-1:2013 using Folin-Ciocalteu reagent (Merck) Gallic acid (monohydrate, purity 98.0%, HiMedia Labs, India) was used as polyphenol standard Briefly, mL diluted Folin–Ciocalteu reagent (10%, v/v) was mixed with 1.0 mL of sample solution After of incubation in the dark at room temperature, mL of aqueous sodium carbonate (7.5%, w/v) was added to the mixture After gentle vibration, the mixture was kept in room temperature for 60 Absorbance was measured at 765 nm using a UV-vis spectrophotometer (Spekol 2000) Total polyphenol content was expressed as grams polyphenols (gallic acid equivalent) per 100 grams of dried leaves (%) Moisture contents of the leaves were determined by using weight loss on drying in the oven at 105oC for hours (TCVN 9738:2013) 2.2.3 Experimental design RSM was applied to evaluate the influence of independent variables on extraction of polyphenols (Mark et al., 2017) using the Design-Expert V11.1.0.1 software (State-Ease lnc., Minneapolis, MN, USA) (Table 1) Table The RSM model applied in the study File Version 11.1.0.1 Study Type Response Surface Design Type I-optimal Design Model Reduced Quadratic Build Time (ms) 9033.00 Coordinate Exchange Subtype Randomized Runs 85 Blocks No Blocks The main factors affecting extraction efficiency, including ethanol concentration (%, A), the extraction temperature (°C, B), sonication time (min, C), material size (mm, D) and solvent/material ratio (E) were selected as independent variables The ranges of values for the variables were choosen on the base of a plemilinary experiment, taking into account the limits of the ultrosonic device The coded values of the experimental factors for the design were presented in Table The complete design was carried out in random order and consisted of 85 combinations (Table 3) Statistical analysis was performed using the Design-Expert V11.1.0.1 software Experimental data were fitted to a secondorder polynomial model where multiple regression analysis and analysis of variance were used to determine fitness of the model and optimal conditions for investigated responses 130 KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 Table Independent variables and their coded and actual values used for optimization Factor Name Units Type A Ethanol concentration % Numeric 30 90 -1 ↔ 30.0 +1 ↔ 90.0 B Sonication time Numeric 10 40 -1 ↔ 10.0 +1 ↔ 40.0 C Extraction temperature o Numeric 30 60 -1 ↔ 30.0 +1 ↔ 60.0 D Material size mm Numeric 0.50 1.00 -1 ↔ 0.5 +1 ↔ 1.0 E Solvent/material ratio Numeric 3.00 6.00 -1 ↔ 3.0 +1 ↔ 6.0 C Minimum Maximum Coded Low RESULTS AND DISCUSSION 3.1 Fitting the response surface models Coded High Table showed polyphenol compounds extracted from C dalatensis leaves ranged from 21.03 to 29.84% A second-order polynomial model describing the correlation between polyphenols yield (TPC, %) and the five variables in this study were obtained in equation below: TPC (%) = 26.60 - 0.11A + 0.45B + 1.11C - 0.17D + 1.00E - 0.46AB -1.18AC + 0.036AD + 0.16AE - 0.20BC - 0.13BD - 0.007BE + 0.12CD + 0.31CE - 0.047DE 2.37A2 + 0.24B2 + 0.15C2 – 0.99E2 Table Design arrangement for extraction and the responses of polyphenols A (%) B C (min) (oC) E D (mm) TPC (%) 27.95 44 30 30 60 0.5 27.95 21.03 45 30 30 30 22.28 24.80 46 90 10 30 0.5 22.54 40 0.5 26.06 47 30 20 40 22.41 10 50 0.5 26.19 48 50 30 30 0.5 24.42 50 40 40 0.5 29.84 49 90 40 50 0.5 25.31 70 30 60 26.82 50 70 10 40 0.5 25.68 90 10 40 0.5 22.79 51 50 40 60 0.5 28.58 50 10 60 28.70 52 50 40 30 0.5 28.07 Run A (%) B C (min) (oC) D E (mm) TPC (%) 50 20 50 0.5 30 10 30 90 10 60 70 20 70 Run 131 KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 Table Design arrangement for extraction and the responses of polyphenols (cont.) Run A (%) B C (min) (oC) D E (mm) TPC Run (%) A (%) B (min) C (oC) D (mm) E TPC (%) 10 50 20 40 24.68 53 90 30 60 21.53 11 90 10 40 22.41 54 50 30 40 0.5 26.56 12 70 40 40 0.5 25.56 55 50 40 30 23.92 13 90 20 40 24.42 56 70 10 30 0.5 23.54 14 70 20 50 26.31 57 90 30 60 0.5 24.42 15 30 30 60 0.5 27.32 58 90 30 30 0.5 23.29 16 70 40 50 0.5 25.43 59 30 20 50 24.55 17 50 40 40 28.20 60 90 40 30 0.5 23.67 18 30 10 40 21.78 61 70 30 40 25.93 19 70 10 60 27.32 62 30 30 40 0.5 22.66 20 30 40 30 0.5 23.29 63 30 40 30 23.67 21 30 10 50 24.05 64 50 10 50 0.5 24.73 22 70 30 50 0.5 22.66 65 50 10 30 23.29 23 90 20 60 23.04 66 50 20 60 28.83 24 50 20 60 0.5 23.42 67 30 40 50 0.5 26.56 25 90 40 40 0.5 24.80 68 30 40 40 22.54 26 70 40 30 25.05 69 50 20 30 0.5 22.41 27 30 10 40 0.5 21.40 70 90 10 50 24.55 28 70 40 60 0.5 24.93 71 90 20 40 0.5 24.05 29 30 20 50 0.5 23.80 72 90 40 30 24.05 30 90 20 30 22.79 73 70 10 50 26.19 31 70 20 60 0.5 25.93 74 70 30 30 0.5 26.19 32 30 20 30 0.5 21.78 75 50 10 40 0.5 26.31 33 50 30 60 0.5 28.96 76 70 30 30 25.18 34 90 30 40 22.54 77 30 40 60 27.45 132 KỶ YẾU HỘI NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 Table Design arrangement for extraction and the responses of polyphenols (cont.) Run A (%) B C D E (min) (oC) (mm) TPC Run (%) A (%) B (min) C (oC) D (mm) E TPC (%) 35 50 30 40 26.06 78 50 40 50 0.5 28.70 36 50 30 30 23.17 79 70 20 50 0.5 27.07 37 90 20 50 0.5 25.05 80 30 40 40 0.5 23.67 38 90 30 50 24.93 81 90 40 60 24.42 39 30 10 60 0.5 26.69 82 30 20 60 26.82 40 70 20 30 25.81 83 50 30 50 27.45 41 70 10 40 22.16 84 30 30 50 0.5 23.67 42 50 40 50 27.70 85 90 30 50 0.5 24.93 43 90 20 60 0.5 25.18 Significance and suitability of the design were then studied using an analysis of the variance (ANOVA, Table 4) According to Table 4, the model F-value of 9.89 and pvalue < 0.0001 implied the model was significant The Lack of Fit F-value of 1.02 and p = 0.5632 implied the Lack of Fit was not significant relative to the pure error Additionally, the degree of freedom for evaluation of lack of fit was 60, much higher than the recommended minimum of for ensuring the model validation The Predicted R² of 0.6895 (Table 5) was in reasonable agreement with the Adjusted R² of 0.7529; i.e the difference was less than 0.2 Adeq Precision measures the signal to noise ratio A ratio greater than is desirable (Mark et al., 2017) Our ratio of 17.1482 indicated an adequate signal This model can be used to navigate the design space Table Analysis of variance (ANOVA) for the investigated systems Source Sum of Squares Df* Mean Square F-Value p-Value Model 270.41 19 14.23 9.89 < 0.0001 significant A-Ethanol concentration 37.35 37.35 25.95 < 0.0001 B-Sonication time 1.98 1.98 1.38 0.2447 C-Extraction temperature 17.26 17.26 11.99 0.0010 D-Material size 3.00 3.00 2.08 0.1536 133 ... NGHỊ KHOA HỌC THƯỜNG NIÊN TRƯỜNG ĐẠI HỌC ĐÀ LẠT NĂM 2018 TỐI ƯU HÓA ĐIỀU KIỆN CHIẾT XUẤT HỢP CHẤT PHENOL TỪ LÁ TRÀ ĐÀ LẠT CAMELLIA DALATENSIS LUONG, TRAN & HAKODA Huỳnh Đình Dũnga, Lữ Hoàng Trúc... polyphenol từ trà mi Đà Lạt (C dalatensis) tối ưu hóa phương pháp quy hoạch thực nghiệm, sử dụng phần mềm Design-Expert.V11.1.0.1 Qua phương pháp tối ưu hóa đáp ứng bề mặt, điều kiện chiết xuất polyphenol... Khoa Hóa học, Trường Đại học Đà Lạt, Lâm Đồng, Việt Nam Khoa Sinh học, Trường Đại học Đà Lạt, Lâm Đồng, Việt Nam * Tác giả liên hệ: Email: dieptt@dlu.edu.vn Tóm tắt Các điều kiện chiết xuất polyphenol