Đề tài Trích ly polyphenol từ trà xanh sử dụng phương pháp trích có hỗ trợ vi sóng

Hội Nghị Khoa Học & Công Nghệ lần 9 Phân ban Công nghệ Hóa học Trang EXTRACTION OF POLYPHENOLS FROM GREEN TEA USING MICROWAVE ASSISTED EXTRACTION METHOD TRÍCH LY POLYPHENOL TỪ TRÀ XANH SỬ DỤNG PHƯƠNG PHÁP TRÍCH CÓ HỖ TRỢ VI SÓNG Pham Thanh Quan, Tong Van Hang, Nguyen Hai Ha, Do Nguyen Tuyet Anh, Truong Ngoc Tuyen Department of Organic Chemical Engineering, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet street, District 10, HCM City, VietNam TÓM TẮT Dịch trích trà xanh được trích từ lá trà xanh sử dụng dung môi trích theo hai phương pháp trích thông thường và phương pháp trích với sự hỗ trợ vi sóng .Một số yếu tố ảnh hưởng như dung môi (rượu-nước), tỉ lệ nguyên liệu/dung môi (1/5-1/15), pH, nhiệt độ trích, thời gian trích và ngâm của hai phương pháp được khảo sát. Ở cùng điều kiện khảo sát, phương pháp trích với sự hỗ trợ vi sóng cho hiệu suất cao với thời gian ngắn hơn (82,6 % trong 360 giây ) phương pháp trích ly thông thường, 62,1% trong 180 phút. Dịch trích trà xanh theo phương pháp trích có hỗ trợ vi sóng có hàm lượng polyphenol ( 36 %) cao hơn phương pháp thông thường. Phương pháp trích có sự hỗ trợ vi sóng hữu hiệu hơn phương pháp trích thông thường về chất lượng, thời gian và chi phí năng lượng. ABSTRACT Green tea extract (GTE) was extracted from raw green tea by using solvent extraction with traditional heating method (SETM) and microwave-assisted extraction (MAE method). Several factors such as solvents (alcohol aquaus), material: solvent ratio (1/5-1/15), pH, extraction temperature, immersion and extraction time of both methods were studied. In same conditions, MAE method gave higher yield in much shorter time than SETM, 82.56% in 360 seconds and 62.14% in 180 minutes, respectively. GTE from MAE method had total polyphenols concentration higher (36%) than that of SETM. Consequently, MAE method was found to be much more effective than SETM in quality of GTE, time and energy consumption. Keywords: Polyphenols, green tea, green tea extract, extraction, microwave assisted extraction. 1. INTRODUCTION Tea (Camellia sinensis) is native to the East Asia region. Camellia Sinensis, a member of the Theaceae family, is an evergreen, usually picked as young shoots in cultivation. Its first recorded use dates from the fourth century A.D. in China [Tong.V.Hang 1985]. Nowaday, tea with its variety products is one of the most widely consumed beverages in the world. Main tea producers concentrated in equatorial regions. Three biggest tea producers and exporters are China, India and Vietnam. Young shoots tea (green tea) contains many polyphenolic compounds, up to 30-40 percent of the extractable solids of dried green tea leaves, with most of the polyphenols being flavanols more commonly known as catechins. The primary catechins in green tea are epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG) (Fig. 1) [Tong.V Hang 1985], [Fujikia et al 2002], [Hemiway et al 1992]. Recently green tea as a healthy beverage has attracted science attention for its anticancer and antioxidation activities [Weisburger 2000]. Polyphenols in green tea are believed as good free radical scavengers. Several clinical studies found them to be active in cancer prevention in several ways and polyphenols have been recently recognized as functionally active molecules, possessing antioxidant, anticancer, antimutagenic 1 Hội Nghị Khoa Học & Công Nghệ lần 9 Phân ban Công nghệ Hóa học . Trang properties, as well as exerting protective effects against cardiovascular and other diseases [Brown, 1999], [Aldini et al 2003], [Gupta et al 2002], [Jun Cai et al 2002], [Azam et al 2004]. Epigalogatechin –3- gallate (EGCG) Epicatechin –3- gallate (ECG) Epicatechin (EG) Epigalocatechin (EGC) Figure 1. Catechins in green tea extracts Vietnam is one of biggest tea producers and exporters in the world, however main export products is raw material or traditional products such as black tea, oolong tea and green tea. Green tea extract is still new in Vietnam market and also new in research and application. HO OH O H H O OH OH CO OH OH OH OH Green tea extract was prepared using several different techniques, including solvent extraction, soxhlet extraction; microwave assisted extraction, solid-phase extraction, and supercritical fluid extraction. Each technique has its own advantages and problems, in small-scale produce, microwave method seems to be a reasonable method that helps reduce time and save energy for producer. In this article, several factors involving in microwave-assisted extraction were studied and compare to those of solvent extraction using traditional heating method. HO OH O H H O OH OH CO OH OH OH 2. MATERIALS AND METHODS Reagents Fresh green tea (picked as shoots with two or three leaves) was purchased from Ladotea (Lam Dong Vietnam). Folin - Ciocalteau agent (Merck), citrate - phosphate pH buffer (Merck), ethanol and other chemical in AP grade HO OH O H H OH OH OH Green tea extraction method Microwave assisted extraction (MAE): Fresh green tea was collected as shoot on fields. 100g of fresh green tea were cut to 1 - 1.5mm size then immerse in solvents (1:5 to 1:15 g/ml) for a certain time (0 - 90 minutes). Then it was transferred to flask, adjusted pH, and brewed in microwave oven (450W) (times: 300- 420s), radiation is done at regular intervals (30s interval) to keep temperature not rise above 70oC. After that, the infusion was let cool down to room temperature, filtered to separate solid and concentrated by rotary vacuum evaporation to 50ml. Final infusion was stored in refrigerator at 4oC. HO OH O H H OH OH OH OH Solvent extraction using traditional heating method (SETH): Extraction procedure was carried out similar to MAE, except extraction time was 180 minutes. 2 Hội Nghị Khoa Học & Công Nghệ lần 9 Phân ban Công nghệ Hóa học Trang Total polyphenols determination Total polyphenols concentrated (TPC) in infusion were spectrophotometrically determined measuring absorption after using Folin-Ciocalteau reagent, using Jenway 6505 UV-VIS Spectrophotometer at 700 nm. GTE composition was identified by TLC. 3. RESULTS AND DISCUSSION Effect of extraction time Fig 2. show that when using MAE extraction yield increases with extraction time and get the highest at 360 seconds, 82.56%, compare to 61,14% of that of SETH (highest yields in 180 minutes extraction). Increasing extraction time in MAE did not help increase yield. In some experiments, when increasing extraction time above 420 seconds, extraction yield dropped below 50%, could be due to effect of long radiation of microwave to polyphenols molecules. 76.000 77.000 78.000 79.000 80.000 81.000 82.000 83.000 84.000 300 360 420 Time (s) Ex tr ac tio n Yi el d (% ) Figure 2. Effect of time to TPC 68.000 70.000 72.000 74.000 76.000 78.000 80.000 82.000 84.000 0 30 60 Immersion time (minute) Ex tr ac tio n yi el d (% ) Figure 3. Effect of immersion time to TPC Effect of immersion time Immersion before extracting help increase extraction yield, but not significant. Immersion gives time to solvent to complete absorbed into material. Yield from 73,64% when not immersion went to 82,56% when immersion in 30 minutes. Fig 3. shows that immersion time doubles (60 minutes), extraction yield slightly decreased to 81.59%. In this factor, SETH showed the same change trend, although with lower yield. 0 50 100 2 2.5 3 3.5 4 pH Ex tr ac tio n Yi el d (% ) Figure 4. Effect of pH to TPC 0 20 40 60 80 100 1:5 1:10 1:15 Material : solvent ratio Ex tr ac tio n Yi el d (% ) Figure 5. Effect of material: solvent ratio to TPC Effect of pH Polyphenols are strong antioxidants, so they are easily oxidated in high pH. Extracting is usually carried out at acid pH because of high antioxidant activities in neutral and base pH. The lower pH is, the better polyphenols are protected, and the higher yield gets. In Fig 4, highest yield at pH 2.5 (82.56%), slightly lower at 2 (79.09%), but continuously decreased above pH 2.5, at pH 6, only 52,50%. The results were similar in SETH with highest yield is 74.57% at pH 3. 3 Hội Nghị Khoa Học & Công Nghệ lần 9 Phân ban Công nghệ Hóa học . Trang Effect of material: solvent ratio REFERENCES 1. Hemingway, R.W, Larks, 1992, Polyphenol plant, Lees, G.L Polyphenols in material go into solvents by diffusion, so extraction yield increase with material: solvent ratio. In Fig. 5, ratio 1:10 gave highest yield (82,56%), 1:5 only 43.28%. But when increasing ratio to 1:15, yield downed to 59,18%, because more time need to heat a large volume of solvents (extraction time in MAE is only 360 seconds). In SETH, ratio lower than 1:5 gave very low yield, material: solvent ratio from 1:5 to 1:10 had not get significant effect to extraction yield because of long extraction time (180 minutes). 2. Tong Van Hang, 1985, Co so sinh hoa va ki thuat che bien tra, Tp.HCM 3. Michael D. Brown, 1999, Green Tea (Camellia Sinensis) Extract and Its Possible Role in the Prevention of Cancer, ND Alternative Medicine Review, Volume 4, Number 5 4. Hirota Fujikia, Masami Suganumab, Kazue Imaic, Kei Nakachic, 2002, Green tea: cancer preventive beverage and/or drug, Cancer Letters 188, 9–13 5. S. Gupta, B. Saha, A.K. Giri, Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review, 2002, Mutation Research 512, 37–65 6. Yuko Yoshida, Masaaki Kiso, Tetsuhisa Goto, 1999, Effeciency of the extraction of catechins from green tea, Food Chemistry 67, 429±433 76 77 78 79 80 81 82 83 84 60o 70o 80o Alcohol Ex tr ac tio n Yi el d (% ) 7. John H. Weisburger, 2000, Tea and health: a historical perspective, Cancer Letters 114 (1997) 315-317 8. Jen – Kun Lin, Yu – Chih Liang, Cancer Chemoprevention by Tea Polyphenols, Vol. 24, No. 1, pp. 1-13. 9. Giancarlo Aldini, Kyung-Jin Yeum, Marina Carini, Norman I. Krinsky, and Robert M. Russell, 2003, (-)-Epigallocatechin-(3)-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma, Biochemical and Biophysical Research Communications 302, 409–414 Figure 6. Effect of solvents to TPC Effect of solvents The most effective solvent in this experiment was ethanol 70o. Extraction yield when using ethanol 70o was 82.56%, higher than using ethanol 80o (81,36%) and ethanol 60o (78.64%). However, these changes did not give much effect to extraction procedure. 10. Yu-Jun Cai, Lan-Ping Ma, Li-Fen Hou, Bo Zhou, Li Yang, Zhong-Li Liu, 2002, Antioxidant effects of green tea polyphenols on free radical initiated peroxidation of rat liver microsomes, Chemistry and Physics of Lipids 120, 109_/117 4. CONCLUSION 11. S. Azam , N. Hadi , N.U. Khan , S.M. Had, 2004, Prooxidant property of green tea polyphenols epicatechin and epigallocatechin- 3-gallate: implications for anticancer properties, Toxicology in Vitro 18 (2004) 555– 561 Compare to solvent extraction using traditional heating method, in solvents, immersion time, pH, material: solvent ratio factors, microwave assisted extraction gave similar changing trend, but with higher yield (36% average). In time consumption, MAE is superior to SETM with extraction time only 360 seconds (6 minutes- and 36 minutes total, including immersion time), while extraction time in SETM is 180 minutes – 210 minutes total. Consequently, MAE is suitable method to extract green tea for further investigation. 4