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48 ผลกระทบของสภาวะการสกัด ปที่ 40 ฉบับที่ 1 (พิเศษ) มกราคม-เมษายน 2552 ว. วิทยาศาสตรเกษตร Wang et al., 2000; Yoa et al., 2006; Yoshida et al., 1999). The main components consisted in tea are polysaccharides, phenolic compounds, vitamin, amino acids and others, among them phenolic compounds have been on focus for their beneficial effects. Due to, it tea has attracted considerable interest (Row และ Jin, 2006). Phenolic compounds which constitutes up to 30% on a dry weight basis of fresh tea leaves are water soluble so can be easily extracted with water, where extraction conditions such as time, temperature, pH, ratio of solvent to material and number of extraction step variously influenced the obtained extract (Labbe et al., 2006; Yoshida et al., 1999). This study investigated extraction of Assam fresh leaves and green tea using water, and varied the tea:water ratio and pH. A multi-step extraction procedure was performed using the optimal tea:water ratio and pH. Materials and Methods Materials Assam fresh leaves and green tea were supplied from Doi Wawee, Chiang Rai, Thailand. Standards of gallic acid, tannic acid, caffeine anhydrous and L-theanine were purchased from Sigma Chemical Co. Study the effect of tea-water ratio and pH Assam fresh tea leaves and green tea were extracted using deionized water with tea:water ratio 1:5, 1:10, 1:20 and 1:30. The pH of water was 4, 5, 6 and 7, adjusted with citric acid. Extraction was performed at 95 o C for 10 min (Uzunalic et al., 2006) in a thermostated waterbath. The aqueous extracts were analyzed for the contents of total polyphenols (Folin-Ciocalteu colorimetric method: ISO 14502-1:2005), tannins (Folin-Ciocalteu colorimetric method: FAO/ IAEA:2000), caffeine (colorimetric method: Yoa et al. (2006)) and total amino acids (colorimetric method: Yoa et al. (2006)) Multi-step extraction Assam fresh tea leaves and green tea were extracted using deionized water (with the optimum tea-water ratio and pH from item 2.2). The residue from the first step of extraction was re-extracted again under the same conditions and this was repeated four times. The four subsequent extracts were then analyzed for the total polyphenols, tannins, caffeine and total amino acids (using procedure as described in item 2.2). Results and Discussion Study the effect of tea-water ratio and pH The fresh tea leaves and green tea contained total polyphenols (21.485 and 20.710% w/w of dried solid), tannins (6.984 and 10.604%), caffeine (4.610 and 4.079%), total amino acids (5.65 and 6.012%), respectively. Table 1 shows compositions from Assam fresh leaves and green tea extracts. A higher ratio of tea:water yielded higher contents of total polyphenols. The tea:water ratio of 1:20 and 1:30 had a better result than other ratios in both tea samples. Water at pH 4 was found to be the most appropriate for extracting total polyphenols from fresh leaves and green tea. Considering about economical aspect, it can be concluded that the best treatment combination to get the maximum amount of total polyphenols extracted from fresh leaves were tea:water ratio 1:20 at pH 4 (12.871%), while in green tea were ratio 1:20 at pH 5 (10.783%). In acid conditions, the total polyphenols extraction is generally better because acids can prevent oxidative changes during extraction. Since phenolic acids usually occur in conjugated forms and are seldom found in free state, it is necessary to do hydrolysis that can rupturing glycosidic linkages to release any bound phenolics (Yoshida et al., 1999).
ว. วิทยาศาสตรเกษตร ปที่ 40 ฉบับที่ 1 (พิเศษ) มกราคม-เมษายน 2552 ผลกระทบของสภาวะการสกัด 49 Table 1 The composition of the extracts extracted from fresh leaves and green tea with various tea:water ratio and pH Total Polyphenols (% w/w dried solid) Tannins (%w/w dried solid) Condition Total Amino Acids (%w/w dried solid) Tea:water pH Fresh Leaves Green tea Fresh Leaves Green tea Fresh Green tea Fresh Ratio Leaves Leaves Green tea 4 9.151 A γ 8.368 b 4.653 a 4.989 e 2.956 f 2.522 b 5.042 A α 4.475 f 1:05 5 8.750 A β 8.408 b 4.818 a 5.681 e 2.804 d 2.422 a 4.577 A α 4.582 f 6 7.380 A αβ 7.140 a 4.808 a 5.037 d 2.572 b 2.219 a 4.524 A α 4.335 cd 7 7.463 A α 7.114 a 4.396 a 4.860 d 2.320 a 2.479 b 4.458 A α 4.050 b 4 12.251 B γ 9.520 e 4.564 a 5.588 f 3.097 h 2.768 d 5.597 B α 4.940 g 1:10 5 10.812 B β 9.510 e 3.960 a 4.671 c 2.993 g 2.369 a 5.517 B α 4.547 f 6 10.126 B αβ 9.071 d 3.923 a 4.273 b 2.903 e 2.376 b 5.437 B α 3.971 b 7 10.104 B α 8.543 c 3.923 a 3.317 a 2.764 c 2.808 c 5.384 B α 3.584 a 4 C γ 12.871 h 10.820 a 4.491 g 5.933 k 4.109 g 3.577 B α 5.625 e 4.471 1:20 5 11.609 C β 10.783 h 4.308 a 4.863 d 4.056 i 3.297 f 5.609 B α 4.243 c 6 11.742 C αβ 10.165 g 4.344 a 5.099 d 4.078 j 3.303 f 5.575 B α 3.888 b 7 10.502 C α 9.457 e 4.180 a 4.907 d 4.078 j 3.188 e 5.564 B α 3.895 b 4 C γ 12.644 h 10.837 a 4.406 d 4.987 m 4.368 h 4.038 B α 5.644 b 3.926 1:30 5 11.780 C β 10.805 h 4.214 a 6.574 h 4.165 m 3.349 f 5.604 B α 4.050 b 6 11.714 C αβ 9.941 f 4.049 a 6.489 h 4.362 m 2.814 d 5.580 B α 3.727 a 7 10.949 C α 8.973 d 4.104 a 6.449 h 4.354 m 2.848 d 5.576 B α 3.631 a Different capital letters indicate significant difference (α = 5 %) among tea:water ratio; different Greek letters indicate significant difference among pH; different lower-case letters indicate significant different among tea:water ratio and pH. Higher tea-water ratio and lower pH tended to give higher tannins obtained in green tea extract, extraction at pH 4 and tea:water ratio 1:20 showed the highest result to get more tannins (5.933%) in the extract. However, in the fresh leaves, ratio and pH have no significance effect to the amount of tannins in the extracts. Tannins classified into hydrolysable tannins and condensed tannins. Hydrolyzable tannins can be hydrolyzed by mild acids or mild bases and also by hot water or enzymes to produce carbohydrate and phenolic acids (Cannas, 2001; Chung et al., 1998). Due to that reason, extraction at lower pH yield higher tannins than that at higher pH in green tea. At higher tea water ratio, amount of caffeine extracted from fresh and green tea tended to be increased. Extraction at the ratio of 1:30 get more caffeine than other ratios in both fresh leaves and green tea. From Table 1, it can be concluded that the maximum amount of caffeine could be obtained by using ratio 1:30 for fresh leaves (4.368%) and ratio 1:30 at pH 4 for green tea (4.038%). Tea to water ratio affected the extraction of total amino acids both in fresh leaves and green tea. With varying tea-water ratio, ratio 1:10, 1:20 and 1:30 had better results than ratio 1:5 in fresh leaves, while in green tea, ratio 1:10 gave a better result than other ratios. Lower pH tended to give better result for extracting amino acids in green tea, but in fresh leaves, pH had no significant difference on these compounds. The most appropriate conditions for extracting total amino acids from fresh leaves were ratio 1:10 at pH 7 (5.384%), while in green tea were ratio 1:10 at pH 4 (4.471%). Multi-step extraction The single step extraction with tea:water ratio 1:20 at pH 5 was found to be the optimum conditions for extracting total polyphenols (10.783%) from green tea. Comparing to the amount of these compounds in raw materials, most of total polyphenols were left in residues. Due to this reason, multi-step extraction procedure was approached. The two steps of extraction were considered to be enough economically for extracting total
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