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(HPLC) 1,1-diphenyl-2-picrylhydrazyl (DPPH) 10 min > 20 min > control > 30 min > 40 min control > 10 min > 20 min > 30 min > 40 min 10 min 36.95% 10 min 40 min
* , layhl@mail.npust.edu.tw 2011 3 10 2011 6 10 8:109-118 (2011) Crop, Environment & Bioinformatics 8:109-118 (2011) 189 Chung-Cheng Rd., Wufeng, Taichung 41362, Taiwan ROC
INTRODUCTION
Tea tree [Camellia sinensis (L.) O. Kuntze], which belongs to Theaceae family and Camellia genus, is the most important economic crop of Theaceae. It had been used as medicine in ancient times and became a daily beverage in the early
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West Han Dynasty (200 BC) in China. In fact, tea drinking has thousands years of history in China, and forms a specific culture (Juan and Chen 1998, Chen1999, Wu et al. 2007). Recently, tea is one of the most popular non-alcohol drinks world-wide, after coffee and cocoa. According to recent studies, many functionally healthful contents were found in tea, and made it known as a health care beverage (Juan and Chen 1998, Baletine 1997, Jain et al. 2006). There are many chemical compounds in tea, including polyphenolic compounds, volatile fragrances, pigments, alkaloids, proteins and free amino acids, carbohydrates, fat, organic acids, and minerals (Yu 1992). Among the components listed above, polyphenolics were the most special and important ones. The content of polyphenolics in tea has been proven its natural antioxidant activity (Tanizawa et al. 1984) and its good prevention efficacy on cardiovascular diseases, tumors forming, anticancer, and anti-antherosclerosis (Che et al. 2005, Farhoosh et al. 2007, Mamati et al. 2006). The content of catechins is the highest and makes up almost 80% of polyphenolic compounds. The major components of catechins include (+)-catechin (C), gallic acid (GA), (-)-epicatechin (EC), (+)gallocatechin (GC), (-)-catechin gallate (CG), (-)gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), and (-)epigallocatechin gallate (EGCG) (Sanderson 1972). Previous studies found that EC, ECG, EGC, and EGCG were transformed into C, CG, GC, and GCG by heat treatment which caused a change of the second position of the structure inducing isomerization (Haginaka et al. 2007, Ito et al. 2003). Therefore, the objective of this study was to quantitatively analyze the antioxidant ability of teas using high performance liquid chromatography (HPLC) and to assess the effect of different time periods of autoclave treatment on quality variation.
Standard samples of caffeine, C, GA, EC, GC, CG, GCG, ECG, EGC, EGCG, quercetin, 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxyltoluene (BHT), and Folin-Ciocalteus phenol reagent were obtained from Sigama Chemical Company (St. Louis, Mo, USA). Scopoletin was purchased from Fluka Chemie AG (Switzerland) and was used as an internal standard. Acetonitrile, methanol, and DMSO (HPLC grade) were obtained from Mallinckrodt, Incorporation (USA), and sodium carbonate and phosphoric acid (analytical-reagent grade) were from Kanto Chemical (Japan). Ultra-pure distilled water with a resistance greater than 18.2 M cm-2 was prepared with a mini-Q system (Millipore, Bedford, MA, USA). All other reagents were of analytical grade.
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200.0g mL-1; C: 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, and 110.0 g mL-1; EC: 1.71, 3.43, 6.87, 13.75, 27.5, 55.0, and 110.0 g mL-1; EGCG: 24.84, 49.68, 99.37, 198.75, 398.5, 795.0, and 1590.0 g mL-1; GCG: 4.21, 8.43, 16.87, 33.75, 67.5, 135.0, and 270.0 g mL-1; ECG: 4.53, 9.06, 18.12, 36.25, 72.5, 145.0, and 290.0 g mL-1; and CG: 0.93, 1.87, 3.75, 7.5, 15.0, 30.0, and 60.0 g mL-1. Each dilution contained 50 g mL-1 of scopoletin, the internal standard solution. After filtering through a 0.45 m membrane filter, an aliquot of 20 L of each concentration solution was injected into the HPLC column for analysis. The calibration line was plotted by using the ratio of the peak areas that corresponded to each standard solution and the internal standard solution on the Y-axis versus each concentration on the X-axis. Linear regression method was used to evaluate the parameters of y = ax + b and the correlation coefficient (r).
2. Accuracy
Each standard stock solution of various concentrations was spiked into methanol solution of tea leaves, and then refluxed at 80oC for 2 h. Internal standard solution was added to each solution to afford a concentration of 50 g mL-1. Then the solution was filtered and subjected to HPLC analysis in triplicates. The recovery (%) was calculated by the equation of [(C3-C2)/C1] 100%, where C1 is the amount of each standard spiked, C2 is the amount of each marker in methanol solution of tea leaves, and C3 is the total amount of each marker in the solution.
Calibration Method
The standard solutions of each marker substance were diluted by 70% methanol to give sequential concentrations of GA: 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, and 100.0 g mL-1; caffeine: 17.81, 35.62, 71.25, 142.5, 285.0, 570.0, and 1140.0 g mL-1; GC: 1.87, 3.75, 7.50, 15.0, 30.0, 60.0, and 120.0 g mL-1; EGC: 3.12, 6.25, 12.50, 25.0, 50.0, 100.0, and
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One gram of each grounded samples was extracted by reflux with 100 mL methanol at 80oC for 2 h. Each solution was filtered and evaporated, and each extract was freeze-dried and stored at -4C for the analysis of antioxidant activity.
11.36 min for GA, 18.80 min for caffeine, 28.99 min for GC, 31.25 min for EGC, 33.32 min for C, 45.47 min for EC, 47.57 min for EGCG, 53.25 min for GCG, 56.37 min for ECG, 68.33 min for CG, and 73.36 min for the internal standard, scopoletin. The peaks of marker substances in the tea sample solutions were qualified by HPLC with photodiode array detector. High purity (more than 0.99) of each peak stood for each marker substance. The results showed that GA, caffeine, GC, EGC, C, EC, EGCG, GCG, ECG, and CG were separated completely and were not interrupted by other compounds under the performed analytical conditions in this study. The result also showed high purification and separation efficacy. Therefore, the conditions described above can be used for quantification of the marker substances.
Calibration Curve
The regression equations and correlation coefficients of calibration lines for those marker substances are listed in Table 2. All calibration curves were in good linear correlation with correlation coefficient of 0.9980~0.9999.
Statistical analysis
All the experiments were triplicate. One-way ANOVA with Duncans multiple range test was used to analyze and compare the data, with P < 0.05 as the limit of significance by SAS (Statistical Analysis System).
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Fig. 1. HPLC chromatograms of marker component (A), Pu-erh tea (B), autoclaved Puerh tea (C), Oolong tea (D), and autoclaved Oolong tea (E). 1: gallic acid; 2: (+)-gallocatechin; 3: (-)-epigallocatechin; 4: (+)-catechin; 5: caffeine; 6: (-)-epicatechin; 7: (-)-epigallocatechin gallate; 8: (-)-gallocatechin gallate; 9: (-)-epicatechin gallate; 10: (-)-catechin gallate; and IS: scopoletin. Table 2. The regression equations of calibration curves of each compounds. Compound GA CA GC EGC C EC EGCG GCG ECG CG Concentration range (g mL-1) 1.56 ~ 100.0 17.81~1,140.0 1.87 ~ 120.0 3.12 ~ 200.0 1.71 ~ 110.0 1.71 ~ 110.0 24.84 ~ 1590.0 4.21 ~ 270.0 4.53 ~ 290.0 0.93 ~ 60.0 Regression equation y = 0.9802x + 0.0013 y = 1.0963x - 0.0703 y = 1.0553x - 0.0082 y = 1.0376x - 0.011 y = 1.0536x - 0.0135 y = 1.1067x - 0.0087 y = 1.0552x-0.1497 y = 1.0973x - 0.0143 y = 1.2638x - 0.0136 y = 1.0063x - 0.0011 r 0.9999 0.9980 0.9996 0.9994 0.9995 0.9996 0.9982 0.9997 0.9998 0.9999 N 3 3 3 3 3 3 3 3 3 3
GA:gallic acid, CA: caffeine, GC: (+)-gallocatechin, EGC: (-)-epigallocatechin, C:(+)-catechin, EC: (-)-epicatechin, EGCG: (-)-epigallocatechin gallate, GCG: (-)-gallocatechin gallate, ECG: (-)-epicatechin gallate, and CG: (-)-catechin gallate.
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Table 3. Relative standard deviations of intra-day, inter-day, and recovery analysis of each compounds. Compound Concentration (g mL-1) 100.00 12.50 1.56 1140.00 142.50 17.81 120.00 15.00 1.88 200.00 25.00 3.13 110.00 13.75 1.72 110.00 13.75 1.72 1590.00 198.75 24.84 270.00 33.75 4.22 290.00 36.25 4.53 60.00 7.50 0.94 R.S.D. % intra-day inter-day (n=3) (n=4) 1.20 1.98 3.08 3.32 0.13 1.16 0.40 1.80 0.26 1.34 0.41 2.04 0.61 1.13 0.23 1.10 0.53 2.25 0.35 1.17 0.41 1.48 0.71 2.99 1.14 3.20 0.92 1.02 0.32 3.92 0.87 2.19 1.88 3.28 0.50 0.90 0.54 1.27 0.16 2.35 0.24 2.06 0.98 1.32 0.37 2.62 0.26 1.98 0.41 1.10 0.18 1.54 0.38 3.33 0.95 3.22 1.19 0.52 1.21 2.31 Recovery Mean S.D. (R.S.D. %) 108.821.65 (1.51) 116.590.06 (0.05) 101.840.77 (0.75) 101.120.89 (0.88) 108.581.19 (1.09) 97.560.18 (0.18) 110.190.29 (0.26) 114.460.62 (0.54) 98.450.04 (0.04) 106.190.82 (0.77) 110.840.33 (0.29) 109.371.14 (1.04) 112.540.95 (0.84) 117.450.25 (0.21) 115.710.09 (0.07) 104.100.57 (0.54) 115.120.73 (0.63) 109.890.69 (0.62) 105.481.32 (1.25) 107.161.03 (0.96) 114.610.99 (0.86) 103.950.83 (0.79) 112.710.61 (0.54) 114.480.83 (0.72) 104.860.52 (0.49) 98.011.21 (1.23) 112.780.75 (0.66) 101.510.69 (0.67) 107.730.32 (0.29) 100.410.44 (0.43)
Gallic acid
(+)-Gallocatechin
(-)-Epigallocatechin
(+)-Catechin
Caffeine
(-)-Epicatechin
(-)-Epigallocatechin gallate
(-)-Gallocatechin gallate
(-)-Epicatechin gallate
(-)-Catechin gallate
S.D.: Standard deviation; R. S. D.: Relative standard deviation. 40 min after autoclaving and the decreasing rates were 61.59, 48.84, 48.91 and 30.96%, respectively. However, contents of GA, GC, C, GCG and CG increased 2.4, 1.9, 1.1, 9.5 and 6 times, respectively. Total catechins reduced 31.36%. Oolong tea had the similar results as that of Puerh tea. The contents of EGCG, EGC, ECG and EC in Oolong tea decreased rapidly in 40 min after autoclaving and the decreasing rates were 40.17, 63.45, 37.49 and 48.99%, respectively; whereas, GA, GC, GCG and CG increased 2.1, 1.8, 15.3 and 1.4 times, respectively. Total catechins reduced 30.6%, whereas, caffeine was not decomposed by autoclaving with time. When autoclaving time reduced, contents of EGCG, EGC, ECG, and EC decreased, while contents of GC, C, GCG, and CG increased, in both Puerh tea and Oolong tea. Previous reports had similar results in the experiments of autoclaving water extract of green tea (Kim et al. 2007, Sun et al. 2004). The major reason behind the increase of GA might be the decomposition of lipid catechins under oxidization or autoclaving (Juan, et al. 1989). However, caffeine was not
Table 4. Contents of compounds of Pu-erh tea and Oolong tea under different time periods of autoclave treatment.
CA GC EGC C EC EGCG GCG ECG CG TC
Time
GA
(min) 30.210.32b 2.430.03b 3.540.03c 3.500.02c 3.090.03d 19.890.16a 0.620.02e 0.690.01b 0.650.01d 0.670.02c 1.770.08e 1.950.01d 36.270.20d 33.560.11e 2.130.02c 40.640.34c 2.670.02b 48.740.64b 0.710.01a 3.470.04a 56.090.93a 0.640.01e 3.910.10d 6.300.06c 7.300.06b 9.800.06a 1.200.06c 1.940.03e 13.820.19e 10.380.17a 1.210.02c 2.170.02c 16.100.17d 8.800.10b 1.250.01b 2.080.04d 17.200.18c 7.240.03c 8.680.04c 8.350.09d 7.250.11e 10.990.24 10.220.85b 7.780.06c 7.270.03cd 6.870.07d 6.450.04a 1.330.01a 3.370.02a 26.470.48b 4.530.01d 10.660.05b 1.410.02c 6.040.07b 1.110.02d 2.810.03b 35.980.53a 1.090.01e 14.190.21a 0.480.01e 1.890.01d 2.150.14c 2.750.03b 2.900.02a nd 0.330.03d 0.840.01c 1.100.02b 1.410.01a 63.11 57.13 44.68 45.65 43.32 93.92 83.53 72.28 66.12 65.18
1.630.03e
2.640.02d 30.800.12a
Puerh
20
3.020.03c
30
3.450.05b 30.710.27a
40
3.840.30a
0.310.01e
Oolong
20
0.460.01c
tea 24.340.14a
30
0.640.04b 24.110.13a
40
0.660.03a
Each sample was triplicated in the test and results were mean (mg mL-1) SD. nd: not detectable.
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decomposed by the longer autoclaving time. When tea leaves were roasted, increase of roasting time at high temperature would decrease catechins content, as in the process of autoclaving, and induced isomerization, oxidized polymerization, and degradation (Wang and Helliwell 2000, Chen et al. 2001). It was ensured that the longer the autoclaving time, the higher was the degree of epicatechins transformed into non-epicatechins. The degree of transformation of epicatechins into non-epicatechins depended on the time period of autoclaving.
100oC, but increased at temperatures higher than 120oC. When roasting temperature higher than 160oC, total phenolic content decreased (Juan et al. 1989). Results indicated that the total phenolic content decreased at 121oC under high pressure.
Fig. 2. Effect of different time periods of autoclave treatments on content of total phenols in Puerh tea and Oolong tea. Each sample was triplicated in the test and data were average (mg g-1 ) SD.
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Fig. 3. DPPH scavenging ability of methanol extracts from Puerh tea and Oolong tea with different time periods of autoclave treatment. extract of Oolang tea control group at 5 g mL-1, when Puerh tea was autoclaved for 10 to 20 min. provide a taste of astringent, while, nonepicatechins provide bitter taste first and sweet flavor later and its astriction was weaker than epicatechins (Sun et al. 2004). Autoclaving and roasting may change color, aroma, and tastes of tea, relating to the change of chemical compounds in tea leaves.
CONCLUSIONS
As a result, autoclave treatment directly affects the transformation from epicatechins to non-epicatechins and indirectly affects the total phenolic content. Longer processing time is disadvantageous to antioxidant ability. Catechins were the source of bitter and astringent taste (Kuo 1999), especially epicatechins, EGCG and ECG. They are the components that formed the quality of tea flavor and affect the taste. Epicatechins
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