Components and Antioxidant Activity in Teas after Autoclave Treatment Research Article

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Components and Antioxidant Activity in Teas after Autoclave Treatment

Bo-Long Chen and Horng-Liang Lay*
Department of Plant Industry, National Pingtung University of Science & Technology, Neipu, Pingtung Hsien 91201, Taiwan ROC
In this study, Puerh and Oolong teas were used as materials to investigate the changes in catechins content and to compare their antioxidant activities, with different time periods of autoclave treatment. Tea samples were extracted with methanol and the quantitative variation of each catechin marker component was analyzed by high performance liquid chromatography. Moreover, antioxidant assays were determined according to total phenolic content and scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radicals. Two kinds of tea showed similar results after autoclave treatment. Catechins were transformed from epicatechins into non-epicatechins. Gallate acid content was increased; however, caffeine was not affected. In antioxidant assays, the antioxidant ability of Puerh tea with time in descent order was 10 min > 20 min > control > 30 min > 40 min and of Oolong tea was control > 10 min > 20 min > 30 min > 40 min. In total phenolic content, Oolong tea was rapidly decreased to 36.95% after 10 minutes of autoclave treatment and Puerh tea was gradually decreased from 10 min to 40 min. Finally, this study showed that autoclave treatment directly affected catechins and indirectly affected total phenolic content. Antioxidant ability of teas decreased with the increase of duration of autoclave treatment. Key words: Tea, Catechins, Antioxidant, Total phenolic content.

(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.

Autoclaving Treatment of Tea Leaves

One hundred grams of Puerh and Oolong tea leaves were put into an autoclave for 0 (control), 10, 20, 30, and 40 min treatments under high pressure (1.5 kg cm-2) and high temperature (121oC). All treatments were triplicated and the average values were used.

Preparation of Sample Solutions

All samples were dried at 60oC for 24 h. One gram of each sample was reflux and extracted with 100 mL methanol at 80oC for 2 h. The solutions were filtered, evaporated, and adjusted up to 25 mL using methanol. Meanwhile internal standard scopoletin was added to each solution to a concentration of 50 g mL-1 and used for subsequent HPLC analysis after filtration through a 0.45 m membrane filter.

Preparation of Standards and Internal Standard Solution

All standard components were weighed and dissolved in 70% methanol to yield sequential concentrations as follows: GA 100.0 g mL-1, caffeine 1140.0 g mL-1, GC 120.0 g mL-1, EGC 200.0 g mL-1, C 110.0 g mL-1, EC 10.0 g mL-1, EGCG 1590.0 g mL-1, GCG 270.0 g mL-1, ECG 290.0 g mL-1, and CG 60.0 g mL-1. They were then prepared as the standard stock solutions. Scopoletin of 50.0 g mL-1 was prepared as the internal standard stock solution.

MATERIALS AND METHODS

Materials
Puerh tea produced in Yun-Nan Province of China was purchased from a local market in Kaohsiung City. Locally made Oolong tea was purchased from Min-Gein Township, Nan-Tou Hsien.

Chemicals and Reagents

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HPLC Measuring System nd Conditions

Analysis was conducted by a Hitachi HPLC system equipped with a degasser (DG-2410), pump (L-7100), UV/VIS detector (L-7420), photodiode array detector (L-4500) and autosampler (L-7200). Peak areas were calculated with the software D-7000 HSM (Hitachi). The reversed phase of an Inertsil 5 ODS-2 column (4.6 mm I.D. 250 mm) was used. The oven temperature set for column was 40oC. The mobile phase consisted of water, 20% acetonitrile aqueous solution, and 100% acetonitrile aqueous solution (each adjusted to pH 3.0 with phosphoric acid), and a linear gradient elution was performed as shown in Table 1. The injection volume was 20 L, with the flow rate of 1.0 mL min-1. The detection wavelength was 220 nm in UV. The samples were quantified by interpolating the linear regression plot obtained from standard solutions. Table 1. Gradient elution program for mobile phase of HPLC anaysis. Time (min) 0 5 15 25 35 45 55 60 70 80 85 90 95 100 H2O 98 85 70 58 58 40 35 35 30 30 0 0 98 98 20% acetonitrile 2 15 30 42 42 60 65 65 70 70 70 10 2 2 100% acetonitrile 0 0 0 0 0 0 0 0 0 0 30 90 0 0

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).

Validation test 1. Precision

Standard stock solutions were diluted with 70% methanol to three different concentrations (Table 1). Intra-day tests (injecting each concentration three times within 24 h) and inter-day tests (injecting each concentration four times over 7 d with each injection separated at least 24 h) were carried out to check reproducibility. The standard deviation (S.D.) and relative standard deviation (R.S.D., %) were calculated.

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.

Each adjusted to pH 3.0 with phosphoric acid.

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

Analysis of Antioxidant Activity 1. Sample Extraction

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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.

2. Total Phenolic Content

Total phenolic content was determined by the Folin-Ciocalteu assay of Li et al. (2006). Briefly, fifty micro-liter sample solution (1 mg mL-1) was mixed with 250.0 L of 10% Folin-Ciocalteu solution, added with 750.0 L of 7.5% (w/v) Na2CO3, and then reacted at room temperature for 2 h in dark. The absorbance was measured at 765 nm using a UV-visible spectrophotometer (ChromTech Co., Taiwan). Total phenolic content was calculated using various concentrations of gallic acid solution (8.13-130.0 g mL-1) from the calibration curve.

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.

3. Free-Radical Scavenging Ability

The scavenging ability of methanol extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH) freeradicals was estimated according to the method of Shimada et al. (1992). Briefly, 2 mL of each test sample was mixed with 0.5 mL of 1 mM DPPH in methanol. The mixture was shaken vigorously and then steadily stayed for 30 min at room temperature in dark. The absorbance of the resulting solution was detected at 517 nm against an aliquot blank. The scavenging ability was calculated as follows: scavenging ability (%) = [(A517 of control A517 of sample) / A517 of control] 100. The higher the scavenging ability value, the higher the antioxidant activity is detected in the methanol extract (Shimada et al. 1992).

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.

Precision and Accuracy

According to the standard solutions with various concentrations shown in Table 3, the relative standard deviations of the intra-day and inter-day were between 0.16 to 3.08 % and 0.52 to 3.92%, respectively. It suggested that the HPLC analysis in this study is stable and reproducible. Recovery rates of the analysis were shown in Table 3, and were all greater than 98.01%, indicating the analysis using HPLC in this study was accurate.

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).

Component Contents in Tea Leaves after Autoclave Treatment

In general, products were sterilized for a period of time at 120C when tea beverages were canned and packaged. It was found that GCG was increased from the isomerization of EGCG while tea beverages were autoclaved (Okumura et al. 2008). In this study, Puerh tea and Oolong tea were autoclaved for 10, 20, 30, and 40 min and were then compared with the control. The results w e r e s ho w n i n T a b l e 4 , i nd i c a t i n g t h a t epicatechins in Puerh tea decreased significantly after autoclaving. Among epicatechins, contents of EGCG, EGC, ECG and EC decreased rapidly in

RESULTS AND DISCUSSION

Separation of Marker Substances by HPLC
HPLC chromatograms of the methanol extracts of Puerh tea and Oolong tea are shown in Fig. 1. The chromatograms indicated the respective retention time for each of components:

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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

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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

10 30.490.28ab 2.540.03b 2.770.03a 2.740.01a 2.130.02e

2.640.02d 30.800.12a

Puerh

20

3.020.03c

tea 30.220.19b 23.250.13b

30

3.450.05b 30.710.27a

40

3.840.30a

0.310.01e

10 24.000.20ab 3.490.03c 3.680.03b 3.830.02a 7.270.03d 7.900.04d 10.220.08c

0.390.05d 23.940.27ab 3.190.03d 13.850.18b

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.

The DPPH Radical-scavenging Capacity of Tea Leaves after Autoclave Treatment

The DPPH free-radical scavenging ability of methanol extracts of Puerh tea and Oolong tea under different autoclaving time periods was shown in Fig. 3. At the concentration of 20 g mL-1, the scavenging ability was higher than 71.64%, no matter how long was the autoclaving time. DPPH scavenging ability depended on the extract concentration. When concentration was 5 g mL-1, DPPH scavenging ability of both extracts decreased to 7.86 to 43.63%. Scavenging ability was lower when Puerh tea and Oolong tea were autoclaved for 30 to 40 min. When comparing Puerh tea and Oolong tea with standard solutions of vitamin C and BHA, all had DPPH scavenging ability of 79.63% at the concentration of 15 to 50 g mL-1. At concentration of 5 g mL-1, the scavenging ability decreased to 22.34%. Control group of Puerh tea had the same free-radical scavenging ability as the methanol

Total Phenolic Content of Tea Leaves after Autoclave Treatment

The total phenolic content of the methanol extract of Puerh tea and Oolong tea under different autoclaving time periods was shown in Fig. 2. Results showed that total phenolic content in Oolong tea was drastically decreased to 36.95% when autoclaved for 10 min. However, total phenols in Puerh tea decreased gradually from 10 to 40 minutes, similar to previous reports (Sun et al. 2004, Wang and Helliwell 2000). Total phenolics content in green tea were decreased when autoclaved. Total phenolic content of Puerh tea and Oolong tea changed little when roasted at

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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