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Available online at www.sciencedirect.com LWT - Food Science and Technology 41 (2008) 1616 e 1623

Available online at www.sciencedirect.com

Available online at www.sciencedirect.com LWT - Food Science and Technology 41 (2008) 1616 e 1623

LWT - Food Science and Technology 41 (2008) 1616e1623

LWT - Food Science and Technology 41 (2008) 1616 e 1623 www.elsevier.com/locate/lwt Effect of different brewing

Effect of different brewing methods on antioxidant properties of steaming green tea

Sheng-Dun Lin a , En-Hui Liu b , Jeng-Leun Mau b , *

a Department of Food and Nutrition, Hungkuang University, Shalu 433, Taichung, Taiwan, Republic of China b Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, Republic of China

Received 10 June 2007; received in revised form 22 October 2007; accepted 23 October 2007

Abstract

The extracts were prepared from cold or hot brewed steaming green tea at different concentrations (2, 6, and 10%), its antioxidant properties studied and potential antioxidant components determined. The yields of hot water extracts (17.49 e28.27%) were significantly higher than those of cold water extracts (11.72e 14.70%). EC 50 values in antioxidant activity determined by the conjugated diene method and reducing power were 2.19 e3.10 and 0.22 e0.28 mg/ml, respectively. EC 50 values in scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals were 29.45 e 43.80 and 2.88e 3.22 mg/ml, respectively. EC 50 values in chelating ability on ferrous ions were 6.45 e13.51 mg/ml. Con- tents of total phenols were 221.71 e330.22 mg/g whereas those of total catechins in cold and hot water extracts were 135.05 e193.14 and 161.57 e195.05 mg/g, respectively. Based on the results obtained, hot water extracts were more effective in antioxidant activity and reducing power. However, cold water extracts were more effective in scavenging ability on DPPH and hydroxyl radicals, and chelating ability on ferrous ions. Summarily, the cold brewing method would be a new alternative way to make a tea. 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Steaming green tea; Antioxidant activity; Reducing power; Scavenging ability; Chelating ability; Phenol, Catechin; Caffeine

1. Introduction

Tea has been consumed for centuries in the forms of unfer- mented (green tea), semi-fermented (oolong), and fermented (black tea) by ancient cultures for its medicinal properties (Ba- lentine, Wiseman, & Bouwens, 1997 ). Black tea is commonly consumed in the West whereas the consumption of green tea is especially popular in Asia, mainly for its health benefits ( Bushman, 1998; Cabrera, Gimenez, & Lopez, 2003; Seeram et al., 2006 ). Many studies have shown that polyphenolic com- pounds extracted from green tea leaves are good antioxidants against lipid peroxidation in phospholipid bilayers ( Terao, Piskula, & Yao, 1994 ), in biological systems ( Guo, Zhao, Li, Shen, & Xin, 1996; Katiyar, Agarwal, & Mukhtar, 1994 ), and against tumourigenesis and DNA damage ( Anderson

* Corresponding author. Tel.: þ886 4 2285 4313; fax: þ886 4 2287 6211. E-mail address: jlmau@dragon.nchu.edu.tw (J.-L. Mau).

et al., 2001; Jankun, Selman, Swiercz, & Skrzypczak-Jankun, 1997 ). Besides, green tea is also reported to reduce serum cho- lesterol levels and inhibit hypertension, mutagenesis, and tumourigenesis in several experiments in vitro and in vivo (Hodgson, Puddey, Burke, Beilin, & Jordan, 1999; Mura- matsu, Fukuyo, & Hara, 1986; Yang & Wang, 1993; Yoko- zawa et al., 1998; Yokozawa, Nakagawa, & Kitani, 2002 ). Several studies have shown that most of antioxidant proper- ties well correlated with polyphenolic compounds rather than ascorbic acid, tocopherol or b-carotene ( Lee, Huang, Liang, & Mau, 2007 ). Recently, the tea prepared by brewing tea leaves in cold water has become a new choice in Taiwan in addition to that traditionally prepared by brewing them in hot water. How- ever, antioxidant properties of the tea brewed in cold water are not available. Therefore, the objectives of this research were to study the effect of cold water and hot water brewing methods on antioxidant properties of steaming green tea. Extracts were pre- pared from cold or hot brewed tea at different concentrations (2, 6, and 10%). Antioxidant properties were assayed in terms

0023-6438/$34.00 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.lwt.2007.10.009

S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623

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of antioxidant activity by the conjugated diene method, reduc- ing power, scavenging abilities on 1,1-diphenyl-2-picrylhydra- zyl (DPPH) and hydroxyl radicals, and chelating abilities on ferrous ions. The contents of potential antioxidant components of cold and hot water extracts were also determined.

2. Materials and methods

2.1. Green tea extract preparation

The steaming green tea was made from the leaves of Camellia sinensis L. (cultivar Shy-Jih-Chun) in the summer season picked from the tea farm in Mingjian, Nantou County, Taiwan. For steam- ing green tea, young leaves were subjected to blanching (95e 100 C, 40e45 s), rolling and drying. The dried tea leaves were groundin a mill (Retsch ultracentrifugal mill and sieving machine, Haan, Germany), and screened through a 60-mesh sieve. The 2, 6 and 10% hot water extracts of steaming green tea (2, 6, and 10HS) were prepared from brewing the ground tea leaf powder (70, 210, 350 g) with 3500 g hot water (90 C) for 20 min and then filtering through Whatman No. 1 filter paper. The filtrate was cooled to room temperature, and lyophilized. The 2, 6 and 10% cold water extracts of steaming green tea (2, 6, and 10CS) were prepared from brewing the ground tea leaf powder (70, 210, 350 g) with 3500 g cold water (4 C) for 24 h, filtered and then freeze-dried. Dry extracts thus obtained were stored at 20 C before use.

2.2. Antioxidant activity

Antioxidant activity was determined by the conjugated diene method ( Lingnert, Vallentin, & Eriksson, 1979 ) and the modi- fied procedure used by Mau, Chao, and Wu (2001) . The antiox- idant activity was calculated as follows: antioxidant activity (%) ¼ [(D A 234 of control D A 234 of sample)/ D A 234 of control] 100%. EC 50 value (mg extract/ml) is the effective concentration at which the antioxidant activity was 50% and was obtained by interpolation from linear regression analysis. Ascorbic acid (Sigma Chemical Co., St. Louis, MO, USA), butylated hydroxyanisole (BHA, Sigma), Planex (Planex-GE, SD BNI Co., Ansan City, Gyeonggi-Do, Korea) and a-tocoph- erol (Sigma) were used for comparison. The specification of Planex: total phenols > 980 mg/g, total catechins > 800 mg/g, EGCG > 450 mg/g, and caffeine < 10 mg/g.

2.3. Reducing power

Reducing power was determined according to the method of Oyaizu (1986) . EC 50 value (mg extract/ml) is the effective concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid, BHA, a -tocopherol and Planex were used for comparison.

2.4. Scavenging ability on 1,1-diphenyl-2-picrylhydrazyl

radicals

Scavenging ability on DPPH radicals was determined according to the method of Shimada, Fujikawa, Yahara, and

Nakamura (1992) . EC 50 value (mg extract/ml) is the effective concentration at which DPPH radicals were scavenged by 50%. Ascorbic acid, BHA, Planex and a -tocopherol were used for comparison.

2.5. Scavenging ability on hydroxyl radicals

Scavenging ability on hydroxyl radicals was determined according to the method of Shi, Dalal, and Jain (1991) . EC 50 value (mg extract/ml) is the effective concentration at which hydroxyl radicals were scavenged by 50%. BHA and Planex were used for comparison.

2.6. Chelating abilities on ferrous ions

Chelating ability was determined according to the method of Dinis, Madeira, and Almeida (1994) . EC 50 value (mg ex- tract/ml) is the effective concentration at which ferrous ions were chelated by 50%. Citric acid (Sigma), ethylenediamine- tetraacetic acid (EDTA, Sigma) and Planex were used for comparison.

2.7. Determination of antioxidant components

Ascorbic acid was determined according to the method of Klein and Perry (1982) . b -Carotene was extracted and ana- lyzed as described by Rundhaug, Pung, Read, and Bertram (1988) . Tocopherols were extracted and analyzed according to the method of Carpenter (1979) . Total phenols were deter- mined according to the method of Taga, Miller, and Pratt (1984) and expressed as mg of gallic acid (Sigma).

2.8. Determination of caffeine and various catechins

Each extract (100 mg) was dissolved in deionized water (5 ml) and the solution was then filtered prior to injection into a HPLC. The HPLC system was the same as for the b - carotene assay. The mobile phase was 0.1% trifluoroacetic acid/acetonitrile, 86:14 (v/v), at a flow rate of 1.0 ml/min, and UV detection was at 280 nm. Contents of caffeine and various catechins were calculated on the basis of the calibra- tion curve of each caffeine, catechin, epicatechin (EC), gallo- catechin (GC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) (all from Sigma).

2.9. Statistical analysis

For each cold or hot water extract from three different treat- ments, three samples were prepared for assays of every antiox- idant attribute and component. The experimental data were subjected to an analysis of variance (ANOVA) for a completely random design (CRD) to determine the Fisher’s least signifi- cant difference at the level of 0.05.

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3. Results and discussion

3.1. Extraction yield

Generally, the yields of cold water extracts (11.72 e 14.70%) from steaming green tea were significantly lower than those of hot water extracts (17.49 e 28.27%) ( Table 1 ). Obviously, cold water was less effective than hot water in ex- tracting water-soluble component out of tea leaves. For cold water and hot water extractions, the yields were lower with higher ratio of tea leaves to water. However, the concentration of dry matters in crude tea infusion made was 2.94, 8.14 and 11.7 mg/ml for 2, 6 and 10CS and 5.65, 13.1 and 17.5 mg/ml for 2, 6 and 10HS, respectively. Although the soluble solids were higher in the tea solution made from higher ratio of tea leaves to water, the yields were lower due to limited solubility. This is in general agreement with the findings in Chao and Chiang (1995) . It seems that the ratio of tea leaves to water played an important role in the extraction yield. Based on the yield of 2CS as the efficacy of 100%, the yield efficacies of 6 and 10CS were 92.3 and 79.7% whereas based on the yield of 2HS as the efficacy of 100%, the yield effica- cies of 6 and 10HS were 77.2 and 61.9%, respectively. The yield efficacies decreased with increased concentrations and the yield efficacies of hot water extracts were lower with increased concentrations. In addition, the yields of hot water extracts were 92.3, 60.8 and 49.2% higher than those of cold water extracts for the corresponding concentrations of 2, 6 and 10%, respectively. When taking the operation and time costs into consideration, the commercial process in the plant might use higher ratio of tea leaves to water to make brewed tea and then diluted thereafter as usual.

3.2. Antioxidant activity

Using the conjugated diene method, antioxidant activities of cold and hot water extracts from steaming green tea were 2.14 e 2.33 and 26.3 e 29.5% at 1 mg/ml, respectively

Table 1 Extraction yield of water extracts from steaming green tea

Extraction method

Extraction % c (w/w)

Cold water extract a

2%

(2CS)

14.70 0.14D 13.57 0.46D 11.72 0.41E

6%

(6CS)

10% (10CS)

Hot water extract b

 

2%

(2HS)

28.27 1.12A 21.82 0.74B 17.49 0.95C

6%

(6HS)

10% (10HS)

a 2, 6 or 10%: tea leaves (70, 210 or 350 g) were extracted with 4 C cold water (3500 g) for 24 h and the filtrate was freeze-dried.

b 2, 6 or 10%: tea leaves (70, 210 or 350 g) were extracted with 95 C hot water (3500 g) for 20 min and the filtrate was freeze-dried.

c Extraction % ¼ (sample extract weight/sample weight) 100%. Each value is expressed as mean standard error (n ¼ 3). Means with different letters within a column are significantly different (P < 0.05).

(Fig. 1 ). At 5 mg/ml, cold and hot water extracts showed high antioxidant activities of 92.4 e 94.9 and 100%, respec- tively. However, antioxidant activities were 100% for BHA and a -tocopherol at 0.5 mg/ml whereas that was 57.5% at 0.5 mg/ml and 95.7% at 1 mg/ml for Planex. Ascorbic acid showed an antioxidant activity of 53.1% at 5 mg/ml. It seems that both water extracts from green tea were more effective in antioxidant activity than ascorbic acid whereas hot water extracts were more effective than cold water extracts. Obviously, Planex containing >98% polyphenols and > 80% catechins was more effective than water extracts, which con- tained some components other than catechins. Ho, Chen, Shi, Zhang, and Rosen (1992) studied antioxidant activities in lard by the Rancimat method and found that the polyphenol extracts from green tea were better than those from semi-fermented and black tea. Graham (1992) showed that catechins were the major components contributing to the antioxidant activity of green tea. Tanizawa, Sazuka, Komatsu, Toda, and Taniko (1983) pointed out that EC exhibited better antioxidant activity than other cate- chins. Lunder (1992) found that the EGCG content of tea leaves correlated well with its antioxidant activity. Chen and Ho (1995)

100 80 60 40 I 20 0 0 5 10 15 20 Antioxidant activity (%)
100
80
60
40
I
20
0
0 5
10
15
20
Antioxidant activity (%)
100 80 60 40 II 20 0 0 5 10 15 20 Antioxidant activity (%)
100
80
60
40
II
20
0
0 5
10
15
20
Antioxidant activity (%)

Concentration (mg/ml)

Fig. 1. Antioxidant activity of water extracts from steaming green tea. Each value is expressed as mean standard error ( n ¼ 3). I: ( B) 2CS, ( ) 6CS, (O ) 10CS, ( : ) ascorbic acid, ( ,) BHA, ( -) Planex, ( P) a-tocopherol; II: ( B) 2HS, ( ) 6HS, ( O) 10HS, (: ) ascorbic acid, (, ) BHA, ( -) Planex, (P ) a -tocopherol.

S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623

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studied antioxidant activities by the Rancimat method and the ef- fectiveness in the inhibition of lipid peroxidation was in the

descending

and Shahidi (1995) compared the antioxidant activities of four types of catechins in a b-caroteneelinoleate model system and found that the effectiveness was in the descending order:

ECG > EGCG w EC > EGC. Summarily, it is obvious that anti- oxidant activities of four types of catechins were not consistent with different assay methods. However, antioxidant activities of cold and hot water extracts exhibited good antioxidant activity.

order: EGCG > ECG > EGC > EC. Amarowicz

Salah et al. (1995) mentioned that the reducing power of flavonoids might be due to their possession of a 2,3-double bond in conjugation with the 4-oxo function in the C ring and a hydroxyl group adjacent to the B ring. In addition, the major components of polyphenols from tea extracts are cate- chins, characterized as flavanoids, which contained a saturated single bond at 2 and 3 positions. It reveals that the reducing power of catechins was not as effective as flavonoids. How- ever, the chemical interactions among various catechins might facilitate the scavenging of free radicals.

3.3. Reducing power

Reducing powers of cold and hot water extracts from steaming green tea were 0.91 e 0.93 and 0.93 e 1.14 at 0.5 mg/ml, respectively ( Fig. 2 ). It seems that the hot water extract 2HS was more effective. However, reducing powers of ascorbic acid, BHA, Planex, and a -tocopherol were 0.93, 0.97, 1.05, and 0.86 at 0.5 mg/ml, respectively. Apparently, both water extracts, ascorbic acid, BHA and Planex were more effective in reducing power than a -tocopherol.

1.4 1.2 1.0 0.8 0.6 I 0.4 0.2 0.0 Absorbance at 700 nm
1.4
1.2
1.0
0.8
0.6
I
0.4
0.2
0.0
Absorbance at 700 nm
0 5 10 15 20 1.4 1.2 1.0 0.8 0.6 0.4 II 0.2 0.0 0
0
5 10
15
20
1.4
1.2
1.0
0.8
0.6
0.4
II
0.2
0.0
0
5 10
15
20
Absorbance at 700 nm

Concentration (mg/ml)

Fig. 2. Reducing power of water extracts from steaming green tea. Each value is expressed as mean standard error (n ¼ 3). I: ( B) 2CS, ( ) 6CS, ( O) 10CS, ( : ) ascorbic acid, (, ) BHA, ( -) Planex, (P ) a-tocopherol; II:

( B) 2HS, ( ) 6HS, (O ) 10HS, ( : ) ascorbic acid, (, ) BHA, ( -) Planex, ( P) a-tocopherol.

3.4. Scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals

At 20 mg/ml, scavenging abilities of cold and hot water extracts from steaming green tea on DPPH radicals were 31.7 e 36.3 and 29.1 e 34.0%, respectively (Fig. 3 ). It seems that both water extracts in scavenging abilities were less effec- tive than ascorbic acid and Planex, which scavenged 41.8 and 69.2% of DPPH radicals at 20 mg/ml, respectively. However, at 0.5 mg/ml, BHA and a -tocopherol showed good scavenging abilities of 93.8 and 96.1%, respectively.

100 80 60 40 20 0 Scavenging ability (%)
100
80
60
40
20
0
Scavenging ability (%)
0 5 10 15 20 100 80 60 40 20 0 0 5 10 15
0 5
10
15
20
100
80
60
40
20
0
0 5
10
15
20
Scavenging ability (%)

Concentration (mg/ml)

Fig. 3. Scavenging ability of water extracts from steaming green tea on 1, 1-dipheny1-2-picrylhydrazyl radicals. Each value is expressed as mean standard error ( n ¼ 3). I: ( B) 2CS, ( ) 6CS, (O ) 10CS, ( : ) ascorbic acid, ( ,) BHA, ( -) Planex, ( P) a-tocopherol; II: ( B) 2HS, ( ) 6HS, ( O) 10HS, ( :) ascorbic acid, (, ) BHA, ( -) Planex, (P ) a -tocopherol.

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With regard to the scavenging ability of cold and hot water extracts on DPPH radicals, catechins seemed not to be a good hydrogen donor and thereby, less effective in terminating the propagation of free radicals. Furthermore, the less effectiveness in scavenging ability might be due to the fact that water extracts did not easily combine or complex with DPPH radicals. Brand- Williams, Cuvelier, and Berset (1995) found that the reaction time of antioxidant and DPPH radicals could be classified into three reaction kinetic types: quick, intermediate and slow types. Many experiments assessed the scavenging ability on DPPH rad- icals after 30 min. However, the results could not reflect the reaction of water extracts and DPPH radicals completely.

3.5. Scavenging ability on hydroxyl radicals

At 5 mg/ml, scavenging abilities of cold and hot water extracts from steaming green tea on hydroxyl radicals were 83.1 e 86.7 and 77.7 e 78.5%, respectively (Fig. 4 ). It seems that cold water extracts were more effective in scavenging abil- ity than hot water extracts but less effective than Planex, which scavenged 100% of DPPH radicals at 5 mg/ml. However, at 20 mg/ml, BHA showed a scavenging ability of 27.7%.

Husain, Cillard, and Cillard (1987) tested scavenging abil- ities of flavanoids on hydroxyl ions and found that the effec- tive ability correlated with the numbers of hydroxyl groups in the B ring. Structurally, catechins showed the presence of ortho -dihydroxyl groups in the B ring and totally, EC, EGC, ECG and EGCG contained 5, 6, 7 and 8 hydroxyl groups. It is speculated that the structure and hydroxyl group number of catechins were responsible for this high scavenging ability. In addition, Shi et al. (1991) reported that caffeine exhibited good hydroxyl radical scavenging ability and attributed the alleged anticarcinogenic properties of caffeine to this ability. These results indicated that water extracts from green tea are effective scavengers for hydroxyl free radicals. Accordingly, it was anticipated that the high scavenging ability of water extracts might possess some antimutagenic properties.

3.6. Chelating ability on ferrous ions

At 10 mg/ml, chelating abilities of cold and hot water extracts from steaming green tea on ferrous ions were 38.9 e 74.3 and 46.7 e 72.4%, respectively ( Fig. 5 ). For both water extracts, 2CS and 2HS showed higher chelating abilities

100 80 60 40 I 20 0 Scavenging ability (%)
100
80
60
40
I
20
0
Scavenging ability (%)
0 5 10 15 20 100 80 60 40 20 II 0 0 5 10
0 5
10
15
20
100
80
60
40
20
II
0
0 5
10
15
20
Scavenging ability (%)

Concentration (mg/ml)

Fig. 4. Scavenging ability of water extracts from steaming green tea on hydroxyl radicals. Each value is expressed as mean standard error ( n ¼ 3). I: ( B) 2CS, ( ) 6CS, ( O) 10CS, ( -) Planex; II: ( B) 2HS, ( ) 6HS, ( O) 10HS, (- ) Planex.

100 80 I 60 40 20 0 Chelating ability (%)
100
80
I
60
40
20
0
Chelating ability (%)
0 5 10 15 20 100 80 II 60 40 II 20 0 0 5
0 5
10
15
20
100
80
II
60
40
II
20
0
0 5
10
15
20
Chelating ability (%)

Concentration (mg/ml)

Fig. 5. Chelating ability of water extracts from steaming green tea on ferrous ions. Each value is expressed as mean standard error ( n ¼ 3). I: ( B) 2CS, ( ) 6CS, (O ) 10CS, ( : ) citric acid, ( ,) EDTA, ( -) Planex; II: ( B) 2HS, ( ) 6HS, ( O) 10HS, ( :) citric acid, ( , ) EDTA, ( -) Planex.

S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623

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than other extracts, indicating that higher ratios of tea leaves to water did not extract more components effective for ferrous ion chelation. Besides, both water extracts at the same ratios were comparable in their chelating ability. Surprisingly, Pla- nex, which contained more than 80% of catechins, exhibited less effective chelating ability of 36.0% at 20 mg/ml. It seems that the components other than catechins in water extracts might contribute most to this chelating ability. However, EDTA showed an excellent chelating ability of 98.1% at a concentration as low as 0.5 mg/ml. Citric acid was not a good chelating agent for ferrous ions and its chelating ability was 7.34% at 20 mg/ml. The chelating ability of tea was attributed to the specific functional groups in its flavanol structure. The adjacent hydroxyl and carbonyl groups in the molecule or hydroxyl groups among molecules could chelate ferrous ion to form

a complex ( Shahidi & Wanasundara, 1992 ). Therefore, the

more the hydroxyl and carbonyl groups in appropriate posi- tions, the higher the chelating ability of the molecule could

exhibit. Since ferrous ions are the most effective pro-oxidants

in the food system ( Yamaguchi, Tatsumi, Kato, & Yoshimitsu,

1988 ), the high ferrous-ion chelating abilities of water extracts

from steaming green tea would be beneficial if they were for- mulated into foods or brewed as a drink.

3.7. EC 50 in antioxidant properties

The antioxidant properties assayed herein were summa- rized in Table 2 , and the results were normalized and expressed as EC 50 values (mg water extract per ml) for com- parison. Effectiveness in antioxidant properties inversely cor- related with EC 50 value. EC 50 values in antioxidant activity by the conjugated diene method were 2.19 e 3.10 mg/ml, in which hot water extracts were more effective than cold water extracts. EC 50 values in reducing power were 0.22e0.28 mg/ml, indicating that both water extracts were extraordinarily effective and comparable. However, the hot water extract 2HS was the most effective. EC 50 values in scavenging ability on DPPH radicals were 29.45 e 43.80 mg/ml whereas those on hydroxyl radicals were 2.88 e 3.22 mg/ml. For scavenging abilities on radicals, cold water extracts were more effective than hot water extracts. With regard to EC 50 values in chelating ability on

ferrous ions, cold water extracts were better than the corre- sponding hot water extracts whereas low extraction ratios of tea leaves to water were more effective than higher ratios. EC 50 values in antioxidant properties were less than 4 mg/ml, except for scavenging ability on DPPH radicals (29.45 e 43.80 mg/ml) and chelating ability on ferrous ions (6.45 e 13.51 mg/ml). From EC 50 values obtained, it can be concluded that hot water extracts from steaming green tea were more effective in antioxidant activity and reducing power. However, cold water extracts were more effective in scavenging ability on DPPH and hydroxyl radicals, and chelat- ing ability on ferrous ions. When the extraction yields were taken into consideration, EC 50 values (mg dried tea leaves per ml) in antioxidant activ- ity were 21.0, 22.3, 26.5, 7.96, 10.2 and 12.5 mg/ml for 2, 6, 10CS, 2, 6 and 10HS, respectively. EC 50 values in reducing power were 1.98, 1.99, 2.30, 0.78, 1.15 and 1.54 mg/ml for 2, 6, 10CS, 2, 6 and 10HS, respectively. EC 50 values in scavenging ability on DPPH radicals were 244, 246, 251, 155, 157 and 194 mg/ml and those on hydroxyl radicals were 19.6, 22.2, 25.6, 11.3, 14.8 and 18.3 mg/ml for 2, 6, 10CS, 2, 6 and 10HS, respectively. EC 50 values in chelating ability on ferrous ions were 43.9, 66.5, 115, 24.1, 52.1 and 69.7 mg/ml for 2, 6, 10CS, 2, 6 and 10HS, respectively. Although BHA and a -tocopherol were good in antioxidant activity, reducing power and scavenging ability on DPPH rad- icals and EDTA was excellent for chelating ferrous ions, they are additives and used or present in mg levels in foods. How- ever, steaming green tea could be used in g levels as food or a food ingredient. Therefore, green tea in human diets might serve as possible protective agents to help human reduce oxi- dative damage, and can be developed as a dietary supplement and functional food in addition to be brewed as a drink.

3.8. Antioxidant components

Naturally occurring antioxidant components, including as- corbic acid and total phenols, were found in all water extracts from steaming green tea. b -Carotene and tocopherols were not found in all water extracts due to their fat-soluble nature. Contents of total phenols were 254.86, 222.04, 221.71, 330.22, 327.29 and 324.76 mg/g whereas those of ascorbic acid were 8.81, 10.39, 11.48, 8.04, 6.95 and 7.68 mg/g

Table 2 EC 50 value of water extracts from steaming green tea in antioxidant properties

EC 50 value a (mg/ml)

2CS

6CS

10CS

2HS

6HS

10HS

Antioxidant activity 3.09 0.05A b 3.03 0.05A 3.10 0.08A 2.25 0.07B 2.22 0.06B 2.19 0.05B

Reducing power

 

0.28 <0.01A 35.91 1.56B 2.88 0.04C 6.45 0.10E

0.27 <0.01A 33.38 1.02B 3.01 0.06B 9.02 00.07C

0.27 <0.01A 29.45 3.73C 3.00 0.03B 13.51 0.08A

0.22 <0.01C 43.80 2.12A 3.18 0.05A 6.82 0.07D

0.25 <0.01B 34.32 1.16B 3.22 0.09A 11.36 0.26B

0.27 <0.01A 33.85 1.92B 3.20 0.03A 12.19 0.68B

Scavenging

ability

on

DPPH radicals

Scavenging

ability

on

OH radicals

Chelating ability on ferrous ions

a EC 50 value, the effective concentration at which the antioxidant activity was 50%; the absorbance was 0.5 for reducing power; the 1,1-diphenyl-2-picryl hy- drazyl (DPPH) or hydroxyl (OH) radicals were scavenged by 50% and ferrous ions were chelated by 50%, respectively. The EC 50 value was obtained by inter- polation from linear regression analysis. EC 50 value of scavenging ability on DPPH radicals was obtained by extrapolation from linear regression analysis.

b Each value is expressed as mean standard error (n ¼ 3). Means with different letters within a row are significantly different ( P < 0.05).

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Table 3 Content of caffeine and various catechins of water extracts from steaming green tea

 

Content (mg/g)

2CS

6CS

10CS

2HS

6HS

10HS

Caffeine

21.84 0.18C b 2.38 0.29D 7.93 0.61B 3.96 0.21C 53.15 0.69B 39.07 0.43D 6.72 0.27C 135.05 2.04D

23.12 1.86C 2.65 0.89D 8.45 1.75B 3.76 0.89C 52.27 1.64B 40.68 1.49D 8.22 0.95B 139.15 7.71D

30.38 0.83A 5.55 1.47C 12.59 0.55A 5.60 0.43B 82.67 1.69A 46.53 0.62C 9.82 1.56B 193.14 2.01A

26.38 0.69B 3.13 0.02A 6.99 0.49B 8.37 0.63A 39.13 0.28D 69.16 1.76B 8.41 0.78B 161.57 1.68C

28.18 0.66B 2.96 0.08AB 7.59 0.62B 9.28 0.59A 43.95 1.67C 72.68 1.99B 13.38 1.54A 178.02 5.50B

31.29 2.01A 2.92 0.09B 8.33 0.73B 10.90 2.14A 44.46 2.72C 83.12 2.54A 14.03 0.82A 195.05 5.56A

Catechin

EC a

ECG

a

EGC

a

EGCG a

GC a

Total catechins

a EC, epicatechin; ECG, epicatechingallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; GC, gallocatechin.

b Each value is expressed as mean standard error (n ¼ 3). Means with different letters within a row are significantly different ( P < 0.05).

for 2, 6, 10CS, 2, 6 and 10HS, respectively. Surprisingly, total phenol contents correlated well with the extraction yields shown in Table 1 . Using 2HS as an example, its extraction yield and total phenol contents were the highest. Although the extraction yield and contents of antioxidant components differed among water extracts, the antioxidant properties assayed were comparable. It seems that the discrepancy in contents of total phenols and ascorbic acid did not cause anti- oxidant properties of water extracts different. Phenols, such as BHT and gallate, were known to be effective antioxidants (Madhavi, Singhal, & Kulkarni, 1996). Due to their scavenging ability on free radicals and chelating abilities on fer- rous ions (Lotito & Fraga, 1998), phenols contained good anti- oxidant properties, antimutagenic properties, and anticancer properties (Ahmad & Mukhtar, 1999). Furthermore, Yen, Duh, and Tsai (1993) found that the antioxidant activity of the meth- anolic extract from peanut hulls correlated with its content of to- tal phenols. Therefore, the high content of total phenols in all water extracts might explain their high antioxidant properties.

3.9. Caffeine and various catechins

Contents of caffeine in cold and hot water extracts from steaming green tea were 21.84 e 30.38 and 26.38 e 31.29 mg/g, respectively (Table 3 ). Obviously, hot water was more effective in extracting caffeine from tea leaves than cold water. Further- more, higher ratios of tea leaves to water extracted more caf- feine than lower ratios. Six catechins were detected in water extracts and contents of individual catechin fluctuated among water extracts and were not consistent with different water extraction and various ratios. Contents of total catechins in cold and hot water extracts were 135.05 e 193.14 and 161.57 e 195.05 mg/g, respectively. Like caffeine, hot water and higher ratios were more effective in extracting total catechins from tea leaves than cold water and lower ratios, respectively. Con- tents of caffeine and total catechins inversely correlated with the extraction yields. However, although contents of caffeine and total catechins were different among water extracts, the antioxidant properties assayed were comparable. EGCG was the major catechin in Planex accounting for more than 450 mg/g and caffeine content was less than 10 mg/g. It seems that caffeine in Planex did not contribute much to its

antioxidant properties. Apparently, content of total phenols (> 980 mg/g) and total catechins (> 800 mg/g) in Planex were 3 e 4 and 4 e 6 folds higher than those in water extracts, respec- tively. Therefore, Planex exhibited better effective in antioxi- dant properties than all water extracts, except for chelating ability on ferrous ions. It reveals that the components responsi- ble for this chelating ability might not be catechins and might be other components, which needed to be investigated further. Several studies ( Balentine et al., 1997; Dufresne & Farn- worth, 2000; Nakane, Hara, & Ono, 1994; Sakanaka & Yama- moto, 1997; Yang, 1999; Yen & Chen, 1994; Yen & Chen, 1996 ) have found that the antioxidant properties, antibacterial, antitoxin, antimutagens, and anti-inflammation of teas were mainly due to phenolic compounds. The antioxidant properties of green tea were the strongest among the various types of teas. Green tea contains considerable amounts of catechins, which contributed most to antioxidant properties. As shown in Table 3 , total phenols accounted for 22 e 33% of water extracts from steaming green tea and the flavanols along with their gallic acid esters (catechins) accounted for 49 e 87% of total phenols. These phenolic compounds usually are the most abundant water-soluble components in the tea ( Balentine et al., 1997 ) and responsible for effective antioxidant properties. In Japan, the tea is usually brewed from green tea using cold or warm water other than hot water. Cold water extracted fewer components than hot water. Furthermore, contents of to- tal phenols, caffeine and total catechins in cold water extracts were lower than those in hot water extracts. However, cold wa- ter extracts were more effective in scavenging ability on DPPH and hydroxyl radicals, and chelating ability on ferrous ions. Based on the results obtained, tea brewed from steaming green tea leaves using cold water could contain fewer components, especially caffeine, but possessed some antioxidant properties higher than tea brewed using hot water. Accordingly, this cold brewing method would be a new alternative way to make a tea.

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