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


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Effect of different brewing methods on


antioxidant properties of steaming green tea
Sheng-Dun Lin a, En-Hui Liu b, Jeng-Leun Mau b,*
b

a
Department of Food and Nutrition, Hungkuang University, Shalu 433, Taichung, Taiwan, Republic of China
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 (17.49e28.27%) were significantly higher than those
of cold water extracts (11.72e14.70%). EC50 values in antioxidant activity determined by the conjugated diene method and reducing power were
2.19e3.10 and 0.22e0.28 mg/ml, respectively. EC50 values in scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl
radicals were 29.45e43.80 and 2.88e3.22 mg/ml, respectively. EC50 values in chelating ability on ferrous ions were 6.45e13.51 mg/ml. Contents of total phenols were 221.71e330.22 mg/g whereas those of total catechins in cold and hot water extracts were 135.05e193.14 and
161.57e195.05 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 unfermented (green tea), semi-fermented (oolong), and fermented
(black tea) by ancient cultures for its medicinal properties (Balentine, 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 compounds 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.: 886 4 2285 4313; fax: 886 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 cholesterol levels and inhibit hypertension, mutagenesis, and
tumourigenesis in several experiments in vitro and in vivo
(Hodgson, Puddey, Burke, Beilin, & Jordan, 1999; Muramatsu, Fukuyo, & Hara, 1986; Yang & Wang, 1993; Yokozawa et al., 1998; Yokozawa, Nakagawa, & Kitani, 2002).
Several studies have shown that most of antioxidant properties 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. However, 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 prepared 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

1617

of antioxidant activity by the conjugated diene method, reducing power, scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (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.

Nakamura (1992). EC50 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. Materials and methods

2.5. Scavenging ability on hydroxyl radicals

2.1. Green tea extract preparation

Scavenging ability on hydroxyl radicals was determined


according to the method of Shi, Dalal, and Jain (1991).
EC50 value (mg extract/ml) is the effective concentration at
which hydroxyl radicals were scavenged by 50%. BHA and
Planex were used for comparison.

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 steaming green tea, young leaves were subjected to blanching (95e
100  C, 40e45 s), rolling and drying. The dried tea leaves were
ground in 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 modified procedure used by Mau, Chao, and Wu (2001). The antioxidant activity was calculated as follows: antioxidant activity
(%) [(DA234 of control  DA234 of sample)/DA234 of
control]  100%. EC50 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-tocopherol (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). EC50 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

2.6. Chelating abilities on ferrous ions


Chelating ability was determined according to the method
of Dinis, Madeira, and Almeida (1994). EC50 value (mg extract/ml) is the effective concentration at which ferrous ions
were chelated by 50%. Citric acid (Sigma), ethylenediaminetetraacetic 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 analyzed as described by Rundhaug, Pung, Read, and Bertram
(1988). Tocopherols were extracted and analyzed according
to the method of Carpenter (1979). Total phenols were determined 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 bcarotene 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 calibration curve of each caffeine, catechin, epicatechin (EC), gallocatechin (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 treatments, three samples were prepared for assays of every antioxidant attribute and component. The experimental data were
subjected to an analysis of variance (ANOVA) for a completely
random design (CRD) to determine the Fishers least significant difference at the level of 0.05.

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

3. Results and discussion


3.1. Extraction yield
Generally, the yields of cold water extracts (11.72e
14.70%) from steaming green tea were significantly lower
than those of hot water extracts (17.49e28.27%) (Table 1).
Obviously, cold water was less effective than hot water in extracting 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 efficacies 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.

(Fig. 1). At 5 mg/ml, cold and hot water extracts showed


high antioxidant activities of 92.4e94.9 and 100%, respectively. 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 contained 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 catechins. Lunder (1992) found that the EGCG content of tea leaves
correlated well with its antioxidant activity. Chen and Ho (1995)
100

Antioxidant activity (%)

1618

80

60

40

20

3.2. Antioxidant activity


0

Using the conjugated diene method, antioxidant activities


of cold and hot water extracts from steaming green tea were
2.14e2.33 and 26.3e29.5% at 1 mg/ml, respectively

Extraction %c (w/w)

Extraction method
Cold water extract
2% (2CS)
6% (6CS)
10% (10CS)
Hot water extractb
2% (2HS)
6% (6HS)
10% (10HS)
a

14.70  0.14D
13.57  0.46D
11.72  0.41E
28.27  1.12A
21.82  0.74B
17.49  0.95C


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

10

15

20

100

Antioxidant activity (%)

Table 1
Extraction yield of water extracts from steaming green tea

80

60

40

II

20

0
0

10

15

20

Concentration (mg/ml)
Fig. 1. Antioxidant activity of water extracts from steaming green tea. Each
value is expressed as mean  standard error (n 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

1619

studied antioxidant activities by the Rancimat method and the effectiveness in the inhibition of lipid peroxidation was in the
descending order: EGCG > ECG > EGC > EC. Amarowicz
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 antioxidant 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.

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 catechins, 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. However, the chemical interactions among various catechins might
facilitate the scavenging of free radicals.

3.3. Reducing power

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


radicals

Reducing powers of cold and hot water extracts from


steaming green tea were 0.91e0.93 and 0.93e1.14 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.

At 20 mg/ml, scavenging abilities of cold and hot water


extracts from steaming green tea on DPPH radicals were
31.7e36.3 and 29.1e34.0%, respectively (Fig. 3). It seems
that both water extracts in scavenging abilities were less effective 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.

1.4

100

Scavenging ability (%)

Absorbance at 700 nm

1.2
1.0
0.8
0.6

0.4
0.2
0.0
0

10

15

80

60

40

20

20

1.4

10

15

20

10

15

20

100

Scavenging ability (%)

Absorbance at 700 nm

1.2
1.0
0.8
0.6
0.4

II

80

60

40

20
0.2
0.0
0

10

15

20

Concentration (mg/ml)

Concentration (mg/ml)

Fig. 2. Reducing power of water extracts from steaming green tea. Each value
is expressed as mean  standard error (n 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.

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 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

1620

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. BrandWilliams, 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 radicals 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

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 38.9e
74.3 and 46.7e72.4%, respectively (Fig. 5). For both water
extracts, 2CS and 2HS showed higher chelating abilities

100

100

80

80

Chelating ability (%)

Scavenging ability (%)

At 5 mg/ml, scavenging abilities of cold and hot water


extracts from steaming green tea on hydroxyl radicals were
83.1e86.7 and 77.7e78.5%, respectively (Fig. 4). It seems
that cold water extracts were more effective in scavenging ability 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 abilities of flavanoids on hydroxyl ions and found that the effective 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.

60

40

20

60

40

20

0
0

10

15

20

100

100

80

80

Chelating ability (%)

Scavenging ability (%)

60

40

II

20

10

15

20

II
60

40

II

20

0
0

10

15

20

Concentration (mg/ml)

10

15

20

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 3).
I: (B) 2CS, ( ) 6CS, (O) 10CS, (-) Planex; II: (B) 2HS, ( ) 6HS,
(O) 10HS, (-) Planex.

Fig. 5. Chelating ability of water extracts from steaming green tea on ferrous
ions. Each value is expressed as mean  standard error (n 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

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, Planex, 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 positions, 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 formulated into foods or brewed as a drink.
3.7. EC50 in antioxidant properties
The antioxidant properties assayed herein were summarized in Table 2, and the results were normalized and
expressed as EC50 values (mg water extract per ml) for comparison. Effectiveness in antioxidant properties inversely correlated with EC50 value. EC50 values in antioxidant activity
by the conjugated diene method were 2.19e3.10 mg/ml, in
which hot water extracts were more effective than cold water
extracts. EC50 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.
EC50 values in scavenging ability on DPPH radicals were
29.45e43.80 mg/ml whereas those on hydroxyl radicals
were 2.88e3.22 mg/ml. For scavenging abilities on radicals,
cold water extracts were more effective than hot water
extracts. With regard to EC50 values in chelating ability on

1621

ferrous ions, cold water extracts were better than the corresponding hot water extracts whereas low extraction ratios
of tea leaves to water were more effective than higher
ratios. EC50 values in antioxidant properties were less than
4 mg/ml, except for scavenging ability on DPPH radicals
(29.45e43.80 mg/ml) and chelating ability on ferrous ions
(6.45e13.51 mg/ml). From EC50 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 chelating ability on ferrous ions.
When the extraction yields were taken into consideration,
EC50 values (mg dried tea leaves per ml) in antioxidant activity 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. EC50 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. EC50 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. EC50 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 radicals and EDTA was excellent for chelating ferrous ions, they
are additives and used or present in mg levels in foods. However, 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 oxidative 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 ascorbic 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
EC50 value of water extracts from steaming green tea in antioxidant properties
EC50 valuea (mg/ml)

Antioxidant activity
Reducing power
Scavenging ability on DPPH radicals
Scavenging ability on OH radicals
Chelating ability on ferrous ions

2CS

6CS

10CS

2HS

6HS

10HS

3.09  0.05Ab
0.28  <0.01A
35.91  1.56B
2.88  0.04C
6.45  0.10E

3.03  0.05A
0.27  <0.01A
33.38  1.02B
3.01  0.06B
9.02  00.07C

3.10  0.08A
0.27  <0.01A
29.45  3.73C
3.00  0.03B
13.51  0.08A

2.25  0.07B
0.22  <0.01C
43.80  2.12A
3.18  0.05A
6.82  0.07D

2.22  0.06B
0.25  <0.01B
34.32  1.16B
3.22  0.09A
11.36  0.26B

2.19  0.05B
0.27  <0.01A
33.85  1.92B
3.20  0.03A
12.19  0.68B

a
EC50 value, the effective concentration at which the antioxidant activity was 50%; the absorbance was 0.5 for reducing power; the 1,1-diphenyl-2-picrylhydrazyl (DPPH) or hydroxyl (OH) radicals were scavenged by 50% and ferrous ions were chelated by 50%, respectively. The EC50 value was obtained by interpolation from linear regression analysis. EC50 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 3). Means with different letters within a row are significantly different (P < 0.05).

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

1622

Table 3
Content of caffeine and various catechins of water extracts from steaming green tea
Content (mg/g)

Caffeine
Catechin
ECa
ECGa
EGCa
EGCGa
GCa
Total catechins
a
b

2CS

6CS

10CS

2HS

6HS

10HS

21.84  0.18Cb
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

EC, epicatechin; ECG, epicatechingallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; GC, gallocatechin.
Each value is expressed as mean  standard error (n 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 antioxidant 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 ferrous ions (Lotito & Fraga, 1998), phenols contained good antioxidant properties, antimutagenic properties, and anticancer
properties (Ahmad & Mukhtar, 1999). Furthermore, Yen, Duh,
and Tsai (1993) found that the antioxidant activity of the methanolic extract from peanut hulls correlated with its content of total 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 21.84e30.38 and 26.38e31.29 mg/g,
respectively (Table 3). Obviously, hot water was more effective
in extracting caffeine from tea leaves than cold water. Furthermore, higher ratios of tea leaves to water extracted more caffeine 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 135.05e193.14 and 161.57e
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. Contents 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
3e4 and 4e6 folds higher than those in water extracts, respectively. Therefore, Planex exhibited better effective in antioxidant properties than all water extracts, except for chelating
ability on ferrous ions. It reveals that the components responsible 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 & Farnworth, 2000; Nakane, Hara, & Ono, 1994; Sakanaka & Yamamoto, 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 22e33% of water extracts from
steaming green tea and the flavanols along with their gallic
acid esters (catechins) accounted for 49e87% 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 total phenols, caffeine and total catechins in cold water extracts
were lower than those in hot water extracts. However, cold water 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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