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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).
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.
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
S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623
1618
80
60
40
20
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
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.
1.4
100
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
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.
S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623
1620
100
100
80
80
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
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. 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
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).
S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623
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