bro

i L

ersniv

rm

Tea has been consumed for centuries in the forms of unfer-

and against tumourigenesis and DNA damage (Anderson

1997). Besides, green tea is also reported to reduce serum cho-lesterol levels and inhibit hypertension, mutagenesis, and

not available. Therefore, the objectives of this research were tostudy the effect of cold water and hot water brewing methods onantioxidant 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

* Corresponding author. Tel.: 886 4 2285 4313; fax: 886 4 2287 6211.E-mail address: jlmau@dragon.nchu.edu.tw (J.-L. Mau).

Available online at www.sciencedirect.com

LWT - Food Science and Technology 4mented (green tea), semi-fermented (oolong), and fermented(black tea) by ancient cultures for its medicinal properties (Ba-lentine, Wiseman, & Bouwens, 1997). Black tea is commonlyconsumed in the West whereas the consumption of green tea isespecially popular in Asia, mainly for its health benefits(Bushman, 1998; Cabrera, Gimenez, & Lopez, 2003; Seeramet al., 2006). Many studies have shown that polyphenolic com-pounds extracted from green tea leaves are good antioxidantsagainst lipid peroxidation in phospholipid bilayers (Terao,Piskula, & Yao, 1994), in biological systems (Guo, Zhao, Li,Shen, & Xin, 1996; Katiyar, Agarwal, & Mukhtar, 1994),

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 thanascorbic acid, tocopherol or b-carotene (Lee, Huang, Liang,&Mau, 2007). Recently, the tea prepared by brewing tea leavesin cold water has become a new choice in Taiwan in addition tothat traditionally prepared by brewing them in hot water. How-ever, antioxidant properties of the tea brewed in cold water are1. Introduction et al., 2001; Jankun, Selman, Swiercz, & Skrzypczak-Jankun,Abstract

The extracts were prepared from cold or hot brewed steaming green tea at different concentrations (2, 6, and 10%), its antioxidant propertiesstudied and potential antioxidant components determined. The yields of hot water extracts (17.49e28.27%) were significantly higher than thoseof cold water extracts (11.72e14.70%). EC50 values in antioxidant activity determined by the conjugated diene method and reducing power were2.19e3.10 and 0.22e0.28 mg/ml, respectively. EC50 values in scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxylradicals 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. Con-tents of total phenols were 221.71e330.22 mg/g whereas those of total catechins in cold and hot water extracts were 135.05e193.14 and161.57e195.05 mg/g, respectively. Based on the results obtained, hot water extracts were more effective in antioxidant activity and reducingpower. However, cold water extracts were more effective in scavenging ability on DPPH and hydroxyl radicals, and chelating ability on ferrousions. 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; CaffeineEffect of differentantioxidant properties

Sheng-Dun Lin a, En-Hua Department of Food and Nutrition, Hungkuang Univ

b Department of Food Science and Biotechnology, National Chung-Hsing U

Received 10 June 2007; received in revised fo0023-6438/$34.00 2007 Swiss Society of Food Science and Technology. Publisdoi:10.1016/j.lwt.2007.10.009ewing methods onf steaming green tea

iu b, Jeng-Leun Mau b,*

ity, Shalu 433, Taichung, Taiwan, Republic of Chinaersity, 250 Kuokuang Road, Taichung 40227, Taiwan, Republic of China

22 October 2007; accepted 23 October 2007

1 (2008) 1616e1623www.elsevier.com/locate/lwthed by Elsevier Ltd. All rights reserved.

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of antioxidant activity by the conjugated diene method, reduc-dra-s on

ferrous ions. The contents of potential antioxidant components

elliasinensis L. (cultivar Shy-Jih-Chun) in the summer season picked

thodof Oyaizu (1986). EC value (mg extract/ml) is the effective

ine-for

comparison.

eter-Pratt

(1984) and expressed as mg of gallic acid (Sigma).

cetic/min,

and UV detection was at 280 nm. Contents of caffeine and

an50

concentration at which the absorbance was 0.5 for reducingpower. Ascorbic acid, BHA, a-tocopherol and Planex wereused for comparison.

2.4. Scavenging ability on 1,1-diphenyl-2-picrylhydrazylradicalsReducing power was determined according to the meAntioxidant activity was determined by the conjugated dienemethod (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(%) [(DA234 of controlDA234 of sample)/DA234 ofcontrol] 100%. EC50 value (mg extract/ml) is the effectiveconcentration at which the antioxidant activity was 50% andwas 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 ofPlanex: total phenols> 980 mg/g, total catechins> 800 mg/g,EGCG> 450 mg/g, and caffeine< 10 mg/g.

2.3. Reducing power2.2. Antioxidant activityfrom the tea farm inMingjian,NantouCounty, Taiwan. For steam-ing green tea, young leaves were subjected to blanching (95e100 C, 40e45 s), rolling and drying. The dried tea leaves wereground in amill (Retsch ultracentrifugalmill and sievingmachine,Haan, Germany), and screened through a 60-mesh sieve. The 2, 6and 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 filteringthrough Whatman No. 1 filter paper. The filtrate was cooled toroom temperature, and lyophilized. The 2, 6 and 10% cold waterextracts of steaming green tea (2, 6, and10CS)were prepared frombrewing the ground tea leaf powder (70, 210, 350 g) with 3500 gcold water (4 C) for 24 h, filtered and then freeze-dried. Dryextracts thus obtained were stored at20 C before use.The steaming green tea was made from the leaves ofCamof cold and hot water extracts were also determined.

2. Materials and methods

2.1. Green tea extract preparationing power, scavenging abilities on 1,1-diphenyl-2-picrylhyzyl (DPPH) and hydroxyl radicals, and chelating abilitieS.-D. Lin et al. / LWT - Food ScienceScavenging ability on DPPH radicals was determinedaccording to the method of Shimada, Fujikawa, Yahara, andFor 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 weresubjected to an analysis of variance (ANOVA) for a completely2.9. Statistical analysisvarious 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 fromSigma).carotene assay. The mobile phase was 0.1% trifluoroaacid/acetonitrile, 86:14 (v/v), at a flow rate of 1.0 ml2.8. 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 injectioninto a HPLC. The HPLC system was the same as for the b-to the method of Carpenter (1979). Total phenols were dmined according to the method of Taga, Miller, and2.7. Determination of antioxidant components

Ascorbic acid was determined according to the method ofKlein and Perry (1982). b-Carotene was extracted and ana-lyzed as described by Rundhaug, Pung, Read, and Bertram(1988). Tocopherols were extracted and analyzed accordingwere chelated by 50%. Citric acid (Sigma), ethylenediamtetraacetic acid (EDTA, Sigma) and Planex were usedScavenging ability on hydroxyl radicals was determinedaccording to the method of Shi, Dalal, and Jain (1991).EC50 value (mg extract/ml) is the effective concentration atwhich hydroxyl radicals were scavenged by 50%. BHA andPlanex were used for comparison.

2.6. Chelating abilities on ferrous ions

Chelating ability was determined according to the methodof Dinis, Madeira, and Almeida (1994). EC50 value (mg ex-tract/ml) is the effective concentration at which ferrous ions2.5. Scavenging ability on hydroxyl radicalsNakamura (1992). EC50 value (mg extract/ml) is the effectiveconcentration at which DPPH radicals were scavenged by50%. Ascorbic acid, BHA, Planex and a-tocopherol wereused for comparison.

1617d Technology 41 (2008) 1616e1623random design (CRD) to determine the Fishers 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 (11.72e14.70%) from steaming green tea were significantly lowerthan those of hot water extracts (17.49e28.27%) (Table 1).Obviously, cold water was less effective than hot water in ex-tracting water-soluble component out of tea leaves. For coldwater and hot water extractions, the yields were lower withhigher ratio of tea leaves to water. However, the concentrationof dry matters in crude tea infusion made was 2.94, 8.14 and11.7 mg/ml for 2, 6 and 10CS and 5.65, 13.1 and 17.5 mg/mlfor 2, 6 and 10HS, respectively. Although the soluble solidswere higher in the tea solution made from higher ratio of tealeaves to water, the yields were lower due to limited solubility.

asedfica-

cies of 6 and 10HS were 77.2 and 61.9%, respectively. The

(Fig. 1). At 5 mg/ml, cold and hot water extracts showedhigh antioxidant activities of 92.4e94.9 and 100%, respec-tively. However, antioxidant activities were 100% for BHAand a-tocopherol at 0.5 mg/ml whereas that was 57.5% at0.5 mg/ml and 95.7% at 1 mg/ml for Planex. Ascorbic acidshowed an antioxidant activity of 53.1% at 5 mg/ml.

It seems that both water extracts from green tea were moreeffective in antioxidant activity than ascorbic acid whereashot 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 lardby the Rancimat method and found that the polyphenol extractsfrom green tea were better than those from semi-fermented andblack tea. Graham (1992) showed that catechins were the majorcomponents contributing to the antioxidant activity of green tea.

Tanizawa, Sazuka, Komatsu, Toda, and Taniko (1983) pointedout that EC exhibited better antioxidant activity than other cate-chins. Lunder (1992) found that the EGCG content of tea leavescorrelated well with its antioxidant activity. Chen and Ho (1995)

2, 6 or 10%: tea leaves (70, 210 or 350 g) were extracted with 4 C cold

C hot

An

tio

xid

an

t activity (%

)

20

40

60

80

100

I

1618 S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623water (3500 g) for 20 min and the filtrate was freeze-dried.c Extraction % (sample extract weight/sample weight) 100%. Eachwater (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 yield efficacies decreased with increased concentrations andthe yield efficacies of hot water extracts were lower withincreased concentrations. In addition, the yields of hot waterextracts were 92.3, 60.8 and 49.2% higher than those of coldwater extracts for the corresponding concentrations of 2, 6and 10%, respectively. When taking the operation and timecosts into consideration, the commercial process in the plantmight use higher ratio of tea leaves to water to make brewedtea and then diluted thereafter as usual.

3.2. Antioxidant activity

Using the conjugated diene method, antioxidant activitiesof cold and hot water extracts from steaming green tea were2.14e2.33 and 26.3e29.5% at 1 mg/ml, respectively

Table 1

Extraction yield of water extracts from steaming green tea

Extraction method Extraction %c (w/w)

Cold water extracta

2% (2CS) 14.70 0.14D6% (6CS) 13.57 0.46D10% (10CS) 11.72 0.41E

Hot water extractb

2% (2HS) 28.27 1.12A6% (6HS) 21.82 0.74B10% (10HS) 17.49 0.95Ca efficacies of 6 and 10CS were 92.3 and 79.7% whereas bon the yield of 2HS as the efficacy of 100%, the yield efThis is in general agreement with the findings in Chao andChiang (1995). It seems that the ratio of tea leaves to waterplayed an important role in the extraction yield.

Based on the yield of 2CS as the efficacy of 100%, the yieldvalue is expressed as mean standard error (n 3). Means with differentletters within a column are significantly different (P< 0.05).0 5 10 15 200

Concentration (mg/ml)

0 5 10 15 20

An

tio

xid

an

t activity (%

)

0

20

40

60

80

100

II

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.

studied antioxidant activities by the Rancimat method and the ef-fectiveness in the inhibition of lipid peroxidation was in thedescending order: EGCG> ECG> EGC> EC. Amarowiczand Shahidi (1995) compared the antioxidant activities of fourtypes of catechins in a b-caroteneelinoleate model system andfound that the effectiveness was in the descending order:ECG> EGCGwEC> EGC. Summarily, it is obvious that anti-oxidant activities of four types of catechins were not consistentwith different assay methods. However, antioxidant activities ofcold and hot water extracts exhibited good antioxidant activity.

3.3. Reducing power

Reducing powers of cold and hot water extracts fromsteaming green tea were 0.91e0.93 and 0.93e1.14 at0.5 mg/ml, respectively (Fig. 2). It seems that the hot waterextract 2HS was more effective. However, reducing powersof 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 weremore effective in reducing power than a-tocopherol.

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

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

At 20 mg/ml, scavenging abilities of cold and hot waterextracts from steaming green tea on DPPH radicals were31.7e36.3 and 29.1e34.0%, respectively (Fig. 3). It seemsthat both water extracts in scavenging abilities were less effec-tive than ascorbic acid and Planex, which scavenged 41.8 and69.2% of DPPH radicals at 20 mg/ml, respectively. However,at 0.5 mg/ml, BHA and a-tocopherol showed good scavengingabilities of 93.8 and 96.1%, respectively.

Ab

so

rb

an

ce at 700 n

m

0.2

0.4

0.6

0.8

1.0

1.2

1.4

n7

I

Scaven

gin

g ab

ility (%

)

40

60

80

100

in

1619S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623Concentration (mg/ml)

0 10 15 20

Ab

so

rb

a

0.0

0.2

0.4

0.6

II

5

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:ce at 0.80 10 15 200.0

00 n

m

1.0

1.2

1.4

5(B) 2HS, () 6HS, (O) 10HS, (:) ascorbic acid, (,) BHA, (-) Planex,(P) a-tocopherol.Concentration (mg/ml)

0 5 10 15 20

Scaven

g

0

20

40

Fig. 3. Scavenging ability of water extracts from steaming green tea on 1,

1-dipheny1-2-picrylhydrazyl radicals. Each value is expressed as meanstandard error (n 3). I: (B) 2CS, () 6CS, (O) 10CS, (:) ascorbicg ab

ility

600 5 10 15 200

20

(%

) 80

100acid, (,) BHA, (-) Planex, (P) a-tocopherol; II: (B) 2HS, () 6HS, (O)10HS, (:) ascorbic acid, (,) BHA, (-) Planex, (P) a-tocopherol.

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

3.5. Scavenging ability on hydroxyl radicals

At 5 mg/ml, scavenging abilities of cold and hot waterextracts from steaming green tea on hydroxyl radicals were83.1e86.7 and 77.7e78.5%, respectively (Fig. 4). It seemsthat cold water extracts were more effective in scavenging abil-ity than hot water extracts but less effective than Planex, whichscavenged 100% of DPPH radicals at 5 mg/ml. However, at20 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 groupsin the B ring. Structurally, catechins showed the presence ofortho-dihydroxyl groups in the B ring and totally, EC, EGC,ECG and EGCG contained 5, 6, 7 and 8 hydroxyl groups. Itis speculated that the structure and hydroxyl group numberof catechins were responsible for this high scavenging ability.In addition, Shi et al. (1991) reported that caffeine exhibitedgood hydroxyl radical scavenging ability and attributed thealleged anticarcinogenic properties of caffeine to this ability.These results indicated that water extracts from green tea areeffective scavengers for hydroxyl free radicals. Accordingly,it was anticipated that the high scavenging ability of waterextracts might possess some antimutagenic properties.

3.6. Chelating ability on ferrous ions

At 10 mg/ml, chelating abilities of cold and hot waterextracts from steaming green tea on ferrous ions were 38.9e74.3 and 46.7e72.4%, respectively (Fig. 5). For both waterextracts, 2CS and 2HS showed higher chelating abilities

ven

gin

g ab

ility (%

)

40

60

80

100

helatin

g ab

ility (%

)

40

60

80

100

I

1620 S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623Concentration (mg/ml)

0 5 10 15 20

Scaven

gin

g ab

ility

0

20

40

60

II

Fig. 4. Scavenging ability of water extracts from steaming green tea on

hydroxyl radicals. Each value is expressed as mean standard error (n 3).(%

) 800 5 10 15 20

Sca

0

20

100

II: (B) 2CS, () 6CS, (O) 10CS, (-) Planex; II: (B) 2HS, () 6HS,(O) 10HS, (-) Planex.Concentration (mg/ml)

0 5 10 15 20

Ch

elatin

g ab

ility (%

)

0

20

40

60

80

II

II

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,1000 5 10 15 20

C

0

20() 6CS, (O) 10CS, (:) citric acid, (,) EDTA, (-) Planex; II: (B)2HS, () 6HS, (O) 10HS, (:) citric acid, (,) EDTA, (-) Planex.

than other extracts, indicating that higher ratios of tea leaves towater did not extract more components effective for ferrousion chelation. Besides, both water extracts at the same ratioswere comparable in their chelating ability. Surprisingly, Pla-nex, which contained more than 80% of catechins, exhibitedless effective chelating ability of 36.0% at 20 mg/ml. It seemsthat the components other than catechins in water extractsmight contribute most to this chelating ability. However,EDTA showed an excellent chelating ability of 98.1% at aconcentration as low as 0.5 mg/ml. Citric acid was nota good chelating agent for ferrous ions and its chelating ability

andom-

ferrous ions, cold water extracts were better than the corre-sponding hot water extracts whereas low extraction ratiosof tea leaves to water were more effective than higherratios. EC50 values in antioxidant properties were less than4 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 beconcluded that hot water extracts from steaming green teawere more effective in antioxidant activity and reducingpower. However, cold water extracts were more effective inscavenging ability on DPPH and hydroxyl radicals, and chelat-

icals2, 6,

10CS, 2, 6 and 10HS, respectively. EC values in chelating

s

as 5

ion

1621S.-D. Lin et al. / LWT - Food Science and Technology 41 (2008) 1616e1623parison. Effectiveness in antioxidant properties inversely cor-related with EC50 value. EC50 values in antioxidant activityby the conjugated diene method were 2.19e3.10 mg/ml, inwhich hot water extracts were more effective than cold waterextracts. EC50 values in reducing power were 0.22e0.28 mg/ml,indicating that both water extracts were extraordinarilyeffective and comparable. However, the hot water extract2HS was the most effective.

EC50 values in scavenging ability on DPPH radicals were29.45e43.80 mg/ml whereas those on hydroxyl radicalswere 2.88e3.22 mg/ml. For scavenging abilities on radicals,cold water extracts were more effective than hot waterextracts. With regard to EC50 values in chelating ability on

Table 2

EC50 value of water extracts from steaming green tea in antioxidant propertie

EC50 valuea (mg/ml)

2CS 6CS

Antioxidant activity 3.09 0.05Ab 3.03 0.05AReducing power 0.28

entsrties

assayed were comparable. It seems that the discrepancy in

en t

0.83A 26.38 0.69B 28.18 0.66B 31.29 2.01A 1 0 0 1 0 1 2epig

eren

ancontents of total phenols and ascorbic acid did not cause anti-oxidant properties of water extracts different.

Phenols, such as BHTand gallate, were known to be effectiveantioxidants (Madhavi, Singhal, & Kulkarni, 1996). Due to theirscavenging ability on free radicals and chelating abilities on fer-rous ions (Lotito & Fraga, 1998), phenols contained good anti-oxidant properties, antimutagenic properties, and anticancerproperties (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 allwater extracts might explain their high antioxidant properties.

3.9. Caffeine and various catechins

Contents of caffeine in cold and hot water extracts fromsteaming green tea were 21.84e30.38 and 26.38e31.29 mg/g,respectively (Table 3). Obviously, hot water was more effectivein extracting caffeine from tea leaves than cold water. Further-the extraction yield and contents of antioxidant compondiffered among water extracts, the antioxidant propefor 2, 6, 10CS, 2, 6 and 10HS, respectively. Surprisingly, totalphenol contents correlated well with the extraction yieldsshown in Table 1. Using 2HS as an example, its extractionyield and total phenol contents were the highest. Although

Table 3

Content of caffeine and various catechins of water extracts from steaming gre

Content (mg/g)

2CS 6CS 10CS

Caffeine 21.84 0.18Cb 23.12 1.86C 30.38Catechin 2.38 0.29D 2.65 0.89D 5.55ECa 7.93 0.61B 8.45 1.75B 12.59ECGa 3.96 0.21C 3.76 0.89C 5.60EGCa 53.15 0.69B 52.27 1.64B 82.67EGCGa 39.07 0.43D 40.68 1.49D 46.53GCa 6.72 0.27C 8.22 0.95B 9.82Total catechins 135.05 2.04D 139.15 7.71D 193.14a EC, epicatechin; ECG, epicatechingallate; EGC, epigallocatechin; EGCG,b Each value is expressed as mean standard error (n 3). Means with diff

1622 S.-D. Lin et al. / LWT - Food Sciencemore, higher ratios of tea leaves to water extracted more caf-feine than lower ratios. Six catechins were detected in waterextracts and contents of individual catechin fluctuated amongwater extracts and were not consistent with different waterextraction and various ratios. Contents of total catechins incold and hot water extracts were 135.05e193.14 and 161.57e195.05 mg/g, respectively. Like caffeine, hot water and higherratios were more effective in extracting total catechins fromtea leaves than cold water and lower ratios, respectively. Con-tents of caffeine and total catechins inversely correlated withthe extraction yields. However, although contents of caffeineand total catechins were different among water extracts, theantioxidant properties assayed were comparable.

EGCGwas themajor catechin in Planex accounting for morethan 450 mg/g and caffeine content was less than 10 mg/g. Itseems that caffeine in Planex did not contribute much to itsantioxidant properties. Apparently, content of total phenols(>980 mg/g) and total catechins (>800 mg/g) in Planex were3e4 and 4e6 folds higher than those in water extracts, respec-tively. Therefore, Planex exhibited better effective in antioxi-dant properties than all water extracts, except for chelatingability on ferrous ions. It reveals that the components responsi-ble for this chelating ability might not be catechins andmight beother 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 weremainly due to phenolic compounds. The antioxidant propertiesof green tea were the strongest among the various types of teas.Green tea contains considerable amounts of catechins, whichcontributed most to antioxidant properties. As shown in Table3, total phenols accounted for 22e33% of water extracts fromsteaming green tea and the flavanols along with their gallicacid esters (catechins) accounted for 49e87% of total phenols.These phenolic compounds usually are the most abundantwater-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 usingcold or warm water other than hot water. Cold water extractedfewer components than hot water. Furthermore, contents of to-tal phenols, caffeine and total catechins in cold water extracts

.47C 3.13 0.02A 2.96 0.08AB 2.92 0.09B

.55A 6.99 0.49B 7.59 0.62B 8.33 0.73B

.43B 8.37 0.63A 9.28 0.59A 10.90 2.14A

.69A 39.13 0.28D 43.95 1.67C 44.46 2.72C

.62C 69.16 1.76B 72.68 1.99B 83.12 2.54A

.56B 8.41 0.78B 13.38 1.54A 14.03 0.82A

.01A 161.57 1.68C 178.02 5.50B 195.05 5.56Aallocatechin gallate; GC, gallocatechin.

t letters within a row are significantly different (P< 0.05).ea

2HS 6HS 10HS

d Technology 41 (2008) 1616e1623were lower than those in hot water extracts. However, cold wa-ter extracts were more effective in scavenging ability on DPPHand hydroxyl radicals, and chelating ability on ferrous ions.Based on the results obtained, tea brewed from steaming greentea leaves using cold water could contain fewer components,especially caffeine, but possessed some antioxidant propertieshigher than tea brewed using hot water. Accordingly, this coldbrewing method would be a new alternative way to make a tea.

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Effect of different brewing methods on antioxidant properties of steaming green teaIntroductionMaterials and methodsGreen tea extract preparationAntioxidant activityReducing powerScavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicalsScavenging ability on hydroxyl radicalsChelating abilities on ferrous ionsDetermination of antioxidant componentsDetermination of caffeine and various catechinsStatistical analysis

Results and discussionExtraction yieldAntioxidant activityReducing powerScavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicalsScavenging ability on hydroxyl radicalsChelating ability on ferrous ionsEC50 in antioxidant propertiesAntioxidant componentsCaffeine and various catechins

References