Research ArticleThe Galloyl Catechins Contributing to Main AntioxidantCapacity of Tea Made from Camellia sinensis in China

Chunjian Zhao12 Chunying Li1 Shuaihua Liu1 and Lei Yang1

1 Key Laboratory of Forest Plant Ecology Ministry of Education Northeast Forestry University Harbin 150040 China2 College of Resources and Environmental Sciences China Agricultural University Beijing 100193 China

Correspondence should be addressed to Chunying Li nefujanealiyuncom

Received 17 May 2014 Accepted 3 August 2014 Published 28 August 2014

Academic Editor Tsutomu Hatano

Copyright copy 2014 Chunjian Zhao et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Total polyphenol content catechins content and antioxidant capacities of green dark oolong and black teas made from Camelliasinensis in China were evaluatedThe total polyphenol content of 20 samples of tea was in the range of 782ndash3236 Total catechinscontent was in the range of 434ndash2427The antioxidant capacity of tea extract was determined by the oxygen radical absorbancecapacity (ORAC) test and the 11-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging test Total polyphenol content catechinscontent and antioxidant capacity decreased in the following order green gt oolong gt black gt dark tea A positive correlation existedbetween the antioxidant capacity and total polyphenol content or catechins content (1198772 = 067ndash087) The antioxidant capacitiesof five major catechins (epigallocatechin gallate (EGCG) epicatechin gallate (ECG) epicatechin epigallocatechin and catechin)were determined by online HPLC DPPH radical-scavenging the antioxidant activity of tea was mainly attributed to the esterifiedcatechins (EGCG or ECG)

1 Introduction

Tea consumption has a long history of more than 2000 yearsOriginating in China and then spreading to Japan Europeand other areas tea has become one of the most popular andfrequently consumed beverages in the world [1] All kindsof tea originate from Camellia sinensis which has the twosubspecies var sinensis (China tea) and var assamica (Assamtea) [2]

InChina six types of teamade from the leaves ofCamelliasinensis including green black oolong dark white and yel-low tea have been classified based on the degree of fermenta-tion and the color of the tea product [1] Green and yellowteas are nonfermented tea and oolong and white teas arecalled semifermented tea while dark and black teas are fullyfermented [3 4]

Of the six types of tea four types (green oolong blackand dark teas) are widely consumed throughout the worldThe manufacturing processes used to prepare the four com-mon types of tea are as follows

(1) Green tea after the fresh tea is picked it is allowedto wither for several hours The tea leaves are then heatedto remove moisture and to denature the enzymes that causeoxidationThe leaves are then kneaded and rolled while beingdried (2) Oolong tea after the fresh leaves are picked theyare left out in the sun to wither for a few hours Then theleaves are brought indoors and are laid out on large bambootrays in a temperature-controlled room to oxidize Whenthe tea has reached the desired level of oxidation the tea isexposed to heat to stop the oxidizationThe tea is then shapedand roasted to dry (3) Black tea the fresh leaves are pickedand allowed to wither in the sun Once they have witheredenough the leaves are then bruised to cause oxidation Theleaves are then put into boxes to oxidize When the leaveshave turned to a dark red-copper color they are fried andthen rolled and shaped (4) Dark tea when the leaves arepicked they are fried kneaded and twisted but instead ofbeing dried as green tea is the leaves are sprinkled with waterand placed in huge piles under cloth to ferment before beingdried The resulting leaves have a dark black color [5]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 863984 11 pageshttpdxdoiorg1011552014863984

2 The Scientific World Journal

O

OH

OH

OH

OH

OH

EGC

O

OH

OH

OH

OH

HO

HO

HO

HO

HO

Catechin

O

OH

OH

OH

OHEC

O

OH

OH

OH

O

OOH

OH

OH

OH

EGCG

ECG

O

OH

OH

OH

O

OOH

OH

OH

Figure 1 Chemical structures of major catechins present in tea

Green tea is popular in China Japan India and inMiddleEastern countries oolong tea is mainly consumed in ChinaJapan and Southeast Asia dark tea is consumed in the south-western regions of China black tea is preferred inmany coun-tries throughout the world

Recent studies have recognized that tea has health-pro-tecting benefits that include antioxidative [6ndash8] antimicro-bial [9 10] antiviral [11 12] and antitumor [13ndash16] activitiesand abilities to prevent diabetes [17] obesity [18] leuke-mia [19] Parkinsonrsquos disease [20] and cardiovascular disease[21]

The radical-scavenging and antioxidant properties of teapolyphenols are frequently cited as important contributors tothe above-mentioned health-protection benefits [22] Nowa-days the content of tea polyphenols is regarded as a qualityindicator of tea [23] Therefore it is desirable to investigatethe total polyphenol contents and antioxidant capacities ofdifferent teas

Catechins are the most important group of active con-stituents in tea polyphenols The major catechins are epigal-locatechin gallate (EGCG) epicatechin gallate (ECG) epicat-echin (EC) epigallocatechin (EGC) and catechin (molecularstructures are shown in Figure 1) [24]

Many researchers have already begun to pay attentionto the correlation between antioxidant capacity and the tea

polyphenol content Anesini et al investigated the total pol-yphenol content and antioxidant capacity of commerciallyavailable tea (Camellia sinensis) in Argentina [25] In theirwork a high correlation was demonstrated between the twoscrutinized properties The antioxidant activity and phenolicprofile in tea and herbal infusions were studied by Atoui et aland their results suggested that black Ceylon tea and Chinesegreen tea infusions can be major sources of polyphenolsthat exhibit important antioxidant behavior [26] Rusak et alinvestigated the phenolic content and antioxidative capacityof green and white tea extracts [24] The results showedthat the antioxidative capacity of the investigated tea extractscorrelated with the phenolic content However in the above-mentioned studies the profiles of catechins in tea were notelucidated and it remains unclear which compounds areresponsible for their antioxidant property

In the present study total polyphenol content and antiox-idant capacity of 20 samples from four types of tea (greendark oolong and black teas) prepared fromCamellia sinensisin China were determined and the correlation between theantioxidant capacity and total polyphenol content or cate-chins was investigated Furthermore the content of individ-ual catechins (EGCG EGC ECG EC and catechin) andtheir contribution to the antioxidant capacity of tea wereelucidated

The Scientific World Journal 3

Table 1 Source and production date of 20 tea samples

Category Product name Source Production date

Green tea

Bi luo chun Jiangsu 2010Huang shan mao feng Anhui 2010Long jing Zhejiang 2011Lu an gua pian Anhui 2010Xin yang mao jian Henan 2010

Oolong tea

Da hong pao Fujian 2010Rou gui Fujian 2010Shui xian Fujian 2010Taiwan oolong Taiwan 2010Tie guan yin Fujian 2010

Black tea

Dian cha Yunnan 2010Henan black tea Henan 2009Qimen black tea Anhui 2010Tanyang gongfu Fujian 2010Zhengshan black tea Fujian 2010

Dark tea

Bai xing fu cha Hunan 2009Qian liang Hunan 2008Liu bao cha Guangxi 2007Qi zi bing Yunnan 2007Sheng pursquoer Yunnan 2009

2 Materials and Methods

21 Tea Samples A total of 20 samples from four types of tea(green dark oolong and black teas)were collected fromDafatea market in Harbin China They included 5 green teas 5oolong teas 5 black teas and 5 dark teas Their identities arelisted in Table 1

22 Chemicals For the determination of the total phenoliccontent (TPC) Folin-Ciocalteu phenol reagents (Sigma-Aldrich USA) gallic acid (GA 975 purity Sigma-AldrichUSA) and anhydrous sodium carbonate (99 purity BeijingChemical Reagents China) were used For the determina-tion of the antioxidant activity 11-diphenyl-2-picrylhydrazyl(DPPH Sigma-Aldrich USA) was used The oxygen radicalabsorbance capacity (ORAC) reagents of 221015840-azobis(2-meth-ylpropionamidine) dihydrochloride (AAPH) 6-hydroxy-2578-tetramethylchromane-2-carboxylic acid (trolox) andfluorescein sodiumwere obtained fromSigma-Aldrich (USA)For the determination of the total catechins content (minus)-epigallocatechin gallate (EGCG ge970) (minus)-epicatechingallate (ECGge98) (minus)-epigallocatechin (EGCge95) (minus)-epicatechin (EC ge98) and (+)-catechin (ge99) standardswere obtained from Sigma-Aldrich (USA) Caffeine (ge98)was obtained from the National Institutes for Food andDrug Control (Beijing China) Acetonitrile acetic acid andEDTAwere of HPLC grade (Sigma-Aldrich USA) Methanolwas of analytical grade (Beijing Chemical Reagents China)Deionized water was prepared using a Milli Q-Plus system(Millipore Germany)

23 Preparation of Tea Extract The preparation of the teaextract was performed according to the method publishedby the International Organization for Standardization (ISO)[23] Each tea sample was ground to a fine powder and 10 gof the powder was extracted with 25mL of methanolwater(7 3 vv) in a water bath at 70∘C for 10min The extractionwas repeated twice and the extracts were combined Afterfiltration through a filter paper and a 045120583mmembrane filter(Millipore) the extract volume was adjusted to 100mL

24 Determination of Total Polyphenols by Folin-CiocalteuMethod The total polyphenol content in 20 samples fromfour types of tea (green dark oolong and black) was deter-mined according to the Folin-Ciocalteu method as describedby ISO 14502-1 [23] with some minor modifications Onemilliliter of the tea extract was diluted with water to 100mLThe diluted extract solution (10mL) was then mixed with50mL of 50 Folin-Ciocalteu reagent After 5min 40mLof 75 Na

2CO3solution was added and mixed Then the

mixture was incubated at room temperature for 60min Theabsorbance of the solution was measured at 765 nm witha Shimadzu UV-2550 UV-Vis spectrophotometer against areagent blank The determination was three times The totalpolyphenol content was expressed as gallic acid equivalents(GAE units g100 g sample)

25 Determination of Catechins by HPLC The content of cat-echins in 20 samples from four types of tea (green darkoolong and black teas) was determined according to the ISO14502-2 method [27] Quantification of the major catechins(EC EGC ECG EGCG and catechin) was performed usingan HPLC system (Jasco Japan) equipped with a 1580 pumpand a 975 UV-Vis detector Chromatographic separationwas achieved with a HiQ Sil C

18V reversed phase column

(250mm times 46mm id) packed with 5 120583m particles andthe column temperature was maintained at 35∘C Mobilephase A was a mixture of 9 acetonitrile and 2 aceticacid with 20120583gmL EDTA Mobile phase B was a mixtureof 80 acetonitrile and 2 acetic acid with 20120583gmL EDTABaseline separation of EC EGC ECG EGCG catechin gallicacid and caffeine was achieved with a binary gradient elutionprogram as follows 100 mobile phase A for 10min thenover 15min a linear gradient to 68 mobile phase A 32mobile phase B and hold at this composition for 10minThe detection wavelength was 278 nm and the flow rate was08mLmin One milliliter of tea extract was diluted withwater to 100mL the diluted extract was filtered througha 045 120583m membrane filter (Millipore) and the filtrate wasinjected into the HPLC apparatus

Standard solutions of the major catechins (EC EGCECG EGCG and catechin) gallic acid and caffeinewere pre-pared by accurately weighing standard powders and dissolv-ing them in 10 acetonitrile with 500120583gmL of EDTA Thestock solutions were stored at 4∘C All standard solutionswere filtered through a 045 120583m membrane filter (Millipore)and the filtrate was injected The total catechins content wasexpressed as a percentage by mass of the sample on a dry

4 The Scientific World Journal

matter basis and was obtained by summation of the EGCcatechin EC EGCG and ECG content

26 Oxygen Radical Absorbance Capacity (ORAC) Assay Theoxygen radical absorbance capacity (ORAC) method is usedto measure the antioxidant capacity of biological samples invitro The ORAC assay was first developed by Cao et al in1993 [28] This approach has been recognized and is now fre-quently used in analysis of polyphenol antioxidant capacity221015840-Azobis(2-amidinopropane) dihydrochloride (AAPH) isthought to produce the peroxyl radical upon heating whichdamages the fluorescent molecule and results in a loss offluorescence [29 30] The fluorescence decay is monitored asfluorescein in the solution decomposes a process that shouldbe slower in the presence of antioxidants Decay curves arerecorded and the area between the two decay curves (withor without antioxidant) is calculated Calculating the areaunder the fluorescence decay curve (AUC) of trolox which isused as a standard antioxidant a standard curve is preparedthat can be used to evaluate the radical-scavenging activity ofother antioxidants in terms of trolox equivalents (TE)

The ORAC assay was performed using a fluorescencespectrophotometer (F-7000 Hitachi Japan) following a pre-viously described procedure [31] with minor modificationSamples were diluted with sodium phosphate buffer (75mMpH 74) Sodium fluorescein solution (600120583L 40 nM) wasadded to 100 120583L of phosphate buffer (blank) to 100 120583Laliquots of trolox solution (125 25 5 10 20 40 and 80 120583M)or to 100120583L of diluted sample The respective mixtures wereincubated for 15min at 37∘C Immediately before initiatingthe measurements 100 120583L of AAPH (150mM) was addedinto the test solution With the excitation filter set to 485 nmand the emission recorded at 535 nm readings were collectedeveryminute for 60minThe results were calculated based onthe differences in AUC between the blank and the sample andwere expressed as in units ofmicromoles TE per 1 g of dry teaThe AUC was calculated as follows

AUC =1198910+ 2sum59

119894=1119891119894+ 11989160

21198910

(1)

where 1198910is the fluorescence reading at initiation of the reac-

tion 119891119894is the fluorescence reading at 119894minutes and 119891

60is the

final measurement Net AUC was calculated as follows

Net AUC = AUCsample minus AUCblank (2)

27 DPPHRadical-ScavengingActivity The antioxidant capac-ity in 20 samples from four types of tea (green dark oolongand black teas) was determined by reduction of the 22-diphenyl-1-picryhydrazyl (DPPH) radical according to thepublishedmethod [32] A stock solutionwas prepared by stir-ring 75mg of DPPH in 1 L of methanol overnight A 075mLaliquot of sample extract was mixed with 15mL of DPPHsolution For each extract a blank of 15mL of methanolwas used without DPPH reagent to correct for any sampleabsorbance at 521 nm Sample and blank absorbance were

recorded at 521 nm after 6min The percentage inhibition ofDPPH was calculated according to the equation below [33]

Inhibition of DPPH () =[1198600minus (1198601minus 119860119904)]

1198600

times 100 (3)

where 1198600is the absorbance of the DPPH solution 119860

1is

the absorbance of the tea extract in the presence of DPPHsolution and 119860

119904is the absorbance of the tea extract solution

without DPPH Each sample was analyzed in triplicateThe EC50 value was calculated by a graphical method

as the concentration that caused 50 inhibition of DPPHThe anti-DPPH efficiency (AE) was also calculated aslog sdot (1EC50) [34]

28 Online HPLC DPPH Screening Analysis of Tea SamplesThe antioxidant activity of EC EGC ECG EGCG cate-chin gallic acid and caffeine was measured using the onlineHPLC DPPH screening method [35] The online HPLCDPPHmethod was developed using a methanolic solution ofDPPH stable free radical (50mgL) The instrumental setupis depicted in Figure 2 The flow of HPLC-separated analytesandDPPH solution was 08mLmin and 02mLmin respec-tively The HPLC-separated analytes reacted with the DPPHsolution in post-column mode which was photometricallydetected as a negative peak at 521 nm The length of the cap-illary (51m times 05mm id) used for the postcolumn reactionwas adjusted to achieve a reaction time of 10minThe HPLCconditions were the same as described above

3 Results and Discussion

31 Total Polyphenol and Catechins Content in Four Typesof Tea Using the Folin-Ciocalteu method total polyphenolcontents in 20 samples from four types of tea were calculatedfrom the standard curve for gallic acid ranging from 10 to50 120583gmL (119910 = 0183119909 minus 0025 1198772 = 09987) The resultsare summarized in Table 2 The contents of catechins in 20samples from four types of tea were determined by HPLCThe retention times were 73min for EGC 95min for cate-chin 153min for EC 165min for EGCG and 221min forECGAt the same time the contents of gallic acid and caffeinewere also determined the retention times were 37min forgallic acid and 124min for caffeine The working calibrationcurves were as follows gallic acid 119910 = 208401119909minus18547 (1198772 =09996 5ndash25120583gmL) EGC 119910 = 2645119909 minus 976 (1198772 = 09989100ndash300 120583gmL) catechin 119910 = 7999119909 minus 1075 (1198772 = 0999150ndash150 120583gmL) caffeine 119910 = 19823119909 minus 1762 (1198772 = 0998750ndash150 120583gmL) EC 119910 = 18574119909 minus 1653 (1198772 = 09990 50ndash150 120583gmL) EGCG 119910 = 29722119909 minus 2678 (1198772 = 09992 100ndash400 120583gmL) ECG 119910 = 16774119909 minus 1546 (1198772 = 09988 50ndash200120583gmL) where 119910 is the peak area of the analyte and 119909 isthe concentration (120583gmL) The results for the catechins aresummarized in Table 2 It can be seen from Table 2 that thecontent of total polyphenols ranged from782 to 3236 g of gal-lic acid equivalent100 g dry tea and the content of catechinsranged from 434 to 2427 (dry tea) Of the selected 20 teasamples Bi luo chun (green tea) had the highest content of

The Scientific World Journal 5

HPLC pump (gradient elution)

Mobile phase Sampler

HPLC pump

DPPHsolution Mixer UV-Vis detector

UV-Vis detector

Liquid waste

ColumnChromatogram at 278nm

Chromatogram at 521nm

Figure 2 Instrumental setup for the HPLC analysis of radical-scavenging compounds using an online reaction with 11-diphenyl-2-picryl-hydrazyl (DPPH)

0

5

10

15

20

25

30

0 10 20 30 40

Cate

chin

s con

tent

()

Content of total polyphenol ()

R2 = 096

Figure 3 The correlation between total polyphenol and catechincontents

polyphenols and catechins Although there was a significantdifference in the content of polyphenols and catechins in thesame type of tea as a whole the contents of total polyphenolsand catechins decreased in the following order green tea gtoolong tea gt black tea gt dark tea

In addition the correlation between catechins and totalpolyphenols in the 20 samples from the four types of teawas tested The results shown in Figure 3 reveal a strongpositive correlation between catechins and total polyphenols(1198772 = 096)

Table 2 lists the contents of individual catechins in thefour types of tea It is apparent that the content of esterifiedcatechins (EGCGor ECG) is higher than that of nonesterifiedcatechins (catechin EC or EGC) In comparison of the fourtypes of tea EGCG content was the highest in green tea

0500

100015002000250030003500

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

ORA

C (120583

mol

TE

g te

a)

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoerFigure 4 Oxygen radical absorbance capacity (ORAC) values ofdifferent samples in four types of tea

(525ndash1439 on dry tea) and in oolong tea (457ndash1308 ondry tea) With the increase in the degree of fermentation theEGCG content gradually decreased and the range of EGCGcontent in dark tea is only 036ndash093 on dry tea Among thefour types of tea the ECG content was the highest in blacktea (338ndash563 on dry tea) and in dark tea (192ndash473 ondry tea)

32 Oxygen Radical Absorbance Capacity (ORAC) in FourTypes of Tea A straight calibration curve was obtained fortrolox antioxidant activity in the concentration range of 125ndash80 120583M 119910 = 036119909 minus 056 where 119909 is concentration oftrolox and 119910 is Net AUC (1198772 = 09983) Figure 4 shows theORAC values of the selected tea samples the ORAC value

6 The Scientific World Journal

Table2Th

econ

tent

oftotalp

olypheno

lstotalcatechinsand

individu

alcatechinsin20

samples

from

four

typeso

ftea

(119899=3)

Samples

oftea

EGC(

)Ca

techin

()

EC(

)EG

CG(

)EC

G(

)Non

esterifi

edcatechins()

Esterifi

edcatechins()

Totalcatechins

()

Total

polyph

enols()

Green

tea

Biluochun

113plusmn006

092plusmn004

184plusmn003

1439plusmn041

599plusmn002

389plusmn013

2038plusmn043

2427plusmn056

3236plusmn093

Huang

shan

mao

feng

094plusmn003

085plusmn004

183plusmn005

1411plusmn047

561plusmn004

362plusmn012

1972plusmn051

2334plusmn063

2686plusmn049

Long

jing

062plusmn002

055plusmn005

108plusmn001

813plusmn039

361plusmn011

225plusmn008

1174plusmn050

1399plusmn058

1817plusmn071

Luan

guap

ian

037plusmn001

027plusmn001

061plusmn003

525plusmn026

185plusmn003

125plusmn005

710plusmn029

835plusmn034

1088plusmn006

Xinyang

mao

jian

096plusmn002

074plusmn003

149plusmn001

1263plusmn023

526plusmn014

319plusmn006

1789plusmn037

2108plusmn043

2694plusmn009

Meanvalue

080plusmn003

067plusmn003

137plusmn003

1090plusmn035

446plusmn007

284plusmn009

1537plusmn042

1821plusmn

051

2304plusmn046

Oolon

gtea

Dah

ongpao

063plusmn002

066plusmn003

074plusmn003

559plusmn022

208plusmn002

203plusmn008

767plusmn024

970plusmn032

1316plusmn009

Rougui

044plusmn002

055plusmn001

052plusmn002

457plusmn009

166plusmn006

151plusmn

005

623plusmn015

774plusmn020

1035plusmn008

Shui

xian

068plusmn001

081plusmn002

090plusmn002

723plusmn031

243plusmn001

239plusmn005

966plusmn032

1205plusmn037

1618plusmn027

Taiw

anwulong

116plusmn002

144plusmn005

153plusmn006

1308plusmn027

417plusmn006

413plusmn013

1725plusmn033

2138plusmn046

2973plusmn004

Tieg

uanyin

068plusmn001

077plusmn002

084plusmn002

686plusmn007

236plusmn001

229plusmn005

922plusmn008

1151plusmn

013

1569plusmn073

Meanvalue

072plusmn002

085plusmn003

091plusmn003

747plusmn019

254plusmn003

247plusmn007

1001plusmn

022

1248plusmn030

1702plusmn024

Blacktea

Diancha

241plusmn009

250plusmn004

043plusmn001

226plusmn003

470plusmn015

534plusmn014

696plusmn018

1230plusmn032

1675plusmn074

Hen

anho

ngcha

158plusmn003

163plusmn006

031plusmn001

155plusmn002

342plusmn001

352plusmn010

497plusmn003

849plusmn013

1153plusmn004

Qim

enho

ngcha

150plusmn003

159plusmn005

028plusmn001

131plusmn

005

338plusmn011

337plusmn009

469plusmn016

806plusmn025

1301plusmn

017

Tanyang

gong

fu17

5plusmn005

197plusmn004

032plusmn001

155plusmn002

419plusmn013

404plusmn010

574plusmn015

978plusmn025

1373plusmn004

Zhengshan

hong

cha

257plusmn009

266plusmn012

045plusmn001

204plusmn007

563plusmn001

568plusmn022

767plusmn008

1335plusmn030

1817plusmn012

Meanvalue

196plusmn006

207plusmn006

036plusmn001

174plusmn004

426plusmn008

439plusmn013

601plusmn012

1040plusmn025

1464plusmn022

Darktea

Baixingfu

cha

022plusmn001

077plusmn001

107plusmn003

036plusmn001

192plusmn003

206plusmn005

228plusmn004

434plusmn009

782plusmn029

Qianliang

028plusmn003

102plusmn004

135plusmn007

050plusmn002

252plusmn005

265plusmn014

302plusmn007

567plusmn021

929plusmn038

Liubaocha

034plusmn001

135plusmn002

187plusmn004

072plusmn002

341plusmn017

356plusmn007

413plusmn019

769plusmn026

1328plusmn060

Qizibing

035plusmn001

148plusmn007

202plusmn007

079plusmn004

395plusmn013

385plusmn015

474plusmn017

859plusmn032

1480plusmn003

Shengpu

rsquoer039plusmn002

197plusmn002

251plusmn009

093plusmn002

473plusmn013

487plusmn013

566plusmn015

1053plusmn028

1629plusmn017

Meanvalue

032plusmn002

132plusmn003

176plusmn006

066plusmn002

331plusmn010

340plusmn011

397plusmn012

736plusmn023

1230plusmn029

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

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Cell BiologyInternational Journal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

2 The Scientific World Journal

O

OH

OH

OH

OH

OH

EGC

O

OH

OH

OH

OH

HO

HO

HO

HO

HO

Catechin

O

OH

OH

OH

OHEC

O

OH

OH

OH

O

OOH

OH

OH

OH

EGCG

ECG

O

OH

OH

OH

O

OOH

OH

OH

Figure 1 Chemical structures of major catechins present in tea

Green tea is popular in China Japan India and inMiddleEastern countries oolong tea is mainly consumed in ChinaJapan and Southeast Asia dark tea is consumed in the south-western regions of China black tea is preferred inmany coun-tries throughout the world

Recent studies have recognized that tea has health-pro-tecting benefits that include antioxidative [6ndash8] antimicro-bial [9 10] antiviral [11 12] and antitumor [13ndash16] activitiesand abilities to prevent diabetes [17] obesity [18] leuke-mia [19] Parkinsonrsquos disease [20] and cardiovascular disease[21]

The radical-scavenging and antioxidant properties of teapolyphenols are frequently cited as important contributors tothe above-mentioned health-protection benefits [22] Nowa-days the content of tea polyphenols is regarded as a qualityindicator of tea [23] Therefore it is desirable to investigatethe total polyphenol contents and antioxidant capacities ofdifferent teas

Catechins are the most important group of active con-stituents in tea polyphenols The major catechins are epigal-locatechin gallate (EGCG) epicatechin gallate (ECG) epicat-echin (EC) epigallocatechin (EGC) and catechin (molecularstructures are shown in Figure 1) [24]

Many researchers have already begun to pay attentionto the correlation between antioxidant capacity and the tea

polyphenol content Anesini et al investigated the total pol-yphenol content and antioxidant capacity of commerciallyavailable tea (Camellia sinensis) in Argentina [25] In theirwork a high correlation was demonstrated between the twoscrutinized properties The antioxidant activity and phenolicprofile in tea and herbal infusions were studied by Atoui et aland their results suggested that black Ceylon tea and Chinesegreen tea infusions can be major sources of polyphenolsthat exhibit important antioxidant behavior [26] Rusak et alinvestigated the phenolic content and antioxidative capacityof green and white tea extracts [24] The results showedthat the antioxidative capacity of the investigated tea extractscorrelated with the phenolic content However in the above-mentioned studies the profiles of catechins in tea were notelucidated and it remains unclear which compounds areresponsible for their antioxidant property

In the present study total polyphenol content and antiox-idant capacity of 20 samples from four types of tea (greendark oolong and black teas) prepared fromCamellia sinensisin China were determined and the correlation between theantioxidant capacity and total polyphenol content or cate-chins was investigated Furthermore the content of individ-ual catechins (EGCG EGC ECG EC and catechin) andtheir contribution to the antioxidant capacity of tea wereelucidated

The Scientific World Journal 3

Table 1 Source and production date of 20 tea samples

Category Product name Source Production date

Green tea

Bi luo chun Jiangsu 2010Huang shan mao feng Anhui 2010Long jing Zhejiang 2011Lu an gua pian Anhui 2010Xin yang mao jian Henan 2010

Oolong tea

Da hong pao Fujian 2010Rou gui Fujian 2010Shui xian Fujian 2010Taiwan oolong Taiwan 2010Tie guan yin Fujian 2010

Black tea

Dian cha Yunnan 2010Henan black tea Henan 2009Qimen black tea Anhui 2010Tanyang gongfu Fujian 2010Zhengshan black tea Fujian 2010

Dark tea

Bai xing fu cha Hunan 2009Qian liang Hunan 2008Liu bao cha Guangxi 2007Qi zi bing Yunnan 2007Sheng pursquoer Yunnan 2009

2 Materials and Methods

21 Tea Samples A total of 20 samples from four types of tea(green dark oolong and black teas)were collected fromDafatea market in Harbin China They included 5 green teas 5oolong teas 5 black teas and 5 dark teas Their identities arelisted in Table 1

22 Chemicals For the determination of the total phenoliccontent (TPC) Folin-Ciocalteu phenol reagents (Sigma-Aldrich USA) gallic acid (GA 975 purity Sigma-AldrichUSA) and anhydrous sodium carbonate (99 purity BeijingChemical Reagents China) were used For the determina-tion of the antioxidant activity 11-diphenyl-2-picrylhydrazyl(DPPH Sigma-Aldrich USA) was used The oxygen radicalabsorbance capacity (ORAC) reagents of 221015840-azobis(2-meth-ylpropionamidine) dihydrochloride (AAPH) 6-hydroxy-2578-tetramethylchromane-2-carboxylic acid (trolox) andfluorescein sodiumwere obtained fromSigma-Aldrich (USA)For the determination of the total catechins content (minus)-epigallocatechin gallate (EGCG ge970) (minus)-epicatechingallate (ECGge98) (minus)-epigallocatechin (EGCge95) (minus)-epicatechin (EC ge98) and (+)-catechin (ge99) standardswere obtained from Sigma-Aldrich (USA) Caffeine (ge98)was obtained from the National Institutes for Food andDrug Control (Beijing China) Acetonitrile acetic acid andEDTAwere of HPLC grade (Sigma-Aldrich USA) Methanolwas of analytical grade (Beijing Chemical Reagents China)Deionized water was prepared using a Milli Q-Plus system(Millipore Germany)

23 Preparation of Tea Extract The preparation of the teaextract was performed according to the method publishedby the International Organization for Standardization (ISO)[23] Each tea sample was ground to a fine powder and 10 gof the powder was extracted with 25mL of methanolwater(7 3 vv) in a water bath at 70∘C for 10min The extractionwas repeated twice and the extracts were combined Afterfiltration through a filter paper and a 045120583mmembrane filter(Millipore) the extract volume was adjusted to 100mL

24 Determination of Total Polyphenols by Folin-CiocalteuMethod The total polyphenol content in 20 samples fromfour types of tea (green dark oolong and black) was deter-mined according to the Folin-Ciocalteu method as describedby ISO 14502-1 [23] with some minor modifications Onemilliliter of the tea extract was diluted with water to 100mLThe diluted extract solution (10mL) was then mixed with50mL of 50 Folin-Ciocalteu reagent After 5min 40mLof 75 Na

2CO3solution was added and mixed Then the

mixture was incubated at room temperature for 60min Theabsorbance of the solution was measured at 765 nm witha Shimadzu UV-2550 UV-Vis spectrophotometer against areagent blank The determination was three times The totalpolyphenol content was expressed as gallic acid equivalents(GAE units g100 g sample)

25 Determination of Catechins by HPLC The content of cat-echins in 20 samples from four types of tea (green darkoolong and black teas) was determined according to the ISO14502-2 method [27] Quantification of the major catechins(EC EGC ECG EGCG and catechin) was performed usingan HPLC system (Jasco Japan) equipped with a 1580 pumpand a 975 UV-Vis detector Chromatographic separationwas achieved with a HiQ Sil C

18V reversed phase column

(250mm times 46mm id) packed with 5 120583m particles andthe column temperature was maintained at 35∘C Mobilephase A was a mixture of 9 acetonitrile and 2 aceticacid with 20120583gmL EDTA Mobile phase B was a mixtureof 80 acetonitrile and 2 acetic acid with 20120583gmL EDTABaseline separation of EC EGC ECG EGCG catechin gallicacid and caffeine was achieved with a binary gradient elutionprogram as follows 100 mobile phase A for 10min thenover 15min a linear gradient to 68 mobile phase A 32mobile phase B and hold at this composition for 10minThe detection wavelength was 278 nm and the flow rate was08mLmin One milliliter of tea extract was diluted withwater to 100mL the diluted extract was filtered througha 045 120583m membrane filter (Millipore) and the filtrate wasinjected into the HPLC apparatus

Standard solutions of the major catechins (EC EGCECG EGCG and catechin) gallic acid and caffeinewere pre-pared by accurately weighing standard powders and dissolv-ing them in 10 acetonitrile with 500120583gmL of EDTA Thestock solutions were stored at 4∘C All standard solutionswere filtered through a 045 120583m membrane filter (Millipore)and the filtrate was injected The total catechins content wasexpressed as a percentage by mass of the sample on a dry

4 The Scientific World Journal

matter basis and was obtained by summation of the EGCcatechin EC EGCG and ECG content

26 Oxygen Radical Absorbance Capacity (ORAC) Assay Theoxygen radical absorbance capacity (ORAC) method is usedto measure the antioxidant capacity of biological samples invitro The ORAC assay was first developed by Cao et al in1993 [28] This approach has been recognized and is now fre-quently used in analysis of polyphenol antioxidant capacity221015840-Azobis(2-amidinopropane) dihydrochloride (AAPH) isthought to produce the peroxyl radical upon heating whichdamages the fluorescent molecule and results in a loss offluorescence [29 30] The fluorescence decay is monitored asfluorescein in the solution decomposes a process that shouldbe slower in the presence of antioxidants Decay curves arerecorded and the area between the two decay curves (withor without antioxidant) is calculated Calculating the areaunder the fluorescence decay curve (AUC) of trolox which isused as a standard antioxidant a standard curve is preparedthat can be used to evaluate the radical-scavenging activity ofother antioxidants in terms of trolox equivalents (TE)

The ORAC assay was performed using a fluorescencespectrophotometer (F-7000 Hitachi Japan) following a pre-viously described procedure [31] with minor modificationSamples were diluted with sodium phosphate buffer (75mMpH 74) Sodium fluorescein solution (600120583L 40 nM) wasadded to 100 120583L of phosphate buffer (blank) to 100 120583Laliquots of trolox solution (125 25 5 10 20 40 and 80 120583M)or to 100120583L of diluted sample The respective mixtures wereincubated for 15min at 37∘C Immediately before initiatingthe measurements 100 120583L of AAPH (150mM) was addedinto the test solution With the excitation filter set to 485 nmand the emission recorded at 535 nm readings were collectedeveryminute for 60minThe results were calculated based onthe differences in AUC between the blank and the sample andwere expressed as in units ofmicromoles TE per 1 g of dry teaThe AUC was calculated as follows

AUC =1198910+ 2sum59

119894=1119891119894+ 11989160

21198910

(1)

where 1198910is the fluorescence reading at initiation of the reac-

tion 119891119894is the fluorescence reading at 119894minutes and 119891

60is the

final measurement Net AUC was calculated as follows

Net AUC = AUCsample minus AUCblank (2)

27 DPPHRadical-ScavengingActivity The antioxidant capac-ity in 20 samples from four types of tea (green dark oolongand black teas) was determined by reduction of the 22-diphenyl-1-picryhydrazyl (DPPH) radical according to thepublishedmethod [32] A stock solutionwas prepared by stir-ring 75mg of DPPH in 1 L of methanol overnight A 075mLaliquot of sample extract was mixed with 15mL of DPPHsolution For each extract a blank of 15mL of methanolwas used without DPPH reagent to correct for any sampleabsorbance at 521 nm Sample and blank absorbance were

recorded at 521 nm after 6min The percentage inhibition ofDPPH was calculated according to the equation below [33]

Inhibition of DPPH () =[1198600minus (1198601minus 119860119904)]

1198600

times 100 (3)

where 1198600is the absorbance of the DPPH solution 119860

1is

the absorbance of the tea extract in the presence of DPPHsolution and 119860

119904is the absorbance of the tea extract solution

without DPPH Each sample was analyzed in triplicateThe EC50 value was calculated by a graphical method

as the concentration that caused 50 inhibition of DPPHThe anti-DPPH efficiency (AE) was also calculated aslog sdot (1EC50) [34]

28 Online HPLC DPPH Screening Analysis of Tea SamplesThe antioxidant activity of EC EGC ECG EGCG cate-chin gallic acid and caffeine was measured using the onlineHPLC DPPH screening method [35] The online HPLCDPPHmethod was developed using a methanolic solution ofDPPH stable free radical (50mgL) The instrumental setupis depicted in Figure 2 The flow of HPLC-separated analytesandDPPH solution was 08mLmin and 02mLmin respec-tively The HPLC-separated analytes reacted with the DPPHsolution in post-column mode which was photometricallydetected as a negative peak at 521 nm The length of the cap-illary (51m times 05mm id) used for the postcolumn reactionwas adjusted to achieve a reaction time of 10minThe HPLCconditions were the same as described above

3 Results and Discussion

31 Total Polyphenol and Catechins Content in Four Typesof Tea Using the Folin-Ciocalteu method total polyphenolcontents in 20 samples from four types of tea were calculatedfrom the standard curve for gallic acid ranging from 10 to50 120583gmL (119910 = 0183119909 minus 0025 1198772 = 09987) The resultsare summarized in Table 2 The contents of catechins in 20samples from four types of tea were determined by HPLCThe retention times were 73min for EGC 95min for cate-chin 153min for EC 165min for EGCG and 221min forECGAt the same time the contents of gallic acid and caffeinewere also determined the retention times were 37min forgallic acid and 124min for caffeine The working calibrationcurves were as follows gallic acid 119910 = 208401119909minus18547 (1198772 =09996 5ndash25120583gmL) EGC 119910 = 2645119909 minus 976 (1198772 = 09989100ndash300 120583gmL) catechin 119910 = 7999119909 minus 1075 (1198772 = 0999150ndash150 120583gmL) caffeine 119910 = 19823119909 minus 1762 (1198772 = 0998750ndash150 120583gmL) EC 119910 = 18574119909 minus 1653 (1198772 = 09990 50ndash150 120583gmL) EGCG 119910 = 29722119909 minus 2678 (1198772 = 09992 100ndash400 120583gmL) ECG 119910 = 16774119909 minus 1546 (1198772 = 09988 50ndash200120583gmL) where 119910 is the peak area of the analyte and 119909 isthe concentration (120583gmL) The results for the catechins aresummarized in Table 2 It can be seen from Table 2 that thecontent of total polyphenols ranged from782 to 3236 g of gal-lic acid equivalent100 g dry tea and the content of catechinsranged from 434 to 2427 (dry tea) Of the selected 20 teasamples Bi luo chun (green tea) had the highest content of

The Scientific World Journal 5

HPLC pump (gradient elution)

Mobile phase Sampler

HPLC pump

DPPHsolution Mixer UV-Vis detector

UV-Vis detector

Liquid waste

ColumnChromatogram at 278nm

Chromatogram at 521nm

Figure 2 Instrumental setup for the HPLC analysis of radical-scavenging compounds using an online reaction with 11-diphenyl-2-picryl-hydrazyl (DPPH)

0

5

10

15

20

25

30

0 10 20 30 40

Cate

chin

s con

tent

()

Content of total polyphenol ()

R2 = 096

Figure 3 The correlation between total polyphenol and catechincontents

polyphenols and catechins Although there was a significantdifference in the content of polyphenols and catechins in thesame type of tea as a whole the contents of total polyphenolsand catechins decreased in the following order green tea gtoolong tea gt black tea gt dark tea

In addition the correlation between catechins and totalpolyphenols in the 20 samples from the four types of teawas tested The results shown in Figure 3 reveal a strongpositive correlation between catechins and total polyphenols(1198772 = 096)

Table 2 lists the contents of individual catechins in thefour types of tea It is apparent that the content of esterifiedcatechins (EGCGor ECG) is higher than that of nonesterifiedcatechins (catechin EC or EGC) In comparison of the fourtypes of tea EGCG content was the highest in green tea

0500

100015002000250030003500

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

ORA

C (120583

mol

TE

g te

a)

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoerFigure 4 Oxygen radical absorbance capacity (ORAC) values ofdifferent samples in four types of tea

(525ndash1439 on dry tea) and in oolong tea (457ndash1308 ondry tea) With the increase in the degree of fermentation theEGCG content gradually decreased and the range of EGCGcontent in dark tea is only 036ndash093 on dry tea Among thefour types of tea the ECG content was the highest in blacktea (338ndash563 on dry tea) and in dark tea (192ndash473 ondry tea)

32 Oxygen Radical Absorbance Capacity (ORAC) in FourTypes of Tea A straight calibration curve was obtained fortrolox antioxidant activity in the concentration range of 125ndash80 120583M 119910 = 036119909 minus 056 where 119909 is concentration oftrolox and 119910 is Net AUC (1198772 = 09983) Figure 4 shows theORAC values of the selected tea samples the ORAC value

6 The Scientific World Journal

Table2Th

econ

tent

oftotalp

olypheno

lstotalcatechinsand

individu

alcatechinsin20

samples

from

four

typeso

ftea

(119899=3)

Samples

oftea

EGC(

)Ca

techin

()

EC(

)EG

CG(

)EC

G(

)Non

esterifi

edcatechins()

Esterifi

edcatechins()

Totalcatechins

()

Total

polyph

enols()

Green

tea

Biluochun

113plusmn006

092plusmn004

184plusmn003

1439plusmn041

599plusmn002

389plusmn013

2038plusmn043

2427plusmn056

3236plusmn093

Huang

shan

mao

feng

094plusmn003

085plusmn004

183plusmn005

1411plusmn047

561plusmn004

362plusmn012

1972plusmn051

2334plusmn063

2686plusmn049

Long

jing

062plusmn002

055plusmn005

108plusmn001

813plusmn039

361plusmn011

225plusmn008

1174plusmn050

1399plusmn058

1817plusmn071

Luan

guap

ian

037plusmn001

027plusmn001

061plusmn003

525plusmn026

185plusmn003

125plusmn005

710plusmn029

835plusmn034

1088plusmn006

Xinyang

mao

jian

096plusmn002

074plusmn003

149plusmn001

1263plusmn023

526plusmn014

319plusmn006

1789plusmn037

2108plusmn043

2694plusmn009

Meanvalue

080plusmn003

067plusmn003

137plusmn003

1090plusmn035

446plusmn007

284plusmn009

1537plusmn042

1821plusmn

051

2304plusmn046

Oolon

gtea

Dah

ongpao

063plusmn002

066plusmn003

074plusmn003

559plusmn022

208plusmn002

203plusmn008

767plusmn024

970plusmn032

1316plusmn009

Rougui

044plusmn002

055plusmn001

052plusmn002

457plusmn009

166plusmn006

151plusmn

005

623plusmn015

774plusmn020

1035plusmn008

Shui

xian

068plusmn001

081plusmn002

090plusmn002

723plusmn031

243plusmn001

239plusmn005

966plusmn032

1205plusmn037

1618plusmn027

Taiw

anwulong

116plusmn002

144plusmn005

153plusmn006

1308plusmn027

417plusmn006

413plusmn013

1725plusmn033

2138plusmn046

2973plusmn004

Tieg

uanyin

068plusmn001

077plusmn002

084plusmn002

686plusmn007

236plusmn001

229plusmn005

922plusmn008

1151plusmn

013

1569plusmn073

Meanvalue

072plusmn002

085plusmn003

091plusmn003

747plusmn019

254plusmn003

247plusmn007

1001plusmn

022

1248plusmn030

1702plusmn024

Blacktea

Diancha

241plusmn009

250plusmn004

043plusmn001

226plusmn003

470plusmn015

534plusmn014

696plusmn018

1230plusmn032

1675plusmn074

Hen

anho

ngcha

158plusmn003

163plusmn006

031plusmn001

155plusmn002

342plusmn001

352plusmn010

497plusmn003

849plusmn013

1153plusmn004

Qim

enho

ngcha

150plusmn003

159plusmn005

028plusmn001

131plusmn

005

338plusmn011

337plusmn009

469plusmn016

806plusmn025

1301plusmn

017

Tanyang

gong

fu17

5plusmn005

197plusmn004

032plusmn001

155plusmn002

419plusmn013

404plusmn010

574plusmn015

978plusmn025

1373plusmn004

Zhengshan

hong

cha

257plusmn009

266plusmn012

045plusmn001

204plusmn007

563plusmn001

568plusmn022

767plusmn008

1335plusmn030

1817plusmn012

Meanvalue

196plusmn006

207plusmn006

036plusmn001

174plusmn004

426plusmn008

439plusmn013

601plusmn012

1040plusmn025

1464plusmn022

Darktea

Baixingfu

cha

022plusmn001

077plusmn001

107plusmn003

036plusmn001

192plusmn003

206plusmn005

228plusmn004

434plusmn009

782plusmn029

Qianliang

028plusmn003

102plusmn004

135plusmn007

050plusmn002

252plusmn005

265plusmn014

302plusmn007

567plusmn021

929plusmn038

Liubaocha

034plusmn001

135plusmn002

187plusmn004

072plusmn002

341plusmn017

356plusmn007

413plusmn019

769plusmn026

1328plusmn060

Qizibing

035plusmn001

148plusmn007

202plusmn007

079plusmn004

395plusmn013

385plusmn015

474plusmn017

859plusmn032

1480plusmn003

Shengpu

rsquoer039plusmn002

197plusmn002

251plusmn009

093plusmn002

473plusmn013

487plusmn013

566plusmn015

1053plusmn028

1629plusmn017

Meanvalue

032plusmn002

132plusmn003

176plusmn006

066plusmn002

331plusmn010

340plusmn011

397plusmn012

736plusmn023

1230plusmn029

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World Journal 3

Table 1 Source and production date of 20 tea samples

Category Product name Source Production date

Green tea

Bi luo chun Jiangsu 2010Huang shan mao feng Anhui 2010Long jing Zhejiang 2011Lu an gua pian Anhui 2010Xin yang mao jian Henan 2010

Oolong tea

Da hong pao Fujian 2010Rou gui Fujian 2010Shui xian Fujian 2010Taiwan oolong Taiwan 2010Tie guan yin Fujian 2010

Black tea

Dian cha Yunnan 2010Henan black tea Henan 2009Qimen black tea Anhui 2010Tanyang gongfu Fujian 2010Zhengshan black tea Fujian 2010

Dark tea

Bai xing fu cha Hunan 2009Qian liang Hunan 2008Liu bao cha Guangxi 2007Qi zi bing Yunnan 2007Sheng pursquoer Yunnan 2009

2 Materials and Methods

21 Tea Samples A total of 20 samples from four types of tea(green dark oolong and black teas)were collected fromDafatea market in Harbin China They included 5 green teas 5oolong teas 5 black teas and 5 dark teas Their identities arelisted in Table 1

22 Chemicals For the determination of the total phenoliccontent (TPC) Folin-Ciocalteu phenol reagents (Sigma-Aldrich USA) gallic acid (GA 975 purity Sigma-AldrichUSA) and anhydrous sodium carbonate (99 purity BeijingChemical Reagents China) were used For the determina-tion of the antioxidant activity 11-diphenyl-2-picrylhydrazyl(DPPH Sigma-Aldrich USA) was used The oxygen radicalabsorbance capacity (ORAC) reagents of 221015840-azobis(2-meth-ylpropionamidine) dihydrochloride (AAPH) 6-hydroxy-2578-tetramethylchromane-2-carboxylic acid (trolox) andfluorescein sodiumwere obtained fromSigma-Aldrich (USA)For the determination of the total catechins content (minus)-epigallocatechin gallate (EGCG ge970) (minus)-epicatechingallate (ECGge98) (minus)-epigallocatechin (EGCge95) (minus)-epicatechin (EC ge98) and (+)-catechin (ge99) standardswere obtained from Sigma-Aldrich (USA) Caffeine (ge98)was obtained from the National Institutes for Food andDrug Control (Beijing China) Acetonitrile acetic acid andEDTAwere of HPLC grade (Sigma-Aldrich USA) Methanolwas of analytical grade (Beijing Chemical Reagents China)Deionized water was prepared using a Milli Q-Plus system(Millipore Germany)

23 Preparation of Tea Extract The preparation of the teaextract was performed according to the method publishedby the International Organization for Standardization (ISO)[23] Each tea sample was ground to a fine powder and 10 gof the powder was extracted with 25mL of methanolwater(7 3 vv) in a water bath at 70∘C for 10min The extractionwas repeated twice and the extracts were combined Afterfiltration through a filter paper and a 045120583mmembrane filter(Millipore) the extract volume was adjusted to 100mL

24 Determination of Total Polyphenols by Folin-CiocalteuMethod The total polyphenol content in 20 samples fromfour types of tea (green dark oolong and black) was deter-mined according to the Folin-Ciocalteu method as describedby ISO 14502-1 [23] with some minor modifications Onemilliliter of the tea extract was diluted with water to 100mLThe diluted extract solution (10mL) was then mixed with50mL of 50 Folin-Ciocalteu reagent After 5min 40mLof 75 Na

2CO3solution was added and mixed Then the

mixture was incubated at room temperature for 60min Theabsorbance of the solution was measured at 765 nm witha Shimadzu UV-2550 UV-Vis spectrophotometer against areagent blank The determination was three times The totalpolyphenol content was expressed as gallic acid equivalents(GAE units g100 g sample)

25 Determination of Catechins by HPLC The content of cat-echins in 20 samples from four types of tea (green darkoolong and black teas) was determined according to the ISO14502-2 method [27] Quantification of the major catechins(EC EGC ECG EGCG and catechin) was performed usingan HPLC system (Jasco Japan) equipped with a 1580 pumpand a 975 UV-Vis detector Chromatographic separationwas achieved with a HiQ Sil C

18V reversed phase column

(250mm times 46mm id) packed with 5 120583m particles andthe column temperature was maintained at 35∘C Mobilephase A was a mixture of 9 acetonitrile and 2 aceticacid with 20120583gmL EDTA Mobile phase B was a mixtureof 80 acetonitrile and 2 acetic acid with 20120583gmL EDTABaseline separation of EC EGC ECG EGCG catechin gallicacid and caffeine was achieved with a binary gradient elutionprogram as follows 100 mobile phase A for 10min thenover 15min a linear gradient to 68 mobile phase A 32mobile phase B and hold at this composition for 10minThe detection wavelength was 278 nm and the flow rate was08mLmin One milliliter of tea extract was diluted withwater to 100mL the diluted extract was filtered througha 045 120583m membrane filter (Millipore) and the filtrate wasinjected into the HPLC apparatus

Standard solutions of the major catechins (EC EGCECG EGCG and catechin) gallic acid and caffeinewere pre-pared by accurately weighing standard powders and dissolv-ing them in 10 acetonitrile with 500120583gmL of EDTA Thestock solutions were stored at 4∘C All standard solutionswere filtered through a 045 120583m membrane filter (Millipore)and the filtrate was injected The total catechins content wasexpressed as a percentage by mass of the sample on a dry

4 The Scientific World Journal

matter basis and was obtained by summation of the EGCcatechin EC EGCG and ECG content

26 Oxygen Radical Absorbance Capacity (ORAC) Assay Theoxygen radical absorbance capacity (ORAC) method is usedto measure the antioxidant capacity of biological samples invitro The ORAC assay was first developed by Cao et al in1993 [28] This approach has been recognized and is now fre-quently used in analysis of polyphenol antioxidant capacity221015840-Azobis(2-amidinopropane) dihydrochloride (AAPH) isthought to produce the peroxyl radical upon heating whichdamages the fluorescent molecule and results in a loss offluorescence [29 30] The fluorescence decay is monitored asfluorescein in the solution decomposes a process that shouldbe slower in the presence of antioxidants Decay curves arerecorded and the area between the two decay curves (withor without antioxidant) is calculated Calculating the areaunder the fluorescence decay curve (AUC) of trolox which isused as a standard antioxidant a standard curve is preparedthat can be used to evaluate the radical-scavenging activity ofother antioxidants in terms of trolox equivalents (TE)

The ORAC assay was performed using a fluorescencespectrophotometer (F-7000 Hitachi Japan) following a pre-viously described procedure [31] with minor modificationSamples were diluted with sodium phosphate buffer (75mMpH 74) Sodium fluorescein solution (600120583L 40 nM) wasadded to 100 120583L of phosphate buffer (blank) to 100 120583Laliquots of trolox solution (125 25 5 10 20 40 and 80 120583M)or to 100120583L of diluted sample The respective mixtures wereincubated for 15min at 37∘C Immediately before initiatingthe measurements 100 120583L of AAPH (150mM) was addedinto the test solution With the excitation filter set to 485 nmand the emission recorded at 535 nm readings were collectedeveryminute for 60minThe results were calculated based onthe differences in AUC between the blank and the sample andwere expressed as in units ofmicromoles TE per 1 g of dry teaThe AUC was calculated as follows

AUC =1198910+ 2sum59

119894=1119891119894+ 11989160

21198910

(1)

where 1198910is the fluorescence reading at initiation of the reac-

tion 119891119894is the fluorescence reading at 119894minutes and 119891

60is the

final measurement Net AUC was calculated as follows

Net AUC = AUCsample minus AUCblank (2)

27 DPPHRadical-ScavengingActivity The antioxidant capac-ity in 20 samples from four types of tea (green dark oolongand black teas) was determined by reduction of the 22-diphenyl-1-picryhydrazyl (DPPH) radical according to thepublishedmethod [32] A stock solutionwas prepared by stir-ring 75mg of DPPH in 1 L of methanol overnight A 075mLaliquot of sample extract was mixed with 15mL of DPPHsolution For each extract a blank of 15mL of methanolwas used without DPPH reagent to correct for any sampleabsorbance at 521 nm Sample and blank absorbance were

recorded at 521 nm after 6min The percentage inhibition ofDPPH was calculated according to the equation below [33]

Inhibition of DPPH () =[1198600minus (1198601minus 119860119904)]

1198600

times 100 (3)

where 1198600is the absorbance of the DPPH solution 119860

1is

the absorbance of the tea extract in the presence of DPPHsolution and 119860

119904is the absorbance of the tea extract solution

without DPPH Each sample was analyzed in triplicateThe EC50 value was calculated by a graphical method

as the concentration that caused 50 inhibition of DPPHThe anti-DPPH efficiency (AE) was also calculated aslog sdot (1EC50) [34]

28 Online HPLC DPPH Screening Analysis of Tea SamplesThe antioxidant activity of EC EGC ECG EGCG cate-chin gallic acid and caffeine was measured using the onlineHPLC DPPH screening method [35] The online HPLCDPPHmethod was developed using a methanolic solution ofDPPH stable free radical (50mgL) The instrumental setupis depicted in Figure 2 The flow of HPLC-separated analytesandDPPH solution was 08mLmin and 02mLmin respec-tively The HPLC-separated analytes reacted with the DPPHsolution in post-column mode which was photometricallydetected as a negative peak at 521 nm The length of the cap-illary (51m times 05mm id) used for the postcolumn reactionwas adjusted to achieve a reaction time of 10minThe HPLCconditions were the same as described above

3 Results and Discussion

31 Total Polyphenol and Catechins Content in Four Typesof Tea Using the Folin-Ciocalteu method total polyphenolcontents in 20 samples from four types of tea were calculatedfrom the standard curve for gallic acid ranging from 10 to50 120583gmL (119910 = 0183119909 minus 0025 1198772 = 09987) The resultsare summarized in Table 2 The contents of catechins in 20samples from four types of tea were determined by HPLCThe retention times were 73min for EGC 95min for cate-chin 153min for EC 165min for EGCG and 221min forECGAt the same time the contents of gallic acid and caffeinewere also determined the retention times were 37min forgallic acid and 124min for caffeine The working calibrationcurves were as follows gallic acid 119910 = 208401119909minus18547 (1198772 =09996 5ndash25120583gmL) EGC 119910 = 2645119909 minus 976 (1198772 = 09989100ndash300 120583gmL) catechin 119910 = 7999119909 minus 1075 (1198772 = 0999150ndash150 120583gmL) caffeine 119910 = 19823119909 minus 1762 (1198772 = 0998750ndash150 120583gmL) EC 119910 = 18574119909 minus 1653 (1198772 = 09990 50ndash150 120583gmL) EGCG 119910 = 29722119909 minus 2678 (1198772 = 09992 100ndash400 120583gmL) ECG 119910 = 16774119909 minus 1546 (1198772 = 09988 50ndash200120583gmL) where 119910 is the peak area of the analyte and 119909 isthe concentration (120583gmL) The results for the catechins aresummarized in Table 2 It can be seen from Table 2 that thecontent of total polyphenols ranged from782 to 3236 g of gal-lic acid equivalent100 g dry tea and the content of catechinsranged from 434 to 2427 (dry tea) Of the selected 20 teasamples Bi luo chun (green tea) had the highest content of

The Scientific World Journal 5

HPLC pump (gradient elution)

Mobile phase Sampler

HPLC pump

DPPHsolution Mixer UV-Vis detector

UV-Vis detector

Liquid waste

ColumnChromatogram at 278nm

Chromatogram at 521nm

Figure 2 Instrumental setup for the HPLC analysis of radical-scavenging compounds using an online reaction with 11-diphenyl-2-picryl-hydrazyl (DPPH)

0

5

10

15

20

25

30

0 10 20 30 40

Cate

chin

s con

tent

()

Content of total polyphenol ()

R2 = 096

Figure 3 The correlation between total polyphenol and catechincontents

polyphenols and catechins Although there was a significantdifference in the content of polyphenols and catechins in thesame type of tea as a whole the contents of total polyphenolsand catechins decreased in the following order green tea gtoolong tea gt black tea gt dark tea

In addition the correlation between catechins and totalpolyphenols in the 20 samples from the four types of teawas tested The results shown in Figure 3 reveal a strongpositive correlation between catechins and total polyphenols(1198772 = 096)

Table 2 lists the contents of individual catechins in thefour types of tea It is apparent that the content of esterifiedcatechins (EGCGor ECG) is higher than that of nonesterifiedcatechins (catechin EC or EGC) In comparison of the fourtypes of tea EGCG content was the highest in green tea

0500

100015002000250030003500

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

ORA

C (120583

mol

TE

g te

a)

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoerFigure 4 Oxygen radical absorbance capacity (ORAC) values ofdifferent samples in four types of tea

(525ndash1439 on dry tea) and in oolong tea (457ndash1308 ondry tea) With the increase in the degree of fermentation theEGCG content gradually decreased and the range of EGCGcontent in dark tea is only 036ndash093 on dry tea Among thefour types of tea the ECG content was the highest in blacktea (338ndash563 on dry tea) and in dark tea (192ndash473 ondry tea)

32 Oxygen Radical Absorbance Capacity (ORAC) in FourTypes of Tea A straight calibration curve was obtained fortrolox antioxidant activity in the concentration range of 125ndash80 120583M 119910 = 036119909 minus 056 where 119909 is concentration oftrolox and 119910 is Net AUC (1198772 = 09983) Figure 4 shows theORAC values of the selected tea samples the ORAC value

6 The Scientific World Journal

Table2Th

econ

tent

oftotalp

olypheno

lstotalcatechinsand

individu

alcatechinsin20

samples

from

four

typeso

ftea

(119899=3)

Samples

oftea

EGC(

)Ca

techin

()

EC(

)EG

CG(

)EC

G(

)Non

esterifi

edcatechins()

Esterifi

edcatechins()

Totalcatechins

()

Total

polyph

enols()

Green

tea

Biluochun

113plusmn006

092plusmn004

184plusmn003

1439plusmn041

599plusmn002

389plusmn013

2038plusmn043

2427plusmn056

3236plusmn093

Huang

shan

mao

feng

094plusmn003

085plusmn004

183plusmn005

1411plusmn047

561plusmn004

362plusmn012

1972plusmn051

2334plusmn063

2686plusmn049

Long

jing

062plusmn002

055plusmn005

108plusmn001

813plusmn039

361plusmn011

225plusmn008

1174plusmn050

1399plusmn058

1817plusmn071

Luan

guap

ian

037plusmn001

027plusmn001

061plusmn003

525plusmn026

185plusmn003

125plusmn005

710plusmn029

835plusmn034

1088plusmn006

Xinyang

mao

jian

096plusmn002

074plusmn003

149plusmn001

1263plusmn023

526plusmn014

319plusmn006

1789plusmn037

2108plusmn043

2694plusmn009

Meanvalue

080plusmn003

067plusmn003

137plusmn003

1090plusmn035

446plusmn007

284plusmn009

1537plusmn042

1821plusmn

051

2304plusmn046

Oolon

gtea

Dah

ongpao

063plusmn002

066plusmn003

074plusmn003

559plusmn022

208plusmn002

203plusmn008

767plusmn024

970plusmn032

1316plusmn009

Rougui

044plusmn002

055plusmn001

052plusmn002

457plusmn009

166plusmn006

151plusmn

005

623plusmn015

774plusmn020

1035plusmn008

Shui

xian

068plusmn001

081plusmn002

090plusmn002

723plusmn031

243plusmn001

239plusmn005

966plusmn032

1205plusmn037

1618plusmn027

Taiw

anwulong

116plusmn002

144plusmn005

153plusmn006

1308plusmn027

417plusmn006

413plusmn013

1725plusmn033

2138plusmn046

2973plusmn004

Tieg

uanyin

068plusmn001

077plusmn002

084plusmn002

686plusmn007

236plusmn001

229plusmn005

922plusmn008

1151plusmn

013

1569plusmn073

Meanvalue

072plusmn002

085plusmn003

091plusmn003

747plusmn019

254plusmn003

247plusmn007

1001plusmn

022

1248plusmn030

1702plusmn024

Blacktea

Diancha

241plusmn009

250plusmn004

043plusmn001

226plusmn003

470plusmn015

534plusmn014

696plusmn018

1230plusmn032

1675plusmn074

Hen

anho

ngcha

158plusmn003

163plusmn006

031plusmn001

155plusmn002

342plusmn001

352plusmn010

497plusmn003

849plusmn013

1153plusmn004

Qim

enho

ngcha

150plusmn003

159plusmn005

028plusmn001

131plusmn

005

338plusmn011

337plusmn009

469plusmn016

806plusmn025

1301plusmn

017

Tanyang

gong

fu17

5plusmn005

197plusmn004

032plusmn001

155plusmn002

419plusmn013

404plusmn010

574plusmn015

978plusmn025

1373plusmn004

Zhengshan

hong

cha

257plusmn009

266plusmn012

045plusmn001

204plusmn007

563plusmn001

568plusmn022

767plusmn008

1335plusmn030

1817plusmn012

Meanvalue

196plusmn006

207plusmn006

036plusmn001

174plusmn004

426plusmn008

439plusmn013

601plusmn012

1040plusmn025

1464plusmn022

Darktea

Baixingfu

cha

022plusmn001

077plusmn001

107plusmn003

036plusmn001

192plusmn003

206plusmn005

228plusmn004

434plusmn009

782plusmn029

Qianliang

028plusmn003

102plusmn004

135plusmn007

050plusmn002

252plusmn005

265plusmn014

302plusmn007

567plusmn021

929plusmn038

Liubaocha

034plusmn001

135plusmn002

187plusmn004

072plusmn002

341plusmn017

356plusmn007

413plusmn019

769plusmn026

1328plusmn060

Qizibing

035plusmn001

148plusmn007

202plusmn007

079plusmn004

395plusmn013

385plusmn015

474plusmn017

859plusmn032

1480plusmn003

Shengpu

rsquoer039plusmn002

197plusmn002

251plusmn009

093plusmn002

473plusmn013

487plusmn013

566plusmn015

1053plusmn028

1629plusmn017

Meanvalue

032plusmn002

132plusmn003

176plusmn006

066plusmn002

331plusmn010

340plusmn011

397plusmn012

736plusmn023

1230plusmn029

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

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Veterinary Medicine International

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Cell BiologyInternational Journal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

4 The Scientific World Journal

matter basis and was obtained by summation of the EGCcatechin EC EGCG and ECG content

26 Oxygen Radical Absorbance Capacity (ORAC) Assay Theoxygen radical absorbance capacity (ORAC) method is usedto measure the antioxidant capacity of biological samples invitro The ORAC assay was first developed by Cao et al in1993 [28] This approach has been recognized and is now fre-quently used in analysis of polyphenol antioxidant capacity221015840-Azobis(2-amidinopropane) dihydrochloride (AAPH) isthought to produce the peroxyl radical upon heating whichdamages the fluorescent molecule and results in a loss offluorescence [29 30] The fluorescence decay is monitored asfluorescein in the solution decomposes a process that shouldbe slower in the presence of antioxidants Decay curves arerecorded and the area between the two decay curves (withor without antioxidant) is calculated Calculating the areaunder the fluorescence decay curve (AUC) of trolox which isused as a standard antioxidant a standard curve is preparedthat can be used to evaluate the radical-scavenging activity ofother antioxidants in terms of trolox equivalents (TE)

The ORAC assay was performed using a fluorescencespectrophotometer (F-7000 Hitachi Japan) following a pre-viously described procedure [31] with minor modificationSamples were diluted with sodium phosphate buffer (75mMpH 74) Sodium fluorescein solution (600120583L 40 nM) wasadded to 100 120583L of phosphate buffer (blank) to 100 120583Laliquots of trolox solution (125 25 5 10 20 40 and 80 120583M)or to 100120583L of diluted sample The respective mixtures wereincubated for 15min at 37∘C Immediately before initiatingthe measurements 100 120583L of AAPH (150mM) was addedinto the test solution With the excitation filter set to 485 nmand the emission recorded at 535 nm readings were collectedeveryminute for 60minThe results were calculated based onthe differences in AUC between the blank and the sample andwere expressed as in units ofmicromoles TE per 1 g of dry teaThe AUC was calculated as follows

AUC =1198910+ 2sum59

119894=1119891119894+ 11989160

21198910

(1)

where 1198910is the fluorescence reading at initiation of the reac-

tion 119891119894is the fluorescence reading at 119894minutes and 119891

60is the

final measurement Net AUC was calculated as follows

Net AUC = AUCsample minus AUCblank (2)

27 DPPHRadical-ScavengingActivity The antioxidant capac-ity in 20 samples from four types of tea (green dark oolongand black teas) was determined by reduction of the 22-diphenyl-1-picryhydrazyl (DPPH) radical according to thepublishedmethod [32] A stock solutionwas prepared by stir-ring 75mg of DPPH in 1 L of methanol overnight A 075mLaliquot of sample extract was mixed with 15mL of DPPHsolution For each extract a blank of 15mL of methanolwas used without DPPH reagent to correct for any sampleabsorbance at 521 nm Sample and blank absorbance were

recorded at 521 nm after 6min The percentage inhibition ofDPPH was calculated according to the equation below [33]

Inhibition of DPPH () =[1198600minus (1198601minus 119860119904)]

1198600

times 100 (3)

where 1198600is the absorbance of the DPPH solution 119860

1is

the absorbance of the tea extract in the presence of DPPHsolution and 119860

119904is the absorbance of the tea extract solution

without DPPH Each sample was analyzed in triplicateThe EC50 value was calculated by a graphical method

as the concentration that caused 50 inhibition of DPPHThe anti-DPPH efficiency (AE) was also calculated aslog sdot (1EC50) [34]

28 Online HPLC DPPH Screening Analysis of Tea SamplesThe antioxidant activity of EC EGC ECG EGCG cate-chin gallic acid and caffeine was measured using the onlineHPLC DPPH screening method [35] The online HPLCDPPHmethod was developed using a methanolic solution ofDPPH stable free radical (50mgL) The instrumental setupis depicted in Figure 2 The flow of HPLC-separated analytesandDPPH solution was 08mLmin and 02mLmin respec-tively The HPLC-separated analytes reacted with the DPPHsolution in post-column mode which was photometricallydetected as a negative peak at 521 nm The length of the cap-illary (51m times 05mm id) used for the postcolumn reactionwas adjusted to achieve a reaction time of 10minThe HPLCconditions were the same as described above

3 Results and Discussion

31 Total Polyphenol and Catechins Content in Four Typesof Tea Using the Folin-Ciocalteu method total polyphenolcontents in 20 samples from four types of tea were calculatedfrom the standard curve for gallic acid ranging from 10 to50 120583gmL (119910 = 0183119909 minus 0025 1198772 = 09987) The resultsare summarized in Table 2 The contents of catechins in 20samples from four types of tea were determined by HPLCThe retention times were 73min for EGC 95min for cate-chin 153min for EC 165min for EGCG and 221min forECGAt the same time the contents of gallic acid and caffeinewere also determined the retention times were 37min forgallic acid and 124min for caffeine The working calibrationcurves were as follows gallic acid 119910 = 208401119909minus18547 (1198772 =09996 5ndash25120583gmL) EGC 119910 = 2645119909 minus 976 (1198772 = 09989100ndash300 120583gmL) catechin 119910 = 7999119909 minus 1075 (1198772 = 0999150ndash150 120583gmL) caffeine 119910 = 19823119909 minus 1762 (1198772 = 0998750ndash150 120583gmL) EC 119910 = 18574119909 minus 1653 (1198772 = 09990 50ndash150 120583gmL) EGCG 119910 = 29722119909 minus 2678 (1198772 = 09992 100ndash400 120583gmL) ECG 119910 = 16774119909 minus 1546 (1198772 = 09988 50ndash200120583gmL) where 119910 is the peak area of the analyte and 119909 isthe concentration (120583gmL) The results for the catechins aresummarized in Table 2 It can be seen from Table 2 that thecontent of total polyphenols ranged from782 to 3236 g of gal-lic acid equivalent100 g dry tea and the content of catechinsranged from 434 to 2427 (dry tea) Of the selected 20 teasamples Bi luo chun (green tea) had the highest content of

The Scientific World Journal 5

HPLC pump (gradient elution)

Mobile phase Sampler

HPLC pump

DPPHsolution Mixer UV-Vis detector

UV-Vis detector

Liquid waste

ColumnChromatogram at 278nm

Chromatogram at 521nm

Figure 2 Instrumental setup for the HPLC analysis of radical-scavenging compounds using an online reaction with 11-diphenyl-2-picryl-hydrazyl (DPPH)

0

5

10

15

20

25

30

0 10 20 30 40

Cate

chin

s con

tent

()

Content of total polyphenol ()

R2 = 096

Figure 3 The correlation between total polyphenol and catechincontents

polyphenols and catechins Although there was a significantdifference in the content of polyphenols and catechins in thesame type of tea as a whole the contents of total polyphenolsand catechins decreased in the following order green tea gtoolong tea gt black tea gt dark tea

In addition the correlation between catechins and totalpolyphenols in the 20 samples from the four types of teawas tested The results shown in Figure 3 reveal a strongpositive correlation between catechins and total polyphenols(1198772 = 096)

Table 2 lists the contents of individual catechins in thefour types of tea It is apparent that the content of esterifiedcatechins (EGCGor ECG) is higher than that of nonesterifiedcatechins (catechin EC or EGC) In comparison of the fourtypes of tea EGCG content was the highest in green tea

0500

100015002000250030003500

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

ORA

C (120583

mol

TE

g te

a)

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoerFigure 4 Oxygen radical absorbance capacity (ORAC) values ofdifferent samples in four types of tea

(525ndash1439 on dry tea) and in oolong tea (457ndash1308 ondry tea) With the increase in the degree of fermentation theEGCG content gradually decreased and the range of EGCGcontent in dark tea is only 036ndash093 on dry tea Among thefour types of tea the ECG content was the highest in blacktea (338ndash563 on dry tea) and in dark tea (192ndash473 ondry tea)

32 Oxygen Radical Absorbance Capacity (ORAC) in FourTypes of Tea A straight calibration curve was obtained fortrolox antioxidant activity in the concentration range of 125ndash80 120583M 119910 = 036119909 minus 056 where 119909 is concentration oftrolox and 119910 is Net AUC (1198772 = 09983) Figure 4 shows theORAC values of the selected tea samples the ORAC value

6 The Scientific World Journal

Table2Th

econ

tent

oftotalp

olypheno

lstotalcatechinsand

individu

alcatechinsin20

samples

from

four

typeso

ftea

(119899=3)

Samples

oftea

EGC(

)Ca

techin

()

EC(

)EG

CG(

)EC

G(

)Non

esterifi

edcatechins()

Esterifi

edcatechins()

Totalcatechins

()

Total

polyph

enols()

Green

tea

Biluochun

113plusmn006

092plusmn004

184plusmn003

1439plusmn041

599plusmn002

389plusmn013

2038plusmn043

2427plusmn056

3236plusmn093

Huang

shan

mao

feng

094plusmn003

085plusmn004

183plusmn005

1411plusmn047

561plusmn004

362plusmn012

1972plusmn051

2334plusmn063

2686plusmn049

Long

jing

062plusmn002

055plusmn005

108plusmn001

813plusmn039

361plusmn011

225plusmn008

1174plusmn050

1399plusmn058

1817plusmn071

Luan

guap

ian

037plusmn001

027plusmn001

061plusmn003

525plusmn026

185plusmn003

125plusmn005

710plusmn029

835plusmn034

1088plusmn006

Xinyang

mao

jian

096plusmn002

074plusmn003

149plusmn001

1263plusmn023

526plusmn014

319plusmn006

1789plusmn037

2108plusmn043

2694plusmn009

Meanvalue

080plusmn003

067plusmn003

137plusmn003

1090plusmn035

446plusmn007

284plusmn009

1537plusmn042

1821plusmn

051

2304plusmn046

Oolon

gtea

Dah

ongpao

063plusmn002

066plusmn003

074plusmn003

559plusmn022

208plusmn002

203plusmn008

767plusmn024

970plusmn032

1316plusmn009

Rougui

044plusmn002

055plusmn001

052plusmn002

457plusmn009

166plusmn006

151plusmn

005

623plusmn015

774plusmn020

1035plusmn008

Shui

xian

068plusmn001

081plusmn002

090plusmn002

723plusmn031

243plusmn001

239plusmn005

966plusmn032

1205plusmn037

1618plusmn027

Taiw

anwulong

116plusmn002

144plusmn005

153plusmn006

1308plusmn027

417plusmn006

413plusmn013

1725plusmn033

2138plusmn046

2973plusmn004

Tieg

uanyin

068plusmn001

077plusmn002

084plusmn002

686plusmn007

236plusmn001

229plusmn005

922plusmn008

1151plusmn

013

1569plusmn073

Meanvalue

072plusmn002

085plusmn003

091plusmn003

747plusmn019

254plusmn003

247plusmn007

1001plusmn

022

1248plusmn030

1702plusmn024

Blacktea

Diancha

241plusmn009

250plusmn004

043plusmn001

226plusmn003

470plusmn015

534plusmn014

696plusmn018

1230plusmn032

1675plusmn074

Hen

anho

ngcha

158plusmn003

163plusmn006

031plusmn001

155plusmn002

342plusmn001

352plusmn010

497plusmn003

849plusmn013

1153plusmn004

Qim

enho

ngcha

150plusmn003

159plusmn005

028plusmn001

131plusmn

005

338plusmn011

337plusmn009

469plusmn016

806plusmn025

1301plusmn

017

Tanyang

gong

fu17

5plusmn005

197plusmn004

032plusmn001

155plusmn002

419plusmn013

404plusmn010

574plusmn015

978plusmn025

1373plusmn004

Zhengshan

hong

cha

257plusmn009

266plusmn012

045plusmn001

204plusmn007

563plusmn001

568plusmn022

767plusmn008

1335plusmn030

1817plusmn012

Meanvalue

196plusmn006

207plusmn006

036plusmn001

174plusmn004

426plusmn008

439plusmn013

601plusmn012

1040plusmn025

1464plusmn022

Darktea

Baixingfu

cha

022plusmn001

077plusmn001

107plusmn003

036plusmn001

192plusmn003

206plusmn005

228plusmn004

434plusmn009

782plusmn029

Qianliang

028plusmn003

102plusmn004

135plusmn007

050plusmn002

252plusmn005

265plusmn014

302plusmn007

567plusmn021

929plusmn038

Liubaocha

034plusmn001

135plusmn002

187plusmn004

072plusmn002

341plusmn017

356plusmn007

413plusmn019

769plusmn026

1328plusmn060

Qizibing

035plusmn001

148plusmn007

202plusmn007

079plusmn004

395plusmn013

385plusmn015

474plusmn017

859plusmn032

1480plusmn003

Shengpu

rsquoer039plusmn002

197plusmn002

251plusmn009

093plusmn002

473plusmn013

487plusmn013

566plusmn015

1053plusmn028

1629plusmn017

Meanvalue

032plusmn002

132plusmn003

176plusmn006

066plusmn002

331plusmn010

340plusmn011

397plusmn012

736plusmn023

1230plusmn029

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World Journal 5

HPLC pump (gradient elution)

Mobile phase Sampler

HPLC pump

DPPHsolution Mixer UV-Vis detector

UV-Vis detector

Liquid waste

ColumnChromatogram at 278nm

Chromatogram at 521nm

Figure 2 Instrumental setup for the HPLC analysis of radical-scavenging compounds using an online reaction with 11-diphenyl-2-picryl-hydrazyl (DPPH)

0

5

10

15

20

25

30

0 10 20 30 40

Cate

chin

s con

tent

()

Content of total polyphenol ()

R2 = 096

Figure 3 The correlation between total polyphenol and catechincontents

polyphenols and catechins Although there was a significantdifference in the content of polyphenols and catechins in thesame type of tea as a whole the contents of total polyphenolsand catechins decreased in the following order green tea gtoolong tea gt black tea gt dark tea

In addition the correlation between catechins and totalpolyphenols in the 20 samples from the four types of teawas tested The results shown in Figure 3 reveal a strongpositive correlation between catechins and total polyphenols(1198772 = 096)

Table 2 lists the contents of individual catechins in thefour types of tea It is apparent that the content of esterifiedcatechins (EGCGor ECG) is higher than that of nonesterifiedcatechins (catechin EC or EGC) In comparison of the fourtypes of tea EGCG content was the highest in green tea

0500

100015002000250030003500

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

ORA

C (120583

mol

TE

g te

a)

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoerFigure 4 Oxygen radical absorbance capacity (ORAC) values ofdifferent samples in four types of tea

(525ndash1439 on dry tea) and in oolong tea (457ndash1308 ondry tea) With the increase in the degree of fermentation theEGCG content gradually decreased and the range of EGCGcontent in dark tea is only 036ndash093 on dry tea Among thefour types of tea the ECG content was the highest in blacktea (338ndash563 on dry tea) and in dark tea (192ndash473 ondry tea)

32 Oxygen Radical Absorbance Capacity (ORAC) in FourTypes of Tea A straight calibration curve was obtained fortrolox antioxidant activity in the concentration range of 125ndash80 120583M 119910 = 036119909 minus 056 where 119909 is concentration oftrolox and 119910 is Net AUC (1198772 = 09983) Figure 4 shows theORAC values of the selected tea samples the ORAC value

6 The Scientific World Journal

Table2Th

econ

tent

oftotalp

olypheno

lstotalcatechinsand

individu

alcatechinsin20

samples

from

four

typeso

ftea

(119899=3)

Samples

oftea

EGC(

)Ca

techin

()

EC(

)EG

CG(

)EC

G(

)Non

esterifi

edcatechins()

Esterifi

edcatechins()

Totalcatechins

()

Total

polyph

enols()

Green

tea

Biluochun

113plusmn006

092plusmn004

184plusmn003

1439plusmn041

599plusmn002

389plusmn013

2038plusmn043

2427plusmn056

3236plusmn093

Huang

shan

mao

feng

094plusmn003

085plusmn004

183plusmn005

1411plusmn047

561plusmn004

362plusmn012

1972plusmn051

2334plusmn063

2686plusmn049

Long

jing

062plusmn002

055plusmn005

108plusmn001

813plusmn039

361plusmn011

225plusmn008

1174plusmn050

1399plusmn058

1817plusmn071

Luan

guap

ian

037plusmn001

027plusmn001

061plusmn003

525plusmn026

185plusmn003

125plusmn005

710plusmn029

835plusmn034

1088plusmn006

Xinyang

mao

jian

096plusmn002

074plusmn003

149plusmn001

1263plusmn023

526plusmn014

319plusmn006

1789plusmn037

2108plusmn043

2694plusmn009

Meanvalue

080plusmn003

067plusmn003

137plusmn003

1090plusmn035

446plusmn007

284plusmn009

1537plusmn042

1821plusmn

051

2304plusmn046

Oolon

gtea

Dah

ongpao

063plusmn002

066plusmn003

074plusmn003

559plusmn022

208plusmn002

203plusmn008

767plusmn024

970plusmn032

1316plusmn009

Rougui

044plusmn002

055plusmn001

052plusmn002

457plusmn009

166plusmn006

151plusmn

005

623plusmn015

774plusmn020

1035plusmn008

Shui

xian

068plusmn001

081plusmn002

090plusmn002

723plusmn031

243plusmn001

239plusmn005

966plusmn032

1205plusmn037

1618plusmn027

Taiw

anwulong

116plusmn002

144plusmn005

153plusmn006

1308plusmn027

417plusmn006

413plusmn013

1725plusmn033

2138plusmn046

2973plusmn004

Tieg

uanyin

068plusmn001

077plusmn002

084plusmn002

686plusmn007

236plusmn001

229plusmn005

922plusmn008

1151plusmn

013

1569plusmn073

Meanvalue

072plusmn002

085plusmn003

091plusmn003

747plusmn019

254plusmn003

247plusmn007

1001plusmn

022

1248plusmn030

1702plusmn024

Blacktea

Diancha

241plusmn009

250plusmn004

043plusmn001

226plusmn003

470plusmn015

534plusmn014

696plusmn018

1230plusmn032

1675plusmn074

Hen

anho

ngcha

158plusmn003

163plusmn006

031plusmn001

155plusmn002

342plusmn001

352plusmn010

497plusmn003

849plusmn013

1153plusmn004

Qim

enho

ngcha

150plusmn003

159plusmn005

028plusmn001

131plusmn

005

338plusmn011

337plusmn009

469plusmn016

806plusmn025

1301plusmn

017

Tanyang

gong

fu17

5plusmn005

197plusmn004

032plusmn001

155plusmn002

419plusmn013

404plusmn010

574plusmn015

978plusmn025

1373plusmn004

Zhengshan

hong

cha

257plusmn009

266plusmn012

045plusmn001

204plusmn007

563plusmn001

568plusmn022

767plusmn008

1335plusmn030

1817plusmn012

Meanvalue

196plusmn006

207plusmn006

036plusmn001

174plusmn004

426plusmn008

439plusmn013

601plusmn012

1040plusmn025

1464plusmn022

Darktea

Baixingfu

cha

022plusmn001

077plusmn001

107plusmn003

036plusmn001

192plusmn003

206plusmn005

228plusmn004

434plusmn009

782plusmn029

Qianliang

028plusmn003

102plusmn004

135plusmn007

050plusmn002

252plusmn005

265plusmn014

302plusmn007

567plusmn021

929plusmn038

Liubaocha

034plusmn001

135plusmn002

187plusmn004

072plusmn002

341plusmn017

356plusmn007

413plusmn019

769plusmn026

1328plusmn060

Qizibing

035plusmn001

148plusmn007

202plusmn007

079plusmn004

395plusmn013

385plusmn015

474plusmn017

859plusmn032

1480plusmn003

Shengpu

rsquoer039plusmn002

197plusmn002

251plusmn009

093plusmn002

473plusmn013

487plusmn013

566plusmn015

1053plusmn028

1629plusmn017

Meanvalue

032plusmn002

132plusmn003

176plusmn006

066plusmn002

331plusmn010

340plusmn011

397plusmn012

736plusmn023

1230plusmn029

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

6 The Scientific World Journal

Table2Th

econ

tent

oftotalp

olypheno

lstotalcatechinsand

individu

alcatechinsin20

samples

from

four

typeso

ftea

(119899=3)

Samples

oftea

EGC(

)Ca

techin

()

EC(

)EG

CG(

)EC

G(

)Non

esterifi

edcatechins()

Esterifi

edcatechins()

Totalcatechins

()

Total

polyph

enols()

Green

tea

Biluochun

113plusmn006

092plusmn004

184plusmn003

1439plusmn041

599plusmn002

389plusmn013

2038plusmn043

2427plusmn056

3236plusmn093

Huang

shan

mao

feng

094plusmn003

085plusmn004

183plusmn005

1411plusmn047

561plusmn004

362plusmn012

1972plusmn051

2334plusmn063

2686plusmn049

Long

jing

062plusmn002

055plusmn005

108plusmn001

813plusmn039

361plusmn011

225plusmn008

1174plusmn050

1399plusmn058

1817plusmn071

Luan

guap

ian

037plusmn001

027plusmn001

061plusmn003

525plusmn026

185plusmn003

125plusmn005

710plusmn029

835plusmn034

1088plusmn006

Xinyang

mao

jian

096plusmn002

074plusmn003

149plusmn001

1263plusmn023

526plusmn014

319plusmn006

1789plusmn037

2108plusmn043

2694plusmn009

Meanvalue

080plusmn003

067plusmn003

137plusmn003

1090plusmn035

446plusmn007

284plusmn009

1537plusmn042

1821plusmn

051

2304plusmn046

Oolon

gtea

Dah

ongpao

063plusmn002

066plusmn003

074plusmn003

559plusmn022

208plusmn002

203plusmn008

767plusmn024

970plusmn032

1316plusmn009

Rougui

044plusmn002

055plusmn001

052plusmn002

457plusmn009

166plusmn006

151plusmn

005

623plusmn015

774plusmn020

1035plusmn008

Shui

xian

068plusmn001

081plusmn002

090plusmn002

723plusmn031

243plusmn001

239plusmn005

966plusmn032

1205plusmn037

1618plusmn027

Taiw

anwulong

116plusmn002

144plusmn005

153plusmn006

1308plusmn027

417plusmn006

413plusmn013

1725plusmn033

2138plusmn046

2973plusmn004

Tieg

uanyin

068plusmn001

077plusmn002

084plusmn002

686plusmn007

236plusmn001

229plusmn005

922plusmn008

1151plusmn

013

1569plusmn073

Meanvalue

072plusmn002

085plusmn003

091plusmn003

747plusmn019

254plusmn003

247plusmn007

1001plusmn

022

1248plusmn030

1702plusmn024

Blacktea

Diancha

241plusmn009

250plusmn004

043plusmn001

226plusmn003

470plusmn015

534plusmn014

696plusmn018

1230plusmn032

1675plusmn074

Hen

anho

ngcha

158plusmn003

163plusmn006

031plusmn001

155plusmn002

342plusmn001

352plusmn010

497plusmn003

849plusmn013

1153plusmn004

Qim

enho

ngcha

150plusmn003

159plusmn005

028plusmn001

131plusmn

005

338plusmn011

337plusmn009

469plusmn016

806plusmn025

1301plusmn

017

Tanyang

gong

fu17

5plusmn005

197plusmn004

032plusmn001

155plusmn002

419plusmn013

404plusmn010

574plusmn015

978plusmn025

1373plusmn004

Zhengshan

hong

cha

257plusmn009

266plusmn012

045plusmn001

204plusmn007

563plusmn001

568plusmn022

767plusmn008

1335plusmn030

1817plusmn012

Meanvalue

196plusmn006

207plusmn006

036plusmn001

174plusmn004

426plusmn008

439plusmn013

601plusmn012

1040plusmn025

1464plusmn022

Darktea

Baixingfu

cha

022plusmn001

077plusmn001

107plusmn003

036plusmn001

192plusmn003

206plusmn005

228plusmn004

434plusmn009

782plusmn029

Qianliang

028plusmn003

102plusmn004

135plusmn007

050plusmn002

252plusmn005

265plusmn014

302plusmn007

567plusmn021

929plusmn038

Liubaocha

034plusmn001

135plusmn002

187plusmn004

072plusmn002

341plusmn017

356plusmn007

413plusmn019

769plusmn026

1328plusmn060

Qizibing

035plusmn001

148plusmn007

202plusmn007

079plusmn004

395plusmn013

385plusmn015

474plusmn017

859plusmn032

1480plusmn003

Shengpu

rsquoer039plusmn002

197plusmn002

251plusmn009

093plusmn002

473plusmn013

487plusmn013

566plusmn015

1053plusmn028

1629plusmn017

Meanvalue

032plusmn002

132plusmn003

176plusmn006

066plusmn002

331plusmn010

340plusmn011

397plusmn012

736plusmn023

1230plusmn029

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World Journal 7

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40Content of total polyphenol ()

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30Catechins content ()

R2 = 077 R2 = 067

ORA

C (120583

mol

TE

g)

ORA

C (120583

mol

TE

g)

Figure 5 Correlation analysis of ORAC values against total polyphenols and catechins in tea

Table 3 Correlation coefficients for correlation of ORAC value andcatechins content in different tea infusions

Catechins content Green tea Oolong tea Black tea Dark teaEGC () 053a 089a 073a 068a

Catechin () 066b 090a 076b 066a

EC () 073c 087a 076b 065a

EGCG () 069d 089a 082c 066a

ECG () 065b 090a 070d 069a

Values marked by the same lowercase superscript letter within the samecolumn are not significantly different (119875 lt 001)

ranged from 90928 to 309251 120583mol TEg Of the selectedteas the antioxidant activity as assessed by theORACmethoddecreased in the following order green tea gt oolong tea gtblack tea gt dark teaHuang shanmao feng (green tea) showedthe strongest antioxidant activity

Figure 5 shows the relationships between theORACvalueand total polyphenols and catechins respectively A positivecorrelation was observed in each case with 1198772 = 067 fortotal polyphenols and1198772 = 077 for catechins In addition thecorrelations between individual catechin content and ORACvalues in 20 sampleswere evaluatedThe correlations betweenORAC value and content of catechin EGC EC EGCGand ECG respectively are shown in Table 3 which shows apositive correlation for each case In green tea the EC contentshowed a relatively higher positive correlation (1198772 = 073)withORACvalues than catechin EGC EGCG and ECG andthe values were significantly different (119875 lt 001) For blacktea the EGCG content showed a relatively higher positivecorrelation (1198772 = 082) with ORAC values than catechinEC EGC andECG and the valueswere significantly different(119875 lt 001) In contrast the correlation coefficients betweenORAC value and content of individual catechin in oolongtea and in dark tea infusions were not significantly different(119875 lt 001)

33 DPPH Radical-Scavenging Activity in Four Types of TeaThe free radical-scavenging activity of the 20 samples of

Table 4 EC50 values (concentration causing 50 inhibition) andanti-DPPH radical efficiency (AE) by tea extract

Sample of teas EC50 (mg teamL) AEGreen tea

Bi luo chun 0148 0830Huang shan mao feng 0142 0848Long jing 0246 0609Lu an gua pian 0203 0693Xin yang mao jian 0166 0780Mean value 0181 0761

Oolong teaDa hong pao 0349 0456Rou gui 0365 0438Shui xian 0290 0538Tai wan wu long 0187 0728Tie guan yin 0299 0525Mean value 0298 0550

Black teaDian cha 0268 0572He nan hong cha 0418 0378Qi men hong cha 0437 0360Tan yang gong fu 0339 0470Zheng shan hong cha 0247 0606Mean value 0342 0489

Dark teaBai xing fu cha 0764 0117Qian liang 0634 0199Liu bao cha 0459 0339Qi zi bing 0372 0430Sheng pursquoer 0359 0444Mean value 0518 0324

the four types of tea was assessed by DPPH assay theresults are shown in Figure 6 All sample extracts showedgood inhibitory activity against the DPPH radical in a dose-dependentmannerTheEC50 andAE values of the tea sampleextracts are summarized in Table 4 where lower EC50 val-ues or higher AE values suggest higher antioxidant activityFor the 20 selected tea samples EC50 was in the range

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

8 The Scientific World Journal

Bi luo chunHuang shan mao fengLong jing

Lu an gua pianXin yang mao jian

Concentration of extract (120583g teamL)

00

200

400

600

800

1000

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(a)

Da hong paoRou guiShui xian

Tai wan wu longTie guan yin

Concentration of extract (120583g teamL)

00

200

400

600

800

00 1000 2000 3000 4000 5000

Inhi

bitio

n (

)

(b)

Dian chaHe nan hong chaQi men hong cha

Tan yang gong fuZheng shan hong cha

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

(c)

Bai xing fu chaQian liangLiu bao cha

Qi zi bing

Concentration of extract (120583g teamL)

00

200

400

600

800

00 2000 4000 6000 8000 10000

Inhi

bitio

n (

)

Sheng pursquoer

(d)

Figure 6 Percentage inhibition of DPPH radical by tea extract for green teas (a) oolong teas (b) black teas (c) and dark teas (d)

0142ndash0764mg teamL and the AE values ranged from0117 to 0848 Generally DPPH radical-scavenging efficiencydecreased in the following order green tea gt oolong tea gtblack tea gt dark teaThe scavenging efficiency ofHuang shanmao feng (green tea) against theDPPH radical was the strong-est its EC50 and AE values were 0142mg teamL and 0848respectively

Figure 7 shows the respective correlations between the AEvalues and the content of total catechins and total polyphe-nols In each case good correlation was observed (1198772 = 087for total catechins 1198772 = 077 for total polyphenols)

The correlations between AE values and the content ofindividual catechins in the 20 tea infusions are summarized inTable 5 significant positive correlation was observed in each

case Among the individual catechins in green tea the EGCGcontent showed a relatively higher positive correlation (1198772 =086) with AE values than those of catechin EC EGC andECG and the values were significantly different (119875 lt 001)

34 Online HPLC DPPH Radical-Scavenging Activity of TeaSamples In the tests of antioxidant capacity in the 20 samplesof tea as measured by the DPPH method it was not clearwhich component played the important role in antioxidantactivity The recent development of the online HPLC DPPHmethodwas used to our advantage in this studyThismethodwhich is now used widely in the testing of food and agricul-tural products and in the pharmaceutical industry [35ndash37]allows the radical-scavenging activity of a single substance

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World Journal 9

012345678

0 10 20 30 40

Ant

i-DPP

H effi

cien

cy

Ant

i-DPP

H effi

cien

cy

Content of total polyphenol ()

012345678

0 10 20 30Catechins content ()

R2 = 087 R2 = 077

Figure 7 Correlation analysis of anti-DPPH efficiency (AE) values versus total polyphenols and catechins in tea

1

35

4

67

2

(V)

(V)

06050504030201

minus022

minus024

minus026

minus028

minus030

minus032

(V)

(V)

0706050504030201

07

minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(a)

13

5

4 6

7

2

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

026022018014010006002

(V)

(V)

minus002minus022

minus024

minus026

minus028

minus030

minus032

0 5 10 15 20 25 30

(min)

(b)

Figure 8 Chromatograms at 278 nm (upper traces) and online DPPH quenching profiles (lower traces) for mixed catechin standards (a) anda tea sample ((b) Bi luo chun) 1 gallic acid 2 EGC 3 catechin 4 caffeine 5 EC 6 EGCG 7 ECG

Table 5 Correlation coefficients between AE and catechins contentin different tea infusions

Catechinscontent Green tea Oolong tea Black tea Dark tea

EGC () 079a 098a 099a 096ab

Catechin () 079a 100bc 100a 094a

EC () 084b 098ab 098ab 096bc

EGCG () 086c 100c 091c 098c

ECG () 079a 100c 098ab 098c

Values marked by the same lowercase superscript letter within same columnare not significantly different (119875 lt 001)

to be measured This allows the calculation of a componentrsquoscontribution to the overall radical-scavenging activity of amixture of antioxidants [37] Thus the radical-scavengingactivities of the major catechins (EGC EC EGCG ECG andcatechin) gallic acid and caffeine were determined by theonline HPLC DPPH screening method

The chromatograms of the mixed catechin standards andthat of a tea sample are shown in Figure 8 illustrating theexcellent baseline separation of EGC EC EGCG ECG

catechin gallic acid and caffeine The areas of the positivepeaks at 278 nm were used for quantitative analysis of ECEGC ECG EGCG gallic acid catechin and caffeine whilethe areas of the negative peaks at 521 nm were used toevaluate the DPPH-scavenging activity The results showedthat while caffeine had almost no DPPH-scavenging activitystrong positive correlations were observed for the contentsof the respective HPLC-separated catechins and for gallicacid (see Figure 9) For the same reaction time (10min) theantioxidant activity as assessed by the online DPPH radical-scavenging method decreased in the following order EGC gtgallic acid gt EGCG gt EC gt ECG gt catechin The contribu-tions of gallic acid and the respective catechins in tea to theDPPH-scavenging activity were calculated based on the arearatios of corresponding negative peaks to the total area of allnegative peaks at 521 nm the results are shown in Figure 10EGCG can be seen as the main contributor to DPPH-scav-enging activity in green and oolong teas while ECG was asignificant contributor in black and dark teas In general theDPPH-scavenging activity can be mainly attributed to theesterified catechins (EGCGor ECG) In addition to gallic acidand catechins other compounds in tea appear to contributeto the DPPH-scavenging activity Based on the results shown

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

10 The Scientific World Journal

05

101520253035404550

000 250 500 750 1000 1250 1500

EGCCatechin

ECEGCG

Are

a of n

egat

ive p

eak

at521

nm

Content of catechins in tea extract (120583gmL)

EGCG y = 2951249x + 1436270

R2 = 100

EC y = 3695605x minus 17628

R2 = 100

EGC y = 4464498x + 34133

R2 = 100

ECG y = 2766780x + 701308

R2 = 099

Catechin y = 2112500x + 115887

R2 = 099

times106

ECG

Figure 9 DPPH-scavenging activities of different catechins

020406080

100

Bi lu

o ch

unH

uang

shan

mao

feng

Long

jing

Lu an

gua

pia

nXi

n ya

ng m

ao ji

an

Da h

ong

pao

Rou

gui

Shui

xia

nTa

i wan

wu

long

Tie g

uan

yin

Dia

n ch

aH

e nan

hon

g ch

aQ

i men

hon

g ch

aTa

n ya

ng g

ong

fuZh

eng

shan

hon

g ch

a

Bai x

ing

fu ch

aQ

ian

liang

Liu

bao

cha

Qi z

i bin

g

Con

trib

utio

n ra

te(

)

Gallic acidCatechinEC

EGCEGCGECG

Green tea Oolong tea Black tea Dark tea

Shen

g pu

rsquoer

Figure 10 Contributions of gallic acid and catechins in tea toDPPH-scavenging activity

in Figure 10 these minor contributions are in the range of182ndash283

4 Conclusions

We conducted a systematic study on the total polyphenoland catechins content as well as antioxidant activity in fourtypes of tea (green dark oolong and black) prepared fromCamellia sinensis in China The highest content of polyphe-nols and total catechins was detected in green tea followedby oolong black and dark tea There was a positive correla-tion between the antioxidant capacity and total polyphenolcontent or catechins content (1198772 = 067ndash087 119875 lt 005) Ingeneral it appears that the longer-fermented teas have lowertotal polyphenol content lower catechins content and lowerantioxidant capacity According to our results green tea in

China is of very high quality in terms of antioxidant capacitywhen compared with the other teas

Results of online HPLC DPPH radical-scavenging activ-ity tests of tea samples showed that gallic acid EGCG ECGEC EGC and catechin were responsible for the antioxidantcapacity of the extracts from tea samples Furthermore theantioxidant capacity of tea extracts was mainly attributedto the esterified catechins (EGCG or ECG) The results alsosuggested that other compounds in tea may also contributeto the antioxidant capacity to a minor degree

While consumers will always rely on personal preferencesrelating to color taste and aroma when selecting tea webelieve that the antioxidant capacity should also be consid-eredThe results of this study should prove useful to any con-sumers who wish to select tea based on antioxidant capacity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the FundamentalResearch Fund for Central Universities (no DL12EA01-04) the National Natural Science Foundation (31200478)China Postdoctoral Science Foundation funded Project(2013M530773) and the Postdoctoral Science-ResearchDevel-opmental Foundation of Heilongjiang Province (no LBH-Q11177)

References

[1] N Xu and Z Chen ldquoGreen tea black tea and semi-fermentedteardquo in TEA Bioactivity and Therapeutic Potential Y S ZhenEd pp 35ndash56 Taylor amp Francis New York NY USA 2002

[2] M K V Carr andW Stephens ldquoClimate weather and the yieldof teardquo inTea Cultivation to Consumption K CWillson andMN Clifford Eds pp 87ndash135 Chapman and Hall London UK1992

[3] V S Lee C R Chen YW Liao J T Tzen andCChang ldquoStruc-tural determination and DPPH radical-scavenging activity oftwo acylated flavonoid tetraglycosides in oolong tea (Camelliasinensis)rdquo Chemical and Pharmaceutical Bulletin vol 56 no 6pp 851ndash853 2008

[4] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

[5] G Z Wang Tea Tea Set and Tea Etiquette of China SciencePress Beijing China 2013

[6] D S Nshimiyimana and Q He ldquoRadical scavenging capacityof Rwandan CTC tea polyphenols extracted using microwaveassisted extractionrdquo Pakistan Journal of Nutrition vol 9 no 6pp 589ndash593 2010

[7] K W Lee H J Lee and C Y Lee ldquoAntioxidant activity of blacktea vs green teardquo Journal of Nutrition vol 132 no 4 pp 785ndash786 2002

[8] H A El-Beshbishy O M Tork M F El-Bab and M A AutifildquoAntioxidant and antiapoptotic effects of green tea polyphenols

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World Journal 11

against azathioprine-induced liver injury in ratsrdquo Pathophysiol-ogy vol 18 no 2 pp 125ndash135 2011

[9] Y Cui Y J Oh J Lim et al ldquoAFM study of the differential inhib-itory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negativebacteriardquo Food Microbiology vol 29 no 1 pp 80ndash87 2012

[10] N C Gordon and D W Wareham ldquoAntimicrobial activity ofthe green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)against clinical isolates of Stenotrophomonas maltophiliardquo Inter-national Journal of Antimicrobial Agents vol 36 no 2 pp 129ndash131 2010

[11] R K Dhiman ldquoThe green tea polyphenol epigallocatechin-3-gallate (EGCG)mdashone step forward in antiviral therapy againsthepatitis C Virusrdquo Journal of Clinical amp Experimental Hepatol-ogy vol 1 no 3 pp 159ndash160 2011

[12] M Nakayama K Suzuki M Toda S Okubo Y Hara and TShimamura ldquoInhibition of the infectivity of influenza virus bytea polyphenolsrdquo Antiviral Research vol 21 no 4 pp 289ndash2991993

[13] M Yokoyama M Noguchi Y Nakao M Ysunaga F Yamasakiand T Iwasaka ldquoAntiproliferative effects of the major tea poly-phenol (minus)-epigallocatechin gallate and retinoic acid in cervicaladenocarcinomardquoGynecologicOncology vol 108 no 2 pp 326ndash331 2008

[14] J D Lambert and R J Elias ldquoThe antioxidant and pro-oxidantactivities of green tea polyphenols a role in cancer preventionrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 65ndash72 2010

[15] R S Murugan R V Priyadarsini K Ramalingam Y Hara DKarunagaran and S Nagini ldquoIntrinsic apoptosis and NF-120581Bsignaling are potential molecular targets for chemopreventionby black tea polyphenols in HepG2 cells in vitro and in arat hepatocarcinogenesis model in vivordquo Food and ChemicalToxicology vol 48 no 11 pp 3281ndash3287 2010

[16] Y Sagara Y Miyata K Nomata T Hayashi and H KanetakeldquoGreen tea polyphenol suppresses tumor invasion and angi-ogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-inducedbladder cancerrdquo Cancer Epidemiology vol 34 no 3 pp 350ndash354 2010

[17] M C Sabu K Smitha and R Kuttan ldquoAnti-diabetic activityof green tea polyphenols and their role in reducing oxidativestress in experimental diabetesrdquo Journal of Ethnopharmacologyvol 83 no 1-2 pp 109ndash116 2002

[18] S Uchiyama Y Taniguchi A Saka A Yoshida and H YajimaldquoPrevention of diet-induced obesity by dietary black tea poly-phenols extract in vitro and in vivordquoNutrition vol 27 no 3 pp287ndash292 2011

[19] L A Beltz D K Bayer J Jansen A L Moss M M Kruthand T A Steele ldquoTea polyphenols and H

2O2effect growth and

cytokine production by normal and leukemic human leuko-cytesrdquo Free Radical Biology and Medicine vol 51 p S83 2011

[20] F Li H Ji and L Shen ldquoAmeta-analysis of tea drinking and riskof parkinsonrsquos diseaserdquo The Scientific World Journal vol 2012Article ID 923464 6 pages 2012

[21] C Lu and L S Hwang ldquoPolyphenol contents of Pu-Erh teas andtheir abilities to inhibit cholesterol biosynthesis in Hep G2 celllinerdquo Food Chemistry vol 111 no 1 pp 67ndash71 2008

[22] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[23] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashPart 1

Content of total polyphenols in teamdashColorimetric methodusing Folin-Ciocalteu reagentrdquo Ref No ISO 14502-12005Cor12006(E) 2006

[24] G Rusak D Komes S Likic D Horzic and M Kovac ldquoPhe-nolic content and antioxidative capacity of green and white teaextracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[25] C Anesini G E Ferraro and R Filip ldquoTotal polyphenolcontent and antioxidant capacity of commercially available tea(Camellia sinensis) in Argentinardquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 9225ndash9229 2008

[26] A K Atoui A Mansouri G Boskou and P Kefalas ldquoTea andherbal infusions their antioxidant activity and phenolic profilerdquoFood Chemistry vol 89 no 1 pp 27ndash36 2005

[27] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins in green teamdashmethod using high-performance liquid chromatographyrdquo ISO 14502-22005Cor12006(E) 2006

[28] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[29] M K Roy M Koide T P Rao T Okubo Y Ogasawara and LR Juneja ldquoORAC and DPPH assay comparison to assess anti-oxidant capacity of tea infusions relationship between total pol-yphenol and individual catechin contentrdquo International Journalof Food Sciences and Nutrition vol 61 no 2 pp 109ndash124 2010

[30] G DuM Li F Ma andD Liang ldquoAntioxidant capacity and therelationshipwith polyphenol andVitaminC inActinidia fruitsrdquoFood Chemistry vol 113 no 2 pp 557ndash562 2009

[31] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal of Agri-cultural and FoodChemistry vol 49 no 10 pp 4619ndash4626 2001

[32] B Tadolini C Juliano L Piu F Franconi and L Cabrini ldquoRes-veratrol inhibition of lipid peroxidationrdquo Free Radical Researchvol 33 no 1 pp 105ndash114 2000

[33] N Chaieb J L Gonzalez M Lopez-Mesas M Bouslama andM Valiente ldquoPolyphenols content and antioxidant capacityof thirteen faba bean (Vicia faba L) genotypes cultivated inTunisiardquo Food Research International vol 44 no 4 pp 970ndash977 2011

[34] S Fujisawa M Ishihara Y Murakami T Atsumi Y Kadomaand I Yokoe ldquoPredicting the biological activities of 2-methox-yphenol antioxidants effects of dimersrdquo In Vivo vol 21 no 2pp 181ndash188 2007

[35] J H Wu C Y Huang Y T Tung and S T Chang ldquoOnline RP-HPLC-DPPH screening method for detection of radical-scav-enging phytochemicals from flowers ofAcacia confusardquo Journalof Agricultural and Food Chemistry vol 56 no 2 pp 328ndash3322008

[36] N Nuengchamnong and K Ingkaninan ldquoOn-line HPLC-MS-DPPHassay for the analysis of phenolic antioxidant compoundsin fruit wine antidesma thwaitesianumMuellrdquo Food Chemistryvol 118 no 1 pp 147ndash152 2010

[37] D Bandoniene and M Murkovic ldquoOn-line HPLC-DPPHscreening method for evaluation of radical scavenging phenolsextracted from apples (Malus domestica L)rdquo Journal of Agricul-tural and Food Chemistry vol 50 no 9 pp 2482ndash2487 2002

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014