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8102019 1-s20-S0963996913001890-main

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Evaluation of antioxidant activity of ten compounds in different tea samples bymeans of an on-line HPLCndashDPPH assay

Yuanting Zhang Qing Li Hang Xing Xuefeng Lu Longshan Zhao Kankan Qu Kaishun Bi

School of Pharmacy Shenyang Pharmaceutical University 103 Wenhua Road Shenyang 110016 PR China

National and Local Joint Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines PR China

a b s t r a c ta r t i c l e i n f o

Article history

Received 18 November 2012Received in revised form 5 March 2013

Accepted 9 March 2013

Keywords

Tea

Antioxidant activity

Free radical scavenging activity

On-line HPLCndashDPPH

Tea (Camellia sinensis) a well-known traditional beverage in China has drawn growing attention due to its var-

ious bene1047297ts to health In this study an on-line assay of coupling high performance liquid chromatography sep-

aration with 11-diphenyl-2-picrylhydrazyl free radical reaction system (HPLCndashDPPH) was applied to evaluate

the antioxidant activity of different teas Twelve main chromatographic peaks were detected in tea and

they were identi1047297ed as gallic acid 3-galloyl-quinic acid theobromine (minus)-gallocatechin (minus)-epigallocatechin

caffeine procyanidin dimer (minus)-epicatechin (minus)-epigallocatechin gallate (minus)-gallocatechin gallate

126-tri-galloyl-glucose and (minus)-epicatechin gallate by comparing their retention times and DAD spectra with

standard compounds respectively or with reference to our previous study DPPH assay showed that ten out of

twelve investigatedcompounds have free radical scavenging activity and their contents were determined or es-

timated Trolox equivalent antioxidant capacities (TEACs) of the ten active components were also determined

EGCGwas themost potent antioxidantwith a TEAC value of 5822 Catechin components were themajor contrib-

utors to the antioxidant activity of tea they decreased during tea fermentation and led to the reduction of anti-

oxidant activity Different tea samples were scienti1047297cally classi1047297ed and their antioxidant activities were

comprehensively evaluated by on-line HPLCndashDPPH assays coupled with principal component analysis and hier-

archicalclustering analysis The resultsindicated thatthe combinationof the on-lineHPLCndashDPPH assay principal

component analysis and hierarchical clustering analysis could be suitable for the bioactivity assessment and va-

riety characterization of tea and its derived products Additionally a simple and rapid method for simultaneouscapture of chemical quantitative analysis and bioactivity evaluation by determining contents of the bioactive

compounds was 1047297rstly established This method could provide a comprehensive evaluation of tea

copy 2013 Published by Elsevier Ltd

1 Introduction

Tea derived from dry leavesof Camelliasinensis (L) O Kuntze is one

of the most widely consumed beverage all over the world In recent

years tea has attracted more attention due to its bene1047297cial health ef-

fects and special 1047298avor and taste Tea contains various groups of

compounds such as polyphenols alkaloids free amino acids proteins

and vitamins Among these chemicals polyphenols are believed to

be the major bioactive components (Kim Goodner Park Choi amp

Talcott 2011 Manian Anusuya Siddhuraju amp Manian 2008) The bio-

activity of polyphenols is mainly represented in terms of antioxidant

activity and free radical scavenging activity (Unachukwu Ahmed

Kavalier Lyles amp Kennelly 2010) The major polyphenols are (minus)-

epigallocatechin gallate (EGCG) (minus)-epigallocatechin (EGC) (minus)-

epicatechin gallate (ECG)(minus)-epicatechin (EC) (minus)-gallocatechin gal-

late (GCG) and (minus)-gallocatechin (GC) The previous pharmacological

studies have demonstrated that tea polyphenols have antitumor

(Lambert amp Yang 2003 Zhang et al 2009) antioxidant (Benzie amp

Szeto 1999 Guo et al 1999) antiarteriosclerotic (Kakuda 2002) anti-

microbial (Bancirova 2010) antimutagenic (Ioannides amp Yoxall 2003)

antiobesity (Kuo et al 2005) anti-in1047298uenza (Furuta Hirooka Abe

Sugata et al 2007) and antidiabetic (Sabu Smitha amp Kuttan 2002) ac-

tivities Actually the health bene1047297ts associated with tea consumption

have been attributed in part to the antioxidantand free radical scaveng-

ing activity of the most abundant tea polyphenols (Chemoprevention

Branch amp Agent Development Committee 1996) Therefore investiga-

tion into antioxidant activity of tea and their polyphenols is very impor-

tant for its quality control Moreover tea is generally consumed in the

form of green black oolong and white tea (Pettigrew 2004) The

processing methods for production of each typeof tea are greatly differ-

ent Different types of tea are categorized based on fermentation degree

(oxidation degree of polyphenols in fresh tea leaves) fermentation de-

gree of green tea is 0 (unfermented) fermentation degree of black tea is

100 (full fermentation) and fermentation degrees of white tea and

oolong tea are about 20ndash30 and 30ndash60 respectively (Engelhardt

2010 Harbowy amp Balentine 1997) Consequently the antioxidant ac-

tivities among them are dissimilar

Food Research International 53 (2013) 847ndash856

Corresponding author Tel +86 24 23986012 fax +86 24 23986259

E-mail address kaishunbisyphugmailcom (K Bi)

0963-9969$ ndash see front matter copy 2013 Published by Elsevier Ltd

httpdxdoiorg101016jfoodres201303026

Contents lists available at ScienceDirect

Food Research International

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e f o o d r e s

8102019 1-s20-S0963996913001890-main


In order to discover natural antioxidants from various tea prod-

ucts the development of qualitative and quantitative analytical tech-

niques is a major task Therefore during the last decade several

analytical methods have been developed for the antioxidant capacity

determination of plants and plant extracts (Magalhatildees Segundo Reis

amp Lima 2008 Sanchez-Moreno 2002 Škrovaacutenkovaacute Mišurcovaacute amp

Machů 2012) most of which are based on the ability of an antioxi-

dant to quench free radicals by hydrogen donation Stable free radical

species such as 22prime

-azinobis (3-ethylbenzthiazoline-6-sulfonic acid)(ABTS+) and 11-diphenyl-2-picrylhydrazyl (DPPHbull) are often used

for the evaluation of free radical scavenging capacity of various anti-

oxidants (Antolovich Prenzler Patsalides McDonald amp Robards

2002) Nonetheless these methods could not determine the bioactive

components in a complex mixture such as plant materials Usually a

conventional procedure for screening and identi1047297cation of free radical

scavengers in herbal extracts is extracting isolating and obtaining the

pure chemical compounds and then evaluating free radical scaveng-

ing capacity of these pure compounds with the guidance of bioassay

The conventional methods used for extraction of compounds from

herbal plants are distillation and solvent extraction and the struc-

tures of the isolated compounds are identi1047297ed by mass spectrometry

and nuclear magnetic resonance spectroscopy which are laborious

and time-consuming (Dong Ye Lu Zheng amp Liang 2011 Yang et

al 2009) Furthermore there is often a loss of antioxidant activity

during the isolation and puri1047297cation process due to the nature of

the extraction solvent and long extraction process (Rodriacuteguez-Rojo

Visentin Maestri amp Cocero 2012) To avoid these problems a method

combining separation and activity evaluation would present a prom-

inent advantage for such investigation Recently rapid and sensitive

on-line HPLC methods (on-line HPLCndashABTS assay on-line HPLCndash

DPPH assay on-line HPLCndashbiochemical detection etc) for analyzing

the bioactivity of individual compounds have been developed

(Ingkaninan de Best van der Heijden Hofte et al 2000 Koleva

Niederlaumlnder amp Van Beek 2000 Niederlaumlnder Van Beek Bartasiute

amp Koleva 2008 Schenk et al 2003 Wu Huang Tung amp Chang

2008) Antioxidant activity determination of on-line HPLCndashDPPH assay

was based on the decrease in absorbance at 517 nm after post-column

reactionof antioxidantsseparated from HPLCwith DPPHbull

and the antiox-idants present in a sample would be easily indicated by negative peaks

(Koleva et al 2000) Moreover free radical scavenging activity was eval-

uated by comparison to the water-soluble synthetic vitamin E derivative

6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) The

on-line HPLCndashDPPH method permits not only rapid selective and rela-

tively simple detection of free radical scavengers but also quantitative

analysis of individual antioxidants in complex mixtures (Niederlaumlnder

et al 2008) Consequently this method is focused on the analyses of

free radical scavenging activities of complex mixtures or matrixes espe-

cially the plant extracts (Nuengchamnong amp Ingkaninan 2010 Wu et

al 2008 Zhang Ding Qi amp Yu 2012)

The purposes of the current study were to evaluate the antioxi-

dant activity of different tea distinguish different tea samples based

on the antioxidant activity 1047297nd out the bioactive compounds whichcontributed to the antioxidant activity of tea and investigate their

contributions by the combination of on-line HPLCndashDPPH method

principal component analysis and hierarchical clustering analysis

2 Materials and methods

21 Chemicals and materials

Sixteen tea samples were collected and summarized in Table 1 The

samples were authenticated by Ying Jia of the Department of Pharma-

cognosy at Shenyang Pharmaceutical University according to the

morphological characteristics of tea Gallic acid theobromine (minus)-

gallocatechin (GC) (minus)-epigallocatechin (EGC) caffeine epicatechin

(EC) (minus

)-epigallocatechin gallate (EGCG) (minus

)-gallocatechin gallate

(GCG) and (minus)-epicatechin gallate (ECG) were purchased from the

National Institutes for Food and Drug Control (Beijing China) HPLC

grade acetonitrile was purchased from Fisher Scienti1047297c (Pittsburgh PA

USA) and HPLC grade methanol was purchased from Yu-wang Chemical

Factory (Shandong China) HPLC grade phosphoric acid was pur-

chased from Beijing Reagent Company (Beijing China) 11-diphenyl-

2-picrylhydrazyl (DPPH) and 6-hydroxy-2578-tetramethylchroman-2-

carboxilic acid (Trolox) were purchased from Sigma-Aldrich (ShanghaiChina) Distilled water prepared from de-mineralized water was used

throughout the experiment

22 Preparation of sample solutions and standard solutions

The tea samples were milled to reach homogeneous particle size

01 g of powder was used to prepare an infusion with 10 mL of puri-1047297ed water at 100 degC twice at 10 min each After that the extracts of

the two times were mixed together and transferred into a 25 mL vol-

umetric 1047298ask and1047297ltered through a 045 μ m membrane prior to an in-

jection into the HPLC system The reference standards of the seven

analytes ie gallic acid GC EGC EC EGCG GCG and ECG were

dissolved in water at 3400 μ gmL 2883 μ gmL 1200 μ gmL

4020 μ gmL 3330 μ gmL 2700 μ gmL and 998 μ gmL respectivelyThe stock standard solution was stored at 4 degC before analysis

23 Preparation of DPPH solution and Trolox solution

The DPPH radical stock solution was prepared in methanol

(6 times 10minus5 molL) immediately before the experiments and kept in

a lightproof container Trolox was dissolved in methanol to a concen-

tration of 4 mmolL and then diluted to appropriate concentration

for the establishment of standard calibration curve

24 HPLC ndashDAD analysis

Analysis of the compounds in tea was performed on a Shimadzu

(Kyoto Japan) HPLC system equipped with a diode array detector

Table 1

Different cultivated regions and DPPH radical scavenging capacities of tea samples

Sample Types and

sub-types

R egions ( mM T roloxg

dry weight)

IC50 (μ gmL)

Green tea

S1 Longjing Zhejiang 2451 plusmn 45a 2513 plusmn 030a

S2 Yunfeng Zhejiang 1419 plusmn 24b 3284 plusmn 041b

S3 Qiandaoyinzhen Zhejiang 2270 plusmn 42c 2715 plusmn 013c

S4 Biluochun Jiangsu 2272 plusmn 40c 2718 plusmn 005c

S5 Rizhaolvcha Shandong 1893 plusmn 37d

2971 plusmn 042d

S6 Xinyangmaojian Henan 2550 plusmn 48e 2458 plusmn 029e

Oolong tea

S7 Tieguanyin Fujian 1419 plusmn 21b 3395 plusmn 042f

S8 Guihuawulong Taiwan 1384 plusmn 17f 3431 plusmn 041f

S9 Alishanwulong Taiwan 1353 plusmn 20g 3572 plusmn 043g

S10 Gaoshanwulong Taiwan 1359 plusmn 25g 3580 plusmn 047g

S11 Milanxiang Guangdong 1321 plusmn 20h 3517 plusmn 038h

S12 Dahongpao Fujian 1396 plusmn 22b 3538 plusmn 051gh

White tea

S13 Xingongyibaicha Fujian 1403 plusmn 16b 3292 plusmn 061b

S14 Shoumei Fujian 1387 plusmn 18f 3308 plusmn 049b

S15 Baimudan Fujian 1472 plusmn 15i 3279 plusmn 059b

S16 Gongmei Fujian 1378 plusmn 13fg 3303 plusmn 046b

Mean of green tea 2143 plusmn 419

A

2777 plusmn 308

A

Mean of oolong tea 1372 plusmn 35B 3506 plusmn 076B

Mean of white tea 1410 plusmn 43C 3296 plusmn 013C

Data are expressed as mean plusmn SD (n = 3) In each column different superscript small

letters indicate signi1047297cant differences ( p b 005) among different tea samples and

different superscript capital letters indicate signi1047297cant differences ( p b 005) among

different tea types

848 Y Zhang et al Food Research International 53 (2013) 847 ndash856

8102019 1-s20-S0963996913001890-main


(DAD) and an LC-10A pump The chromatographic separation was

carried out on a Kromasil C18 column (250 mm times 46 mm 5 μ m

Zhonghuida Co Ltd China) maintained at 35 degC The UV absorbance

was monitored at 210 nm The mobile phase was acetonitrile (A) and

005 phosphoric acid aqueous solution (B) with a gradient program

as follows 0ndash8 min linear gradient 5ndash8 A 8ndash28 min linear gradi-

ent 8ndash10 A 28ndash40 min 10 A isocratic elution 40ndash55 min linear

gradient 10ndash14 A 55ndash80 min linear gradient 14ndash24 A and

80ndash

90 min 24 A isocratic elution at a 1047298

ow rate of 1 mLmin All in- jection volumes of samples and standard solutions were 20 μ L

The calibration curves and linear equations of peak area concen-

tration ratios were determined using the aforementioned HPLC

program conditions for the seven standard analytes The limit of de-

tection (LOD) and the limit of quanti1047297cation (LOQ) were determined

at a signal-to-noise ratio (SN) of about 3 and 10 respectively by a fur-

ther diluting of the standard solutions In addition precision repeat-

ability stability for 12 h and recovery were all carried out to validate

the HPLC method

25 Off-line spectrophotometric DPPH assay

The antioxidant capacities of sixteen tea samples were evaluated by

off-line DPPH radical scavenging assay The method is based on the re-

duction of the relatively stable radical DPPH to the formation of a non

radical form in the presence of hydrogen donating antioxidant The

tea samples showed antioxidant activity by the reduction of purple

colored DPPH to the yellow colored diphenylpicrylhydrazine deriva-

tives DPPH radical scavenging capacity was estimated according to

Brand-Williams Cuvelier and Berset (1995) and Shyu and Hwang

(2002) with slight modi1047297cations In the assay 1 mL of diluted extract

was mixed with 2 mL of 01 mmolL solution of DPPH in methanol

The mixture was incubated in the dark at room temperature for

30 min and the absorbance at 517 nm was measured All tests were

performed in triplicate The scavenging capacity was calculated as

(1 minus AsAc) times 100 where Ac is the absorbance of the control and As

is the absorbance of the tested sample after 30 min Trolox was used

as standard Free radical scavenging capacities of tea samples were

expressed as mM Trolox equivalent and IC50 values (concentration of samples required to scavenge 50 of DPPH radicals)

26 On-line HPLC ndashDADndashDPPH assays

On-line HPLCndashDADndashDPPH assays were employed to investigate

and evaluate the free radical scavenging activity of compounds with

antioxidation in tea samples On-line HPLCndashDPPH method combines

a separation technique with fast post-column chemical detection

that can rapidly pinpoint the active compounds in complex mixtures

The separated analytes reacted post-column with the DPPH solution

and compounds with antioxidation would bleach of the latter In

the second high performance liquid chromatography the DPPH radi-

cal solution acted as mobile phase and as a background at 517 nm

The reaction of antioxidants with DPPH radical would reduce the con-centration of DPPH radical and lower the absorbance and then pro-

vide a negative peak on a constant background signal while for

those without antioxidants effects would not change the constant

background signal (Niederlaumlnder et al 2008)

This on-line assay was performed by using the method introduced

by Koleva et al (2000) and Niederlaumlnder et al (2008) with slight

modi1047297cations as shown in Fig 1 The extracts (20 μ L) were injected

into an HPLC system HPLC separation was carried out as described

in the previous section HPLC eluated compounds arrived to a

T-junction where the methanolic DPPH solution with the concentra-

tion of 6 times 10minus5 molL was delivered via another LC pump with a

1047298ow rate of 08 mLmin After the eluates mixed with DPPH solution

in a reaction coil (10 m times 0254 mm id PEEK tubing) the negative

peaks were measured at 517 nm by DAD

27 Quantitative analysis of individual compounds in tea and antioxidant

activity

Gallic acid GC EGC EC EGCG GCG and ECG were all quanti1047297ed by

reference to their individual calibration curves while 3-galloyl-quinic

acid was quanti1047297ed by estimation using gallic acid as standard and

procyanidin dimer and 126-tri-galloyl-glucose were quanti1047297ed by

estimation using (minus)-epicatechin as standard because their standard

references were unavailableAntioxidant activity was evaluated by the areas of negative peaks

at 517 nm and quanti1047297ed by reference to a Trolox standard calibra-

tion curve as an equivalent antioxidant concentration (μ M) The

Trolox equivalent antioxidant capacity (TEAC) was de1047297ned as the

concentration of Trolox (mM) having the same activity as 1 mM of

the test compound All measurements were performed in triplicate

The theoretical Trolox equivalent concentration was calculated

according to Karaman Tuumltem Başkan and Apak (2010) with slight

modi1047297cations In this study the theoretical Trolox equivalent concen-

tration was calculated by multiplying the content of the investigated

compound with its TEAC value Theoretical Trolox equivalent concen-

tration (μ M) = content (μ M) times TEAC

28 Statistical data analysis

Principal component analysis (PCA) was used for separating inter-

relationships into statistically independent this analysis was useful in

regression analysis to mitigate the problem of multi-collinearity and

to explore the relations among the independent variables which

allowed the identi1047297cation of the primary predictors with minimal

multicollinearity (Wold 1987) Here the PCA was performed on dif-

ferent tea samples by SIMCA-P + 120 software (Umetrics Sweden)

Hierarchical cluster analysis (HCA) is a multivariate analysis tech-

nique that is used to sort samples into groups (Kannel Lee Kanel amp

Khan 2007) Here different tea samples were analyzed by using SPSS

statistics software (SPSS 170 for Windows SPSS Inc USA) and an

HCA analysis was performed on the results The within-groups linkage

method was applied using cosine as a measure of dissimilarity for the

interval data The rescaled distance cluster reveals the relative distancebetween the combined clusters Analysis of variance (ANOVA) was

performed by SPSS 170 software (SPSS Inc USA)

3 Results and discussion

31 Method validation

The characteristics of the calibration curves including regression

equations correlation coef 1047297cients (r ) and linear ranges as well as

LODs and LOQs were listed in Table 2 All the analytes showed good

linearity (r gt 0999) over the tested concentration ranges The LOD

and LOQ ranged from 006 to 091 μ gmL and 020 to 284 μ gmL re-

spectively The relative standard deviation (RSD) values obtained for

the precision repeatability and stability tests were all less than 25as given in Table 2 which showed that the system was reliable for

chemical analysis of tea samples The recovery method percentage

ranged from 953 to 973 with RSD less than 22 as shown in

Table 2 Considering the results the method was accurate enough

32 Evaluation of total antioxidant capacity of tea extracts by off-line

DPPH assay

DPPH radical scavenging capacities of tea samples were shown in

Table 1 The antioxidant activities of different tea samples were

compared with that of Trolox and were expressed as mM of Trolox

equivalent per g of dry weight The Trolox equivalents among the

different tea samples ranged from 1321 mM Trolox for Milanxiang

(S11) to 2550 mM Trolox for Xinyangmaojian (S6) showing a 19

849Y Zhang et al Food Research International 53 (2013) 847 ndash856

8102019 1-s20-S0963996913001890-main


fold difference in antioxidant activity Green teas possessed signi1047297-

cantly ( p b 005) higher mean Trolox equivalents (2143 mM Trolox)

than white teas (1410 mM Trolox) and oolong teas (1372 mM

Trolox) Meanwhile IC50 values of different tea samples were also de-

termined The lower IC50 value implies the higher antioxidant activi-

ty Green teas (IC50 values in the range of 2458ndash3284 μ gmL) also

exhibited signi1047297cantly ( p b 005) higher antioxidant activity than

white teas (IC50 values in the range of 3279ndash3308 μ gmL) and

oolong teas (IC50 values in the range of 3395ndash3580 μ gmL) (Table 1)

Comparable DPPH results from investigations by Unachukwu et al

(2010) on green teas have mean DPPH IC50 value of 2326 μ gmL slight-

ly lower than that obtained in our investigation however the prepara-

tion methods of tea sample solutions slightly differed Manian et al

(2008) observed mean DPPH IC50 value for green tea extracts of

1950 μ gmL with a different extraction method and solvent These re-

sults indicated that all the tea samples possessed a good free radical

scavenging capacity and the antioxidant capacities of the three types

of tea mentioned above were signi1047297cantlydifferent ( p b 005) General-

ly the processing methodsfor production of thethree types of tea were

different Green tea is an unfermented tea (degree of fermentation is 0)

which is particularly rich in polyphenol compounds Nevertheless

white tea and oolong tea are partially fermented teas their degrees of

fermentation are about 20ndash30 and 30ndash60 respectively the polyphe-nolic compounds contained in them were partially oxidized during fer-

mentation (Harbowy amp Balentine 1997 Hsiao Chen amp Cheng 2010)

Kim et al (2011) and Unachukwu et al (2010) reported that antioxi-

dant activity of tea samples was positively correlated with the amount

of phenolic compounds Therefore green tea revealed higher antioxi-

dant activity than white tea and oolong tea did

33 On-line HPLC ndashDPPH quantitative analysis of individual compounds

in tea and antioxidant activity

The HPLC separated analytes reacted with DPPH radical post-

column and the reduction was detected as a negative peak at

517 nm whereas phenolic compounds in tea samples were detected

at 210 nm In Fig 2 the chromatographic analysis of tea sample

(S5) extract together with the respective chromatogram after the

DPPH reaction depicted as negative peaks were presented The anti-

oxidant components are easily indicated from the induced bleaching

without the need of laborious isolation procedures As a result twelve

chromatographic peaks were detected in tea (Fig 2) and 10 peaks

(1ndash2 4ndash5 7ndash12) were detected as negative using the DPPH assay

which indicated that these components had free radical scavenging

activity Nine of the twelve detected chromatographic peaks were

identi1047297ed as gallic acid (1) theobromine (3) GC (4) EGC (5) caffeine

(6) EC (8) EGCG (9) GCG (10) and ECG (12) by comparing their re-tention times and DAD spectra with standard compounds respective-

ly Another three peaks were identi1047297ed as 3-galloyl-quinic acid (2)

Fig 1 Schematics of the on-line HPLCndashDPPH assay

Table 2

Calibration curves LOD LOQ precision repeatability and recoveries of the developed method

Analyte Regression equation r Linear range (μ gmL) LODa (μ gmL) LOQ b (μ gmL)

Gallic acid y = 1848 times 105 x minus 814 times 104 09992 1700ndash3400 006 020

GC y = 1529 times 105 x minus 1738 times 105 09991 1442ndash2883 014 042

EGC y = 1675 times 105 x minus 1000 times 106 09994 6000ndash1200 038 117

EC y = 1393 times 105 x minus 874 times 104 09998 2010ndash4020 052 153

EGCG y = 1347 times 105 x minus 2000 times 106 09994 1665ndash3330 091 284

GCG y = 1455 times 105 x minus 2165 times 105 09991 1350ndash2700 020 061

ECG y = 1490 times 105

xminus

5604 times 105

09996 4992ndash

998 043 125

Analyte Precision Repeatability Stability Recoverye

RSDc () SEd RSD () SE RSD () SE Mean plusmn SD () SE

Gallic acid 08 001 2 001 19 001 960 plusmn 105 035

GC 1 001 17 002 18 002 953 plusmn 107 036

EGC 12 006 22 014 19 012 963 plusmn 110 037

EC 1 002 19 006 19 006 958 plusmn 207 069

EGCG 14 018 16 037 19 045 965 plusmn 100 034

GCG 11 001 19 003 21 002 973 plusmn 126 042

ECG 13 005 25 015 2 012 972 plusmn 174 058

Probability level 005a LOD limit of detectionb LOQ limit of quanti1047297cationc RSD relative standard deviation where n = 6d SE standard errore

Recovery () = 100 times (amount foundminus

original amount)amount spiked (n = 9)


8102019 1-s20-S0963996913001890-main


procyanidin dimer (7) and 126-tri-galloyl-glucose (11) by reference

to our previous study (Song Li Guan Wang amp Bi 2012) (Fig 3)

Trolox equivalent antioxidant capacities (TEACs) of individual anti-

oxidants were obtained by injecting standard solution of appropriate

concentration into the on-line HPLCndashDPPH system and those of

3-galloyl-quinic acid procyanidin dimer and 126-tri-galloyl-glucose

were got by injecting tea sample (S5) extract into the same system

Trolox equivalent antioxidant capacities of individual antioxidants are

presented in Table 3AsitcanbeseeninTable 3 the TEAC values ranged

from 03210 for EC to 5822 for EGCG re1047298ecting an 18-fold difference in

scavenging DPPH radical capacity among the 10 tested compounds as

presented in Table 3 The antioxidant activity order of individual com-

pounds was EGCG gt procyanidin dimer gt 126-trigalloylglucos gtEGC gt 3-galloyl-quinic acid gt ECG gt GCG gt gallic acid gt GC gt EC

according to theTEAC valuewhichwas analogous to the resultreported

by Nanjo et al (1996) and Unno Yayabe Hayakawa and Tsuge (2002)

EGCG was the most potent antioxidant with TEAC value of 5822

procyanidin dimer also revealed apparent antioxidant activity with

TEAC value of 4937

The different antioxidant capacity exhibited by polyphenolic com-

pounds is consistent with their chemical structure in regard to the

number and position of phenolic hydroxyl groups The results

presented in Table 3 showed that DPPH radical scavenging effects of

procyanidin dimer and 126-tri-galloyl-glucose were quite strong be-

cause of plenty of hydroxyl groups present in them The results also

showed that DPPH radical scavenging effects of EGC and EGCG were

rather stronger or their reaction speeds with DPPH radical werefaster than other catechins Unno et al (2002) also proved that

EGCG and EGC made a particularly high contribution to the total su-

peroxide radical scavenging capacity of green tea extract among all

the catechin constituents It is known that hydroxyl substitution is

necessary for the antioxidant activity of a 1047298avonoid (Rice-Evans

Miller amp Paganga 1996) Cao So1047297c and Prior (1997) proved that in

general compounds with more hydroxyl substitutions on the B ring

might reveal stronger antioxidant activity Consequently the higher

DPPH radical scavenging activity of EGC and EGCG could be attributed

to three hydroxyls on their B ring Noda Kaneyuki Mori and Packer

(2002) and Bansal et al (2011) also proved that hydroxyl groups at

3prime 4prime and 5prime positions in the B ring are contributing to their activity

and adjacent hydroxylation at 3prime and 4prime positions in the B ring are

also important In the present study the DPPH radical scavenging

effects of galloylated catechins (EGCG ECG GCG) were stronger

than those of nongalloylated catechins (EGC EC GC) EGC which

has an ortho-trihydroxyl group in the B ring revealed more effective

radical scavenging effect than EC which has an ortho-dihydroxyl

group in the B ring A similar tendency in the scavenging effects of

EGCG GCG ECG EGC GC and EC on superoxide radical was observed

by Unno et al (2002) and the scavenging effects of EGCG ECG EGC

and EC on peroxyl radical hydroxyl radical and peroxynitrite were

observed by Kang et al (2010) As a consequence it is suggestedthat the attachment of a galloyl ester in the C ring andor the presence

of ortho-trihydroxyl group in the B ring contributes to the strong rad-

ical scavenging activity of the compounds The importance of the

galloyl group andor trihydroxyl group has also been noticed in the

case of superoxide radical scavenging (Guo et al 1999 Unno et al

2002) singlet oxygen scavenging 22prime-azobis (2-amidinopropane)

hydrochloride (AAPH) radical scavenging DPPH radical scavenging

(Guo et al 1999) and the scavenging of ABTS radical cation (Salah

et al 1995) Furthermore the antioxidant activities of catechins

might have a certain relationship with the orientation of substituent

at 3 position in the C ring In the present study the TEAC value of

EGCG was higher than that of GCG ( p b 005) and the TEAC value of

EGC was higher than that of GC ( p b 005) Analogical result was

reported by Unno et al (2002) It was probably because α orientation

of substituent at 3 position in the C ring showed stronger antioxidant

activity than β orientation In other words catechin compounds re-

vealed stronger antioxidant activity because of their many hydroxyl

groups and adjacent hydroxyl groups in the B ring

The quanti1047297cation data of individual antioxidants and contents of

the investigated compounds in tea were presented in Table 4 and

Table 5 respectively which showed that the Trolox equivalent con-

centration and the contents of the investigated compounds varied

greatly in different tea samples and revealed signi1047297cant differences

( p b 005) among green tea (S1ndashS6) oolong tea (S7ndashS12) and white

tea (S13ndashS16) The relative contribution of individual active constitu-

ents to the overall scavenging activity was evaluated which can be

estimated by Trolox equivalent concentration of individual antioxi-

dants Table 4 showed clearly that EGCG was the major contributor

to the free radical scavenging activity in tea which was responsiblefrom 6756 to 7918 of the total antioxidant activity It was probably

because EGCG not only had the highest TEAC value of 5822 but also

possessed the highest concentration from 4658 to 5643 of the total

amount of the tested ten components which were presented in

Table 5 Nanjo Mori Goto and Hara (1999) also found EGCG to be

the most effective scavenger among tea catechins for the superoxide

anion hydroxyl radical and DPPH radical In previous study it was

proved that intensity of antioxidant peaks depend on both types

and amounts of the antioxidant compounds present in the sample

(Nuengchamnong et al 2005) EGC procyanidin dimer and ECG

also contributed greatly to the overall antioxidant capacity of the

tea extracts (from 4000 to 2484 3967 to 7219 and 2009 to

7489 respectively) since both TEAC values and concentrations of

them were relatively high among the tested components exceptEGCG

Furthermore the correlation between off-line and on-line DPPH

assays was investigated The Trolox equivalent concentrations and

DPPH IC50 values of each tea extract obtained from the off-line exper-

iment were compared with the total Trolox equivalent concentrations

of ten free radical scavengers in tea extracts got from the on-line

assay respectively The total Trolox equivalent concentrations got

from the on-line assay were positively correlated (r = 092) with

the Trolox equivalents obtained from the off-line assay (Fig 4A) and

negatively correlated (r = minus0932) with DPPH IC50 values obtained

from the off-line assay (Fig 4B) indicating that the results obtained

from the on-line HPLCndashDPPH assay had a clear correlation with the

antioxidant activity of tea extracts and that these compounds were

the main components responsible for the antioxidant activity of tea

Fig 2 HPLC chromatograms of tea sample (S5) detected at 210 nm (A) and 517 nm

(B negative peaks) Negative peaks indicate antioxidant activity The identi1047297ed peaks in-

clude 1 mdash gallic acid 2 mdash 3-galloyl-quinic acid 3 mdash theobromine 4 mdash GC 5 mdash EGC 6 mdash

caffeine 7 mdash procyanidin dimer8 mdashEC9mdashEGCG 10mdashGCG11ndash126-tri-galloyl-glucose

and 12 mdash

ECG


8102019 1-s20-S0963996913001890-main


extracts The above results suggested that this on-line HPLCndashDPPH

method was a simple and ef 1047297cient tool to pinpoint the antioxidants

in tea and could be used to evaluate the antioxidant activity of tea

and its derived products

34 Results of PCA and HCA analysis

In order to analyze the relationship of the sixteen tea samples PCA

was carried out based on the Trolox equivalent concentrations of the

ten bioactive components In Fig 5A the PCA score plot showed

that all the samples could be divided into three groups ie oolong

tea (S7ndashS12) green tea (S1ndashS6) and white tea (S13ndashS16) Hierarchi-

cal clustering analysis of the sixteen tested samples was also

performed based on the Trolox equivalent concentrations of the ten

bioactive components The results presented in Fig 5B showed clearly

that sixteen tested samples were appropriately divided into two main

clusters The samples of oolong tea (S7ndashS12) were grouped as one

distinct cluster (Cluster I) The samples of green tea and white tea

Fig 3 Chemical structures of the investigated compounds in tea

Table 3

Trolox equivalent antioxidant capacities of individual antioxidants

Analyte Trolox equivalent

concentration (μ M)

Concentration

(μ M)

TEAC1

Gallic acid 980 plusmn 15 7994 plusmn 064 1226 plusmn 0013a

3 -galloyl-quinic a cid 15 59 plusmn 20 95 1 plusmn 08 1 63 9 plusmn 001 8b

GC 4312 plusmn 056 3766 plusmn 038 1145 plusmn 0014c

EGC 3665 plusmn 54 1567 plusmn 19 2338 plusmn 0035d

Procyanid in dimer 25 07 plusmn 38 50 7 8 plusmn 060 4 93 7 plusmn 005 9e

EC 1778 plusmn 029 5540 plusmn 054 03210 plusmn 00042f

EGCG 1692 plusmn 21 2906 plusmn 41 5822 plusmn 0086g

GCG 3253 plusmn 037 2356 plusmn 026 1381 plusmn 0012h

126-tri-galloyl-glucose 1137 plusmn 10 3620 plusmn 051 3141 plusmn 0029i

ECG 1321 plusmn 12 903 plusmn 12 1463 plusmn 0013 j

Data are expressed as mean plusmn SD (n = 3) Different superscript small letters indicate

signi1047297cant differences ( p b 005) among different analytes1 Trolox equivalent antioxidant capacity (TEAC) de1047297ned as the concentration of

Trolox (mM) having the same activity as 1 mM of the test compound


8102019 1-s20-S0963996913001890-main


(S1ndashS6 and S13ndashS16) shared a close similarity and were grouped as

Cluster II however a detail analysis revealed that another two clus-

ters were developed The samples of green tea from S1 to S6 could

be grouped as one cluster (Cluster II-1) and the samples of white

tea could be grouped as another cluster (Cluster II-2) ie S13 to

S16 This grouping was in agreement with the result of PCA

Based on the results above oolong tea green tea and white tea

were divided as Cluster I Cluster II-1 and Cluster II-2 respectively

The data of determination were analyzed by using analysis of vari-ance (ANOVA) followed by an analysis of least signi1047297cant difference

( p b 005) among the means the Trolox equivalent concentrations

of the ten investigated compounds ( p b 005) in these clusters of tea

samples were signi1047297cantly different

The total Trolox equivalent concentrations of free radical scavengers

in these clusters were signi1047297cantly different ( p b 005) 3057ndash3655 μ M

for thesamples of Cluster I (oolong tea) 3975ndash4871 μ M for the samples

of Cluster II-1 (green tea) and 3164ndash3781 μ M for those of Cluster II-2

(white tea) (Table 4) This was in accordance with the result obtained

from the off-line DPPH assay in which the antioxidant capacities of

the three types of tea were also signi1047297cantly different (p b 005) We

have known that green tea isan unfermented tea and is rich inpolyphe-

nol compounds Yen and Chen (1995) found that the higher contentsof

phenolic compounds in green tea might be contributed by the presence

of catechins such as catechin GC GCG EGC ECG and EGCG Kim et al

(2011) reported that four major tea catechins including EGCG EGC

EC and ECG decreased during tea fermentation because galloyl groups

of EGCG andor ECG were cleaved during oxidative fermentation This

was conformable to our results In the present study the Trolox equiv-

alent concentrations and contents of EC EGCG GCG and ECG in oolong

tea and white tea were signi1047297cant lower ( p b 005) than those in green

tea (Tables 4 amp 5) We have proved that white tea and oolong tea re-

vealed lower antioxidant activity than green tea did in the off-line as-says Thus the results above indicated that these catechin components

contributed to the antioxidant activity of tea which was in agreement

with Kim et al (2011) the reduction of these catechins during tea fer-

mentation could lead to thedecreaseof antioxidant activity Other poly-

phenol compounds such as 3-galloyl-quinic acid procyanidin dimmer

and 126-tri-galloyl-glucose also decreased in white tea and oolong

tea showing that they are also responsible for the antioxidant activity

of tea These also suggested that the on-line HPLCndashDPPH assays could1047297nd out the bioactive compounds which contributed to the antioxidant

activity and evaluate their antioxidant activity So based on the above

data analysis we could 1047297nd out the bioactive compounds which con-

tributed to the antioxidant activity of tea distinguish different tea

samples and evaluate their antioxidant activity by the combination of

principal component analysis hierarchical clustering analysis and the

Table 4

Trolox equivalent concentrations (μ M) of individual antioxidants in tea

Sample1 Gallic acid 3-Galloyl-quinic acid GC EGC Procyanidin dimer

S1 6498 plusmn 078a 1952 plusmn 29a 3167 plusmn 053a 2634 plusmn 52a 2695 plusmn 43a

S2 5333 plusmn 060b 1192 plusmn 18b 2024 plusmn 035bc 2287 plusmn 45b 2666 plusmn 41a

S3 6577 plusmn 081a 1575 plusmn 22c 1701 plusmn 028c 1916 plusmn 38c 3060 plusmn 47b

S4 5968 plusmn 073c 1505 plusmn 22c 2449 plusmn 041b 2925 plusmn 57d 2513 plusmn 39a

S5 3150 plusmn 037d 1559 plusmn 21c 3170 plusmn 052a 3742 plusmn 74e 2507 plusmn 40a

S6 4729 plusmn 055e 1904 plusmn 28a 3251 plusmn 054a 3861 plusmn 76e 2643 plusmn 40a

S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

S8 1896 plusmn 022f 7401 plusmn 093e 4150 plusmn 069d 6534 plusmn 130g NDc

S9 1656 plusmn 020f 4422 plusmn 062f 4939 plusmn 083ef 7368 plusmn 142f NDc

S10 1857 plusmn 024f 5985 plusmn 073d 4714 plusmn 080e 7687 plusmn 151fh NDc

S11 3177 plusmn 041d 5614 plusmn 069d 5147 plusmn 087f 5965 plusmn 118g NDc

S12 3302 plusmn 040d 5241 plusmn 058d 5004 plusmn 083f 7165 plusmn 141f NDc

S13 6117 plusmn 076cg 4213 plusmn 056f NDg 1348 plusmn 25i 1891 plusmn 30d

S14 5676 plusmn 065c 2389 plusmn 033g NDg 2130 plusmn 41b 1741 plusmn 27de

S15 7866 plusmn 094h 4197 plusmn 060f NDg 1600 plusmn 30 j 1500 plusmn 23f

S16 7926 plusmn 095h 3780 plusmn 045f NDg 1315 plusmn 23i 1698 plusmn 25e

Mean of S1ndashS6 5376 plusmn 1028A 1615 plusmn 230A 2627 plusmn 668A 2894 plusmn 761A 2681 plusmn 202A

Mean of S7ndashS12 2535 plusmn 808B 5742 plusmn 982B 4719 plusmn 392B 6963 plusmn 619B NDB

Mean of S13ndashS16 6896 plusmn 969C 3645 plusmn 861C NDC 1598 plusmn 357C 1708 plusmn 161C

Sample EC EGCG GCG 126-tri-galloyl-glucose ECG Total

S1 2856 plusmn 034a 3182 plusmn 54a 6707 plusmn 107a 1039 plusmn 21a 2921 plusmn 52a 4498 plusmn 72a

S2 2348 plusmn 028b 2875 plusmn 46b 3983 plusmn 064b 1434 plusmn 29b 2051 plusmn 36b 3975 plusmn 63b

S3 2621 plusmn 030ac

3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c

S4 2303 plusmn 028b 3047 plusmn 50a 4771 plusmn 075c 1304 plusmn 26b 2094 plusmn 36b 4236 plusmn 68c

S5 2595 plusmn 028c 2863 plusmn 44b 3561 plusmn 058d 1137 plusmn 23c 2085 plusmn 37b 4091 plusmn 64b

S6 3296 plusmn 039d 3423 plusmn 57c 6465 plusmn 100a 1180 plusmn 22c 3116 plusmn 55c 4871 plusmn 77d

S7 2127 plusmn 024be 2650 plusmn 43d 2447 plusmn 042e NDd 1183 plusmn 20d 3655 plusmn 57e

S8 2087 plusmn 023e 2195 plusmn 36e 2308 plusmn 037ef NDd 1107 plusmn 19dh 3138 plusmn 46fg

S9 2226 plusmn 025be 2085 plusmn 33e 2317 plusmn 039ef NDd 7992 plusmn 14e 3057 plusmn 48f

S10 2303 plusmn 025b 2091 plusmn 35e 2191 plusmn 035f NDd 6496 plusmn 10f 3095 plusmn 47f

S11 2244 plusmn 026be 2390 plusmn 40f 2289 plusmn 035ef NDd 1375 plusmn 23g 3309 plusmn 51g

S12 2187 plusmn 022be 2155 plusmn 37e 2378 plusmn 039e NDd 1034 plusmn 17h 3156 plusmn 48fg

S13 1231 plusmn 013f 2568 plusmn 43d 3079 plusmn 050g 7335 plusmn 14ef 2519 plusmn 43i 3364 plusmn 53g

S14 1412 plusmn 018f 2350 plusmn 42f 5017 plusmn 079c 6914 plusmn 14eg 2127 plusmn 37b 3164 plusmn 50fg

S15 1330 plusmn 014f 2994 plusmn 48ab 4677 plusmn 076c 7842 plusmn 16f 2183 plusmn 39b 3781 plusmn 61e

S16 1398 plusmn 016f 2538 plusmn 45d 3386 plusmn 053dg 6592 plusmn 13g 2171 plusmn 37b 3287 plusmn 54g

Mean of S1ndashS6 2670 plusmn 367A 3075 plusmn 209A 4908 plusmn 1060A 1180 plusmn 167A 2587 plusmn 324A 4318 plusmn 291A

Mean of S7ndashS12 2196 plusmn 079B 2261 plusmn 201B 2322 plusmn 086B NDB 1025 plusmn 263B 3176 plusmn 203B

Mean of S13ndashS16 1343 plusmn 083C 2613 plusmn 242C 4040 plusmn 851C 7171 plusmn 541C 2250 plusmn 131C 3429 plusmn 229C

ND not detectedData are expressed as mean plusmn SD (n = 3) In each column different superscript small letters indicate signi1047297cant differences ( p b 005) among different tea samples and different

superscript capital letters indicate signi1047297cant differences ( p b 005) among S1ndashS6 S7ndashS12 and S13ndashS161 Sample number as listed in Table 1


8102019 1-s20-S0963996913001890-main


quanti1047297cation of free radical scavenging capacity The present study

provided a scienti1047297c classi1047297cation of different tea varieties which

would become guidance to the evaluation of the bioactivities of tea

PCA and HCA of the sixteen tea samples were also performed

based on the theoretical Trolox equivalent concentrations of the ten

bioactive components In Fig 6A the PCA score plot showed that

all the samples could be divided into three parts ie oolong tea

(S7ndashS12) green tea (S1ndashS6) and white tea (S13ndashS16) That was in

agreement with the result of the above PCA which based on the

Trolox equivalent concentrations In Fig 6B the result of HCA showedthat all the samples could be divided into two clusters ie Cluster I

(S7ndashS12) and Cluster II (S1ndashS6 and S13ndashS16) and Cluster II could

be further divided into Cluster II-1 (S1ndashS6) and Cluster II-2 (S13ndash

S16) This result was identical to the above hierarchical clustering

analysis which is based on the Trolox equivalent concentrations

The data of determination were also analyzed by using analysis of

variance (ANOVA) followed by an analysis of least signi1047297cant differ-

ence ( p b 005) among the means the theoretical Trolox equivalent

concentrations of the ten investigated compounds ( p b 005) in

these clusters of tea samples were signi1047297cantly different

The above results indicated that we could evaluate free radical

scavenging capacity by the theoretical Trolox equivalent concentra-

tion instead of the determination of the Trolox equivalent concentra-

tion The theoretical Trolox equivalent concentration was calculated

as content times TEAC Thus we can simply and rapidly evaluate the

free radical scavenging capacity by determining contents of the inves-

tigated compounds In terms of instrumental setup we can use only

one high performance liquid chromatograph to simultaneously cap-

ture chemical quantitative determination and antioxidant activity

evaluation by determining contents of the bioactive compounds

which can reduce analysis time and materials As described above

the polyphenol was the major bioactive ingredient in antioxidant ac-

tivity and other health bene1047297cial activities its content could affect the

quality and pharmacological properties of tea to a large extent Con-sequently the simultaneous obtainment of chemical quantitative

analysis and bioactivity evaluation by determining contents of the

bioactive compounds in one single run could provide a comprehen-

sive evaluation of tea This method could be applied to routine analy-

sis of tea and its related products

4 Conclusions

In the present study different tea samples were scienti1047297cally clas-

si1047297ed based on their antioxidant activities and their antioxidant activ-

ities were comprehensively evaluated by the combination of principal

component analysis hierarchical clustering analysis and the on-line

HPLCndashDPPH assays The bioactive compounds which contributed

to the antioxidant activity of tea were found out and their Trolox

Table 5

Contents (mgg) of 10 investigated compounds in tea


S1 2281 plusmn 0045a 929 plusmn 017a 2118 plusmn 0036a 903 plusmn 019a 7566 plusmn 0150ab

S2 1871 plusmn 0036b 7437 plusmn 0142b 1219 plusmn 0020a 828 plusmn 019b 7482 plusmn 0150ab

S3 2303 plusmn 0044a 834 plusmn 015c 0937 plusmn 0015a 6019 plusmn 0133c 7916 plusmn 0152b

S4 2115 plusmn 0044c 7838 plusmn 0150b 1663 plusmn 0027a 975 plusmn 021a 7331 plusmn 0148a

S5 1135 plusmn 0022d 819 plusmn 015c 2656 plusmn 0045a 1310 plusmn 029d 7344 plusmn 0148a

S6 1590 plusmn 0029e 1005 plusmn 018d 2197 plusmn 0036a 1285 plusmn 028d 7418 plusmn 0150a

S7 1188 plusmn 0021dg

2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc

S8 04870 plusmn 00096f 2978 plusmn 0054e 2680 plusmn 0046a 1715 plusmn 037g NDc

S9 05140 plusmn 00102f 2662 plusmn 0050f 3126 plusmn 0053a 2429 plusmn 053ef NDc

S10 05440 plusmn 00108f 2781 plusmn 0052f 3023 plusmn 0050a 2539 plusmn 055f NDc

S11 1086 plusmn 0021g 3028 plusmn 0058e 3233 plusmn 0055a 1967 plusmn 042h NDc

S12 1166 plusmn 0023dg 2339 plusmn 0044g 3245 plusmn 0053a 1818 plusmn 041h NDc

S13 3768 plusmn 0075h 2266 plusmn 0041g 06360 plusmn 0011a 889 plusmn 019a 5300 plusmn 0110d

S14 2038 plusmn 0040c 1295 plusmn 0024h 0837 plusmn 0014a 877 plusmn 017a 4962 plusmn 0091e

S15 2617 plusmn 0049i 2040 plusmn 0039g 0926 plusmn 0019a 5725 plusmn 0139c 4383 plusmn 0084e

S16 2686 plusmn 0051i 2098 plusmn 0039g 07150 plusmn 0011a 6483 plusmn 0148i 4933 plusmn 0089e



Mean of S13ndashS16 2778 plusmn 0671C 1924 plusmn 0413C 07825 plusmn 01273C 7468 plusmn 1346C 4884 plusmn 0376C

Sample EC EGCG GCG 126-tri-galloyl-glucose ECG


S2 5082 plusmn 0095bf 6073 plusmn 096b 3223 plusmn 0057b 6564 plusmn 0124b 1646 plusmn 039b

S3 5925 plusmn 0113c 6088 plusmn 095b 3120 plusmn 0058b 4794 plusmn 0088c 2131 plusmn 053a

S4 4980 plusmn 0099b 5985 plusmn 096b 4238 plusmn 0078c 6570 plusmn 0120b 1578 plusmn 038b

S5 5866 plusmn 0112c 5872 plusmn 095bh 3239 plusmn 0057b 5761 plusmn 0112ad 1521 plusmn 037b

S6 813 plusmn 015d 7609 plusmn 122c 5125 plusmn 0056d 5999 plusmn 0113d 2359 plusmn 054a

S7 4808 plusmn 0096b 5045 plusmn 080d 1960 plusmn 0038e NDe 923 plusmn 022c

S8 4266 plusmn 0080e 4841 plusmn 076dg 1676 plusmn 0035f NDe 869 plusmn 017cd

S9 5257 plusmn 0097f 4360 plusmn 069e 1923 plusmn 0037e NDe 6507 plusmn 0142e

S10 5206 plusmn 0103f 3839 plusmn 062f 2017 plusmn 0040e NDe 5060 plusmn 0117f

S11 5072 plusmn 0091bf 4683 plusmn 070g 1786 plusmn 0036f NDe 835 plusmn 016d

S12 4717 plusmn 0094b 4225 plusmn 071e 1885 plusmn 0038e NDe 7908 plusmn 0183g

S13 2692 plusmn 0051g 5126 plusmn 082d 2472 plusmn 0044g 3439 plusmn 0068f 1920 plusmn 043h

S14 3418 plusmn 0069h 5622 plusmn 094bhi 4164 plusmn 0076c 3392 plusmn 0067f 1659 plusmn 035b

S15 3006 plusmn 0055i 5694 plusmn 095bhi 4152 plusmn 0078c 3881 plusmn 0068g 1723 plusmn 039b

S16 3161 plusmn 0054i 5455 plusmn 097i 3642 plusmn 0066h 3339 plusmn 0065f 1849 plusmn 041h


Mean of S7ndashS12 4887 plusmn 0241B 4489 plusmn 421B 1873 plusmn 0124B NDB 7621 plusmn 1526B

Mean of S13ndashS16 3070 plusmn 0303C 5474 plusmn 246C 3598 plusmn 0465C 3501 plusmn 0234C 1767 plusmn 1063A




8102019 1-s20-S0963996913001890-main


equivalent antioxidant capacities (TEACs) and contributions to the

antioxidant activity were investigated We found that catechin com-

ponents especially EGCG contributed greatly to the antioxidant activ-

ity of tea they decreased during tea fermentation and led to the

reduction of antioxidant activity Moreover we established a simple

and rapid method for simultaneous capture of chemical quantitative


bioactive compounds for the 1047297rst time This method could provide a

comprehensive evaluation of tea and its derived products

Acknowledgment

This study was 1047297nancially supported by the Program for Liaoning

Innovative Research Team in University (item number LT2012018

Key Technologies in Quality Control of Traditional Chinese Medicines)

References

Antolovich M Prenzler P D Patsalides E McDonald S amp Robards K (2002)Methods for testing antioxidant activity Analyst 127 183ndash192

Bancirova M (2010) Comparison of the antioxidant capacity and the antimicrobial ac-tivity of black and green tea Food Research International 43 1379ndash1382

Bansal P Paul P Nayak P G Pannakal S T Zou J H Laatsch H et al (2011)Phenolic compounds isolated from Pilea microphylla prevent radiation-inducedcellular DNA damage Acta Pharmaceutica Sinica B 1 226ndash235

Benzie I F amp Szeto Y T (1999) Total antioxidant capacity of teas by the ferric reducingantioxidant power assay Journal of Agricultural and Food Chemistry 47 633ndash636

Brand-Williams W Cuvelier M E amp Berset C (1995) Use of a free radical method toevaluate antioxidant activity Lebensmittel-Wissenschaft und Technologie 28 25ndash30

Cao G So1047297c E amp Prior R L (1997) Antioxidant and prooxidant behavior of 1047298avonoidsStructurendashactivity relationships Free Radical Biology amp Medicine 22 749ndash760

Chemoprevention Branch amp Agent Development Committee (1996) Clinical develop-ment plan Tea extracts green tea polyphenols epigallocatechin gallate Journal of Cellular Biochemistry 63 236ndash257

Dong J J Ye J H Lu J L Zheng X Q amp Liang Y R (2011) Isolation of antioxidantcatechins from green tea and its decaffeination Food and Bioproducts Processing 89 62ndash66

Engelhardt U H (2010) 323 mdash Chemistry of tea Comprehensive Natural Products II 3999ndash1032

Furuta T Hirooka Y Abe A Sugata Y Ueda M Murakami K et al (2007) Concisesynthesis of dideoxyepigallocatechingallate (DO-EGCG) and evaluation of itsanti-in1047298uenza virus activity Bioorganic amp Medicinal Chemistry Letters 17 3095ndash3098

Guo Q Zhao B Shen S Hou J Hu J amp Xin W (1999) ESR study on the structure ndash

antioxidant activity relationship of tea catechins and their epimers Biochimica et Biophysica Acta 1427 13ndash23

Harbowy M E amp Balentine D A (1997) Tea chemistry Critical Reviews in Plant Sceience 16 415ndash430

Hsiao H Y Chen R L C amp Cheng T J (2010) Determination of tea fermentationdegree by a rapid micellar electrokinetic chromatography Food Chemistry 120

632ndash

636

Fig 4 (A) Correlation between the total Trolox equivalent concentration got from

the on-line assay (μ M) and the Trolox equivalent obtained from the off-line assay

(mM Troloxg) (B) Correlation between the total Trolox equivalent concentration

got from the on-line assay (μ M) and DPPH IC50 values obtained from the off-line

assay (mM Troloxg)

Fig 6 (A) PCA score plot and (B) HCA dendrogram of 16 tea samples (S1 to S16 as

shown in Table 1) based on the theoretical Trolox equivalent concentrations of ten

bioactive components


shown in Table 1) based on the Trolox equivalent concentrations of ten bioactive

components


8102019 1-s20-S0963996913001890-main


Ingkaninan K de Best C M van der Heijden R Hofte A J P Karabatak B Irth Het al (2000) High-performance liquid chromatography with on-line coupled UVmass spectrometric and biochemical detection for identi1047297cation of acetylcholines-terase inhibitors from natural products Journal of Chromatography A 872 61ndash73

Ioannides C amp Yoxall V (2003) Antimutagenic activity of tea Role of polyphenolsCurrent Opinion in Clinical Nutrition and Metabolic Care 6 649ndash656

Kakuda T (2002) Neuroprotective effects of the green tea components theanine andcatechins Biological amp Pharmaceutical Bulletin 25 1513ndash1518

Kang K W Oh S J Ryu S Y Song G Y Kim B -H Kang J S et al (2010) Evalu-ation of the total oxy-radical scavenging capacity of catechins isolated fromgreen tea Food Chemistry 121 1089ndash1094

Kannel P R Lee S Kanel S R amp Khan S P (2007) Chemometric application inclassi1047297cation and assessment of monitoring locations of an urban river system Analytica Chimica Acta 582 390ndash399

Karaman Ş Tuumltem E Başkan K S amp Apak R (2010) Comparison of total antioxidantcapacity and phenolic composition of some apple juices with combined HPLCndash

CUPRAC assay Food Chemistry 120 201ndash1209Kim Y Goodner K L Park J -D Choi J amp Talcott S T (2011) Changes in antioxidant

phytochemicals and volatile composition of Camellia sinensis by oxidation duringtea fermentation Food Chemistry 129 1331ndash1342

Koleva I I Niederlaumlnder H A G amp Van Beek T A (2000) An on-line HPLC method fordetection of radical scavengingcompoundsin complexmixtures Analytical Chemistry72 2323ndash2328

Kuo K L Weng M S Chiang C T Tsai Y J Lin-Shiau S Y amp Lin J K (2005)Comparative studies on the hypolipidemic and growth suppressive effects of oolong black pu-erh and green tea leaves in rats Journal of Agricultural andFood Chemistry 53 480ndash489

Lambert J D amp Yang C S (2003) Cancer chemopreventive activity and bioavailabilityoftea andtea polyphenolsMutation Research Fundamentaland Molecular Mechanismsof Mutagenesis 523ndash524 201ndash208

Magalhatildees L M Segundo M A Reis S amp Lima J L (2008) Methodological aspectsabout in vitro evaluation of antioxidant properties Analytica Chimica Acta 613 1ndash19

Manian R Anusuya N Siddhuraju P amp Manian S (2008) The antioxidant activityand free radical scavenging potential of two different solvent extracts of Camelliasinensis (L) O kuntz Ficus bengalensis L and Ficus racemosa L Food Chemistry107 1000ndash1007

Nanjo F Goto K Seto R Suzuki M Sakai M amp Hara Y (1996) Scavenging effects of tea catechins and their derivatives on 11-diphenyl-2-picrylhydrazyl radical FreeRadical Biology amp Medicine 21 895ndash902

Nanjo F Mori M Goto K amp Hara Y (1999) Radical scavenging activity of tea cate-chins and their related compounds Bioscience Biotechnology and Biochemistry63 1621ndash1623

Niederlaumlnder H A G Van Beek T A Bartasiute A amp Koleva I I (2008) Antioxidantactivity assays on-line with liquid chromatography Journal of Chromatography A1210 121ndash134

Noda Y Kaneyuki T Mori A amp Packer L (2002) Antioxidant activities of pomegran-ate fruit extract and its anthocyanidins Delphinidin cyanidin and pelargonidin

Journal of Agricultural and Food Chemistry 50 166ndash171Nuengchamnong N de Jong C F Bruyneel B Niessen W M A Irth H amp

Ingkaninan K (2005) HPLC coupled on-line to ESI-MS and a DPPH-based assayfor the rapid identi1047297cation of anti-oxidants in Butea superba Phytochemical Analysis16 422ndash428

Nuengchamnong N amp IngkaninanK (2010)On-lineHPLCndashMSndashDPPHassayfor theanal-ysis of phenolic antioxidant compounds in fruit wine Antidesma thwaitesianumMuell Food Chemistry 118 147ndash152

Pettigrew J (2004) The tea companion A connoisseurs guide (1st ed) PhiladelphiaPa Running Press Book Publishers 160

Rice-Evans C A Miller N J amp Paganga G (1996) Structurendashantioxidant activity rela-tionships of 1047298avonoids and phenolic acids Free Radical Biology amp Medicine 20933ndash956

Rodriacuteguez-Rojo S Visentin A Maestri D amp Cocero M J (2012) Assisted extractionof rosemary antioxidants with green solvents Journal of Food Engineering 10998ndash103

Sabu M C Smitha K amp Kuttan R (2002) Anti-diabetic activity of green tea polyphe-nols and their role in reducing oxidative stress in experimental diabetes Journal of Ethnopharmacology 83 109ndash116

Salah N Miller N J Paganga G Tijburg L Bolwell G P amp Rice-Evans C (1995) Poly-phenolic 1047298avanols as scavengers of aqueous phase radicals and as chain-breakingantioxidants Archives of Biochemistry and Biophysics 322 339ndash346

Sanchez-Moreno C (2002) Methods used to evaluate the free radical scavengingactivity in foods and biological systems Food Science and Technology International8 121ndash137

Schenk T Appels N M G M van Elswijk D A Irth H Tjaden U R amp van der Greef J (2003) A generic assay for phosphate-consuming or -releasing enzymes coupledon-line to liquid chromatography for lead 1047297nding in natural products AnalyticalBiochemistry 316 118ndash126

Shyu Y S amp Hwang L S (2002) Antioxidantactivity of the crude extract of lignan gly-cosides from unroasted Burma black sesame meal Food Research International 35357ndash365

Škrovaacutenkovaacute S Mišurcovaacute L amp Machů L (2012) Antioxidant activity and protectinghealth effects of common medicinal plants Advances in Food and Nutrition Research67 75ndash139

Song M T Li Q Guan X Y Wang T J amp Bi K S (2012) A novel HPLC method toevaluate the quality and identify the origins of Longjing green tea AnalyticalLetters httpdxdoiorg101080000327192012704532

Unachukwu U J Ahmed S Kavalier A Lyles J T amp Kennelly E J (2010) White andgreen teas (Camellia sinensis var sinensis) variation in phenolic methylxanthineand antioxidant pro1047297les Journal of Food Science 75 C541ndashC548

Unno T Yayabe F Hayakawa T amp Tsuge H (2002) Electron spin resonance spectro-scopic evaluation of scavenging activity of tea catechins on superoxide radicalsgenerated by a phenazine methosulfate and NADH system Food Chemistry 76 259ndash265

Wold S (1987) Principal component analysis Chemometrics and Intelligent LaboratorySystems 2 37ndash52

Wu J H Huang C Y Tung Y T amp Chang S T (2008) Online RP-HPLCndashDPPH screen-ing method for detection of radical-scavenging phytochemicals from 1047298owers of

Acacia confusa Journal of Agricultural and Food Chemistry 56 328ndash332YangZ Tu YBaldermann SDong FXu Y amp WatanabeN (2009) Isolation and iden-

ti1047297cation of compounds from the ethanolic extract of 1047298owers of the tea (Camelliasinensis) plant and their contribution to the antioxidant capacity LWT mdash Food Scienceand Technology 42 1439ndash1443

Yen G C amp Chen H Y (1995) Antioxidantactivity of various tea extracts in relation totheir antimutagenicity Journal of Agriculture and Food Chemistry 47 23ndash32

Zhang L Ding X P Qi J amp Yu B Y (2012) Determination of antioxidant activity of tea by HPLCndashDPPH Journal of China Pharmaceutical University 43 236ndash240

Zhang Y M Han G G Fan B Zhou Y F Zhou X Wei L et al (2009) Green tea(minus)-epigallocatechin-3-gallate down-regulates VASP expression and inhibitsbreast cancer cell migration and invasion by attenuating Rac1 activity European

Journal of Pharmacology 606 172ndash179


8102019 1-s20-S0963996913001890-main


In order to discover natural antioxidants from various tea prod-

ucts the development of qualitative and quantitative analytical tech-

niques is a major task Therefore during the last decade several

analytical methods have been developed for the antioxidant capacity

determination of plants and plant extracts (Magalhatildees Segundo Reis

amp Lima 2008 Sanchez-Moreno 2002 Škrovaacutenkovaacute Mišurcovaacute amp

Machů 2012) most of which are based on the ability of an antioxi-

dant to quench free radicals by hydrogen donation Stable free radical

species such as 22prime

-azinobis (3-ethylbenzthiazoline-6-sulfonic acid)(ABTS+) and 11-diphenyl-2-picrylhydrazyl (DPPHbull) are often used

for the evaluation of free radical scavenging capacity of various anti-

oxidants (Antolovich Prenzler Patsalides McDonald amp Robards

2002) Nonetheless these methods could not determine the bioactive

components in a complex mixture such as plant materials Usually a

conventional procedure for screening and identi1047297cation of free radical

scavengers in herbal extracts is extracting isolating and obtaining the

pure chemical compounds and then evaluating free radical scaveng-

ing capacity of these pure compounds with the guidance of bioassay

The conventional methods used for extraction of compounds from

herbal plants are distillation and solvent extraction and the struc-

tures of the isolated compounds are identi1047297ed by mass spectrometry

and nuclear magnetic resonance spectroscopy which are laborious

and time-consuming (Dong Ye Lu Zheng amp Liang 2011 Yang et

al 2009) Furthermore there is often a loss of antioxidant activity

during the isolation and puri1047297cation process due to the nature of

the extraction solvent and long extraction process (Rodriacuteguez-Rojo

Visentin Maestri amp Cocero 2012) To avoid these problems a method

combining separation and activity evaluation would present a prom-

inent advantage for such investigation Recently rapid and sensitive

on-line HPLC methods (on-line HPLCndashABTS assay on-line HPLCndash

DPPH assay on-line HPLCndashbiochemical detection etc) for analyzing

the bioactivity of individual compounds have been developed

(Ingkaninan de Best van der Heijden Hofte et al 2000 Koleva

Niederlaumlnder amp Van Beek 2000 Niederlaumlnder Van Beek Bartasiute

amp Koleva 2008 Schenk et al 2003 Wu Huang Tung amp Chang

2008) Antioxidant activity determination of on-line HPLCndashDPPH assay

was based on the decrease in absorbance at 517 nm after post-column

reactionof antioxidantsseparated from HPLCwith DPPHbull

and the antiox-idants present in a sample would be easily indicated by negative peaks

(Koleva et al 2000) Moreover free radical scavenging activity was eval-

uated by comparison to the water-soluble synthetic vitamin E derivative

6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) The

on-line HPLCndashDPPH method permits not only rapid selective and rela-

tively simple detection of free radical scavengers but also quantitative

analysis of individual antioxidants in complex mixtures (Niederlaumlnder

et al 2008) Consequently this method is focused on the analyses of

free radical scavenging activities of complex mixtures or matrixes espe-

cially the plant extracts (Nuengchamnong amp Ingkaninan 2010 Wu et

al 2008 Zhang Ding Qi amp Yu 2012)

The purposes of the current study were to evaluate the antioxi-

dant activity of different tea distinguish different tea samples based

on the antioxidant activity 1047297nd out the bioactive compounds whichcontributed to the antioxidant activity of tea and investigate their

contributions by the combination of on-line HPLCndashDPPH method

principal component analysis and hierarchical clustering analysis

2 Materials and methods

21 Chemicals and materials

Sixteen tea samples were collected and summarized in Table 1 The

samples were authenticated by Ying Jia of the Department of Pharma-

cognosy at Shenyang Pharmaceutical University according to the

morphological characteristics of tea Gallic acid theobromine (minus)-

gallocatechin (GC) (minus)-epigallocatechin (EGC) caffeine epicatechin

(EC) (minus

)-epigallocatechin gallate (EGCG) (minus

)-gallocatechin gallate

(GCG) and (minus)-epicatechin gallate (ECG) were purchased from the

National Institutes for Food and Drug Control (Beijing China) HPLC

grade acetonitrile was purchased from Fisher Scienti1047297c (Pittsburgh PA

USA) and HPLC grade methanol was purchased from Yu-wang Chemical

Factory (Shandong China) HPLC grade phosphoric acid was pur-

chased from Beijing Reagent Company (Beijing China) 11-diphenyl-

2-picrylhydrazyl (DPPH) and 6-hydroxy-2578-tetramethylchroman-2-

carboxilic acid (Trolox) were purchased from Sigma-Aldrich (ShanghaiChina) Distilled water prepared from de-mineralized water was used

throughout the experiment

22 Preparation of sample solutions and standard solutions

The tea samples were milled to reach homogeneous particle size

01 g of powder was used to prepare an infusion with 10 mL of puri-1047297ed water at 100 degC twice at 10 min each After that the extracts of

the two times were mixed together and transferred into a 25 mL vol-

umetric 1047298ask and1047297ltered through a 045 μ m membrane prior to an in-

jection into the HPLC system The reference standards of the seven

analytes ie gallic acid GC EGC EC EGCG GCG and ECG were

dissolved in water at 3400 μ gmL 2883 μ gmL 1200 μ gmL

4020 μ gmL 3330 μ gmL 2700 μ gmL and 998 μ gmL respectivelyThe stock standard solution was stored at 4 degC before analysis

23 Preparation of DPPH solution and Trolox solution

The DPPH radical stock solution was prepared in methanol

(6 times 10minus5 molL) immediately before the experiments and kept in

a lightproof container Trolox was dissolved in methanol to a concen-

tration of 4 mmolL and then diluted to appropriate concentration

for the establishment of standard calibration curve

24 HPLC ndashDAD analysis

Analysis of the compounds in tea was performed on a Shimadzu

(Kyoto Japan) HPLC system equipped with a diode array detector

Table 1

Different cultivated regions and DPPH radical scavenging capacities of tea samples

Sample Types and

sub-types

R egions ( mM T roloxg

dry weight)

IC50 (μ gmL)

Green tea

S1 Longjing Zhejiang 2451 plusmn 45a 2513 plusmn 030a

S2 Yunfeng Zhejiang 1419 plusmn 24b 3284 plusmn 041b

S3 Qiandaoyinzhen Zhejiang 2270 plusmn 42c 2715 plusmn 013c

S4 Biluochun Jiangsu 2272 plusmn 40c 2718 plusmn 005c

S5 Rizhaolvcha Shandong 1893 plusmn 37d

2971 plusmn 042d

S6 Xinyangmaojian Henan 2550 plusmn 48e 2458 plusmn 029e

Oolong tea

S7 Tieguanyin Fujian 1419 plusmn 21b 3395 plusmn 042f

S8 Guihuawulong Taiwan 1384 plusmn 17f 3431 plusmn 041f

S9 Alishanwulong Taiwan 1353 plusmn 20g 3572 plusmn 043g

S10 Gaoshanwulong Taiwan 1359 plusmn 25g 3580 plusmn 047g

S11 Milanxiang Guangdong 1321 plusmn 20h 3517 plusmn 038h

S12 Dahongpao Fujian 1396 plusmn 22b 3538 plusmn 051gh

White tea

S13 Xingongyibaicha Fujian 1403 plusmn 16b 3292 plusmn 061b

S14 Shoumei Fujian 1387 plusmn 18f 3308 plusmn 049b

S15 Baimudan Fujian 1472 plusmn 15i 3279 plusmn 059b

S16 Gongmei Fujian 1378 plusmn 13fg 3303 plusmn 046b

Mean of green tea 2143 plusmn 419

A

2777 plusmn 308

A

Mean of oolong tea 1372 plusmn 35B 3506 plusmn 076B

Mean of white tea 1410 plusmn 43C 3296 plusmn 013C

Data are expressed as mean plusmn SD (n = 3) In each column different superscript small

letters indicate signi1047297cant differences ( p b 005) among different tea samples and

different superscript capital letters indicate signi1047297cant differences ( p b 005) among

different tea types


8102019 1-s20-S0963996913001890-main










80ndash










the HPLC method












































activity















































DPPH assay








8102019 1-s20-S0963996913001890-main
















































Table 2










xminus

5604 times 105

09996 4992ndash

998 043 125




GC 1 001 17 002 18 002 953 plusmn 107 036

EGC 12 006 22 014 19 012 963 plusmn 110 037

EC 1 002 19 006 19 006 958 plusmn 207 069

EGCG 14 018 16 037 19 045 965 plusmn 100 034

GCG 11 001 19 003 21 002 973 plusmn 126 042

ECG 13 005 25 015 2 012 972 plusmn 174 058





8102019 1-s20-S0963996913001890-main



















TEAC value of 4937

























































































and 12 mdash

ECG


8102019 1-s20-S0963996913001890-main



















Table 3




Concentration

(μ M)

TEAC1















8102019 1-s20-S0963996913001890-main













































Table 4









S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

















3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c




















8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main










80ndash










the HPLC method












































activity















































DPPH assay








8102019 1-s20-S0963996913001890-main
















































Table 2










xminus

5604 times 105

09996 4992ndash

998 043 125




GC 1 001 17 002 18 002 953 plusmn 107 036

EGC 12 006 22 014 19 012 963 plusmn 110 037

EC 1 002 19 006 19 006 958 plusmn 207 069

EGCG 14 018 16 037 19 045 965 plusmn 100 034

GCG 11 001 19 003 21 002 973 plusmn 126 042

ECG 13 005 25 015 2 012 972 plusmn 174 058





8102019 1-s20-S0963996913001890-main



















TEAC value of 4937

























































































and 12 mdash

ECG


8102019 1-s20-S0963996913001890-main



















Table 3




Concentration

(μ M)

TEAC1















8102019 1-s20-S0963996913001890-main













































Table 4









S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

















3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c




















8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main
















































Table 2










xminus

5604 times 105

09996 4992ndash

998 043 125




GC 1 001 17 002 18 002 953 plusmn 107 036

EGC 12 006 22 014 19 012 963 plusmn 110 037

EC 1 002 19 006 19 006 958 plusmn 207 069

EGCG 14 018 16 037 19 045 965 plusmn 100 034

GCG 11 001 19 003 21 002 973 plusmn 126 042

ECG 13 005 25 015 2 012 972 plusmn 174 058





8102019 1-s20-S0963996913001890-main



















TEAC value of 4937

























































































and 12 mdash

ECG


8102019 1-s20-S0963996913001890-main



















Table 3




Concentration

(μ M)

TEAC1















8102019 1-s20-S0963996913001890-main













































Table 4









S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

















3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c




















8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main



















TEAC value of 4937

























































































and 12 mdash

ECG


8102019 1-s20-S0963996913001890-main



















Table 3




Concentration

(μ M)

TEAC1















8102019 1-s20-S0963996913001890-main













































Table 4









S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

















3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c




















8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main



















Table 3




Concentration

(μ M)

TEAC1















8102019 1-s20-S0963996913001890-main













































Table 4









S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

















3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c




















8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main













































Table 4









S7 3323 plusmn 036d

5789 plusmn 081d

4357 plusmn 073d

7058 plusmn 139f

NDc

















3059 plusmn 51a

3959 plusmn 061b

987 plusmn 19a

2774 plusmn 48a

4239 plusmn 67c




















8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main







































4 Conclusions







Table 5










2970 plusmn 0055e

3181 plusmn 0054a

2302 plusmn 050e

NDc




































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main











Acknowledgment




References















632ndash

636





assay (mM Troloxg)






components


8102019 1-s20-S0963996913001890-main











































8102019 1-s20-S0963996913001890-main











































1-s2.0-S0963996913001890-main

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