Eeaw

BLa

b

a

ARRAA

KCHMPTT

1

bscfaea

pa

0d

Journal of Chromatography A, 1216 (2009) 3223–3231

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

fficient procedure for isolating methylated catechins from green tea andffective simultaneous analysis of ten catechins, three purine alkaloids,nd gallic acid in tea by high-performance liquid chromatographyith diode array detection

ing Hua, Lin Wanga, Bei Zhoua, Xin Zhanga, Yi Suna, Hong Yea,iyan Zhaoa, Qiuhui Hua, Guoxiang Wangb, Xiaoxiong Zenga,∗

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaLife Science Laboratory Center, Nanjing Agricultural University, Nanjing 210095, China

r t i c l e i n f o

rticle history:eceived 30 October 2008eceived in revised form 3 February 2009ccepted 9 February 2009vailable online 13 February 2009

eywords:atechinPLC-DADethylated catechin

urine alkaloideaoyopearl HW-40S column chromatography

a b s t r a c t

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC),(−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3′′Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of greentea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. Inaddition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerizationand semi-preparative HPLC chromatography. With the prepared catechin standards, an effective andsimultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea wasdeveloped in the present study. Using an ODS-100Z C18 reversed-phase column, fourteen compoundswere rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) andmethanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods

(40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins,as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most componentsat the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peakarea variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rateswere generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has beenreadily achievable with commonly used chromatography equipments in the present study, which will

linica

facilitate the analytical, c

. Introduction

Tea (Camellia sinensis L.) is one of the most widely consumedeverages in the world. A lot of epidemiological and preclinicaltudies have demonstrated that drinking tea may reduce the risk ofancer and cardiovascular disease [1,2]. Moreover, other biologicalunctions of tea have also been reported, such as anti-inflammation,nti-oxidation, anti-allergy, and anti-obesity [3,4]. These beneficialffects have been attributed mainly to the presence of polyphenols

nd purine alkaloids in tea [4,5].

Tea contains many natural polyphenols. Recently, more than 96olyphenolic components have been identified from 41 green teasnd 25 fermented teas by Lin et al. [6]. Among them, gallic acid (GA)

∗ Corresponding author. Fax: +86 25 84396791.E-mail address: zengxx@njau.edu.cn (X. Zeng).

021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.02.020

l, and other studies of tea catechins.© 2009 Elsevier B.V. All rights reserved.

and tea catechins, mainly (−)-epicatechin (EC), (−)-epicatechin gal-late (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechingallate (EGCG) are the major functional components in tea. Theyinvolve in many biological activities such as anti-oxidative, anti-carcinogenic, anti-microbial, anti-viral, and anti-atherosderoticproperties [7–11]. It has also been reported that epimerization ofEC, EGC, EGCG, and ECG occurred at the C-2 position during theprocessing of tea and preparation of tea infusion for drink withhot water, resulting in the formation of their corresponding iso-mers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechingallate (GCG), and (−)-catechin gallate (CG), respectively [12–14].These epimers of tea catechins have been attracted much attention

because they may be active or more active as the major tea catechins[15–17]. In addition, unique O-methylated forms of EGCG, havingpotent inhibitory activities to allergies in vitro and in vivo, have beenreported to present in limited Oolong tea and green tea [18,19].Accordingly, great attention has been paid to the O-methylated

3224 B. Hu et al. / J. Chromatogr. A 1216 (2009) 3223–3231

uctur

dpimtetstotc

imaAm

Fig. 1. Chemical str

erivatives of tea catechins for their anti-allergic properties andotential application [20–22]. The chemical structures of the orig-

nal tea catechins and their derivatives are illustrated in Fig. 1. Theajor purine alkaloids in tea include caffeine, theobromine, and

heophylline. Although there are some reports about the adverseffects of excessive intake of caffeine [23], it has been reportedhat caffeine has various physiological effects on various bodyystems including the central nervous system, cardiovascular, gas-rointestinal, respiratory, and renal systems [24]. Furthermore, theral administration of caffeine can decrease the numbers of lungumors in A/J mice [25]. These components are also the importanthemical components affecting the quality and taste of tea [26,27].

In order to investigate the health-promoting function and qual-

ty of tea, there is a demand for rapid and effective analytical

ethods for its major chemical components, which should be suit-ble across a wide range of research and practical applications.lthough modern liquid chromatography (LC) techniques includingonolith column, capillary liquid chromatography (cLC) and ultra-

es of tea catechins.

high-pressure liquid chromatography (UHPLC) have considerablepotential in or have already actualized speed and effective analysisof tea components [28–30], Separation by high-performance liquidchromatography (HPLC) followed by UV or electrochemical detec-tion is the most widely used method for analysis of tea polyphenolsand purine alkaloids [31–35]. However, most HPLC methods havebeen only developed for the determination of major tea catechinsdue to the lack of tea catechin standards, especially O-methylatedcatechins. Up to now, just a few HPLC methods have been reportedto determine simultaneously all catechins in tea [36–38]. In most ofthem, the contents of methylated derivatives or epimers of tea cat-echins are quantified by reference to relative catechin. In addition,the analysis time for one sample (generally requiring 40–105 min)

is too long, resulting in relative low efficiency.

Ready availability of tea catechin standards, therefore, wouldfacilitate its analytical, clinical and other studies. The methylatedcatechin standards are not commercially available now for they areconcentrated in limited tea cultivars, and their concentrations are

gr. A 1

mduchfbmro

cgsfta(

2

2

(opUglwo(Tw(toDQUadtMr

tCwtwpdEts

2

uHppM

B. Hu et al. / J. Chromato

uch lower than those of major catechins. At present, methodseveloped for purification of catechins include Sephadex LH-20 col-mn chromatography, preparative or semi-preparative HPLC, andounter-current chromatography [18,19,39,40]. Just a few of themave been developed for the preparation of methylated catechins

rom tea using Sephadex LH-20 column or sillic gel column com-ined with preparative (semi-preparative) HPLC. However, theseethods are needed to be improved since they are time-consuming,

equire several steps of isolation and use of large amounts of toxicrganic solvents.

Herein, simple and efficient procedures for preparation of teaatechins especially methylated catechins with high purity fromreen tea have been described. Then with the prepared catechintandards, we report a rapid and simultaneous analytical methodor the analysis of fourteen components in tea, i.e. GA, four majorea catechins, four of their epimers, two methylated catechins,nd three purine alkaloids using HPLC with diode array detectionHPLC-DAD).

. Experimental

.1. Reagents, standards and samples

Standards of EGC (>98%), EGCG (>98%), EC (>98%), and ECG>98%) were purchased from Funakoshi (Tokyo, Japan). Standardsf GA (>98%), caffeine (>98%), theobromine (>98%), and theo-hylline (>98%) were obtained from Sigma–Aldrich (St. Louis, MO,SA). Standards of C, GC, CG, GCG, (−)-3-O-methyl epicatechinallate (ECG3′Me), and (−)-epigallocatechin 3-O-(3-O-methyl) gal-ate (EGCG3′′Me) were prepared according to reported methods

ith some modifications as described in Section 2.3 [41,42]. Toy-pearl HW-40S and Diaion HP20 resins were purchased from TosohTokyo, Japan) and Mitsubishi Chemical (Tokyo, Japan), respectively.ea samples of green tea, Oolong tea, black tea, and pu-erh teaere obtained from Orihiro (Saitama, Japan), Qimen Tea Factory

Anhui, China), and Yiliang Tea Factory (Yunnan, China), respec-ively. HPLC grade of methanol and acetonitrile (MeCN) werebtained from Hanbon Science and Technology (Jiangsu, China).istilled deionized water (ddH2O) was produced using a Milli-Ultrapure water-purification systems (Millipore, Bedford, MA,

SA). Formic acid solution (pH 2.5) was prepared by diluting formiccid in ddH2O (2:998, v/v). Acetic acid solution was prepared byiluting glacial acetic acid in ddH2O (2:98, v/v). 80% Ethanol solu-ion was prepared by mixing ethanol with ddH2O (80:20, v/v).

eCN solution was prepared by mixing MeCN with ddH2O in aatio of 1:1 (v/v).

For peak identification, standard curve, and limit of detec-ion (LOD), solutions of GA, EGC, GC, EC, C, EGCG, GCG, ECG,G, EGCG3′′Me, ECG3′Me, caffeine, theobromine, and theophyllineere prepared in acetic acid solution. In order to assess the effec-

iveness of HPLC separation, mixture of standards mentioned aboveas also prepared in acetic acid solution. Serial dilutions wereerformed with acetic acid solution to produce the following stan-ard solutions: 100 �g/mL to 1.25 �g/mL for EGC, EC, EGCG, ECG,GCG3′′Me, ECG3′Me, and caffeine standard curves and 100 �g/mLo 50 ng/mL for GA, GC, C, GCG, CG, theobromine, and theophyllinetandard curves.

.2. Preparation of tea infusion for HPLC-DAD analysis

Aqueous infusions of green, Oolong, black, and pu-erh teas were

sed as the source of GA, tea catechins, and purine alkaloids ofPLC-DAD analysis. Firstly, all of these teas were ground into teaowders by a mill. Then, tea infusions of each sample were pre-ared by using two different methods. One was pouring 250 mL 50%eCN solution over 5 g tea powders and brewing for 40 min with

216 (2009) 3223–3231 3225

stirring (120 rpm) at 30 ◦C in dark (method A). Another one waspouring 250 mL boiling ddH2O over 5 g tea powders and brewingin a water bath at 90 ◦C for 40 min in dark (method B). After brewing,20 mL of each infusion was diluted with 10 mL acetic acid solutionin order to bring down pH and stabilize polyphenols prior to anal-ysis. And the acidified solutions were centrifuged at 3000 rpm for10 min on an Avanti J-25 centrifuge (Beckman Coulter, Fullerton,CA, USA) at 4 ◦C. The supernatants were immediately analyzed byHPLC-DAD. At the same time, all aliquots of each supernatant werestored at −80 ◦C in a ultra-low-temperature refrigerator (Ultra-LowTechnology of Kendro Laboratory Products, Asheville, NC, USA) untilanalysis.

2.3. Preparation of C, GC, GCG, CG, EGCG3′′Me, and ECG3′Me

For preparation of monomers of tea catechins, aqueous infu-sion of green tea was prepared by method A as described inSection 2.2. After brewing, the whole infusion was acidified andcentrifuged. The supernatant was collected and partitioned withan equal volume of chloroform. The resulting aqueous layer waspartitioned twice with equal volume of ethyl acetate. The ethylacetate phase containing tea catechins was concentrated using arotary evaporator (Heidolph, German) under vacuum. The resultingresidue was dissolved in distilled water and applied to a column(30 cm × 1.6 cm) of HP-20 resin. After reaching adsorptive satura-tion, the column was first washed by distilled water with 4 timesof bed volume, and then was eluted by 95% ethanol–water solu-tion (95:5, v/v) with 3 times of bed volume. The effluent of ethanolsolution was collected and concentrated to afford the crude extractused for further purification.

The crude extract was dissolved and loaded onto a Toy-opearl HW-40S column (50 cm × 5.0 cm) pre-equilibrated with 80%ethanol in water at a flow rate of 3.5 mL/min. The column was elutedwith the same solution, and the elution was monitored by mea-suring the absorbance at 280 nm and auto-collected at 8 mL/tube.The eluted fractions were analyzed by HPLC-DAD then each frac-tion was collected, concentrated, and loaded again onto a columnof Toyopearl HW-40S treated as described above. As results, thefractions containing the desired catechins were concentrated andfreeze-dried by a Freeze-Dry System (Labconco, Kansas City, MO,USA) respectively.

Epimers of C, GC, GCG, and CG were prepared by heat-epimerization of EC, EGC, EGCG and ECG, respectively [41]. Briefly,monomers of EC, EGC, ECG, and EGCG obtained as described abovewere dissolved in ddH2O and autoclaved for 7 min at 123 ◦C, respec-tively. After filtration through a 0.45-�m cellulose filter, the filtratewas applied to semi-preparative HPLC column of Zorbax SB-C18(250 mm × 9.4 mm, 5 �m, Agilent, Santa Clara, CA, USA) using anAKTA purifier (GE Healthcare Biosciences, Piscataway, NJ, USA). Incase of C, GC, and GCG, the column was eluted with 13% (75 mL),13% (38 mL), and 17% (63 mL) of MeCN solution containing formicacid solution (pH 2.5), respectively, at a flow rate of 2 mL/min. ForCG, the column was eluted with a linear gradient as follows at aflow rate of 1.8 mL/min: 0–25 mL, 70% formic acid solution (pH 2.5,A) and 30% methanol (B); 25–50 mL, A from 70% to 40%, B from 30%to 60%; 50–60 mL, 40% A and 60% B; 60–65 mL, A from 40% to 70%,B from 60% to 30%. The fractions were auto-collected (1 mL/tube)and analyzed by HPLC-DAD. The fractions corresponding to C, GC,GCG, and CG were evaporated and lyophilized, respectively.

2.4. Structural identification of prepared standards

The structures of prepared monomers were confirmed by elec-trospray ionization time-of-flight mass spectrometry (ESI-TOF-MS)and 1H NMR. 1H NMR spectrum was recorded in D2O as solvent witha Bruker DRX-500 spectrometer operated at 300 K. Chemical shifts

3 gr. A 1216 (2009) 3223–3231

(w

2

(m6s(w(gS02wH

2usD

2

ptE1tyut

uhtwtTts

eidHt

2

Vd

3

3E

wE

Fig. 2. Representative elution profile of extract from green tea using 50% acetonitrilesolution at 30 ◦C (method A) by HPLC-DAD (A). The mobile phase consisted of A-formic acid solution (pH 2.5), B-methanol. Elution was performed with the lineargradient as follows: 0–15 min, A from 82% to 40%, B from 18% to 60%. System flow

226 B. Hu et al. / J. Chromato

ı) are given in ppm, and J values are given in Hz. ESI-TOF-MS spectraere recorded on an Applied Biosystems mass spectrometer.

.5. HPLC-DAD analysis

HPLC-DAD analysis was done on an Agilent 1100 series HPLCdwell volume, 1.2 mL) consisted of a model G1379A degasser, a

odel G1311A pump with a low-pressure gradient mixer (G1311-9701), a model G1316A column oven, and a model G1315B DADystem. The separation was achieved on a TSKgel ODS-100Z column150 mm × 4.6 mm, 5 �m, Tosoh). The temperature of column ovenas set at 40 ◦C. The mobile phase consisted of formic acid solution

pH 2.5, A) and methanol (B). Elution was performed with a linearradient as follows: 0–15 min, A from 82% to 40%, B from 18% to 60%.ystem flow rate was 1.0 mL/min. The sample was filtered through.45 �m cellulose filter prior to injection. The injection volume was0 �L. The typical system operating pressure range was 80–105 bar,hich is within the allowable system operating pressure for AgilentPLC (approximately 150 bar).

The DAD acquisition wavelength was set in the range of00–600 nm. Chromatographic data was collected and integratedsing Agilent Chemstation software. Calibration plots were con-tructed with authentic standards by plotting peak areas from theAD absorbance signal at 280 nm versus standard concentrations.

.6. HPLC-DAD calibration and measurement of variability

Calibration curves for all authentic standards were prepared byerforming HPLC-DAD analysis in triplicate on six incremental dilu-ions of each standard, ranging from 100 �g/mL to 1.25 �g/mL forGC, EGCG, EC, ECG, EGCG3′′Me, ECG3′Me and caffeine, and from00 �g/mL to 50 ng/mL for GA, GC, GCG, C, CG, theobromine, andheophylline. LOD was determined by performing HPLC-DAD anal-sis in triplicate on incrementally diluted solutions of each standardntil the ratio of signal (peak height) to noise at 280 nm was reducedo below 3:1.

Recovery rates of the quantified constituents were determinedsing green and Oolong teas that the respective chemical contentsad been predetermined by this HPLC method. In each case, a mix-ure of standards with 0.5%, 1% or 2% of the weight of dry tea powderas spiked into tea sample, and then the tea sample was subjected

o the preparing procedure of infusion as described in Section 2.2.he aliquots of infusion were analyzed by HPLC-DAD. Therefore,he recovery was calculated by comparing the found amount of thetandards to that of added.

Intraday variation (repeatability) of analytical response wasvaluated by performing HPLC-DAD analysis on aliquots of eachnfusion five times on the same day. Interday variation (repro-ucibility) of analytical response was assessed by performingPLC-DAD analysis on freshly thawed aliquots of each infusion in

riplicate on 3 consecutive days.

.7. Data analysis

Statistical analysis was performed on SAS Software for Window8. Relative standard deviation (RSD) was defined as sample stan-ard deviation divided by sample mean, multiplied by 100%.

. Results and discussion

.1. Isolation, purification and identification of ECG3′Me,

GCG3′′Me, C, GC, CG, and GCG

In the present study, monomers of tea catechins with high purityere isolated and purified from green tea, and then the obtained EC,

GC, ECG, and EGCG were used for the preparation of C, GC, CG, and

rate was 1.0 mL/min. The typical system operating pressure range was 80–105 bar.Refer to Table 1 for peak identities; Toyopearl HW-40S chromatographic separationof catechins in green tea (B); HPLC-DAD chromatograms of the fractions separatedby Toyopearl HW-40S column (C).

GCG by using heat-epimerization according to the reported method[41].

The HPLC chromatogram of green tea infusion prepared bymethod A is shown in Fig. 2A. Peaks 4, 7, 8, 9, and 12 were identifiedas EGC, EGCG, caffeine, EC, and ECG according to the retention time(tR) and online DAD spectra of authentic standards (Table 1). Twounknown compounds (peaks U1 and U2) appeared. Thus, the crudeextract of green tea was loaded on to a column of Toyopearl HW-40Scolumn. After isolation by Toyopearl HW-40S column chromatogra-phy, 6 fractions (F1–F6) were obtained and analyzed by HPLC-DADas shown in Fig. 2B and C. As results, F1, F3, F5, and F6 were EC, EGC,ECG, and EGCG, respectively. F2 and F4 contained the unknown com-pounds U2 (89.1%) and U1 (87.5%), respectively. Each fraction wasconcentrated under vacuum, loaded again onto the column of Toy-opearl HW-40S and treated as done described above, affording high

purity (>97%) of EC, EGC, ECG, EGCG, U1, and U2, respectively. Thestructures of U1 and U2 were characterized by ESI-TOF-MS and 1HNMR.

Positive-mode MS for U1 gave a molecular ion signal at m/z 495.2for [M+Na]+ and m/z 473.2 for [M+H]+. The results indicate that

B. Hu et al. / J. Chromatogr. A 1216 (2009) 3223–3231 3227

Table 1Summary of data used to identify and quantify major components of tea infusions eluted by the HPLC-DAD methods: HPLC-DAD observed retention time (tR), molecularweight (MW), ionized molecule ([M+Na]+) observed in ESI-TOF-MS, calibration curve regression equation, calibration curve coefficient of determination (R2), limit of detection(LOD), and linear range (LR).

Peak Identification Calibration

Compouda tR (min) MW (g mol−1) [M+Na]+ (m/z) Regression equationb R2 LODc LRd (�g/mL)

(ng) (pmol)

1 GA 3.0 126.1 – y = 45.762x + 7.7941 0.9997 0.04 0.32 0.05–1002 GC 3.3 306.3 329.1 y = 4.248x − 2.0958 0.9998 1.7 5.55 0.05–1003 Theobromine 4.1 180.2 – y = 34.909x + 18.898 0.9993 0.17 0.94 0.05–1004 EGC 5.1 306.3 – y = 3.0585x + 0.0744 1.0000 2.4 7.84 1.25–1005 C 5.4 290.3 313.6 y = 34.832x − 14.387 0.9999 0.28 0.96 0.05–1006 Theophylline 5.7 180.2 – y = 53.420x + 15.537 0.9997 0.11 0.61 0.05–1007 EGCG 6.7 458.4 – y = 40.523x − 40.300 1.0000 0.16 0.35 1.25–1008 Caffeine 7.2 194.2 – y = 56.012x + 6.3172 0.9998 0.1 0.52 1.25–1009 EC 7.4 290.3 – y = 27.059x + 13.604 0.9998 0.2 0.69 1.25–100

10 GCG 7.8 458.4 481.1 y = 17.338x − 43.494 0.9992 0.89 1.94 0.05–10011 EGCG3′′Me 8.3 472.2 495.2 y = 27.202x − 23.362 0.9996 0.36 0.76 1.25–10012 ECG 9.0 442.6 – y = 38.675x − 18.577 1.0000 0.2 0.72 1.25–10013 CG 9.7 442.6 465.6 y = 46.294x − 26.875 0.9995 0.33 1.19 0.05–10014 ECG3′Me 10.5 456.2 479.2 y = 77.812x − 60.722 0.9998 0.4 0.88 1.25–100

Refer to Fig. 2A for analytical conditions of HPLC-DAD analysis.a Identification and calibration data for GA, EGC, GC, EGCG, GCG, EC, C, ECG, CG, EGCG3′′Me, ECG3′Me, caffeine, theobromine, and theophylline were obtained using authentic

standards.b y, peak area; x, the amount of each analytical injected (mg/L).c LOD represents total ng on-column (calculated by multiplying the solution concentration by the injection volume, 20 �L), and represent HPLC-DAD detection values. LOD

iline re

tmsaciE

65(EU(H24

aaeswotN3dG

aofeiopp

based on comparison of peak retention times (tR) and online DADspectra of the authentic standards (Table 1). In addition, we foundthat EGC, EGCG, EC, ECG, EGCG3′′Me, and ECG3′Me were predom-inant in tested green and Oolong teas. C, GC, CG, and GCG were

s also represented as total moles-on-column (pmol).d LR represents the concentration range over which the least-squares regression

he molecular weight of U1 is 472.2, corresponding to that of O-ethylated EGCG. In the 1H NMR spectrum of U1, characteristic

ignal at 3.650 ppm (3 H, s) for methyl group (OCH3) was assigned,nd all other physical data as shown in next paragraph are identi-al to those of EGCG and EGCG3′′Me [19,43,44]. Therefore, U1 wasdentified as EGCG3′′Me. In a similar manner, U2 was identified asCG3′Me [18,32,45].

Data for U1: 1H NMR, � 6.931 (1 H, s, H-2′′), 6.827 (1 H, s, H-6′′),.475 (2 H, s, H-2′, H-6′), 6.044 (1 H, d, H-8), 6.015 (1 H, d, H-6),.457 (1 H, s, H-3), 4.960 (1 H, d, H-2), 3.572 (1 H, bs, 3-OH), 3.6503 H, s, OCH3), 2.907 (1 H, dd, H-4 ax), and 2.865 (1 H, dd, H-4 eq);SI-MS, m/z 495.2 for [M+Na]+ and m/z 473.2 for [M+H]+. Data for2: 1H NMR, � 6.940 (1 H, s, H-6′), 6.880 (2 H, s, H-2′′, H-6′′), 6.778

1 H, s, H-2′), 6.718 (1 H, s, H-3′), 6.049 (1 H, d, H-8), 6.021 (1 H, d,-6), 5.467 (1 H, s, H-3), 5.033 (1 H, s, H-2), 3.652 (3 H, s, OCH3),.935 (1 H, dd, H-4 ax), and 2.843 (1 H, dd, H-4 eq); ESI-MS, m/z79.2 for [M+Na]+ and 457.2 for [M+H]+.

For preparation of C, GC, CG, and GCG, the EC, EGC, ECG,nd EGCG obtained as described above were dissolved in ddH2Ond treated by heat-epimerization, respectively. After heat-pimerization, the solution was separated by AKTA purifier withemi-preparative ODS-C18 column. Fractions of C, GC, CG, and GCGere collected, concentrated, and lyophilized to afford monomers

f C, GC, CG, and GCG with high purity (>98%), respectively. In addi-ion, the structures of C, GC, CG, and GCG were confirmed by 1HMR and ESI-TOF-MS. ESI-MS data for C, GC, CG and GCG were m/z13.6, 329.1, 465.6 and 481.1 for [M+Na]+, respectively. And 1H NMRata of C, GC, CG, and GCG are all identical to those of C, GC, CG, andCG, respectively [46].

In summary, monomers of EC, EGC, ECG, EGCG, EGCG3′′Me,nd ECG3′Me were obtained by two-time separation by Toy-pearl HW-40S column chromatography. The eluent (80% ethanol)or chromatography was simple. Therefore, it is a simple and

fficient procedure for preparation of tea catechin monomersncluding O-methylated catechins compared with reported meth-ds [18,19]. In addition, monomers of C, GC, CG, and GCG wererepared from EC, EGC, ECG, and EGCG by heat-epimerization andurification.

lating peak area to standard concentration exhibited R2 ≥ 0.999.

3.2. HPLC-DAD separation and peak identification

HPLC separation of standard mixture of GA, EGC, GC, EGCG, GCG,EC, C, ECG, CG, EGCG3′′Me, ECG3′Me, caffeine, theobromine, andtheophylline was achieved using a TSKgel ODS-100Z column in15 min by a linear gradient elution of formic acid solution (pH 2.5)and methanol (Fig. 3). The representative chromatograms of infu-sions from, Oolong, black, and pu-erh teas prepared by method Aare shown in Fig. 4, and that of green tea is the same to Fig. 2A whereU1 and U2 have been identified as EGCG3′′Me and ECG3′Me, respec-tively. Elution profiles of GA, EGC, GC, EGCG, GCG, EC, C, ECG, CG,EGCG3′′Me, ECG3′Me, caffeine, theobromine, and theophylline intea samples were similar to those observed for their correspondingstandards. Therefore, peak identities in tea infusions were assigned

Fig. 3. Elution profiles of mixed authentic standards. Refer to Fig. 2 for elutionconditions. Refer to Table 1 for peak identities.

3228B.H

uet

al./J.Chromatogr.A

1216(2009)

3223–3231

Fig.4.R

epresen

tativeelu

tionp

rofiles

of50%

acetonitrile

extractedaqu

eous

infu

-sion

sfrom

,Oolon

gtea

(A),black

tea(B

),and

pu

-erhtea

(C)

byH

PLC-D

AD

analysis.

Refer

toFig.2

forelu

tioncon

dition

s.Refer

toTable

1for

peak

iden

tities.

main

lyd

etectedin

teain

fusion

prep

aredby

meth

odB

.G

Aw

asth

em

aincom

pon

ent

inblack

and

pu

-erhteas.A

lthou

ghcaffein

eoccu

rredin

allkind

softea

with

relativeh

ighcon

tent,th

eobromin

ew

asn

otd

etectedin

the

testedtea

samp

les.

3.3.Standard

curves,limit

ofdetection,andlinear

range

Mean

peak

areasfrom

triplicate

HPLC

-DA

Dan

alysesof

aran

geof

solution

concen

trations

were

used

tod

etermin

ecalibration

curves

foreach

stand

ard,w

hich

was

defi

ned

asth

eleast-squ

aresregression

line

relating

absorbance

peak

areaat

280n

mto

sam-

ple

concen

tration.C

oefficien

tsof

determ

ination

(R2)

assh

own

inTable

1w

ere>0.999

forcalibration

curves

ofallstand

ards.

Forcalcu

lating

the

LOD

,eachstan

dard

solution

was

increm

en-

tallyd

iluted

un

tilthe

ratioofsign

al(peak

heigh

t):noise

at280

nm

was

redu

cedto

below3:1.LO

Dfor

eachstan

dard

was

then

defi

ned

asth

econ

centration

thatresu

ltedin

asign

al:noise

ratioof3:1.LO

Ds

form

oststan

dard

sw

ereap

proxim

atelyon

the

range

of0.1–1.0n

g;EG

Ch

adth

eh

ighest

LOD

at2.4

ng,

followin

gw

asG

Cat

1.7n

g.A

nd

GA

had

the

lowest

LOD

at0.04

ng

(Table1).

Linear

range

represen

tsth

econ

centration

range

overw

hich

the

least-squares

regressionlin

erelatin

gp

eakarea

tostan

dard

concen

trationexh

ib-ited

R2≥

0.999.Th

elin

earran

gew

as2

orders

ofm

agnitu

de

for

Table 2Intradaya and interdayb of repeatability of peak areas of tea infusions and recoveriesc for determination of GA, catechins and purine alkaloids.

Peak Compound Green tea Oolong tea Black tea Pu-erh tea

IntradayRSD (%)

InterdayRSD (%)

Recovery(%)

RecoveryRSD (%)

IntradayRSD (%)

InterdayRSD (%)

Recovery(%)

RecoveryRSD (%)

IntradayRSD (%)

InterdayRSD (%)

IntradayRSD (%)

InterdayRSD (%)

1 GA 1.3 2.5 102.9 3.3 3.2 4.8 94.8 2.9 0.3 1.8 0.3 2.72 GC 2.1 3.2 96.3 3.1 5.8 8.3 91.4 1.3 11.9 13.9 0.5 6.13 Theobromine 3.8 5.0 94.8 2.9 4.6 4.7 93.6 3.9 2.7 3.7 0.3 2.04 EGC 2.0 3.8 98.3 2.0 3.0 1.8 98.8 1.6 2.5 4.1 - -5 C 2.9 4.3 99.5 3.4 4.1 7.3 95.5 4.4 4.3 6.2 0.9 4.76 Theophylline – – – – – – – – – – – –7 EGCG 1.1 2.6 95.9 1.4 1.4 2.4 98.2 2.7 1.7 2.3 – –8 Caffeine 0.2 1.9 99.5 2.0 0.6 1.9 99.2 1.2 0.2 1.8 0.2 1.49 EC 1.9 2.7 95.3 2.0 0.6 2.6 97.5 2.6 2.5 5.0 3.4 6.1

10 GCG 3.5 5.0 - – 2.1 4.5 88.7 3.5 3.5 6.0 – –0.9 4.9 4.6 96.9 2.0 – – – –2.5 2.1 1.8 97.3 1.7 1.9 2.3 2.6 5.23.1 2.1 4.4 – – 7.6 11.1 – –2.7 1.4 5.4 94.4 3.5 – – – –

tracted by boiling ddH2O (method B). (Data of the tea infusions extracted by 50% acetonitrile solution (method A) are not shown.)he same day.fusion performed on 3 consecutive days; triplicate analyses on the same day were averaged to give single data points used to assess variation among

11 EGCG3′′Me 2.9 5.4 104.512 ECG 1.5 2.4 99.213 CG 3.0 5.5 100.314 ECG3′Me 2.3 6.1 97.7

Refer to Fig. 2A for HPLC-DAD analytical conditions. The tea infusions were exa Intraday RSDs represent data from five analyses of each infusion within tb Interday RSDs represent averaged data from triplicate analyses of each in

days.c Average of recoveries at three spiked levels.

gr. A 1

Em(

3

ituRiiaptw

cbRsiGh[aed

por4

3

pedbm

TC

GGTECTECEGEECE

R

B. Hu et al. / J. Chromato

GC, EC, EGCG, ECG, EGCG3′′Me, ECG3′Me, caffeine, and 4 orders ofagnitude for GA, GC, C, GCG, CG, theobromine, and theophylline

Table 1).

.4. Repeatability, reproducibility, and recovery

Repeatability of analytical response was quantified by perform-ng HPLC-DAD analysis on aliquots of each infusion five times onhe same day. For each infusion, peak areas of the five runs weresed to calculate the RSD for each compound within the same day.eproducibility of analytical response was quantified by perform-

ng HPLC-DAD analysis on freshly thawed aliquots of each infusionn triplicate on 3 consecutive days. For each infusion, the mean peakreas of triplicate analyses on each day were treated as single dataoints to calculate the RSD for each compound among days. Theea infusions used for determining repeatability and reproducibilityere prepared by method B.

Both intraday and interday RSDs were generally <6% for theompounds detected in green, Oolong, black, and pu-erh teas,ut were significantly higher in Oolong tea (Table 2). IntradaySDs were generally smaller than interday RSDs for all the testedamples. Generally, compounds with low responses (theobrominen green tea; GC, EGCG3′′Me, and theobromine in Oolong tea;C and CG in black tea; GC, ECG, and C in pu-erh tea) had theighest RSDs. Similar results were reported in Neilson’s study26]. The relatively low RSDs for GA, most catechins, and purinelkaloids (except theobromine) in both intraday and interdayxperiments indicate that this method is both repeatable and repro-ucible.

The recovery rates were investigated with the tea infusion pre-ared by method A in order to avoid the possible heat degradationf standards in case of extraction with boiling water. As results, theecovery rates were within the range of 92–106% with RSD less than.39% (Table 2).

.5. Analysis of the composition of tea infusions

Using this HPLC-DAD analytical method, the contents of GA,

urine alkaloids, and tea catechins in green, black, Oolong, and pu-rh teas were determined. The tea infusions were prepared by twoifferent methods as mentioned in Section 2.2. Quantification wasased on mean peak areas for each compound obtained for assess-ent of intraday variability (n = 5). The results as shown in Table 3

able 3oncentrationsa of major components of tea infusionsb measured by HPLC-DAD analysis.

Green tea Oolong tea

AEc (mg/250 mL) WEc (mg/250 mL) AE (mg/250 mL) WE (mg/250

A 2.85 ± 0.05 6.07 ± 0.09 4.47 ± 0.07 8.97 ± 0.32C 29.8 ± 0.5 43.7 ± 0.9 16.1 ± 0.4 33.4 ± 1.8hebromine 4.19 ± 0.06 3.93 ± 0.25 4.88 ± 0.12 4.66 ± 0.34GC 318 ± 3 236 ± 5 203 ± 2 151 ± 6

11.6 ± 0.2 11.7 ± 0.3 7.42 ± 0.27 7.78 ± 0.23heophylline – – – –GCG 167 ± 2 86.3 ± 0.9 208 ± 2 129 ± 2affeine 118 ± 0 106 ± 0 119 ± 0 116 ± 1C 31.3 ± 0.4 22.2 ± 0.5 28.3 ± 0.1 27.5 ± 0.2CG – 43.4 ± 1.1 – 55.9 ± 0.9GCG3′′Me 92.6 ± 1.2 50.1 ± 1.3 22.6 ± 0.4 17.0 ± 0.6CG 79.2 ± 1.9 38.3 ± 0.6 65.3 ± 0.5 38.9 ± 0.8G 4.87 ± 0.08 6.17 ± 0.10 – 5.83 ± 0.06CG3′Me 15.2 ± 0.3 8.95 ± 0.12 6.94 ± 0.03 5.51 ± 0.02

efer to Fig. 2A for HPLC-DAD elution and detection conditions. Refer to Table 1 for identia Values reported as mean ± SD (n = 5) obtained from intraday variation (repeatability)b Infusions were prepared by 50% acetonitrile solution (method A) and boiling ddH2O (c AE and WE represent tea infusions obtained by 50% acetonitrile solution (method A) a

216 (2009) 3223–3231 3229

are general in agree with those of previous reports [31,32,36–38].Green tea contained higher levels of catechins than Oolong, black,and pu-erh teas; the contents of catechins of black and pu-erhteas were very low. On the contrary, levels of GA were higher inOolong, black, and pu-erh teas than that of green tea. Green tea(non-fermented), Oolong tea (partially fermented), and black tea(fully fermented) are the three major commercial types of tea hencediffer in their chemical compositions. Pu-erh tea, produced mainlyin Yunnan province of China, is a microbial fermented tea madefrom the leaves of large-leaf species tea plant [47]. Green tea isderived directly from inactivating by steaming or microwave anddrying the fresh tea leaves, thus, the chemical composition of greentea is very similar to that of fresh tea leaves. For Oolong, black,and pu-erh teas, most of catechins are oxidized and polymerizedby endogenerous or microbial enzymes during the manufacturingprocess. Accordingly, Oolong, black, and pu-erh teas contain lesscatechins compared with green tea. As Oolong tea is a partially fer-mented tea, it has higher levels of catechins than black and pu-erhteas. The fermentation process also increases the liberation of GAfrom ECG and EGCG, resulting higher levels of GA in Oolong, black,and pu-erh teas.

EGCG3′′Me and ECG3′Me were detected in green tea and Oolongtea, and the contents of EGCG3′′Me were relatively higher thanthat of ECG3′Me. However, both black and pu-erh teas did not con-tain detectable O-methylated catechins. Interestingly, the contentof EGCG in this Japanese green tea was comparatively lower thanEGC. Generally, EGCG is the dominant component of catechins ingreen tea. Here, it might be related to the unique tea species. It isalso possible that the synthesized EGCG might be used as the sub-strate of EGCG3′′Me biosynthesis in fresh tea leaves. A similar resulthas been reported by Chiu et al. [48].

Some epimers of catechin were detected in aqueous tea infu-sions extracted by hot water. Catechin has two asymmetric carbonatoms (C-2 and C-3) in the C ring, and epimers of epicatechinswith the carbon at the C-2 position are exclusively detected in teainfusion [36,38]. It can be seen obviously from Fig. 5 that epimer-ization of EC, EGC, EGCG, and ECG occurred at the C-2 positionduring extraction with hot water (method B). We found that the

concentrations of the epimers corresponding to the epicatechinswere much higher in tea infusions prepared by method B thanin infusions prepared by method A (Table 3). The percent valuesof GC/EGC, C/EC, GCG/EGCG, and CG/ECG in infusion prepared bymethod B were 18.5%, 52.9%, 50.3% and 16.1%, respectively, and the

Black tea Pu-erh tea

mL) AE (mg/250 mL) WE (mg/250 mL) AE (mg/25 mL) WE (mg/250 mL)

17.1 ± 0.1 30.2 ± 0.1 7.84 ± 0.14 12.5 ± 0.1– 29.4 ± 4.1 – 15.7 ± 0.1

5.91 ± 0.22 4.48 ± 0.19 23.9 ± 0.1 19.2 ± 0.194.9 ± 2.2 49.5 ± 1.2 13.0 ± 0.2 –9.95 ± 0.41 10.7 ± 0.4 3.65 ± 0.03 6.19 ± 0.04

– - – –32.7 ± 1.0 18.7 ± 0.2 – –166 ± 1 151 ± 0 212 ± 1 193 ± 01.80 ± 0.14 1.41 ± 0.43 8.40 ± 0.05 7.44 ± 0.02

– 14.5 ± 0.1 – –– – – –

24.3 ± 0.2 15.8 ± 0.2 4.35 ± 0.08 3.95 ± 0.044.35 ± 0.11 5.25 ± 0.07 – –

– – – –

fication, calibration and standard curve data.assessment of analytical response for each infusion.method B) respectively over 5 g tea powder for 40 min with stirring (120 rpm).nd by boiling ddH2O (method B), respectively.

3230 B. Hu et al. / J. Chromatogr. A 1

Fac

v6oaidTdbo

4

wtcofscbpOwfrtet

[

[

[

[

[

[

[[[[

[[[

[[

ig. 5. Representative elution profiles of green tea infusions prepared using 50%queous acetonitrile at 30 ◦C (A) or hot water at 90 ◦C (B). Refer to Fig. 2 for elutiononditions. Refer to Table 1 for peak identities.

alues in infusion prepared by method A were 9.40%, 37.1%, 0, and.20%, respectively. With regard to purine alkaloids, concentrationsf caffeine were higher in tea infusions prepared by method A. Inddition, a significant increase of GA concentration could be seenn tea infusions prepared by method B. It might be caused by heategradation of EGCG and ECG during preparation of tea infusion.herefore, extraction by 90 ◦C ddH2O provides an easy way to pro-uce more available epimers of major catechins and GA. Extractiony 50% MeCN solution can represent the real chemical compositionsf tea.

. Conclusion

Monomers of EGC, EGCG, EC, ECG, EGCG3′′Me, and ECG3′Meith high purity (>97%) were successfully prepared from green

ea by just two times separation with Toyopearl HW-40S columnhromatography eluted by 80% ethanol. In addition, monomersf C, GC, GCG, and CG with high purity (>98%) were preparedrom EC, EGC, EGCG, and ECG, obtained by heat-epimerization andemi-preparative HPLC chromatography as described above. Thehemical structures of the prepared monomers were all confirmedy ESI-TOF-MS and 1H NMR. They are quite simple and efficientrocedures for the preparation of tea catechin monomers including-methylated catechins. Furthermore, fourteen compounds in teaere rapidly separated within 15 min by a linear gradient elution of

ormic acid solution (pH 2.5) and methanol using an ODS-100Z C

18eversed-phase column. It represented roughly a 2.5–7-fold reduc-ion in total analysis time (15 min, as opposed to 40–105 min) fromxisting analytical methods for analysis of purine alkaloids, GA,ea catechins including their O-methylated derivatives and epimers

[

[[

216 (2009) 3223–3231

of epicatechins. All data of calibration, LOD, repeatability, repro-ducibility, and recovery rate demonstrate that this rapid HPLC-DADmethod is repeatable, reproducible, sensitive, and practical. Finally,our results suggest that extraction with method B provides an easyway to produce more available epimers of epicatechins accompa-nying with less purine alkaloids from tea leaves, and extractionwith method A is suitable for determination of the actual chemicalcompositions of tea.

Acknowledgements

This work was supported by a grant-in-aid from 863 Program,Ministry of Science and Technology of China under 2007AA10Z351and 2007AA100403, a grant-in-aid from National Key TechnologyR&D Program of China under 2006BAD27B04 and a grant-in-aidfrom Natural Science Fund of Jiangsu Province under BK2008339.

References

[1] C.S. Yang, P. Maliakal, X. Meng, Annu. Rev. Pharmacol. Toxicol. 42 (2002) 25.[2] P.M. Kris-Etherton, C.L. Keen, Curr. Opin. Lipidol. 13 (2002) 41.[3] Y. Fujimura, H. Tachibana, K. Yamada, FEBS Lett. 556 (2004) 204.[4] N. Khan, H. Mukhtar, Life Sci. 81 (2007) 519.[5] D.S. Wheeler, W.J. Wheeler, Drug Dev. Res. 61 (2004) 45.[6] L.-Z. Lin, P. Chen, J.M. Harnly, J. Agric. Food Chem. 56 (2008) 8130.[7] C.G. Fraga, IUBMB Life 59 (2007) 308.[8] J.M. Song, K.H. Lee, B.L. Seong, Antivir. Res. 68 (2005) 66.[9] S. Bettuzzi, M. Brausi, F. Rizzi, G. Castagnetti, G. Peracchia, A. Corti, Cancer Res.

66 (2006) 1234.[10] S.-H. Kim, H.-J. Park, C.-M. Lee, I.-W. Choi, D.-O. Moon, H.-J. Roh, H.-K. Lee, Y.-M.

Park, FEBS Lett. 580 (2006) 1883.[11] C. Pasten, N.C. Olave, L.H. Zhou, E.M. Tabengwa, P.E. Wolkowicz, H.E. Grenett,

Thromb. Res. 121 (2007) 59.[12] H.F. Wang, K. Helliwell, Food Chem. 70 (2000) 337.[13] H. Bao, H. Ren, H. Endo, Y. Takagi, T. Hayashi, Food Chem. 86 (2004) 517.[14] R. Ito, A. Yamamoto, S. Kodama, K. Kato, Y. Yoshimura, A. Matsunaga, H.

Nakazawa, Food Chem. 83 (2003) 563.[15] I. Ikeda, M. Kobayashi, T. Hamada, K. Tsuda, H. Goto, K. Imaizumi, A. Nozawa, A.

Sugimoto, T. Kakuda, J. Agric. Food Chem. 51 (2003) 7303.[16] J.Y. Chung, C. Huang, X. Meng, Z. Dong, C.S. Yang, Cancer Res. 59 (1999)

4610.[17] L.R. Fukumoto, G. Mazza, J. Agric. Food Chem. 48 (2000) 3597.[18] R. Saijo, Agric. Biol. Chem. 46 (1982) 1969.[19] M. Sano, M. Suzuki, T. Miyase, K. Yoshino, M. Maeda-Yamamoto, J. Agric. Food

Chem. 47 (1999) 1906.20] Y. Fujimura, D. Umeda, S. Yano, M. Maeda-Yamamoto, K. Yamada, H. Tachibana,

Biochem. Biophys. Res. Commun. 364 (2007) 79.[21] M. Maeda-Yamamoto, N. Inagaki, J. Kitaura, T. Chikumoto, H. Kawahara, Y.

Kawakami, M. Sano, T. Miyase, H. Tachibana, H. Nagai, T. Kawakami, J. Immunol.172 (2004) 4486.

22] Y. Fujimura, H. Tachibana, M. Maeda-Yamamoto, T. Miyase, M. Sano, K.J. Yamada,J. Agric. Food Chem. 50 (2002) 5729.

23] I. Hindmarch, U. Rigney, N. Stanley, P. Quinlan, J. Rycroft, J. Lane, Psychopharma149 (2000) 216.

24] M.U. Eteng, E.U. Eyong, E.O. Akapanyung, M.A. Agiang, C.Y. Aremu, Plant FoodsHum., Nutr. 51 (1997) 231.

25] G. Lu, J. Liao, G.-Y. Yang, K. Reuhl, X. Hao, C.S. Yang, Cancer Res. 66 (11) (2006)494.

26] Q.S. Chen, J.W. Zhao, H.D. Zhang, X.Y. Huang, X.Y. Wang, Anal. Chim. Acta 572(2006) 77.

27] J.H. Thorngate, A.C. Noble, J. Sci. Food Agric. 67 (1995) 531.28] H.F. Zou, X.D. Huang, M.L. Ye, Q.Z. Luo, J. Chromatogr. A 954 (2002) 5.29] I. Molnár-Perl, Zs. Füzfai, J. Chromatogr. A 1073 (2005) 201.30] Z. Spáicl, L. Novkoává, P. Solich, Talanta 76 (2008) 189.

[31] A.P. Neilson, R.J. Green, K.V. Wood, M.G. Ferruzzi, J. Chromatogr. A 1132 (2006)132.

32] X.R. Yang, C.X. Ye, J.K. Xu, Y.M. Jiang, Food Chem. 100 (2007) 1132.33] A. Kotani, N. Miyashita, F. Kusu, J. Chromatogr. B 788 (2003) 269.34] M. Álamo, L. Casado, V. Hernández, J.J. Jiménez, J. Chromatogr. A 1049 (2004)

97.35] Y. Zuo, H. Chen, Y. Deng, Talanta 57 (2002) 307.36] D.M. Wang, J.L. Lu, A.Q. Miao, Z.Y. Xie, D.P. Yang, J. Food Compos. Anal. 21 (2008)

361.[37] E. Nishitani, Y.M. Sagesaka, J. Food Compos. Anal. 17 (2004) 675.

38] M. Sano, M. Tabata, M. Suzuki, M. Degawa, T. Miyase, M. Maeda-Yamamoto,

Analyst 126 (2001) 816.39] N.S. Kumar, M. Rajapaksha, J. Chromatogr. A 1083 (2005) 223.40] Q. Du, P. Wu, Y. Ito, Anal. Chem. 72 (2000) 3363.

[41] J. Haginaka, H. Tabo, M. Ichitani, T. Takihara, A. Sugimoto, H. Sambe, J. Chro-matogr. A 1156 (2007) 45.

gr. A 1

[

[[[

B. Hu et al. / J. Chromato

42] B. Zhou, L. Wang, W. Li, Y. Sun, H. Ye, X. Zeng, Chin. J. Anal. Chem. 36 (2008) 494(in Chinese).

43] G.L. Gall, I.J. Colquhoun, M. Defernez, J. Agric. Food Chem. 52 (2004) 692.44] A.L. Davis, Y. Cai, A.P. Davies, J.M. Lewis, Magn. Reson. Chem. 34 (1996) 887.45] T. Kohri, M. Suzuki, F. Nanji, J. Agric. Food Chem. 51 (2003) 5561.

[

[

[

216 (2009) 3223–3231 3231

46] R. Seto, H. Nakamura, F. Nanjo, Y. Hara, Biosci. Biotechnol. Biochem. 61 (1997)1434.

47] S.-C. Wu, G.-C. Yen, B.-S. Wang, C.-K. Chiu, W.-J. Yen, L.-W. Chang, P.-D. Duh,LWT-Food Sci. Technol. 40 (2007) 506.

48] F.-L. Chiu, J.-K. Lin, J. Agric. Food Chem. 53 (2005) 7035.