Presence of two forms of methylated (–)-epigallocatechin-3-gallate in green tea

Ryszard Amarowicz1 and Fereidoon Shahidi2

1 Introduction

To date, the main catechins from green tea have been(–)-epicatechin (EC), (–)-epigallocatechin (EGC), (–)-epicate-chin-3-gallate (ECG), and (–)-epigallocatechin-3-gallate(EGCG). Biological activity of these compounds has beenobserved in many experiments. For example, green tea cate-chins may inhibit carcinogenesis of the small intestine tract [1]and lung tumorigenesis of rat [2]. Antimutagenic effects ofcatechins was reported by Yen and Chen [3]. Many studieshave shown that catechins possess strong antioxidant activity[4–6]. Phytochemical investigations of plants such as Ourateasp. [7], Kokona zelyanica [8], Cinnamomum cassis, C. abtusi-folium and Lindea umbellata [9], and Prinostemma aspera[10] resulted in the isolation and identification of methylatedcatechins. The presence of methylated ECG and EGCG ingreen tea was first noted by Saijo [11]. The present work wasundertaken in order to isolate new methylated catechins fromgreen tea.

2 Materials and methods

Chinese green tea leaves were obtained from Anhui Pro-vince (Chinese People’s Republic). Crude catechins wereextracted from 50 g of green tea leaves, obtained using 500 mlof hot water (808C) while stirring over a 1 h period [12]. Pre-liminary purification of catechins was achieved by means ofcounter-current chromatography. Solvent systems employedwere water :chloroform (1:1 v/v) and water :ethyl acetate (1:1v/v) [13]. Column chromatography of the so obtained materialwas carried out on a Sephadex LH-20 column (6063.0 cm)equilibrated with methanol. Crude catechins (0.8 g) were dis-

solved in 8 mL of methanol; fractions (10 mL) were thenacquired using a fraction collector. The presence of individualcatechins in fractions eluted from the Sephadex LH-20 columnwas monitored by TLC on silica gel plates (Sigma, St. Louis,MO, USA) with a chloroform: methanol :water mobile phase(65 :35:10, v/v/v, lower phase) [14] and with vanillin-hydro-chloric reagent for detection [15]. Standards of EC, EGC,ECG and EGCG were prepared according to Amarowicz andShahidi [16]. For separation of catechins from a Sephadex LH-20 fraction which showed the presence of EGCG, a Shimadzusemipreparative HPLC system was used: LC-6A pump, SPD-6AV UV-VIS spectrophotometric detector, SCL-6B systemcontroler and CR 501 Chromatopac. Conditions of separationswere as follows: semipreparative Hibarm prepacked column RT(106250 mm) with Lichrosorb RP-18 (7 lm) (Merck, Darm-stadt, Germany); mobile phase, water :dimethylformamide :methanol :acetic acid (157 :40:2 :1 v/v/v/v) [17]; flow rate,3 mL/min; injection volume, 500 lL; the detector was set at280 nm. The electrospray (ES) mass spectra (positive andnegative ion mode) were recorded with a Micromass VG-Quat-tro II quadrupole-hexapole-quadrupole mass spectrometer(Micromass, Cheshire, UK). The 1H NMR spectrum wasobtained at 300 MHz with a GN-300 spectrometer (GeneralElectric, Palo Alto, CA, USA) and recorded at room tempera-ture in DMSO-d6. Chemical shifts were reported relative totetramethylsilane as an internal standard.

3 Results and discussion

The semipreparative HPLC chromatogram of a SephadexLH-20 fraction containing EGCG is depicted in Fig. 1. Themain peak was EGCG and it eluted at a retention time of 27.5min, based on the HPLC operating conditions employed. Com-pound(s) that give a positive reaction to the vanillin-hydrochlo-ric reagent eluted from the column at a retention time earlierthan that of EGCG: 22.5 min. The mass spectra of the sepa-rated compound(s) are shown in Fig. 2; they were characterisedwith a positive ion [M + Na]+ at m/z of 495, and a negative ion[M –H]– at m/z of 471. Consequently, the Mr of the separatedcompound(s) was believed to be 472. The same Mr was attrib-uted to epigallocatechin-3-(3-O-methylgallate) which wasseparated by Saijo [11]. The daughter ion from 471 in the ES-

mass spectrum appeared at an m/z of 125, which thereforeexcludes the possibility of any methylation in the B-ring. Onthe other hand, the presence of a methoxy group in the gallatering moieties was confirmed from the daughter ion of gallateat an m/z of 183. The fragment ion with an m/z of 413 (i. e., in

Nahrung/Food 47 (2003) No. 1, pp. 21 –23 i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0027-769X/2003/0101-0021$17.50+.50/0 21

Crude catechins extract from Chinese green tea were fractionatedusing Sephadex LH-20 column chromatography. The fraction contain-ing (–)-epigallocatechin-3-gallate (EGCG) was then subjected to asemipreparative high-performance liquid chromatography (HPLC).Using a mobile phase of water : dimethyl formamide :methanol : acetic

acid (157 :49 :2 :1 v/v/v/v), the mixture of two methylated catechinswas separated and isolated. According to mass spectrometry (MS) andnuclear magnetic resonance (1H-NMR) date, these compounds wereidentified as (–)-epigallocatechin-3-(3-O-methylgallate) and (–)-epi-gallocatechin-3-(4-O-methylgallate).

Correspondence: Dr. R. Amarowicz, Division of Food Science, Insti-tute of Animal Reproduction and Food Research of Polish Academy ofSciences, ul. Tuwima 10, P.O. Box 55, PL-10-718 Olsztyn, Poland1Division of Food Science, Institute of Animal Reproduction and FoodResearch of Polish Academy of Sciences, Alsztyn, Poland2Department of Biochemistry, Memorial University of Newfoundland,St. John’s, NF, CanadaE-mail: amaro@pan.olsztyn.plFax: +48-89-524-01-24

Abbreviations: EC, (–)-epicatechin; ECG, (–)-epicatechin-3-gallate;EGC, (–)-epigallocatechin; EGCG, (–)-epigallocatechin-3-gallate

Keywords: Green tea / High-performance liquid chromatography /Mass spectrometry / Methylated catechins / Nuclear magnetic reson-ance

Amarowicz et al.

22 Nahrung/Food 47 (2003) No. 1, pp. 21–23

the positive ion mode) was attributed to the loss of a methoxygroup from [M + H]+.In the 1H-NMR spectrum of the separated compound(s), the

singlets at d 5.82 and 5.92 ppm were assigned to H-6 and H-8in the A-ring, respectively, while the singlet at d 6.40 ppm wasfrom H-29 and H-69 in the B-ring, and the singlet at d 6.96 ppmwas due to H-299 and H-699 in the 3-O-gallolyl ring. The multi-plet observed at d 2.65 to 2.92 ppm was assigned to two pro-tons at position 4 of the C-ring, while the singlet at d 4.98 ppmand multiplet at d 5.52 ppm were assigned to H-2 and H-3 inthe C-ring, respectively. The broad signal from 1OH wasnoted at d 9.0 ppm. Two singlets at d 3.72 and 3.76 ppm wereassigned as two methoxy groups. Since the MS data showedthat EGCG was methylated with only one carbon atom, wepostulated that the catechin isolated by semipreparative HPLCis actually a mixture of two derivatives of EGCG with a meth-oxy group attached to the carbon atom at either position 3 or 4in the gallolyl ring (Fig. 3, 4). Due to similar polarities, theretention times of these compounds could be identical. Saijo[11] observed in his study that the 1H-NMR spectrum ofEGCG with a methylated gallate ring indicated the presence ofa methoxy group at d 3.82 ppm. It is interesting that an investi-gation of methylated catechins from Cinnamonum cassia byMorimoto et al. [9] revealed a very similar chemical shift forthe methoxy moieties at position 5 (d 3.72 ppm) and 7 (d 3.77ppm) in the A-ring. Kamal et al. [8] reported a chemical shiftat d 3.76 ppm for the methoxy groups in the methylated gallatering of methyl epigallocatechin isolated from Kokoona zeyla-nica.In conclusion, in our investigation we confirmed the pre-

sence of methylated EGCG in green tea using MS and1H NMR data, whereas Saijo [11] had used infrared (IR) and1H NMR spectroscopies in his study. Furthermore, we notedthat methylation of EGCG can occur on the carbon atoms ateither position 3 or 4 of the gallolyl ring.

Figure 1. Semipreparative chromato-gram of the fraction separated on aSephadex LH-20 column; the fraction ofmethylated catechins is marked with “X”.

Figure 2. ES-MS spectra of compounds from fraction “X” obtainedusing semipreparative HPLC; (A) scan in positive ion mode, (B) scanin negative mode; (C) daughter ions of 471 ES–.

Figure 3. 1H-NMR spectrum of com-pounds from fraction “X”; the range of dis attributed to methoxy group.

Figure 4. Chemical structure of obtained methylated catechins:(1) R1 = CH3, R2 = H; (2) R1 = H , R2 = CH3.

Methylated EGCG in green tea

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Received November 8, 2001Accepted September 18, 2002