ANTIOXIDANT ACTIVITY OF GREEN TEA CATECHINS IN A ?-CAROTENE-LINOLEATE MODEL SYSTEM

ANTIOXIDANT ACTIVITY OF GREEN TEA CATECHINS IN A 0-CAROTENE-LINOLEATE MODEL SYSTEM

RYSZARD AMAROWICZ1 and FEREIDOON SHAHIDI*

Depanment of Biochemistry Memorial University of Newfoundland

St. John's, Newfoundland, Canada AIB 3x9

Received for Publication October 7, 1994 Accepted for Publication December 22, 1994

ABSTRACT

A crude mixture of catechins was isolatedfiom Chinese green tea leaves using a hot water extraction. Individual catechins were then separated by chromatographic means using Sephadex LH-20 followed by semi-preparative HPLC. lhe antioxidant activity of crude and individual catechins was then determined in a @-carotene-linoleate model system. Results indicated that (-)-epicatechin-3-gallate (ECG) possessed the strongest antioxidative activity and (-)- epigallocatechin (EGC) showed the weakest effect. The antioxidative eficacy of (-)-epicatechin (EC) and (-)-epigallocatechin-3-gallate (EGCG) was similar and in between those of ECG and EGC. Furthermore, the antioxidant activity of a reconstituted catechin mixture in the proportions present in the crude extract was lower than that of the crude mixture itself, thus indicating that noncatechin components in the mixture possessed their own antioxidant activity or acted synergistically with the catechins.

INTRODUCTION

Green tea leaves contain a number of biologically active catechins (Fig. 1) which have been the subject of intense investigation in many laboratories. These studies have revealed that green tea catechins may inhibit carcinogenesis of the small

'On leave of absence from Department of Food Science, Centre for Agrotechnology and Veterinary Sciences, Polish Academy of Sciences, Olsztyn, Poland. *To receive correspondence.

Journal of Food Lipids 2 (1995) 47-56. All Rights Reserved. 0 Copyright 1995 by Food & Nutrition Press, h c . , Trumbull, Connecticut. 47

48 RYSZARD AMAROWICZ and FEREIDOON SHAHIDI

intestinal tract (It0 et al. 1992) and lung tumorigenesis (Chung et al. 1992; Yang and Hong 1993) of rat. Their active inhibition of bacterial mutation (Kada et al. 1985) and inhibition of HIV reverse transcriptase activity (Nakane and On0 1990) have also been reported. The preventive effects of tea catechins against halitosis (Ui et al. 1991) and dental caries (Sakanaka m al. 1992) are also well recognized.

Several studies have compared the antioxidant activity of tea catechins (Ka- jimoto 1963; Tanizawa et al. 1983, Matsuzaki and Hara 1985; Yoshigioka et al. 1991; Lunder 1992). The reported results, however, are often contradictory. For example Matsuzaki and Ham (1985) have shown that (-)epi-gallocatechin-3-galalte (EGCG) has the strongest antioxidant activity, while Tanizawa et al. (1983) reported that (-)epi-catechin (EC) showed the strongest activity among catechins. Kajimoto (1963) reported that (-)epi-gallocatechin (EGC) was the strongest antioxidative component of green tea. In all experiments reported, the activity of catechins was tested in oil systems.

However, no detailed studies have been performed to examine the antioxidant properties of individual catechins in a &carotene-linoleate system. Thus, the present study was undertaken in order to isolate and investigate the activity of crude and individual green tea catechins in this model system, which is frequently used in studies of natural antioxidants and emulsified systems.

MATERIALS AND METHODS

A crude mixture of catechins from Chinese green tea leaves, obtained from Anhui Province, was prepared using a hot water extraction. Preliminary purifica- tion of catechins was achieved using counter-current chromatography. Solvent

OH

I H ’ OH

FIG. 1, CHEMICAL STRUCTURES OF GREEN TEA CATECHINS (-)epi-catechin (EC), R,, R2 = H; (-)epi-gallocatechin (EGC). R, = OH, R2 = H; (-)epi-catechin-3-galIate (ECG), R, = H, RZ = 3,4,5-trihydmxybe~yl; and (- )epi-gallocatechin-3-gallate (EGCG), R, = OH, Rz = 3,4,5-trihydroxybenzoyl.

ANTIOXIDANT ACTIVITY OF CATECHINS 49

systems employed were water-chloroform (1 : 1 , v/v) and water-ethyl acetate (1 : 1 , v/v) (Matsuzaki and Hara 1985). The crude catechins were then separated on a Sephadex LH-20 column (2.5 cm, i.d. X 45 cm) which was equilibrated with chloroform-methanol-petroleum ether (1 :2: 1 , v/v/v) (Robertson and Bendal 1983).

Fractions, 20 ml each, were collected and their absorbance read at 280 nm. Absorbance values at 500 nm were also measured after color development with vanillin reagent (Price er al. 1978). As well, each fraction was examined by TLC using silica gel plates (Sigma, St. Louis, MO) and chloroform-methanol-water (65:35:10, v/v/v) as the developing system (Tanizawa er al. 1983). To visualize the catechins, plates were sprayed with a vanillin-hydrochloric acid reagent (Bate- Smith 1953).

Individual catechins were separated from the Sephadex-isolated fractions by semi-preparative HPLC. A Shimadzu (Japan) chromatographic system was used and consisted of a LC-6A pump, SPDdAV UV-VIS spectrophotometric detector, SCLdB system controller, CR 501 Chromatopac, and a Hibar pre-packed column RT (10 mm X 250 mm) with Lichrosorb RP-18 (7 pm) (Merck, Darm- stadt, Germany). The mobile phase was water-acetonitrile-methanol-acetic acid (79.5: 18:2:0.5 vlvlvlv) (Saijo 1982). The flow rate was 3 ml/min, and the injection volume was 500 pl.

Individual catechins so obtained and characterized by proton NMR spectroscopy (Amarowicz and Shahidi 1995) were used as standards in the analysis mixtures of crude catechins under the conditions given below. Specifics of the Shimadzu chromatographic system were provided earlier in this section. A CSL-Spherisorb- ODS-2 analytical column (4.5 mm X 250 mm) (Chromatographic Sepcialities, Inc., Brockville, ON, Canada) was used. The mobile phase was water- dimethylformamide-methanol-acetic acid (157:40:3: 1 , v/v/v/v) (Hofler and Cog- gon 1976) and the flow rate was 1.5 ml/min with an injection volume of 20 pl. For both preparative and analytical methods, the detector wavelength was set at 280 nm.

Antioxidant activity of crude and pure catechins was determined by measuring the coupled oxidation of ,&carotene and linoleic acid (Miller 1971) with specifics described below. Approximately 2 mg of /3-carotene were dissolved in 10 ml of chloroform. A 3 ml portion of this solution was pipetted into a volumetric flask. After removal of chloroform under nitrogen, 20 mg of linoleic acid, 200 mg of Tween 40, and 50 ml of oxygenated distilled water were added to the flask with vigorous stirring. Aliquots (4.5 ml) of this emulsion were transferred into a series of test tubes containing dissolved catechins in 0.5 ml of distilled water. In each series, 2.0 mg or 0.5 mg of crude catechins or 0.5 mg of butylated hydroxyanisolle (BHA), 0.5 mg of individual catechins, or 0.3 mg of a reconstituted mixture of individual catechins in the same proportion present in a crude catechin mixture was used. In the control tube, pure water was used instead of catechin solutions.


As emulsion was added to each tube, the zero time absorbance was read at 470 nm. The emulsion was then kept in a water bath at 50C and absorbance readings were recorded over a 120-min period at 15 min intervals.

RESULTS AND DISCUSSION

The chromatogram of crude catechins following Sephadex LH-20 column separation using a chloroform-methanol-petroleum ether (1 :2: 1, v/v/v) mixture as the mobile phase is shown in Fig. 2. Four maxima for the optical density at 500 nm belonging to EG, EGC, (-)epicatechin-3-gallate (ECG) and EGCG were observed (Amarowicz and Shahidi 1995). The above solvent system was superior to that of ethanol, which was used previously for a similar study (Amarowicz and Shahidi 1995). The high purity of the isolated catechins was confirmed by a semi-preparative HPLC separation (Fig. 3).

FIG. 2. SEPARATION OF CRUDE CATECHINS ON A SEPHADEX LH-20 COLUMN


f D d

GCG

20 40 60 Time ( min )

FIG. 3. CHROMATOGRAM OF THE ANALYTICALLY- SEPARATED PURE CATECHINS OBTAINED FROM

PREPARATIVE HPLC SEPARATION AND MIXING SEPHADEX LH-20 FRACTIONS FOLLOWED BY SEMI-

Antioxidant activity of crude catechins (2 mg) in a 0-carotene-lineolate model system was quite remarkable (Fig. 4). The absorbance of the model system at 470 nm containing crude catechins after 120 min was 60% when compared with that of butylated hydroxyanisole @HA) at a similar level. The lower quantity (0.5 mg) of the mixture was not effective in preventing the bleaching of pcarotene. The crude catechins (Shahidi et al. 1995) were found to be a more effective antioxidant than crude extracts of canola, mustard and flax, but certain individual phenolic fractions of the latter extracts possessed higher antioxidant activity than that of the crude catechins themselves (Amarowicz et al. 1993, 1994; Shahidi et al. 1994; Wanasundara et al. 1994).

The antioxidant activity of individual catechins in a 0-carotene-linoleate model system showed the following trend (Fig. 5):

ECG > EGCG = EC > EGC

The molecular weights of EC, EGC, ECG and EGCG are 290, 306,442 and 458 daltons, respectively. The strongest antioxidant activity observed for EGC in this work lends further support to the findings of Terro et al. (1994). These authors reported that the antioxidant activity of EGC and EGCG in unilamellar


'* '*\

'o\o \

'*\*

- -- 0 Control

2.0 mg of crude catechins 0.5 mg of crude catechins

15 30 45 60 75 90 105 120 Time min 1

RG. 4. ANTIOXIDANT ACTNITY OF CRUDE CATECHINS

EGC 0 Control

I I 1

15 30 45 60 75 90 105 120 Time ( m i n )

FIG. 5. ANTIOXIDANT ACTIVITY OF INDIVIDUAL CATECHINS (0.5 mg)


liposomes was lower than EC and ECG. They further concluded that the antioxidant activity of flavonoids containing a pyrogallol moiety in their B-ring was lower than flavonoids possessing other constituents. Yoshioka ef af. (1991) have reported that the rate of decrease in the intensity of ESR signals was highest for EGC and lowest for ECG, thus indicating that ECG was a powerful antioxidant. A similar conclusion was reached when considering time-dependence of chemiluminescence studies.

Figure 6 displays the content of individual catechins in the crude catechin mixture as determined by HPLC. In each gram of the crude extract 300 mg of cakhins were present, which is only half the amount reported by Matsuzaki and Hara (1985). The preparation of the individual catechins in the crude extract was EC, 17.2%; EGC, 39.2%; ECG, 7.6%; and EGCG, 36%. The dominance of EGC and ECGC in green tea catechins was also noted by Price and Spitzer (1993).

Finally, the antioxidant activity of crude catechin extracts in a P-carotene- linoleate model system was examined (Fig. 7). It was observed that the crude extracts had a much higher antioxidant activity than the reconstituted mixture of individual catechins containing a similar amount of catechins. This indicated that

n L 0 2 c

iCG

ECG

P

20 40 60 Time ( min )

FIG. 6. CHROMATOGRAM OF HPLC DETERMINATION OF INDMDUAL CATECHINS IN CRUDE CATECHINS MIXTURE

54

0.1

RYSZARD AMAROWICZ and FEREIDOON SHAHIDI

' 0.3 mg of pun catcchins

I I 4 I I I I I , 15 30 45 60 75 90 105 120

Time ( m h )

FIG. 7. COMPARISON OF THE ANTIOXDANT ACTIVITY OF CRUDE CATECHINS AS SUCH AND RECONSTITUTED CATECHIN MIXTURES WITH THE SAME

TOTAL CONTENT OF CATECHINS

other noncatechin components present in the crude extracts possessed antioxidant activity or else they acted synergistically with the catechins. In other words, the presence of noncatechin components in the crude extracts has an accentuating effect on the antioxidant activity of crude catechins. Furthermore, the use of decolorized crude extracts in various applications is recommended.

ACKNOWLEDGMENTS

We are grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support.

REFEWNCES

AMAROWICZ, R. and SHAHIDI, F. 1995. Rapid chromatographic method for separation of individual catechins from green tea. Food Res. Int. (Submitted.)

AMAROWICZ, R., WANASUNDARA, J., SHAHIDI, F. andKARAMAC, M. 1994. Antioxidant activity of hydrophilic phenolic fractions of flaxseed. In


Proceedings of the EuroFood Tox W: Bioactive Substances in Food of Plant Origin Conference, pp. 166-171, Sept. 22-24, Olsztyn, Poland.

AMAROWICZ, R., WANASUNDARA, U., WANASUNDARA, J. and SHAHIDI, F. 1993. Antioxidant activity of ethanolic extracts of flaxseed in 0-carotene-linoleate model system. J. Food Lipids I , 11 1-1 17.

BATE-SMITH, E.C. 1953. Colour reaction of flowers attributed to (a) flavanols and (b) carotenoid oxides. J. Exper. Bio. 4, 1-7.

CHUNG, F., XU, Y., HO, C.T. and HAN, C. 1992. Protection against tobacco- specific nitrosamine-induced lung tumorigenesis by green tea and its components. In Phenolic Compounds in Food and Their Effects on Health. 11. An- tioxidants and Cancer Prevention, (M.-T. Huang, C.-T. Ho and C.Y. Lee, eds.) ACS Symp. Ser. 506, pp. 300-307, Am. Chemical SOC., Washington, D.C.

HOEFLER, A.C. and COGGON, P. 1976. Reversed-phase high-performance liquid chromatography of tea constituents. J. Chromatogr. 129, 460-463.

ITO, N., HIROSE, M. and SHIRAI, T. 1992. Carcinogenicity and modification of carcinogenic response by plant phenols. In Phenolic Compounds in Food and n e i r Effects on Health. 11. Antioxidants and Cancer Prevention, (M.-T. Huang, C.-T. Ho and C.Y. Lee, eds.) ACS Symp. Ser. 506, pp. 269-281, Am. Chemical SOC., Washington, D.C.

KADA, T., KANEKO, K., MATSUZAKI, S., MATSUZAKI, T. andHARA, Y. 1985. Detection and chemical identification of natural bioantimutagens: A case of the green tea factor. Mutation Res. 150, 127-132.

KAJIMOTO, G. 1963. Antioxidative components and antiseptic components in tea leaves. I. Antioxidant action and antiseptic action of materials extracted from leaves with alcohol and water. Nippon Shokuhin Kogyo Gakkaishi 10,

LUNDER, T.L. 1992. Catechins of green tea: Antioxidant activity. In Phenolic Compounds in Food and Their Effects on Health. ZZ. Antioxidants and Cancer Prevention, (M.-T. Huang, C.-T. Ho and C.Y. Lee, eds.) ACS Symp. Ser. 506, pp. 114-120, Am. Chemical SOC. , Washington, D.C.

MATSUZAKI, T.L. and H A M , Y. 1985. Antioxidative activity of tea leaf catechins. Nippon Nogeikagaku Kaishi 59, 129-134.

NAKANE, H. and ONO, K. 1990. Differential inhibitory effects of some catechins derivatives on the activities of HIV reverse transcriptase and cellular deox- yribonucleic acid and RNA polymerase. Biochemistry 28, 2841-2845.

PRICE, M.L., VAN SCOYOC, S. and BUTLER, L.G. 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J. Agric. Food Chem. 26, 1214-1218.

PRICE, W. E . and SPITZER, J . C . 1993. Variations in the amounts of individual flavanols in a range of green teas. Food Chem. 47, 271-276.

13-20.


ROBERTSON, A. and BENDALL, D.S. 1983. Production and HPLC analysis of black tea theaflavins and thmbigins during in v i m oxidation. Phytochemistry 22, 883-887.

SAIJO, R. 1982. Isolation and chemical structures of two new catechins from fresh tea leaf. Agric. Biol. Chem. 46, 1969-1970.

SAKANAKA, S., SHIMURA, N., AIZAWA, M., KIM, M. andYAMAMOT0, T. 1992. Preventive effects of green tea polyphenols against dental caries in conventional rats. Biosci. Biotechnol. Biochem. 56, 592-594.

SHAHIDI, F., WANASUNDARA, U. and AMAROWICZ, R. 1994. Natural antioxidants from low-pungency mustard flour. Food Res. Int. 27,489493.

SHAHIDI, F., WANASUNDARA, U. and AMAROWICZ, R. 1995. Isolation and partial characterization of oilseed phenolics and evaluation of their antioxidant activity. In Advances in Food Science and Nutrition (G. Charalam- bous, ed.), Elsevier, Amsterdam.

TANIZAWA, H., SAZUKA, Y., KOMATSU, A., TODA, S. and TANIKO, Y. 1983. A new efficacy test of antioxidant based on air-oxidation of lineolic. Chem. Pharm. Bull. 31, 4139-4143.

TERRO, J., PISKULA, M. andYAO, Q. 1994. Protectiveeffect ofepicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers. Arch. Biochem. Biophys. 308, 278-284.

UI, M., YASUDA, H., SHIBATA, M., MARUYAMA, T., HORITA, H. and YASUDA, T. 1991. Effect of tea catechins for halitosis and their application to chewing gum. Nippon Shokuhin Kogyo Gakkaishi 38, 1098-1102.

WANASUNDARA, U., AMAROWICZ, R. and SHAHIDI, F. 1994. Isolation and identification of an antioxidative component in canola. J. Agric. Food Chem. 42, 1285-1290.

YANG, C.S. and HONG, J.-Y. 1993. Inhibition of nitrosamine-induced tumorigenesis by diallyl sulfide and tea. In Food and Cancer Prevention: chemical Md Biological Aspects, (K.W. Waldron, I.T. Johnson and G.R. Fen- wick, ds.) pp. 247-252, Royal Society of Chemistry, Cambridge.

YOSHIOKA, H. et al. 1991. Formation of radicals and chemiluminescence during the autoxidation of tea catechins. Agric. Biol. Chem. 55, 2117-2723.

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ANTIOXIDANT ACTIVITY OF GREEN TEA CATECHINS IN A ?-CAROTENE-LINOLEATE MODEL SYSTEM