PREVENTIVE

MEDICINE

Antioxidative

21, 520-525 (1992)

Effect of Polyphenol Extract Various Chinese Teas’,*

Prepared

from

CHI-TANG Ho, PH.D.,~ QINYUN CHEN, B.S., HUANG SHI, B.S., KE-QIN ZHANG, B.S., AND ROBERT T. ROSEN, PH.D. Department of Food Science and the Cenrer for Advanced Jersey Agricultural Experiment Station, Rutgers University,

Food Technology, New Brunswick,

Cook New

College, New Jersey 08903

Methods. Twelve different types of Chinese teas, including green, semifermented, and black tea, were studied for their antioxidant activities and active components. Compositions of (- )-epicatechin, ( -)-epigallocatechin, ( -)-epicatechin gallate, (-)-epigallocatechin gallate, and gallic acid were identified by fast atom bombardment-mass spectrometry and high-performance liquid chromatography-mass spectrometry and quantified by highperformance liquid chromatography. Antioxidant activities in lard were measured by the Rancimat method. Results. The results showed that both yields of polyphenol extract and antioxidant activities varied with different tea processing methods. It was found that (-)-epigallocatechin gallate, (-)-epigallocatechin, and (-)-epicatechin gallate inhibited soybean lipoxygenase at the IC,, values ranging from 10 to 20 p.M. 0 IWZ Academic FWSS, IX.

INTRODUCTION

In China, tea has been considered a crude medicine for 4,000 years. Different kinds of pharmaceutical effects such as protection of blood vessels, reduction of serum cholesterol levels, and prevention of atherosclerosis were reported as an integrated effect (I). It has been reported that the death rate from cancer, especially stomach cancer in both sexes, in Shuzuoka Prefecture is much lower than the average for Japanese people. Green tea is a staple product and the main beverage in this area (2). Recently, the antimutagenicity and anticarcinogenicity of green tea were studied in both China and Japan. In China, an antioxidative fraction of green tea extract was prepared. The green tea antioxidants inhibited mutagenicity of AFB, , MeIQ, BaP, cigarette smoke condensate, and extracts of fried fish in Salmonella tryphymurium and/or V79 cells (3). A significant inhibitory effect of green tea antioxidant on malignant transformation was observed in BALA/3T3 cells treated by BaP, X rays, or MCA/TPA separately (1, 4). In Japan, Matsuzaki and Hara (5) and Namiki and Osawa (6) isolated and identified six catechins from green tea. Four catechins, (-)-epicatechin (EC), ( - )-epigallocatechin (EGC), ( - )-epicatechin gallate (ECG), and ( - )epigallocatechin gallate (EGCG), when isolated have antioxidative characteristics ’ Presented at the First International Symposium on the Physiological and Pharmacological Effects of Camellia sinensis (Tea), March 4-6, 1991, American Health Foundation, New York City. Jointly sponsored by the Tea Council and the National Tea Association. ’ New Jersey Agricultural Experiment Station Publication No. 1020.5-l-91, supported by state funds. 3 To whom requests for reprints should be addressed. 520 0091-7435192 $5.00 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

PROCEEDINGS:

PHYSIOLOGICAL

AND

PHARMACOLOGICAL

EFFECTS

OF TEA

521

when tested in vegetable oils (5). Most recently, EGCG, the main polyphenolic constituents of extract of green tea, has been shown to inhibit tumor promotion by teleocidin in two-stage carcinogenesis on mouse skin (7) and found to inhibit tumor promotion in the gastrointestinal tract in the model system of mouse duodenal carcinogenesis by ENNG (8). There is no report on the antioxidative components and properties of tea other than green tea. The present paper reports the study on the polyphenol composition of various Chinese teas and their antioxidant properties. MATERIALS Preparation

of Polyphenol

Extract

from

AND METHODS Tea

One hundred grams of tea was extracted three times with 300 ml of methanol at 50°C for 3 hr. Solvent was removed from the combined extract with a rotary evaporator. The extract was dissolved in 500 ml of water and extracted three times with 200 ml hexane and three times with 200 ml chloroform to remove chlorophyll and caffeine. The aqueous phase was extracted three times with 200 ml ethyl acetate, and the ethyl acetate was evaporated under reduced pressure and the residue redissolved in 300 ml water and freeze-dried to obtain the polyphenol extract. HPLC

Analysis

of Tea Extract

Chromatographic analyses were performed on a Whatman Partisil 5 ODS-2 HPLC column (12.5 cm X 4.6 mm, i.d.). The mobile phase was acetic acid/ acetonitrile/dimethylformamide/water (3/l/15/81) flowing at 0.8 ml/min. A Varian Model 5000 liquid chromatograph with a Model 2050 variable wavelength uv detector set at 280 nm was used. Mass

Spectrometric

Analysis

of Tea Components

EC and EGC were identified by liquid chromatography-mass spectrometry. A Vestec 201 LC-mass spectrometer operated in the positive ion discharge mode was used. The technique used was thermospray (chemical ionization). No column was used in the analysis and the sample was delivered by flow injection. The solvent system was 90% methanol in water. ECG and EGCG were identified by fast atom bombardment (FAB)-mass spectrometry. FAB-mass spectra were obtained on a VG 7070E mass spectrometer. The matrix used was “magic bullet,” a mixture of 3: 1 dithiothreitohdithioerythritol. Spectra were obtained in the positive ion mode. Evaluation

of the Antioxidant

Activity

by the Rancimat

Method

Pure lard (pork fat, Hatfield Packing Co.) without any additives was used as the substrate to evaluate the antioxidant activity of tea extracts and tea components. The test samples were prepared by mixing the tea extract or component with lard in 0.02% concentration by weight. A 670 Rancimat (Metrohm AC, Switzerland) was used. A 2.5-g portion of each test sample was loaded into the reaction vessel

522

HO

ET AL.

cylinder. Six different samples were conducted in one batch. The air supply was maintained at 20 ml/min and the heating temperature at 100°C throughout the experiment. Lipoxygenase

Assay

Lipoxygenase (Sigma, Type V) activity was analyzed by using linoleic acid (Sigma, final concentration, 1.2 mM) as the substrate in a 0.1 M pH 8.5 Tris buffer at 22°C. The absorbance at 234 nm was recorded as a function of time on a U-31 10 uv spectrophotometer. A sample containing all of the reagents except the enzyme solution was used as the blank control. Various concentrations of inhibitor were added to the enzyme and the mixture was incubated for 5 min. The residual enzyme activity was then measured as described above. RESULTS

AND DISCUSSION

Twelve tea products including six green teas, four semifermented teas, and two black teas were obtained from China. Table 1 shows the yields of polyphenol extract prepared from these tea products. The yields of polyphenols were consistently higher in green teas. Black teas contained the least amount of ethyl acetate-soluble polyphenols. The yields of polyphenols in semifermented teas varied the most probably due to the varying degrees of fermentation. The compositions of polyphenol extracts were analyzed by HPLC and are also listed in Table 1. Caffeine and gallic acid were identified by comparing their retention times with that of a pure standard. EC and EGC were identified by LC-mass spectrometry. Figure 1 shows the LC-mass spectrum of EGC. ECG and EGCG were identified by FAB-mass spectrometry using the magic bullet as the matrix. Figure 2 shows the FAB-mass spectrum of EGCG. Green tea polyphenol

YIELD

Green tea GT-1 GT-2 GT-3 GT-4 GT-5 GT-6 Semifermented SFT-1 SFT-2 SFT-3 SFT-4 Black tea BT-1 BT-2

TABLE 1 OF POLYPHENOL EXTRACT

AND COMPOSITION Yield %

Caffeine %

9.6 6.2 6.2 7.9 8.5 8.2

9.7 15.8 15.6 14.1 6.2 16.0

3.9 3.3 8.2 6.0 2.3 2.3

GA %

FROM

CHINESE

TEAS

EC %

ECG %

EGC %

EGCG %

Others %

4.4 5.7 7.9 6.4 0.7 3.4

8.4 4.1 3.7 4.4 4.4 3.4

8.7 17.4 15.6 16.2 12.5 16.7

8.6 2.3 2.4 3.0 12.1 1.4

31.2 49.3 49.6 51.0 49.1 52.8

29.3 5.4 5.2 5.1 15.0 6.5

9.4 11.4 12.1 11.1

4.4 3.3 5.2 5.4

3.8 4.4 6.2 4.8

11.4 8.0 4.3 9.3

2.5 4.3 4.9 2.5

19.9 26.6 30.2 21.0

49.3 42.2 37.1 45.9

12.2 33.4

26.5 36.4

11.4 3.4

14.5 0.8

2.7 4.3

6.8 4.3

26.0 17.3

tea

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EFFECTS OF TEA

OH

90

OH

80 70

OH

40 30 169 ZO-

291

150

175

225

200

FIG.

250

275

300

325

350

375

400

MASS 1. LC-mass spectrum of ( -)-epigallocatechin.

extracts contained the highest concentration of EGCG, whereas the black teas contained relatively higher amounts of gallic acid. The antioxidant activities of polyphenol extracts and purified components were measured by the Rancimat method. The Rancimat method is both simpler and easier compared with the traditional active oxygen method (AOM). The Rancimat method measures the conductivity changes caused by formation of small free fatty acid molecules when the fats and oils are oxidized under elevated temperatures and accelerated aeration. Experimental results (9, 10) showed that the Rancimat method and the AOM method correlated well over a range of temperature (lOO200°C) on a variety of fats and oils. The induction times of lard with tea extract added are shown in Table 2. The 100

288

90

OH

a0 70

HO

60 _ 459 50 _ 40 _

OH

221

250 FIG.

300

350

400

2. FAB-mass spectrum of (-)-epigallocatechin

450

gallate.

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ET AL.

TABLE ANTIOXIDATIVE

ACTIVITY

2 OF TEA EXTRACT

Sample

Induction time (hr)

Control Green tea GT-1 GT-2 GT-3 GT-4 GT-5 GT-6 Semifermented tea SFT-1 SFT-2 SFT-3 SFT-4 Black tea BT-1 BT-2 Tea component Gallic acid Caffeine EC EGC ECG ECGC

4.86 45.45 46.08 54.50 45.50 41.60 40.80 38.82 21.61 15.50 25.30 25.10 46.80 63.30 4.80 10.50 52.90 31.60 55.50

longer induction times suggest stronger antioxidant activities. In general, green tea extracts showed stronger antioxidant activities than the semifermented tea extracts. The antioxidant activities of six purified components of tea were also measured. Gallic acid, EGCG, and EGC had strong antioxidant activities and the caffeine had no antioxidant activity. It is obvious that the strong antioxidant activities of green tea are mainly due to the higher content of EGCG. The good antioxidant activities of black tea extracts may be due to gallic acid which is more abundant in black tea extracts. The metabolism of arachidonic acid to lipid peroxides and various other lipoxygenase products is believed to be significant in carcinogenesis (11). It appears to play an important role in tumor promotion because inhibitors of arachidonic acid metabolism have been observed to inhibit promotion (12). We have measured the TABLE INHIBITION

OF SOYBEAN

LIPOXYGENASE

Compound (-)-Epicatechin ( -)-Epicatechin gallate ( - )-Epigallocatechin ( - )-Epigallocatechin gallate

3 BY GREEN

TEA COMPONENTS

G,

(CLIM) 140 18 21 10

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lipoxygenase-inhibitory activities of isolated green tea polyphenol components. EGCG, ECG, and EGC displayed KS0 values toward soybean 154ipoxygenase enzyme ranging from 10 to 21 IJAM(Table 3). EC is, on the other hand, inactive. The present study shows that the polyphenol composition varied greatly with different processing methods. It also suggested that possible mechanisms through which tea polyphenols function biologically are as antioxidant and lipoxygenase inhibitors. REFERENCES 1. Cheng S, Wang Z, Ho CT. In: Current Medicine in China. Beijing: The People’s Medical Publishing House, 1988: 165-172. 2. Oguni I, Nasu N, Yamamoto S, Nomura T. On the antitumor activity of fresh green tea leaf. Agric Biol Chem 1988; 52:1,879-1,880. 3. Cheng SJ, Ho CT, Lou HZ, Bao YD, Jian YZ, Li MH, Ciao YN, Zhu GF, Bai JF, Guo SP, Li XQ. A preliminary study on the antimutagenicity of green tea antioxidants. Acta Biol Exp Sinica 1986; 19427-431. 4. Cheng SJ, Ho CT, Feng J, Bai JF, Guo SP, Li XQ. Inhibitory effect of green tea polyphenols on mutagenesis and carcinogenesis. Carcinog Teratog Mutagen 1989; 1:1-4. 5. Matsuzaki T, Hara Y. Antioxidative activity of tea leaf catechins. Nippon Nogeikagaku Kaishi 1985; 59:129-134.

6. Namiki M, Osawa T. In: Shankel DM, Hartman PE, Kada T, Hollaender A, eds. Antimutagenesis and Anticarcinogenesis Mechanisms. New York: Plenum, 1986: 131-142. 7. Yoshizawa S, Horiuchi T, Fujiki H, Yoshida T, Okuda T, Sugimura T. Antitumor promoting activity of (-)-epigallocatechin gallate, the main constituent of “tannin” in green tea. Phytother Res 1987; 1:44-47. 8. Fujita Y, Yamane T, Tanaka M, Kuwata K, Okuzumi J, Takahashi T, Fujiki H, Okuda T. Inhibitory effect of (- )-epigallocatechin gallate on carcinogenesis with N-ethyl-N’nitrosoguanidine in mouse duodenum. Jpn J Cancer Res 1989; 80:503-505. 9. Laubli MW, Bruttel PA. Determination of the oxidative stability of fats and oils: Comparison between the active oxygen method (AOCS Cd12-57) and the Rancimat method. JAm Oil Chem Sot 1986; 63~792-795. 10. Zhang KQ, Bao Y, Wu P, Rosen RT, Ho CT. Antioxidative components of Tanshen (Salvia miltiorrhizu Bang). J Agric Food Chem 1990; 38:1,194-1,197. 11. Powles TJ, Bockman RS, Honn KV, Ramwell P. First International Conference on Prostaglandins and Cancer, Prostaglandins and Related Lipids, Vol. 2. New York: Liss, 1982. 12. Belman S, Solomon J, Segal A, Block E, Barany G. Inhibition of soybean hpoxygenase and mouse skin tumor promotion by onion and garlic components. J Biochem Toxicol 1989; 4151-160. Received August 21, 1991 Revised February 14, 1992 Accepted February 14, 1992

Antioxidative effect of polyphenol extract prepared from various Chinese teas.

Twelve different types of Chinese teas, including green, semifermented, and black tea, were studied for their antioxidant activities and active compon...
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