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Tea phenols: Antioxidant effectiveness of teas, tea components, tea fractions and their binding with lipoproteins
Nutrition Research. Vol. 18, No. 6, pp. 1067-1075.1998 Copyright @ 1998 Elsevier Science Inc. F’tinted in the USA. All rights reserved 0271-5317/98 $19.00+ .OO ELSEVIER
PI1SO271-5317(98)00089-X
TEA PHENOLS: ANTIOXIDANT EFFECTIVENESS OF TEAS, TEA COMPONENTS, FRACTIONS AND THEIR BINDING WITH LIPOPROTEINS Joe A. Vinson. Ph.D.. Yousef A. Dabbagh, M.A. Department of Chemistry, University of Scranton, Scranton,
TEA
PA
18510
Phenols in tea are responsible for _. its antioxidant The pure catechins and phenolic acids found activity. in tea are more powerful than the antioxidant vitamins C, E and S-carotene in an in vitro lipoprotein oxidation model. Comparison of the tea fractions indicated that both catechins and theaflavins contribute to the teas' antioxidant characteristics. Black and green teas were not significantly different in phenol content, in antioxidant strength as measured by I&, or in antioxidant potential as measured by the phenol antioxidant index (PAOXI). The PAOXI of teas was significantly Tea catechins and higher than grape juices and wines. both green and black tea exhibited potent lipoproteinbound antioxidant activity. Phenol antioxidants from tea were calculated to be a large source of antioxidants in the U.S. diet. 0 1998 Elsevia Sciencelx. Key Words: Tea, Catechins, Antioxidant, Lipoproteins, Lipid Peroxidation
The most popular beverage in the world is tea prepared from leaves of Camellia sinensis. There are two major varieties, sinensis and assamica and tea is cultivated in more than 30 countries. The composition of tea varies with season, the age of the leaf, climate and horticultural practices. To make green tea, the leaves are chopped, rolled and quickly steamed or heated For black tea the leaves which inactivates polyphenol oxidase. are kept warm for 6 hours and the leaf polyphenols, especially Oolong tea is the catechins, are oxidized and condensed. intermediate between green and black tea and is produced by This process retains a heating in air for one or two hours. considerable amount of the original catechins of green tea and By tradition the Orient, also produces some unique products. North Africa and the Middle East favor green tea and the rest of the world drinks black tea. Oolong tea production is confined to China and Taiwan. Black tea accounts for 80% of the world tea crop (1). Phone To whom requests for reprints should be addressed. 717-941-7551, Fax 717-941-7510, email [email protected]. 1067
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J.A. VINSON and Y.A. DABBAGH
Since the introduction of the oxidation theory of atherogenesis (2), there has been increased interest and research into the effect of antioxidants on heart disease. Cholesterol as low density lipoprotein (LDL) and very low density lipoprotein (VLDL) is rendered atherogenic by oxidation that causes an accelerated foam cell development and plague formation. The effect of dietary antioxidants on atherosclerosis was reviewed in 1994 (3). The mechanisms by which food antioxidants are protective for heart disease have recently been reviewed (4). Flavonoids in the Dutch diet were inversely associated with risk of coronary heart disease and tea provides most of the flavonoids in their diet (5). A more recent epidemiology study of seven countries showed a cross-cultural association of flavonoid consumption and a corresponding reduction in heart disease risk (6). The Japanese, who average 7 cups of tea/day, have an extremely low incidence of heart disease despite a high percentage of smokers. Pharmaceutical benefits of tea such as reduction in serum cholesterol level and systolic blood pressure have been reported in epidemiological studies in Japan (7) and Norway (8). Catechin has been shown to inhibit the in vitro oxidation of LDL (9) and green tea polyphenols decreased the oxidation of LDL induced by macrophages (10). Tea catechins protected LDL cholesterol and apoB protein fragmentation by cupric ions (11). These compounds also protected LDL from oxidation by metmyoglobin (12). The present research was done to ascertain the effectiveness of tea components by the following: compare compounds present in teas with other antioxidants, I:; compare teas with other high phenolic beverages, (c) determine which purified tea fraction was most responsible for teas' antioxidant activity, and (d) investigate possible binding of tea antioxidants with lipoproteins.
Beverages and teas were obtained from local supermarkets and liquor stores. World blend tea was prepared from commercial grade tea leaves and provided by Lipton Tea Company. Teas were prepared by extracting 12.5 g of tea for 5 minutes with 2 successive 500 ml portions of boiling water. This is a typical concentration for human consumption (1). Purified tea fractions were obtained from Lipton Tea Company and made up to the same 7.25 mg/lO ml of water, before dilution. Black concentration, and green tea extracts were water extracts and black and green Catechin, gallic acid, polyphenols were ethyl acetate extracts. chlorogenic acid and vitamins were purchased from Sigma Chemical Company and pure tea catechins were gifts from Lipton Tea Total polyphenol content of teas, tea fractions and Company. beverages was measured with the Folin-Cocialteu reagent (Sigma) using catechin as the standard. LDL + VLDL, were isolated by affinity column Lipoproteins, from the plasma of a single normocholesterolemic individual who was not consuming antioxidant supplements. The procedure for isolation of the lipoproteins, incubation with the oxidant cupric ion, measurement of lipid peroxidation, determination of the
TEA PHENOL ANTIOXIDANTS
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concentration of antioxidant for 50% inhibition (I&), and calculation of the phenol antioxidant index (PAOXI) has been previously described (13). PAOXI = total phenols/IC,, and is a combined measure of both quality and quantity of phenol antioxidants. All analyses were done in duplicate. Binding was investigated by single ex vivo spiking experiments (14). Plasma from a single normolipemic individual was spiked with either pure compounds or teas at a phenol concentration of 50, 100 and 200 I.~Mand allowed to equilibrate for 1 hour at room temperature. The isolation and oxidation of the lipoproteins, including any bound antioxidants, were described above. Conjugated diene formation at 37' was automatically monitored at 234 nm in a Genesys 5 spectrophotometer (Milton Roy, Rochester, N.Y.) equipped with an Application II Softcard for kinetic analysis. This allows a total of 8 samples to be analyzed simultaneously. Lag times, a measure of lipoprotein-bound antioxidant activity, were determined graphically by extrapolation of the propagation part of the curve to the linear extension of the time zero absorbance.
A comparison of pure antioxidants and tea fractions was done in Table 1. The tea catechins were much better antioxidants, i.e., a lower ICsO, than the vitamins C, E and the provitamin B-carotene in this lipoprotein oxidation model. The best antioxidant, epigallocatechin gallate, was 30 times more potent on a mole basis than the phenolic vitamin tocopherol. We have found that many natural polyphenols and flavonoids were much better antioxidants than vitamins (15). Of the tea fractions the order of activity/mole phenol was green tea polyphenols > black tea polyphenols > decaffeinated green tea extract - green tea extract > theaflavins > decaffeinated black tea extract - black tea extract. In Table 2 are shown the antioxidant effectiveness of some common juice beverages known to be high in flavonoids, such as wines and grape juice as compared with teas. Teas had the highest phenol content of all the beverages including red wine. The best antioxidant (i.e., the lowest IC,,) among the green and black teas was the world blend green tea. The best black and green teas were stronger antioxidants than the best grape juices or wines. The average I& of green tea was 0.52 + 0.33 PM and 0.62 + 0.05 for oolong tea and 0.86 + 0.32 PM for black tea. The PAOXI of teas was very much higher than other beverages including red wine. The average PAOXI of green teas was 37258 + 27370, oolong teas 26067 + 3364 and for black teas 25586 + 5254. There was no significant difference for the 3 types of teas in any of these antioxidant parameters or in total phenols. The polyphenols in tea, often called tannins, are well known for their binding properties. Indeed a common method to analyze total tannins is the precipitation of a protein such as albumin and the measurement of the protein content in the supernatant or pellet (16). Recently it was shown that tea catechins bound strongly to cholesterol and fatty acids (17). Lipoprotein-bound antioxidant activity of vitamin E and the tea component epigallocatechin gallate were measured and compared with the
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J.A. VINSON and Y.A. DABBAGH
Effectiveness
TABLE 1 of Pure Antioxidants
and Tea Fractions
Antioxidant/Vitamin ICso CAM VITAMINS 4.30 p-carotene 2.40 vitamin E 1.25 vitamin C TEA PHENOLS 1.25 gallic acid 0.67 catechin 0.30 chlorogenic acid 0.19 epicatechin epicatechin gallate 0.14 epigallocatechin 0.10 epigallocatechin gallate 0.08 TEA FRACTIONS black tea extract (46.32% PP*) 0.59 decaffeinated black tea extract (47.08% PP) 0.59 theaflavins 0.29 green tea extract (46.19% PP) 0.22 decaffeinated green tea extract (49.15% PP) 0.22 purified black tea polyphenols (91.38% PP) 0.16 0.13 purified green tea polyphenols (95.22% PP) *Percent of solids as polyphenols (PP) in the tea fractions and tea extracts was analyzed by Matthew E. Harbowy of T. J. Lipton Company. world blend black and green teas and the results displayed in Figure 1. Black tea was a better bound antioxidant than green tea at the low concentration but the reverse was true at higher concentration. Overall the two types of tea were not significantly different in their lipoprotein-bound antioxidant activity nor were they significantly different from tocopherol or epigallocatechin gallate. Tea's lipoprotein-bound antioxidant activity, as measured by the % change in lag time, correlated with the concentration of polyphenols (Pearson correlation coefficient r = 0.896) for green tea and for black tea (r = 0.966).
Catechins in tea are able to act as antioxidants by chelating with metal ions including cupric ions (18, 19). However, the cupric ion concentration in this in vitro model is too high (25 PM) compared with the micro and submicromolar concentrations of phenols to allow for chelation; the mechanism is thus a free radical scavenging one. In examining the antioxidant activity of the pure compounds, the tea phenols were better antioxidants than tocophenol, the phenolic vitamin, as also seen by Hara (20). The individual tea phenols, catechins and phenolic acids, were also better antioxidants than vitamin C. Since ascorbic acid is the best vitamin antioxidant in plasma (21), tea compounds once absorbed, could contribute significantly to the antioxidants in the plasma. The order of antioxidant activity of the catechins was the same as that seen by Ho (22) for air-induced lard oxidation by the Rancimat method, namely
TEA PHENOL ANTIOXIDANTS
Comparative
Antioxidant
TABLE 2 Effectiveness
of Juices
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and Teas
Beverage Phenol(pM) PAOXI IC,,(pM)* JUICES grape juices (n=6)** concord 6671 0.65 10263 white wines (n=2) riesling 715 0.77 929 red wines (n=5) petite syrrah 9573 0.45 21273 prune juice (n=2) 5628 0.30 18760 pineapple juice (n=2) 4516 1.39 3249 GREEN TEAS world blend 17400 0.23 75652 Japanese 12665 0.35 36185 Japanese 12622 0.51 24759 Chinese 12315 0.99 12439 OOLONG TEAS Chinese 16531 0.60 27551 Chinese 16209 0.57 28436 Chinese 14885 0.67 22216 BLACK TEAS world blend 11987 0.38 31544 Ceylon 22356 1.07 20901 darjeeling 28455 1.00 28455 Chinese 21443 1.00 21443 of tea fractions and beverages the *For I&" determinations phenol concentrations as measured by the Folin reagent in /.LMunits were used. **n = number of different types of juices tested for phenol content: the one with the highest value was then tested for I&, and PAOXI. epigallocatechin gallate > epigallocatechin > epicatechin gallate The strongest single antioxidant epigallocatechin > epicatechin. gallate is also the major catechin component of both black and green tea (23). The highly purified tea fractions' antioxidant effectiveness (IC,,) was strongly inversely correlated with the % polyphenol This was not the case with the teas and content (1:= -0.999). beverages in Table 2 which was probably due to the contribution of other constituents acting as antioxidants. Lunder found that the antioxidant activity of different teas correlated well with the epigallocatechin gallate content (24). Caffeine did not seem to have an antioxidant effect in this model as the caffeinated extracts had the same antioxidant power as the corresponding Theaflavins, the products of oxidation of decaffeinated samples. catechins, were still very strong antioxidants and compared very favorably with the other tea fractions. Theaflavins were stronger antioxidants than the vitamins and even some catechins for hydroperoxide-induced liver homogenate oxidation (25). Simic found that theaflavin reacted with superoxide radicals over 10 times faster than EGCG (26). It appears that the antioxidant activity of the tea is produced by both the catechins and the oxidation products such as theaflavins. Similarly, the LDL antioxidant activity of wine was the result of the many phenolic constituents (27). The similar concentrations of polyphenols in green and black tea as found using the Folin assay were also shown using Polyclar
J.A. VINSON and Y.A. DABBAGH
1072
E c= 150 z? -I .c s 100 !?? 2 50 *0 0
50 uM
q Vitamin E
100 uM Phenol Concentration mEGCG
q
GreenTea
200 uM
n
BlackTea
Figure 1 Percent increase in LDL + VLDL lag time compared with control for antioxidants added to plasma at 3 different concentrations. Antioxidants were allowed to equilibrate with lipoproteins and the lower density lipoproteins isolated and oxidized with cupric ions. EGCG = epigallocatechin gallate.
a
AT absorbables as the assay procedure (23). Green teas were stronger antioxidants than black teas as measured by I& and also contained a greater dose of effective antioxidants on a given volume basis as determined by PAOXI. Oolong teas were intermediate between green and black tea. However, there was no significant difference between the types of teas for any of the antioxidant parameters. Only a small antioxidant difference, 17% higher for green tea as compared to black tea, was found by Yoshino (25). The greater catechin content of green tea is apparently countered by the greater theaflavins, thearubigens and phenolic acids content of black tea (1). The equivalence of the antioxidant parameters of both teas is also found in the similarity of the health benefits of black and green tea (28). In this study tea was compared with grape juice and wine and found other fruit juices commonly consumed superior as an antioxidant; had much lower phenol content and PAOXI than tea. The results shown here show the antioxidant effectiveness of Of course, the tea compounds, fractions and the teas themselves. question to be answered is whether they are in viva antioxidants. Both There is no doubt that tea phenols are absorbed by humans. green and black tea consumption produced increases in plasma The first in viva experiment demonstrating polyphenols (29, 30). tea as antioxidant has shown that both green and black tea increased the plasma antioxidant activity of humans 41-48% Our spiking results following consumption of 300 ml of tea (31). suggest that the consumption of tea should enrich lipoproteins with powerful tea polyphenol antioxidants that would provide
TEA PHENOL ANTIOXIDANTS
1073
protection against oxidation in vivo. The average intake of tea is about lg/day/person in the USA (32). Since the polyphenols in tea correspond to 20-30% of solids, the total tea phenols ingestion is between 200-300 mg/day. This dose is much greater than the total RDA of vitamins C, E and P-carotene that is 70 mg/day (33) and thus tea phenols are calculated to be a large source of dietary antioxidants.
ACKNOWLEDGMENTS Support for this research was provided by grants from Lipton Tea Company and the Faculty Research and Development Fund of the University of Scranton. The authors wish to thank Jinhee Jang for experimental data on the vitamins and Jihong Yang for the juice data. REFERENCES 1. 2.
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GRAHAM, H.N. Green tea composition, consumption and phenol chemistry. Preventive Med. 21: 334-350, 1992. STEINBERG, D., PARTHASARATHY, S., CAREW, T.E., KHOO, J.C. and WITZUM, J.L. Beyond cholesterol: modification of low density lipoprotein that increases its atherogenicity. New EDg. J. I.&d. u: 915-924, 1989. DUELL, P.B. Dietary antioxidants and atherosclerosis. Curr. Diab., 1st &., 251-259, 1994. KINSELLA, J.E., FRANKEL, E., GERMAN, B. and KANNER, J. Possible mechanisms for the protective role of antioxidants in wine and plant foods. Food Technol. 47: 85-89, 1993. HERTOG, M.G.L., FESKENS, E.J.M., HOLLMAN, P.C.H., KATAN, M.B. and KROMHOUT, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zuphen elderly study. Lank& 342: 1007-1011, 1993. HERTOG, M.G.L., KROHOUT, D., ARVANIS, C., BLACKBURN, H., BUZINA, R., FIDANZA, F., GIAMPAOLI, S., JANSEN, A., MERLOTTI, A., NEDJKOVIC, S., PEKKARINEN, M., BOZIDAR, S.S., TOSHIMA, H., FESKENS, F.J.M., HOLLMAN, P.C.H. and KATAN, M.B. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch. Intern. Med. l5.5: 381-386, 1995. KONO, S., SHINCHI, K., IKEDA, N., YANAI, F. and IMANISHI, K. Green tea consumption and serum lipid profiles: a crosssectional study in Northern Kyushu, Japan. Preventive Med. 21: 526-531, 1992. STENSVOLD, I., TVERDAY, A., SOLVOLL, K. and FOSS, O.P. Tea consumption. Relationship to cholesterol, blood pressure and coronary and total mortality. Preventive Med. 21: 546-553, 1992. MANGIAPANE, H., THOMSON, J., SALTER, A., BROWN, S., BELOL, G.D. and WHITE, D.A. The inhibition of oxidation of low density lipoprotein by (+)-catechin, a naturally occurring flavonoid. Biochem.. a: 445-450, 1992. ZHENHUA, D., YUAN, C., MEI, Z. and YUNZHONG, F. Inhibitor effect of China green tea polyphenol on the oxidative modification of low density lipoprotein by macrophages. Sci. Bes. u: 767-768, 1991. MIURA, S., WATANABE, J., TOMITA, T., SANO, M. And TOMITA, I. The inhibitory effects of tea polyphenols (flavan-3-ol-
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derivatives) on cupric ion mediated oxidative modification of low density lipoprotein. Bial.. u: 15671572, 1994. SALAH, N., MILLER, N.J., PAGANGA, G., TIJBURG, L., BOLWELL, G.P. and RICE-EVANS, C. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chainbreaking antioxidants. Arch. vBiaahvs. 322: 339-342, 1995. VINSON, J.A. and HONTZ, B.A. Phenol antioxidant index; comparative antioxidant effectiveness of red and white wines. J- 4gric. Foad. &: 401-403, 1995a. VINSON, J.A., JANG, J., DABBAGH, Y.A., SERRY, M.M. and CAI, S. Plant polyphenols exhibit lipoprotein-bound antioxidant activity using an in vitro oxidation model for heart disease. J. Agric. Food. u: 2798-2799, 199533. VINSON, J.A., DABBAGH, Y.A., SERRY, M.M. and JANG, J. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric. Food. 4_3: 2800-2802, 1995c. . A Guide tQ HARBONNE, J.B. In Pbvtowda. Chapman and Hall, M&--n !k&rkgws of Plant # New York, 1988, pp. 87-88. IKEDA, I., IMASOTO, Y., SASAKI, E., NAKAYAMA, M., NAGAO, H., TAKEO, T., YAYABE, F. and SUGANO, M. Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats. Biochim. . 141-146, 1992. ac;ta 1122: VAN ACKER, S.A.B.E., VAN DEN BERG, D.-J., TROMP, M.N.J.L., GRIFFIOEN, E.H., VAN BENNEKM, W.P., VAN DER JIJGH, W.J.F. Structural aspects of antioxidant activity and BAST, A. of flavonoids. Free. Med. 2a: 331-342, 1996. MILLER, N.J., CASTELLUCCIO, C., TIJBURG, L. and RICE-EVANS, C. The antioxidant properties of theaflavins and their gallate esters-radical scavengers or metal chelators? nT,etts. m: 40-44, 1996. Prophylactic functions of tea polyphenols. In: HARA, Y. Phv-for PI-P. C.T. Ho, T. Osawa, M.T. Huang and R.T. Rosen, (eds). American Chemical Society, Washington, D.C., 1994, pp. 34-50. Ascorbate is an FREI, B., ENGLAND, L. and AMES, B.N. outstanding antioxidant in human blood plasma. Proc.. Sci. 86: 6377-6386, 1989. HO, C.T., CHEN, Q., SHI, H., ZHANG, K.Q. and ROSEN, R.T. Antioxidant effect of polyphenol extract prepared from various Chinese teas. Preventive Med. 21: 520-525, 1992. WANG, Z.Y., HUANG, M.T., LOU, Y-R., XIE, J-G., REUHL, K.R., NEWMARK, H.I., Ho, C.T., YANG, C.S. and Inhibitory effects of black tea, green tea, CONNEY, A.H. decaffeinated black tea and decaffeinated green tea on ultraviolet B light-induced skin carcinogenesis in 7-12dimethylbenz[a]anthracene-initiated SKH-1 mice. Cancer m. s: 3428-3435, 1994. Catechins of green tea. In Phenalic LUNDER, T.L. on Health : M.T. Huang, C.T. Ho, C.Y. Lee, (eds). American Chemical Society, Washington, D.C., 1992, pp. 114-119. YOSHINO, K., HARA, Y., SANO, M. and TOMITA, I. Antioxidative effects of black tea theaflavins and thearubigin
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28. 29.
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31. 32. 33.
on lipid peroxidation of rat liver homogenates induced by tert-butyl hydroperoxide. Biol.. 1u: 146-149, 1994. JOVANOIC, S.V., HARA, Y., STEENKEN and SIMIC, M.G. Antioxidant potential of theaflavins. A pulse radiolysis study. w. JJ.2: 5337-5343, 1997. SIMONETTI, P., PIETTA, P. and TESTOLIN, G. Polyphenol content and total antioxidant potential in Italian wines. ic. Food. 45: 1152-1155, 1997. DREOSTI, I.E. Bioactive ingredients: antioxidants and Nutr.Revs. 5/&: 551-558, 1996. polyphenols in tea. HE, Y.H. and KIES, C. Green and black tea consumption by Impact on polyphenol concentrations in feces, humans. blood and urine. mt Fooda Hum. Nuti. u: 221-229, 1994. Lee, M.-J., WANG, Z.-Y., LI, H., CHEN, C., SUN, Y., Analysis of GOBBO, S., BALENTINE, D.A. and Yang, C.S. plasma and urinary tea polyphenols in human subjects. Prey. 4: 393-399, 1995. SERAFINI, M., GHISELLI, A. and FERRO-LUZZI, A. Red wine, I,ancet m: 626, 1994. tea and antioxidants. WHO International Agency for Research on Cancer. m Riw. u: 207-271, 1991. National Research Council Food and Nutrition Board. Recommended Daily Allowances, 10th ed., National Academy Press, 1989, Washington, D.C.
Acceptedforpublication February 21, 1998
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TEA PHENOLS: ANTIOXIDANT EFFECTIVENESS OF TEAS, TEA COMPONENTS, FRACTIONS AND THEIR BINDING WITH LIPOPROTEINS Joe A. Vinson. Ph.D.. Yousef A. Dabbagh, M.A. Department of Chemistry, University of Scranton, Scranton,
TEA
PA
18510
Phenols in tea are responsible for _. its antioxidant The pure catechins and phenolic acids found activity. in tea are more powerful than the antioxidant vitamins C, E and S-carotene in an in vitro lipoprotein oxidation model. Comparison of the tea fractions indicated that both catechins and theaflavins contribute to the teas' antioxidant characteristics. Black and green teas were not significantly different in phenol content, in antioxidant strength as measured by I&, or in antioxidant potential as measured by the phenol antioxidant index (PAOXI). The PAOXI of teas was significantly Tea catechins and higher than grape juices and wines. both green and black tea exhibited potent lipoproteinbound antioxidant activity. Phenol antioxidants from tea were calculated to be a large source of antioxidants in the U.S. diet. 0 1998 Elsevia Sciencelx. Key Words: Tea, Catechins, Antioxidant, Lipoproteins, Lipid Peroxidation
The most popular beverage in the world is tea prepared from leaves of Camellia sinensis. There are two major varieties, sinensis and assamica and tea is cultivated in more than 30 countries. The composition of tea varies with season, the age of the leaf, climate and horticultural practices. To make green tea, the leaves are chopped, rolled and quickly steamed or heated For black tea the leaves which inactivates polyphenol oxidase. are kept warm for 6 hours and the leaf polyphenols, especially Oolong tea is the catechins, are oxidized and condensed. intermediate between green and black tea and is produced by This process retains a heating in air for one or two hours. considerable amount of the original catechins of green tea and By tradition the Orient, also produces some unique products. North Africa and the Middle East favor green tea and the rest of the world drinks black tea. Oolong tea production is confined to China and Taiwan. Black tea accounts for 80% of the world tea crop (1). Phone To whom requests for reprints should be addressed. 717-941-7551, Fax 717-941-7510, email [email protected]. 1067
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J.A. VINSON and Y.A. DABBAGH
Since the introduction of the oxidation theory of atherogenesis (2), there has been increased interest and research into the effect of antioxidants on heart disease. Cholesterol as low density lipoprotein (LDL) and very low density lipoprotein (VLDL) is rendered atherogenic by oxidation that causes an accelerated foam cell development and plague formation. The effect of dietary antioxidants on atherosclerosis was reviewed in 1994 (3). The mechanisms by which food antioxidants are protective for heart disease have recently been reviewed (4). Flavonoids in the Dutch diet were inversely associated with risk of coronary heart disease and tea provides most of the flavonoids in their diet (5). A more recent epidemiology study of seven countries showed a cross-cultural association of flavonoid consumption and a corresponding reduction in heart disease risk (6). The Japanese, who average 7 cups of tea/day, have an extremely low incidence of heart disease despite a high percentage of smokers. Pharmaceutical benefits of tea such as reduction in serum cholesterol level and systolic blood pressure have been reported in epidemiological studies in Japan (7) and Norway (8). Catechin has been shown to inhibit the in vitro oxidation of LDL (9) and green tea polyphenols decreased the oxidation of LDL induced by macrophages (10). Tea catechins protected LDL cholesterol and apoB protein fragmentation by cupric ions (11). These compounds also protected LDL from oxidation by metmyoglobin (12). The present research was done to ascertain the effectiveness of tea components by the following: compare compounds present in teas with other antioxidants, I:; compare teas with other high phenolic beverages, (c) determine which purified tea fraction was most responsible for teas' antioxidant activity, and (d) investigate possible binding of tea antioxidants with lipoproteins.
Beverages and teas were obtained from local supermarkets and liquor stores. World blend tea was prepared from commercial grade tea leaves and provided by Lipton Tea Company. Teas were prepared by extracting 12.5 g of tea for 5 minutes with 2 successive 500 ml portions of boiling water. This is a typical concentration for human consumption (1). Purified tea fractions were obtained from Lipton Tea Company and made up to the same 7.25 mg/lO ml of water, before dilution. Black concentration, and green tea extracts were water extracts and black and green Catechin, gallic acid, polyphenols were ethyl acetate extracts. chlorogenic acid and vitamins were purchased from Sigma Chemical Company and pure tea catechins were gifts from Lipton Tea Total polyphenol content of teas, tea fractions and Company. beverages was measured with the Folin-Cocialteu reagent (Sigma) using catechin as the standard. LDL + VLDL, were isolated by affinity column Lipoproteins, from the plasma of a single normocholesterolemic individual who was not consuming antioxidant supplements. The procedure for isolation of the lipoproteins, incubation with the oxidant cupric ion, measurement of lipid peroxidation, determination of the
TEA PHENOL ANTIOXIDANTS
1069
concentration of antioxidant for 50% inhibition (I&), and calculation of the phenol antioxidant index (PAOXI) has been previously described (13). PAOXI = total phenols/IC,, and is a combined measure of both quality and quantity of phenol antioxidants. All analyses were done in duplicate. Binding was investigated by single ex vivo spiking experiments (14). Plasma from a single normolipemic individual was spiked with either pure compounds or teas at a phenol concentration of 50, 100 and 200 I.~Mand allowed to equilibrate for 1 hour at room temperature. The isolation and oxidation of the lipoproteins, including any bound antioxidants, were described above. Conjugated diene formation at 37' was automatically monitored at 234 nm in a Genesys 5 spectrophotometer (Milton Roy, Rochester, N.Y.) equipped with an Application II Softcard for kinetic analysis. This allows a total of 8 samples to be analyzed simultaneously. Lag times, a measure of lipoprotein-bound antioxidant activity, were determined graphically by extrapolation of the propagation part of the curve to the linear extension of the time zero absorbance.
A comparison of pure antioxidants and tea fractions was done in Table 1. The tea catechins were much better antioxidants, i.e., a lower ICsO, than the vitamins C, E and the provitamin B-carotene in this lipoprotein oxidation model. The best antioxidant, epigallocatechin gallate, was 30 times more potent on a mole basis than the phenolic vitamin tocopherol. We have found that many natural polyphenols and flavonoids were much better antioxidants than vitamins (15). Of the tea fractions the order of activity/mole phenol was green tea polyphenols > black tea polyphenols > decaffeinated green tea extract - green tea extract > theaflavins > decaffeinated black tea extract - black tea extract. In Table 2 are shown the antioxidant effectiveness of some common juice beverages known to be high in flavonoids, such as wines and grape juice as compared with teas. Teas had the highest phenol content of all the beverages including red wine. The best antioxidant (i.e., the lowest IC,,) among the green and black teas was the world blend green tea. The best black and green teas were stronger antioxidants than the best grape juices or wines. The average I& of green tea was 0.52 + 0.33 PM and 0.62 + 0.05 for oolong tea and 0.86 + 0.32 PM for black tea. The PAOXI of teas was very much higher than other beverages including red wine. The average PAOXI of green teas was 37258 + 27370, oolong teas 26067 + 3364 and for black teas 25586 + 5254. There was no significant difference for the 3 types of teas in any of these antioxidant parameters or in total phenols. The polyphenols in tea, often called tannins, are well known for their binding properties. Indeed a common method to analyze total tannins is the precipitation of a protein such as albumin and the measurement of the protein content in the supernatant or pellet (16). Recently it was shown that tea catechins bound strongly to cholesterol and fatty acids (17). Lipoprotein-bound antioxidant activity of vitamin E and the tea component epigallocatechin gallate were measured and compared with the
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J.A. VINSON and Y.A. DABBAGH
Effectiveness
TABLE 1 of Pure Antioxidants
and Tea Fractions
Antioxidant/Vitamin ICso CAM VITAMINS 4.30 p-carotene 2.40 vitamin E 1.25 vitamin C TEA PHENOLS 1.25 gallic acid 0.67 catechin 0.30 chlorogenic acid 0.19 epicatechin epicatechin gallate 0.14 epigallocatechin 0.10 epigallocatechin gallate 0.08 TEA FRACTIONS black tea extract (46.32% PP*) 0.59 decaffeinated black tea extract (47.08% PP) 0.59 theaflavins 0.29 green tea extract (46.19% PP) 0.22 decaffeinated green tea extract (49.15% PP) 0.22 purified black tea polyphenols (91.38% PP) 0.16 0.13 purified green tea polyphenols (95.22% PP) *Percent of solids as polyphenols (PP) in the tea fractions and tea extracts was analyzed by Matthew E. Harbowy of T. J. Lipton Company. world blend black and green teas and the results displayed in Figure 1. Black tea was a better bound antioxidant than green tea at the low concentration but the reverse was true at higher concentration. Overall the two types of tea were not significantly different in their lipoprotein-bound antioxidant activity nor were they significantly different from tocopherol or epigallocatechin gallate. Tea's lipoprotein-bound antioxidant activity, as measured by the % change in lag time, correlated with the concentration of polyphenols (Pearson correlation coefficient r = 0.896) for green tea and for black tea (r = 0.966).
Catechins in tea are able to act as antioxidants by chelating with metal ions including cupric ions (18, 19). However, the cupric ion concentration in this in vitro model is too high (25 PM) compared with the micro and submicromolar concentrations of phenols to allow for chelation; the mechanism is thus a free radical scavenging one. In examining the antioxidant activity of the pure compounds, the tea phenols were better antioxidants than tocophenol, the phenolic vitamin, as also seen by Hara (20). The individual tea phenols, catechins and phenolic acids, were also better antioxidants than vitamin C. Since ascorbic acid is the best vitamin antioxidant in plasma (21), tea compounds once absorbed, could contribute significantly to the antioxidants in the plasma. The order of antioxidant activity of the catechins was the same as that seen by Ho (22) for air-induced lard oxidation by the Rancimat method, namely
TEA PHENOL ANTIOXIDANTS
Comparative
Antioxidant
TABLE 2 Effectiveness
of Juices
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and Teas
Beverage Phenol(pM) PAOXI IC,,(pM)* JUICES grape juices (n=6)** concord 6671 0.65 10263 white wines (n=2) riesling 715 0.77 929 red wines (n=5) petite syrrah 9573 0.45 21273 prune juice (n=2) 5628 0.30 18760 pineapple juice (n=2) 4516 1.39 3249 GREEN TEAS world blend 17400 0.23 75652 Japanese 12665 0.35 36185 Japanese 12622 0.51 24759 Chinese 12315 0.99 12439 OOLONG TEAS Chinese 16531 0.60 27551 Chinese 16209 0.57 28436 Chinese 14885 0.67 22216 BLACK TEAS world blend 11987 0.38 31544 Ceylon 22356 1.07 20901 darjeeling 28455 1.00 28455 Chinese 21443 1.00 21443 of tea fractions and beverages the *For I&" determinations phenol concentrations as measured by the Folin reagent in /.LMunits were used. **n = number of different types of juices tested for phenol content: the one with the highest value was then tested for I&, and PAOXI. epigallocatechin gallate > epigallocatechin > epicatechin gallate The strongest single antioxidant epigallocatechin > epicatechin. gallate is also the major catechin component of both black and green tea (23). The highly purified tea fractions' antioxidant effectiveness (IC,,) was strongly inversely correlated with the % polyphenol This was not the case with the teas and content (1:= -0.999). beverages in Table 2 which was probably due to the contribution of other constituents acting as antioxidants. Lunder found that the antioxidant activity of different teas correlated well with the epigallocatechin gallate content (24). Caffeine did not seem to have an antioxidant effect in this model as the caffeinated extracts had the same antioxidant power as the corresponding Theaflavins, the products of oxidation of decaffeinated samples. catechins, were still very strong antioxidants and compared very favorably with the other tea fractions. Theaflavins were stronger antioxidants than the vitamins and even some catechins for hydroperoxide-induced liver homogenate oxidation (25). Simic found that theaflavin reacted with superoxide radicals over 10 times faster than EGCG (26). It appears that the antioxidant activity of the tea is produced by both the catechins and the oxidation products such as theaflavins. Similarly, the LDL antioxidant activity of wine was the result of the many phenolic constituents (27). The similar concentrations of polyphenols in green and black tea as found using the Folin assay were also shown using Polyclar
J.A. VINSON and Y.A. DABBAGH
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E c= 150 z? -I .c s 100 !?? 2 50 *0 0
50 uM
q Vitamin E
100 uM Phenol Concentration mEGCG
q
GreenTea
200 uM
n
BlackTea
Figure 1 Percent increase in LDL + VLDL lag time compared with control for antioxidants added to plasma at 3 different concentrations. Antioxidants were allowed to equilibrate with lipoproteins and the lower density lipoproteins isolated and oxidized with cupric ions. EGCG = epigallocatechin gallate.
a
AT absorbables as the assay procedure (23). Green teas were stronger antioxidants than black teas as measured by I& and also contained a greater dose of effective antioxidants on a given volume basis as determined by PAOXI. Oolong teas were intermediate between green and black tea. However, there was no significant difference between the types of teas for any of the antioxidant parameters. Only a small antioxidant difference, 17% higher for green tea as compared to black tea, was found by Yoshino (25). The greater catechin content of green tea is apparently countered by the greater theaflavins, thearubigens and phenolic acids content of black tea (1). The equivalence of the antioxidant parameters of both teas is also found in the similarity of the health benefits of black and green tea (28). In this study tea was compared with grape juice and wine and found other fruit juices commonly consumed superior as an antioxidant; had much lower phenol content and PAOXI than tea. The results shown here show the antioxidant effectiveness of Of course, the tea compounds, fractions and the teas themselves. question to be answered is whether they are in viva antioxidants. Both There is no doubt that tea phenols are absorbed by humans. green and black tea consumption produced increases in plasma The first in viva experiment demonstrating polyphenols (29, 30). tea as antioxidant has shown that both green and black tea increased the plasma antioxidant activity of humans 41-48% Our spiking results following consumption of 300 ml of tea (31). suggest that the consumption of tea should enrich lipoproteins with powerful tea polyphenol antioxidants that would provide
TEA PHENOL ANTIOXIDANTS
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protection against oxidation in vivo. The average intake of tea is about lg/day/person in the USA (32). Since the polyphenols in tea correspond to 20-30% of solids, the total tea phenols ingestion is between 200-300 mg/day. This dose is much greater than the total RDA of vitamins C, E and P-carotene that is 70 mg/day (33) and thus tea phenols are calculated to be a large source of dietary antioxidants.
ACKNOWLEDGMENTS Support for this research was provided by grants from Lipton Tea Company and the Faculty Research and Development Fund of the University of Scranton. The authors wish to thank Jinhee Jang for experimental data on the vitamins and Jihong Yang for the juice data. REFERENCES 1. 2.
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Acceptedforpublication February 21, 1998
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