inhibitory effect of green tea on the growth of ... · green tea leaves (special gunpowder type)...

[CANCER RESEARCH 52, 6657-6665, December 1, 1992]

Inhibitory Effect of Green Tea on the Growth of Established Skin Papillomas in M i c e 1

Zhi Y. Wang, Mou-Tuan Huang, Chi-Tang Ho, Richard Chang, Wei Ma, Thomas Ferraro, Kenneth R. Reuhl, Chung S. Yang, and Allan H. Conney 2 Laboratory for Cancer Research, Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway 08855 [Z. Y. W., M-T. H., R. C., W. M., T. F., C. S. Y., A. H. C.]; Department of Food Science, Cook College, Rutgers, The State University of New Jersey, New Brunswick 08901 [C-T. H.]; and Neurotoxicology Laboratories, Department of Pharmacology and Toxicology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway 08855 [K. R. R.], New Jersey

A B S T R A C T

In 10 separate experiments, mice with established chemically induced or UV light-induced skin papillomas were treated continuously with green tea in the drinking water or with i.p. injections of a green tea polyphenol fraction or (-)-epigallocatechin gallate three times a week for 4-10 weeks. Partial tumor regression or >90% inhibition of tumor growth, as measured by changes in tumor volume per mouse, was observed in 5 experiments, and marked inhibition of tumor growth (46-89%) was observed in 5 additional experiments. Treatment of the mice with green tea or green tea constituents had an inhibitory effect on body weight increases in several but not all of the studies. Examination of the data from all ten experiments revealed that complete tumor regression occurred in 14 of 346 papilloma-bearing mice (4%) that were treated with green tea in the drinking water or with i.p. injections of green tea constituents, whereas none of the 220 papilloma-bearing control mice treated with only vehicle exhibited complete tumor regression. These observations indicate that oral administration of green tea, i.p. administration of a green tea polyphenol fraction, or i.p. administration of (-)-epigailocatechin gallate inhibited the growth and/or caused the regression of established experimentally induced skin papillomas.

I N T R O D U C T I O N

Studies during the past few years have indicated an inhibitory effect of green tea or green tea consti tuents on tumorigenesis in mice and rats (1-11). Recent chemoprevent ion studies from our laboratory demonstra ted that oral adminis t ra t ion of green tea as the sole source of drinking water inhibited UVB-induced 3 ini t iat ion of skin tumors in SKH-1 mice, TPA-induced promot ion of skin tumors in UVB-ini t ia ted SKH-1 mice, UVB- induced formation of skin tumors in SKH-1 mice previously init iated with DMBA, NNK-induced lung tumors in A/J mice, and N-ni t rosodiethylamine-induced forestomach and lung tumors in A/J mice (8, 9). In addit ion to decreasing the incidence and number of skin tumors in DMBA-ini t ia ted mice treated with UVB, oral adminis t ra t ion of green tea during the UVB treatment regimen resulted in markedly decreased tumor size (8). A similar inhibitory effect of orally administered green tea on the size of skin tumors was observed in DMBA-ini t ia ted mice that were given green tea orally during promot ion with T P A : In both of these chemoprevent ion studies, tumors from animals treated with green tea together with UVB or TPA were

Received 6/30/92; accepted 9/24/92. The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

I Supported in part by Grants CA49756 and ES05022 from the NIH. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: UVB, ultraviolet B light; HPLC, high-performance

liquid chromarography; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMBA, 7,12-dimethylbenz[a]anthracene; NNK, 4-(methylnitrosamino)- 1-(3-pyridyl)-l- butanone.

4 M-T. Huang, Z. Y. Wang, T. Ferraro, C. S. Yang, and A. H. Conney, unpub- lished observations.

only 15-25% as large as tumors from the carcinogen-treated positive controls. These observations suggested that green tea inhibited the formation and/or growth of skin tumors. In the present study, we investigated the possible inhibitory effects of the adminis t ra t ion of green tea and green tea consti tuents on the growth of previously established papil lomas in tumor-bearing mice. We found that green tea and some of its const i tuents had a marked inhibitory effect on the growth of preformed skin papillomas. In some experiments, regression of skin tumors was also observed.

M A T E R I A L S A N D M E T H O D S

Materials. DMBA was purchased from the Sigma Chemical Co. (St. Louis, MO). TPA was purchased from the LC Services Co. (Woburn, MA). Green tea leaves (special gunpowder type) were ex- ported by the China National Native Produce and Animal By-Products Import and Export Corporation, Zhejiang Tea Branch (Zhejiang, China) and purchased from the Kam Kuo Co. (New York, NY). Instant green tea powder (dehydrated water extract) was supplied by the Thomas J. Lipton Co. (Englewood Cliffs, N J). Purified reverse osmosis water was used for the preparation of green tea samples.

Animals. Female CD-I mice (6-8 weeks old) were purchased from Charles River Laboratories (Kingston, NY). The animals were kept in our animal facility for at least I week before use. Mice were given water and Purina Laboratory Chow 5001 diet (Ralston-Purina Co., St. Louis, MO) ad libitum, and the animals were kept on a 12-h light, 12-h dark cycle.

Preparation and Composition of Green Tea. Green tea leaves (12.5 g, special gunpowder type) were added to 500 ml of freshly boiled water and were steeped for 15 min. The infusion was cooled to room temperature in an ice bath and then filtered. The tea leaves were similarly extracted a second time, and the two filtrates were combined to obtain a 1.25% green tea water extract (1.25 g tea leaves/100 ml water). The amount of solids present in the 1.25% green tea extract was determined to be 4.0 mg/ml by drying the samples in an air convection oven (18 h at 65~ Ten g of instant green tea powder (lyophilized water extract) from the Thomas J. Lipton Co. were added to 1 liter of warm water (60~ to make 1% instant green tea solution (10 mg green tea solids/ ml). In some experiments the tea solutions were diluted 1:1 with water (0.5% instant green tea solution; 5.0 mg of tea solids/ml). The compo- sitions of the 1.25% green tea water extract and the 1% instant green tea solution were determined by HPLC as described earlier (8). The con- centrations of polyphenolic compounds and caffeine in typical samples of these tea preparations are shown in Table 1. These tea solutions were administered to the mice ad libitum as their sole source of drinking water.

Preparation and Composition of Green Tea Polyphenol Fraction. One hundred g of green tea leaves were extracted 3 times with 300 ml of methanol at 50~ for 3 h, and the samples were filtered after each extraction. Solvent was removed from the combined extract with a vacuum rotary evaporator. The residue was dissolved in 500 ml of water (50~ and extracted 3 times with 200 ml hexane and 3 times with 200 ml chloroform. The aqueous phase was extracted 3 times with 180 ml

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INHIBITORY EFFECT OF GREEN TEA ON THE GROWTH OF SKIN PAPILLOMAS

Table I Composition of typical green tea preparations a

1.25% green tea water extract 0.5% instant green tea solution

Component mg/ml % of total solids mg/ml % of total solids

Green tea polyphenol fraction

% of dry solids

Total solids 4.0 100 5.0 100 Total catechins 0.98 24 1.33 27

Catechins (-)-Epigallocatechin gallate 0.49 12 0.53 11 (-)-Epigallocatechin 0.32 8 0.46 9 (-)-Epicatechin gallate 0.10 2 0.14 3 (-)-Epicatechin 0.05 1 0.13 (+)-Catechin 0.02 0.6 0.06

Gallic acid 0.01 0.3 0.005

Caffeine 0.28 7 0.28

3 1

0.1

6

100 82

49 11 14 6 2

0.3

0.8 a Green tea samples were prepared as described in "Materials and Methods."

ethyl acetate, and the ethyl acetate was evaporated under reduced pres- sure. The residue was redissolved in 300 ml water and lyophilized to obtain 8-9 g of dried green tea polyphenol fraction. The composition of the green tea polyphenol fraction was determined by HPLC as described elsewhere (8) and is shown in Table 1.

Preparation of (-)-Epigallocatechin Gallate. Two g of green tea polyphenol fraction in 15 ml of 95% ethanol were applied to a chro- matography column (50 mm x 600 mm) that was packed with 200 g of Sephadex LH-20 gel. The polyphenolic compounds were eluted with 95% ethanol (flow rate = 2.2 ml/min). (-)-Epicatechin gallate, (-)-epigallocatechin gallate, and (-)-epigallocatechin were monitored at 280 nm and were collected after elution for about 12, 16, and 21 h, respectively. Appropriate fractions were pooled and freeze dried. (-)-Epi- gallocatechin gallate was identified by UV spectra and by fast atom bombardment-mass spectrometry with a VG 7070E high-resolution mass spectrometer (VG Analytical, Ltd., Manchester, England). The fast atom bombardment matrix was a 3:1 mixture of dithiothreitol: dithioerythritol, commonly known as "magic bullet." An Ion Tech (Manchester, England) saddle field fast atom bombardment gun pro- duced the bombarding xenon atoms. Spectra were obtained in the positive ion mode. The purity of the (-)-epigallocatechin gallate was 98% as determined by HPLC.

Procedure for the Formation of Skin Papillomas. In experiments 1-4 and 6-9, female CD-1 mice (6 weeks old) were treated once topically with 200 nmol of DMBA in 200 #1 acetone. One week later, the animals were treated with 5 nmol of TPA in 200 #1 acetone twice weekly. After 10-14 weeks of treatment with TPA, some animals had small papillomas with an average tumor volume of 0.9-3 mm3/papilloma (experiments 1, 2, 6, and 7). After 25-30 weeks of treatment with TPA, many of the animals had large papillomas, and the average volume was 9.5-42.5 mm3/papilloma (experiments 3, 4, 8, and 9). In experiments 5 and 10, female CD-1 mice (6 weeks old) were treated with 180 mJ/cm 2 of UVB once a day for 10 days as previously described (8). One week later, the mice were treated topically with 16 nmol of TPA in 200 ~1 acetone twice a week for 30 weeks. These animals had large papillomas, and the average volume was 27-30 mm3/papilloma for the mice in experiment 5 and 206-266 mm3/papilloma for the mice in experiment 10. A summary of the protocols for these experiments is shown in Table 2.

Treatment of Mice with Skin Papillomas. After the generation of papillomas in each of the 10 experiments, tumor-bearing mice were distributed into parallel groups with similar tumor characteristics (number of tumors per mouse, size, etc.). One group served as vehicle- treated controls and the other groups were treated with green tea or green tea constituents. One week after the last application of TPA, the tumor-bearing animals in each experiment were distributed into different groups as described above and were treated with green tea as their sole source of drinking water (Table 3 and Fig. 1, experiments 1-5) or with i.p. injections of green tea polyphenol fraction or (-)-epigallocatechin gallate three times a week (Table 4 and Fig. 2, experiments 6-10). The number of tumors and their size were recorded for 4-10 weeks.

Tumor volume was determined by measuring the three-dimensional size of all tumors and by using the average of the three measurements to calculate radius. Tumor volume was calculated by the formula

41rr 3 Volume=

where r = radius. A summary of the protocols for these experiments is shown in Table 2. For experiments 2 and 7, the skin was removed and fixed in 10% buffered formalin and embedded in Paraplast. Six-~m sections were stained with hematoxylin-eosin for histopathological examination.

R E S U L T S

Composition of Different Green Tea Preparat ions. The com- posit ions of typical samples of 1.25% green tea water extract, 0.5% instant green tea solution, and the dried green tea polyphenol fraction are shown in Table 1. The compounds that were measured (catechins, gallic acid, and caffeine) in both the 1.25% green tea water extract and the 0.5% instant green tea solution accounted for about 32% of the total green tea solids present. Catechins accounted for 24 -27% of the total solids. Storage of the 1.25% green tea water extract or the 0.5% instant green tea for 48 h at room temperature in these studies resulted in only a small decrease (8-12%) in the concentrat ion of tea catechins (data not shown).

Inhibi tory Effect of Administrat ion p.o. of Green Tea on the Growth of Skin Tumors. In five separate studies, we evaluated the effect of adminis t ra t ion p.o. of green tea on the growth and regression of previously established experimental ly induced skin papil lomas in mice. The results of these studies are shown in Table 3 and Fig. 1 (experiments 1-5). Animals with small tumors (average volume = 0.9-3.3 mm 3) were studied in experiments 1 and 2, and animals with larger tumors (average volume = 9.5-42.5 mm 3) were studied in experiments 3-5. Changes with t ime in mean body weight, number of tumors per mouse, volume per tumor, and tumor volume per mouse are shown in Fig. 1. The later parameter was used as a measure of total tumor burden per mouse.

Adminis t ra t ion p.o. of green tea in the drinking water for 6 -10 weeks partially inhibited body weight increases in experiments 1-4, but this did not happen in experiment 5 (Table 3; Fig. 1A).

The mean number of papil lomas per mouse generally decreased in both control animals and in animals treated orally with green tea for 6 -10 weeks (Table 3; Fig. 1B). An exception

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Table 2 Protocols for studying the effect of administration of green tea or green tea constituents on the growth of preformed skin papillomas

Formation of skin Treatment of Experiment papillomas tumor-bearing mice a

1 DMBA (200 nmol) + Green tea water extract TPA (5 nmol) twice a (0.63-1.25%) as the week for 10 weeks sole source of drinking

water for 6 weeks

DMBA (200 nmol) + TPA (5 nmol) twice a week for 10 weeks

Instant green tea (0.5-1.0%) as the sole source of drinking water for 10 weeks


Green tea water extract (1.25%) as the sole source of drinking water for 6 weeks


UVB (180 mJ/cm 2) once daily for 10 days + TPA (16 nmol) twice a week for 30 weeks




Green tea polyphenol fraction (50-75 mg/kg, i.p.) 3 times a week for 8 weeks



(-)-Epigallocatechin gallate (25-50 mg/kg, i.p.) 3 times a week for 8 weeks

Green tea polyphenol fraction (50 mg/kg, i.p.) 3 times a week for 4 weeks

10


UVB (180 mJ/cm 2) once daily for 10 days + TPA (16 nmol) twice a week for 30 weeks

Green tea polyphenol fraction (50 mg/kg, i.p.) 3 times a week for 4 weeks

Green tea polyphenol fraction (50-75 mg/kg, i.p.) 3 times a week for 6 weeks

a Treatment of mice with green tea or green tea constituent was initiated 1 week after discontinuing TPA.

was experiment 5, where the number of papillomas per mouse in the control group continued to increase, and the number of tumors per mouse in the tea group decreased.

The administration p.o. of green tea to tumor-bearing mice inhibited the growth of papillomas (volume per tumor) in each of 5 separate experiments (Table 3; Fig. 1C). The results showed a consistent trend of increasing differences in tumor size between control and green tea-treated mice with time (Fig. 1C).

The administration p.o. of green tea had a strong inhibitory effect on the increase in total tumor volume per mouse (Table 3; Fig. 1D). Tumor volume per mouse is a calculated measure of the total tumor burden per mouse obtained by multiplying the number of tumors per mouse by the volume per tumor at each time point. With the number of tumors per mouse generally declining with time, and the volume per tumor largely increasing with time, tumor volume per mouse thus represents a rea- sonable estimate of total tumor burden at each time point. While administration p.o. of green tea inhibited the growth of tumors (tumor volume per mouse) in all 5 experiments, the data

suggested a greater inhibitory effect of green tea administration on the growth of large tumors (experiments 3-5) than on the growth of small tumors (experiments 1 and 2). The data on time trends suggested that control and oral green tea groups had increasing differences in total tumor volume per mouse with increasing time (Fig. 1D). Administration of 0.63% green tea water extract for 6 weeks (experiment 1), 1.25% green tea water extract for 6 weeks (experiment 1), 0.5% instant green tea for 10 weeks (experiment 2), 1% instant green tea for 10 weeks (experiment 2), 1.25% green tea water extract for 6 weeks (experiment 3), and 1.25% green tea water extract for 6 weeks (experiment 5) inhibited the increase in tumor volume per mouse by 42, 50, 54, 89, 71, and 94%, respectively (see Table 3). For these calculations, we compared the percentage increase in tumor volume per mouse in tumor-bearing control animals treated with vehicle with the percentage increase in tumor volume per mouse in tumor-bearing mice treated with tea or its constituents. In experiment 4, administration p.o. of green tea caused partial tumor regression. In this experiment, tumor volume per mouse increased 431% in control mice and decreased 26% in mice treated with 1.25% green tea in the drinking water for 6 weeks.

Inhibitory Effect of i.p. Administration Of Green Tea Polyphenol Fraction or (-)-Epigallocatechin Gallate on the Growth of Skin Papillomas. We evaluated the effects of i.p. injections of a green tea polyphenol fraction or (-)-epigallocatechin gallate on the growth and regression of skin papillomas in mice in 5 separate experiments, and the results of these studies are shown in Table 4 and Fig. 2 (experiments 6-10). Animals with small tumors (volume = 1.0-2.1. mm 3) were utilized in experiments 6 and 7, whereas animals with larger tumors (volume = 20-266 mm 3) were utilized in experiments 8-10.

Intraperitoneal administration of green tea constituents 3 times a week had an inhibitory effect on body weight gain in experiments 6-8, but there was little or no effect on body weight changes in experiments 9 and 10 (Table 4; Fig. 2//).

The number of papillomas per mouse in the vehicle-treated control groups declined or remained approximately constant during the 4-8-week experimental period, except for the control (saline-injected) group of experiment 7, where the number of tumors per mouse continued to increase during the study period. In experiments 6-9, i.p. administration of the green tea polyphenol fraction or (-)-epigallocatechin gallate decreased the number of papillomas per mouse (Table 4; Fig. 2B).

The i.p. injection of green tea polyphenol fraction or ( - ) - epigallocatechin gallate 3 times a week markedly inhibited tumor growth as measured by changes in volume per papilloma in experiments 6-10 (Table 4; Fig. 2C). As with administration p.o. (Table 3 and Fig. 1, experiments 1-5), there was a greater inhibitory effect of i.p. administration of green tea constituents on the growth of large tumors (Table 4 and Fig. 2, experiments 8-10) than on the growth of small tumors (Table 4 and Fig. 2, experiments 6 and 7). In addition, there was a trend toward increasing differences with time in tumor size between green tea-treated mice and saline-treated controls (Fig. 2).

Data on tumor volume per mouse (volume per tumor x number of tumors per mouse) for experiments 6-10 are shown in Table 4 and Fig. 2D. In each experiment, i.p. administration of green tea constituents 3 times a week for 4-8 weeks inhibited the increase in tumor volume per mouse (total tumor burden) as compared to saline-injected controls. The i.p. administration of green tea constituents had a stronger inhibitory effect (tumor

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Table 3 Effect of administration p.o. of green tea on the growth and regression of skin papillomas in mice

Volume per papilloma Tumor volume per mouse Body weight (g) Papillomas per mouse (ram 3) (mm 3)

Dura- No. tion of % % % %

Treatment a (wk) mice Initial Final change Initial Final change Initial Final change Initial Final change

Experiment 1, DMBA/TPA Water 6 30 34.1 -+ 0.7 35.9 -+ 0.7 5.3 16.4 + 1.8 13.7 -+ 2.0 -17 3.3 -+ 0.3 20.5 _+ 2.5 521 54.1 + 9.2 281 -+ 101 420 0.63%GT 6 31 34.9-+0.7 35.9-+0.6 2.9 16.1 + 1.9 12.9-+ 1.5 -20 2.7+0.2 11.6_+ 1.2 320 43.5_+8.7 150-+34 245 1.25%GT 6 29 35.9_+0.7 33.1 -+0.4 -7.8 15.6-+2.0 11.5-+ 1.6 -26 2.6+0.2 11.2+ 1.6 331 41.4+8.2 129+33 212

Experiment 2, DMBA/TPA Water 10 30 30.6-+0.4 36.1 -+0.8 18.0 7.5+0.5 6.2-+0.6 -17 0.9-+0.1 71.6_+ 11.1 7856 6.5+ 1.0 444_+ 128 6731 0.5% IGT 10 30 30.7 -+ 0.4 33.4 -+ 0.3 8.8 9.2 + 0.8 6.3 -+ 0.8 -32 0.9 + 0.1 41.9 + 7.1 4556 8.3 - 1.8 264 + 72 3081 1.0% IGT 10 30 31.6 -+ 0.4 33.9 -+ 0.5 7.3 9.7 -+ 0.8 4.2 _+ 0.7 -57 1.1 -+ 0.1 21.9 -+ 3.3 1891 10.7 + 1.3 92 -+ 24 760

Experiment 3, DMBA/TPA Water 6 7 40.5 _+ 2.0 43.3 -+ 4.4 6.9 5.6 -+ 0.8 4.3 _+ 0.7 -23 9.5 _+ 2.1 36.7 -+ 10.6 286 53.2 -+ 19.8 158 -+ 47 197 1.25% GT 6 7 43.8-+2.3 40.1_+1.7 -8.5 5.0-+1.1 3.6-+0.5 -28 9.7-+1.9 21.2-+5.6 118 48.5_+8.9 76_+39 57

Experiment 4, DMBA/TPA Water 6 21 42.0_+ 1.4 46.1 +0.2 9.8 8.0+ 1.8 5.8-+ 1.0 -28 42.5+9.0 311.0+85.6 632 340_+95 1804-+528 431 1.25% GT 6 21 41.1 -+ 1.1 42.2 + 0.8 2.7 8.3 + 0.8 5.8 _+ 0.7 -30 33.0 + 5.2 35.0 -+ 6.5 6 274 -+ 94 203 -+ 35 -26

Experiment 5, UVB/TPA Water 6 8 39.0 + 2.3 36.8 + 1.8 -5.6 3.9 + 1.5 4.4 + 2.1 13 30.4 + 16.5 61.4 + 26.2 102 119 + 63 270 _+ 101 127 1.25% GT 6 8 38.6+1.6 37.3+1.3 -3.4 4 .4+1.4 3.4_+1.4 -23 26.6+6.7 37.0+7.5 39 117+37 126_+54 8

a Female CD-I mice (6 weeks old) were initiated with DMBA or UVB and promoted with TPA as described in "Materials and Methods" and in Table 2. One week after stopping TPA administration, tumor-bearing mice were treated with a green tea water extract (GT) or instant green tea (IGT) as their sole source of drinking water for 6-10 weeks. In experiments 1 and 2, there was a stepwise increase in concentration from 25, 50, 75, and 100% of full-strength green tea on days 0, 2, 4 and 6, respectively, followed by full strength green tea thereafter. Full-strength green tea was used throughout experiments 3-5. Measurements of body weight and tumor characteristics were made at the start and conclusion of tea administration. Each value represents the mean + SE. 1.25% GT and 0.5% IGT contained 4.0 and 5.0 mg, respectively, of green tea solids/ml.

volume per mouse) on large tumors (experiments 8-10) than on small tumors (experiments 6 and 7).

The i.p. injection of green tea polyphenol fraction (50 mg/kg) 3 times a week for 8 weeks (experiment 6), green tea polyphenol fraction (75 mg/kg) 3 times a week for 8 weeks (experiment 6), (-)-epigallocatechin gallate (25 mg/kg) 3 times a week for 8 weeks (experiment 7), (-)-epigallocatechin gallate (50 mg/kg) 3 times a week for 8 weeks (experiment 7), or green tea polyphenol fraction (50 mg/kg) 3 times a week for 4 weeks (experiment 9) inhibited the increase in tumor volume per mouse by 68, 90, 71, 69, and 100%, respectively (see Table 4). Intraperitoneal injections of green tea polyphenol fraction caused partial tumor regression (tumor volume per mouse) in experiments 8 and 10 (Table 4; Fig. 2D). In experiment 8, the total tumor volume per mouse for control animals increased from 330 mm 3 at the start of the study to 491 mm 3 at the end of the study (49% increase), whereas the total tumor volume per mouse in animals injected i.p. with green tea polyphenol fraction (50 mg/kg) 3 times a week for 4 weeks decreased from 312 mm a at the start of the study to 199 mm 3 at the end of the study (36% decrease). In experiment 10, the total tumor volume per mouse for control animals increased from 412 mm 3 at the start of the study to 976 mm 3 at the end of the study (137% increase), whereas the total tumor volume per mouse was decreased from 379 mm 3 at the start of the study to 235 mm a at the end of the study (38% decrease) in mice injected i.p. with green tea polyphenol fraction (50 mg/kg) 3 times a week for 6 weeks. In mice injected with green tea polyphenol fraction (75 mg/kg) 3 times a week for 6 weeks, the total tumor volume per mouse was decreased from 478 mm 3 at the start of the study to 138 mm 3 at the end of the study (71% decrease).

Effects of Green Tea and Green Tea Constituents on the Formation of Large Tumors and on Tumor Regression. We evaluated the effects of administration of vehicle, green tea, or green tea constituents on the number of mice with complete

tumor regression, the number of mice that developed one or more large tumors (>100 mm3), and the total number of large tumors per group in all of the papilloma-bearing mice described in experiments 1-10 (Table 5). The results of this evaluation revealed that fewer animals treated with green tea developed large tumors than did vehicle-treated controls and that green tea-treated animals had fewer large tumors than vehicle-treated control mice (Table 5). The mean number of large tumors per mouse for all 10 experiments at the start of vehicle administration was 0.19, and this increased to 0.93 large tumors/mouse after treatment with vehicle for 4-10 weeks (Table 5). In parallel groups of animals, the mean number of large tumors per mouse for all 10 experiments at the start of tea administration was 0.20, and this increased to 0.42 large tumors/mouse after treatment of the animals with green tea or green tea constituents for 4-10 weeks (Table 5). The results described here indicate that treatment of papilloma-bearing mice with green tea or green tea constituents strongly inhibited tumor growth and inhibited the formation of large papillomas (>100 mm3). Exam- ination of the data from all ten experiments revealed that complete tumor regression occurred in 14 of the 346 mice (4%) that were treated with green tea or green tea constituents for 4-10 weeks, whereas none of the 220 parallel vehicle-treated control mice had complete regression of their tumors.

Histopathology Studies. Histology studies with representa- tive tumor-bearing animals from experiments 2 and 7 revealed papillomas with keratosis and parakeratosis. The papillomas from animals treated with instant green tea in the drinking water for 10 weeks or with i.p. injections of (-)-epigallocatechin gallate for 8 weeks (experiments 2 and 7) could not be distin- guished histologically from tumors obtained from vehicle- treated control animals. Lesions from both experimental groups ranged in size from small raised nodules to large, horny papillary lesions connected to the dermis by a thin stalk. There were no significant effects of administration p.o. of instant

6660




A

530

1 0] O 0.63% GT ~ 1,25% GT

0 1 . . . . . . . 0 2 4 6

A

~ 30 " " ........

"~ 20

1 O~ 0 0,5% IGT I ~ 1.0% IGT

01 ~ ......... 0 2 4 6 8 1~0 '

A

40 ..........

~3o

~ 20

I e 1.25% (IT !

01 . . . . . . . 0 2 4 6

A 5 0

[ .......... M ,~ 4 0 ~

30]101 ....... .l~ ....... Water ~ 20

!

0 I . . . . . . . 0 2 4 6

A

..~ 40

�9 ~ 30

20

10 ....... ~ ....... Water

0 t . . . . . . . 0 2 4 6

Experiment 1; DMBA/TPA B

2 ~ 24

$ 1 , ~ 20

~ 1 2 ~ 16 ~L 12

,. 4

0 2 4 6 ' ~ 0

C

./"

Experiment 2; DMBA/TPA B ,-, C

o

[~ 8 ....... .- ~ 60 / t R 6 ~ ."

40 ~"

~ 20

0 2 4 6 8 10' ~ 0~" + , '~ . . . . . . .

Experiment 3; DMBA/TPA B C

m 50

i 6 40

4 .... 30 ..:"

20 "'

~ 10 .........

O" ~ ' d 6 ' o

D 40

30 ..

~. 20 / /

! l0 ...... "

i �9 m-'- D '601 50

40 .."

30 ..

20 /:

i 0 2 4 6 8 10 [-

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2o

15 ..

~ 0 "/

i 5 .

[-, Experiment 4; DMBA/TPA

1 B C ~ 2 5 0 ~ D

4~176 I +.+,oo+ t +'~176 1 ..,iI ~ 1500

200 ~ 100ff /:":

o 2 4 6 ~ ' i ' i " 6 ' ~ "2 ' i ' ~ '

Experiment 5; UVB/TPA B C 8 ---

........ 60 .+

o . ~ , ~ ,

Weeks of oral administration

[-,

40 ~ 3O

Fig. 1. Time course for the effect of administration p.o. of green tea on the growth and regression of skin papillomas in mice.

green tea or i.p. injections of (-)-epigallocatechin gallate on tumor blood supply, tumor cell size, or number of mitotic figures. No cytotoxic effects of the administration of instant green tea or (-)-epigallocatechin gallate were observed.

D I S C U S S I O N

In the present investigation, we studied the possibility that green tea may have an inhibitory effect on the growth of established papillomas in mouse skin. We treated tumor-bearing mice with green tea in the drinking water or with i.p. injections of a green tea polyphenol fraction or with i.p. injections of (-)-epigallocatechin gallate for 4-10 weeks. Partial regression of skin tumors or >90% inhibition of further tumor growth, as measured by changes in tumor volume per mouse, was observed

in 5 separate studies (Tables 3 and 4, experiments 4, 5, and 8-10). Marked inhibition of tumor growth was observed in 5 additional studies (Tables 3 and 4, experiments 1-3, 6, and 7). Although a substantial inhibitory effect of green tea or green tea constituents on the growth of small skin papillomas was observed (Tables 3 and 4, experiments 1, 2, 6, and 7), the data suggest that there was an even greater effect (tumor regression or >90% inhibition of tumor growth) in 5 of the 6 experiments done in mice with large papillomas (Tables 3 and 4, experiments 4, 5, and 8-10). Additional research is needed to confirm this observation. The results of our studies indicated that treatment of mice with green tea or green tea constituents had an inhibitory effect on body weight increases in several but not all experiments (Tables 3 and 4). A possible effect of green tea on body weight gain is important, because earlier studies have

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Table 4 Effect of i.p. administration of green tea constituents on the growth and regression of skin papillomas in mice

Trea tment a

Volume per papil loma Tumor volume per mouse Body weight (g) Papil lomas per mouse (mm a) (mm a)

Dura- No. tion of % % % % (wk) mice Initial Final change Init ial Final change Initial Final change Initial Final change

Experiment 6, DMBA/TPA Saline 8 30 31.2_+0.4 34.7_+0.5 11.2 19.2_+1.3 16.1• -16 1 .0• 21.3• 2030 19.2_+2.5 343-+78 1686 GTP (50 mg/kg) 8 30 30.4 • 0.5 32.8 _+ 0.6 7.9 21.1 • 1.6 13.6 • 1.5 -36 1.0 _+ 0.l 10.0 _+ 0.9 900 21.1 -+ 2.3 136 -+ 24 545 GTP (75 mg/kg) 8 20 31.3 • 0.5 32.1 • 0.5 2.6 15.4 _+ 1.9 7.7 • 1.3 -50 1.2 _+ 0.1 6.3 • 1.4 425 18.5 _+ 5.8 49 + 16 165

Experiment 7, DMBA/TPA Saline 8 25 32.9 + 0.4 36.8 -+ 0.9 11.9 6.4 -+ 0.9 8.3 _+ 1.2 30 2.1 + 0.5 120.8 _+ 19.8 5652 13.4 -+ 4.0 1003 -+ 238 7385 EGCG (25 mg/kg) 8 26 31.2 -+ 0.3 34.0 _+ 0.4 9.0 7.3 • 0.9 5.6 _+ 0.9 -23 1.6 • 0.2 47.4 _+ 9.5 2863 11.7 _+ 2.1 265 _+ 70 2165 EGCG (50 mg/kg) 8 26 33.4 -+ 0.5 35.8 • 0.7 7.2 8.5 • 0.8 6.2 • 0.7 -27 1.8 • 0.2 58.1 -+ 9.0 3128 15.3 _+ 2.1 360 + 84 2253

Experiment 8, DMBA/TPA Saline 4 28 33.8_+0.8 37.1 • 9.8 10.1 + 1.4 9 .0+ 1.7 -11 32.7_+ 8.1 54.6_+ 14.3 67 330-+92 491 _+ 149 49 GTP (50 mg/kg) 4 29 35.7 • 0.5 35.4 -+ 0.6 -0.8 13.7 • 1.5 8.5 -+ 1.1 -38 22.8 + 4.3 23.4 -+ 3.4 3 312 -+ 75 199 • 54 -36

Experiment 9, DMBA/TPA Saline 4 22 37.1 + 0.9 39.1 + 1.1 5.4 6.7 + 1.2 5.7 ___ 0.8 -15 20.1 _+ 3.4 74.2 + 7.7 269 135 + 27 423 + 104 214 GTP (50 mg/kg) 4 21 34.4 _ 0.7 36.0 • 0.7 4.7 8.9 • 1.6 6.5 _+ 1.5 -27 24.0 _+ 4.3 32.6 • 5.6 36 214 + 51 212 _+ 54 -1

Experiment 10, UVBFFPA Saline 6 19 40.3 + 0.7 37.5 • 1.3 -7 .0 2.0 • 0.2 1.8 + 0.2 -10 206 + 59 543 + 156 164 412 -+ 105 976 + 270 137 GTP (50 mg/kg) 6 19 40.7 + 1.0 37.4 • 1.0 -8.1 1.8 + 0.2 1.7 + 0.2 - 6 210 _+ 45 138 + 31 -34 379 + 82 235 + 70 -38 GTP (75 mg/kg) 6 19 39.4 -+ 0.7 37.5 _+ 0.9 -4 .8 1.8 _+ 0.2 1.6 -+ 0.3 -11 266 + 53 86 + 23 -68 478 - 114 138 + 41 -71

a Female CD-1 mice (6 weeks old) were initiated with DMBA or UVB and promoted with TPA as described in "Materials and Methods" and in Table 2. Tumor-bearing mice were then treated i.p. with a green tea polyphenol fraction (GTP) or (-)-epigallocatechin gallate (EGCG) 3 times a week for 4 -8 weeks. The indicated dose is the amount of tea consti tuent administered for each treatment. Each value represents the mean + SE.

shown an inhibitory effect of calorie or diet restriction on TPA-induced tumor promotion and on tumor size (12, 13). In experiments 5, 9, and 10, there was little or no effect of green tea or the green tea polyphenol fraction on body weight, but these treatments still caused tumor regression or a marked inhibition of tumor growth. These observations suggest that the inhibitory effects of green tea and green tea constituents on tumor growth are not directly related to their inhibitory effects on body weight gain. Longer-term experiments and variation of dose regimens are needed to clarify this point.

In all 10 experiments of our study, the time course trends indicated increased differences with time between controls and green tea-treated mice in volume per tumor and in tumor volume per mouse (Figs. 1 and 2) during the 4-10-week period of green tea administration. It would be interesting and important to determine whether these differences magnify, diminish, or plateau in studies of longer duration. Longer-term tea-induced growth inhibition or regression of tumors would be pertinent to the potential design of human chemoprevention studies.

The 1.25% green tea water extract used in our study is roughly comparable in composition to human green tea bever- ages. However, given as the total drinking water of mice in experiments 1, 3, 4, and 5, this administration represents a higher intake level than ordinary tea drinking by humans. The i.p. doses administered in experiments 6-10 are far higher than human ingestion, and these doses may approach maximum tolerated levels for mice. In one experiment, a single i.p. injection of 100 mg of green tea polyphenol fraction/kg body weight killed some of the mice that were given this high dose.

Further studies are needed to determine if green tea administration can inhibit the growth of microscopic to grossly ob- servable papillomas, reduce the number of papillomas by increasing resolution or disappearance of tumors, and/or inhibit the conversion of papillomas to carcinomas. Preliminary data described here suggest that green tea may produce the complete regression of tumors in a small percentage of tumor-bearing mice. Further studies, of longer duration and variable dosage

regimens, are needed to determine if this effect can be magni- fied. Studies by others have indicated an inhibitory effect of orally or i.p. administered green tea or green tea polyphenol fraction on the growth of transplanted Ehrlich ascites tumor cells or sarcoma 180 cells (14-16). In one study, administration p.o. of a green tea infusion (5 g tea leaves/100 ml water) twice a day starting 1 week before i.p. injection of Ehrlich ascites tumor cells and continuing for an additional 8 days resulted in a 41% inhibition in tumor growth (14). In another study, administration p.o. of 400-800 mg/kg of a decaffeinated instant green tea powder once a day for 4 days starting 24 h after s.c. inoculation of ICR mice with sarcoma 180 cells resulted in a 50-59% inhibition of tumor growth during a 21-day interval (15). In another study, i.p. or s.c. injections of a green tea polyphenol fraction or (-)-epigallocatechin gallate into mice that were inoculated s.c. with cells from a solid Ehrlich tumor or with cells from a sarcoma 180 tumor inhibited tumor growth by 20-56% (16). Feeding a green tea polyphenol fraction in the diet also was reported to inhibit the growth of sarcoma 180 cells that were inoculated into mice (16). An inhibitory effect of the tea components on body weight was observed in some but not all of the above studies. The results of our studies with established skin tumors (Tables 3-5) and the studies described above with transplanted tumor cells indicate that green tea and constituents of green tea have an inhibitory effect on tumor growth and can cause tumor regression.

The mechanism(s) of the inhibitory effect of green tea administration on tumor growth is unknown. Topical application of a green tea polyphenol fraction to mouse skin inhibits TPA-induced proliferation of epidermal cells, TPA-induced increases in ornithine decarboxylase activity, TPA-induced hydrogen peroxide generation, and TPA-induced inflammation (11). Several studies have indicated a relationship between cell proliferation and elevated levels of ornithine decarboxylase activity, and recent studies have shown elevated levels of polyamines and ornithine decarboxylase activity in papillomas (17). It would be of interest to determine whether administration of green tea to

6662




A "~>, 4 0 ~

""30 ........... " ............

"~ 20

~ 101 O 50 mg/kg GTF ] r 75 mg/kg GTP I

01 . . . . . . . . . 0 2 4 6 8

A

Experiment 6; DMBA/TPA

2 i 25

~12 ~ 10 . . ~ ' / ~ o ~

, >. 0 0 8 ~ ~

Experiment 7; DMBA/TPA B C

::1 ~ 6 ' ~ ' ~ 1 ' ~ ' 8 '

~ D

t ....... . ....... S~no ~''1-- 10"[ O 25 mg/kg EGCG

21 r 50 mg/kg EGCG 0t . . . . . . . . .

o 2 4 6 8 o ~ ) , ~ . . ~ , ~ , 8

A 40 ....................

20

10[ r 50 mg/kg GTP 0~ . . . . . 0 1 2 3 4 5

~ 1501 ,~ 120(~ T

v " ~ 900~ .."~

60~ ."]~""

~ o ~ - . . . . . ~ ~ , - . , . 0 2 4 6 8 ~ 0 2 4 6 8

Experiment 8; DMBA/TPA B C ~ D

60 60 o=12 "" ~'

............. . ....

Ol ' , , , , ~ . . . . . , 0 1 2 3 4 5 00 i 2 3 4 5 ~ [- Experiment 9; DMBA[I'PA

A B

~ 3 0 ~ 4 0 ...................... ~-..-.-~ i 8 ~ 1 0 ~

o ~ ....... �9 ~ ....... Saline fl _" 50 mg/kg GTP

o i 2 3 3, ~ 0 1 2 3 4 5

A B

~4o ~ 2 ......................

~. 20t "~ I ...... *" ...... sai~

I -_ 75 rng/kg GTP 0~ . . . . . . . 0 2 4 6 0 2 4 6

C ~ D

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01 OW 0 1 . . . . 2 3 4 5 o 1 2 3 4 ., [-. Experiment 10; UVB/I'PA

C 800

6o01 I .......

2ooT r �9 ~ ,~

Weeks of intraperitoneal administration

D

,~ 0! . . . . . - . 0 2 4 6 [-

Fig. 2. Time course for the effect of i.p. administration of green tea constituents on the growth and regression of skin papillomas in mice.

tumor-bearing mice decreased the elevated levels of polyamines and ornithine decarboxylase activity in these tumors. (-)-Epi- gallocatechin gallate reduces the binding of the tumor promoters TPA and teleocidin to their protein kinase C receptor (1), and this may decrease signal transduction and cellular proliferation. The free radical scavenging properties of the catechin polyphenols in green tea (18-20) may also play a role in their antitumor effects. Free radical scavengers prevent cellular prooxidant states that are induced by chemical carcinogens, radiation, and tumor promoters. We have shown that topical administration of a green tea polyphenol fraction inhibits TPA- induced formation of hydrogen peroxide in mouse epidermis (11). Other laboratories have shown that (-)-epigallocatechin gallate inhibits myeloperoxidase activity that contributes to the generation of cellular hydrogen peroxide in vivo (21) and that (-)-epigallocatechin gallate inhibits TPA-induced formation of

hydrogen peroxide in human polymorphonuclear cells in vitro (22). Free radical scavengers can protect against oxidative damage to membrane phospholipids, DNA, and the immunological system (23-25). The topical application of (-)-epigallocatechin gallate inhibits TPA-induced inflammation (11) and oxidative damage to epidermal DNA (21).

Enhancement of immunosurveillance is another possible mechanism for inhibiting the formation and growth of tumors. Certain chemical carcinogens (benzo[a]pyrene, DMBA, and 3-methylcholanthrene), radiation (UV and X-rays), and promoters (TPA) have been shown to damage the immune system (26-29). Yi et al. (14) reported that the number of a-naphthyl acetate esterase-positive blood T-lymphocytes (marker for stim- ulation of the immune system) was significantly increased by administration p.o. of green tea in Ehrlich ascites tumor-bearing animals (14). Furthermore, a green tea polyphenol fraction

6663




Table 5 Inhibitory effect of green tea and its constituents on the formation of large tumors in papilloma-bearing mice

% of mice Total no. of large No. of large No. of mice with large tumors tumors per group tumors per mouse

Treatment a Initial Final Initial Final Initial Final Initial Final

Experiment 1, DMBA/TPA Water 31 30 0 20 0 14 0 0.47 0.63% GT 31 31 0 7 0 3 0 0.10 1.25% GT 30 29 0 7 0 5 0 0.17

Experiment 2, DMBA/TPA Water 30 30 0 50 0 43 0 1.40 0.5% IGT 31 30 0 37 0 18 0 0.60 1.0% IGT 31 30 0 23 0 11 0

Experiment 3, DMBA/TPA Water 7 7 0 43 0 3 0 0.43 1.25% GT 7 7 0 43 0 3 0 0.43

Experiment 4, DMBA/TPA Water 21 21 24 52 5 13 0.24 0.62 1.25% GT 21 21 29 29 14 8 0.67 0.38

Experiment 5, UVB/TPA Water 8 8 25 63 2 5 0.25 0.63 1.25% GT 8 8 25 25 2 2 0.25 0.63

Experiment 6, DMBA/TPA Saline 31 30 0 47 0 25 0 0.83 GTP (50 mg/kg) 31 30 0 23 0 7 0 0.23 GTP (75 mg/kg) 20 20 0 10 0 2 0 0.10

Experiment 7, DMBA/TPA Saline 26 25 0 72 0 48 0 1.92 EGCG (25 mg/kg) 26 26 0 54 0 19 0 0.73 EGCG (50 mg/kg) 27 26 0 62 0 32 0 1.23

Experiment 8, DMBA/TPA Saline 28 28 21 32 12 17 0.42 0.61 GTP (50 mg/kg) 30 29 20 31 11 9 0.37 0.31

Experiment 9, DMBA/TPA Saline 22 22 32 55 8 17 0.36 0.77 GTP (50 mg/kg) 21 21 38 29 12 8 0.57 0.38

Experiment 10, UVB/TPA Saline 24 19 78 68 17 20 0.71 1.05 GTP (50 mg/kg) 25 19 74 37 15 11 0.6 0.58 GTP (75 mg/kg) 25 19 74 26 18 7 0.72 0.36

Summary (10 experiments) Control 228 220 18 48 44 205 0.19 0.93 Green tea 364 346 17 29 72 145 0.20 0.42

a Female CD-1 mice with tumors were treated with a green tea water extract (GT) p.o., instant green tea (IGT) p.o., green tea polyphenol fraction (GTP) i.p., or (-)-epigallocatechin gallate (EGCG) i.p., as described in Tables 3 and 4. The number of large tumors (>100 mm 3) was determined for each experiment before and after treatment with vehicle or green tea component.

was reported to stimulate the formation of tumor necrosis factor-like activity (16). Generally, immune function is lower a n d the spleen size is larger in tumor-bearing animals than in non-tumor-bear ing animals. In an earlier study (8) and in the present study, we found that mice with skin tumors had a larger spleen than did control animals with no tumors. We found that tumor-bearing mice treated with instant green tea in the drinking water or with i.p. injections of a green tea polyphenol fraction or (-)-epigal locatechin gallate had.a decreased spleen size (experiments 2, 6, and 7). 5 Tumor-bearing mice treated with vehicle, 0.5% instant green tea, or 1% green tea in the drinking water for 10 weeks had a spleen weight of 219 _+ 40 (SE), 148 + 11, and 120 + 6 mg/mouse, respectively (experiment 3). Control non-tumor-bearing mice had a spleen weight of 83 + 7 mg/mouse. Addit ional studies on possible effects of green tea and its consti tuents on spleen function and on the immune system are needed.

s Z. Y. Wang, M-T. Huang, C-T. Ho, C. S. Yang, and A. H. Conney, unpub- lished observations.

Studies during the past few years have demonstra ted that administrat ion of green tea or green tea consti tuents to rodents inhibits chemically induced and UV-induced tumorigenesis. Adminis t ra t ion p.o. of green tea or a green tea polyphenol fraction inhibits N-ni t rosodiethylamine-induced forestomach and lung tumors in A/J mice (9), NNK-induced lung tumors in

A/J mice (9), UVB-induced skin tumors in SKH-1 mice (8), DMBA-induced skin tumors in Sencar mice (3), TPA-induced promot ion of skin tumors in SKH-1 mice (9), and N-nitrosomethylbenzylamine-induced esophageal tumors in rats (5). It has also been shown that administrat ion p.o. of black tea inhibits N-ni t rosomethylbenzylamine-induced esophageal tumors in rats (5) and NNK-induced lung tumors in mice (9 ) .

In addition to these observations, the administrat ion p.o.

of (-)-epigal locatechin gallate (major catechin in green tea) inhibits N-ethyl-N'-nitro-N-nitrosoguanidine-induced duodenal tumors in C57BL/6J mice (4) and NNK-induced lung tumors in A/J mice (10). The broad effects of green tea as a cancer chemoprevent ive agent in many different animal models is of

6664




c o n s i d e r a b l e in te res t . I t is poss ib le t ha t s o m e or all o f the cance r

c h e m o p r e v e n t i v e effects o f g r een tea are r e la ted to its i n h i b i t o r y

a c t i o n o n t u m o r g r o w t h . A l t h o u g h the re have been several ep-

i d e m i o l o g i c a l s tud ies o n t h e effects o f t ea i n g e s t i o n o n h u m a n

c a n c e r (30), t h e resu l t s o f these s tud ies are n o t conc lus ive , a n d m o r e ex tens ive r e sea rch is n e e d e d . T h e s tud ies r e p o r t e d here

d e m o n s t r a t e a m a r k e d i n h i b i t o r y effect o f g r een tea a d m i n i s -

t r a t i o n o n the g r o w t h o f an e s t ab l i shed e x p e r i m e n t a l l y i n d u c e d

n o n m a l i g n a n t sk in p a p i l l o m a in mice . A d d i t i o n a l w o r k is

n e e d e d to d e t e r m i n e w h e t h e r the a d m i n i s t r a t i o n o f g reen tea o r

its m a j o r c o n s t i t u e n t s can inh ib i t t he p r o g r e s s i o n o f p a p i l l o m a s

to c a r c i n o m a s a n d to d e t e r m i n e w h e t h e r o r n o t a d m i n i s t r a t i o n

o f g reen tea can inh ib i t t he g r o w t h or e n h a n c e the r eg re s s ion o f c a r c i n o m a s .

A C K N O W L E D G M E N T S

We thank Dr. Douglas Balentine for the H P L C analysis and dry weight determinations with green tea samples and Harold Newmark, Deborah Bachorik, and Diana Lim for their excellent help in the preparation of this manuscript.

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1992;52:6657-6665. Cancer Res Zhi Y. Wang, Mou-Tuan Huang, Chi-Tang Ho, et al. Skin Papillomas in MiceInhibitory Effect of Green Tea on the Growth of Established

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