Chapter 21
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Inhibitory Effect of Green Tea on Tumorigenesis and Tumor Growth in Mouse Skin 1
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Allan H. Conney , Zhi Yuan Wang , Chi-Tang Ho , Chung S. Yang , and Mou-Tuan Huang 1
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Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, P.O. Box 789, Piscataway, NJ 08855-0789 Department of Food Science, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903 2
Topical application of a green tea polyphenol fraction inhibited 12-Otetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin. Oral administration of green tea inhibited ultraviolet Β lightinduced tumorigenesis in mouse skin, and tumors from tea-treated animals were smaller than tumors from water-treated control animals. Oral administration of green tea to papilloma-bearing mice inhibited tumor growth and in some experiments caused a decrease in tumor size. Green tea is an important beverage in Asian countries such as China and Japan, whereas black tea is more popular in North America and Europe. Recent investigations have shown that constituents of green tea have an inhibitory effect on teleocidin-induced and 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumori genesis in mouse skin and on N-ethyl-A'-nitro-N-nitrosoguanidine-induced tumorigenesis in mouse duodenum (1-3). Additional studies have shown an inhibitory effect of green and black tea on N-nitrosomethylbenzylamine-induced esophageal tumors in rats (4) and an inhibitory effect of green tea on ultraviolet Β light (UVB)-induced and 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumors in mice (5-7). In the present report, we summarize our studies showing an inhibitory effect of a topically applied green tea polyphenol fraction on tumor promotion by TPA and an inhibitory effect of orally administered green tea on U V B induced tumorigenesis in mouse skin. We also describe an inhibitory effect of orally administered green tea on the growth of experimentally-induced papillomas in mouse skin. Materials and Methods Animals. Female CD-1 or SKH-1 (hairless) mice (6 to 8 weeks old) were purchased from Charles River Laboratories (Kingston, N Y ) . The animals were kept in our animal facility for at least 1 week before use. Mice were given water and food ad libitum and kept on a 12 hour light, 12 hour dark cycle. Purina Laboratory Chow 5001 diet (Ralston-Purina Co., St. Louis, MO) was used for all studies. 0O97-6156/92/0507-O284$06.00/0 © 1992 American Chemical Society
Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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21. CONNEY ET AL.
Green Tea, Tumorigenesis, and Tumor Growth
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Preparation and Composition of Green Tea Polyphenol Fraction. One hundred grams of green tea leaves were extracted three times with 300 ml of meth anol at 50°C for 3 hours, 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°C) and extracted three times with 200 ml hexane and three times with 200 ml chloroform. The aqueous phase was extracted three times with 180 ml ethyl acetate, and the ethyl acetate was evaporated under reduced pressure. The residue was redissolved in 300 ml water and lyophilized to obtain 8 to 9 g of green tea polyphenol fraction. H P L C analysis of the green tea polyphenol fraction revealed the following composition: (-)epigallocatechin gallate (49.5%), (-)-epicatechin gallate (11%), (-)-epicatechin (6.1%), (-)-epigallocatechin (11.5%) and caffeine (7.6%). Preparation and Composition of Green Tea Aqueous Extract. Green tea leaves (12.5 g) were added to 500 ml of boiling water and were steeped for 15 minutes. The infusion was cooled to room temperature in an ice bath and then filtered. The tea leaves were 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 resulting clear solution is similar to tea brews consumed by humans. In some experiments, the 1.25% green tea water extract was diluted 1:1 with water to make a 0.63% green tea extract. During specified experimental periods, the tea extract was given ad libitum as the sole source of drinking water. The amount of solids present in the 1.25% green tea extract was determined to be 4.69 mg per ml by drying samples in an air convection oven (18 hours at 65°C) and weighing the dry residue. Analysis of the green tea extract by H P L C indicated that the total green tea solids in the extract contained (-)-epigallocatechin gallate (15.1%), (-)-epigallocatechin (6.9%), (-)-epicatechin gallate (3.0%), (-)-epicatechin (1.8%), (+)-catechin (0.5%), caffeine (8.1%) and theobromine (0.4%). The compounds that were measured accounted for 36% of the total green tea solids present in the water extract. Storage of the green tea water extract for 72 hours at room temperature resulted in only a small decrease (8 to 12% ) in the concentration of tea catechins. Ultraviolet Light. Ultraviolet lamps (FS72T12-UVB-HO) that emit ultraviolet Β light (UVB) (280 to 320 nm; 75 to 80% of total energy) and ultraviolet A light (UVA) (320 to 375 nm; 20 to 25% of total energy) were obtained from the Voltare Co. (Fairfield, CT). The dose of U V B was quantified with an U V B Spectra 305 Dosimeter that was obtained from the Daavlin Co. (Bryan, OH). The radiation was further calibrated with an IL1700 Research Radiometer/Photometer from Inter national Light Inc. (Neburgport, M A ) . For exposure to U V (UVA+UVB), 10 mice were housed in a 25.4 cm (width) χ 45.7 cm (length) plastic box. Six boxes (without tops) were placed under eight U V lamps; 50.8 cm (width) χ 182.9 cm (length), and the boxes were systematically rotated during the course of the study to compensate for possible small differences in flux at various positions under the lamps. The distance between the U V lamps and the backs of the mice or the U V B detector was 43.2 cm. The amount of exposure to U V B was controlled by a Spectra 305 Dosimeter. The exposure time for a 180 mJ/cm2 dose of U V B was 130 to 160 sec. Although all data are expressed as exposure to U V B , some additional exposure to U V A also occurred as indicated above.
Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II
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Results and Discussion Inhibitory Effect of Topically Applied Green Tea Polyphenol Fraction on T P A - i n d u c e d T u m o r Promotion in Mouse S k i n . Mice that were initiated with 200 nmol of D M B A and promoted with 5 nmol of TPA twice weekly for 20 weeks developed 20.7 skin tumors per mouse, and 93% of the mice had skin tumors (Table I). Topical application of 3.6 mg of green tea polyphenol fraction together with each treatment of TPA markedly inhibited the percentage of mice with tumors and the number of tumors per mouse (Table I).
Table I. Effect of Topical Application of a Green Tea Polyphenol Fraction on TPA-induced Tumor Promotion in Mouse Skin 12 Weeks Tumors Percent per mice mouse with tumors
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0* D M B A + Acetone D M B A + TPA 15.3 ± 2.4 D M B A + TPA + GTP (3.6 mg) 0.6 ± 0.4
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0 90 17
20 Weeks Tumors Percent per mice mouse with tumors
0* 20.7 ± 2.9 1.2 ± 0.5
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0 93 27
Female CD-I mice were treated topically with 200 nmol of D M B A in 200 μΐ acetone followed a week later by acetone, TPA (5 nmol) or TPA (5 nmol) together with a green tea polyphenol fraction (GTP, 3.6 mg) in acetone twice weekly for 20 weeks. Skin tumors with a diameter greater than 1 mm were recorded. The data were taken from ref. 7 and represent the mean ± S.E. from 30 mice per group. Statistically different from D M B A + TPA group (p
