D-Glucose and (-)-Epigallocatechin Gallate - American Chemical Society

Chapter 25

Penta-O-Galloyl-β-D-Glucose and (-)-Epigallocatechin Gallate Cancer Preventive Agents 1

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on August 27, 2015 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch025

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S. Yoshizawa , T. Horiuchi , M . Suganuma , S. Nishiwaki , J . Yatsunami , S. Okabe , T. Okuda , Y. Muto , K. Frenkel , W. Troll , and H. Fujiki 2

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Cancer Prevention Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan Faculty of Medicine, Gifu University School of Medicine, Tsukasa-machi, Gifu 500, Japan Department of Environmental Medicine, New York University Medical Center, New York, NY 10016 2

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Penta-O-galloyl-β-D-glucose ( 5 G G ) , obtained by methanolysis of tannic acid and (-)-epigallocatechin gallate (EGCG), the main constituent of Japanese green tea, were polyphenols which strongly inhibited the specific H-12-O-tetradecanoylphorbol-13-acetate (TPA) binding to the phorbol ester receptors in a particulate fraction of mouse skin. Based on the evidence, these polyphenols were thought to inhibit tumor promotion of TPA on mouse skin. 5GG and E G C G inhibited tumor promotion of teleocidin, one of the TPA-type tumor promoters on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA) in two-stage carcino genesis experiments. Moreover, E G C G inhibited tumor promotion of okadaic acid, which acts differently on cells than the TPA-type tumor promoters. The mechanisms of action of E G C G were studied in relation to the reduction of specific binding of the tumor promoters, H - T P A and H-okadaic acid to their receptors in cell membrane. A single application of 5 mg E G C G decreased immediately to the minimum level the specific binding of H-tumor promoters. The effect was understood to be a sealing of the membrane by E G C G , due to EGCG-protein interaction, resulting in reduction of protein phosphorylation in cells. In addition, E G C G inhibited H O formation by TPA-activated human polymorpho nuclear leukocytes. Since E G C G is a non-toxic compound, ingested in green tea in every day life in Japan, we extended the study of E G C G as a cancer preventive agent. This paper also reviews inhibitory effects of E G C G on duodenal carcinogenesis of male C 5 7 B L / 6 mice and develop ment of spontaneous hepatoma in male C3H/HeN mice. We think E G C G is a practical cancer chemopreventive agent to be implemented in every day life. 3

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Current address: Ehime University School of Medicine, Ehime 791-02, Japan Corresponding author 0097-6156/92/0507-0316$06.00/0 © 1992 American Chemical Society

In Phenolic Compounds in Food and Their Effects on Health II; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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Polyphenols and Phorbol Ester Receptor Binding Our study of E G C G for the purpose of cancer prevention started with the anticarcinogenic study of polyphenols derived from medicinal plants and drugs. About 30 polyphenols were first tested to find whether they shared the same phorbol ester receptor in a particulate fraction of mouse skin as TPA. Most of the polyphenols inhibited the specific H -T P A binding dose-dependendy, but some did not. Thirty polyphenols were roughly classified into four classes according to the ED50 values for inhibition (Fig. 1). The ED50 values of the Class 1 polyphenols were about one thousand times more than that of TPA (1). Table 1 lists some representatives of the 30 polyphenols, according to classification. 5GG , E G C G , pedunculagin, chebulinic acid and buddledin A were included in Class 1 with ED50 values of 1.7 uM. Since the structures of these five compounds are unrelated to that of TPA (Fig. 2), we thought that they might interact with the phorbol ester receptor in the cell membrane differently from TPA. Polyphenols have the ability to precipitate watersoluble proteins, such as a hemoglobin solution. The precipitability of hemoglobin by geraniin was taken as a standard and the potencies of various polyphenols were compared with their result with geraniin and these values were called the relative astringency to geraniin. The relative astringency to geraniin was used to estimate the biochemical effect of polyphenols that was caused by polyphenol-protein interaction (2). Some polyphenols showed similar values in two parameters, the relative astringency and inhibition of specific binding of H -T PA to the receptor (Fig. 3). From these results, 5GG from hydrolyzable tannins and E G C G from condensed tannins were selected for studying inhibition of tumor promotion in two-stage carcinogenesis experiments. Polyphenols of Class 4, such as (+)-catechin, ellagic acid and methyl gallate, which did not bind to the receptor, were deleted from further experiments.


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5 G G and Its Inhibition of Tumor Promotion of Teleocidin We first had to obtain 5GG on an order of gram amounts sufficient to carry out the inhibition of tumor promotion experiment. 25 g of tannic acid, which was isolated from a gall, Schisandraefructuss,was incubated in 500 ml of methanol:acetate buffer (9:1) at pH 6.0 at 40 °C for 6 hours. This methanolysis cleaved the depside linkage between 5GG and the galloyl group and released 5GG and the galloyl residue. According to the procedure summarized in Table 2, 8 g of 5GG were pre cipitated, after the methanol layer was diluted to 2% methanol solution and placed at 4 °C. The purity of 5GG was determined by H P L C on a T S K gel silica 60 column with n-hexane: methanol: tetrahydrofurane:formic acid (55:33:11:1) containing 450 mg/L oxalic acid as a solvent. U V absorption was monitored at 280 nm and resulted in 95% purity. This method was quick and applicable to large scale purification. Inhibition by 5GG of tumor promotion was studied in a two-stage carcinogenesis experiment on skin of 8 week-old female CD-I mouse. Initiation was carried out by a single application of 100 μ£ D M B A and tumor promotion was achieved by repeated applications of 2.5 μg teleocidin, twice a week (3). In the experimental group, 5 mg of 5GG were applied topically 15 min before each treatment with teleocidin. The 5GG treatment reduced the percentages of tumor-bearing mice from 100% to 53% and the average numbers of tumors per mouse from 3.3 to 0.9 in week 20 (4). The evidence encouraged us next to pursue an experiment with E G C G .



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P H E N O L I C C O M P O U N D S I N F O O D A N D T H E I R E F F E C T S O N H E A L T H II

Concentration ( η M )

Fig. 1. Classification of polyphenols according to their ED50 values of inhibition of specific H - T P A binding to a particulate fraction of mouse skin. 3

Table 1

Classification of Various Polyphenols by Phorbol Ester Receptor Binding Class 1 ( E D = 1.7 uM) Penta-O-galloyl-p-D-glucose (-)-Epigallocatechin gallate Pedunculagin Chebulinic acid Buddledin A 50

Class 2 (ED o = 30uM) Rugosins D and Ε Coriariin A Comusiin A Nobotaniin C 5

Class 3 (ED o = 340uM) Tellimagrandin I Tellimagrandin Π 5

Class 4 (no inhibition) (+)-Catechin Ellagic acid Methyl gallate



25. Y O S H I Z A W A E T A L .

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Penta-Ο- Galloyl-β-D- Glucose

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Chebulinic acid

Buddledin A with that of TPA.


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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II

Relative astringency to geraniin 1.5

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Polyphenols

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Inhibition of specific binding of H-TPA ( % ) 50 100 3

( + )-Catechin Ellagic acid Di-O-Caffeoylquinic acid Downloaded by UNIV OF CALIFORNIA SAN DIEGO on August 27, 2015 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch025

Procyanidin B-2 Chebulinic acid EGCG 5GG Ss-tannin 1 Procyanidin B-2 digallate

Fig. 3. Comparative values of biochemical effects of polyphenols on relative astringency to geraniin and inhibition of specific binding of H - T P A to a mouse skin particulate fraction. The precipitability of hemoglobin by geraniin was taken as a standard. 3

Table 2

Purification of Penta-O-galloyl-P-D-glucose

Tannic acid

(25 g)

I Methanolysis |— Extracted with ether Aqueous layer [—Extracted with ethyl acetate Ethyl acetate layer (12 g) |— Partition with dichloromethane:methanol Methanol layer

(2:3)

[—Dilute to 2% methanol solution Precipitate of penta-O-galloyl-p-D-glucose

(8 g)


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E G C G and Its Inhibition of Tumor Promotion of Okadaic Acid


In 1987, we first reported that E G C G inhibited tumor promotion of teleocidin on mouse skin (1). This paper first reports the inhibitory effects of E G C G on tumor promotion of okadaic acid, which acts differently than teleocidin (5). The E G C G used in the experiment was isolated from Japanese green tea leaves and was made up of E G C G (85%), (-)-epicatechin (10%) and (-)-epicatechin gallate (5%), as described previously (1). According to the two-stage carcinogenesis experiment, initiation was carried out by a single application of 100 ug D M B A on skin of female CD-I mouse and tumor promotion was achieved by repeated applications of 1 μg okadaic acid on the same area of the initiated skin, twice a week (5). In the experimental group, 5 mg E G C G was applied topically before each treatment with okadaic acid. As Figure 4 shows, E G C G treatment completely inhibited tumor promotion of okadaic acid up to week 20 of tumor promotion, with regard to the percentages of tumor-bearing mice and the average numbers of tumors per mouse. Our results indicated that E G C G inhibited tumor promotion of two tumor promoters, okadaic acid in this experiment, and teleocidin in the previous experiment, with different mechanisms of action (5-7). Okadaic acid inhibits dephosphorylation of phosphoserine and phosphothreonine through inhibition of protein phosphatase 1 and 2A activities (8), resulting in an increase of phosphoproteins (9), whereas teleocidin activates protein kinase C and produces phosphoproteins in larger amounts than in the usual state (3). In both cases, the increase of phosphoproteins might act as a signal for tumor promotion. Since E G C G inhibits both tumor promotion pathways, we studied their receptor bindings using H-okadaic acid and H-TPA. Okadaic acid binds to protein phos phatases 1 and 2A (10,11), whereas TPA and teleocidin bind to the phorbol ester receptor (3). These two different receptors are present in a membrane fraction of mouse skin. When mouse skin was treated with a single application of 5 mg E G C G , both the specific binding of H - T P A and that of H-okadaic acid decreased immediately and reached a minimum in 5 to 10 min (Fig. 5). The levels gradually returned to normal. In previous experiments of tumor promotion, E G C G was usually applied 15 min before each treatment with a tumor promoter. Therefore, the results of Figure 5 indicate that okadaic acid or teleocidin had been applied to the skin which was at the weakest condition for receptor binding. Based on the evi dence, E G C G treatment possibly inhibited the interaction of tumor promoters with their receptors, resulting in reduction of protein kinase activity, as well as inhibiting the process of tumor promotion after tumor promoter receptor binding. Figure 6 is a schematic illustration of the suggested mechanisms of action of E G C G . We think E G C G first induces the sealing of the membrane, due to EGCG-protein interaction, which explains the reduction of specific binding of the tumor promoters to their receptors in the cell membrane. This sealing consequently causes inhibition of phosphorylation in cells, although phosphorylation originally acts as a signal for tu mor promotion. In addition, it is generally accepted that a tumor promoter generates several active oxygen species. Since E G C G is a strong antioxidant, E G C G was expected to inhibit the effect of TPA. E G C G clearly inhibited H2O2 formation by TPA-activated human polymorphonuclear leukocytes, indicating that E G C G partly acts by suppressing oxyradical formation (12). In general, we assume that a membrane sealed by E G C G might additionally interrupt the interaction of various growth factors and hormones with their receptors in the membrane through autocrine and paracrine, subsequently resulting in a specific inhibition of the cell growth. 3

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ooo

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ÇKX3

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è

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ςχί ρ

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Weeks of promotion Fig. 4.

Inhibition by E G C G on tumor promotion of the suggested okadaic acid. Groups treated with D M B A and okadaic acid 0) and with D M B A and okadaic acid plus E G C G (·).

100*-

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Fig. 5. Inhibition of specific binding of H -T P A and H-okadaic acid to a mouse skin particulate fraction after a single application of E G C G . H - T P A (#) and H-okadaic acid 0). 3

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Tumor promoter

Fig. 6.

A schematic illustration of the suggested mechanisms of action of E G C G , through a sealed cell membrane.


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Anticarcinogenic Effects of E G C G E G C G was effective in inhibition of carcinogenesis in other organs than the skin. The results are briefly reviewed. A solution of either 0.05% or 0.1% E G C G was significantly effective in inhibition of spontaneous hepatoma development in male C3H/HeN mice (Muto et al., manuscript in preparation). The effects might be related to the evidence that E G C G inhibits lipid peroxidation in liver mitochondria (13). As we previously reported, a solution of 0.005% E G C G significantly inhibited duodenal carcinogenesis of male C57BL/6 mice, which had been induced by N-ethyl-N'-nitro-N-nitrosoguanidine (14). However, E G C G in drinking water did not inhibit development of spontaneous thymic lymphoma in female A K R mice. Bladder cancer in female C3H/HeN mice induced by N-butyl-N-(4-hydroxylbutyl)nitrosamine was also not inhibited (Ohtani et al., unpublished result). Moreover, the effects of E G C G on carcinogenesis in various organs, such as the colon and liver, are under investigation by scientists in Japan. Since inhibition of chemical carcinogenesis induced by large amounts of potent carcinogens is not a suitable test system to screen for a cancer preventive agent (15), we have to estimate the effects of E G C G based on various carcinogenesis experiments under different conditions. It is an important task to find out how we should apply E G C G for the prevention of human cancers, or what kinds of cancers are preventable by E G C G treatment. Drinking green tea should be evaluated as one of the most practical methods of cancer prevention available at present.

Acknowledgments This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, a grant for the Program for a Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan, and by grants from the Foundation for promotion of Cancer Research, the Uehara Memorial Life Science Foundation and the Princess Takamatsu Cancer Research Fund. We thank Dr. T. Sugimura at National Cancer Center for his encouragement of the work. Literature Cited 1. Yoshizawa, S.; Horiuchi, T.; Fujiki, H.; Yoshida, T.; Okuda, T.; Sugimura, T. Phytotherapy Res. 1987, 1, 44. 2. Okuda, T.; Mori, K.; Hatano, T. Chem. Pharm. Bull. 1985, 33, 1424. 3. Fujiki, H.; Sugimura, T. Adv. in Cancer Res. 1987, 49, 223. 4. Fujiki, H.; Yoshizawa, S.; Horiuchi, T.; Suganuma, M.; Yatsunami, J.; Nishiwaki, S.; Okabe, S.; Matsushima, R.N.; Okuda, T.; Sugimura, T. Preventive Medicine, 1991, in press. 5. Fujiki, H.; Suganuma, M.; Sugimura, T. Envir. Carcino. Revs. 1989 C7(l), 1. 6. Sassa, T.; Richter, W.W.; Uda, N.; Suganuma, M.; Suguri, H.; Yoshizawa, S.; Hirota, M.; Fujiki, H. Biochem. Biophys. Res. Commun. 1989, 159, 939. 7. Fujiki, H.; Tanaka, Y.; Miyake, R.; Kikkawa, U.; Nishizuka, Y.; Sugimura, T. Biochem. Biophys. Res. Commun. 1984, 120, 339.




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8. Takai, Α.; Bialojan, C.; Troschka, M.; Rüegg, J. C. FEBS Lett. 1987, 217, 81. 9. Yatsunami, J.; Fujiki, H.; Suganuma, M.; Yoshizawa, S.; Eriksson, J. E.; Olson, M.O.J.; Goldman, R.D. Biochem. Biophys. Res. Commun. 1991, 177, 1165. 10. Suganuma, M.; Suttajit, M.; Suguri, H.; Ojika, M.; Yamada, K.; Fujiki, H. FEBS Lett. 1989, 250, 615. 11. Nishiwaki, S.; Fujiki, H.; Suganuma, M.; Ojika, M.; Yamada, K.; Sugimura, T. Biochem. Biophys. Res. Common. 1990, 170, 1359. 12. Zhong, Z.; Tius, M.; Troll, W.; Fujiki, H.; Frenkel, K. Proceedings of the AACR, 1991, 32, 127. 13. Okuda, T.; Kimura, Y.; Yoshida, T.; Hatano, T.; Okuda, H.; Arichi, S. Chem. Pharm. Bull. 1983, 31, 1625. 14. Fujita, Y.; Yamane, T.; Tanaka, M.; Kuwata, K.; Okuzumi, J.; Takahashi, T.; Fujiki, H.; Okuda, T. Jpn. J. Cancer Res. 1989, 80, 503. 15. Ashby, J.; Morrod, R.S. Nature 1991, 352, 185. RECEIVED January 13, 1992


D-Glucose and (-)-Epigallocatechin Gallate - American Chemical Society

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