Comparative Study of Ellagic Acid and Its Analogues as

Chapter 24

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Comparative Study of Ellagic Acid and Its Analogues as Chemopreventive Agents against Lung Tumorigenesis Andre Castonguay, Mohamed Boukharta, and Guylaine Jalbert Laboratory of Cancer Etiology and Chemoprevention, School of Pharmacy, Laval University, Quebec G1K 7P4, Canada

The polyphenol ellagic acid inhibits lung tumorigenesis induced by a nicotine-derived nitrosamine in A / J mice. This inhibition was related to the logarithm of the dose of ellagic acid added to the diet. The biodistribution of ellagic acid was studied in mice gavaged with ellagic acid. Pulmonary levels of ellagic acid reach a maximum 30 min after gavage and were directly proportional to the dose between 0.2 and 2.0 mmol EA/kg b.w. Ellagitannins extracted from pomegranate are hydrolyzed extensively in mice leading to the excretion of ellagic acid in the feces and urine. Feeding mice pomegranate ellagitannins (10 g/kg diet) did not inhibit lung tumorigenesis.

Nicotine accounts for 90% of the alkaloids present in tobacco. Levels of nicotine in mainstream smoke of cigarette range from 0.1 to 3 mg/cigarette (1). Cigarettes also deliver about 0.3 mg of minor alkaloids. Among those is nornicotine. Combustion of cigar and pipe tobacco also yields nicotine (2). Thus nicotine is an essential component in tobacco products and there are many lines of evidence that it is the pharmacological reinforcer in tobacco smoke (3). Freshly harvested tobacco leaves contain between 0.6 and 14 mg nitrate per cigarette. During the curing and processing of tobacco leaves, reaction of nicotine and nornicotine with nitrate produces N-nitrosarnines (4), one of which is 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK). Levels of N N K in mainstream smoke range from 4 to 1700 ng per cigarette (5). Tumorigenic assays of N N K in laboratory animals have revealed a remarkable specificity of N N K for lung tissues (6). These observations led Hoffmann and Hecht to conclude that N N K is important in the etiology of tobacco smoke-induced lung cancer (7). It is estimated that humans consume as much as 1 g of plant phenols per day (8). Ellagic acid is a polyphenol generated from ellagitannins present in grapes, strawberries raspberries, and pomegranate, which are normally consumed by humans (9). This phenol is one of most promising chemopreventive agent likely to reduce the risk of human cancer and which could be introduced in human intervention trials (10). The aim of this study was to characterize the chemopreventive

0097-6156/94/0546-0294$06.00/0 © 1994 American Chemical Society In Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

24. CASTONGUAY ET AL.

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efficacy of ellagic acid in lung tumorigenesis and to compare the efficacies of E A and ellagitannins, the precursors of EA. Materials and Methods


Chemicals. N N K was synthesized as previously described (77). Its purity was higher than 98.5% as determined by reverse phase HPLC (72). E A was purified by crystallization in pyridine and drying at 40°C for 3 days. Lung Adenoma Assay. In a first bioassay, tumor response was studied as a function of the dose of E A . Female A/J mice (6 to 7 weeks old) were housed in groups of 5 in cages with wire bottoms. They were fed ad libitum sl powdered A I N 76A diet (Teklad, Madison, WI) or AIN-76A diet with EA, starting 2 weeks before carcinogen treatment and throughout the experiment (25 weeks). The initial concentration of N N K was 62.4 μg/ml and was adjusted thereafter for each cage according to water consumption. The cumulative dose of N N K was 9.1 mg per mouse. Five groups received E A at doses of 0, 0.06, 0.25, 1.00 or 4.00 g/kg diet. A l l the mice were necropsied 16 weeks after the end of the carcinogen treatment. In a second bioassay, the efficacies of E A and pomegranate ellagitannin were compared. The first group of 10 mice was fed control diet only. Three groups of 25 mice were given a cumulative dose of 9.1 mg of N N K per mouse. Two of these groups were fed ΕA (4 g/kg diet) or pomegranate ellagitannins (10 g/kg diet) starting 2 weeks before carcinogen treatment and throughout the bioassay. Biodistribution of EA. The biodistribution of E A was studied as a function of the dose and time after gavage with a suspension of EA. Six groups of 3 mice were fasted for 24 hours. They were gavaged with 0.5 ml of a suspension containing 0, 0.2, 0.5, 1.0, 1.5 or 2 mmol EA/kg b.w. in distilled water and sacrificed 30 min later. Seven groups of 3 mice were fasted for 24 hours and gavaged with 0.5 ml of a suspension containing 2 mmol EA/kg b.w. They were sacrificed 10, 15, 30, 45, 60, 120 or 240 min later. Liver (300 mg) or lung tissues (100 mg) were homogenized in 2 ml 1 M N a H P 0 (pH 3) for 15 sec. After the addition of 20 ml of a mixture of acetone and ethyl acetate (1:2, v:v) the homogenization was continued for 2 min. The homogenate was centrifuged (12,000 g, 15 min., 4°C) and a 5 ml aliquot of the supernatant was evaporated to dryness under nitrogen at room temperature and dissolved in 100 μΐ methanol. A 20 μΐ aliquot of this solution was injected on a μBondapak Q g column (10 μπι, 0.4 χ 30 cm). The E A was eluted with 2% formic acid in methanol:water (2:3, ν:v). The column was washed with methanol for 12 min before each assay. The flow rate was 1 ml/min and E A was detected at 254 nm. 2

4

Pomegranate Extraction. Pomegranate peels were extracted 3 times with 80% aqueous acetone. Concentration of the extracts under reduced pressure left a brown precipitate, which was removed by filtration. The filtrate was applied to a Sephadex LH-20 column. After washing with water, the column was eluted with 80% aque ous acetone. The solvent was evaporated and the residue was dried over P2O5. A n aliquot of the residue was analyzed by HPLC on μBondapak Q g 10 μπι. Solvent A was a 2% aqueous solution of formic acid and solvent Β was 2% solution of formic acid in methanol:water (3:7; v:v). The solvent gradient was as follows: 100% solvent A for 3 minutes, then linear to 100% solvent Β in 72 minutes.

In Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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A 1 mg aliquot of the residue was dissolved in 2 ml methanol/water (1:1, v:v), mixed with 0.75 ml concentrated HC1 and heated at 100°C for two hours. The hydrolysate was analyzed by HPLC as described for pomegranate extract. Analysis of Feces and Urine. Two groups of five A / J mice were fasted for 6 hours. Group 1 received 0.5 ml of a pomegranate ellagitannins solution in 10% Aca cia (588 mg/kg b.w.). Group 2 received E A (147 mg/kg b.w.). The feces and urine were collected for 24 hours. Feces were extracted with methanol and analyzed by HPLC as described above. Levels of Ε A and ellagitannins in feces were determined by interpolation from calibration curves which were constructed by extracting feces from untreated mice to which were added known amounts of Ε A or ellagitannins. The extraction of free E A from urine was carried out as described for lung tissues. Results The inhibition of lung tumorigenesis was studied at four dose levels of E A . Feeding mice 4 g EA/kg diet reduced the lung tumor multiplicity by 54% (Figure 1). In mice fed a diet containing 0.06-4.00 g EA/kg diet, the lung multiplicity was inversely proportional to the logarithm of the dose of E A (r = 0.992). On the other hand, pomegranate ellagitannins had no effect on tumor multiplicity (Table I). 2

1 6

r

14 L

2h 0

l

ι

.01

.1

I

1

10

[EA] in diet (g/Kg)

Figure 1. Inhibition of NNK-induced lung tumors by E A at various concen trations. Data are the means ± SE from 23 to 25 mice. The curve was fit by the least square method and the data analyzed by linear regression (r = 0.992). 2

A HPLC assay of E A in EA-treated mice was developed. Tissues from mice gavaged with Ε A were extracted and the extract fractionated by HPLC. H P L C profiles of lung tissues extracts from EA-treated and untreated mice are compared in Figure 2. In Ε A treated mice, we observed a peak which co-eluted with E A and was base separated. The limit of detection of this assay was 0.4 nmol/g tissue.



24.

CASTONGUAY ET AL.


0

5

10

15

20

RETENTION TIME (min)

Figure 2. HPLC profiles of lung extracts. Panel A : Untreated mice; panel B : E A added to lung tissues from untreated mice before extraction; panel C: mice gavaged with 2 mmol EA/kg and sacrificed 30 min later.


297

298



Table I. Effects of Ellagic Acid and Pomegranate Ellagitannins on NNK-induced Lung Tumors in A/J Mice Total dose of N N K (mg/mouse)

Chemopreventive agent

Number of tumors per mouse

None 9.1 9.1 9.1

None None Ellagic acid 4 g/kg diet Pomegranate ellagitannin 10 g/kg diet

0.6 ± 0.2 6.8 ± 0 . 8 3.9 ± 0.4* 9.1 ± 2 . 0

a

l

mean ± SE, η = 25, *p1^Λ J

I

40

20

60

TIME (minutes)

π

1 20

1

1

1 40

1

r

60

TIME (minutes)

Figure 5. Panel A : HPLC profile of pomegranate extract. Panel B : H P L C profile of a pomegranate extract hydrolyzed with concentrated HC1. Bold arrows: ellagittannins, plain arrows: ellagic acid.



24. CASTONGUAY ET AL.


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process of carcinogenesis. Chemopreventive agents can be divided into two major classes: synthetic compounds (including pharmaceuticals) and naturally occurring compounds. The latter class has the advantages of being more acceptable to the public and more likely to be introduced quickly in clinical trial. It is noteworthy that most on-going intervention trials in chemoprevention of lung cancer involve vitamin Β12, Ε, A and β-carotene (provitamin A) found in food and beverages (15). Ellagitannin is a family of chemically complex substances characterized by the presence of one or more hexahydroxydiphenoyl groups. This group is cleaved during hydrolysis and undergoes intramolecular esterification. The resulting dilactone, ellagic acid, has been investigated for its efficacies to inhibit chemically induced carcinogenesis in laboratory animals. At high concentrations, it is generally effective against a wide variety of carcinogens in various tumor models (16-20). In this study we confirmed that Ε A is effective in preventing lung tumorigenesis. The efficacy of E A was dose related. Studies of the mechanism of action of E A have concluded that it could inhibit phase I enzymes involved in the activation of procarcinogens (21-22). According to Das et al. (23), ellagic acid induces hepatic glutathione S-transferase, a phase II enzyme responsible for the detoxification of some carcinogen-generated electrophiles. Ellagic acid may also scavenge oxygen radicals involved in oxidative destruction of membrane lipids and involved in tumor promotion (24). The relative importance of various hypotheses on the mechanism of action of E A remains to be determined. In this study, we have demonstrated that localization of Ε A in lung tissues is dose related. Our results demonstrated that localization of E A or its metabolites in lung tissues coincide with the inhibition of tumorigenesis. We have recently reported that pulmonary localization of E A could be increased by including Ε A in a β-cyclodextrin polymer (25). The chemopreventive properties of E A have been investigated extensively in spite of the fact that Ε A is not present as such in living plants (26). In contrast, the ellagitannins that are the precursors of E A have received little attention as chemopreventive agents. Recently, Gali et al. (27) showed that two ellagitannins, castagalagin and vescalagin, reduced TPA-induced ODC activity and hydro peroxide production in mouse epidermis. Geraniin and other ellagitannins exhibit strong antimutagenic properties (28). The major ellagitannins present in pomegra nate are granatins A and Β (29). The results of this study demonstrated for the first time that pomegranate ellagitannins are metabolized extensively in A/J mice. This metabolism leads to the hydrolysis of the hexahydroxydiphenoic group and its cyclization to EA. These results suggest that pomegranate is a natural source of EA, a non-toxic lung tumorigenesis chemopreventive agent. A relatively high dietary dose might be necessary, however, to achieve significant inhibition of lung tumori genesis. Acknowledgments This study was supported by a grant from the National Cancer Institute of Canada. Literature Cited 1. Hoffmann, D.; Hecht, S. S. In Handbook of Experimental Pharmacology; Cooper, C.; Grover, L . P., Eds.; Springer-Verlag: Berlin, 1990, Vol 94/I; pp 63-102.



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2. Brunnemann, K . D., Hoffmann, D., Wynder, E. L.; Gori, G. B . Proceedings of 3rd World Conference on Smoking and Health; US Department of Health, Education and Welfare: Washington DC [DHEW Publication No. (NIH) 761221], 1976; pp 441-449 3. Jaffe, J. H . In Nicotine Psychopharmacology, Molecular, Cellular, and Behavioral aspects; Wonnacott, S.; Russell, M . A . H . ; Stolerman, I. P.; Oxford University Press: New York, 1990; pp 1-37 4. Fischer, S.; Spiegelhalder, B.; Eisenbarth, J.; Preussmann, R. Carcinogenesis 1990, 11, 723-730. 5. Fischer, S.; Spiegelhalder, B.; Preussmann, R. Arch. Geschwulstforsch. 1990, 3, 169-177. 6. Hecht, S. S.; Hoffmann, D. Carcinogenesis 1988, 9, 875-884. 7. Hecht, S. S.; Hoffmann, D. Cancer Surv. 1989, 8, 273-294. 8. Kühnau, J. World Rev. Nutr. Diet 1976, 24, 117-191. 9. Bate-Smith, E. C. In The Pharmacology of Plant Phenolics Fairbairn, J. W., Ed.; Academic Press: New York, 1959; pp 133-147. 10. Kelloff, G. J.; Malone, W. F.; Boone, C. W.; Sigman, C. C.; Fay, J. R. Semin. Oncol. 1990, 17, 438-455. 11. Hecht, S. S.; Chen, C. B.; Dong, M.; Ornaf, R. M . ; Hoffmann, D.; Tso, T. C. Beitr. Tabakforsh. 1977, 9, 1-6. 12. Castonguay, Α.; Lin, D.; Stoner, G. D.; Radok, P.; Furuya, K.; Hecht, S. S.; Schut, H. A. G.; Klaunig, J. E. Cancer Res. 1983, 43, 1223-1229. 13. U . S. Surgeon General The Health Consequences of Smoking: Cancer U . S. Department of Health and Human Services: Washington, D.C. (NIH Publ. No. 82-50179), 1982. 14. Ramström, L . M. In Tobacco, a Major International Health Hazard Zaridze, D.; Peto, R., Eds. IARC Sci. Publi., 1986, Vol. 74; 135-142. 15. Castonguay, A. Cancer Res. 1992, 52; 2641s-2651s. 16. Tanaka, T.; Iwata, H.; Niwa, K.; Mori, Y.; Mori, H . Jpn. J. Cancer Res. 1988, 79, 1297-1303. 17. Mukhtar, H.; Das, M.; Bickers, D. R. Cancer Res. 1986, 46, 2262-2265. 18. Mukhtar, H.; Das, M.; Del Tito, B., Jr; Bickers, D. R. Biochem. Biophys. Res. Commun. 1984, 119, 751-757. 19. Lesca, P. Carcinogenesis 1983, 4, 1651-1653. 20. Mandai, S.; Stoner, G. D. Carcinogenesis 1990, 11, 55-61. 21. Teel, R. W.; Dixit, R.; Stoner, G. D. Carcinogenesis, 1985, 6, 391-395. 22. Mandal, S.; Shivapurkar, Ν. M.; Galati, A . J.; Stoner, G. D. Carcinogenesis 1988, 9, 1313-1316. 23. Das, M.; Bickers, D. R.; Mukhtar, H. Carcinogenesis 1985, 6, 1409-1413. 24. Osawa, T.; Ide, Α.; Su, J-D.; Namiki, M . J. Agric. Food Chem. 1987, 35, 808812. 25. Boukharta, M.; Jalbert, G.; Castonguay, A. Nutr. Cancer 1992, 18, 181-189. 26. Okuda, T.; Mori, K., Hatano; T. Phytochemistry 1980, 19, 547-551. 27. Gali, H . U . ; Perchellet, E. M.; Bottari, V.; Hemingway, R. W.; Scalbert, Α.; Perchellet, J. P. Proc. Am. Assoc. Cancer Res. 1992, 33, 162. 28. Okuda, Y.; Mori, K.; Hayatsu, H. Chem. Pharm. Bull. 1984, 32, 3755-3758. 29. Tanaka, T.; Nonaka, G.; Nishioka, I., Chem. Pharm. Bull. 1990, 38, 24242428. RECEIVED September 7, 1993


Comparative Study of Ellagic Acid and Its Analogues as

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