Chapter 17
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Inhibition of Oral Carcinogenesis by Green Coffee Beans and Limonoid Glucosides 1
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E. G. Miller , A. P. Gonzales-Sanders , A. M . Couvillon , J . M . Wright , Shin Hasegawa , Luke Κ. T. Lam , and G. I. Sunahara 2
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Department of Biomedical Sciences, Baylor College of Dentistry, Dallas, TX 75246 Fruit and Vegetable Chemistry Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 263 South Chester Avenue, Pasadena, CA 91106 Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455 Nestle Research Centre, CH-1800, Vevey, Switzerland BP 353 2
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The hamster cheek pouch model was used to test green coffee beans, defatted green coffee beans, green coffee bean oil, and three limonoid glucosides for antineoplastic activity. The cancer chemo preventive activity of green coffee beans was found to be due to chemicals in the oil, probably kahweol and cafestol, and to one or more chemicals in the defatted portion of the bean. Using a technique developed in this laboratory, it was found that the oil induced increased glutathione S-transferase activity; the defatted beans did not. In a separate experiment, one of the limonoid glucosides, limonin 17-β-D-glucopyranoside, was found to inhibit, by approximately 55%, the development of oral tumors. This chemical had no affect on the glutathione S-transferase activity of oral epithelial cells. This research on the cancer chemopreventive activity of green coffee beans is an outgrowth of previous work in Dr. Lee Wattenberg's laboratory at the University of Minnesota. Their results showed that the addition of green coffee beans (20%) to the diet of experimental animals inhibited by approximately 60% the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors (1). Fur ther studies (2) led to the isolation of two cancer chemopreventive agents from green coffee beans. The compounds, kahweol and cafestol, are diterpenoids that are usually found as esters of fatty acids. The free alcohols are chemically similar, differing from each other by one extra double bond that is found in one of the sixsided rings in kahweol (3). The antineoplastic activity of these two coffee bean constituents was demonstrated in a rat model for mammary carcinogenesis. It was found that oral intubation of the two diterpenes once a day for 3 days prior to a single dose of the carcinogen resulted in a diminished (40%) neoplastic response (4). Each dose 0097-6156/94/0546-0220$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.
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17. MILLER ET AL.
Green Coffee Beans and Limonoid Glucosides
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contained 60 mg of the diterpene. The kahweol and cafestol were dissolved in cottonseed oil. Additional studies with kahweol and cafestol suggest that both compounds are type A blocking agents that induce increases in glutathione ^-transferase (GST) activity (5). Since kahweol is the more potent inducer of GST activity, it is assumed that kahweol is the more potent inhibitor of carcinogenesis. The experiments with kahweol (5) also showed that certain features in its chemical structure are critical to its ability to stimulate GST activity. One of these structural features is a furan ring, the other is the extra double bond. This laboratory has continued the research on the cancer chemopreventive activity of green coffee beans (6,7). Using the hamster cheek pouch model for oral carcinogenesis, it was found that a diet containing 20% green coffee beans (Colombian) inhibited the formation of DMBΑ-induced epidermoid carcinomas by more than 95% (6). With the help of Dr. Wurzner, Dr. Liardon, and Mr. Bertholet at the Nestlé Research Centre, we were able to obtain large quantities of a 50:50 mixture of kahweol and cafestol. Using this mixture we prepared a diet that approximated the kahweol and cafestol content of a diet containing 20% green coffee beans (Colombian). This experiment showed that when the modified diet was fed to hamsters, it inhibited the development of D M B Α-induced oral tumors by approximately 35-40% (7). Green Coffee Beans and Green Coffee Bean Fractions In summary, the data from these experiments (6,7) indicate that green coffee beans might contain additional cancer chemopreventive agents. To test this possibility, we went back to the green coffee bean and to two fractions of the green coffee bean, green coffee bean oil and the defatted green coffee bean. In the Colombian green coffee bean, the green coffee bean oil accounts for approximately 15% (w/w) of the whole bean. The oil contains kahweol and cafestol as well as other plant oils and lipids. After the oil is removed, the remainder of the green coffee bean (85%) is the defatted green coffee bean. This fraction is essentially devoid of kahweol and cafestol. For the experiment, 64 female Syrian golden hamsters (Lak:LVG strain) weighing 80-90 g were purchased from the Charles River Breeding Laboratories (Wilmington, MA). The animals were housed in stainless steel cages in a temperaturecontrolled room (22°C) with a 12 hour light-dark cycle. Water and food were furnished ad libitum. The hamsters were given 10 days to adjust to their new surroundings. During this time they were fed a Purina Lab Chow specifically formulated for small rodents. The animals were then weighed and separated into four equal groups. The hamsters in group 1 were fed the Purina Lab Chow. The hamsters in groups 2, 3, and 4 were fed the same diet supplemented with 15% green coffee beans (group 2), 12.75% defatted green coffee beans (group 3), or 2.25% green coffee bean oil (group 4). After the animals adjusted to their respective diets, the treatments with the carcinogen were initiated. The left buccal pouches of all of the animals in each group were inverted and painted 3 χ weekly with a 0.5% solution of D M B A dissolved in heavy mineral oil. Each application with a camel hair brush places approximately 0.05 ml of the D M B A solution on the surface of the pouch. After a total of 36 applications the treatments were discontinued. One week later all of the animals were sacrifice by inhalation of an ethyl ether overdose. The pouches were excised. Tumors, when present grossly, were counted and measured (length, width, In Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
and height). The sum of these three measurements divided by six was used to calculate an average radius for each tumor. By using the formula for the volume of a sphere, 4/3πΓ , an approximation of the volume of each tumor was determined. A simple sum of the volume of each tumor in one pouch was defined to be the animal's tumor burden (7-9). Once the measurements had been taken the excised pouches were fixed in 10% formalin, embedded in paraffin, processed by routine histological techniques, and stained with hematoxylin and eosin. The microscopic and macroscopic observations were used to determine tumor incidence, number, mass, and type. Student's ί-test was used to assess the significance of the data.
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Tumor Data. Two animals died before the end of the experiment. Both of these animals were excluded from the study. At the end of the experiment, the number of hamsters in the four groups was 16 (group 1), 15 (group 2), 15 (group 3), and 16 (group 4). A l l of the hamsters in groups 1 and 4, 12 of the 15 hamsters in group 2, and 13 of the 15 hamsters in group 3 had visible tumors. Most of the animals, 15/16 in group 1, 10/15 in group 2, 13/15 in group 3, and 14/16 in group 4 had multiple tumors. The total number of tumors ranged from a low of 39 for groups 2 and 3 to a high of 75 for group 1. The values for tumor radii, which were calculated from the three measurements, ranged from 0.5 to 5.0 mm. The values for tumor volume ranged from 0.5 to 524 mm . The data for average tumor number, average tumor burden, and average tumor mass are given in Table I. As illustrated, the treatment with green coffee beans (group 2) led to a 45% reduction in average tumor number and a 55% reduction in average tumor mass. Tumor burden was reduced by 70%. A similar comparison between groups 1 and 3 showed that the diet containing defatted green coffee beans reduced average tumor number by 45% and the average tumor mass by 25%. The decrease in average tumor burden was 55%. The same comparison for groups 1 and 4 (green coffee bean oil diet) yielded a 25% reduction in average tumor number, a 55% reduction in average tumor mass, and a 60% reduction in average tumor burden. 3
Table I. Effect of Dietary Green Coffee Beans and Green Coffee Bean Fractions on Oral Carcinogenesis Group
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Number of Animals 16 15 15 16
Number of Tumors 3
4.7 ± 0.5 2.6 ±0.6**** 2.6 ±0.4**** 3.5 ±0.6*
Tumor Burden (mm ) 3
145 ± 4 1 43 ± 15*** 62 ± 34* 57 ± 1 5 * *
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Tumor Mass (mm )
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30.9 16.5 23.8 16.3
SOURCE: Adapted from ref. 10. Values are means ± S.E. Values for average tumor mass were calculated by dividing the values for average tumor burden by corresponding values for average tumor number. Statistically different from Group 1: *, p
