Chapter 7
The Anticancer Activity of Coffee Beans E. G. Miller , A. P. Gonzales , A. M . Orr , W. H . Binnie , and G. I. Sunahara 1
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Department of Biomedical Sciences, Baylor College of Dentistry, Texas A& M University System, 3302 Gaston Avenue, Dallas, TX 75246 Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada 1
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Earlier studies have shown that green coffee beans (GCB), defatted GCB, and GCB oil can inhibit the development of cancer in animals. The purpose of this study was to see what effect roasting might have on this activity. Sixty-eight hamsters were separated into 4 groups. The animals in group 1 were fed a normal diet, while the hamsters in groups 2, 3, and 4 were fed the same diet supplemented with 15% roasted coffee beans (RCB), 12.75% defatted RCB, or 2.25% RCB oil. The pouches of the hamsters were painted 36 times (3 x weekly) with a 0.5% solution of the carcinogen, 7,12-dimethylbenz[a]anthracene. The results showed that each of the special diets significantly reduced tumor burden. The mean values in mm were 238 (1), 29 (2), 38 (3), and 115 (4). 3
General Introduction In less than 400 years coffee has gone from a regional drink consumed primarily in Arab countries to an international beverage consumed throughout the world. Today more than five million tons are produced annually in approximately 50 coffee-growing countries. In international trade, green coffee beans are the second most important commodity in the world. In most of the industrialized countries of the world, coffee drinkers on average consume 3-5 cups per day. In 1991, the average was 3.4 cups per day for consumers in the United States (i). A variety of factors contribute to the chemical mixture that is labeled a cup of coffee. Some of these factors include the soil where the coffee is grown, the variety of the coffee plant, the degree of roasting, and the way the coffee is prepared. With so many variables, it is not surprising that each cup of coffee contains several hundred different chemicals. Some chemicals have been identified, others await discovery. Interest in the chemical composition of coffee partially stems from the fact that a large number of these chemicals contribute to the unique aroma and
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© 2000 American Chemical Society
57 flavor of the beverage. A second reason is that a cup of coffee has very little nutritional value. Consumption is instead linked to a stimulant, caffeine. Since coffee contains at least one pharmacologically active agent, it has been postulated that coffee might contain other chemicals, which might affect the health and well being of the consumer.
Mammary Carcinogenesis Work on the cancer chemopreventive activity of green coffee beans and coffee bean chemicals was initiated in the early 1980's in Dr. Lee Wattenberg's laboratory at the University of Minnesota In the first experiment, it was shown that the addition of 20% green coffee beans to the diets of the experimental animals inhibited by approximately 50-60% the development of carcinogen-induced mammary tumors (2). Further research led to the isolation of two antineoplastic agents (3,4). The chemicals, kahweol and cafestol, are both diterpenes that are structurally similar. The only difference is an additional double bond that is found in one of the rings in kahweol (4). The cancer chemopreventive activity of these two plant oils was demonstrated in a rat model for mammary carcinogenesis. Repeated exposures to a mixture of kahweol and cafestol followed by a single dose of the carcinogen resulted in a diminished (40%) neoplastic response (5). Additional studies with kahweol and cafestol indicated that both of these chemicals are blocking agents that induce increases in glutathione S-tiansferase activity (6). The glutathione S-transferase enzymes form one of the major enzyme systems in the body for the safe removal of complex chemical waste, including carcinogens, from the body (2).
Oral Carcinogenesis In the late 1980's, we initiated a series of experiments on the antineoplastic activity of green coffee beans, green coffee bean fractions, and coffee chemicals. In each experiment, the hamster cheek pouch model for oral carcinogenesis was utilized (7), The results from the first experiment showed that the addition of 20% green coffee beans to the diets of the experimental animals inhibited by 90-95% the development of the carcinogen-induced oral carcinomas (8). The second experiment tested the possibility that kahweol and cafestol might be responsible for the antineoplastic activity of green coffee beans seen in this tumor model. With the help of scientists at the Nestle Research Centre in Verschez-les-Blanc, Switzerland, we were able to obtain a 50:50 mixture of kahweol and cafestol. Using this mixture a test diet was constructed that approximated the kahweol and cafestol content of a diet containing 20% green coffee beans. The results of this experiment (0) showed that this special diet inhibited the development of carcinogen-induced oral carcinomas by only 35%. The data from the experiment with the diet containing kahweol and cafestol suggested that green coffee beans might contain other anticancer agents. To test
58 this possibility an experiment was conducted with green coffee beans and two green coffee beanfractions,green coffee bean oil and defatted green coffee beans. In the Colombian green coffee bean, the oil accounts for approximately 15% of the whole coffee bean. Essentially all of the kahweol and cafestol is in the oil fraction. The residual material accounting for 85% of the green coffee bean is the defatted green coffee bean fraction. The three special diets in this experiment contained either 15% green coffee beans, 12.75% defatted green coffee beans, or 2.25% green coffee bean oil. Each of these special diets significantly inhibited the development of the carcinogen-induced oral tumors. The extent of the inhibition rangedfrom70% with whole green coffee beans, to 55% with defatted green coffee beans, to 60% with green coffee bean oil (10,11). The results of these experiments indicate that the cancer chemopreventive activity of green coffee beans is more complex than originally thought Instead of two cancer chemopreventive agents, green coffee beans appear to contain multiple chemicals with antineoplastic activity. Some of the chemicals with biological activity are in the oil; others are in the defatted portion of the bean. The objective of the experiment described in this paper was to see what effect, if any, roasting might have on the cancer chemopreventive activity of coffee beans. During roasting, the beans are heated to 200-230°C and kept at this temperature for 10-15 minutes. This process leads to obvious changes in color and size, as well as, multiple changes in chemical composition. For example, the chemicals that give coffee its unique aroma are formed during the roasting process. A brief report of this research has been published (12).
Roasted Coffee Beans and Roasted Coffee Bean Fractions For the experiment, 80 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 wire-mesh cages of stainless steel in a temperature-controlled room (22°C) with a 12:12 hour light-dark cycle. Throughout the experiment water and food were furnished ad libitum. After arriving, the hamsters were given 10 days to acclimate. During this time, all of the animals were fed a Purina Lab Chow (St. Louis, MO) specifically formulated for small rodents. After this initial period of adjustment, the hamsters were weighed and randomly divided into one of four equal groups (20 animals per group) and placed on one of four diets. The hamsters in group 1 remained on the Purina Lab Chow. The hamsters m groups 2,3, and 4 received the same Purina Lab Chow supplemented with either 15% whole roasted coffee beans (group 2), 12.75% defatted roasted coffee beans (group 3), or 2.25% roasted coffee bean oil (group 4). One batch of whole roasted Colombian coffee beans was used to prepare the ingredients used in the diets for groups 2, 3, and 4. On a weight to weight basis, 15% of the Colombian roasted coffee bean is the roasted coffee bean oil fraction and 85% is the defatted roasted coffee bean fraction.
59 All of the diets were given in powdered form. The diets for the hamsters in groups 2, 3, and 4 were prepared weekly. The animals remained on their respective diets for the rest of the experiment The hamsters were given an additional week to adjust to their diets. Seventeen animals were then selected from each group. The left buccal pouches of these animals were painted 3 χ weekly with a 0.5% solution of the carcinogen, 7,12-dimethylbenz[a]anthracene (Sigma Chemical Co., St Louis, MO). Heavy mineral oil was used to prepare this solution. The 3 remaining animals served as controls. The left buccal pouches of these animals were painted 3 χ weekly with heavy mineral oil. A #5 earners-hair brush was used to paint the liquids on the pouches. Each application places approximately 50 μΐ of liquid on the surface of the pouch (P). After a total of 36 applications, the hamsters were sacrificed by inhalation of an overdose of carbon dioxide. The left buccal pouches were excised and tumors, when present, were counted and measured (length, width, and height). The sum of the three measurements divided by six was used to calculate an average radius for each tumor. This number was then used to determine an approximate volume for the tumor. Since the tumors are exophytic and spherical in shape, the formula for the volume of a sphere, 4/3πτ , was used for this calculation. The sum of the volumes of all of the tumors in a pouch was determined and defined to be the animal's total tumor burden (9, 13). After the gross tumor data were collected, the pouches were mounted on heavy paper and fixed in 10% formalin. The tissues, including the pouches from the control animals, were embedded in paraffin, processed by routine histological techniques, and stained with hematoxylin and eosia The Student's t-test and Chisquare analysis were used to analyze the significance of the tumor data. These procedures are similar to the procedures used in our earlier experiments with green coffee beans, green coffee beanfractions,and isolated coffee chemicals (8-11). 3
Tumor Data Three hamsters died before the end of the experiment These deaths occurred relatively early before the animals had a chance to develop tumors. All of these hamsters were excludedfromthe study. At the end of the experiment, there were 16 experimental animals in groups 1, 2, and 3 and 17 hamsters in group 4. The tumor incidence data are given in Table I. As indicated, 15 of the 16 animals on the regular chow (group 1) had gross tumors. In 12 of these animals, multiple tumors (2-10) were found. Six of the pouches from the animals on the special diets (groups 2, 3, and 4) were free of visible tumors. Multiple tumors (2-9) were common in two of the three groups (11/16 in group 3 and 13/17 in group 4). In group 2, only 6 of the 16 pouches contained multiple tumors (2-5). The high and low values for the total number of tumors were 70 for group 1 and 29 for group 2. The data for average tumor number radii, and burden are given in Table Π. As illustrated, each of the special diets had an effect on tumor number. Comparing groups 2,3, and 4 to group 1, it can be seen that the inhibition in average tumor
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Table L Tumor Incidence Group
No. of Animals
1 2 3 4
16 16 16 17
No. of Tumor Bearing Animals 15 13 14 16
(94%) (81%) (88%) (94%)
Total No. ofTumors 70 29 45 66
SOURCE: Adaptedfromreference 12. number ranged from a low of 10% for the animals on the roasted coffee bean oil diet (group 4), to 35% for the animals on the defatted roasted coffee bean diet (group 3), to 60% for the animals on the whole roasted coffee bean diet (group 2). Only the differences between groups 1 and 2 were significant (Student's t-test). The three diets also had an effect on average tumor radii. Here the extent of the inhibition rangedfroma low of 20% for group 4 to 35% for groups 2 and 3.
Table Π. Average Tumor Number, Radii, and Burden
Group
Avg. No. of Tumor f
1 2 3 4
4.4 ± 0 . 7 1.8±0.4 2.8 ± 0 . 6 3.9 ± 0 . 6
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Avg. Tumor Radii (mm) 2.35 1.55 1.50 1.90
Avg. Tumor Burden (mm )* 3
238 ± 6 5 29±15 38±16 115 ± 3 3 ° b
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Values are means ± S.E. Statistically differentfromGroup 1, p
