Effects of Coffee Cherry, the Residue Left after Removal of the Beans from the Coffee Fruit, on Mammary Glands, Automatic Behavior and Related Parameters in Mice
(Accepted for publication October 25, 2000)
Abstract: To clarify the mechanisms of the anti-mammary tumor activity of coffee cherry (CC), the residue left after the removal of beans from the fruit, the effects in SHN mice of CC on plasma and urine component levels, mammary gland growth, spontaneous motor activity and several related parameters were examined. Hot water extract of CC was given to 2-month-old mice in drinking water (0.5%) for 60 days. The treatment prevented the elevation of plasma and urine levels of alanin amino-transferase and asparate aminotransferase, indicating that CC can protect against metabolic abnormality, which is a cause of the high mammary tumor susceptibility of SHN mice. It also resulted in an inhibition of the formation of precancerous mammary hyperplastic alveolar nodules. Neither food and water intake nor spontaneous motor activity was affected by CC. The findings provide novel information on the mechanism of the protective effect of CC on mammary tumorigenesis and confirm the usefulness of CC as a safe chemopreventive agent of mammary and other types of tumors.
Coffee cherry (CC: the residue left after removal of beans from the coffee fruit) is mostly discarded: a small percentage is used locally as a health drink. We previously found that the hot water extract of CC markedly inhibited both the development (Nagasawa et al., 1995) and the growth (Nagasawa et al., 1996) of spontaneous mammary tumors in mice. It is well known that changes in the target tissue are important in the development of lesions and that emotional or mental state has some kind of effect on most diseases with long latent periods. In this respect, several natural products influence spontaneous motor activity, a reflection of the emotional or mental condition, relaxed or stressful (Nagasawa et al., unpublished). Furthermore, the general metabolic activity is considered to relate to mammary tumor potential in mice (Yasuda et al., 1996). Irradiation with far-infrared rays, which inhibited markedly the spontaneous mammary tumorigenesis of mice (Nagasawa et al., 1999b, 2000b; Udagawa et al., 1999) also suppressed the formation of precancerous hyperplastic aleveolar nodules in mammary tissue (Nagasawa et al., 2000b), stimulated spontaneous motor activity (Nagasawa et al., 2000b; Udagawa and Nagasawa, 2000a) and ameliorated the metabolism (Nagasawa et al., 2000c; Udagawa and Nagasawa, 2000a).
Thus, in this study, we examined the effects of CC on mammary glands, spontaneous behavior and plasma and urine component levels in young SHN mice given CC for limited periods as a step in clarifying the mechanisms of the protective role of CC in mammary tumorigenesis.
Materials and Methods
Coffee Cherry (CC)
Dry matter of CC (500 g) donated by Toarco JAYA (Jakarta, Indonesia) was soaked in distilled water (5 liter) and left at room temperature for 7 days with occasional shaking every day. This was repeated 5 times and the supernatants were pooled and dried in vacuo at 45 [degrees] C. The dried extract was diluted with tap water at the concentration of 0.5% (5 g/1 liter) and given as drinking water.
Animals and Treatments
A high mammary tumor strain of SHN/Mei virgin mice (Nagasawa et al., 1976; Staats, 1976) maintained by full-sib mating was used. At 2 months of age, the mice were divided into two groups, the control and the experimental groups, given tap water and CC solution, respectively. Throughout the experiments, mice were kept in plastic cages with wood shavings (16 x 28 x 13 cm), 4 each, maintained in a windowless animal room, which was air-conditioned (22 [+ or -] 0.5 [degrees] C and 60-70% relative humidity) and artificially illuminated (14 hr of light from 0500 hr to 1900 hr). The diet (Lab MR Breeder, Nihon Nosan Kogyo KK, Yokohama, Japan) and water were provided ad libitum.
The motor activity of each animal was measured after 30 days of treatments and shortly thereafter blood and urine were taken from each of these mice. This procedure was repeated for each of animals at 60 days and they were then sacrificed. All procedures were carried out according to the NIH Guide for the Care and Use of Laboratory Animals, USA (Natl. Res. Council, 1996).
Spontaneous Motor Activity
After 30 and 60 days of CC treatment (3 and 4 months of age), spontaneous motor activity was measured by Supermex (Muromachi Kikai, Tokyo, Japan) (Masuno et al., 1995), a sensor monitor which was mounted above the cage to detect changes in heat across multiple zones of the cage through an array of Freshnel lenses. The body heat radiated by an animal was detected with paired IR pyroelectric detectors on the sensor head of the monitor. In this way, the system could monitor and count all spontaneous movements, both vertical and horizontal, including locomotion, rearing, head-movement, etc. All counts were automatically totaled and recorded at an hourly interval.
Mice were individually placed in the measurement cages (15 x 28 x 11 cm) in the measurement room 24 hr before measurement so as to become accustomed to the conditions.
Food and Water Intakes
Beginning at 1 week of treatment, food and water intakes were estimated from the differences in the weights of the food box and water bottle, respectively, for 3 consecutive days and finally expressed in terms of g/mouse/day.
Urine Component Levels
A few days after each measurement of motor activity, urine was collected by
the gentle pressing of the bladder and centrifuged at room temperature at 2,000 xg for 15 min. The supernatant was stored at -70 [degrees] C. The component levels were determined by Abaxis (EA-4: Daiichi Pure Chemical Co, Ltd, Tokyo, Japan).
Plasma Component Levels
A few days after each urine collection, mice were fasted for 18 hr and heparinized blood was collected by puncture of the orbital vein under light ether anesthesia and centrifuged at 2,000 xg at room temperature for 20 min. The plasma was stored at -70 [degrees] C. Plasma component levels were determined by Abaxis.
Body and Organ Weights
Each mouse was weighed at the start of the experiment, just after the measurement of motor activity and at killing by decapitation under light ether anesthesia immediately after the 2nd bleeding.
At autopsy, anterior pituitary, adrenals, ovaries, thymus, spleen, heart, lung, pancrease, liver and kidneys were immediately removed and weighed.
Normal and Preneoptastic Growth of Mammary Glands
At autopsy, the unilateral third thoracic mammary gland was prepared for wholemount evaluation and examined at 10-fold magnification. The degree of normal end-bud formation was rated from 1 to 7 in increments of 1 (Nagasawa et al., 1980) and the area bound by the tops of ducts with straight lines was also measured by computerized digitizer (Model LA-535; PIAS, Tokyo, Japan) as an index of duct growth.
The preneoplastic mammary hyperplastic alveolar nodules (HAN) were enumerated and the area of each HAN was measured.
The statistical significance p [is less than] 0.05) of the difference between groups in each parameter was evaluated by Student’s t-test.
Spontaneous Motor Activity (Figure 1)
[Figure 1 ILLUSTRATION OMITTED]
While the activity in the dark phase differed little between 30 and 60 days of treatment, the activity in the light phase and thus total activity were higher at 60 days than 30 days in both the control and CC groups, which agreed with our previous observation (Nagasawa et al., 2000a). However, the activity at all phases differed only slightly between groups at either 30 or 60 days of treatment.
Food and Water Intakes
Food and water intakes were 3.5-4.0 and 5.5-6.5 g/day/mouse, respectively, in both groups, and differed little (data not shown).
Plasma Component Levels (Figure 2)
[Figure 2 ILLUSTRATION OMITTED]
Plasma levels of alanine aminotransferase (ALT), asparate aminotransferase (AST) and blood urea nitrogen at 30 days of treatment differed little between the control and CC groups, which corresponds to the levels of ICR, most popular and normal mice. Meanwhile, at 60 days, these levels were elevated in the control, but were the same as at 30 days in the CC group.
The plasma cholesterol level at 60 days, which declined in the control, was maintained at the normal (ICR) level in the CC group.
No marked changes between 30 and 60 days and between groups were observed in the other plasma component levels.
Urine Component Levels (Table 1)
Table 1. Urinary Component Levels in Each Group (Mean [+ or -] SEM)
period (days) 30
Group (4)(a) (4)
Albumin NC ND
Alkaline phosphatase 14.8 [+ or -] 1.5 12.5 [+ or -] 0.6
Alanine aminotransferase 56.0 [+ or -] 10.0 51.3 [+ or -] 5.6
Amylase 5.0 ND
(U/l) [5.0] (1/4)(d)
Aspartate aminotransferase 25.8 [+ or -] 3.6 24.5 [+ or -] 1.8
Globulin(e) ND ND
Total bilirubin ND ND
Urea nitrogen ND ND
Calcium 4.1, 4.1 ND
(mg/dl) [4.0] (2/4)
Cholesterol ND 24
(mg/dl)  (1/4)
Creatinine 1.3, 2.3 1.4 [+ or -] 0.4
(mg/dl) [0.2] (2/4) (3/4)
Glucose 31.5 [+ or -] 3.3 37.0 [+ or -] 4.2
Total protein ND ND
period (days) 60
Group (4) (4)
Albumin ND ND
Alkaline phosphatase 13.0 [+ or -] 1.1 13.0 [+ or -] 0.6
Alanine aminotransferase 65.5 [+ or -] 6.7 46.0 [+ or -] 2.4(*)
Amylase 16.3 [+ or -] 6.6 10.3 [+ or -] 2.7
(U/l) [5.0] (3/4) (3/4)
Aspartate aminotransferase 30.0 [+ or -] 3.4 22.8 [+ or -] 0.5(*)
Globulin(e) ND ND
Total bilirubin ND 0.2
(mg/dl) [0.1] (1/4)
Urea nitrogen ND ND
Calcium 4.6, 4.2 5.0
(mg/dl) [4.0] (2/4) (1/4)
Cholesterol ND ND
Creatinine 5.8 [+ or -] 2.6 10.4 [+ or -] 3.2
(mg/dl) [0.2] (3/4) (3/4)
Glucose 58.3 [+ or -] 14.9 27.3 [+ or -] 1.9(*)
Total protein ND ND
(a) Number of animals.
(b) Detectable limit. All samples were not detected.
(d) Number of detected samples/Number of samples.
(e) Globulin = Total protein-Albumin.
(*) Significantly different from the corresponding control at p < 0.05.
Elevations from 30 to 60 days of treatment in the urine levels of ALT and AST in the control were prevented in the CC group.
Growth of Normal and Preneoptastic Mammary Glands (Figure 3)
[Figure 3 ILLUSTRATION OMITTED]
The rating as an index of mammary end-bud formation was apparently lower in the CC group than the control. Furthermore, the number of precancerous HAN of the CC group was approximately one/third that of the control.
Weights of Body and Organs
All parameters differed little between the control and the CC groups except body weight after 30 days of treatment and pancreatic weight at autopsy, which was significantly lower and higher, respectively, in the CC group (data not shown).
ALT, AST and blood urea nitrogen are generally used as indices of the functions of liver, kidney and other organs, of which abnormality results in their elevation in the circulation. In this study, the elevation of both plasma and urine levels of ALT and AST was normalized nearly to the ICR levels by the CC treatment. Low metabolic function is a cause of the high mammary tumor susceptibility of SHN mice used in this study (Yasuda et al., 1996). These findings indicate that CC protects against mammary tumorigenesis through bringing functional and metabolic activity near to the normal levels and support our previous hypothesis that the protective role of CC in mammary tumorigenesis is principally ascribable to its `normalization effects’ (Nagasawa et al., 1999a; Udagawa and Nagasawa 2000b). The decreased number of HAN on CC treatment obtained in this study is also ascribed to this normalization effect of CC.
Spontaneous motor activity at all phases differed little between the control and the CC groups. This strongly suggests that CC had little affect on the emotional/mental state. CC treatment had no marked effect on food and water intakes, or weights of body and major organs. CC reportedly decreased body weight significantly, which may prevent obesity (Nagasawa et al., 1995). This would principally be due to the difference in the treatment period.
The observations in this study provide information as to the mechanism of the protective effect of CC on mammary tumorigenesis and confirmed the usefulness of CC as a safe chemopreventive agent for mammary and other types of tumors.
We thank Mr. Takashi Ohara, Previous General Advisor of P.T. Toarco JAYA, Jakarata, Indonesia, for CC. Our thanks are also due to T. Nakajima of this laboratory for his help.
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Hiroshi Nagasawa(*)(1), Erika Yada(2), Yoko Udagawa(1) and Hideo Inatomi(3) (1) Experimental Animal Research Laboratory and (3) Laboratory of Natural Products Chemistry, Meiji University, Kawasaki, Japan, and (2) Laboratory of Applied Genetics, Department of Animal Resource Science, Graduate School of Agricultural Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan (*) Corresponding address
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