logo Nutrition Research, Vol. 20, No. 11,
pp. 1557-1567, 2000
Copyright © 2000 Elsevier Science Inc.
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Effects Of The Coral Calcium As An Inhibitory Substance Against Colon Cancer And Its Metastasis In The Lungs

Material & Methods


  • (i) Survival (growth) rates of the cancer cells in acidic culture media and alkaline culture media, in vitro, and (ii) the effect of Coral Calcium concentration against Colon 26 cancer cells in comparison with calcium carbonate, in vitro.
Cancer cell strains (i.e.. mouse Colon 26 cancer cells) generated following repeated administration of N-methyl-N-nitroso-urethan to the rectum of BALB/C mice (purchased from Japan Clea Strain) were used.  Study groups consisted of five groups with pH 6.0, pH 6.5, pH 7.0, pH 7.2 and pH 7.5. The group with pH 7.2, being identical to the pH value in the human body, was used as a control group. Using edible efflorescent coral powders (Coral Calcium* supplied from Coral Biotech. Co., Ltd, Fig. 1), the pH values of the culture media were adjusted to the designated values.  That is, aqueous solutions of Coral Calcium (500 mg of Coral Calcium per dL) were initially prepared. The solutions were adjusted to pH 7.0 with 1N NaOH, respectively, and adjusted to pH 6.0, pH 6.5, pH 7.0, pH 7.2 and pH 7.5 with 1N HCl, respectively. To each pH group, a fetal cow serum (FCS) was added to make a concentration of 10%.

*Coral Calcium is a powder of natural coral which has formed a reef and effloresced for several hundred million years to ten thousand years. Coral Calcium as collected as a natural, edible efflorescent coral from the bottom of the sea in the southwest islands or the Okinawa island of Japan, and was processed as an edible powdered product. Coral Calcium is a product registered as No. 428 in a list of food ingredients specified by the Ministry of Health and Welfare in Japan, in 1996. As shown in Fig. 1, Coral Calcium is a mixture of calcium carbonate and various trace elements. Calcium (Ca) only accounts for 35 w/w% of the total amount of Coral Calcium.


FIG. 1   Analytical Values (%) Of Coral Calcium Powder
  Sample 1 (100 g)* Sample 2 (100 g)* Sample 3 (100 g)* Sample 4 (100 g)*
  mg w/w% mg w/w% mg w/w% mg w/w%
Calcium (Ca) 35800 35.8 35200 35.2 35800 35.8 35100 35.1
Sodium (Na) 29 0.29 280 0.28 290 0.29 390 0.39
Magnesium (Mg) 2180 2.18 2090 2.09 2280 2.28 2690 2.69
Potassium (K) 7 0.007 8 0.0008 6 0.0006 7 0.007
Phosphorus (P) 47 0.047 32 0.032 62 0.062 49 0.049
Iron (Fe) 26 0.026 47 0.047 25 0.025 34 0.034
Chlorine (Cl) 17 0.017 15 0.015 23 0.023 19 0.019
Sulfur (S) 200 0.20 230 0.23 190 0.19 180 0.18
Silicon (Si) 90 0.09 110 0.11 230 0.23 180 0.16
Aluminum (Al) 32 0.032 35 0.035 30 0.03 33 0.033
Strontium (Sr) 34 0.034 31 0.031 21 0.021 38 0.038
Chromium (Cr) 2.2 0.0022 1.5 0.0015 2.3 0.0023 1.9 0.0019
Cobalt (Co) 1.2 0.0012 1.1 0.0011 1.5 0.0015 1.3 0.0013
Nickel (Ni) 1.3 0.0013 1.5 0.0015 0.9 0.0009 1.2 0.0012
Manganese (Mn) 1.1 0.0011 1.3 0.0013 1.4 0.0014 1.3 0.0013
Copper (Cu) 0.6 0.0006 0.59 0.00059 0.56 0.00056 0.54 0.00054
Zinc (Zn) 0.5 0.0005 0.58 0.00058 0.54 0.00054 0.6 0.0006
Molybdenum (Mo) 0.05 0.00005 0.05 0.00005 0.06 0.00006 0.055 0.000055
Cadmium (Cd) ND ND ND ND ND ND ND ND
Total Mercury (Hg) ND ND ND ND ND ND ND ND
         
Microorganisms        
Total viable aerobic count < 3 X 102/g < 3 X 102/g < 3 X 102/g < 3 X 102/g
Mould - Fungi < 1 X 10/g < 1 X 10/g < 1 X 10/g < 1 X 10/g
Yeast - Fungi Negative Negative Negative Negative
E. Coli Negative Negative Negative Negative
Salmonellae Negative Negative Negative Negative
Vibrioparahaemolyticus Negative Negative Negative Negative
         
Polychlorobiphenyl (PCB) ND Not detected Not detected Not detected
Residual agricultural chemicals ND Not detected Not detected Not detected
Arsenic (as AS2O3) 1.4 ppm 1.8 ppm 1.3 ppm 1.5 ppm
Lead 0.3 ppm 0.5 ppm 0.6 ppm 0.8 ppm
* Measured by an atomic absorption spectrophotometer. Components other than the above components are trace elements, each of which was below the detection limit, and are carbon dioxide and oxygen combined.   ND: Not detected.


The subcultivation medium contained RPMI-1640 liquid medium supplemented with 0.2% NaHCO3, 10% FCS, 100U/mL-100 µg/mL of P-S, and 25mM HEPES. The Colon 26 cancer cells (1 X 105) used were separated from the mice bearing Colon 26 cancer, serially transferred by the subcutaneous inoculation (0.2 µL/body) in a dorsal portion of the mouse. The serial passage leads to a decrease in activity of the Colon 26 cancer cells. For this reason, the colon cancer strains (Colon 26) were separated from mice having cancer, at the time when the study was carried out. Each group containing the cells was incubated for 120 hrs. at 37°C in 5%CO2. The survival (growth) rate (%) of the Colon 26 cancer cells in each group was calculated — as a ratio of the number of Colon 26 cancer cells in each group to that of the pH 7.2 group. In addition, the morphological findings of the cells were observed by a microscope.
  • Prophylactic in vivo effects of Coral Calcium (as a cancer-inhibitory substance) against the metastasis of cancer in the lungs, following inoculation of Colon 26 cancer cells into mice.
    - This study is supported by three control in vivo studies.
BALB/C mice (purchased from Japan Clea Strain) at the age of 7 weeks were used.
Study groups in mice:
Group A: Administration of 100mg of Coral Calcium per kg, 300 µL/body
Group B: Administration of 5 mg of Coral Calcium per kg. 300 µL/body
Using Coral Calcium supplied from Coral Biotech. Co., Ltd., Fig.1, 100mg of Coral Calcium per kg, 300 µL/body was orally administered once a day for 30 consecutive days to each of 28 mice in group A, while 5 mg of Coral Calcium per kg, 300 µL/body was orally administered once a day for 30 consecutive days to each of 28 mice in group B.

On Day 30 after the administration, mouse colon cancer cell strains (Colon 26, 1 x 105 cells/200µL for each mouse) were inoculated into the tall vein (i.v.) of each mouse, after which the coral powders were continuously administered to each mouse until sacrificed. On Day 7 after the inoculation of the cancer cells, in half of the mice in each group, the activity (%) of natural killer cells was measured by 51Cr release method and the number (%) of macrophages was also measured. On Day 14 after the inoculation, in the remaining half of the mice in each group, metastatic lesions of cancer cells in the lungs were measured, along with histopathological and hematological examinations. Statistical analyses were performed according to student t-test, Dunnet method, and Kruskal-Wallis method.


Abstract   |   Introduction   |   Materials & Methods   |   Results & Discussion   |   References