JPH10212228A - Carcinogenesis or neoplasm metastasis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a carcinogenesis or neoplasm metastasis inhibitor, which is derived from a naturally occurring substance, and shows little toxicity and excellent effects of inhibiting carcinogenesis and neoplasm metastasis, by using, as the active ingredient, a specific, naturally-occurring carotenoid, in particular that presents in foods. SOLUTION: This carcinogenesis or neoplasm metastasis inhibitor uses zeaxanthin as the active ingredient. Zeaxanthin is found in corn, alfalfa or the like, and algae. In particular, it is richly contained in blue-green algae, and Spirulina algae are the most preferable stock to produce zeaxanthin. It is recommended to extract zeaxanthin from the algae and purify the extract so as to contain only a limited quantity of impurities, e.g. to a purity of 95% or higher. It is also recommended to have a content of the active ingredient in the inhibitor normally in a range from 0.01 to 100wt.%. The daily dose rate is 5 to 50mg/kg as zeaxanthin in the case of oral administration while 0.1 to 10mg/kg in the case of parenteral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a carcinogenesis or tumor metastasis inhibitor containing zeaxanthin as an active ingredient.

[0002]

2. Description of the Related Art At present, surgical treatment, radiation therapy, chemotherapy (drug administration) and the like are generally performed as treatments for cancer. Of these, as chemotherapy,
Therapeutic methods of administering an agent that directly acts on tumor cells to kill tumor cells are widely applied, and there are many proposals regarding antitumor agents used in this type of therapy. However, this type of drug that acts on tumor cells kills tumor cells and also acts on normal cells, so that it has a high therapeutic effect on cancer, but has the disadvantage of having very strong side effects.

[0003] These methods are effective means for removing or killing the primary tumor, but in most cases when the primary tumor is found, it has been completely cured because it has already metastasized to other tissues or organs. It is extremely difficult to eradicate. In addition, in chemotherapy, serious side effects are often observed due to large doses, which is an important issue in cancer treatment.

[0004] Therefore, as a new antitumor agent, a drug that enhances the immune ability of normal cells and indirectly suppresses the growth of tumor cells has attracted attention, and for example, krestin, interferon, and the like have been developed. Although these drugs have a weak effect on tumor cells, they have the advantage of having few side effects. More recently, carotenoids have been shown to effectively inhibit the growth of tumor cells. For example, regarding β-carotene, “Oncology 39, 33-37, 1982” and “BIOCHEM
ICAL AND BIOPHYSICAL RESEARCH COMMUNICATION, 1130〜
1135, 1986, regarding α-carotene,
Japanese Patent Application Laid-Open No. 2004-0909, lycopene,
There is a report in 227970 and the like.

[0005] On the other hand, various studies have been made on the physiological effects of algae, especially cyanobacteria, especially spirulina used in foods.
r. Report Int., 28, 519, 1983, Journal of Japanese Society of Nutrition and Food Science, 37, 323, 1984, Nutr. Report Int, 37,
1329, 1988), blood pressure elevation inhibitory effect (Journal of Women's Nutrition University, 21, 63, 1990), intestinal microflora improvement (Chiba Prefectural College of Health and Science, Vol. 5, No. 2, 27) P., 1987
Years), renal dysfunction inhibitory action (108th, 109th, 110th)
Annual Pharmaceutical Society of Japan, 1988, 1989, 1990), alcohol metabolism-improving effect (Journal of Japan Society of Nutritional Food, 47, 395, 199)
4 years), immunostimulatory effect (J. Nutr. Sci. Vitaminol., Volume 40,
431, 1994), antiviral activity (PHYTOTHERAPY RES)
EARCH, Vol. 7, p. 76, 1993).

[0006] In particular, Japanese Patent Application Laid-Open No. 54-73108 discloses a "cancer drug" containing cyanobacterial spirulina as a main component.
Is disclosed. This is intended for a water-soluble substance in Spirulina, and is completely different from the present carcinogenesis inhibitor in the active ingredient and the action mechanism. As described above, β-carotene, α-carotene and lycopene are known as carotenoid-based carcinogenesis inhibitors, and in particular, β-carotene has been reported to promote lung cancer, and it has a low toxicity. Further development of more effective carcinogenesis inhibitors is expected.

[0007]

An object of the present invention is to provide an agent for inhibiting carcinogenesis or tumor metastasis derived from natural products which has a low toxicity and an excellent effect of suppressing carcinogenesis and tumor metastasis. is there.

[0008]

Means for Solving the Problems The present inventors have found that the natural world,
In particular, carotenoids present in foods have been studied extensively for carcinogenesis and tumor metastasis suppression, especially for substances with anti-promoter activity, and among them, zeaxanthin has a strong carcinogenesis suppression and tumor metastasis suppression effect. The present invention has been completed.

That is, the present invention provides (1) a carcinogenesis inhibitor containing zeaxanthin as an active ingredient, (2) a carcinogenesis inhibitor containing zeaxanthin extracted from algae as an active ingredient, and (3) an algae being a cyanobacterium ( (4) a cyanobacterium according to the genus Spirulina, (4) a tumor metastasis inhibitor comprising zeaxanthin as an active ingredient, and (6) an algae. (7) the tumor metastasis inhibitor according to (6), wherein the algae are cyanobacteria, and (8) the cyanobacteria are algae of the genus Spirulina (7). )).

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Zeaxanthin used in the present invention is known to be contained in higher plants (corn, alfalfa, etc.) and algae (cyanobacteria, green algae, red algae, etc.). Zeaxanthin is particularly abundant in cyanobacteria. As blue-green algae, Spirulina (Spirina)
ulina), Fischerel (Fischerel)
la) genus, Anabaena genus, Nostoc genus, Cynechocystis (Cynech)
ocystis), Synechococcus (Cynechoccus)
coccus), Tripothrix (Tolypoth)
rix) genus.

Of these, algal bodies belonging to the genus Spirulina, which are produced on an industrial scale, whose safety has been confirmed, and are edible, are the most preferable as a raw material for obtaining zeaxanthin. In addition, as an agent for suppressing carcinogenesis or tumor metastasis, it is preferable to extract and purify zeaxanthin in alga bodies and use them in a state with few impurities, rather than using alga bodies themselves, and to use zeaxanthin having a purity of 95% or more. Is more preferred.

Zeaxanthin used in the present invention can be obtained from the above-mentioned higher plants and algae by an extraction method using an organic solvent, for example, alcohols such as ethanol and methanol, ketones such as acetone, chloroform, ethers and esters.
Alternatively, a crude extract extracted by a critical point extraction method using carbon dioxide gas or the like, or a purified zeaxanthin obtained by further purifying the extract may be used. Usually, 50 to 150 mg of zeaxanthin is contained in 100 g of Spirulina algae. Zeaxanthin is subjected to air oxidation and photolysis, and therefore it is preferable to handle and store it in a cool and dark place under an inert gas atmosphere.

The agent for inhibiting carcinogenesis or tumor metastasis of the present invention can be administered orally or parenterally (for example, intravenously, etc.), and can be prepared into a formulation suitable for each administration method upon administration. Such preparations can be prepared in the form of tablets, capsules, granules, fine granules, powders, troches, injections, emulsions, suspensions, syrups and the like depending on the use.

In preparing these, for example, non-toxic excipients, binders, disintegrants, lubricants, preservatives, antioxidants, isotonic agents, buffers which are commonly used in this type of drug are used. ,
Coating agent, flavoring agent, solubilizer, base, dispersant,
It can be formulated by a known method using additives such as a stabilizer and a colorant. Further, the content of the active ingredient of the present invention in such a preparation varies depending on the dosage form, but it is generally desirable that the active ingredient is contained in a concentration of 0.01 to 100% by weight.

The dosage of the carcinogenesis or tumor metastasis inhibitor of the present invention can be varied over a wide range depending on the kind of the target human or other warm-blooded animals, the severity of the symptoms, the judgment of the doctor, etc., but is generally effective. As an ingredient, in the case of oral administration, 0.5 to 50 mg of zeaxanthin per day per kg of body weight,
Preferably, 0.5 to 20 mg, and in the case of parenteral administration, 0.1 to 10 mg, preferably 0.5 to 5 mg, per day per kg of body weight is administered. The above dose can be administered once or several times a day. These can be changed appropriately according to the severity of the patient's symptoms and the diagnosis of a doctor.

The inhibitor of carcinogenesis or tumor metastasis comprising zeaxanthin of the present invention as an active ingredient exhibits an inhibitory effect at the stage of promoting carcinogenesis and exhibits an inhibitory effect in an antioxidant manner. No, preventing liver cancer, gastric cancer, pancreatic cancer, cervical cancer, lung cancer, colon cancer, breast cancer, brain tumor, leukemia, laryngeal cancer, esophageal cancer, etc. Cancer, cervical cancer, lung cancer,
It is effective in suppressing metastasis to other organs such as liver cancer, breast cancer, brain tumor, leukemia, laryngeal cancer, and esophageal cancer.

The carcinogenesis or tumor metastasis inhibitor containing zeaxanthin of the present invention as an active ingredient is preferably high in purity from the viewpoint of ensuring the safety of pharmaceuticals. However, other impurities accompanying zeaxanthin obtained from a natural product are not preferred. Since they are carotenoids similar to zeaxanthin, safety is generally not impaired if the purity is 95% or more.

The agent for suppressing carcinogenesis or tumor metastasis of the present invention has an effect of preventing spontaneous carcinogenesis and suppresses metastasis of generated cancer to other organs. Has the effect of making the cancer treatment method more effective.

[0019]

EXAMPLES The present invention will be described below with reference to examples.
This is only one mode, and the present invention is not limited to the embodiment.

(Example of producing zeaxanthin) Spirulina 10
400 g of methanol is added to 0 g,
Overnight to extract zeaxanthin. Spirulina algae were filtered off under nitrogen pressure of 0.1 kg / cm ² , and 400 g of methanol was further added to the obtained residue.
Over time, zeaxanthin was extracted. The obtained filtrate was combined with the previously obtained methanol extract, and methanol in the methanol extract was distilled off under a nitrogen atmosphere under reduced pressure to obtain a concentrate having a solid content of 12.1 g.

In this concentrate, 40 g of methanol and KOH5
g was added and a saponification reaction was performed at 64 ° C to 65 ° C for 2 hours. After saponification, methanol was distilled off under reduced pressure, and the obtained concentrate was dissolved by adding 50 g of water and 100 g of ethyl acetate, and liquid-liquid extraction was performed to obtain an ethyl acetate extract. Ethyl acetate in the ethyl acetate extract was distilled off under reduced pressure to obtain 2.25 g of a solid content as a carotenoid fraction. The carotenoid fraction was subjected to silica gel column chromatography (silica gel C-200, 50 g) and eluted with n-hexane / ethyl acetate = 3/1 to 2/1 to separate zeaxanthin from other carotenoids.

The obtained zeaxanthin-containing fraction is subjected to silica gel column chromatography (silica gel C-20).
0, 40 g), and n-hexane / ethyl acetate =
Eluted at 1/1. The obtained zeaxanthin fraction was concentrated under reduced pressure under a nitrogen atmosphere to precipitate crystals. The crystals were filtered off and dried in vacuo at room temperature to give zeaxanthin crystals 47.
mg was obtained.

The obtained zeaxanthin crystals were subjected to high performance liquid chromatography LC-6A manufactured by Shimadzu Corporation.
Column (Wakosil 60-5, 4.6 m
mx 150mm), detection wavelength 450nm, column temperature 4
0 ° C., mobile phase; n-hexane: acetone = 4: 1 (v /
v) As a result of analysis at a flow rate of 1.0 ml / min, the purity of zeaxanthin was 98.7%. In the following experiment, a sample obtained by a production test using 100 times the amount of the zeaxanthin production example described above was used.

(Example 1) Spontaneous hepatocarcinogenesis inhibitory effect C3H / H, which is known to cause spontaneous hepatocarcinogenesis
e Male mice (control group: 14 animals, test group: 12 animals) were used, and they were allowed to freely ingest feed / drinking water for 40 weeks, and then sacrificed, and the number of tumors generated in the liver was counted.
After suspending 20 mg of zeaxanthin (purity 98.5%) obtained in the same manner as in the above-mentioned example of producing zeaxanthin in 1 ml of dimethyl sulfoxide (DMSO), the beverage was adjusted to a final concentration of 0.005%. Added to water (test group). On the other hand, the same amount of DMSO alone was added to the control group.
The results are shown in Table 1.

[0025]

[Table 1]

From the results shown in Table 1, the number of tumor-generating individuals and the average number of tumors per mouse are clearly suppressed in the test group (suppression rate: 95%), and the effect of zeaxanthin on spontaneous carcinogenesis is clear. In the same experimental system, β
-Carotene has been shown to have no effect.

(Example 2) Inhibitory effect on colon cancer liver metastasis BALB / c mice were laparotomized, and mouse Colon 26 colon cancer high liver metastasis strain 2 × 10 ⁴ was injected into the portal vein. Zeaxanthin (4 μg / mouse in Test Example 1, 40 μg in Test Example 2)
/ Mouse, 400 μg / mouse in Test Example 3) and all-trans retinoic acid (A
(TRA, 160 μg / mouse) was repeated twice with one cycle of daily oral administration for 5 days after tumor inoculation and a 2-day washout. Twenty days later, the mice were sacrificed and the number of visible tumor nodules in the liver was counted. The results are shown in Table 2. * In the table indicates a significant difference of p <0.005 (Whitney's U-test).

[0028]

[Table 2]

From the results shown in Table 2, the number of visible tumor nodules in the liver was decreased in a dose-dependent manner in the zeaxanthin administration group, and liver metastasis of zeaxanthin to colon cancer was clearly suppressed. In addition, an increase in liver weight observed with liver metastasis of the tumor is also suppressed. Incidentally, similar results are obtained from ATRA.
It was also observed in the administration group.

Example 3 Inhibitory Effect of Basement Membrane Infiltration The mechanism of zeaxanthin in Example 2 for inhibiting liver metastasis of colon cancer was examined using a transwell chamber.
Using a chamber divided into two layers by a membrane filter having a pore diameter of 8 μm, the upper surface of the filter was coated with 5 μg of matrigel (basement membrane reconstituted substrate), and the lower surface was coated with 1 μg of fibronectin. 2x
B16-BL6 melanoma cells at a cell density of 10 ⁵ cells / 100 μl were added to the upper layer of the chamber, and zeaxanthin 0.05
The cells were cultured at 37 ° C. in a 5% CO ₂ incubator for 4 hours in the presence of ５００500 μM. After completion of the culture, the number of cells that had infiltrated and transferred to the lower surface of the filter was counted under an optical microscope. Table 3 shows the results. In the table, Comparative Example shows a system to which only DMSO used for sample preparation of Test Example was added. In the table, * indicates p <0.05, and ** indicates p <0.00.
5 (Whitney's U-test) shows a significant difference.

[0031]

[Table 3]

From the results shown in Table 3, zeaxanthin inhibited the invasion of basement membrane of melanoma cells in a concentration-dependent manner. From these results, inhibition of invasion of cancer cells into the basement membrane is considered as one of the mechanisms for suppressing liver metastasis of colon cancer.

Example 4 Mouse Acute Toxicity Test Using a stomach probe for mice, zeaxanthin (purity 9
8.5%) in mice (ICR male, male weight 20-25)
g, n = 3) at 1000 or 2000 mg / kg by oral gavage. After oral administration, the animals were bred in cages for 7 days, the presence or absence of dead animals and the general condition were observed, and the approximate 50% lethal dose (LD50: m
g / kg). As a result, the zeaxanthin L
D50 was 2000 mg / kg or more, which proved to be extremely high in safety.

[0034] Zeaxanthin (98.5% purity) crystals were crushed in a mortar, lactose was added thereto, and the mixture was sufficiently mixed while being crushed with a pestle to prepare a 5% powder.

[0035]

In a mortar, zeaxanthin (purity 98.5)
%) Was mixed and ground with an equal amount of starch. This with lactose,
The starch residue was added and mixed. Separately 30mg of gelatin
Then, 1 ml of purified water was added to the mixture, and the mixture was heated and dissolved. After cooling, 1 ml of ethanol was added to the mixture to prepare a gelatin solution. The gelatin solution was added to the above mixture, kneaded, granulated, and dried. And sized.

[0037]

A 5 mg tablet was prepared in a pot using 20 times the amount of the above ingredients. That is, 100 mg of zeaxanthin (98.5% purity) crystals were pulverized, and lactose and starch were added thereto and mixed. Starch paste was added to the above blend, kneaded and granulated. After drying, talc and magnesium stearate were mixed and tableted by a conventional method.

[0039]

155 g of soybean oil and 5 g of zeaxanthin (purity: 98.5%) were put into an emulsifier and mixed, and 20 g of beeswax and 20 g of glycerin fatty acid ester were added thereto, and the mixture was uniformly emulsified and dispersed. According to a conventional method, this dispersion 200m
g was encapsulated with 150 mg of gelatin.

[0041]

Industrial Applicability The present invention can provide a natural-derived carcinogenesis or tumor metastasis inhibitor having a low toxicity and an excellent carcinogenesis inhibitory and tumor metastasis inhibitory effect.

Claims (8)

[Claims] 1. A carcinogenesis inhibitor comprising zeaxanthin as an active ingredient. 2. A carcinogenesis inhibitor comprising zeaxanthin extracted from algae as an active ingredient. 3. The carcinogenesis inhibitor according to claim 2, wherein the algae are cyanobacteria. 4. The carcinogenesis inhibitor according to claim 3, wherein the blue-green algae is a genus Spirulina. 5. A tumor metastasis inhibitor comprising zeaxanthin as an active ingredient. 6. A tumor metastasis inhibitor comprising zeaxanthin extracted from algae as an active ingredient. 7. The tumor metastasis inhibitor according to claim 6, wherein the algae are cyanobacteria. 8. The tumor metastasis inhibitor according to claim 7, wherein the cyanobacterium is an algae of the genus Spirulina.