JP2002121132A - Carcinogenic inhibitor and method for inhibiting carcinogenesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a carcingenic inhibitor usable as a carcinogenic prophylactic agent or a relapse prophylactic agent for cancer or as an active ingredient of a medicine, a food additive, a food or a beverage and having high safety and to provide a method for inhibiting carcinogenesis. SOLUTION: This carcinogenic inhibitor comprises an L-ascorbic acid derivative and a tocopherol derivative. The method for inhibiting the carcinogenesis is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a carcinogenesis inhibitor comprising an L-ascorbic acid derivative and a tocopherol derivative.

[0002]

2. Description of the Related Art Various studies have been made on the treatment of cancer. In particular, a great deal of research and development has been conducted on therapeutic agents, and some agents are actually used in medical practice. However, there is still no silver bullet that cures cancer, and currently used cancer therapeutics such as anticancer drugs or immunosuppressants have a problem of weak therapeutic effects and strong side effects.

[0003] The problems with such therapeutic drugs after cancer development are that it is difficult to cause the drugs to act specifically on cancer cells without affecting normal cells, and that the drugs of chemically synthesized substances are not effective. Due to inherent safety issues.

[0004] In recent years, search for a cancer-suppressing substance has been particularly regarded as important from the viewpoint of suppressing the occurrence of cancer separately from therapeutic agents after cancer has occurred. If an effective cancer-suppressing substance can be extracted or synthesized and used as a drug to prevent carcinogenesis, it is particularly possible to prevent hereditary cancer or occupational cancer. Such drugs are expected to greatly contribute to the maintenance of health in future new medical treatments and daily life.

It is preferable that such a carcinogen suppressor can be easily taken in daily life. Furthermore, it is a necessary condition that the compound is effective even when administered orally and has no side effects. However, to date, there has been no example in which a specific carcinogen-suppressing substance having no problem in safety is used as a drug for preventing or treating carcinogenesis.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has been developed to prevent hereditary or occupational cancer, or prevent cancer due to other causes, or to treat cancer after treatment. An object of the present invention is to provide a carcinogenesis inhibitor that is effective for preventing recurrence of cancer and a method for suppressing carcinogenesis using the same.

[0007]

Means for Solving the Problems The present inventors have widely searched for a safe compound that has a carcinogenesis-suppressing action and does not adversely affect the human body. Alternatively, an L-ascorbic acid derivative which is easily converted to L-ascorbic acid and a tocopherol derivative which is easily converted to tocopherol in a culture solution exhibit a good carcinogenesis-suppressing effect at a low concentration. For example, the inventors have found that a composition containing an L-ascorbic acid-2-phosphate ester derivative and a tocopherol derivative as L-ascorbic acid derivatives exhibits a carcinogenesis-suppressing action, and have completed the present invention.

That is, the present invention relates to the following matters. [1] A carcinogenesis inhibitor comprising an L-ascorbic acid derivative and a tocopherol derivative. [2] The carcinogenesis inhibitor according to the above [1], which reduces intracellular active oxygen and / or free radicals and suppresses cell carcinogenesis. [3] The above-mentioned [1], wherein the L-ascorbic acid derivative is L-ascorbic acid-2-phosphate or a salt thereof.
Or the carcinogenesis inhibitor according to [2].

[4] The salt of L-ascorbic acid-2-phosphate is sodium, potassium, magnesium,
The cancer suppressant according to the above [3], which is a salt of calcium, aluminum, trimethylamine, triethylamine, pyridine, picoline, N, N-dibenzylethylenediamine, ethanolamine, diethanolamine, tris (hydroxymethyl) aminomethane or dicyclohexylamine. [5] The above-mentioned [1], wherein the suppression rate of focus formation that appears when cultured in a tumor-promoting medium on animal cells into which UV, mutagenic substances or artificially introduced oncogenes have been introduced is 10% or more. ] The carcinogenesis inhibitor according to any one of [4] to [4]. [6] The carcinogenesis inhibitor according to any one of [1] to [5], which is used for suppressing transformation of skin cells including skin cancer.

[7] The carcinogenesis inhibitor according to any one of the above [1] to [6], which is used for oral administration, external preparation or injection. [8] A food additive containing the carcinogenesis inhibitor according to any of [1] to [7]. [9] A food containing the food additive according to the above [8].

[10] The food according to the above [9], which is a confectionery. [11] The food according to the above [9], which is a beverage. [12] A method for suppressing carcinogenesis, which comprises ingesting a cancer-suppressing agent according to any of the above [1] to [7] into a mammal orally or parenterally. [13] The method of suppressing carcinogenesis according to the above [12], wherein the daily intake is 0.001 to 10,000 mg / kg body weight.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. In the present invention, “carcinogenesis suppression” means to suppress the generation of cancer cells.

More specifically, when an animal cell into which UV (ultraviolet rays), a mutagenic substance or an oncogene is artificially introduced and initiated is cultured for 11 days in a tumor promoter medium, transformed foci appearing (focus) ) Is suppressed.

The carcinogenesis inhibitor of the present invention is characterized by reducing intracellular active oxygen and free radicals and suppressing the process of cell carcinogenesis. In the present invention, suppression of cell carcinogenesis broadly includes progress of the process of cell carcinogenesis.

Epidemiological studies have shown that most human carcinogenesis is due to environmental factors. At least two mechanically distinct processes, initiation and promotion, are involved in canceration by environmental chemical factors (chemical substances), and each process is induced by chemicals called initiator and promoter. It is considered. Initiation causes irreversible damage at the genetic level by the initiator,
It is thought to be the step of inducing normal cells into potential tumor cells. On the other hand, promotion is a stage in which the repetitive stimulation of the promoter promotes the growth of the potential tumor cells, reveals the tumor cells, and further induces the cells to become cancerous.

This mechanism is called "two-stage carcinogenesis theory",
Berenblum (Cance
r Res. , 1, 44, 1941), which is useful for simplifying and understanding complex carcinogenesis mechanisms. Although carcinogenesis in humans with a long life expectancy is said to be more complex and appears to be a multi-stage carcinogenesis, it is basically thought that both initiation and promotion processes are involved.

In recent years, various promoters have been found in the environment along with a large number of initiators such as carcinogens and mutagens. The best way to prevent carcinogenesis is to remove these carcinogens, but it is impossible to completely eliminate various carcinogens that are widely distributed in the environment.

Therefore, various studies have been made to prevent carcinogenesis or to enhance the defense mechanism on the living body side. As one means,
Attention has been focused on the chemical inhibition of the two-stage carcinogenesis process described above. That is, if either one of the initiation and promotion processes or both processes can be inhibited at the same time, it is considered that canceration will eventually be suppressed.

In general, since it is considered that there is already a potential tumor cell which cannot return to a normal cell in adulthood, it is necessary to prevent the cell from continuously touching the promoter or to actively suppress the action of the promoter. These and other measures are effective in preventing carcinogenesis.

It has been reported that active oxygen and / or free radicals play an important role in the process of promotion of carcinogenesis. For example, TPA (12-O-tetradecanoylphorbol-13-acetate), which is a typical promoter, is known to have effects such as induction of ornithine decarboxylase and plasminogen activator, and enhancement of phospholipid metabolism. Have been. Active oxygen is involved in these reactions, and the reaction is suppressed by the active oxygen remover.

Furthermore, it has been reported that TPA, mezerein, phenobarbital, bile acids, asbestos, etc., increase the production of active oxygen by direct radical reaction of anthraline, benzoyl peroxide, hydrogen peroxide, etc. via cell membranes. . Thus, in the process of promoting carcinogenesis, active oxygen and / or free radicals play an important role. Therefore, continuous removal of active oxygen or free radicals is effective in suppressing carcinogenesis.

In the carcinogenesis inhibitor of the present invention, the L-ascorbic acid derivative is not particularly limited as long as it is selected according to the intended use and expected effects. For example, substituents at the 2- and 3-positions are expected to have immediate effect. In this case, a substituent (such as a phosphate group in the case of human) that is rapidly hydrolyzed by an enzyme in serum or cell membrane is desirable. Substituents that are hydrolyzed (such as glycosyl groups in humans) are desirable.

Specific examples include L-ascorbic acid-2-phosphate, L-ascorbic acid-2-glucoside and the like. Further, the substituents at the 5-position and the 6-position are desirably substituents which are rapidly taken into cells (in the case of humans, esters of higher fatty acids such as palmitic acid and lauric acid in addition to hydroxyl groups). Specifically, L-ascorbic acid-2-phosphate-6-palmitate, L-ascorbic acid-2-phosphate-6-stearate, L-ascorbic acid-2-phosphate-6-laurate, L- Ascorbic acid-
2-glucoside-6-palmitate and the like.

In the present invention, unless otherwise specified, L-ascorbic acid derivatives include salts. Such L-ascorbic acid derivatives include:
For example, when BALB / c 3T3 cells derived from mouse whole fetuses pretreated with 20-methylcholanthrene (MCA) are cultured in a medium containing phorbol 12-myristate 13 acetate (TPA) and an L-ascorbic acid derivative for 11 days. And L-ascorbic acid derivatives and the like, which can achieve the same or higher effect of suppressing the expression of transformed foci (focus) at a concentration of 30 μM or less in the medium as compared with the case of using 100 μM sodium L-ascorbate in the medium. Can be

Salts of L-ascorbic acid-2-phosphate (including various pyrophosphates and triphosphates), such as alkali metals, are more stable than L-ascorbic acid in water. Since it is easily soluble, it is suitable for use in the present invention.

The carcinogenesis inhibitor of the present invention has an inhibitory effect on the formation of foci which appears when cultured in a carcinogenesis promoter medium on animal cells which are initiated by introducing UV, mutagenic substances or artificially introduced oncogenes. It is 10% or more, and preferably, when cultured in a carcinogen promoter medium in an animal cell initiated with a mutagenic substance, the rate of suppression of focus formation that appears is 30% or more. In general, a carcinogenesis inhibitor is used as an injection or an oral administration agent, and therefore, it is desired that these agents have high solubility in water.

The L-ascorbic acid-2-phosphate ester and its salt used in the present invention have higher solubility in water when a water adduct or a water adduct of crystallization is used as an active ingredient, and have a monovalent property. Considering, for example, that the salt has higher solubility in water than the divalent salt, the salt is not particularly limited.However, in order to maintain better solubility, when the active ingredient of the present invention is a solid, water content In the case of water of crystallization, the content is preferably 1 to 50% by weight, and in the case of water of crystallization, water molecules in the range of 1 to 20 hydrate are preferably retained.

Examples of the salt of an acid such as 2-phosphate ester or 2-sulfate ester which is an L-ascorbic acid derivative used in the present invention include sodium salt, potassium salt, magnesium salt, calcium salt, aluminum salt and the like. Further, examples of the organic base include trimethylamine, triethylamine, pyridine, picoline, N, N-dibenzylethylenediamine, ethanolamine, diethanolamine, trishydroxymethylaminomethane, dicyclohexylamine and the like. Among these, L
The sodium and magnesium salts of -ascorbic acid-2-phosphate have low cytotoxicity and high biocompatibility, and are suitable as the carcinogenesis inhibitor of the present invention.

Further, in the present invention, the combined use of the above-mentioned L-ascorbic acid derivative and a tocopherol derivative enhances the effect of suppressing carcinogenesis. In the present invention, the tocopherol derivative preferably includes tocopherol phosphate, tocopherol sulfate, tocopherol glucosyl ester, and the like, and salts thereof. Particularly, α-tocopherol phosphate sodium salt is preferred from the viewpoints of the combined effect with ascorbic acid, water solubility and safety.

Desirable tocopherol derivatives include:
For example, when BALB / c 3T3 cells derived from mouse whole fetuses pretreated with 20-methylcholanthrene (MCA) were cultured for 11 days in a medium containing phorbol 12-myristate 13 acetate (TPA) and a tocopherol derivative, Examples of the tocopherol derivative include a tocopherol derivative which can suppress the expression of transformed foci (focus) at a concentration in a medium of 3 μM or less as compared with the case of using 10 μM tocopherol acetate. Salts of tocopherol phosphates (including various phosphoric acids) such as alkali metals are more suitable for use in the present invention because they are more soluble in water than tocopherol acetate.

The L-ascorbic acid derivative itself can serve as a carcinogenesis inhibitor, and the L-ascorbic acid derivative in the preparations and compositions
-The content of the ascorbic acid derivative is not particularly limited,
Generally, 0.01 to 98% by weight is preferable, and 0.5 to 50% by weight.
% Is more preferred. The carcinogenesis inhibitor of the present invention has low toxicity and can be administered orally or parenterally to mammals including humans. Particularly, oral administration, administration as an external preparation or injection is preferable.

The dosage form of the carcinogenesis inhibitor of the present invention is not particularly limited, and is orally administered, tablet, powder, liquid, suppository, external preparation,
Ointments, patches, eye drops, intravenous injections, powders, granules, tablets, dragees, capsules, pills, suspensions, ampules, injections, isotonic solutions, etc., and can be formulated into any pharmaceutical product It is. It can also be formulated as a pharmaceutically acceptable inert carrier or as a mixture with diluents and / or other pharmacological agents, or in dosage unit form.

Further, according to a well-known pharmaceutical production method, it may be formulated as a complex. For example, the main components of the carcinogenesis inhibitor of the present invention are EPA461, 427 for the purpose of increasing the solubility in water to promote absorption and enhance pharmacological activity.
In the method described in Japanese Patent Application Laid-Open Publication No. H10-264, it may be used as a complex with cyclodextrin or maltosyl-cyclodextrin.

The carcinogenesis inhibitor of the present invention is usually obtained by freeze-drying these active ingredients to form a solid mixture. One of the active ingredients is an aqueous solution, and the other active ingredient is freeze-dried. It may be in the form of a kit or a kit in which each active ingredient is separately formulated.

In the present invention, a method according to a known pharmaceutical production method (the 13th revised Japanese Pharmacopoeia,
USP24, etc.). Also,
The active ingredient can be separately formulated and administered using a pharmaceutically acceptable diluent, excipient, or the like, if desired.

When the preparation of the present invention is a solution, a water-soluble agent such as distilled water; a water-soluble preparation such as physiological saline and Ringer's solution; and an oil-based solvent such as sesame oil, corn oil, olive oil, etc. Prepared by

At this time, if desired, a solubilizing agent such as sodium salicylate and sodium acetate; a buffer such as sodium citrate and glycerin; an isotonic agent such as glucose; a stabilizer such as human serum albumin and polyethylene glycol; Preservatives such as alcohols and phenols; additives such as soothing agents such as benzalkonium chloride and procaine acetate can also be used.

If desired, for example, diluents, excipients, binders, disintegrants, colorants, stabilizers, extenders, wetting agents, surfactants, lubricants, dispersants, buffers, Use pharmacologically and pharmaceutically acceptable additives such as flavoring agents, flavoring agents, fragrances, preservatives, dissolution aids, solvents, coating agents, dragees, etc., or use those formulated with these. You can also.

The diluent used for preparing the carcinogenesis inhibitor of the present invention is not particularly limited as long as it is pharmaceutically acceptable. However, it refers to a material other than the present invention, and may be a solid, semi-solid, liquid or ingested. Capsules, dextrin inclusions, microcapsules using phospholipids, and the like, and various examples are available.

The carcinogenesis inhibitor obtained by the present invention comprises:
It may be manufactured in any known manner, for example by mixing the active ingredient with a diluent, for example into granules, and then shaping the composition into, for example, tablets. It should be sterile, especially when used as a parenteral drug,
If necessary, it should be isotonic with blood. Parenteral administration includes, for example, percutaneous injection including intravenous drip intramuscular injection, and transanal (suppository) administration.

Compositions for oral administration further include tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, sprays and the like. Such a composition itself is produced by a known method, and lactose, starch, sucrose, magnesium stearate, or the like is used as a carrier or excipient.

For parenteral administration, for example, injections,
It is used as suppositories, patches, eye drops, external preparations and the like.
The injection is usually filled in a suitable ampoule. Dosage forms include, for example, rectal suppositories, vaginal suppositories and the like, and external preparations include, for example, ointments, nasal administration agents, transdermal administration agents and the like.

When used as an external preparation, the composition of the present invention can be made into a solid, semi-solid or liquid solvent according to a known method. For example, as the solid, the composition of the present invention may be used as it is or as an excipient (for example,
Glycols, mannitol, starch, microcrystals, cellulose and the like. ), A thickener (for example, natural gums, cellulose derivatives, acrylic polymers, etc.) are added and mixed to form a powdery composition. When used as a liquid, it is an oily or aqueous suspension as in the case of injection. In the case of a semi-solid form, an aqueous or oily gel or an ointment is preferred.

In addition, these are all carbonic acid, phosphoric acid,
PH adjusting solutions such as citric acid, hydrochloric acid and sodium hydroxide; preservatives such as paraoxybenzoic acid esters, chlorobutanol and benzalkonium chloride may be added. When used as a suppository, the composition of the present invention is converted into an oily or aqueous solid, semi-solid, or liquid suppository according to a known method.

The L-ascorbic acid derivative intake in the carcinogenesis inhibitor of the present invention depends on age, sex, body weight, dosage form,
In the case of oral administration, suppository administration, external preparations, etc., it is usually 0.001 to 8500 per adult per day, depending on the administration form.
mg / kg body weight, preferably 0.01-1000 mg / kg
kg, more preferably in the range of 0.1-100 mg / kg, and in the case of intravenous injection or infusion, usually per adult daily,
0.025-200 mg / kg, preferably 0.25-
100 mg / kg can be administered once or in several divided doses.

In the method for suppressing carcinogenesis of the present invention, the amount of tocopherol derivative in the carcinogenesis inhibitor varies depending on age, sex, body weight, dosage form and administration form. In this case, the daily dose is usually 0.001 to 5000 mg / kg body weight per adult, preferably 0.01 to 500 mg / kg, more preferably 0.1 to 500 mg / kg.
In the case of intravenous injection and infusion, usually 0.025 to 200 mg / k per day for an adult.
g, preferably 0.25 to 100 mg / kg, can be administered once or in several divided doses.

In the method for suppressing carcinogenesis of the present invention, the daily adult intake of the carcinogenesis inhibitor is 0.001 to 10,000.
mg / kg body weight is effective, preferably 0.01-1.
000mg / kg, more preferably 0.1-200mg
/ Kg.

In the present invention, the composition ratio of the L-ascorbic acid derivative and the tocopherol derivative is not particularly limited, and varies depending on the target animal.
5-5: 95 is preferred, and 95: 5-40: 60 is more preferred.

The carcinogenesis inhibitor of the present invention can be used not only for humans (humans), but also for other mammals (eg, mice, rats, pigs, foxes, cats, rabbits, dogs, cows, horses, goats, monkeys) Etc.), and as a pharmaceutical or food additive, has the effect of preventing carcinogenesis in these animals.

The composition itself or a mixture with other ingredients can be used as a dietary supplement. The other components to be mixed are not particularly limited as long as they are not denatured by mixing, and include foods and drinks.
Foods and drinks are not particularly limited, and include, for example, chocolate, gum, yogurt, soft drinks, coffee, tea, alcoholic drinks, biscuits, bread, jelly, and the like.

In the production of foods or beverages, various substances can be added as needed. For example, sucrose, fructose, sugars such as glucose, sugar alcohols such as xylitol, amino acids, citric acid, lactic acid, malic acid, flavonoids, other antioxidants such as catechin,
Any substance used in normal production of foods, such as gelatin, vitamins, pigments, flavors, calcium preparations, glycerin fatty acid esters, pectin, etc., can be appropriately blended and used.

The range of application of the carcinogenesis inhibitor of the present invention includes various malignant and benign tumors such as melanoma, lymphoma, gastrointestinal cancer, lung cancer, esophagus cancer, stomach cancer, colorectal cancer, rectal cancer, and colon cancer. , Ureteral tumor, gallbladder cancer, bile duct cancer, breast cancer, liver cancer, pancreatic cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, ovarian cancer, Uterine cancer, prostate cancer, thyroid cancer, brain tumor, Kaposi's sarcoma, hemangiomas, leukemia, polycythemia vera, neuroblastoma, retinoblastoma, myeloma, cystoma, sarcoma, osteosarcoma, skin cancer, Basal cell carcinoma,
Examples include skin appendage cancer, skin metastasis cancer, and skin melanoma, and are particularly effective in controlling skin cancer, skin appendage cancer, skin metastasis cancer, skin melanoma, and the like. Further, the carcinogenesis inhibitor of the present invention is effective not only in preventing malignant tumors but also in preventing benign tumors.

[0053]

The present invention will be more specifically described below with reference to examples showing the carcinogenesis-suppressing action of a carcinogenesis inhibitor containing an ascorbic acid derivative and a tocopherol derivative, a production example of the therapeutic agent of the present invention, and a formulation example of foods and beverages. However, the present invention is not limited by these.

In this example, L-ascorbic acid-2
-Magnesium phosphate and L-ascorbic acid-2
-Sodium phosphate was synthesized by the method described in JP-A-10-17580. L-ascorbic acid-2-glucoside was extracted from UV White (manufactured by Shiseido Co., Ltd.). The tocopherol phosphate used in this example was synthesized according to the method described in detail in WO 97/14705. Other reagents were commercially available.

(Example 1) Cell transformation test using BALB / c 3T3 cells (1): When using 12-O-tetradecanoylphorbol-13-acetate as a carcinogenesis promoter BALB / c 3T3 cells In the step transformation test method, L for focus formation by a carcinogen (20-methylcholanthrene (MCA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a carcinogen promoter). -The inhibitory effect on ascorbic acid derivatives and / or tocopherol derivatives was observed.

The test was carried out according to the usual two-step transformation test of BALB / c 3T3 cells (Tsuchiya and Umeda, Car
ciogenesis, 8, 1887-1894, 1
995). 60m using MEM + 10% FBS medium
1 x 1 BALB / c 3T3 cells per m dish
0 ^four were seeded (10 dish / group).

After the cells were seeded and cultured for 24 hours, MC
A was added and an initiation treatment was performed for 72 hours. After removing the initiator, DME / F12 + 1% ITES
Culture was performed using a + 2% FBS medium while changing the medium twice a week.

Further, TP was carried out for 11 days from 7 days after the cell seeding.
A (100 ng / ml) was used for carcinogenesis promotion processing. Sodium L-ascorbate, L-ascorbic acid-2-phosphate, L-ascorbic acid-2
-Glucoside, L-ascorbin-2-sulfate (medium concentration of 30 or 100 µM) tocopherol acetate (medium concentration of 3 or 10 µM) or α-tocopherol phosphate sodium (medium concentration of 3
Or 10 μM) was added every day when the medium was replaced from the 7th day of cell seeding. Twenty-five days after cell seeding, the cells were fixed with methanol and stained with Giemsa, and the generated foci were counted.

The focus is determined by 1) layering of cells,
2) Basophilic staining, 3) Infiltration of surrounding monolayer cells,
4) Focuses satisfying the condition of a diameter of 2 mm or more were counted, and the effect of suppressing focus formation is shown in Table 1.

[0060]

[Table 1]

From these results, it is found that sodium L-ascorbate, L-ascorbic acid-2-glucoside or L-ascorbic acid
Ascorbin-2-sulfate (100 μl concentration in the medium)
M), L-ascorbic acid-2-phosphate magnesium salt or sodium salt (concentration 1 in the medium)
00 μM) was confirmed to have a focus formation inhibitory effect, and sodium α-tocopherol phosphate (10 μM in the medium) was also confirmed to have a focus formation inhibitory effect. Did not. On the other hand, L-ascorbic acid-2-phosphate magnesium salt (100 μM in the medium) and α-tocopherol sodium phosphate (10 μM in the medium)
The combined use of M) completely inhibited focus formation.

(Example 2) Cell transformation test using BALB / c 3T3 cells (2): When phorbol-12,13-deacetate or tumor necrosis factor-α was used as a carcinogenesis promoter BALB / c 3T3 cells In a two-step transformation test, carcinogenic substances (20-methylcholanthrene (MCA) as initiator, phorbol-12,13-deacetate (PDD) or tumor necrosis factor (TNF-α) as tumor promoter) The inhibitory effect of the L-ascorbic acid derivative and / or the tocopherol derivative on the formation of foci caused by the F. was observed.

The tests were carried out according to the usual two-step transformation test of BALB / c 3T3 cells (Tsuchiya and Umeda, Car
ciogenesis, 8, 1887-1894, 1
995). 60m using MEM + 10% FBS medium
1 x 1 BALB / c 3T3 cells per m dish
0 ^four were seeded (10 dish / group). After inoculating the cells and culturing for 24 hours, MCA was added and the cells were subjected to an initiation treatment for 72 hours. After removing the initiator, DM
Using E / F12 + 1% ITES + 2% FBS medium,
Culture was performed while changing the medium twice a week.

In addition, after 7 days from the cell seeding, the PD
D (0.3 ng / ml) or TNF-α (3 ng / m
Carcinogenesis promotion processing was performed in l). Sodium L-ascorbate, L-ascorbic acid-2-phosphate, L-ascorbic acid-2-glucoside, L-
Ascorbin-2-sulfate (medium concentration 100
μM), tocopherol acetate, and sodium α-tocopherol phosphate (medium concentration: 10 μM) were added from day 7 of cell seeding every time the medium was replaced. Cell seeding 2
Five days later, the cells were fixed with methanol and stained with Giemsa, and the generated foci were counted.

The focus is determined by 1) layering of cells,
2) Basophilic staining, 3) Infiltration of surrounding monolayer cells,
4) Focuses satisfying the condition of a diameter of 2 mm or more were counted, and the focus formation suppression rates are shown in Tables 2 and 3.

[0066]

[Table 2]

[0067]

[Table 3]

From these results, it was found that sodium L-ascorbate, L-ascorbic acid-2-glucoside or L-ascorbic acid
Ascorbin-2-sulfate (100 μl concentration in the medium)
M), L-ascorbic acid-2-phosphate magnesium salt or sodium salt (concentration 1 in the medium)
00 μM) was confirmed to have a focus formation inhibitory action, and sodium α-tocopherol phosphate (10 μM in the medium) was also confirmed to have a focus formation inhibitory action, but did not completely inhibit focus formation. .

On the other hand, magnesium L-ascorbic acid-2-phosphate (medium concentration: 100 μM) and α-tocopherol sodium phosphate (medium concentration: 10 μM)
The combined use of M) completely inhibited focus formation.

(Example 3) Rat mid-term carcinogenesis test: When 2-acetylaminofluorine was used as a carcinogenesis promoter 30 male 9-week-old F344 rats were used, and MF powder (Oriental Yeast Co., Ltd.) was used as a basic feed. Was used for breeding. Rats were divided into two groups (15 / group),
At the start of the test (day 0), the carcinogen diethylnitrosamine (DEN) was administered to rats in each group at a rate of 20 mg / kg body weight.

To the rats of the first group, L-ascorbic acid-2-magnesium phosphate was dissolved in distilled water so as to have a concentration of 1 mM, and administered with drinking water 3 days before DEN administration. To the rats in the second group, α-tocopherol sodium phosphate was dissolved in distilled water to a concentration of 0.1 mM, and administered with drinking water 3 days before DEN administration. For rats in the third group, L-ascorbic acid-2-magnesium phosphate was dissolved in distilled water to 1 mM and α-tocopherol sodium phosphate to 0.1 mM.
Drinking water was administered from 3 days before EN administration. The rats in the fourth group were allowed to freely take distilled water.

For rats in any of Groups 1 to 4 for 2 weeks from the start of the test, 2 weeks and 3 weeks, 2-acetylaminofluorine (2-AA), a carcinogen, was used.
F) was given at a concentration of 0.01% mixed with the basal powder feed. On the first day of the third week, 2/3
A partial hepatectomy was performed to amplify the liver lesions.

On the first day of the fifth week, all rats were sacrificed and the liver was removed. Tissue specimens of each liver were prepared. Using the tissue sample of the rat liver, the presence or absence of fetal glutathione-S-transferase (GST-P), which is an indicator of a precancerous lesion of the liver, was examined by immunohistological staining using an avidin-biotin complex method.

The stained tissue was analyzed with an image analyzer. The number of GST-P-positive cell nests and the average area thereof were compared for the livers of rats of Groups 1 to 3 and Group 4 and L-ascorbic acid-2-magnesium phosphate and α-tocopherol phosphate were compared. The carcinogenesis inhibitory effect of sodium and its combined effect were evaluated. Table 4 shows the results.

[0075]

[Table 4]

The liver weight relative to the final body weight and total body weight of the rats showed no particular difference in any group, and toxicity due to L-ascorbic acid-2-magnesium phosphate and sodium α-tocopherol phosphate was observed. Did not.

In the search results of GST-P-positive cell nests shown in Table 4, it was found that the first group of L-ascorbic acid-2-magnesium phosphate administration group and the second group of α-tocopherol sodium phosphate administration group. The number and the area were smaller than those of the control rats to which distilled water of Group 4 was administered. Therefore, it was found that L-ascorbic acid-2-magnesium phosphate and α-tocopherol phosphate sodium clearly have an effect of suppressing carcinogenesis. On the other hand, the combined use of magnesium L-ascorbic acid-2-phosphate and sodium α-tocopherol phosphate showed a remarkable effect of suppressing carcinogenesis.

Next, production examples of various pharmaceuticals will be described.

(Example 4) Tablet containing L-ascorbic acid derivative and tocopherol derivative After mixing the components according to the composition shown in Table 5, the mixture was tableted with a tableting machine to produce a tablet having a diameter of 8 mm and a weight of 200 mg. .

[0080]

[Table 5]

(Example 5) Granules containing L-ascorbic acid derivative and tocopherol derivative The tablet produced in Example 1 was crushed, sieved and
Granules of 顆粒 50 mesh were produced.

(Example 6) Capsule containing L-ascorbic acid derivative and tocopherol derivative After mixing the components according to the composition shown in Table 6, the mixture was encapsulated in a gelatin capsule to prepare a capsule.

[0083]

[Table 6]

[0086] In the following, chocolates, gums containing L-ascorbic acid derivatives and tocopherol derivatives,
Made candy and beverages. At the same time, chocolates, gums, candy and beverages not containing the L-ascorbic acid derivative and the tocopherol derivative were produced as comparative subjects, and visually inspected and tasted using these, and the effects on the color tone, flavor and taste were compared and studied.

Evaluation was carried out by blindly inspecting and tasting ten testers, and when there were eight or more testers who answered "Same", the addition of L-ascorbic acid derivative and tocopherol derivative Was determined to have no effect on color tone, fragrance, or taste.

Example 7 Chocolate According to the composition shown in Table 7, chocolate containing an L-ascorbic acid derivative and a tocopherol derivative was produced.

[0087]

[Table 7]

As a result of comparison with chocolate containing no L-ascorbic acid derivative and tocopherol derivative, the effect of magnesium L-ascorbic acid-2-phosphate and sodium α-tocopherol phosphate on the color, aroma and taste of chocolate Was not.

Example 8 Gum According to the composition shown in Table 8, a gum containing an L-ascorbic acid derivative and a tocopherol derivative was produced.

[0090]

[Table 8]

This was visually inspected and tasted to compare it with a gum containing no L-ascorbic acid derivative and a tocopherol derivative.
-Magnesium ascorbic acid-2-phosphate and
The color, flavor and taste of the gum were not affected by the sodium α-tocopherol phosphate.

Example 9 Candy According to the composition shown in Table 9, a candy containing an L-ascorbic acid derivative and a tocopherol derivative was produced.

[0093]

[Table 9]

This was compared with a candy not containing the L-ascorbic acid derivative and the tocopherol derivative by visual inspection and tasting, and the blended magnesium L-ascorbic acid-2-phosphate and sodium α-tocopherol phosphate were compared. Candy color, aroma,
There was no effect on taste.

Example 10 Beverage According to the composition shown in Table 10, a beverage containing an L-ascorbic acid derivative and a tocopherol derivative was produced.

[0096]

[Table 10]

This was compared with a beverage not containing the L-ascorbic acid derivative and the tocopherol derivative by visual inspection and tasting. The blended magnesium salt of L-ascorbic acid-2-phosphate and α-tocopherol phosphate sodium were used. Had no effect on the color, flavor or taste of the beverage.

[0098]

According to the present invention, a highly safe carcinogenesis inhibitor and a method for suppressing carcinogenesis containing an L-ascorbin derivative and a tocopherol derivative are provided. The carcinogenesis inhibitor of the present invention can be used as a carcinogenesis prevention or cancer recurrence prevention agent. Furthermore, the carcinogenesis inhibitor of the present invention can be used as an active ingredient in medicines, foods and beverages for cancer prevention, and is effective in a wide range of fields.

Claims (13)

[Claims] 1. A carcinogenesis inhibitor comprising an L-ascorbic acid derivative and a tocopherol derivative. 2. The carcinogenesis inhibitor according to claim 1, wherein intracellular active oxygen and / or free radicals are reduced to suppress cell carcinogenesis. 3. The method according to claim 1, wherein the L-ascorbic acid derivative is L-ascorbic acid-2-phosphate or a salt thereof. 4. The salt of L-ascorbic acid-2-phosphate ester is sodium, potassium, magnesium, calcium, aluminum, trimethylamine, triethylamine, pyridine, picoline, N, N-dibenzylethylenediamine, ethanolamine, diethanolamine,
The carcinogenesis inhibitor according to claim 3, which is a salt of tris (hydroxymethyl) aminomethane or dicyclohexylamine. 5. The method according to claim 1, wherein the rate of suppression of focus formation, which appears when cultured in a tumor-promoting medium in an animal cell into which UV, mutagenic substances or artificially introduced oncogenes are introduced, is 10% or more. Item 5. The carcinogenesis inhibitor according to any one of Items 1 to 4. 6. The carcinogenesis inhibitor according to claim 1, which is used for suppressing transformation of skin cells containing skin cancer. 7. The carcinogenesis inhibitor according to claim 1, which is used for oral administration, external preparation or injection. 8. A food additive containing the carcinogenesis inhibitor according to any one of claims 1 to 7. 9. A food containing the food additive according to claim 8. 10. The food according to claim 9, which is a confectionery. 11. The food according to claim 9, which is a beverage. 12. A method for suppressing carcinogenesis, comprising ingesting a cancer-suppressing agent according to any one of claims 1 to 7 orally or parenterally into a mammal. 13. The method for suppressing carcinogenesis in humans according to claim 12, wherein the daily intake is 0.001 to 10,000 mg / kg body weight.