JP2002308768A - Vascularization agent, cytostatic agent, tube formation inhibitor and fgf inhibitor and food or food additive including tocotrienol as an active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vascularization inhibitor, a cytostatic agent, a tube formation inhibitor, and an FGF inhibitor and food or food additive that has high inhibitory actions and high safety. SOLUTION: The objective vascularization inhibitor, cytostatic agent inhibitor, tube formation inhibitor and FGF inhibitor and food or food additive include tocotrienol as an active ingredient. These drugs and foods are useful as a prophylactic and therapeutic agent for vascularization-dependent diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an angiogenesis inhibitor, a cell growth inhibitor, a tube formation inhibitor and a fibroblast growth factor (FGF) inhibitor containing tocotrienol as an active ingredient, and a food containing tocotrienol. Or it relates to food additives.

[0002]

2. Description of the Related Art Angiogenesis is a phenomenon in which new blood vessels are formed from existing blood vessels, and comprises the following steps.
(1) Angiogenic factors transmit angiogenic signals to endothelial cells, which digest the nearby basement membrane and extracellular matrix.
(2) Vascular endothelial cells migrate and proliferate. (3) Vascular endothelial cells form a lumen (angiogenesis) and form a capillary network. This angiogenesis can be caused by solid tumors, diabetic retinopathy, rheumatoid arthritis, hemangiomas, periodontal disease, scleroderma, glaucoma, psoriasis,
It is observed to occur when the disease develops and progresses in lesions such as age-related macular degeneration, and is thought to promote the progress of each pathological condition. Various angiogenesis inhibitors have been developed for the treatment and prevention of the above-mentioned various diseases, but none have been put to practical use yet, and it is desired to search for a compound having a safe angiogenesis inhibitory action without side effects. ing.

[0003] Incidentally, a synthetic inhibitor of a heat shock protein having a molecular weight of 47 kDa containing tocopherol or a derivative thereof as an active ingredient is known (Japanese Patent Application Laid-Open No. 9-45556). The derivatives include tocotrienol homologs, ie, α-, β-, γ- and δ-tocotrienol. The present invention is intended for the treatment of diseases exhibiting extracellular matrix production, such as cirrhosis and scleroderma, wherein the fibrosis associated with these diseases is due to an increase in collagen synthesis, and the heat shock protein It has been shown that the synthesis of collagen increases with the increase in expression of. Furthermore, he pointed out that collagen synthesis in the basement membrane plays an important role also in angiogenesis, and this synthesis inhibitor is used for diseases based on abnormal growth of angiogenesis, ie, diabetic retinopathy, glaucoma, rheumatoid arthritis. And so on. However, in the present invention, α-tocopherol which is vitamin E is particularly preferred, and only α-tocopherol is shown in Examples. As for tocotrienol, it is merely listed, and no specific content is given.

[0004] On the other hand, it has been said that tocopherols do not have an inhibitory effect on angiogenesis, but the present inventors have also confirmed them through comparative experiments described later in this specification.

[0005]

DISCLOSURE OF THE INVENTION The present invention has no problem in safety, and exhibits angiogenesis inhibitory action, cell growth inhibitory action, lumen formation inhibitory action and FGF inhibitory action which are far superior to α-tocopherol. An object of the present invention is to provide an inhibitor, and thereby contribute to treatment of a disease based on abnormal growth of angiogenesis.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, tocotrienol
In particular, they have found that β-, γ-, and δ-tocotrienol have far superior angiogenesis, cell growth, tube formation, and FGF inhibitory effects than α-tocopherol, and based on this finding, The invention has been completed.

That is, the present invention relates to β-, γ- or δ-
Angiogenesis inhibitor containing tocotrienol as an active ingredient, β
Cell growth inhibitor containing-, γ- or δ-tocotrienol as an active ingredient, tube formation inhibitor containing β-, γ- or δ-tocotrienol as an active ingredient, β-, γ- or δ-tocotrienol as an active ingredient Fibroblast growth factor,
It is intended to provide a food containing α-, β-, γ- or δ-tocotrienol and a food additive containing α-, β-, γ- or δ-tocotrienol.

[0008]

DETAILED DESCRIPTION OF THE INVENTION There are four homologs of tocotrienol, α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol. All of them exhibit an angiogenesis inhibitory action, a cell growth inhibitory action, a lumen formation inhibitory action and an FGF inhibitory action, but β-tocotrienol, γ-tocotrienol and δ-tocotrienol have a remarkably large action as compared with α-tocotrienol. Demonstrate. Each of these tocotrienols has an L-form and a D-form, but any of these can be used, and a mixture of two or more of the above four homologues may be used. In this case, α-tocotrienol may or may not be present as long as an effective amount of the active ingredient β-tocotrienol, γ-tocotrienol and / or δ-tocotrienol is present in the mixture. Good thing. Therefore, even if α-tocotrienol is contained after extraction and separation from a naturally occurring raw material, the effect is not offset by the interaction, so that there is no particular limitation. Rather, the amount of α-tocotrienol contained in the mixture may be selected in view of the effect as a medicine, the ease of taking and ingesting, and the cost due to repeated purification.

[0009] The tocotrienol may be in the form of an ester capable of releasing tocotrienol in vivo. This ester is preferably an ester with a saturated or unsaturated fatty acid.

[0010] Tocotrienol can be obtained by methods such as compression of natural products, extraction from natural products, and synthesis. Generally, it is extracted from the pericarp and / or seed of a palm plant.
Tocotrienol obtained from natural product extracts is a mixture of multiple tocotrienol homologs.

[0011] Natural products can be produced according to known methods. For example, water can be added to the extract for separation, and the oil layer can be separated and purified by column chromatography to prepare tocotrienol containing no or only trace amounts of tocopherols. The ratio and content of each tocotrienol homolog can be measured by a conventional method, for example, by liquid chromatography (HPLC).

Each homologue of tocotrienol is commercially available as α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol.

Alternatively, the extracted and concentrated tocotrienol is further subjected to column chromatography by an ordinary method to obtain α-
It can be isolated and purified into each component of tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol.

Each inhibitor of the present invention may be composed of only tocotrienol, or may contain other components according to the use and the like. Examples of other components include water and palm oil. The amount of water or palm oil used varies depending on the dosage form and is not particularly limited. However, the active ingredients β-tocotrienol and γ
-Tocotrienol, δ-tocotrienol can be contained in any proportion as long as an effective amount is present. For example, when used as a liquid, 0.01 to 99% by weight,
Preferably it is 0.01 to 90% by weight.

The inhibitors of the present invention can be used as therapeutic and prophylactic agents for diseases in which their inhibitory effects are effective. The target disease varies depending on the mechanism of onset, but angiogenesis inhibitors include solid tumors, diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, neovascular glaucoma, chronic Rheumatoid arthritis, hemangiomas, periodontal disease, scleroderma, psoriasis, etc., cell growth inhibitors are solid tumors, diabetic retinopathy, rheumatoid arthritis, hemangiomas, periodontal disease, scleroderma, glaucoma, Psoriasis, age-related macular degeneration, etc., and tube formation inhibitors include solid tumors, diabetic retinopathy, rheumatoid arthritis, hemangiomas, periodontal disease, scleroderma, glaucoma, psoriasis, age-related macula. Degeneration, etc., and FGF inhibitors
Solid tumors, diabetic retinopathy, rheumatoid arthritis, hemangiomas, scleroderma, glaucoma, psoriasis, age-related macula It is effective for degeneration and the like.

These therapeutic and prophylactic agents include animals other than humans, for example, domestic animals such as cattle, horses, pigs and sheep, chickens,
It can also be used for poultry such as quail and ostrich, reptiles, birds, pets such as small mammals, and cultured fish.

The pharmaceutical composition of the present invention may be prepared in a conventional manner, for example, by preparing tablets, sublingual tablets, pills, suppositories, powders, powders, fine granules,
It can be formulated into granules, capsules, microcapsules, injections, emulsions, patches and the like. For example, tablets are uniformly mixed with a pharmacologically acceptable carrier and tableted, and powders, powders, and granules are prepared by dissolving the drug and the carrier into a solution or suspension. It can be produced by drying by spray drying or freeze drying.

Powders, powders, fine granules, granules and tablets are excipients such as lactose, glucose, sucrose and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; Alcohol, hydroxypropylcellulose, binders such as gelatin, surfactants such as fatty acid esters,
It can be produced using a plasticizer such as glycerin.

If necessary, other antioxidants may be added to the pharmaceutical composition of the present invention. The antioxidant is not particularly limited, and may be applied as long as it has an antioxidant effect. For example, vitamin A such as retinol and 3,4-didehydroretinol, vitamin C such as vitamin B, D-ascorbic acid and L-ascorbic acid;
Vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, vitamin E acetate, vitamin E succinate, vitamin E phosphate
, Coenzyme Q, flavonoids, tannins, ellagic acid, polyphenols, radical inhibitors, hydroperoxide decomposers, metal chelators, active oxygen scavengers, α-
One or more selected from the group consisting of carotene, carotenes including β-carotene, γ-carotene, and δ-carotene, tocoquinone, and pharmaceutically acceptable salts thereof, and mixtures thereof. .

Injectable preparations may contain an active ingredient, if necessary, such as a pH adjuster, a buffer, a solubilizer, a suspending agent, a tonicifying agent, a stabilizing agent,
It can be formulated by a conventional method in the presence of a preservative or the like.

[0021] If necessary, a freeze-dried preparation can be prepared by a conventional method.

Examples of the suspending agent include polysorbate 80, methylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, gum arabic and powdered tragacanth. Examples of the solubilizer include polysorbate 80, hydrogenated polyoxyethylene castor oil, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester.
Examples of the stabilizer include sodium sulfite and sodium metasulfite. Examples of the preservative include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like.

The content of β-, γ- or δ-tocotrienol in each inhibitor of the present invention (in the case of two or more types, the total amount) is 0.01 to 99% by weight, preferably 1 to 99% by weight. Can be contained.

The dosage of each inhibitor of the present invention varies depending on the type of disease, age, sex, body weight, degree of symptoms, administration method, etc. of the patient, and is not particularly limited. About 3000 mg, preferably 100 to 30
About 00 mg is orally or parenterally administered in about 1 to 4 times a day.

The tocotrienol used in the present invention is
It is known that acute toxicity is of course safe, without noticeable chronic toxicity.
8-96021).

When the active ingredient of the present invention is used in foods,
Any of the above-mentioned effective amounts of the isomers of β-, γ-, δ-tocotrienol of tocotrienol and mixtures thereof can be used in an appropriate combination. The tocotrienol may be prepared as it is, in a form diluted with an oil or the like, in a milky form, or in a form to which a carrier generally used in the food industry is added.

In the emulsion form, for example, tocotrienol or a mixture thereof is added to the oil phase, and glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, glycerol, dextrin, rapeseed oil, soybean oil, corn oil are added. To the aqueous phase, L-ascorbic acid or an ester or salt thereof, such as locust bean gum, gum arabic or gelatin, and flavonoids such as hesperidin, rutin, quercetin, catechin, thianidine, etc. Alternatively, it can be prepared by adding and emulsifying a polyphenol or a mixture thereof.

The form of the beverage is a non-alcoholic beverage or an alcoholic beverage. As non-alcoholic beverages, for example, non-carbonated beverages such as carbonated beverages, fruit juice beverages, nectar beverages, soft drinks, sports beverages, tea, coffee, cocoa, etc. Examples include general food forms.

Specific examples of foods and drinks include the following. Western confectionery (pudding, jelly, gummy candy, candy, drop, caramel, chewing gum, chocolate, pastry, butter cream, custard groom, cream puff, hot cake, bread, potato chips, french fries,
Popcorn, biscuits, crackers, pies, sponge cakes, castellas, waffles, cakes, donuts, biscuits, cookies, rice crackers, oysters, rice balls, steamed buns, candy, etc., dried noodle products (macaroni, pasta),
Egg products (mayonnaise, fresh cream), drinks (functional drinks, lactic acid drinks, lactic acid bacteria drinks, concentrated milk drinks, fruit drinks,
Fruitless drinks, pulp drinks, transparent carbonated drinks, carbonated drinks with fruit juice, carbonated drinks with fruit), luxury items (green tea, black tea, instant coffee, cocoa, canned coffee drinks),
Dairy products (ice cream, yogurt, milk for coffee, butter, butter sauce, cheese, fermented milk, processed milk), pastes (marmalade, jam, flower paste, peanut paste, fruit paste, fruit syrup), meat products (Ham, sausage, bacon, dry sausage, beef jerky, lard), seafood (fish ham, fish sausage, kamaboko,
Chikuwa, hang pen, dried fish, dried bonito, mackerel, boiled, sea urchin, salted squid, squid, dried fish mirin, dried shellfish,
Smoked products such as salmon), boiled boiled fish (small fish, shellfish, wild vegetables, mushrooms, kelp), curries (instant curry, retort curry, canned curry), seasonings (miso, powder, end miso, soy sauce, powdered soy sauce) , Moromi, fish sauce, sauce, ketchup, oyster sauce, solid bouillon, grilled meat sauce, curry roux, stew mushroom, soup mushroom, dashi mushroom, paste, instant soup, sprinkle, dressing, salad oil), fried products (oil fried) , Fried sweets, instant ramen), soy milk,
Margarine, shortening and the like.

The above food and drink can be processed and manufactured by blending tocotrienol with raw materials of general foods according to a conventional method.

The amount of tocotrienol to be added to the above food and drink depends on the form of the food and is not particularly limited, but is usually preferably 0.001 to 10%. The amount of tocotrienol to be used is adjusted so that the amount of tocotrienol used per day is necessary to suppress the onset or progression of angiogenesis. As far as it is known, 10 to 3,000 m as tocotrienol per adult daily intake
g, preferably 100 to 1,000 mg, but the amount can be appropriately selected by the user depending on the form of the food.

The above food and drink can also be used as functional foods, dietary supplements or health foods. The form is not particularly limited.For example, as a food production example, proteins such as milk protein, soy protein, egg albumin, etc. having a high nutritional value with amino acid balance, their degraded products, egg white oligos In addition to peptides, soybean hydrolysates, etc., mixtures of amino acids alone can be used in accordance with conventional methods. Further, it can be used in the form of a soft capsule, a tablet or the like.

Examples of dietary supplements or functional foods include liquid foods, semi-digestive nutritional foods, component nutritional foods, which contain sugars, fats, trace elements, vitamins, emulsifiers, flavors and the like.
Examples include processed forms of drinks, capsules, enteral nutritional supplements and the like. In the above-mentioned various foods, for example, foods and drinks such as sports drinks and nutritional drinks, in order to improve the nutritional balance and flavor, furthermore, amino acids, vitamins, nutrient additives such as minerals and sweeteners, spices, flavors , A dye or the like can be blended.

In order to stabilize tocotrienol, which is an active ingredient of the drug or food of the present invention, antioxidants such as tocopherol, L-ascorbic acid, BHA, and rosemary extract can be used in a conventional manner. .

The food and drink of the present invention can be applied to feed for livestock, poultry and pets. For example, it can be blended with dry dog food, dry cat food, wet dog food, wet cat food, semi-moist dock food, poultry feed, livestock feed such as cows and pigs. The feed itself can be prepared according to a conventional method.

[0036]

EXAMPLES First, the cells used, culture conditions, and samples used (tocotrienol, homologs of tocotrienol, α-tocopherol and fibroblast growth factor (FGF)) used in the examples, and How to use will be described.

MTT is 2-4,3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, and LDH is Lactate Deh.
ydrogenase, MEM is an abbreviation for Minimum Essential Medium. WST is 2- (4-I
(odophenyl) -3- (4-nitropheny)
l) -5- (2,4-disulfophenyl) -2
-H-tetrazolium monosodium
It is an abbreviation for salt.

(1) Cells used Normal bovine aorta-derived endothelial cells (Normal Bovi)
ne Aortic Endothelial Cel
l: BAEC) was purchased from Dainippon Pharmaceutical Co., Ltd. from Cell Systems.

(2) Culture conditions Cells were cultured in MEM (Sigma) (MEM + 10% FB) supplemented with 10% (v / v) bovine serum (FBS) and penicillin-streptomycin (20 IU / ml each).
With S) was prepared as a 1 × 10 ⁵ cells / ml, using a 60mm dish which had been coated with type I collagen, and cultured in a 5% CO ₂ incubator (37 ° C.), 2 to 3 days It was passaged every other time.

(3) Sample used (3-1) Tocotrienol mixture: Tocotrienol component is α-tocotrienol 18.9% by weight, γ-
Tocotrienol 22.6% by weight, δ-tocotrienol 1.2% by weight, α-tocopherol 0.0% by weight, and 45.3% by weight (mainly water or palm oil) were used.

(3-2) Tocotrienol homolog: commercially available α-tocotrienol, β-tocotrienol, γ-tocotrienol
Tocotrienol and δ-tocotrienol (trade name “Tocotrienol”, DL-form, CALBIO)
CHEM) was used after dissolving in ethanol.

(3-3) α-tocopherol: commercially available α
-Tocopherol (manufactured by Wako) was dissolved in ethanol and used.

(3-4) Fibroblast growth factor (Fibr
oblast Growth Factor (FGF):
A commercial product (manufactured by Sigma) is replaced by MEM (manufactured by Sigma)
And used.

Each of the above samples was prepared by adding MEM (hereinafter, MEM + 2%) containing 2% (v / v) bovine serum (FBS) and penicillin-streptomycin (20 IU / ml each).
FBS).

Examples 1 to 5 Tocotrienol is BAE
Effect on C cell proliferation The effect of BAEC on cell proliferation was determined by MTT assay. The confluently grown BAECs were treated with trypsin to give a 1 × 10 ⁴ cells / ml suspension using MEM + 10% FBS. 96-well microplate (4
Cells) at 100 μl / well and then CO 2
Incubated overnight in ₂ incubators. Each w
After removing the culture solution in the cell, the medium containing the sample (M
(EM + 2% FBS) was replaced with 100 μl and incubation was continued. For those after 96 hours, the medium was changed once after 48 hours. At 0, 24, 48 and 96 hours after the addition of the medium containing the sample, each microplate was taken out, and the MTT solution [3 mg / ml PB
S (-)] at 50 μl / well, and then CO ₂
Incubated for 3 hours in incubator. PB
After adding S (-) to each well, the liquid was aspirated, and then 200 μl of isopropyl alcohol containing 0.04 mol / l hydrochloric acid was added. After one night in the dark,
The absorbance of each well was measured at 595 nm and 655 nm (dual) using a microplate reader.

As a sample, a tocotrienol mixture,
The MTT assay was performed using DL-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, and DL-δ-tocotrienol. The sample without addition was designated as control group A.

Comparative Example 1 Assay was carried out in the same manner as in Example 1 except that DL-α-tocotrienol in Example 1 was replaced with α-tocopherol.

Table 1 shows the types, amounts, assay times, and results of the samples of Examples 1 to 5 and Comparative Example 1. In the table, n = 8 and the result of the MTT assay is Mea.
Indicated by n ± SE.

[0049]

[Table 1]

DL-β-tocotrienol, DL-γ-
Tocotrienol and DL-δ-tocotrienol are 10
An inhibitory effect is seen by adding μM.

On the other hand, DL-α-tocotrienol is 10
The addition of μM was equivalent to the control group. Also,
No improvement was observed for α-tocopherol even when 100 μM was added.

Examples 6 to 11 FG of tocotrienol
F Inhibition Test Effect of Tocotrienol Mixture, DL-α on FGF Activity
-Tocotrienol, DL-β-tocotrienol, D
The effects of L-γ-tocotrienol and DL-δ-tocotrienol were tested in the MTT assay in the same manner as in Examples 1 to 5. The sample without addition of sample was designated as control group B.

Table 2 shows the types and amounts of the samples used in Examples 6 to 11, the incubation time, and the results.

Comparative Example 2 A test was conducted in the same manner as in Example 7 except that α-tocopherol was used in place of DL-α-tocotrienol in Example 7.

Table 2 shows the types of samples, the amounts used, the incubation times, and the results of Comparative Example 2. still,
In the table, n = 8, the result of the MTT assay is Mean ± SE
Indicated by

[0056]

[Table 2]

Examples 12-14 Angiogenesis Assay BAECs were cultured between two layers of type I collagen gel and the luminal network formed was evaluated.

The confluently grown BAECs were trypsinized and 1 × 10 5 with MEM + 10% FBS.
A suspension of ⁵ cells / 1.5 ml was used. 1.5 ml / ml in a 12-well microplate coated with type I collagen
After seeding the cells in a well, they were incubated overnight in a CO ₂ incubator. The following reagents were placed in a tube and mixed quickly under ice-cooling [Vitrogen c
ollagen (collagen) 8 vol.
1N NaOH 1vol, × 10MEM (GIBCO
BRL) 1 vol]. The culture solution was removed by suction, and the collagen prepared above was dispensed on the cells at 0.5 ml / well. This was left in a CO ₂ incubator for about 30 minutes to gel. Medium containing sample (MEM
+ 2% FBS) 1.5 ml and then every other day
Incubation was continued while changing the edium.

By observing the change of the cells with a microscope, taking a picture, and counting the number of branches of the cells forming the lumen,
The tube formation was evaluated.

Table 3 shows the types of the samples of Examples 12 to 14, the amounts used, and the results thereof (the number of luminal cells / cm ² ). A sample to which no sample was added was used as a control group C. In the table, the results of n = 4 and the number of tube forming cells / cm ² are shown as Mean.
Shown by ± SE.

[0061]

[Table 3]

From the above results, the use of 2 μM or more of tocotrienol reduced the number of tube-forming cells,
It can be seen that tube formation cells are not seen at all with the addition amount of μM.

Examples 15 to 20 Tube formation inhibition test DL-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, DL-δ-tocotrienol were used instead of the tocotrienol mixture of Examples 12 to 14. A tube formation inhibition test was performed in the same manner as in Example 12 above.

Table 4 shows the types and amounts of the samples of Examples 15 to 20 and the results thereof (the number of lumen forming cells / cm ² ). A sample to which no sample was added was used as a control group D. In the table, the results of n = 4 and the number of tube forming cells / cm ² are shown as Mean.
Shown by ± SE.

Comparative Example 3 A tube formation inhibition test was performed in the same manner as in Example 15 except that α-tocopherol was used instead of DL-α-tocotrienol in Example 15 described above.

Table 4 shows the types of the samples of Comparative Example 3, the amounts used, and the results thereof (the number of luminal cells / cm ² ).

[0067]

[Table 4]

From the results shown in Table 4 above, DL-δ-tocotrienol completely inhibited tube formation at a dose of 10 μM, and DL-γ-tocotrienol and the mixture of tocotrienol also sufficiently inhibited tube formation. DL-α-tocotrienol was equivalent to the control group at the addition amount of 10 μM, but when the addition amount was increased, tube formation was inhibited at 30 μM or more and completely inhibited at 50 μM. on the other hand,
Α-Tocopherol used as a comparative example did not inhibit tube formation even at an addition amount of 100 μM.

Examples 21-26 Inhibition test of tube formation induced by FGF Tube formation induced by FGF was measured using a mixture of tocotrienol, DL-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, DL- −δ−
It was tested whether tocotrienol inhibited. The sample without addition was designated as control group E. FGF was added at the same time as tocotrienol.

Table 5 shows the types and amounts of the samples of Examples 21 to 26 and the results (number of cells forming the lumen / cm ² ). In the table, the results of n = 4 and the number of tube forming cells / cm ² are shown by Mean ± SE.

Comparative Example 4 In place of DL-α-tocotrienol in Example 22, α-
The test was conducted in the same manner as in Example 22 except that tocopherol was used.

Comparative Example 4 Table 5 shows the types of the samples, the amounts added, and the results thereof (the number of luminal cells / cm ² ).

[0073]

[Table 5]

The results in Table 5 show that DL-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, and DL-δ-tocotrienol inhibit FGF-induced tube formation. Especially DL-
β-tocotrienol and DL-δ-tocotrienol completely inhibited tube formation at 10 μM.

Examples 27 to 29 Measurement of LDH activity In order to evaluate the cytotoxicity against BAEC by a sample, the free LDH activity in the medium was measured using an LDH-cytotoxicity test kit (manufactured by Wako).

Confluently grown BAECs were trypsinized and 5 × 10 5 with MEM + 10% FBS.
A suspension of ⁴ cells / ml was used. After seeding the cells at 100 μl / well in a 96-well microplate, CO ₂ was added.
Incubated overnight in incubator. Each we
The medium containing the sample (MEM
+ 2% FBS) and the incubation was continued. MEM as negative control (NC)
+ 2% FBS, 0.2% Tween2 dissolved in MEM + 2% FBS as a positive control (PC)
0 was used. After 48 hours, the medium in the wells is
Each of the kits was transferred to another new well, and 50 μl of the coloring reagent of the kit was added thereto, followed by incubation at room temperature for 45 minutes. The reaction was stopped by adding 100 μl of 0.5N HCl, the absorbance at 595 nm was measured, and the cytotoxicity was determined by the following formula. Cytotoxicity = (SN) / (PN) × 100 (%) S = absorbance of sample N = absorbance of negative control P = absorbance of positive control

The types and amounts of the samples in Examples 27 to 29, and the results thereof [Cytotoxicity (% of contro
l)] is shown in Table 7. Control group F without sample
And In the table, n = 4, and the cell injury rate was Mean ±
Indicated by SE.

Comparative Examples 5 to 7 Tests were carried out in the same manner as in Example 27 except that α-tocopherol was used instead of the tocotrienol mixture of Example 27.

The types and amounts of the samples of Comparative Examples 5 to 7
Table 6 shows the results [cell damage rate (% of control)].

[0080]

[Table 6]

From the results shown in Table 6, it can be seen that the cytotoxic effect of tocotrienol is stronger than that of tocopherol.

Examples 30 to 34 In place of the tocotrienol mixture of Example 27, D
The cell injury rate (% of control) was determined in the same manner as in Example 27 except that each homologue of L-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, and DL-δ-tocotrienol was used. .
Table 8 shows the types of the samples of Examples 30 to 34, the amounts added, and the results [cytotoxicity (% of control)]. The group without addition was used as a control group G. In the table, n =
5. Cell injury rate (% of control) is Mean
Indicates ± SE.

Comparative Example 8 A test was conducted in the same manner as in Example 30 except that α-tocopherol was used instead of DL-α-tocotrienol in Example 30.

[0084]

[Table 7]

From the results in Table 7, it can be seen that DL-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, and DL-δ-tocotrienol damage cells.

On the other hand, administration of α-tocopherol at 100 μM was equivalent to that of the control group, and there was no cytotoxic effect.

Examples 35-40 DL-α-tocotrienol against FGF, DL-β
-Tocotrienol, DL-γ-tocotrienol, D
The cytotoxic effects of L-δ-tocotrienol and tocotrienol mixtures were tested. The test method was according to Examples 27 to 29 except that FGF was added.

The types of the samples of Examples 35 to 40 and the results thereof ((cell damage rate (% of control)) are shown in Table 8. The sample without additives was used as control group H.
In the table, n = 4, cytotoxicity rate (% of control)
Is shown.

Comparative Example 9 The cytotoxicity was determined in the same manner as in Example 36 except that α-tocopherol was used instead of DL-α-tocotrienol in Example 36.

The types of the samples of Examples 35 to 40 and Comparative Example 9 and the results thereof [Cell damage rate (% of contr)
ol)] is shown in Table 8.

[0091]

[Table 8]

The following table summarizes the effects of tocotrienol on cell growth, tube formation and cytotoxicity of FGF.

[0093]

[Table 9]

In the table, △ indicates promotion, / indicates increasing tendency, → indicates no change, ↓ indicates suppression, ↓↓ indicates strong suppression,-indicates no toxicity, + indicates no toxicity.
Somewhat toxic, ++ indicates toxic.

From the results in Table 9, it can be seen that the mixture of tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol and DL-δ-tocotrienol was 10 μM each.
Indicates a tube formation inhibitory effect (β = δ> γ). DL-α
-From the results of the tube formation inhibition test, it is considered that the use of tocotrienol at 10 μM or more can provide a tube formation inhibitory effect.

On the other hand, no inhibitory effect was observed for α-tocopherol even at 100 μM.

Examples 41-60 When tocotrienol is B
Effect on AEC cell proliferation The effect of tocotrienol on BAEC cell proliferation was examined at various concentrations. For this purpose, cell viability was quantified using WST-1, which can perform high-sensitivity measurement, instead of MTT which is a conventional method for measuring cell proliferation.

[0098] The confluently grown BAECs were trypsinized and 2x1 using MEM + 10% FBS.
0 ⁴ was cells / ml of cell suspension. 100 cells in a 96-well microplate coated with type I collagen
After seeding at μl / well, the cells were incubated in a CO ₂ incubator for 24 hours. Medium alone was incubated without seeding cells as a blank. Each wel
The medium in the sample (MEM)
+ 2% FBS) and the incubation was continued. Twenty-four hours after the addition of the sample medium, the microplate was removed and the WST-1 solution was added at 10 μl / w.
After the addition in the well, the mixture was incubated in a CO ₂ incubator. After 3 hours, using a microplate reader, 450 nm and 655 nm for each well.
(Dual) absorbance was measured.

WST was performed using DL-α-tocotrienol, DL-β-tocotrienol, DL-γ-tocotrienol, and DL-δ-tocotrienol as a sample.
-1 assay was performed. The sample without addition was designated as control group I.

The types of the samples of Examples 41 to 60
The amount used and the result [Cell viability (% of contr)
ol)] is shown in Table 10. Cell viability = (SB) / (CB) × 100 (%) S = absorbance of sample C = absorbance of control group B = absorbance of blank In the table, n = 9, WST-1 assay Result is Mea
Shown as n ± SD. TC indicates tocotrienol.

[0101]

[Table 10]

Tocotrienol showed a concentration-dependent inhibitory effect. Furthermore, DL-α-tocotrienol <DL-
γ-tocotrienol <DL-β-tocotrienol <
A strong inhibitory effect was observed in the order of DL-δ-tocotrienol. That is, the inhibitory effect of tocotrienol on endothelial cell proliferation varies greatly depending on structural isomerism.

The results in Table 10 are shown, and the 50% growth inhibitory concentration (IC ₅₀ ) was determined from this figure. The vertical axis of the figure is% of control, the horizontal axis is the concentration (μM), − ● −
Indicates DL-α-tocotrienol,-■-indicates DL-β-tocotrienol,-△-indicates DL-γ-tocotrienol, and -x- indicates DL-δ-tocotrienol. Table 11 shows the results.

[0104]

[Table 11]

Formulation Example 1 Tablet An oral tablet having the following composition was produced by a conventional method. Tocotrienol mixture 50mg Lactose 50mg Corn starch 14mg Polyvinylpyrrolidone 5mg Magnesium stearate 1mg

Formulation Example 2 Tablet An oral tablet having the following composition was produced by a conventional method. DL-β-tocotrienol 50mg lactose 50mg corn starch 14mg polyvinylpyrrolidone 5mg magnesium stearate 1mg

Formulation Example 3 Injection for muscle 100 mg of DL-β-tocotrienol was dissolved in 1 ml of purified corn oil to prepare an injection for intramuscular injection.

Formulation Example 4 Injection for muscle 100 mg of DL-δ-tocotrienol was dissolved in 1 ml of purified corn oil to give an injection for intramuscular injection.

Formulation Example 5 Injection for intravenous injection 50 mg of tocotrienol was emulsified and dispersed in 1 ml of purified water using 5 mg of polyoxyethylene sorbitan monostearate, and 0.2 mg of propylparaben was added thereto. It was used as an injection.

[0110]

Industrial Applicability According to the present invention, an angiogenesis inhibitor, a cell growth inhibitor, a tube formation inhibitor, an FGF inhibitor, a food or a food additive containing tocotrienol as an active ingredient can be provided.

[Brief description of the drawings]

FIG. 1 illustrates the numerical values of Table 1. The vertical axis in the figure is% of
control, the horizontal axis is concentration (μM),-●-is DL-
α-tocotrienol,-■-is DL-β-tocotrienol,-△-is DL-γ-tocotrienol,-×-
Represents DL-δ-tocotrienol.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 17/06 A61P 17/06 19/02 19/02 27/02 27/02 29/00 101 29/00 101 35/00 35/00 C07D 311/72 101 C07D 311/72 101 (72) Inventor Hiroo Miyazawa 7-17-7 Takamori, Izumi-ku, Sendai-shi, Miyagi F term (reference) 4B018 MD26 MD48 ME08 ME09 ME10 4C062 FF16 4C086 AA01 AA02 BA09 MA04 MA35 MA66 NA14 ZA33 ZA89 ZA96 ZB15 ZB26 4C088 AB83 AC04 AC05 BA08 MA02 MA35 MA66 NA14 ZA33 ZA67 ZA89 ZA96 ZB15 ZB26 ZC35

Claims (6)

[Claims] 1. An angiogenesis inhibitor comprising β-, γ- or δ-tocotrienol as an active ingredient. 2. A cell growth inhibitor comprising β-, γ- or δ-tocotrienol as an active ingredient. 3. A tube formation inhibitor comprising β-, γ- or δ-tocotrienol as an active ingredient. 4. A fibroblast growth factor inhibitor comprising β-, γ- or δ-tocotrienol as an active ingredient. 5. Food containing α-, β-, γ- or δ-tocotrienol 6. A food additive containing α-, β-, γ- or δ-tocotrienol.