JP2002308777A - Composition having vascularization inhibitory effect - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition having no toxicity on a body when a component derived from an edible mushroom is administered within a common intake range for a mushroom and exhibiting a vascularization inhibitory effect, which is an antitumor effect having high safety. SOLUTION: The composition having a vascularization inhibitory effect contains ergosterol and a cyclodextrin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a composition having an inhibitory effect on angiogenesis, and more particularly to the effective use of newly elucidated physiological functions of ergosterol contained in basidiomycetes and the like. More specifically, the present invention relates to a composition having an angiogenesis inhibitory action, comprising ergosterol and cyclodextrin. This composition is used as a medicine or health food having an angiogenesis inhibitory action.

[0002]

BACKGROUND OF THE INVENTION Angiogenesis is the development of human or animal embryos,
While occurring in normal physiological conditions, such as ovulation or placental formation by the female sexual cycle, or in healthy conditions, such as wound healing, inflammation, and other repair processes, capillaries rapidly increase and increase, causing severe tissue damage. It is known to occur in many pathological conditions and the like that cause damage.

[0003] Malignant tumors, diabetic retinopathy, rheumatism and the like are known as diseases in which angiogenesis is involved in the etiology or deterioration of the disease state. Of these, the relationship between malignant tumors and angiogenesis has been extensively studied, and it has been elucidated that angiogenesis is particularly important in metastasis and prognosis of malignant tumors. In recent years, several substances showing angiogenesis inhibitory activity have been reported, for example, sulfated polysaccharide, shark cartilage, platelet factor-4 (PF-4), pentosan polysulfate,
TNP-470 (an analog of the fungus product fumagillin), a tissue metalloproteinase inhibitor (TIM
P), minocycline, demethylzeaylral and the like are known. However, these angiogenesis inhibitory actions cannot be said to be sufficient, and the development of a substance having a better angiogenesis inhibitory action is eagerly desired.

By the way, mushrooms are known to have various physiological functions, for example, an immunopotentiating action, a blood pressure and blood sugar lowering action, an antioxidant action, and the like. Widely used as medicine or health food.

The following are known as physiologically active substances of these mushrooms. (1) β- (1 → 3) → glucan, β- (1 → 6) → glucan protein complex, acidic heteroglycan, xyloglycan, heteroglycan protein complex, RNA protein complex, glycoprotein (lectin) Activation of immune cells such as macrophages and complement, activation of reticuloendothelial system function, promotion of induction of cytokines such as interferon, Biological response modifi
It acts as ers (BRM) and has an antitumor effect, a life-prolonging effect, an antiviral effect, and a hypoglycemic effect due to immunopotentiation. (2) The ceresterol derivative and ergosterol oxidized derivative have a cancer cell growth inhibitory effect due to cytotoxicity, based on the results of the HeLe cell growth inhibition test. (3) Dietary fiber / indigestible β-glucan, heteropolysaccharide, and chitin have a cancer-preventing effect by adsorbing and excreting carcinogens. (4) Dietary fibers and unsaturated fatty acids, such as linoleic acid, that constitute lipids have a decrease in blood pressure, a decrease in cholesterol, and an improvement in arteriosclerosis. (5) Ergosterol has a vitamin D ₂ effect by light irradiation.

As described above, sterols such as ceresterol derivatives and ergosterol oxidized derivatives have been found to have an anticancer effect. However, this is a mechanism different from the angiogenesis inhibitory effect, and mushrooms (particularly ergosterol) ) Had no angiogenesis inhibitory effect.

[0007]

Therefore, in addition to the function of enhancing the immunity as a physiological action of mushrooms and the function as a dietary fiber, angiogenesis which is nontoxic to living organisms and is a highly safe antitumor effect It is desired that the inhibitory action be exerted in the normal range of mushroom intake. An object of the present invention is to solve such a problem.

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have been conducting research for many years on an efficient method of ingesting mushroom components. Can be solubilized by inclusion in cyclodextrin, thereby finding a significant angiogenesis inhibitory effect,
It was confirmed that the extract obtained by adding cyclodextrin to the organic solvent extract of mushrooms or the extract obtained by adding cyclodextrin to mushrooms exhibited the above-mentioned effects effectively, and completed the present invention. I came to.

[0009] That is, the present invention relates to a composition having an angiogenesis inhibitory action, comprising ergosterol and cyclodextrin.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. Ergosterol according to the present invention is contained in basidiomycetes in nature, and mushroom is a common name for its fruiting body. The ergosterol according to the present invention is at least selected from the group consisting of shiitake mushroom, oyster mushroom, nameko, maitake, enokitake, bunashimeji, hatakeshimeji, agaricus mushroom, mesimakobushi mushroom, mannen mushroom (reishi), matsutake mushroom, kawara mushroom and chiyorimai mushroom. It is derived from one type of mushroom. Therefore, those which can be applied to the present invention as mushrooms containing ergosterol include, for example, shiitake mushrooms,
Pleurotus, Nameko, Maitake, Enokitake, Bunashimeji, Hatakeshimeji, Agaricus mushroom, Meshimakobu mushroom, Mannen mushroom (reishiba), Matsutake mushroom, Kawara mushroom, Chiyoraimai mushroom, and the like. Used. All of the above mushrooms are distributed on the market in large quantities as edible mushrooms, and artificial mushroom cultivation methods have been established for many mushrooms. The present invention is not limited to natural ones,
Commercially cultivated products can also be suitably used. Agaricus mushroom, Mesimakobu mushroom, Mannen mushroom (Lingzhi), Matsutake mushroom, Kawara mushroom, Chiyoraimai mushroom, etc., for which no artificial cultivation method has been established, can also be used in the same manner.

There are no particular restrictions on the form of the mushrooms.
Raw mushrooms, dried products obtained by subjecting the same to a drying method such as sun drying and hot air drying, or extracts obtained by extracting them with an organic solvent or the like are used. By mixing mushrooms containing ergosterol with cyclodextrin, a composition having an angiogenesis inhibitory effect can be obtained. This composition is expected to be widely used as medicines, health foods and the like.

In a preferred embodiment of the composition of the present invention, a fat-soluble fraction containing ergosterol obtained by extracting the above mushroom with an organic solvent by a conventional method is used as ergosterol. For example, an appropriate amount of water and / or an organic solvent is added to the raw mushroom, its dried product, or its crushed product, heated as necessary, and extracted under non-reflux or reflux. Examples of the organic solvent used for extracting mushrooms include methanol, ethanol, propanol, acetone, chloroform, and mixtures thereof. After the extraction, the extract and the solid may be separated, and the extract may be collected by distilling off the extraction solvent from the extract under reduced pressure. As for the extraction temperature, in the case of water, it is preferably 90 ° C. or higher, particularly preferably about 95 ° C. In the case of an organic solvent, it can be extracted at room temperature, and may be appropriately heated as needed. The extract may be in the form of an extract, but is preferably in a dry state from the viewpoint of storage stability and the like, and is preferably ground and powdered as necessary.

The cyclodextrin (hereinafter sometimes abbreviated as CD) that can be used in the present invention includes α- and β- in which 6 to 8 cyclically linked glucoses have α-1,4 bonds. , Γ-CD, and branched CDs in which a glucosyl group, a maltosyl group, or the like is bonded to the CD ring at α-1,6 in order to improve the solubility or physical properties of these CDs.
Or galactosyl-, mannosyl-, in which a galactosyl group, a mannosyl group, or N-acetylglucosamine is bonded to the CD ring of these CDs or branched CDs directly or to a glucosyl group in a side chain portion of the ring by β- or α-bond , N-acetylglucosaminyl-branched CD, and glucuronylglucosyl CD in which a sugar residue at the terminal of the side chain of the branched CD is oxidized. Also, a mixture of these can be used.

Since cyclodextrin can solubilize ergosterol, when the composition of the present invention is obtained, the above-mentioned mushrooms are not extracted in advance, and for example, commercially available raw mushrooms or dried products are used as they are. May be used. In addition, dried mushrooms or extracts are suitable enzymes,
For example, those treated with a glycolytic enzyme such as amylase or glucanase can also be used. Residues obtained by extracting β-glucan by treating mushrooms with water or hot water can also be used. In addition, when extracting ergosterol from mushrooms, CD is added and extraction is performed, and the obtained extract can be used as the composition of the present invention.

The composition according to the present invention may be prepared by mixing an extract or mushroom containing ergosterol with cyclodextrin, and the mixing ratio thereof is preferably from 1 to 100 (molar ratio) per ergosterol content of 1. It is. Preferably, after adding a cyclodextrin solution to the extract containing ergosterol, kneading or homogenizing,
The desired composition can be obtained by drying by a known method. Also, after adding a cyclodextrin solution to the raw mushrooms and dried mushrooms, homogenize,
It is dried in the same manner as described above, or the organic solvent such as ethanol is added thereto to enhance the recovery efficiency, and the liquid layer obtained by separating the solid and the liquid layer is concentrated as an extract, or the dried product thereof. Can also be obtained. Furthermore, a method of adding cyclodextrin when decoctioning the dried mushrooms is also effective, whereby a composition containing ergosterol and cyclodextrin can be obtained.

Although the composition according to the present invention has various aspects as described above, these can be administered to humans or animals (livestock, pets) together with conventional pharmaceutical carriers. The administration form is not particularly limited, and may be appropriately selected and used as needed to be used as an oral preparation such as tablets, capsules, granules, fine granules, powders, and the like, and parenteral injections, suppositories, and the like. Agents.

In the case of oral preparations, the preparations are prepared in a conventional manner using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch and inorganic salts. In addition, components such as oligosaccharides, lactic acid bacteria, chitin / chitosan, shark cartilage, ginkgo biloba extract, and DHA may be used alone or in combination as long as the effects of the composition of the present invention are not impaired. In this type of preparation, a binder, a disintegrating agent, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a flavor, and the like can be used as appropriate in addition to the excipients. Specific examples of each of these substances are as follows.

Binders: starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose,
Ethyl cellulose, polyvinylpyrrolidone, macrogol and the like.

Disintegrators: starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose and the like.

Surfactants: sodium lauryl sulfate, soy lecithin, sucrose fatty acid ester, polysorbate 8
0 magnitude.

Lubricants: talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.

Fluidity promoter: light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

The composition of the present invention can also be used as suspensions, emulsions, syrups, elixirs, etc. These various forms may contain flavoring agents, coloring agents and the like, if desired. You may make it contain.

In order to use the composition of the present invention as a medicine or health food, etc., to exert the desired effect, the dosage when taken orally depends on the age, weight, purpose of use and the like of humans and animals. There is no particular limitation. However,
In the case of humans, mushroom extract per adult is usually 10m per day
g to 10,000 mg, preferably 50 mg to 50 mg
It is considered appropriate to take 00 mg in several divided doses. Since the composition of the present invention contains a component derived from edible mushrooms as a main component, there is no problem in safety even when ingested for a long period of time.

[0025]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1 Solubilization of ergosterol by CD Various CD aqueous solutions (2% (v / v) or 5% (v / v)
v)) 10 mg of ergosterol is added to 100 ml or 100 ml of water, and TK mixer (manufactured by Tokushu Kika Kogyo)
And homogenized at room temperature under conditions of 3000 rpm for 5 minutes. Next, the mixture was filtered through a 0.45 μm membrane filter, and 40 ml of ether was added to the filtrate to extract the enclosed or free ergosterol. The ether layer was collected and dried under reduced pressure to give 0.5 mL of acetonitrile.
and analyzed by HPLC. Dissolve 5 mg of ergosterol in 20 ml of acetonitrile and add 10 mg
A standard curve was prepared using the double-fold dilution as a standard solution.
The amount of dissolution was calculated from the height of the peak obtained by analyzing the sample. In addition, HPLC analysis conditions are as follows.

Column: Asahi-pak ODP-50 6D 6.0 × 150 mm Eluent: acetonitrile / methanol = 90/10 (v
/ V) Flow rate: 0.8 ml / min Temperature: 35 ° C Detection: UV 265 nm

The results are shown in Table 1. As is clear from the table, the amount of ergosterol dissolved in the control water was 0, but the amount of ergosterol dissolved increased in the case of the aqueous CD solution. In particular, maltosyl-β-CD (in the table, G2
-Β-CD) was the highest.

[0028]

[Table 1] Table 1 Isoelite P: CD brand name, manufactured by Shiosui Minato Sugar Co., Ltd. HANKS: Uses HANKS buffer

Example 2 To 1 kg of dried Agaricus mushroom was added 2 L of chloroform: methanol (= 1: 1), and the mixture was extracted under reflux for 1 hour. This operation was repeated three times to obtain a chloroform: methanol extraction fraction. Next, the solvent was distilled off from this fraction under reduced pressure to obtain 250 g of a chloroform: methanol extract. To 10 g of this extract is added 1 L of a 10% maltosyl-β-CD solution.
In addition, the mixture was homogenized with a TK mixer and freeze-dried to obtain 110 g of a chloroform: methanol extract / CD composition.

Chloroform: 200 g of methanol extract
Was fractionated into an acetone-soluble fraction (160 g) and an acetone-insoluble fraction (40 g). The acetone-soluble fraction was further fractionated into a hexane-insoluble fraction and a hexane-soluble fraction, and the hexane-insoluble fraction was subjected to silica gel chromatography,
Elution with% ethyl acetate / hexane. The eluted fraction was evaporated under reduced pressure to remove the solvent, and further recrystallized from methanol to obtain 2 g of ergosterol. A 10% maltosyl-β-CD solution is added to this ergosterol for 100 m.
and homogenized using a TK mixer. Next, this was freeze-dried to obtain 12 g of an ergosterol / CD composition. To 200 mg of this composition, 140 mg of lactose as an excipient, 60 mg of corn starch, 15 mg of low-substituted hydroxypropylcellulose as a disintegrant, and 3 mg of magnesium phosphate as a lubricant were mixed well and then tableted. This was submerged in keratin and cocoa butter, immersed in a keratin solution, dried and coated with a film to produce tablets.

Example 3 2 L of purified water was added to 1 kg of dried agaricus mushroom, and hot water extraction was performed while heating and refluxing for 2 hours. After the extraction, the extraction residue was recovered by centrifugation, and the residue was subjected to an extraction operation while heating and refluxing with a 50% ethanol and 5% maltosyl-β-CD solution for 2 hours. Next, centrifugation was performed to remove insolubles, and the extract was freeze-dried to obtain 300 g of an ethanol: CD extraction composition. Dextrin was added instead of CD, and the same operation was performed to obtain an ethanol: dextrin extract (control).

Example 4 Maltosyl-β-CD100 was added to 1 kg of enokitake fruit body.
g and 2 L of 50% ethanol were added, and an extraction operation was performed while heating and refluxing for 2 hours to obtain an extract. After this operation was further repeated twice, the extracts were combined. Next, the solvent was distilled off under reduced pressure, and the obtained extract was freeze-dried to obtain 120 g of Enokitake mushroom extract. Similarly, shiitake mushroom, oyster mushroom, nameko, maitake, bunashimeji, hatakeshimeji,
120 g, 110 g, 110 g, 120 g of the extract of Meshimakobu mushroom, Kawara mushroom and Chiyoremai mushroom, respectively.
115 g, 123 g, 118 g, 115 g and 120 g were obtained.

Example 5 The method of Passaniti et al. (Laboratory Invest., 67, 519)
-528 (1992)), the effect of ergosterol on angiogenesis induced by Matrigel Matrix was examined. That is, the following subjects were transplanted under cooling into the abdomen of 5-week-old C57BL / 6 female mice at a rate of 0.5 ml each, and the Matrigel Matrix was taken out 5 days after the transplantation to observe the state of neovascularization. . Matrigel Matrix was lyophilized and weighed. In addition, I took out Matrigel
After adding 1 ml of pure water to the Matrix, homogenizing with a polytron and centrifuging at 2,000 rpm for 5 minutes, the supernatant was filtered through a 0.2 μm filter, and the amount of hemoglobin was measured using “Hemoglobin Test Wako” (Wako Pure Chemical). Was measured.

Normal group: Matrigel Matrix (Becton Dikin
control group: Matrigel Matrix, 64 units heparin, 1 ng / m
l Acid fibroblast growth factor (hereinafter sometimes abbreviated as FGF) Ergosterol addition group 1: Ergosterol 10 mg / kg,
Matrigel Matrix, 64 units Heparin, 1 ng / ml FGF Ergosterol group 2: Ergosterol 20 mg / kg,
Matrigel Matrix, 64 units heparin, 1 ng / ml FGF Ergosterol is 2% maltosyl-β-CD
(Phosphate buffer). Heparin and FGF were prepared aseptically with 1% BSA (bovine serum albumin).

The obtained results are shown in FIGS. As is clear from the figure, the control group significantly promoted angiogenesis and increased the weight of the Matrigel Matrix and the amount of hemoglobin as compared with the normal group. On the other hand, in the ergosterol administration group, angiogenesis was suppressed in a concentration-dependent manner, and Matrig
The increase in el Matrix weight and hemoglobin content was suppressed. This test revealed that ergosterol itself has an inhibitory effect on angiogenesis.

Example 6 Tumor Neovascular Inhibition Test Using Mice A test was performed by the dosal air sac method. That is, a millipore filter was attached to both sides of a plastic ring having a diameter of 14 mm, and 1.5 × 1
Culture medium or DMEM solution of 0 ⁶ cells / ml in LLC (Lewis lung carcinoma cells) (Normal group) to create a chamber in 200μl injected. Mice (C57BL / 6 female, 5 weeks old) were divided into 6 mice per group, and this chamber was inserted subcutaneously at the back of the mice.
From the next day, 2% β-CD for 4 days for 4 groups of mice
Ergosterol (0, 5, 10, or 20 mg / kg) suspended in the solution was administered intraperitoneally. Ergosterol 0 mg / kg administration group is a control group.

On the sixth day, the skin on the chamber was shaved to evaluate the degree of inhibition of new blood vessels. On the other hand, the other group (normal group) was a group into which a chamber into which DMEM solution was injected was inserted, and this group contained 2% β without ergosterol.
Only the -CD solution was administered, and the degree of inhibition was similarly evaluated on the sixth day. Each time the neovascularization promoting substance from the tumor permeates the Millipore filter, new blood vessels are observed growing on the surface in contact with the chamber. The degree of inhibition of the growth of new blood vessels was evaluated according to the following evaluation criteria. Table 2 shows the results.

[0038]

[Table 2] Table 2

As is apparent from Table 2, the control group promoted angiogenesis remarkably with the proliferation of LLC compared to the normal group. In contrast, the ergosterol-administered group suppressed angiogenesis in a concentration-dependent manner. In the group without CD performed for comparison, no effect was confirmed at all when the dose of ergosterol was in the above range, and even when suspended in 5% gum arabic, the dose of ergosterol was 100 mg / mg.
The effect could only be confirmed with kg or more. From this, it was found that it is effective to use ergosterol together with CD.

Example 7 A mouse into which a chamber prepared by injecting 200 μl of a culture solution of LLC (Lewis lung cancer cells) or DMEM solution (normal group) at 1.5 × 10 ⁶ cells / ml as in Example 6 was inserted. Next, the ethanol: CD extract composition or ethanol: dextrin extract prepared in Example 3 (control)
Was intraperitoneally administered at a dose of 20 mg / kg for 5 days.
On the day, the degree of inhibition was evaluated. As a result, marked angiogenesis was observed in the control group, but almost no angiogenesis was observed in the ethanol: CD extract composition administration group.

Example 8 A mouse into which a chamber prepared by injecting 200 μl of a culture solution of LLC (Lewis lung cancer cell) or DMEM solution (normal group) at 1.5 × 10 ⁶ cells / ml as in Example 6 was inserted. In addition, chloroform: methanol extract composition of Agaricus mushroom obtained in Example 2, Enokitake mushroom extract, Shiitake mushroom extract, Oyster mushroom extract, Nameko extract, Maitake mushroom extract, Bunashimeji extract, Hatakemushi extract object,
100 mg / kg or 1000 mg of Mesimakobu mushroom extract, Kawara mushroom extract or Chiyoremai mushroom extract, respectively
mg / kg was orally administered for 7 days, and on day 8, the degree of angiogenesis was observed. In addition, a control to which no mushroom extract was administered was observed in the same manner as in Example 6. Table 3 shows the results.

[0042]

[Table 3] Angiogenesis inhibitory effect of mushroom extract / CD composition

As is evident from Table 3, the chloroform: methanol extract of Agaricus mushroom exhibited a high angiogenesis inhibitory effect. The effect was also observed with other mushroom extracts. In addition, there was no change in body weight, thymus, spleen, or epididymal adipose tissue weight by such oral administration, and no side effects such as a decrease in leukocytes were observed.

[0044]

The composition having an angiogenesis inhibitory action provided by the present invention has high safety without any toxicity to living organisms, since it contains a component derived from edible mushrooms as a main component. Therefore, by administering this composition in the range of normal intake, it is possible to inhibit the angiogenesis effect which is an antitumor effect, a drug useful for prevention and treatment of lifestyle-related diseases such as cancer, It is expected to be used as a health food.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of measuring the weight of Matrigel Matrix in each group in Example 5.

FIG. 2 is a diagram showing the results of measuring the amount of hemoglobin in each group in Example 5.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 105 A61P 43/00 105 // A23L 1/30 A23L 1/30 B Z (72) Inventor Takumichi Okuda 1174-17 Takako-cho, Matsuyama-shi, Ehime (72) Inventor Yoshiyuki Kimura 520 Fujo-ya, Yajo-machi, Nakagyo-ku, Kyoto-shi, Kyoto 520 (72) Inventor Eriko Kikuchi Oguro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa 13-46 Inside Yokohama International Bio Research Institute Co., Ltd. (72) Inventor Takeshi Kamei 5-13-11 Yokowari, Fuji City, Shizuoka Prefecture Inside Fuji Bio Co., Ltd. (72) Inventor Hitoshi Hashimoto 13-46 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Takeo Ishihara 2-6-8 Hitotsubashi, Chiyoda-ku, Tokyo Tommy Building B.H. (Ref.) NA02 NA10 NA11 ZA36 ZB26

Claims (4)

[Claims] 1. A composition having an angiogenesis inhibitory action, comprising ergosterol and cyclodextrin. 2. The ergosterol is at least selected from the group consisting of shiitake mushrooms, oyster mushrooms, nameko, maitake, enokitake, bunashimeji, hatakeshimeji, agaricus mushroom, mesimakobushi mushroom, mannen mushroom (reishi), matsutake mushroom, kawara mushroom and chiyoraimai mushroom. The composition according to claim 1, which is derived from one kind of mushroom. 3. The mushroom is water or methanol, ethanol,
3. The composition according to claim 1, wherein the composition is extracted with an organic solvent selected from propanol, acetone, chloroform and a mixture thereof. 4. The composition according to claim 1, wherein the cyclodextrin is β-cyclodextrin, maltosyl-β-cyclodextrin, or a mixture containing these.