JP2008143835A - Vascular endothelial cell proliferation inhibitor and/or vascularization inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vascular endothelial cell proliferation inhibitor and/or vascularization inhibitor containing phosphatidylinositol as an active ingredient. <P>SOLUTION: This new vascular endothelial cell proliferation inhibitor and/or vascularization inhibitor containing the phosphatidylinositol as an active ingredient is provided. Also, a functional food and cosmetic having the vascular endothelial cell proliferation inhibitory function and/or vascularization inhibitory function, and containing the phosphatidylinositol as an active ingredient are provided. Further, the use of the phosphatidylinositol for preparing a composition having the vascular endothelial cell proliferation inhibitory effect and/or vascularization inhibitory effect, and the method for inhibiting the vascular endothelial cell proliferation and/or vascularization by taking and/or applying the phosphatidylinositol are also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a vascular endothelial cell growth inhibitor and / or an angiogenesis inhibitor. More specifically, the present invention relates to a vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor containing phosphatidylinositol as an active ingredient.

Vascular endothelial cells are a single layer of cell that forms the intima by lining the lumen of a blood vessel. Vascular endothelial cells mediate the transport of necessary components such as nutrients in the blood to the tissue, prevent the unnecessary passage of a large amount of components, the function of circulating blood smoothly without coagulation, Has the action of preventing the presence or absence when the blood vessel is disconnected, the regulating action of keeping the tension of the blood vessel constant, the action of controlling the inflammation by producing factors involved in inflammation, the action of controlling the pathology in the blood vessel, etc. Yes.
Intravascular cells are also important factors in angiogenesis. Angiogenesis occurs in the order of digestion of the basement membrane by proteases produced by vascular endothelial cells, migration / proliferation of vascular endothelial cells, and then lumen formation of vascular endothelial cells. Angiogenesis is thought to be caused by various growth factors, cytokines, arachidonic acid metabolites, monobutyrin and the like. Known growth factors that cause angiogenesis include fibroblast growth factor, tumor growth factor, vascular endothelial growth factor (VEGF), and vascular permeability factor. Among them, VEGF is considered to be the most important angiogenic factor because the receptor exists only in the vascular endothelium and acts selectively on the vascular endothelium.
In addition, angiogenesis is a phenomenon in which blood vessels newly grow and is not only involved in the formation of the circulatory system and tissue formation in the vertebrate embryonic stage, but also in the luteinization and placenta formation in the sexual cycle of mature individuals. And so on. Thus, angiogenesis occurs in a healthy state and plays an important role in tissue recovery. However, in many chronic diseases including diabetes, it is also known that the number of capillaries increases to cause serious damage to tissues.
Diseases involving angiogenesis include macular degeneration, malignant tumor growth and metastasis, diabetic retinopathy, neovascular glaucoma, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis and the like.
For example, macular degeneration is a disease that produces new blood vessels from the choroid. Choroidal neovascularization occurs mostly in the center of the fundus called the macular region, and the choroidal neovascularization has the property of easily leaking water and bleeding, so water under the retina and retinal pigment epithelium. And blood accumulates. HYPERLINK "http://health.goo.ne.jp/medical/search/10A70500.html" if it accumulates under the retina, causes retinal pigment epithelium detachment if it accumulates under the retinal pigment epithelium The function of the retina is impaired. As a result, things appear distorted (metastasis), things appear small (microvision), the center is difficult to see (central dark spot), etc., appearing as early symptoms, and in many cases, visual acuity gradually decreases To do. In addition, sudden loss of visual acuity may occur when sudden bleeding occurs suddenly.
When a malignant tumor grows, the tumor cell itself induces angiogenesis by an angiogenesis promoting factor in order to obtain nutrients and oxygen necessary for the growth of the tumor cell. When a malignant tumor metastasizes to another organ or site, angiogenesis is induced, and tumor cells move along the bloodstream. In the case of diabetic retinopathy, in the case of diabetic retinopathy, capillaries are clogged by viscous blood from diabetes, causing bleeding and edema in the retina, which becomes chronic and causes oxygen and nutrient deficiencies in the retina. Therefore, new blood vessels are generated on the retina and nervous system papillae, and a fibrous tissue is formed around them. This fiber tissue pulls up the retina (retinal detachment), tears the blood vessels of the retina, causes bleeding (vitreous hemorrhage), and eventually leads to severe visual impairment and blindness.
As described above, angiogenesis caused by proliferation of vascular endothelial cells is deeply involved in the onset and progression of various diseases. Therefore, for the purpose of treating these diseases, substances that inhibit vascular endothelial cell proliferation and / or angiogenesis. Many searches have been conducted so far. Examples of substances and drugs having an action of inhibiting vascular endothelial cell proliferation and / or angiogenesis include sulfated polysaccharides (Patent Document 1), ascorbic acid ethers and related compounds (Patent Document 2), and thiazole derivatives (Patent Document 3). Shark cartilage extract (chondroitin and mucopolysaccharide) (Patent Document 4). However, until now, substances proposed as substances that inhibit the action of inhibiting vascular endothelial cell proliferation and / or angiogenesis have not been able to exert practically satisfactory effects. For this reason, it has been required to develop a highly safe substance that more strongly inhibits vascular endothelial cell proliferation and / or angiogenesis.
As a method for treating cancer, which is one of diseases associated with vascular endothelial cell proliferation and / or angiogenesis, surgical methods, radiation therapy, chemotherapy (drug administration) and the like are generally performed. Among these, as chemotherapy, treatment methods that administer drugs that act directly on tumor cells and kill tumor cells are widely applied, and proposals for antitumor agents to be applied to such treatment methods are Many. However, since such a drug kills tumor cells and also acts on normal cells, it has a high therapeutic effect on cancer, but has a drawback that side effects are very strong. In addition, even if such a drug works on a cell-by-cell basis, it does not reach the deep part of the tumor mass, so it is not a fundamental solution. Furthermore, cancers have different properties depending on their organs, and it is not uncommon for the selection and administration methods of antitumor agents to differ, and antitumor agents that are effective against various types of cancers are desired.
Here, many phosphatidylinositols have been reported to be contained in soybean lecithin and deeply involved in cell signal transmission. In recent years, it has been reported that intake of phosphatidylinositol decreases the triacylglycerol (TG) concentration in blood and increases the HDL-C (high density lipoprotein cholesterol) concentration (Non-patent Document 1).
However, there is no report about the vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor as described above using phosphatidylinositol.
JP-A-63-119500 Japanese Patent Laid-Open No. 58-131978 Japanese Examined Patent Publication No. 6-62413 JP-A-10-147534 Jim W. Burgess et al., Journal of Lipid Research (46) 350-355

  An object of the present invention is to provide a new drug that inhibits vascular endothelial cell proliferation and / or angiogenesis. It is another object of the present invention to provide a therapeutic agent for diseases associated with vascular endothelial cell proliferation and / or angiogenesis.

As a result of various studies on phosphatidylinositol, the present inventors have found that phosphatidylinositol has a vascular endothelial cell growth inhibitory activity and / or angiogenesis inhibitory activity, and has completed the present invention.
That is, the present invention
(1) A vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor containing phosphatidylinositol as an active ingredient,
(2) The vascular endothelial cell growth inhibitor and / or the angiogenesis inhibitor according to the above (1), wherein the phosphatidylinositol is a phosphatidylinositol having a purity in a total phospholipid of 50 mol% or more,
(3) a functional food having a vascular endothelial cell growth inhibitory activity and / or angiogenesis inhibitory activity, comprising phosphatidylinositol as an active ingredient,
(4) The functional food according to (3) above, wherein the phosphatidylinositol is phosphatidylinositol having a purity of 50 mol% or more in the total phospholipids,
(5) Use of phosphatidylinositol for preparing a composition having an inhibitory effect on vascular endothelial cell proliferation and / or angiogenesis.
(6) A method for inhibiting vascular endothelial cell proliferation and / or angiogenesis, which comprises ingesting and / or applying phosphatidylinositol,
(7) Cosmetics having an effect of improving macular degeneration, containing phosphatidylinositol as an active ingredient,
(8) The vascular endothelial cell proliferation according to (1) or (2) above, wherein the phosphatidylinositol is obtained by allowing an enzyme derived from Candida having phospholipase B activity to act on soybean-derived phospholipid. Inhibitors and / or angiogenesis inhibitors.
About.
Furthermore, the present invention provides
(9) A medicine containing phosphatidylinositol as an active ingredient,
(10) A therapeutic agent for a disease associated with vascular endothelial cell proliferation and / or angiogenesis, comprising phosphatidylinositol as an active ingredient,
(11) A therapeutic agent for macular degeneration, increase or metastasis of malignant tumor, diabetic retinopathy, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or osteoarthritis, comprising phosphatidylinositol as an active ingredient,
About.

ADVANTAGE OF THE INVENTION According to this invention, the new vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor which contain phosphatidylinositol as an active ingredient are provided. This vascular endothelial cell growth inhibitor and / or a drug containing an angiogenesis inhibitor as an active ingredient is used as a therapeutic agent for various diseases associated with angiogenesis such as macular degeneration, tumor growth, rheumatoid arthritis, diabetic retinopathy and the like. be able to. Furthermore, since the vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor or therapeutic agent of the present invention has low toxicity, it is highly safe.
In addition, functional foods and cosmetics having a vascular endothelial cell growth inhibitory action and / or angiogenesis inhibitory action containing phosphatidylinositol as an active ingredient, and further a composition having a vascular endothelial cell growth inhibitory action and / or angiogenesis inhibitory action The use of phosphatidylinositol to prepare a product, and the method of inhibiting vascular endothelial cell proliferation and / or angiogenesis by ingesting and / or applying phosphatidylinositol can be provided.

The phosphatidylinositol used in the present invention is not limited in its origin and production method as long as it is a phosphatidylinositol that can be taken or edible as a medicine or food, but from the soybean phospholipid to the hot ethanol method (Japanese Patent Laid-Open No. 2000-300186) ) Or those produced using phospholipase B that does not substantially act on phosphatidylinositol from soybean phospholipids.
Specific examples of such phospholipase B (PLB) include the following (1) to (4).
(1) An enzyme derived from the genus Candida having PLB activity and having the following physicochemical properties.
1) Action: Action of hydrolyzing phospholipids into free molar ratio of 2 fatty acids and glyceryl phosphorylcholine in equimolar ratio 2) Molecular weight: 53,000 ± 3,000 (by SDS electrophoresis)
3) Isoelectric point: pH 4.21 ± 0.2
4) Optimal pH: pH around 5.5 to 6.5 5) pH stability: around pH 5 to 9 (treated at 37 ° C. for 90 minutes)
6) Stability: 55 ° C. (treated at pH 5 for 10 minutes)
7) Substrate specificity: 10% or less of the activity ratio to phosphatidylinositol in the case of phosphatidylcholine (2) Phospholipase B containing enzyme fraction A or enzyme fraction B obtained by the following steps.
1) Step of culturing Candida cylindrasse (Rugosa) 2) Step of concentrating the culture solution of Candida cylindrasse (Rugosa) 3) Step of precipitating enzyme with organic solvent 4) Crude enzyme obtained by step of 3) Step 5) Purifying the solution by hydrophobic chromatography Step 5) Separating and purifying the enzyme obtained in step 4) into enzyme fraction A or enzyme fraction B by ion exchange chromatography (3) Amino acid sequence of SEQ ID NO: 1 Phospholipase B having
(4) Phospholipase B having the amino acid sequence of SEQ ID NO: 2 in the sequence listing.

  The use of phosphatidylinositol in the present invention is not particularly limited as long as it is used for preparing the composition for inhibiting vascular endothelial growth and / or inhibiting angiogenesis of the present invention. Examples of the composition for inhibiting vascular endothelial growth and / or inhibiting angiogenesis of the present invention include a drug containing phosphatidylinositol as an active ingredient; a vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor containing phosphatidylinositol as an active ingredient A therapeutic agent for vascular endothelial cell proliferation and / or angiogenesis-related disease containing phosphatidylinositol as an active ingredient; a vascular endothelial cell proliferation inhibitory action and / or angiogenesis inhibitory action containing phosphatidylinositol as an active ingredient Food: Cosmetics containing phosphatidylinositol as an active ingredient and having an inhibitory effect on vascular endothelial cell proliferation and / or angiogenesis, and the like. Furthermore, it may be a functional dietary supplement having a vascular endothelial cell growth inhibitory action and / or angiogenesis inhibitory action containing phosphatidylinositol as an active ingredient.

  In the present invention, the content of phosphatidylinositol in a pharmaceutical or functional food is not limited as long as it is an amount that can inhibit vascular endothelial cell growth inhibition and / or angiogenesis inhibition. The lower limit of inositol intake is preferably 1 mg or more, more preferably 10 mg or more, further preferably 30 mg or more, particularly preferably 50 mg or more, and particularly preferably 100 mg or more. Moreover, as an upper limit, 50000 mg or less is preferable, 10000 mg or less is more preferable, 1000 mg or less is further more preferable, 500 mg or less is especially preferable, 200 mg or less is very especially preferable.

  The therapeutic agent for diseases associated with vascular endothelial cell proliferation and / or angiogenesis of the present invention contains phosphatidylinositol as an active ingredient in the same manner as the vascular endothelial cell proliferation inhibitor and / or angiogenesis inhibitor of the present invention described above. . Diseases associated with vascular endothelial cell proliferation and / or angiogenesis include macular degeneration, malignant tumor growth and metastasis, diabetic retinopathy, neovascular glaucoma, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis, etc. It is done. The medicament of the present invention is useful for the treatment of these diseases, and particularly useful for the treatment of macular degeneration with vascular endothelial cell proliferation and / or angiogenesis, especially age-related macular degeneration.

  The pharmaceutical administration route of the present invention may be either oral administration or parenteral administration. The dosage form is appropriately selected depending on the administration route. For example, injections, infusions, powders, granules, tablets (including chewable tablets, quick-disintegrating tablets), soft capsules, hard capsules, pills, enteric solvents, troches, liquids for internal use, suspensions, emulsions, syrups, Liquid preparations for external use, poultices, nasal drops, ear drops, eye drops, inhalants, ointments, creams, lotions, gels, adhesives, suppositories, enteral nutrients and the like can be mentioned. This can be used alone or in combination depending on the symptoms. In these preparations, auxiliary agents usually used in the pharmaceutical preparation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and corrigents are used as necessary. .

  These preparations are prepared according to conventional methods. However, since phosphatidylinositol is poorly soluble in water, it can be dissolved in non-hydrophilic organic solvents such as vegetable oils and animal oils, or emulsifiers, dispersants or surfactants. Along with the agent and the like, a homogenizer (high-pressure homogenizer) may be used by dispersing and emulsifying in an aqueous solution. Further, in order to enhance the absorption of phosphatidylinositol, it is possible to use or eliminate the excipient (gum arabic, dextrin, casein, etc.), and use the finely divided average particle system to about 1 micron. .

  Additives that can be used for formulation include animal oils such as soybean oil, safflower oil, olive oil, germ oil, sunflower oil, beef tallow, sardine oil, polyethylene glycol, propylene glycol, glycerin, sorbitol, etc. Surfactant such as polyhydric alcohol, sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, purified water, lactose, starch, crystalline cellulose, D-mannitol, maltose, lecithin, gum arabic, dextrin, Examples include excipients such as sorbitol liquid and sugar liquid, sweeteners, colorants, pH adjusters, and fragrances. The liquid preparation may be dissolved or suspended in water or other appropriate medium when taken. Tablets and granules may be coated by a known method.

  When administered in the form of an injection, it can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, transdermally, intraarticularly, within the bursa, intravesicular, intraperiosteally, sublingually, orally. In particular, intravenous administration or intraperitoneal administration is preferred. Intravenous administration may be either drip administration or porous administration.

Examples of functional foods having vascular endothelial cell growth inhibitory action and / or angiogenesis inhibitory action containing the phosphatidylinositol of the present invention as active ingredients include phosphatidylinositol-containing supplements (dispersed, granule, soft capsule, hard capsule, tablet, chewable, etc. Tablets, quick-disintegrating tablets, syrups, liquids, etc.), phosphatidylinositol-containing beverages (tea, carbonated beverages, lactic acid beverages, sports beverages, etc.), phosphatidylinositol-containing confectionery (gummy, jelly, gum, chocolate, cookies, candy, etc.), phosphatidyl Inositol-containing oil, phosphatidylinositol-containing oil and fat food (mayonnaise, dressing, butter, cream, margarine, etc.), phosphatidylinositol-containing ketchup, phosphatidylinositol-containing sauce , Phosphatidylinositol-containing liquid food, phosphatidylinositol-containing dairy products (milk, yogurt, cheese, etc.), phosphatidylinositol-containing breads, phosphatidylinositol-containing noodles (udon, soba, ramen, pasta, yakisoba, kishimen, somen, hiyamugi, Beef noodles), phosphatidylinositol-containing soups (miso soup, corn soup, consomme soup, etc.), phosphatidylinositol-containing sprinkles and the like.
In addition, the functional food as used in the field of this invention is a health functional food, and is the food which satisfy | filled the standard standards etc. which the country set in consideration of safety | security, effectiveness, etc. among health foods. It is permitted to display health functions and nutritional functions, and is classified into “special health foods” and “nutrient functional foods” depending on the purpose and function of the food.

  Examples of cosmetics having a vascular endothelial cell growth inhibitory action and / or angiogenesis inhibitory action containing the phosphatidylinositol of the present invention as an active ingredient include, for example, cosmetics for improving macular degeneration, particularly age-related macular degeneration, etc. Is mentioned. The form is not particularly limited, and examples thereof include creams, emulsions, lotions, microemulsion essences, haptic agents, bathing agents, and the like.

  The composition for inhibiting vascular endothelial growth and / or inhibiting angiogenesis of the present invention includes various nutrients and various vitamins as necessary (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D). , Vitamin E, vitamin K, etc.), various minerals (magnesium, zinc, iron, sodium, potassium, selenium, titanium oxide, etc.), dietary fiber, various sugars (cellulose, dextrin, chitin, etc.), various polyunsaturated fatty acids (Arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, etc.), various conjugated fatty acids (conjugated linoleic acid, conjugated linolenic acid, conjugated arachidonic acid, conjugated DHA, conjugated EPA, conjugated DPA, etc.), various phospholipids ( Lecithin, phosphatidic acid, phosphatidylserine, phosphatidylethanol Amine, phosphatidylglycerol, phosphatidylcholine, phosphatidyl DHA, etc.), various glycolipids (such as cerebroside), various carotenoids (β-carotene, lycopene, astaxanthin, β-cryptoxanthin, capsanthin, lutein, zeaxanthin, etc.), various flavonoids ( Quercetin, luteolin, isoflavone, etc.), various amino acids (glycine, serine, alanine, glutamine, valine, leucine, isoleucine, lysine, arginine, aspartic acid, glutamic acid, tryptophan, phenylalanine, histidine, proline, methionine, cysteine, etc.) Various nutrients (reduced coenzyme Q10, carnitine, sesamin, α-lipoic acid, inositol, D-kaileunositol, pinitol, taurine Glucosamine, chondroitin sulfate, S-adenosylmethionine, curcumin, γ-oryzanol, glutathione, γ-aminobutyric acid, synephrine, pyrroloquinoline quinone, catechin, capsaicin, etc.), various dispersants, stabilizers such as various emulsifiers, various sweet tastes The ingredients (sorbitol, sucrose, etc.), various flavoring ingredients (citric acid, malic acid, etc.), flavor, royal jelly, honey, beeswax, propolis, agaricus, ginseng, bioperine, etc. can be blended. Moreover, you may mix | blend herbs, such as peppermint, bergamot, camomile meal, and lavender. Moreover, you may mix | blend raw materials, such as theanine, a dehydroepiandosterone, and melatonin.

  The vascular endothelial growth inhibitory activity and angiogenesis inhibitory activity of the present invention can be measured by co-culturing human umbilical vein endothelial cells and fibroblasts and analyzing the effect of the composition on lumen formation using angiogenesis quantification software. . For example, it is carried out by culturing human vascular endothelial cells using a commercially available angiogenesis kit and observing the effect of the composition on the formation of lumens. Other examples include the Madrigel method.

  Although this invention is demonstrated based on a reference example, a formulation example, a foodstuff manufacture example, a cosmetics manufacture example, and an Example, this invention is not limited only to a following example.

[Reference Example 1] Method for producing phospholipase B used for the production of phosphatidylinositol A 30 liter jar fermenter was charged with 3% cotton wool flour, 0.3% sodium chloride, 0.2% dipotassium phosphate, 0.2 Candida cylindrasse ATCC 14830 strain cultured in 20 liters of sterilized medium consisting of 1% potassium phosphate, 0.1% magnesium sulfate and 0.3% antifoaming agent in the same medium (28 ° C, 4 days) 100 ml of the solution was inoculated aseptically. Cultivation was performed at 28 ° C. with aeration of 20 liters of sterile air for 1 minute while stirring at 300 rpm for 1 minute. The maximum activity (0.2 U / ml) of PLB after 50 hours was confirmed.
The obtained culture broth was centrifuged at 5000 rpm for 10 minutes to obtain 16 liters of supernatant. The supernatant was concentrated by ultrafiltration. The precipitate formed by adding 9 liters of cold acetone to 3 liters of the resulting concentrated solution was centrifuged at 5000 rpm for 10 minutes to collect the precipitates, and 10 mM Tris-HCl buffer solution (hereinafter referred to as 2M sodium chloride) , Abbreviated as Tris-HCl) (pH 7.5). Insoluble matter was removed by centrifugation, and PLB was adsorbed through octyl sepharose (bed volume; 300 ml) packed in advance in a column. Next, the column was washed with 10 mM Tris-HCl (pH 7.5) containing 2 M sodium chloride and 10 mM Tris-HCl (pH 7.5). PLB adsorbed on the column was eluted with 10 mM Tris-HCl (pH 7.5) containing 2% adekatol SO120 (purification by hydrophobic chromatography). Next, the eluate (1 liter) was passed through a DEAE-Sepharose column (bed volume; 200 ml) pre-equilibrated with 10 mM Tris-HCl (pH 7.5) to adsorb PLB, and the column was washed with the above buffer solution. The enzyme was eluted with a 0-0.3M sodium chloride gradient. PLB enzyme fraction A was obtained when sodium chloride was about 0.1 M, and PLB enzyme fraction B was obtained when sodium chloride was about 0.25 M (separation and purification by ion exchange chromatography).
When the N-terminal amino acid sequences of enzyme fraction A and enzyme fraction B were specified by an amino acid sequencer, the amino acid sequence of enzyme fraction A was the sequence shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence was found to be the sequence shown in SEQ ID NO: 2 in the sequence listing. Furthermore, the homology analysis with GENEX WIN 5.2 (manufactured by Software Co., Ltd.) analyzed the homology of enzyme fraction B with enzyme fraction A. As a result, it was found to be 88.5%.

[Reference Example 2] Method for measuring PLB activity The enzyme activity of PLB can be confirmed by measuring GPC produced from lecithin by the enzyme method. That is, 1 M MES-NaOH buffer (hereinafter abbreviated as MES-NaOH) (pH 6) 0.05 ml, 10 mM egg yolk phosphatidylcholine dissolved in 3% Triton X100 0.05 ml, 1 M calcium chloride 0.025 ml, 0.2% TODB0 .05 ml, 0.2% 4-aminoantipyrine 0.05 ml, 50 U / ml monoglyceride lipase 0.1 ml, 300 U / ml glycerophosphate oxidase 0.1 ml, 6 U / ml GPC phosphodiesterase 0.025 ml, 100 U / ml peroxidase PLB diluted with 10 mM Tris-HCl (pH 7.5) containing 0.05% BSA and diluted with the same buffer after pre-heating 0.5 ml of the reaction solution consisting of 0.05 ml at 37 ° C. for 2 to 3 minutes 25 μl of solution (0.03-0.15 U / ml) The reaction was started by adding 10 ml, and exactly 10 minutes later, 1 ml of 0.5% SDS was added to stop the reaction, and the absorbance at 550 nm was measured.
In addition, the activity which releases 1 micromol GPC in 1 minute was made into 1 unit.

[Reference Example 3] Method for measuring lipase activity The lipase activity was measured by converting a monoglyceride produced using diglyceride as a substrate into glycerin with a monoglyceride lipase and then measuring by an enzymatic method. Namely, 0.1 ml of 1M MES-NaOH (pH 6.0), 0.05 ml of 10 mM 1,2 diglyceride dissolved in 3% Triton X100, 0.025 ml of 0.05 M calcium chloride, 0.025 ml of 0.05 M magnesium chloride, 0.05 MATP0 .05 ml, 10 U / ml monoglyceride lipase 0.05 ml, 5 U / ml glycerokinase 0.05 ml, 400 U / ml glycerophosphate oxidase 0.025 ml, 100 U / ml peroxidase 0.025 ml, 0.3% TOOS 0.025 ml, 0. 25 μl of an enzyme solution (0.03 to 0.15 U / ml) diluted with 10 mM Tris-HCl containing 0.05% BSA was added to 0.5 ml of a reaction solution consisting of 0.025 ml of 3% 4-aminoantipyrine to carry out the reaction. After 10 minutes at 37 ° C The reaction was stopped with 0.5% SDS and the absorbance at 550 nm was measured. One unit of lipase activity was defined as the activity of releasing 1 micromole of glycerin per minute.

[Reference Example 4] Purification of PLB Fraction A by Gel Filtration Chromatography The PLB active enzyme fraction A obtained in Reference Example 1 was concentrated using a centrifugal ultrafiltration apparatus. The concentrated solution was subjected to gel filtration chromatography (Superdex 75) pre-equilibrated with 10 mM Tris-HCl (pH 7.5) containing 0.5 M sodium chloride, and the active fraction was separated at a flow rate of 0.5 ml per minute. Obtained.
In each fraction, PLB activity and lipase activity were determined by the methods of Reference Examples 2 and 3, respectively, and the protein concentration was measured by absorbance at 280 nm. The results of PLB activity, lipase activity, and protein concentration in each fraction are shown in FIG.
As a result, an elution pattern in which the protein concentration measured at 280 nm, the lipase activity, and the PLB activity coincided was obtained, and it was found that the proteins having the lipase activity and the PLB activity were the same enzyme protein.

[Reference Example 5] Various properties of PLB 5-1 (optimum pH for reaction)
Among the reaction solution compositions shown in Reference Example 2, acetate buffer (hereinafter abbreviated as Acetate), phosphatidylinositol PES-NaOH buffer (hereinafter abbreviated as phosphatidylinositol PES-NaOH), or MES-NaOH as a buffer solution. The enzyme activity of enzyme fraction A at each pH was measured, and the relative activity in each buffer relative to the activity when MES-NaOH pH 6.0 was used was determined. The results are shown in FIG.
As shown in FIG. 2, it was expected that MES-NaOH showed the maximum enzyme activity around pH 6, and phosphatidylinositol PES-NaOH and Acetate also showed the maximum enzyme activity around pH 6.

5-2 (pH stability)
The enzyme fraction A was dissolved at 10 ° C. in various buffer solutions of 10 mM of Acetate, MES-NaOH, Tris-HCl, or Bicine-NaOH buffer (hereinafter abbreviated as Bicine-NaOH) at 37 ° C. Then, the PLB activity was measured based on the method of Reference Example 2, and the relative residual activity at various pHs relative to the activity at Acetate pH 5 was determined. The results are shown in FIG.
As shown in FIG. 3, it was found to be stable in a wide range from pH 5 to pH 8.

5-3 (Thermal stability)
Enzyme fraction A is dissolved in 10 mM MES-NaOH (pH 6.0) so as to be 5 U / ml, and the PLB activity is measured after heating for 10 minutes in the temperature range of 0 to 70 ° C. by the method of Reference Example 2. did. The results are shown in FIG. The relative residual activity when the PLB activity of the enzyme fraction A stored refrigerated was 100% was 90% or more after the treatment up to 55 ° C., and it was found that the enzyme was stable to heat.

5-4 (Substrate specificity)
According to the PLB activity measurement method described in Reference Example 2, PLB (enzyme fraction A and enzyme fraction B) as two types of phospholipid processing agents obtained by the ion exchange chromatography of Reference Example 1 were used for various phosphorus to PC. Relative activity for lipids was measured. The results are shown in Table 1. As a result, the relative activity of phosphatidylinositol for PC in both enzyme fraction A and enzyme fraction B is lower than that of other phospholipids, and the phospholipid processing agent has a low substrate-specific activity in phosphatidylinositol among phospholipids. It turns out that there is sex. From the results in Table 1, it can be seen that a mixture of both fractions can also be used as the phospholipid processing agent since both fractions have PLB activity.

5-5 (Molecular weight)
In purified enzyme fraction A obtained by the method described in Reference Example 1, a single band was obtained by SDS polyacrylamide electrophoresis. The molecular weight obtained using a protein with a known molecular weight as a marker was 53 kilodaltons.

5-6 (Isoelectric point)
For the purified enzyme fraction A obtained by the method described in Reference Example 1, the isoelectric point for enzyme fraction A was measured by isoelectric focusing performed by preparing a pH gradient using carrier ampholite. Further, the protein concentration was measured by absorbance at 280 nm. The relationship between the isoelectric point of enzyme fraction A and the enzyme concentration is shown in FIG. As a result, the pH measured by the absorbance at 280 nm, the lipase activity and the PLB activity completely coincided, and the pH value (isoelectric point) was 4.21. As can be seen from FIG. 5, PLB, lipase, and protein showed exactly the same peak, and it became clear that PLB and lipase are the same enzyme protein.

[Reference Example 6] Quantification method of phosphatidylinositol 1M Tris-HCl (pH 8) 0.1 ml, 10 mM NAD 0.05 ml, 10 mM magnesium chloride 0.05 ml, 2% Triton X100 0.05 ml, 50 U / ml, phosphatidylinositol specific To 0.5 ml of coloring solution composed of 0.05 ml of phospholipase C, 0.05 ml of 50 U / ml alkaline phosphatase, 0.05 ml of 50 U / ml inositol dehydrogenase, 0.05 ml of 5% nitrotetrazolium blue, and 0.15 ml of purified water Then, 0.02 ml of a phosphatidylinositol solution prepared by dissolving a test substance (1 to 3 mg / ml) containing phosphatidylinositol in 2% Triton X100 was added, reacted at 37 ° C. for 10 minutes, and 0.5% SDS0. 5 Quenched with l, the absorbance was measured at 550nm. The amount of phosphatidylinositol was quantified using a known concentration of inositol aqueous solution as a calibrator.

[Reference Example 7] Method for producing phosphatidylinositol 40 grams of soybean-derived phospholipid was stirred and suspended in 400 ml of 10 mM Acetate (pH 5.8) and then 1400 units of PLB (enzyme fraction A obtained in Reference Example 1). The reaction was started at 45 ° C. After 20 hours, the reaction was stopped and 200 ml of ethanol and 400 ml of hexane were added and stirred for 1 hour. The hexane layer and the aqueous layer were separated by centrifugation, and the organic solvent layer was recovered. The collected organic solvent was concentrated under reduced pressure, 150 ml of acetone was added to the concentrate, and the resulting precipitate was collected and dried to obtain a powder containing 8.2 grams of phosphatidylinositol at a high concentration. As a result of measuring the purity of the obtained phospholipid by a standard oil analysis method (established by the Japan Oil Chemists' Society, 1996), it was 86.8 mol% in the total phospholipid.

[Food Production Example 1] Functional Margarine Containing Phosphatidylinositol (PI) After adding PI obtained in Reference Example 7 to vegetable oil so as to be 5% by weight of margarine, it is stirred uniformly with an emulsifier and the like. Margarine was prepared by a conventional method.

[Food Production Example 2] Functional bread containing phosphatidylinositol (PI) 1 g of PI obtained in Reference Example 7, 15 g of sugar, 2 g of salt and 5 g of fat powdered milk are dissolved in 70 g of hot water, and 2 chicken eggs are added and mixed well. It was. This was added to a mixture of 130 g of wheat flour and 2 g of dry yeast, kneaded by hand, then kneaded with about 30 g of butter to make 30 roll bread dough. Next, after fermenting, a beaten egg was applied to the surface and baked in an oven at 180 ° C. for 15 minutes to obtain a roll.

[Food Production Example 3] Functional Udon containing Phosphatidylinositol (PI) To 400 g of wheat flour, 200 g of water, 2 g of PI obtained in Reference Example 7 and 20 g of sodium chloride were added, and the kneaded well. Thereafter, the dough was stretched and cut into widths of about 6 mm to produce udon.

[Food Production Example 4] Functional beverage containing phosphatidylinositol (PI) 30 g of PI obtained in Reference Example 7 was suspended in 5 times the amount of olive oil and heated to 50 ° C. to obtain an oil phase. 10 g of glycerol fatty acid ester as an emulsifier was added to 90 g of glycerin and heated to 70 ° C. to dissolve. The previous oil phase was gradually added to this solution with stirring. The mixed solution was subjected to high-pressure emulsification using an emulsifier to obtain an emulsified composition. 180 ml of water was added to 20 g of this emulsified composition and stirred to obtain a PI-containing beverage.

[Formulation Example 1] Phosphatidylinositol (PI) -containing tablet PI 120 g obtained in Reference Example 7
Crystalline cellulose 330g
Carmellose-calcium 15g
Hydroxypropylcellulose 10g
60 ml of purified water

The above composition was blended and dried by a conventional method, 10 g of magnesium stearate was added, and tableting was performed to obtain 100 mg tablets containing 20 mg of PI per tablet.

[Formulation Example 2] Phosphatidylinositol (PI) -containing soft capsule The PI obtained in Reference Example 7 was suspended in 5 times the amount of olive oil, mixed well to be homogeneous, and then filled with a capsule filling machine. A capsule with a content of about 300 mg was obtained.

[Cosmetics production example] Phosphatidylinositol (PI) -containing cream (functional cosmetics)
The PI obtained in Reference Example 7 was added to white petrolatum so as to be 10% by weight and stirred uniformly with a fragrance and the like to prepare a cream by a normal method.

Example 1 Vascular Endothelial Cell Growth Inhibition and / or Angiogenesis Inhibitory Activity Angiogenesis inhibition experiments were performed as follows using an angiogenesis kit (Angiogenesis Kit, manufactured by Kurabo Industries).
Human vascular endothelial cells and fibroblasts are co-cultured at an optimal concentration, and samples in the growth stage at the initial stage of tube formation are used as samples to obtain a PI concentration of 3 μg / ml, 10 μg / ml, 30 μg / ml. After adding to the wells and culturing for 11 days (change the medium containing the sample after 4, 7, and 9 days), tube formation was stained with Mouse anti-human CD31 and Goat anti-mouse IgG AlkP Conjugate, It was observed with a microscope. After taking five micrographs near the center of each well, these results were quantitatively evaluated using angiogenesis quantification software (Angiogenesis Image Analyzer, Kurabo Industries). As a control, one without PI was used. Evaluation is 4 of Tube area (total blood vessel area), Length (total length of lumen network), Path (number of branches forming path network), Joint (branch point forming number of joint (number of joints)) The relative value (%) with respect to the control value was determined for each item, and the results are shown in Fig. 6, 7, 8, and 9. As is clear from these figures, PI is vascular endothelial cell proliferation depending on the concentration. And / or inhibited angiogenesis.

[Example 2]
This effect can be examined by administering the composition of the present invention to an age-related macular degeneration patient and examining the macular before and after administration.

  According to the present invention, a vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor containing phosphatidylinositol as an active ingredient is provided. This vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor is useful for the treatment of various diseases associated with angiogenesis such as age-related macular degeneration, tumor growth, rheumatoid arthritis, diabetic retinopathy and the like.

The chromatographic pattern by gel filtration chromatography of PLB as a phospholipid processing agent of the present invention based on Reference Example 4 is shown. The optimal pH of PLB as a phospholipid processing agent of the present invention based on Reference Example 5 is shown. The result of pH stability of PLB as a phospholipid processing agent of the present invention based on Reference Example 5 is shown. The result of the thermal stability of PLB as a phospholipid processing agent of the present invention based on Reference Example 5 is shown. The result of the isoelectric focusing of PLB as a phospholipid processing agent of the present invention based on Reference Example 5 is shown. The influence which PI has on Tube area (total lumen area) is shown. The effect of PI on Length (total length of luminal network) is shown. The effect of PI on Joint (the number of branch points (joints) forming a lumen network) is shown. The influence of PI on Path (the number of branches (paths) forming a lumen network) is shown.

Claims (8)

  A vascular endothelial cell growth inhibitor and / or angiogenesis inhibitor comprising phosphatidylinositol as an active ingredient.   The vascular endothelial cell proliferation inhibitory action and / or vascular endothelial cell proliferation inhibitor and / or angiogenesis inhibitor according to claim 1, wherein the phosphatidylinositol is phosphatidylinositol having a purity of 50 mol% or more in the total phospholipid.   A functional food having vascular endothelial cell growth inhibitory activity and / or angiogenesis inhibitory activity, comprising phosphatidylinositol as an active ingredient.   The functional food according to claim 3, wherein the phosphatidylinositol is phosphatidylinositol having a purity of 50 mol% or more in the total phospholipid.   Use of phosphatidylinositol for preparing a composition having an inhibitory effect on vascular endothelial cell proliferation and / or angiogenesis.   A method for inhibiting vascular endothelial cell proliferation and / or angiogenesis, comprising administering phosphatidylinositol.   A cosmetic comprising phosphatidylinositol as an active ingredient and having an effect of improving macular degeneration.   The vascular endothelial cell growth inhibitor and / or blood vessel according to claim 1 or 2, wherein the phosphatidylinositol is obtained by allowing an enzyme derived from Candida having phospholipase B activity to act on soybean-derived phospholipid. Angiogenesis inhibitor.