JP2013133311A - Neovascularization inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a neovascularization inhibitor containing melanoidin as an active ingredient, which is contained in a fermented essence obtained by the symbiotic fermentation of a natural raw material with lactic acid bacteria and yeast.SOLUTION: The neovascularization inhibitor includes the melanoidin as an active ingredient, which is contained in the fermented essence of polysaccharides, vegetables and fruits, and seaweeds with lactic acid bacteria and yeast. The melanoidin contained in the fermented essence, obtained by fermenting brown rice as the polysaccharides, papaya as the vegetables and fruits, and sea tangle and Nemacystus decipiens as the seaweeds for six months with the lactic acid bacteria and the yeast, and removing the microbial cells, has the neovascularization inhibiting effect of 1.5 times as high as that of suramin.

Description

  The present invention relates to an angiogenesis inhibitor containing melanoidin as an active ingredient.

  Angiogenesis is a phenomenon observed under normal physiological conditions such as luteinization, ovulation, embryogenesis, placenta formation, and wound healing. It is limited to.

  The mechanism of angiogenesis is considered as follows if it is described in terms of malignant tumors. First, vascular endothelial growth factor (hereinafter simply referred to as VEGF) is produced from malignant tumor cells and binds to the VEGF receptor on the endothelial cell membrane. This binding stimulates the cells to release matrix metalloprotease (hereinafter referred to as MMP), which is a type of proteolytic enzyme. MMP degrades and destroys the basement membrane and extracellular matrix of blood vessels and enhances vascular permeability, whereby vascular endothelial cells migrate, proliferate, and adhere to the extracellular matrix. By this adhesion, vascular endothelial cells differentiate to form vascular cavities and generate new linear blood vessels. The periphery of the new blood vessel is supported by smooth muscle cells and blood vessel wall cells and becomes a stable blood vessel.

  In addition to the above-mentioned malignant tumors, diseases that cause angiogenesis or contribute to deterioration of the disease include diabetic retinopathy, neovascular glaucoma, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis, and warts There are obstructive diseases such as arteriosclerosis and myocardial infarction.

  Since angiogenesis is involved in the onset and progression of various diseases such as malignant tumors, various angiogenesis inhibitors have been developed for the purpose of treatment and prevention thereof. For example, there is a composition for inhibiting angiogenesis containing Melissa leaf extract and either Ginkgo biloba extract or ticlopidine (Patent Document 1). Melissa leaf extract is obtained by extracting and concentrating dried Melissa leaf with aqueous alcohol. Melissa leaf extract has an inhibitory activity on the proteolytic activity of MMP, which can inhibit angiogenesis.

  There is also an angiogenesis inhibitor made of a compound having a specific structure called RKB-3384HS-A produced from Aspergillus (Patent Document 2). In the examples, RKB-3384HS-A suppresses the chemotaxis of HUVEC cells induced by VEGF at a concentration of 1-30 μg / ml, thereby inhibiting angiogenesis.

  There is also an angiogenesis inhibitor comprising a compound called RQN-18690A isolated from a culture solution of actinomycetes belonging to the genus Streptomyces (Patent Document 3). In the examples, RQN-18690A inhibits HUVEC cell chemotaxis induced by VEGF at a concentration of 0.08 μg / ml by 50%.

  Furthermore, an angiogenesis inhibitor (Patent Document 4) containing ganoderic acid F having a specific structure obtained from Ganoderma lucidum as an active ingredient, or an angiogenesis inhibitor containing glutamic anhydride extracted from a child of basidiomycetes as an active ingredient There is also (patent document 5). In Examples, Matrigel matrix containing acidic fibroblast growth factor (hereinafter aFGF) added to the right abdomen subcutaneously of a mouse was transplanted with a combination of aFGF and the above components, and the weight of Matrigel after a predetermined period and hemoglobin in Matrigel. The amount is being measured. These compounds show an anti-angiogenic effect due to a small increase in the weight of Matrigel and the amount of hemoglobin.

  On the other hand, microorganisms that are harmless and beneficial to humans, animals, crops and the natural environment are called useful microorganisms, and are photosynthetic bacteria, lactic acid bacteria, yeasts, actinomycetes, filamentous fungi, basidiomycetes, Bacillus subtilis Etc. are known. Photosynthetic bacteria produce antioxidants, amino acids, saccharides, and various physiologically active substances using light as an energy source, and lactic acid and acetic acid produced by lactic acid bacteria can suppress the growth of harmful bacteria because of their strong antibacterial activity. In addition, yeast can enhance the activity of other useful bacteria in addition to changing sugar to alcohol. Furthermore, actinomycetes produce antibacterial substances, and filamentous fungi can degrade and saccharify persistent carbohydrates such as fiber. Fermentation using such useful microorganisms is an essential process for the production of sake, miso, soy sauce, and other fermented foods.

  It is known that soy sauce, miso, and the like, which are one of such fermented foods, contain compounds collectively called melanoidins, which are Maillard reaction products of reducing sugars and amino compounds. Although the production route has not been fully elucidated, the D-glucoside produced from the saccharide becomes an Amadori compound via enaminol, and is produced by condensation polymerization reaction with an amino compound using oson or furfural as an intermediate. It is considered. Melanoidin is an aggregate of various substances because different products can be produced depending on the type of reducing sugar or amino compound.

  Melanoidins are known to have antioxidant activity. Antioxidants can eliminate toxic active oxygen and various free radicals, so protease is added to the miso charging mixture to increase the content of melanoidin with excellent antioxidant power, and to produce miso with high antioxidative activity A technique called “Patent Document 6” has been developed.

  There is also a technique using melanoidin as an interleukin-12 production promoter (Patent Document 7). Since interleukin-12 has a high allergic reaction inhibitory effect, it is said that allergic reaction can be effectively suppressed by promoting production of interleukin-12 by melanoidin. In the examples, D-glucose and ε-amino-N-caproic acid were refluxed in distilled water at 95 ° C. for 4 hours to obtain melanoidin.

Japanese Patent No. 4173733 Japanese Patent No. 3913542 Japanese Patent No. 4452787 Japanese Patent No. 4506079 Japanese Patent No. 4681801 JP 2006-333776 A JP 2005-225833 A

  As described above, malignant tumors, diabetic retinopathy, and the like cause blood vessels to be regenerated as they progress, and angiogenesis contributes to the worsening of the disease state. Therefore, a drug capable of inhibiting angiogenesis is effective not only as a therapeutic agent for such diseases but also as a prophylactic agent. Diseases for which angiogenesis inhibitors are used include diabetic retinopathy, neovascular glaucoma, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis, atherosclerosis, obstructive diseases such as myocardial infarction, etc. There are many chronic diseases over the long term. The angiogenesis inhibitor described in Patent Document 1 uses a ginkgo biloba extract, but ginkgo biloba is not a food. Further, the compounds described in Patent Documents 2 to 4 are not themselves foods. Therefore, it is desired to develop a novel food-derived angiogenesis inhibitor that can be used for the prevention of malignant tumors and disease caused by angiogenesis and diabetic retinopathy.

  On the other hand, melanoidin is a Maillard reaction product of a reducing sugar and an amino compound, and is a fermentation product produced in fermentation processes such as miso and soy sauce. As shown in Patent Document 6, melanoidin obtained from the fermentation product is expected to have an antioxidant effect, and as shown in Patent Document 7, melanoidin produced by heating with a saccharide and an amino acid is expected to promote interleukin-12 production. Has been. However, nothing is known about the anti-angiogenic effect of melanoidin.

  In view of the above-described present situation, an object of the present invention is to provide an angiogenesis inhibitor containing melanoidin produced from food as a raw material as an active ingredient.

  The inventors of the present invention examined the effects of melanoidin in detail, and found that melanoidin extracted from a symbiont fermentation product of yeast and lactic acid bacteria has an angiogenesis inhibitory effect, and completed the present invention. It is not known that brown melanoidin is produced without heating due to the co-fermentation of lactic acid bacteria and yeast, and such an excellent angiogenesis inhibitory effect is not known at all.

  That is, the present invention provides an angiogenesis inhibitor containing melanoidin as an active ingredient.

  The present invention also provides the angiogenesis inhibitor, wherein the melanoidin is an extract from a fermented extract obtained by co-fermenting lactic acid bacteria and yeast.

  The present invention also provides the angiogenesis inhibitor, wherein the melanoidin is obtained by subjecting a polysaccharide, a vegetable / fruit, and a seaweed to the co-fermentation.

  Moreover, this invention provides the said antiangiogenic agent whose said vegetables and fruits are papayas.

  The present invention also provides the angiogenesis inhibitor as described above, wherein the polysaccharide is rice, rice bran and / or brown rice.

  According to the present invention, an angiogenesis inhibitor containing melanoidin as an active ingredient is provided.

It is a figure which shows the chromatogram of the gel filtration HPLC of the methanol elution fraction of the styrene-type synthetic adsorbent adsorbate of the fermentation extract manufactured in Manufacturing Example 2. 6 is a diagram showing a UV spectrum of a brown substance obtained in Production Example 2. FIG. 2 is a diagram showing a UV spectrum of melanoidin obtained in Production Example 1. FIG. In Example 1, it is a figure which shows the captured image after dye | staining a lumen | bore about the well which culture | cultivated only the culture medium. In Example 1, it is a figure which shows the captured image after dye | staining a lumen | bore about the well which added suramin and culture | cultivated. In Example 1, it is a figure which shows the captured image after dye | staining a lumen | bore about the well which added the fermentation extract 1% dilution liquid and culture | cultivated. In Example 1, it is a figure which shows the picked-up image after dye | staining a lumen | bore about the well which added and fermented extract 10% dilution liquid and culture | cultivated. In Example 1, it is a figure which shows the captured image after dye | staining a lumen | bore about the well which added the melanoidin obtained by manufacture example 2, and was cultured. In Example 1, it is a figure explaining the angiogenesis inhibitory effect of the melanoidin of this invention. (A) is the result of evaluating the area of the lumen, and (B) is the result of evaluating the length of the lumen.

  The present invention is an angiogenesis inhibitor containing melanoidin as an active ingredient. Since melanoidin is a general term for the reaction product of a saccharide and an amino compound, the melanoidin varies depending on the type of saccharide or amino compound. In addition, since fermentation is a process of processing organic compounds by the action of useful microorganisms, various melanoidins are produced if different microorganisms are used. According to the present invention, at least melanoidin produced by syrup fermentation of lactic acid bacteria and yeast has an angiogenesis inhibitory effect.

  The melanoidin used in the present invention is an extract from a fermented extract obtained by co-fermenting lactic acid bacteria and yeast. Melanoidin can be produced by using at least lactic acid bacteria and yeast. Lactic acid bacteria produce lactic acid from carbohydrates, but the protein contained in the culture broth is broken down into peptides and amino acids by the lactic acid produced in the fermentation broth. In addition, lactic acid bacteria having amylase activity can decompose amylase contained in rice and brown rice into monosaccharides. Yeast produces alcohol and carbon dioxide from sugars under anaerobic conditions, but yeast containing proteolytic enzymes can break down proteins in the culture medium into peptides and amino acids, and the sugar that yeast ingests in the body. Can produce vitamins and amino acids. In the present invention, at least lactic acid and yeast may be used. Further, koji molds, Bacillus subtilis, filamentous fungi, photosynthetic bacteria, etc. are contained in the fermentation system as long as the effects of the present invention are not impaired. Also good. It is difficult to remove all bacteria attached to the raw material.

  In the present invention, “Kyoto fermentation” refers to fermentation performed by co-culturing lactic acid bacteria and yeast, and “fermentation extract” is obtained by removing microorganisms and raw material residues from the fermentation solution of Koji fermentation. .

  Nutrients for culturing lactic acid bacteria and yeast are polysaccharides, vegetables and fruits, and seaweed. As the polysaccharide, polysaccharides derived from natural raw materials such as rice, rice bran, brown rice, sake lees, distilled lees, wheat straw, beer lees, rice flour, wheat flour, starch and potatoes are preferred. More preferred are rice, rice bran, brown rice, sake lees, and rice flour, and particularly preferred are rice, rice bran and / or brown rice. Since brown rice and rice bran contain germs containing amylase, starch amylose and amylopectin can be converted into glucose, maltose, and oligosaccharide, and smooth fermentation using lactic acid bacteria and yeast using polysaccharides as raw materials Can do.

  The kind of vegetables and fruits is not particularly limited, but those containing a lot of minerals and enzymes can be preferably used. Those containing saccharides and proteins are preferred. For example, papaya, pineapple, apple, orange, banana, radish, carrot, cabbage, mushroom and the like can be suitably used. Papaya is preferable. Papaya contains a large amount of proteolytic enzymes such as papain, can break down proteins in the culture medium into peptides and amino acids, and participates in active melanoidin formation. The papaya is not limited to ripe as long as it is edible, and may be an immature green papaya. The amount of enzyme of green papaya is particularly preferable because it has a higher content than mature papaya.

  There are no particular limitations on the seaweed, and brown algae such as kelp, hijiki, mozuku, and young cloth, red algae such as cassava and primrose, green algae such as aosa, aonori, casanori, cactus, cypress, and mill are preferably used. More preferably, it is brown algae. In particular, kelp, hijiki and mozuku are preferred. These seaweeds contain amino acids in addition to minerals and polysaccharides.

The lactic acid bacteria and yeast used in the present invention only need to be able to finally produce melanoidin in the fermented extract by mash fermentation. Examples of lactic acid bacteria include Lactobacillus genus, Bifidobacterium genus, Enterococcus genus, Lactococcus genus, Pediococcus genus, Leuconostoc genus, Streptococcus genus, and the like, which can be appropriately selected according to the raw materials used. In the present invention, since polysaccharides derived from natural raw materials such as rice, rice bran, brown rice, sake lees, distilled lees, wheat straw, beer lees, rice flour, wheat flour, starch are used, it is possible to use lactic acid bacteria that can assimilate sugars. preferable. Examples of lactic acid bacteria that can assimilate saccharides include lactic acid bacteria such as Lactobacillus, Streptococcus, and Leuconostoc.
Lactobacillus casei (Lactobacillus casei), Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus amylophilus (Lactobacillus amylophilus), Lactobacillus cilophilus (Lactobacillus cilophilus) Lactobacillus sanfrancisco, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus paracasei, etc.
Examples of Streptococcus lactic acid bacteria include Streptococcus faecalis and Streptococcus faecium.
Examples of lactic acid bacteria belonging to the genus Leuconostoc include Leuconostoc mesenteroides and Leuconostoc lactis.

  Examples of yeast include Saccharomyces genus, Candida genus, Tolropsis genus, Digosaccharomyces genus, Schizosaccharomyces genus, Debariomyces genus, Geotrichum genus, Wickelhamia genus and Ferromyces genus. In the present invention, Saccharomyces genus that is a budding yeast, Schizosaccharomyces genus and Candida genus that are fission yeasts can be preferably used. Saccharomyces cerevisiae is preferable.

  In order to symbiotically ferment using lactic acid bacteria and yeast, all the above-mentioned raw materials are charged at once in the lactic acid bacteria and yeast syrup medium, and each raw material is uniformly adjusted to adjust the fermentation stage of each raw material. May be separately charged into a culture solution of lactic acid bacteria or yeast and pre-fermented, and then mixed and fermented together. It can be suitably selected according to the types of lactic acid bacteria and yeast, and the types of polysaccharides, vegetables / fruits, seaweeds and the like as nutrients for these.

The lactic acid bacteria and yeast used in the present invention are pre-cultured. The concentration of the lactic acid bacteria in the preculture solution is 10 ⁵ to 10 ¹⁰ cells / mL, and the concentration of the yeast in the preculture solution is 10 ⁵ to 10 ¹⁰ cells / mL. In addition, the preparation amount of a raw material (wet weight) is 0.01-100 w / v% of a preculture liquid, More preferably, it is 0.1-30 w / v%. The mixing ratio of vegetables and fruits to 100 parts by mass of polysaccharide is 1 to 100 parts by mass, more preferably 3 to 50 parts by mass. Moreover, the mixture ratio of the seaweed with respect to 100 mass parts of polysaccharides is 0.1-50 mass parts, More preferably, it is 1-30 mass parts.

A method for preparing a fermented extract using brown rice and rice bran as polysaccharides, papaya as vegetables and fruits, and kelp and mozuku as seaweed will be described. First, steamed brown rice is added to a syrup culture solution of lactic acid bacteria and yeast. As lactic acid bacteria to be used, lactic acid bacteria capable of assimilating polysaccharides are used. Thereby, the polysaccharide contained in brown rice can be decomposed into sucrose, glucose and the like. Lactic acid bacteria decompose polysaccharides and produce lactic acid. Since yeast has excellent acid resistance, it can grow in lactic acid acidic fermentation broth and produce amino acids and the like.
Separately from the above brown rice, papaya and seaweed are separately charged in a syrup culture medium of lactic acid bacteria and yeast, and then prefermented. Since papaya contains many proteolytic enzymes and the like, the components contained in the fermentation broth are further decomposed by these enzymes. When the degree of decomposition in each fermented liquid becomes substantially uniform, these are mixed and fermented together.

  Fermentation can be appropriately selected according to the type of microorganism used, the type of raw material, the amount of raw material charged, etc., and is generally 5 to 100 weeks, more preferably 10 to 50 weeks, and particularly preferably 20 to 40 weeks. is there. The above is the time from the start to the end of fermentation. Moreover, fermentation temperature is 20-40 degreeC. As the fermentation progresses, a culture solution of lactic acid bacteria or a culture solution of yeast may be added.

  Yeast and lactic acid bacteria can both grow under anaerobic conditions and aerobic conditions, and the products differ depending on the presence or absence of oxygen. For example, yeast produces alcohol using sugar as a raw material under anaerobic conditions, while lactic acid bacteria produce lactic acid under aerobic conditions. In the present invention, it is preferable to increase the activity of lactic acid bacteria in the first half of the fermentation, and to control the activity of other microorganisms in the second half of the fermentation. This is because by controlling the first half of fermentation to aerobic conditions, it is possible to sterilize miscellaneous bacteria mixed by lactic acid produced by lactic acid bacteria. Such control can be performed by controlling pH, the amount of dissolved oxygen, and temperature. Thereby, long-lasting fermentation can be performed with lactic acid bacteria and yeast.

  The end of the fermentation can be known by the brown color of the fermentation broth. In addition, you may age | cure | ripen a fermented liquor on the conditions which do not supply oxygen after completion | finish of fermentation.

  In the above method, polysaccharides, vegetables / fruits, and seaweed are separately pre-fermented with a lactic acid bacteria / yeast syrup medium, but the total amount of polysaccharides, vegetables / fruits, and seaweeds is It may be prepared in a pre-fermented solution with yeast and co-cultured, and the polysaccharide is pre-fermented with a lactic acid bacterium and yeast co-cultured solution, and vegetables, fruits and seaweed are pre-fermented with a lactic acid bacterium culture solution. Fermentation liquid may be mixed and fermented together. Melanoidin is considered to be produced by microorganisms that coexist after the raw material is decomposed into reducing sugars and amino compounds by microorganisms. Therefore, it is not necessary that the entire fermentation process is symbiotic fermentation. In addition, in a fermentation process, preparation water may be added and a bacterial solution may be added as needed.

  After fermentation or subsequent aging, lactic acid bacteria, yeast and other microorganisms are removed by filtration, centrifugation, etc. to obtain a fermented extract. The pH is generally 4 to 8.5.

  The fermentation extract can be treated with an ion exchange resin, a synthetic adsorption resin, or the like to separate and recover the fermentation product melanoidin. An organic acid or the like contained in the fermented extract can be removed with an ion exchange resin, a synthetic adsorption resin, or the like to obtain brown melanoidin. As the ion exchange resin to be used, sulfonic acid groups, phosphoric acid groups, carboxymethyl groups, diethylamino groups, quaternary aminoethyl groups, and the like can be used as ion exchange groups, and both cation exchange resins and anion exchange resins can be used. Both can be used. Melanoidin can also be obtained by using a synthetic adsorption resin (Diaion, Sepabead, etc., manufactured by Mitsubishi Chemical Corporation).

  The melanoidin used in the present invention has a molecular weight of 500 to 3000. This melanoidin was found to have an angiogenesis inhibitory effect, as shown in the Examples described later.

  As an administration route of the angiogenesis inhibitor containing the melanoidin of the present invention as an active ingredient, oral, intravenous, intraperitoneal, subcutaneous, intramuscular, intraarterial, transdermal, rectal, nasal, ocular and topical administration, etc. can do. A suitable administration route can be selected according to the type of disease and the progress of the disease. The dose varies depending on the administration route, and in the case of oral administration, it is 1 to 100 mg / day, and can be taken divided into a plurality of times per day.

  The angiogenesis inhibitor of the present invention may be prepared in any form. For example, injectable preparations (intrinsic solutions, suspensions or emulsions), topical preparations (ointments, patches, sprays, solutions, etc.), oral dosage forms (tablets, capsules, soft capsules, water-soluble medicines, Pills, granules) and the like.

  The angiogenesis inhibitor of the present invention has long-term chronic effects such as diabetic retinopathy, neovascular glaucoma, inflammatory skin diseases, rheumatoid arthritis, osteoarthritis, atherosclerosis, obstructive diseases such as myocardial infarction, etc. It can be used as a prophylactic agent for diseases.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

(Production Example 1)
A solution containing 2 mol of glucose, 2 mol of glycine and 0.2 mol of sodium hydrogen carbonate was adjusted to pH 6.8. Melanoidin was synthesized by refluxing at 95 ° C. for 7 hours. After cooling the reaction solution, dialysis was performed for 1 week using a dialysis membrane (trade name “UC36-32-100”, manufactured by Viscose). The internal solution was lyophilized and pulverized in a mortar to form a powder. About the aqueous solution of this powdery substance, UV spectrum was measured using JASCO V-570. The results are shown in FIG. In the UV spectrum, no absorption peak was observed at a specific location, and the absorption gradually decreased from a wavelength of 300 nm to 700 nm.

(Production Example 2)
Lactobacillus casei, Lactobacillus plantarum, Lactobacillus lactis were used as lactic acid bacteria, and Saccharomyces cerevisia was used as yeast. A solution obtained by prefermenting this with a culture solution made of rice bran was used as a seed-fermented fermentation solution. Steamed brown rice was added to an inoculum fermentation solution containing lactic acid bacteria and yeast, and primary fermentation was performed under conditions that promoted production of lactic acid bacteria. Similarly, papaya and seaweed (kombu and mozuku) were separately added to the inoculum fermentation solution containing lactic acid bacteria and yeast, and primary fermentation was performed in the same manner as brown rice. The raw materials used were 25 parts by mass of papaya and 8 parts by mass of seaweed with respect to 100 parts by mass of steamed brown rice. Then, each symbiotic fermentation broth was mixed. Fermentation was continued under conditions where the amount of dissolved oxygen was adjusted to promote the activity of bacteria other than lactic acid bacteria. In addition, in the fermentation process, pure water or an inoculum fermentation liquid was added as needed. The end point of the fermentation was determined as the fermentation broth became clear and brown as the raw material progressed. It took 6 months from the start of primary fermentation to the end of fermentation. Subsequently, it was aged for 6 months from the end of fermentation. Bacteria were removed from the fermented liquid after aging using a filter having a diameter of 0.45 μm to obtain a fermented extract. The pH was 6.0.

  The fermented extract was passed through a styrene-based synthetic adsorbent (manufactured by Mitsubishi Chemical Corporation, trade name “Diaion HP-20”) to adsorb the fermented product contained in the fermented extract.Then, the column was washed with water, Elution with methanol was carried out to fractionate a brown methanol elution.

  This brown methanol elution fraction was subjected to gel filtration HPLC analysis under the following conditions. A gel filtration chromatogram is shown in FIG.

HPLC analysis conditions Pump: manufactured by SHIMADZU, LC-10ADvp
Degasser: manufactured by SHIMADZU, DGU-14A
Auto injector: manufactured by SHIMADZU, SIL-10ADvp
System controller: SHIMADZU, SCL-10Avp
Diode array detector: manufactured by SHIMADZU, SPD-M10Avp
Evaporative light scattering detector: manufactured by SHIMADZU, ELSD-LT
Column: Shodex OH pak SB-803 HQ
Flow rate: 0.8mL / min
Mobile phase: 100 mM phosphate buffer

  When the molecular weight distribution of the brown fermentation product was measured under the same conditions as the above HPLC measurement conditions using pullulan as a molecular weight marker, a calibration curve was created thereby, and a size exclusion chromatography method for obtaining a relative molecular weight distribution was examined. ~ 3000.

  Subsequently, the UV spectrum of the brown substance was measured. The measurement was performed using JASCO V-570. The results are shown in FIG. In the UV spectrum, no absorption peak was observed at a specific location, and the absorption gradually decreased from a wavelength of 200 nm to 700 nm. This spectrum was similar in waveform to the melanoidin of Production Example 1.

  For brown fermentation products, protein, polysaccharides and monosaccharides are colorimetric using a spectrophotometer, organic acids are ion chromatographed, amino acids are amino acid analyzers, and lipids are gas chromatographed. Analysis. Proteins, polysaccharides, monosaccharides, organic acids, amino acids, and lipids were not detected in this fraction. Therefore, the main component of the methanol elution fraction was melanoidin.

Example 1
An angiogenesis kit (trade name “Angiogenesis Kit KZ-1000” manufactured by Kurashiki Boseki Co., Ltd.) was used. In a 24-well plate attached to the kit, human umbilical vein vascular endothelial cells (HUVEC) and human fibroblasts are embedded. A medium was prepared by adding VEGF-A (trade name “KZ-1350”, manufactured by Kurashiki Boseki Co., Ltd.) to a final concentration of 10 ng / ml to the angiogenesis medium-2 attached to the kit.

  1% diluted fermented extract obtained by diluting the fermented extract obtained in Production Example 2 to 1% with purified water, 10% diluted fermented extract diluted to 10% of the same, and melanoidin obtained in Production Example 2 (final in purified water). (Prepared to a concentration of 100 μg / mL) was diluted 10-fold with the above medium and filtered through a 0.22 μm filter to obtain a test solution. As a negative control, a medium containing 50 μM suramin was used. Three wells were used for each test solution, and 500 μl of the test solution was added to each well. In addition, what added only the culture medium to the well was made into the control.

After adding the test solution, the medium was changed on the 4th, 7th, and 9th days after the start of the culture, and the culture was performed at 37 ° C. in a 5% CO ₂ atmosphere for 10 days. On day 11 of culture, cells were fixed with 70% ethanol. After fixation, the wells were washed with 1% BSA / PBS, and then anti-CD31 antibody was added. Subsequently, the well was washed with 1% BSA / PBS, and then an alkaline phosphatase-labeled anti-IgG antibody was added. After washing the wells with distilled water, 5-bromo-4-chloro-3-indolylphosphatase was added to stain the formed lumen. After washing with distilled water and air-drying, three images were taken for each well. Captured images are shown in FIGS. The captured image was analyzed by angiogenesis quantitative software (Kurashiki Boseki Co., Ltd., Ver. 2) to evaluate the area and length of the lumen. The results are shown in Table 1 and FIG.

  As shown in FIG. 5, in the well administered with suramin as a negative control, a stained band-like lumen was observed, but the amount of lumen was smaller and the tube was smaller than the well containing only the medium (FIG. 4). There were few cavity branches. On the other hand, the well (FIG. 8) to which melanoidin obtained in Production Example 2 was administered did not have a band-like lumen, and an angiogenesis inhibitory effect was observed more than suramin. In addition, in the fermented extract 1% diluted solution (FIG. 6) and the fermented extract 10% diluted solution (FIG. 7), the amount of stained lumen was small compared to the well containing only the medium, and an anti-angiogenic effect was observed. In addition, as shown in FIG. 9, the angiogenesis inhibitory effect of suramin, 1% diluted solution of fermented extract, and 10% diluted solution of fermented extract was substantially the same.

  As shown in Table 1, in the wells to which suramin was administered, the area of the lumen was reduced to 50% and the length was reduced to 70% as compared with the well containing only the medium. In addition, both the 1% diluted extract of the fermented extract and the 10% diluted fermented extract had a reduced lumen area of about 50% and a length of about 70% compared to the well containing only the medium. Dependency was not observed. On the other hand, the melanoidin obtained in Production Example 2 has a reduced lumen area of 8% and a length of 13% compared to the well containing only the medium, and an extremely strong angiogenesis inhibitory effect was observed.

  The present invention uses melanoidin extracted from a fermentation product of natural products as an angiogenesis inhibitor, and can be used for prevention and treatment of malignant tumors and the like.

Claims (5)

  An angiogenesis inhibitor comprising melanoidin as an active ingredient.   The angiogenesis inhibitor according to claim 1, wherein the melanoidin is an extract from a fermented extract obtained by co-fermenting lactic acid bacteria and yeast.   The angiogenesis inhibitor according to claim 2, wherein the melanoidin is obtained by fermenting the polysaccharide, vegetables / fruits, and seaweed with the co-fermentation.   The angiogenesis inhibitor according to claim 3, wherein the vegetables and fruits are papayas.   The angiogenesis inhibitor according to claim 3 or 4, wherein the polysaccharide is rice, rice bran and / or brown rice.