JP2007084504A - Composition containing fermentation product of barley as active ingredient and having neovascularization inhibitory action - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition having an excellent neovascularization inhibitory action from an inexpensive and relatively easily available material not having a problem on safety, preferably distillation residual liquids of barley Shochu (white distilled liquor), by a simple treatment method suitable for practical production in a factory scale without passing through a troublesome purification process. <P>SOLUTION: This composition for inhibiting neovascularization contains the fermentation product of barley, preferably an ingredient originated from distillation residual liquids of barley Shochu, as active ingredient. The composition is a composition for treating or preventing diseases having neovascularization to be inhibited, concretely diseases caused by abnormal neovascularization in tumors or cancers, chronic inflammations or retinopathy. The composition is in a form selected from the group consisting of food additives, food materials, foods, drinks, medicines/quasi-drugs, and feeds for inhibiting the neovascularization. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a composition having an angiogenesis-inhibiting action, comprising as an active ingredient a component derived from barley subjected to fermentation. More specifically, the present invention relates to a composition (fermented barley extract) obtained by fractionating a substance obtained by subjecting barley to fermentation, preferably a substance contained in a barley shochu distillation residue, more preferably a barley shochu distillation residue. ), An active ingredient, a composition for treating or preventing a disease that should inhibit angiogenesis, preferably a composition in the form of a food material, food or drink, feed or the like.

  Cancer has been the leading cause of death in Japan since 1981. Among the three major causes of death, only cancer has consistently increased and the number of deaths continues to increase. According to the Ministry of Health, Labor and Welfare's demographic statistics, out of 97,331 deaths in 2001, 300,658 died from cancer, and one in three died from cancer. It will be. For such diseases, the main treatment currently performed in hospitals is symptomatic treatment in which cancer cells are directly attacked by various therapies such as drugs, chemistry, and physics. In addition, there is a problem that normal cells are damaged, and as a result, the patient dies as a result of a decrease in immunity and natural healing power. With this background, the demand for safe treatments with few side effects is increasing day by day, and more emphasis is placed on prevention than disease treatment.

  In recent years, the application of angiogenesis inhibition as a safe cancer treatment has attracted attention. By producing angiogenesis-promoting substances, cancer cells have the property of spreading blood and feeding nutrients and oxygen to their cells, supplying enough blood, and repeating explosive growth. However, cancer treatment applying an angiogenesis inhibitory action is to prevent the proliferation of cancer cells by blocking the blood pathway to the cancer cells. That is, to put it simply, cancer cells are attacked.

  In recent years, it has been reported that the action of inhibiting angiogenesis has the effect of preventing and safely treating not only cancer but also rheumatoid arthritis, diabetes, heart disease and other various diseases (Non-patent Document 1 ~ 3) Early provision of a substance having an anti-angiogenic action is strongly demanded.

  Regarding an active ingredient derived from a natural product having an angiogenesis inhibitory action, Patent Document 1 discloses an angiogenesis inhibitor, a cell growth inhibitor, and a lumen formation inhibitor containing tocotrienol obtained from palm skin and seeds as an active ingredient. Agents and FGF inhibitors and foods or food additives are described, and Patent Document 2 describes a food composition for inhibiting angiogenesis including shiitake mycelium extract. Patent Documents 3 and 4 describe that a novel compound obtained by culturing a filamentous fungus and obtained from the culture solution has an angiogenesis inhibitory effect.

  By the way, barley, a gramineous plant, has been indispensable for humankind since prehistoric times, and has been loved as a healthy food, as described in old Japanese medical books. Various studies have been conducted on the physiologically active function of barley or barley fermented with microorganisms.

  Patent Document 5 discloses that an extract obtained from a crushed fraction of barley is extracted with an aqueous solvent, so that an extract mainly from the husk fraction is useful for bioactivity, that is, immune enhancement, blood pressure lowering, and blood flow improvement. Functional food materials that have physiological activity such as action, angiotensin I converting enzyme inhibitory action, and antibacterial action are described. However, this invention is an extract extracted from the bark grain fraction, its active ingredient is a readily water-soluble substance, has a molecular weight of 500,000 or less, the main component is a molecular weight of 100,000 or less, a protein content of 3 to 30%, It is rich in water-soluble ferulic acid and p-coumaric acid.

  As an invention using a barley shochu distillation residue as a by-product in the production of shochu using barley as a raw material, Patent Document 6 discloses a liquid component obtained by solid-liquid separation of the barley shochu distillation residue, and an alkali is added to the liquid component. To add an alkali-soluble fraction, neutralize the alkali-soluble fraction with an acid to obtain a neutral soluble fraction, and add the ethanol to the neutral soluble fraction. It is described that an ethanol-insoluble fraction containing acid, protein, and hemicellulose has a fatty liver inhibitory action, and Patent Document 7 obtains a liquid component by solid-liquid separation of a barley shochu distillation residue, It is described that an organic solvent-insoluble fraction collected by adding an organic solvent to the liquid component has an inhibitory effect on leukemia cell growth. Patent Document 8 discloses from a barley shochu distillation residue, Patent Document Obtained in the same way as 7. The organic solvent-insoluble fraction, it is described that the activation of natural killer cells.

  Patent Document 9 includes a non-adsorbed fraction obtained by solid-liquid separation of a barley shochu distillation residue and applying the liquid to a synthetic adsorbent, and the non-adsorbed fraction has an average chain. A plurality of peptides having a length of 3.0 to 5.0 are contained, and these peptides have an amino acid composition of glutamic acid 24 to 38%, glycine 4 to 20%, aspartic acid 5 to 10%, proline 4 to 9%, and serine 4 to 8%, and has a suppressive action and curative action on alcoholic liver damage and has an excellent taste. Products and methods for their production are described. Patent Document 10 describes a pharmaceutical composition in which the composition obtained in the same manner as Patent Document 9 has an onset suppressing action and a healing action against alcoholic liver injury, and a method for producing the same.

  Further, in Patent Document 11, a barley shochu distillation residue is subjected to solid-liquid separation to obtain a liquid component, which is subjected to an ion exchange treatment to obtain an ion-exchange resin non-adsorbed fraction. It is described that the organic solvent-insoluble fraction obtained by subjecting the adsorbed fraction to ultrafiltration treatment to obtain a concentrated liquid and adding an organic solvent to the concentrated liquid has an antioxidant action.

Eur.J.Cancer., 32A, 2534-2539 (1996) Nature Med., 1, 27-33 (1995) Immunity., 12, 121 (2000) JP 2002-308768 A JP 2004-196791 A JP 2003-183249 A JP 2004-262881 A JP 2002-371002 A Japanese Patent Laid-Open No. 2001-145472 JP 2003-73294 A JP 2003-73295 A Japanese Patent Laid-Open No. 2004-112 Japanese Patent Laid-Open No. 2004-2266 Japanese Patent Laid-Open No. 2004-238452

  As mentioned above, barley and fermented barley have been confirmed to have various physiological activities, but the angiogenesis inhibitory action is not included in the physiological activities, and barley inhibits angiogenesis. It has not been known so far to exhibit an action and to exhibit a stronger action by subjecting barley to fermentation.

  The present invention is a product obtained by subjecting barley, which has no safety problems and is relatively easily available, to fermentation, preferably a barley shochu distillation residue, and does not go through complicated purification steps and is practically used on a factory scale. Providing a composition having an excellent angiogenesis inhibitory action by a simple treatment method suitable for production, preferably a composition for treating or preventing a disease that should inhibit angiogenesis, more specifically a food It is an object of the present invention to provide a product selected from the group consisting of additives, food materials, food and drink, pharmaceuticals / quasi drugs, and feed.

  As a result of diligent research to solve the above problems, the present inventors have found that an excellent angiogenesis inhibitory action exists on cereal-derived components, particularly barley-derived components, and a stronger effect is achieved by fermentation. I found out that there was an invention. In the present invention, the composition obtained by fractionating from the barley shochu distillation residue has a significant angiogenesis inhibitory action, thereby newly providing a composition having angiogenesis inhibitory activity with fewer side effects. Can be provided.

That is, the gist of the present invention is a composition for inhibiting angiogenesis of the following (1) to (10).
(1) A composition for inhibiting angiogenesis, characterized in that a component derived from fermentation of barley is used as an active ingredient for inhibiting angiogenesis.
(2) The composition for inhibiting angiogenesis according to claim 1, wherein the component derived from fermentation of barley is a barley shochu distillation residue.
(3) The composition for inhibiting the angiogenesis of (1) or (2), wherein the component derived from fermentation of the barley is selected from the group consisting of fermented barley extract and fermented barley fiber.
(4) The fermented barley extract is a composition obtained by fractionating a barley shochu distillation residue, a lactic acid bacteria culture solution using the barley shochu distillation residue as a medium, and a Bacillus natto culture using the barley shochu distillation residue as a medium. The composition for inhibiting the angiogenesis of (3) which is chosen from the group which consists of a liquid, and the said fermented barley fiber is a fermented barley fiber alkali extract.
(5) The composition obtained by fractionating the barley shochu distillation residue is a non-adsorbed fraction treated with barley shochu distillation residue synthetic adsorbent, an adsorbed fraction treated with barley shochu distillation residue, and barley The composition for inhibiting angiogenesis according to (4), which is one or more compositions selected from the group consisting of ethanol precipitation fractions from shochu distillation residue.
(6) The composition for inhibiting angiogenesis is a composition for treating or preventing a disease for which angiogenesis is to be inhibited, for inhibiting angiogenesis according to any one of (1) to (5). Composition.
(7) The composition for inhibiting angiogenesis according to (6), wherein the disease to inhibit angiogenesis is a disease caused by abnormal angiogenesis in tumor or cancer, chronic inflammation or retinopathy.
(8) The composition is in a form selected from the group consisting of food additives for inhibiting angiogenesis, food materials, food and drink, pharmaceuticals / quasi-drugs, and feeds (1) to (7 ) A composition for inhibiting angiogenesis.
(9) The composition for inhibiting angiogenesis according to (8), wherein the food or drink is a functional food, a nutritional supplement or a health food or drink for inhibiting angiogenesis.
(10) The composition for inhibiting angiogenesis according to (8), wherein the feed is a feed for livestock, poultry and pets for inhibiting angiogenesis.

  The present invention has no problem in safety, is cheap and is relatively easily available material barley that has been subjected to fermentation, preferably from the barley shochu distillation residue, without going through complicated purification steps, factory scale A composition having an anti-angiogenic activity with few side effects, preferably a composition for treating or preventing a disease that should inhibit angiogenesis, more specifically, by a simple treatment method suitable for actual production in the plant, more specifically, The thing of the form chosen from the group which consists of a food additive, a foodstuff material, food-drinks, a pharmaceutical and quasi-drug, and feed can be provided.

Barley to be subjected to fermentation of the present invention may be either barley or bare barley, or may be either Nijo barley or Rojo barley. Colored barley with pigments deposited on the grain surface may also be used. Although it does not restrict | limit especially as a form of barley, It is preferable to use the whole grain, such as brown wheat. In addition, processing such as roasting, milling, and pressure biasing may be added.
In addition, when attaching | subjecting barley to fermentation, although the microorganisms to be used are not specifically limited, yeast, lactic acid bacteria, natto, or koji molds can be used. In particular, examples of yeast used in the fermentation process for preparing fermented barley extract include brewer's yeast, sake yeast, shochu yeast, wine yeast, and baker's yeast. It is desirable to use shochu yeast.

The above composition of the present invention is a product obtained by subjecting barley, which is an inexpensive and relatively easily available material, to fermentation, preferably from a barley shochu distillation residue, without any problem in safety. It is obtained by a simple processing method suitable for actual production at a factory scale, and its application to living bodies can be used for food, drink, pharmaceuticals, fertilizer, feed and skin external preparations, and has an excellent angiogenesis inhibitory effect. You can expect to get. The composition of the present invention has an active ingredient derived from a natural product having an angiogenesis inhibitory action, and by applying angiogenesis inhibition based on the active ingredient, for example, leads to a safe cancer treatment method. Therefore, the action of inhibiting angiogenesis is a functional composition having an effect of preventing and safely treating not only cancer but also rheumatoid arthritis, diabetes, heart disease and other various diseases.
That is, the composition for inhibiting angiogenesis according to the present invention is a composition for treating or preventing a disease that should inhibit angiogenesis. The disease that should inhibit angiogenesis is a target as long as it is a disease in which angiogenesis plays an important role in the pathogenesis. Thus, diseases that should inhibit angiogenesis are diseases caused by abnormal angiogenesis in tumors or cancer, chronic inflammation or retinopathy. More specifically, diseases that should inhibit angiogenesis include, for example, solid tumors, myelomas, hemangiomas and other tumors or cancers that occur in various tissues; rheumatoid arthritis, psoriasis, osteoarthritis, etc. Diseases such as, but not limited to, chronic inflammation; or retinopathy such as age-related macular degeneration, diabetic retinopathy, neovascular glaucoma; In the present invention, it is preferable to target various tumors or cancers as diseases for inhibiting angiogenesis.

  In order to examine the angiogenesis inhibitory activity of the composition of the present invention in vitro, vascular endothelial cells and fibroblasts are cultured under culture conditions in the presence of VEGF, an angiogenesis-inducing factor, as necessary. Thus, it is possible to confirm whether or not angiogenesis is suppressed by adding the composition of the present invention to the culture in which the growth state at the initial stage of tube formation is created by co-culture. Inhibition of angiogenesis can be performed, for example, by staining a lumen to examine whether or not the lumen formation is suppressed. As a kit for investigating the inhibitory effect of angiogenesis in vitro, an angiogenesis kit (manufactured by KURABO) can be used, and an anti-CD31 antibody or an anti-von Willebrand factor antibody can be used to stain a lumen. Luminal staining kits (KURABO, manufactured by Kurabo Co., Ltd.) that use antibodies can be used.

  The composition for inhibiting angiogenesis according to the present invention is characterized in that a component derived from fermenting barley is used as an active ingredient for inhibiting angiogenesis. The substance exhibiting an angiogenesis inhibitory action contained in the component derived from the fermentation of barley is preferably a substance contained in the barley shochu distillation residue. The substances contained in the barley shochu distillation residue are a composition obtained by fractionating the barley shochu distillation residue (fermented barley extract), a lactic acid bacteria culture solution using the barley shochu distillation residue as a medium, and a barley shochu distillation residue. Is a substance contained in a composition selected from the group consisting of a Bacillus natto culture solution and a fermented barley fiber alkali extract.

  As a method for obtaining a composition obtained by fractionating a barley shochu distillation residual liquid, a method for obtaining a solid by solid-liquid separation using a screw press, a liquid obtained by solid-liquid separation, and a synthetic adsorbent There is a method of performing purification by using a column or a column of an ion exchange resin, followed by lyophilization or insolubilization with an organic solvent. Further, there is a method in which the liquid obtained by solid-liquid separation is used for a culture medium such as lactic acid bacteria or natto bacteria, and the culture liquid is freeze-dried. Therefore, the composition obtained by fractionating the barley shochu distillation residue is the barley shochu distillation residue synthetic adsorbent-treated non-adsorbed fraction, the barley shochu distillation residue synthetic adsorbent-treated adsorbent fraction, and the barley shochu distillation residue. Examples thereof include one or more compositions selected from the group consisting of a liquid ion exchange resin-treated non-adsorbed fraction and a barley shochu distillation residual liquid ethanol precipitation fraction.

The composition obtained by fractionation from the barley shochu distillation residue is obtained by the following steps.
First, barley straw is produced. After absorbing 40% (w / w) of barley and steaming for 40 minutes, it is allowed to cool to 40 ° C and inoculated with 1 kg of seed meal (birch) per ton of barley, 38 ° C, RH 95% for 24 hours, 32 Barley koji can be produced by holding at ℃ and RH92% for 20 hours. To the barley koji produced by the above method, water and shochu yeast yeast are added to obtain primary mash, and the obtained primary mash is subjected to 5-day fermentation (first stage fermentation). Next, in the secondary charging, water and barley koji produced by the above method are added to the primary mash after the first stage fermentation, and subjected to 11 days of fermentation (second stage fermentation). The fermentation temperature is 25 ° C for both the first and second charge. The secondary mash after the second stage fermentation is subjected to simple distillation by a conventional method to obtain a barley shochu and barley shochu distillation residual liquid of 100% koji ratio.

  The barley shochu distillation residue obtained above may use the solid content obtained by solid-liquid separation (fermented barley fiber alkali extract powder) as it is, or mix the liquid content with dextrin and freeze-dry. What is done (fermented barley extract powder) can also be used. Alternatively, the non-adsorbed component (fermented barley extract S powder) can be used by passing the liquid through a synthetic adsorbent column, and the fraction (fermented barley extract P powder) from which the adsorbed component has been eluted can be used with an organic solvent. The insoluble fraction (ethanol precipitated fraction powder) can also be used as insoluble by treatment. Furthermore, before processing with an organic solvent, it can be used by passing through an ion exchange resin column, or after passing through an ion exchange resin column, it can also be used by passing through an ultrafiltration membrane.

  As the synthetic adsorbent used above, an aromatic, aromatic modified or methacrylic synthetic adsorbent can be used. Specific examples of such a synthetic adsorbent include Amberlite XAD-4, Amberlite XAD-16, Amberlite XAD-1180 and Amberlite XAD-2000 manufactured by Organo Corporation, and Sepa beads manufactured by Mitsubishi Chemical Corporation. Aromatic (also called styrene) synthetic adsorbents such as SP850 and Diaion HP20, Amberlite XAD-7 from Organo Corporation, and Methacrylic (or Diaion HP2MG from Mitsubishi Chemical Corporation) (or Aromatic modified synthetic adsorbents such as synthetic adsorbents and Sephapez SP207 manufactured by Mitsubishi Chemical Corporation can be fried. Specific examples of suitable ion exchange resins used in the above include strong acid cation exchange resins IR-120, IR-120B, Amberlite 200CT, weak acid cation exchange resins IRC50 and IRC76 manufactured by Organo, Examples include strong acid cation exchange resins Diaion SK1B, SK104, and PK208 manufactured by Mitsubishi Chemical Corporation and weak acid cation exchange resins WK10 and WK40.

  As the organic solvent used above, ethanol is desirable, but it is most desirable to add ethanol so that the final concentration is 75% by volume.

  As another form, the barley shochu distillation residue can be used as a culture medium such as lactic acid bacteria or natto bacteria, and the culture liquid can be freeze-dried (lactic acid bacteria culture powder, natto culture liquid powder). it can. That is, fermented barley extract is not only a composition obtained by fractionating barley shochu distillation residue, barley shochu distillation residue, but also lactic acid bacteria culture solution, barley shochu distillation residue using barley shochu distillation residue as a medium. Selected from the group consisting of Bacillus natto culture solution and fermented barley fiber alkali extract.

  The lactic acid bacteria used above are not particularly limited, but lactic acid bacteria belonging to Lactococcus lactis subsp. Lactis are preferred. Specifically, Lactococcus lactis NCDO497, Lactococcus lactis NIZO R5, Lactococcus lactis ATCC 7962 and Lactococcus lactis ATCC11454, Lactococcus lactis NIZO 22186, Lactococcus lactis NRRL-B-18583, Lactococcus lactis NCFB2118, Lactococcus lactisococ lactis NIZO 221186, Lactococcus lactis IO-1 (JCM7638), Lactococcus lactis subsp. Ishizaki Chizuka (JCM11180), Lactococcus lactis subsp. Ishizaki Yasaka 5B (JCM11181), Lactococcus lactis subsp. Ishizaki Yasaka 7B (JCM11182), Lactococcus lactis subsp. Ishizaki Yasaka 8B (JCM11183), and Lactococcus lactis subsp. Ishizaki Yasaka 9B (JCM11184) can be mentioned as a preferable one.

  The Bacillus natto used in the above is not particularly limited, and examples include the commercially available natto bacillus belonging to Bacillus Subtillis.

A composition obtained by subjecting barley to fermentation and / or a barley shochu distillation residue is composed of a food additive, a food material, a food and drink, a pharmaceutical / quasi-drug, and a feed for inhibiting angiogenesis. It is of the form chosen. Utilizing the functionality of the composition, it has become possible to develop healthy foods and drinks, foods containing nutritional foods and drinks for patients, and feeds for domestic animals such as livestock, poultry and fish.
That is, the said food / beverage products are a functional food, a nutritional supplement, or a health food / beverage product for inhibiting angiogenesis. The feed is a feed for livestock, poultry and pets for inhibiting angiogenesis. Specifically, the food material containing the barley fermentation mentioned above and the barley shochu distillation residue can be used in any form of food, beverage or feed.
In the case of using the above-described functionality of the composition for inhibiting angiogenesis of the present invention, the content is not particularly limited, but is appropriately adjusted depending on the degree of intended function, usage mode, usage amount, etc. For example, it is 0.001-100 mass%. The composition for inhibiting the angiogenesis can be used for the human body, other foods and drinks, pharmaceuticals, feeds and external preparations for skin. It can also be taken orally or applied to the skin. Can be incorporated into oral and parenteral products according to conventional methods and used in various fields such as seasonings, food additives, food ingredients, food and drink, health food and drink, topical skin preparations, pharmaceuticals and feed Can do. For example, when blended with food or drink, food or drink for treating or preventing a disease that should inhibit angiogenesis can be provided. From the effect of prevention, it can be expected to be used as health food, nutritional food and the like. In addition, it can be used for livestock and / or fish feed and feed. By using it for the human body and other foods and drinks, pharmaceuticals, fertilizers, feeds and external preparations for skin, it is possible to obtain an effect of treating or preventing a disease that should inhibit angiogenesis.
Furthermore, it can be easily obtained from the barley shochu distillation residue obtained by fermentation of barley, which is preferable from the viewpoint of cost and effective utilization of resources.

When the composition of the present invention is used for food, it can be prepared as it is, in a form diluted with oil, a milk form meal, or a form to which a carrier generally used in the food industry is added. Also good.
In the form of an emulsion, for example, a composition is added to the oil phase, and liquid fats such as glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, glycerol, dextrin, rapeseed oil, soybean oil, corn oil, etc. L-ascorbic acid or an ester or salt thereof, for example, gum such as locust bean gum, gum arabic or gelatin, for example, flavonoids such as hesperidin, rutin, quercetin, catechin, thianidine or polyphenols or the like It can be prepared by adding a mixture and emulsifying.

  The form of the beverage is a non-alcoholic beverage or an alcoholic beverage. Non-alcoholic beverages include, for example, non-carbonated beverages such as carbonated beverages, fruit juice beverages, and nectar beverages, soft drinks, sports beverages, tea, coffee, cocoa, and the like, and in the form of alcoholic beverages spirits, liqueurs, chuhai, The form of general foods, such as fruit liquor, barley liquor, Happoshu, and medicinal liquor, can be mentioned.

  Specific examples of food and drink include the following. Pastry (pudding, jelly, gummy candy, candy, drop, caramel, chewing gum, chocolate, pastry, butter cream, custard cream, cream puff, hot cake, bread, potato chips, french fries, popcorn, biscuits, crackers, pie, sponge Cakes, castella, waffles, cakes, donuts, biscuits, cookies, rice crackers, rice crackers, rice cakes, manju, candy, etc., dried noodle products (macaroni, pasta), egg products (mayonnaise, fresh cream), beverages (functional beverages, Lactic acid beverages, lactic acid bacteria beverages, concentrated dairy beverages, fruit juice beverages, fruit juice beverages, pulp beverages, transparent carbonated beverages, carbonated beverages with fruit juice, fruit colored carbonated beverages), luxury products (green tea, tea, instant coffee, cocoa, canned Coffee Milk), dairy products (ice cream, yogurt, coffee milk, butter, butter sauce, cheese, fermented milk, processed milk), pastes (marmalade, jam, flower paste, peanut paste, fruit paste, fruit syrup) , Livestock meat products (ham, sausage, bacon, dry sausage, beef jerky, lard), seafood products (fish meat ham, fish sausage, salmon, chikuwa, hampen, dried fish, bonito, bonito, boiled, sea urchin Salted fish, plums, dried fish, shellfish, salmon products such as salmon), boiled fish (small fish, shellfish, wild vegetables, salmon, kelp), curry (improvised curry, retort curry, canned curry), seasoning (Miso, powdered miso, soy sauce, powdered soy sauce, moromi, fish sauce, sauce, ketchup, oyster sauce, solid bouillon List of grilled meat sauce, curry roux, stew sauce, soup sauce, soup stock, paste, instant soup, sprinkle, dressing, salad oil), fried products (fried food, fried confectionery, instant ramen), soy milk, margarine, shortening, etc. be able to.

  The said food and drink can be processed and manufactured by mix | blending a composition with the raw material of a general food according to a conventional method.

  Although the compounding quantity of the composition to the said food-drinks changes with forms of food and is not specifically limited, Usually, 0.001 to 20% is preferable.

  The above food and drink can also be used as functional foods, nutritional supplements or health foods. The form is not particularly limited, and examples of food production include milk proteins with high amino acid balance, soy protein, and proteins such as egg albumin, degradation products thereof, and egg white oligos. In addition to peptides, soybean hydrolysates, and the like, mixtures of amino acids alone can be used according to conventional methods. It can also be used in the form of a soft capsule, a tablet or the like.

  Examples of dietary supplements or functional foods include liquid foods, semi-digested nutritional foods, ingredient nutritional foods, drinks, capsules, and sucrose containing sugars, fats, trace elements, vitamins, emulsifiers, and fragrances. Processing forms such as enteral nutrients can be mentioned. In the above-mentioned various foods, for example, foods and drinks such as sports drinks and nutritional drinks are further supplemented with nutritional additives such as amino acids, vitamins and minerals, sweeteners, spices and fragrances to improve nutritional balance and flavor. , Pigments and the like can also be blended.

  In order to stabilize the composition of the present invention, an antioxidant such as tocopherol, L-ascorbic acid, BHA, rosemary extract and the like can be used in combination according to a conventional method.

The composition of the present invention can be applied to feed for livestock, poultry and pets. For example, it can mix | blend with livestock feeds, such as dry dog food, dry cat food, wet dog food, wet cat food, semi-moist dock food, poultry feed, cattle, and pigs. The feed itself can be prepared according to a conventional method.
These therapeutic agents and preventive agents are non-human animals, for example, domestic mammals such as cattle, horses, pigs and sheep, poultry such as chickens, quails and ostriches, reptiles, birds and pets such as small mammals, It can also be used for farmed fish.

  Below, each composition using the barley shochu distillation residue liquid component as a material is prepared and the angiogenesis inhibitory effect is described, but the scope of the present invention is not limited to these examples.

[Experiment 1] Angiogenesis inhibition experiment and TOF-MS measurement <Sample name>
No. 1-1: Fermented barley extract powder (50% dextrin)
No. 1-2: Fermented barley extract _S powder (50% dextrin)
No. 1-3: Fermented barley extract _P powder
No. 1-4: Fermented barley extract EI
No. 1-5: Fermented barley extract EI-C
No. 1-6: Fermented barley extract U
No. 1-7: Fermented Barley Extract EI-CU
No. 1-8: Fermented barley extract CU
No. 1-9: Lactic acid bacteria culture solution
No. 1-10: Bacillus natto culture solution (75% dextrin)
No. 1-11: Fermented barley fiber alkali extraction

<Preparation method of sample used in experiment>
The sample used in this experiment has a balance with other experiments, and the powdered sample is redissolved and used in the experiment. However, there is no problem even if it is used in the experiment as it is in a liquid state. In addition, when powdered as described below, there is also a product added with an equivalent amount of dextrin as an extractant. The dextrin content is weight percent concentration (W / W).

[Barley shochu distillation residual powder] (fermented barley extract powder)
Barley (70% refined) was used as a raw material.
Manufacture of rice bran: 40% (w / w) absorption of barley, steamed for 40 minutes, allowed to cool to 40 ° C, inoculated with 1 kg seed barley (birch) per ton of barley, 38 ° C, RH 95% Barley koji was produced by holding for 24 hours at 32 ° C. and RH 92% for 20 hours.
Manufacture of barley shochu and barley shochu distillation residue: In the first charge, add 3.6 kiloliters of water and 1 kg (wet weight) of shochu yeast culture as yeast to the barley koji (3 tons as barley) produced by the above method. Primary mash was obtained, and the obtained primary mash was subjected to fermentation for 5 days (first stage fermentation). Next, in the secondary charging, 11.4 kiloliters of water and barley koji (6 tons as barley) produced by the above method were added to the primary mash after the first stage fermentation for 11 days (2nd stage fermentation). (Fermentation of the eyes). The fermentation temperature was set to 25 ° C. for both the primary charge and the secondary charge. The secondary mash after the second stage fermentation was subjected to single distillation by a conventional method to obtain 10 kiloliters of barley shochu and 15 kiloliters of barley shochu distillation residue. The obtained barley shochu distillation residue was centrifuged at 8000 rpm for 10 minutes to obtain 5 L of barley shochu distillation residue. A dextrin equivalent to the solid content of the liquid was mixed and lyophilized.

[Barley shochu distillation residual liquid synthetic adsorbent-treated non-adsorbed fraction powder] (fermented barley extract S powder)
The barley shochu distillation residue obtained in the distillation process of barley shochu production is centrifuged at 8000 rpm for 10 minutes to obtain a liquid content of the barley shochu distillation residue, and 25 L of this liquid and 10 L of deionized water are added in this order. It is composed of a flow-through liquid that exhibits non-adsorbability with respect to the synthetic adsorbent of the column by passing through a column (resin capacity 10 L) packed with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Co., Ltd. The non-adsorbed fraction was collected. The solid content of the obtained non-adsorbed fraction was mixed with an equivalent amount of dextrin and freeze-dried using a vacuum freeze dryer to obtain 2400 g of a freeze-dried product.

[Barley Shochu Distillation Residue Synthetic Adsorbent Processed Adsorbed Fraction Powder] (Fermented Barley Extract P Powder)
The barley shochu distillation residue obtained in the distillation process of barley shochu production is centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue, and 25 L of the resulting liquid is synthesized by Organo Contact with a column (resin capacity 10 L) packed with adsorbent Amberlite XAD-16 to obtain an adsorbed fraction adsorbed on the column, and further contact 10 L of deionized water with the column adsorbed the adsorbed fraction After removing the obtained eluate, 10 L of 1 (wt / vol)% sodium hydroxide solution and 10 L of deionized water were brought into contact with the column in this order, so that 20 L of the eluate consisting of the adsorbed fraction was collected. After contacting 20 L of the eluate with a column (resin capacity: 10 L) packed with a strongly acidic cation exchange resin IR-120B manufactured by Organo, the liquid was freeze-dried.

[Barley shochu distillation residual liquid ethanol precipitation fraction powder] (each EI fraction)
<Fermented barley extract EI>
The barley shochu distillation residue obtained in the distillation process of barley shochu production is centrifuged under the conditions of 8000 rpm and 10 min to obtain 5 L of barley shochu distillation residual liquid, and the resulting liquid has a Brix degree of 25 After concentration with a vacuum distillation machine, 3 volumes of ethanol was added and centrifuged at 8000 rpm for 10 min to collect an ethanol-insoluble fraction. The ethanol-insoluble fraction was freeze-dried.
<Fermented barley extract EI-C>
The barley shochu distillation residue obtained in the distillation process of barley shochu production is centrifuged at 8000 rpm for 10 min to obtain a barley shochu distillation residue liquid component, and 25 L of liquid and 10 L of deionized water are sequentially added in this order. It is composed of a flow-through liquid that exhibits non-adsorbability with respect to the cation exchange resin of the column by performing adsorption separation treatment through a column (resin capacity: 10 L) filled with the cation exchange resin Amberlite 200CT Na made by Organo. The non-adsorbed fraction was collected. Concentrate the obtained liquid with a vacuum still until the Brix degree reaches 60, add 3 volumes of ethanol, and centrifuge at 8000 rpm for 10 min to collect an ethanol-insoluble fraction. Fractions were lyophilized.
<Fermented barley extract EI-CU>
The barley shochu distillation residue obtained in the distillation process of barley shochu production was centrifuged at 8000 rpm for 10 min to obtain a barley shochu distillation residue liquid, and the resulting liquid was adjusted to a Brix degree of 10, The liquid content 1L adjusted to Brix degree 10 was passed through a column packed with 500 ml capacity of Amberlite 200CT Na (strongly acidic cation exchange resin) manufactured by Organo, to obtain a non-adsorbed fraction on the ion exchange resin, Fraction non-adsorbed on the obtained ion exchange resin was subjected to a concentration treatment with an ultrafiltration membrane centramate omega 10K (fraction molecular weight 10,000) manufactured by Nippon Pole Co., Ltd. to obtain a concentrated solution. Concentrate the liquid with a vacuum still until the Brix degree is 40, add 3 volumes of ethanol, centrifuge at 8000 rpm for 10 min to collect the ethanol-insoluble fraction, and remove the ethanol-insoluble fraction. Freeze-dried.

[Fermented barley extract U]
The barley shochu distillation residue obtained in the distillation process of barley shochu production was centrifuged at 8000 rpm for 10 min to obtain a barley shochu distillation residue liquid, and the resulting liquid was made by Nippon Pole Co., Ltd. Concentrated liquid was obtained by concentrating with ultrafiltration membrane Centramate Omega 10K (fraction molecular weight 10,000), and the resulting liquid was freeze-dried.
[Fermented barley extract CU]
The barley shochu distillation residue obtained in the distillation process of barley shochu production was centrifuged at 8000 rpm for 10 min, and the barley shochu distillation residue liquid was converted to Organo Amberlite 200CT Na (strongly acidic cation exchange resin). The fraction not adsorbed to the ion exchange resin is obtained through a column packed with the cation exchange resin, and the fraction not adsorbed to the obtained ion exchange resin is ultrafiltered membrane Centramate Omega 10K (min. A concentrated solution was obtained by concentration treatment with a molecular weight of 10,000), and the obtained liquid was freeze-dried.

[Lactic acid bacteria culture medium using barley shochu distillation residue]
-The barley shochu distillation residue obtained in the distillation process of barley shochu production was centrifuged at 8000 rpm for 10 minutes to obtain a liquid content of the barley shochu distillation residue, and the liquid content was diluted with water to The pH was adjusted to 4 and 3.6% by weight of glucose was added. After adjusting the pH to 5.5 with sodium hydroxide, the mixture was sterilized at 121 ° C. for 15 minutes to obtain a culture medium for lactic acid bacteria.
・ Pre-culture of lactic acid bacteria capable of producing nisin
Inoculate 50 μl of a stock strain of Lactococcus lactis IO-1 in 10 ml of TGC medium and incubate at 37 ° C. for 18 hours to obtain a culture solution. Inoculate 10 ml of the culture solution in 100 ml of CMG medium and incubate at 37 ° C. for 3 hours. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
・ Main culture of lactic acid bacteria capable of producing nisin
Into a 2 L jar fermenter, 500 ml of the lactic acid bacteria culture medium and 25 ml of the lactic acid bacteria preculture were introduced, and batch culture was performed under the conditions of a stirring speed of 250 rpm, a culture temperature of 30 ° C., a culture time of 24 hours, and a pH of 5.5. The lactic acid bacteria culture solution was freeze-dried.

[Natto culture medium using barley shochu distillation residual liquid]
-The barley shochu distillation residue obtained in the distillation process of barley shochu production was centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue, and sodium hydroxide was added to the liquid to The pH value was adjusted to 7.0. The obtained preparation solution was sterilized to obtain a culture medium for Bacillus natto.
-Dissolve 10g of natto precultured meat extract and 10g of peptone in 1L of distilled water, add sodium hydroxide to the resulting solution to adjust its pH value to 7.0, and then sterilize at 121 ° C for 15 minutes Processing was performed to obtain a meat extract medium. The meat extract medium (5 ml) and commercially available natto bacteria (Miyagino 1 platinum ear) were introduced into a test tube, stirred, and cultured with shaking at 42 ° C. for 15 hours to obtain a pre-cultured natto bacteria culture solution.
・ Main culture of Bacillus natto
Into a 2 L jar fermenter, 1 L of the above natto bacteria culture medium and 5 ml of the above natto bacteria preculture were introduced, and cultured for 14 days under the conditions of an aeration rate of 0.2 vvm, a stirring speed of 300 rpm, and a culture temperature of 42 ° C. A dextrin of 3 times the solid content of the Bacillus natto culture was mixed and freeze-dried.

[Fermented barley fiber alkali extract]
Barley shochu moromi was produced using 1 ton of 70% barley barley, fermented / ripened for about 2 weeks according to a conventional method, and alcohol was separated by a single distiller to obtain 1.5 kL of barley shochu distillation residue. The obtained barley shochu distillation residue 1 kL was subjected to solid-liquid separation using a screw press, and 50 kg of barley groove convention was obtained from the obtained solid. The obtained barley groove was dried with a drum dryer and then pulverized with a roll mill to obtain 5.5 kg of barley groove powder (composition). 50 g of the barley groove powder was suspended in 1 L of 2% Ca (OH) ₂ aqueous solution, stirred at room temperature for 6 hours, adjusted to pH 7.0 with HCL, and filtered through filter paper. The filtrate was lyophilized.

<Preparation of experimental samples, experimental methods and results>
1. Experimental sample
1-1 Sample preparation
1-1-1 For angiogenesis inhibition experiment Samples 1-1 to 1-11 mentioned above were weighed 1000 μg each, dissolved in 1 ml medium separately, sterilized by filtration (0.22 μm), and diluted 10 times (2 And samples having a concentration of 10 μg / ml to 100 μg / ml were prepared.
1-1-2 For TOF-MS measurement test After dissolving the above sample Nos. 1-1 to 1-4 in water at a concentration of 10 mg / ml, 1 mg / ml, 100 μg / ml, 10 μg / ml Dilute to concentrations of 1 μg / ml and 0.1 μg / ml.
* 1-11 has low solubility.

2. Experimental method and results
2-1 Angiogenesis inhibition experiment Human vascular endothelial cells and fibroblasts were co-cultured at the optimal concentration, and each sample (1-1-1) was added to the cells in the proliferative state at the initial stage of tube formation for 11 days. After culturing (changing 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 and then observed under a microscope. The formed lumen-like network structure was evaluated for the angiogenesis inhibitory effect. The obtained results are shown in FIG. As is clear from FIG. 1, the fermented barley fiber alkaline extract and the fermented barley extract P significantly inhibited tube formation.

2-2 TOF-MS measurement test
2-2-1 Measurement conditions Measurement was performed in the reflector mode under both positive ion and negative ion conditions. Beta-cyclodextrin (MW 1135.12) was used as a measurement reference substance, and calibration was performed using DNBA dimmer (positive ion mode 273, negative ion mode 307) and beta-cyclodextrin.
2-2-2 Sample preparation A 10 mg / ml 2, 5-dihydroxybenzoic acid (DHBA) aqueous solution or 20% ethanol aqueous solution was used for the matrix. The sample solution of each concentration was mixed 1: 1 or 1: 2 with the matrix solution, and 1 μl of each mixed solution was applied onto a measurement plate, allowed to air dry, and then measured in negative ion mode.
In the measurement in positive ion mode, polysaccharides generally have [M + Na] + or [M + K] as the main peak. Therefore, referring to a report of MALDI-TOF MS measurement of cyckodextrin derivatives [B. Chankvetadze et al., Carbohydrate Research, 287, 139-555 (1996)], sample solution: matrix solution: 30 mM NaCl aqueous solution = 1: 1: A mixed solution of 1 was also prepared and used for measurement in positive ion mode.
As a result, the same spectral pattern was observed for the four samples (not shown).
In positive ion mode, peaks were observed from the peak around 650 Da to about 3000 Da at intervals of about 210 Da, and the peak intensity decreased as the molecular weight increased. In negative ion mode, the same behavior was observed at an interval of about 210 Da from the peak of about 580 Da.
Like the 645 Da peak in the positive ion mode and the 576 Da peak in the negative ion mode, the peak interval showing close values in the positive / negative mode is approximately 69 Da, 69 Da corresponds to approximately 3Na +, and the NaCl aqueous solution at the time of sample preparation This is probably due to the addition of.
The following two points are considered as the possibility that peaks are observed at an interval of 210 Da.
1) The sugar-modified portion corresponding to 210 Da is cleaved with laser irradiation.
2) A mixture of homopolysaccharides composed of sugar equivalent to 210 Da.
Although the possibility about the above 1) was examined, the sugar modification corresponding to 210 Da was investigated and could not be found. On the other hand, regarding the possibility of 2), the molecular weight of the sugar having a carboxyl group (—COOH) in the modified part was very close to 209 Da.
[reference]
-NMR Measurement In order to examine the possibility of the above 2), 1H-NMR at 7 mg / ml in heavy water was measured for Sample 2-1 (not shown). When the measurement results were compared with the spectrum of beta-cyclodextrin, a very complex signal was observed at 3-5 ppm in addition to a signal that was thought to be derived from sugar at 1-3 ppm. In addition, a signal is also observed at 6.5-7.5 ppm, suggesting that a nickname compound having an amide proton or aromatic ring is included, and it is difficult to estimate the structure of the polysaccharide.
From the current results, it is inferred that the sample contains a polysaccharide having a molecular weight of about 600 to 3000 Da and a low molecular weight compound. Since compounds of 3000 Da or more may exist, we plan to examine the measurement conditions and continue measurement.

[Experiment 2] Angiogenesis inhibition experiment <Sample name>
As shown in Table 1.
A1, A2: Negative control
A3, A4: Positive control
D1-D3: Barley shochu distillation residual liquid synthetic adsorbent treated adsorption fraction powder (fermented barley extract P powder)

<Preparation method of sample used for angiogenesis inhibition experiment>
The sample used in this experiment has a balance with other experiments, and the powdered sample is redissolved and used in the experiment. However, there is no problem even if it is used in the experiment as it is in a liquid state. In addition, when powdered as described below, there is also a product added with an equivalent amount of dextrin as an extractant. The dextrin content is weight percent concentration (W / W).

[Barley Shochu Distillation Residue Synthetic Adsorbent Processed Adsorbed Fraction Powder] (Fermented Barley Extract P Powder)
The sample described in paragraph 0045 above was used.

<Preparation of experimental samples, experimental methods, and results>
1. Experimental sample
1-1 Sample preparation
1-1-1 For angiogenesis inhibition experiment Each sample is weighed 1000 μg, dissolved separately in 1 ml medium, sterilized by filtration (0.22 μm), diluted 10 times (3 times), and Table 1 (Vessels) A sample having a concentration of 10 μg / ml to 1000 μg / ml as shown in the Breakdown of Newborn Inhibitory Effect Evaluation Test Samples) was prepared. Similarly, negative control and positive control samples shown in Table 1 were prepared.
2. Experimental methods and results
2-1 Angiogenesis inhibition experiment Human vascular endothelial cells and fibroblasts were co-cultured at the optimum concentration, and each sample (1-1-1, negative control, positive control) was in a proliferative state at the initial stage of tube formation. After 11 days of culture (change the medium containing the sample after 4, 7, 9 days), tube formation was stained with Mouse anti-human CD31 and Goat anti-mouse IgG AlkP Conjugate, and then observed under a microscope . The formed lumen-like network structure was evaluated for the angiogenesis inhibitory effect.
The obtained results are shown in Table 2 and FIGS. The effect of inhibiting angiogenesis was determined by taking photographs of vascular tissues as digital data and measuring the areas of several randomly selected blood vessel portions (black portions). That is, the smaller the numerical value in the AREA column in Table 2, the higher the effect of inhibiting angiogenesis.

  The effect of inhibiting angiogenesis was evident in fermented barley extract P powder.

Experiment 3: Angiogenesis inhibition experiment <sample name>
As shown in Table 3.

Preparation method of samples (1) to (9) used in the angiogenesis inhibition experiment The sample used in this case has a balance with other experiments, and the powdered sample is redissolved and used in the experiment. There is no problem even if it is used in the experiment in the liquid state. In addition, when powdered as described below, there is also a product added with an equivalent amount of dextrin as an extractant. The dextrin content is weight percent concentration (W / W).

[Barley Shochu Distillation Residue Synthetic Adsorbent Processed Adsorbed Fraction Powder] (Fermented Barley Extract P Powder)
See paragraph 0044 above.

[Preparation of experimental samples, experimental methods, and results]
1. Experimental sample
1-1 Sample preparation
1-1-1 For angiogenesis inhibition experiment Each sample is weighed in 1000μg, dissolved separately in 1ml medium, sterilized by filtration (0.22μm), diluted 10 times (3 times), from 10μg / ml A sample with a concentration of 1000 μg / ml was prepared.

2. Experimental method and results
2-1 Angiogenesis inhibition experiment Human vascular endothelial cells and fibroblasts were co-cultured at the optimal concentration, and each sample was added to those in the proliferative state at the initial stage of lumen formation, and cultured for 11 days (4, 7, 9 The medium containing the sample was changed after day), and the tube formation was stained with Mouse anti-human CD31 and Goat anti-mouse IgG AlkP Conjugate and then observed under a microscope. As evaluation criteria, 10 ng / ml of VEGF-A that promotes tube formation, 50 μM of Suramin that inhibits tube formation were similarly added, and control was not added. The formed lumen-like network structure was evaluated for the angiogenesis inhibitory effect.
Method: Measured with a kit manufactured by Kurabo Industries. The lumen formation state (area, length, number of branches) at four points was observed. Using a system in which VEGF as an angiogenic factor and Suramin as an angiogenesis inhibitor coexist as a control, the luminal formation state in the presence of VEGF and each sample was compared.
(Comparison of angiogenesis inhibitory activity of Suramin and sample)
Sample addition concentration: 10, 100, 1000 μg / mL
(Suramin is all constant concentration; 50μM)
Results: The results obtained are shown in Table 3 (numerical data on the angiogenesis inhibitory effect) and FIG.

  From the results shown in Table 3 and FIG. 4, 100 μg of fermented barley extract P completely inhibited tube formation.

  The composition of the present invention is a cereal that has been indispensable for humankind since prehistoric times, and it has been applied to fermented barley, a gramineous plant that has been loved as a healthy food, as described in old Japanese medical books. It applies angiogenesis inhibition based on the active ingredient derived from the product, and is highly available as a functional food material.

It is a photograph replaced with drawing which shows the influence which the fermented barley extract of Example 1 has on angiogenesis. FIG. 5 is a photomicrograph in place of a drawing for explaining a state of lumen formation using VEGF-A and Suramin 50 μM in Example 2. FIG. A1; VEGF-A, A2; VEGF-A, A3; Suramin, A4; Suramin It is a microscope picture instead of drawing explaining the mode of lumen formation using the fermented barley extract P powder of Example 2 and describing the mode of lumen formation of a control cultured without addition. Fermented barley extract P powder addition amount D1; 10μg, D4; medium only 4 is a drawing for explaining the results of an angiogenesis inhibition test of Example 3. FIG.

Claims (10)

  A composition for inhibiting angiogenesis, characterized in that a component derived from fermentation of barley is used as an active ingredient for inhibiting angiogenesis.   The composition for inhibiting angiogenesis according to claim 1, wherein the component derived from fermentation of barley is a barley shochu distillation residue.   The composition for inhibiting angiogenesis according to claim 1 or 2, wherein the component derived from fermentation of barley is selected from the group consisting of fermented barley extract and fermented barley fiber.   The fermented barley extract comprises a composition obtained by fractionating a barley shochu distillation residue, a lactic acid bacteria culture solution using the barley shochu distillation residue as a medium, and a Bacillus natto culture solution using the barley shochu distillation residue as a medium. 4. The composition for inhibiting angiogenesis according to claim 3, wherein the fermented barley fiber is selected from the group and the fermented barley fiber alkali extract.   The composition obtained by fractionating the barley shochu distillation residue is a non-adsorbed fraction treated with barley shochu distillation residue synthesized adsorbent, a barley shochu distilled residue synthesized adsorbent treated adsorbent, and a barley shochu distilled residue. The composition for inhibiting angiogenesis according to claim 4, which is one or more compositions selected from the group consisting of liquid ethanol precipitation fractions.   6. The composition for inhibiting angiogenesis according to any one of claims 1 to 5, wherein the composition for inhibiting angiogenesis is a composition for treating or preventing a disease for which angiogenesis is to be inhibited.   The composition for inhibiting angiogenesis according to claim 6, wherein the disease to inhibit angiogenesis is a disease caused by abnormal angiogenesis in a tumor or cancer, chronic inflammation or retinopathy.  The composition according to any one of claims 1 to 7, wherein the composition is in a form selected from the group consisting of food additives for inhibiting angiogenesis, food materials, food and drink, pharmaceuticals / quasi drugs, and feed. A composition for inhibiting angiogenesis.   The composition for inhibiting angiogenesis according to claim 8, wherein the food or drink is a functional food, a nutritional supplement or a health food or drink for inhibiting angiogenesis.   The composition for inhibiting angiogenesis according to claim 8, wherein the feed is a feed for livestock, poultry or pets for inhibiting angiogenesis.