JP2006022033A - Neovascularization inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a neovascularization inhibitor which can effectively inhibit neovascularization and has improved safety for living bodies. <P>SOLUTION: This neovascularization inhibitor capable of being used in the fields of foods, medicines and the like is disclosed. The neovascularization inhibitor contains an extract originating from at least one alga selected from the group consisting of Laminaria japonica in Laminariaceae, Phaeophyceae; Eisenia bicyclis, Eisenia arborea, Ecklonia stlonifera, Ecklonia kurome, Ecklonia cava, Undaria pinnatifida, Undaria peterseniana, Undaria undarioides, Alaria praelomga, and Alaria crassifolia in Alaria, Phaeophyceae; Sargassum fuluvellum, Hizikia fusiforme, and Sargassum horneri in Sargassaceae, Phaeophyceae; Nemacystus decipieus and Cladosiphon okamuranus in Spermatochnaceae, Phaeophyceae; and algae in Gigartinaceae, Gigartinales, Phaeophyceae, as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an angiogenesis inhibitor, and more specifically, prevents or treats angiogenesis diseases such as cancer growth or metastasis, diabetic retinopathy, rheumatoid arthritis, etc. by inhibiting angiogenesis in vivo. The present invention relates to an angiogenesis inhibitor obtained.

  Angiogenesis plays an important role in development, wound healing, and the like. On the other hand, angiogenesis is known to be involved in angiogenic diseases such as cancer growth or metastasis, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and psoriasis.

  Angiogenesis is understood as the process by which new blood vessels are formed from existing blood vessels. Angiogenesis occurs through the migration, proliferation and differentiation of endothelial cells after the production of proteases by vascular endothelial cells.

  Regarding the relationship between the above-mentioned angiogenic diseases and angiogenesis, in recent years, ligands such as VEGF (Vascular Endothelial Growth Factor), angiopoietin, which are specific factors that act on vascular endothelial cells and promote angiogenesis, and The receptor system has been clarified as it was discovered one after another. Therefore, suppressing angiogenesis is considered effective for the prevention and treatment of these diseases.

Conventionally, various blood vessel inhibitors for the purpose of suppressing such angiogenesis are known. As such a vascular inhibitor, for example, a K ⁺ channel blocker is contained as an active ingredient as a substance that inhibits the action of endothelium-derived hyperpolarizing factor (EDHF) and suppresses angiogenesis. (Patent Document 1), a compound containing a carboline derivative as an active ingredient (Patent Document 2), a compound containing a compound produced by a specific strain belonging to the genus Aspergillus (Patent Document 3), docosapenta Those containing enoic acid (DPA) or a derivative thereof as an active ingredient (Patent Document 4) and those containing polyalkoxyflavonoids as an active ingredient are known (Patent Document 5).

  However, such conventional anti-angiogenic agents are highly cytotoxic and are likely to cause side effects, require many production steps until products are obtained, or are intended to prevent or treat the above-mentioned angiogenic diseases On the other hand, it has been pointed out that the inhibitory effect on angiogenesis is still not sufficient, and there are concerns about practicality. In particular, since prevention or treatment of such diseases requires continuous treatment over a long period of time, an angiogenesis inhibitor that can achieve both effective inhibition of angiogenesis and avoidance of side effects is desirable. Has been.

JP 2003-38441 A JP 2003-321363 A JP 2003-183249 A JP 2003-308765 A JP 2004-083417 A

  An object of the present invention is to provide an angiogenesis inhibitor that can more effectively suppress angiogenesis and has improved safety with respect to a living body. There is.

  The present invention relates to brown algae (Aminifera), laminaria japonica, brown algae (Eisenia bilocris), red sea urchin (Eisenia arborea), vine (Eckneria ck). (Ecklonia cava), sea turtle (Undaria pinnatifida), green sea turtle (Undaria peterenia), brown sea bream (Undaria unarrioides), brown sea turtle (Aldaria peas) ), Hinoki (Hizikia fusiforme), and red mosquito (Sargassum horneri); brown alga (Megacystus depipieus) and Okinawa moss (Cladosifonus algae); An angiogenesis inhibitor comprising, as an active ingredient, an extract derived from at least one seaweed selected from the group consisting of;

  In a preferred embodiment, the extract is an extract obtained from a supernatant of 50 (v / v)% or more C1-C3 alcohol solution containing the hot water crude extract derived from the seaweed.

  In a more preferred embodiment, the extract is obtained by subjecting the concentrate of the supernatant to column chromatography using 40 (v / v)% to 90 (v / v)% C1-C3 alcohol aqueous solution. It is an extract derived from the obtained fraction.

  In an even more preferred embodiment, the adsorbent used in the column chromatography is a styrene-divinylbenzene adsorbent.

  The present invention is an angiogenesis inhibitor containing an extract derived from seaweed as an active ingredient, and the extract contains fluorotannin in a proportion of 15% by weight or more based on the weight of solids contained in the extract. Contains an angiogenesis inhibitor.

  The present invention is also an angiogenesis inhibitor containing phlorotannin as an active ingredient.

  In a preferred embodiment, the fluorotannin is derived from seaweed.

The present invention is also a method for producing an angiogenesis inhibitor,
Brown algae (Epidonia varneur), brown algae (Eisenia biloclis), brown turtle (Eisenia arborea) , Seaweed (Undaria pinnatifida), green sea turtle (Undaria peterseniana), Japanese flounder (Undaria uniaroides), brown squirrel (Alaria promagida) H zikia fusiforme, and red moss (Sargassum horneri); brown algae moths (Nemacytus decipieus) and Okinawa mosquito (Cladosifon okamuranus); Extracting at least one selected seaweed from hot water at 60 ° C. to 100 ° C. to obtain a hot water crude extract;
Combining the C1 to C3 alcohol or the C1 to C3 alcohol aqueous solution with the hot water crude extract to obtain an alcohol liquid containing 50% (v / v)% or more of the alcohol; Sorting step;
A method comprising

  In a preferred embodiment, the step of further concentrating the supernatant to obtain a concentrate of the supernatant; and the concentration of the supernatant of 40 (v / v)% to 90 (v / v)% Passing through a column using an aqueous C1-C3 alcohol solution.

  In a further preferred embodiment, the column contains a styrene-divinylbenzene adsorbent.

  ADVANTAGE OF THE INVENTION According to this invention, while being able to suppress angiogenesis more effectively, the safety | security with respect to a biological body can also be improved. For this reason, the angiogenesis inhibitor of the present invention can be more routinely used, and versatility can be enhanced in various fields such as the food field and the pharmaceutical field. The angiogenesis inhibitor of the present invention is also used for the growth and metastasis of solid tumors (gastric cancer, colon cancer, lung cancer, pancreatic cancer, etc.), retina such as diabetic retinopathy, macular degeneration, retinal vein occlusion, retinal artery occlusion It can be used for the purpose of prevention or treatment of diseases, neovascular glaucoma, rheumatism, rheumatoid arthritis, various inflammatory diseases such as psoriasis, pathological conditions such as atherosclerosis and myocardial infarction.

  First, the first angiogenesis inhibitor of the present invention will be described.

The first angiogenesis inhibitor of the present invention contains an extract derived from seaweed as an active ingredient.

  Examples of the seaweed used in the present invention include the brown algae Kombu Kombu family, the brown algae Kombu Ogura family, the brown algae Hiba mata Hidawara family, the brown algae Nagamatsu and Mizushi family, or the red algae Suginori Examples include seaweeds belonging to the Suginori family.

  Specific examples included in the seaweed include the brown algae Kombu Kombu (Laminaria japonica), the brown algae Osmophila Alame (Eisenia bicyclis), Sagarame (Eisenia arborea), tsuru arame stlonifera), chromate (Ecklonia kurome), beforehand (Ecklonia cava), seaweed (Undaria pinnatifida), Aowakame (Undaria peterseniana), spread (Undaria undarioides), Ainu wakame ((Alaria praelomga), and Chigaiso (Alaria crassifolia); brown algae Hibamata Hondawara Honda Sahara (Sa rgasssum fluvellum, hijiki fusiforme, and red spider (Sargassum horneri); brown algae (Nemacytus decipeius) and red moss As the seaweed used in the present invention, a fraction of an extract having a relatively large production amount of seaweed itself and easily available and having an inhibitory action on angiogenesis described later is obtained. Alame, chrome, seaweed, and hinoki are preferred because they are easily treated.

  In the present specification, the seaweed includes the whole grass of these seaweeds themselves or at least one part including roots, stems, and leaves. The seaweed forms used in the present invention include raw ones, fragments, strips or powders. Furthermore, the seaweed used in the present invention includes both dry matter and raw state.

  In the present invention, the extract means an extract obtained by extracting the seaweed as described above using water, a polar or nonpolar solvent, or a mixture thereof as an extraction solvent under appropriate conditions. The form of the extract is not particularly limited, and includes an extract, or a powder or paste obtained by concentrating or drying the extract by means usually used by those skilled in the art.

  The seaweed-derived extract used in the present invention is preferably extracted from water and / or alcohol (for example, methanol, ethanol, or propanol) derived from the kind of seaweed as described above, more preferably It was obtained from the supernatant of the C1-C3 alcohol liquid containing the hot water crude extract derived from the kind of seaweed. In particular, “what was obtained from the supernatant of a C1-C3 alcohol solution containing a seaweed-derived hot water crude extract” obtained a hot water crude extract from the seaweed through an operation as described below, It can be obtained by fractionating the supernatant from 80% (v / v)% or more of C1-C3 alcohol solution combined with C1-C3 alcohol or C1-C3 alcohol aqueous solution.

In the first angiogenesis inhibitor of the present invention, the extract is preferably 15% by weight or more, more preferably 18% by weight to 100% by weight of fluorotannin based on the weight of the solid content contained in the extract. %, Even more preferably from 30% to 99% by weight, even more preferably from 60% to 99% by weight, even more preferably from 60% to 99% by weight.
Here, the term “weight of solids contained in the extract” as used herein means that the liquid component constituting the extract is removed (for example, evaporated) to remain as a solid component. Refers to the weight of the substance to obtain. When the extract satisfies the phlorotannin content in such a range, the inhibitory action on angiogenesis is remarkably improved. The term “fluoro tannin” used in the present specification is a kind of polyphenol generally called seaweed tannin, and refers to a high molecular compound including the entire fluoro tannin having phloroglucinol as a basic skeleton unit. Say. The fluorotannin that can be used in the present invention includes any one kind of the fluorotannins or a mixture of a plurality of the fluorotannins.

  Next, the second angiogenesis inhibitor of the present invention will be described.

  The second angiogenesis inhibitor of the present invention contains an extract derived from seaweed as an active ingredient, and the extract contains 15% by weight or more of fluorotannin, preferably based on the weight of solids contained in the extract. Is contained in a proportion of 18 wt% to 100 wt%, more preferably 30 wt% to 99 wt%, even more preferably 60 wt% to 99 wt%, and even more preferably 60 wt% to 99 wt%.

  In the second angiogenesis inhibitor of the present invention, it is important that phlorotannins contained in seaweed are contained in the above range. Therefore, as long as this content range is satisfied, the type of seaweed that can be used is not particularly limited. Examples of seaweed that can be used as the extract contained in the second angiogenesis inhibitor of the present invention include seaweeds belonging to brown algae or red algae. In addition, more specific examples of these brown algae or red algae include the brown algae Kombu Kombu family, the brown algae Kombu Komiso family, the brown algae Hiba or Hamawara family, the brown algae Nagamatsu and Mizukumaku family. In addition, seaweeds belonging to the family of red seaweeds are also not particularly limited.

  More specific examples of seaweed that can be used in the second angiogenesis inhibitor of the present invention include those similar to the seaweed used in the first angiogenesis inhibitor of the present invention.

  In the first and second angiogenesis inhibitors of the present invention, the seaweed-derived extract used for the inhibitor is not particularly limited, and is produced, for example, as follows.

  First, a hot water crude extract is produced by immersing the seaweed in hot water for a predetermined time.

  The quantity ratio between the seaweed and hot water in this immersion is not particularly limited. For example, 1 liter to 10 liters, more preferably 2 liters to 5 liters of hot water is used per 100 g of seaweed (dry weight). used.

  The temperature (extraction temperature) of the hot water used is preferably 60 ° C to 100 ° C, more preferably 80 ° C to 100 ° C, and even more preferably 90 ° C to 100 ° C. In addition, while the seaweed is immersed, for the purpose of facilitating extraction of the active ingredient used in the present invention, it is heated using means known to those skilled in the art so as not to lower the temperature of hot water. It is preferable to maintain the temperature by doing so.

  The time of immersion in hot water (extraction time) varies depending on the extraction temperature to be used, and thus is not necessarily limited. For example, when hot water is maintained at approximately 100 ° C., preferably 1 minute to 120 minutes, more preferably Is from 10 to 60 minutes. By performing the extraction time within such a range, the active ingredient used in the present invention can be extracted more efficiently, and excessive extraction of unnecessary materials can be prevented.

  After the immersion, for example, it is allowed to cool to room temperature, and seaweed is removed by filtration or centrifugation. Thus, a hot water crude extract can be obtained. In addition, the obtained hot water crude extract may be derived from an aqueous layer from which the organic layer has been removed by combining with an organic solvent such as hexane or chloroform for the purpose of removing impurities in advance. Further, the obtained hot water crude extract may be used as it is for the steps described below, or, if necessary, a dry solid or a paste obtained by evaporating water using a means known to those skilled in the art. It may be used in the form of a thing.

  Next, the C1-C3 alcohol or the C1-C3 alcohol aqueous solution is combined with the crude hot water extract, preferably 50 (v / v)% or more, more preferably 60 (v / v)% to 98 (v / v). A C1-C3 alcohol solution having a% alcohol concentration is prepared. Here, the concentration of the C1-C3 alcohol solution that can be prepared can be set to any concentration by those skilled in the art, preferably within such a range. That is, the content of components that suppress angiogenesis important in the present invention may vary depending on the type of seaweed used, the site of use, the production area, the collection time, the storage state after collection, and the like. In addition, an error may occur in the obtained component content even in a series of extraction operations described later. Therefore, those skilled in the art. According to such conditions, the concentration of the alcohol solution that can be prepared can be arbitrarily set within this range.

  More specifically, the crude hot water extract is combined with an alcohol having 1 to 3 carbon atoms or an aqueous alcohol solution (hydrous alcohol) having 1 to 3 carbon atoms prepared to a predetermined concentration. Thus, an alcohol liquid having the alcohol concentration as described above is prepared. Examples of alcohols having 1 to 3 carbon atoms that can be used in such operations include methanol, ethanol and propanol, and combinations thereof. In consideration of further improving the safety to the living body, it is preferable to use ethanol. The amount of such alcohol or hydrous alcohol used is not particularly limited because it varies depending on the amount of hot water crude extract to be combined.

  Thereafter, the supernatant of the C1-C3 alcohol solution is collected.

  By preparing an alcohol solution set to the alcohol concentration, a substance insoluble in the alcohol solution may precipitate. The supernatant is collected for the purpose of removing this precipitate, and the supernatant can be removed by methods well known to those skilled in the art (for example, filtration or centrifugation).

  Thus, the seaweed-derived extract used in the present invention can be produced. The obtained seaweed-derived extract may be purified for the purpose of enhancing the anti-angiogenic activity in the present invention. This purification is performed by, for example, concentrating the supernatant obtained above using a means well known to those skilled in the art, and then preferably 40 (v / v)% to 90 (v / v) )%, More preferably 50 (v / v)% to 80 (v / v)%, by passing through a column using an aqueous solution of C1-C3 alcohol (preferably ethanol). The adsorbent useful for this column chromatography is preferably an aromatic adsorbent, and a more specific example is a styrene-divinylbenzene adsorbent. The styrene-divinylbenzene adsorbent is commercially available from Mitsubishi Chemical Corporation under the trade name Diaion HP20, for example. In the present invention, for the purpose of further enhancing the angiogenesis inhibitory effect, preliminary purification using water (for example, distilled water) is performed on the column before the purification using the C1-C3 alcohol aqueous solution. It is preferable to carry out. Further, the preliminary purification using water is preferably performed in a plurality of times.

  By performing the chromatography, it is possible to extract a fraction having an enhanced angiogenesis inhibitory effect. The obtained fraction can be used as it is as an angiogenesis inhibitor of the present invention.

  The angiogenesis inhibitor of the present invention can be used for any purpose, including those intended for oral administration or ingestion, those intended for percutaneous absorption, and those intended for external preparations for skin.

  The angiogenesis inhibitor of the present invention may also contain other components depending on the purpose in addition to the seaweed-derived extract as an active ingredient. Examples of other ingredients that may be included in the present invention include water; alcohol; processed meat products; rice, wheat, corn, potatoes, sweet potatoes, soy, kombu, wakame, tengusa and other common food materials and their powders; Sugars such as starch, starch syrup, lactose, glucose, fructose, sucrose, mannitol; general food additives such as spices, sweeteners, edible oils, vitamins; surfactants; excipients; coloring agents; preservatives; Coating aids; lactose; dextrin; corn starch; sorbitol; crystalline cellulose; polyvinyl pyrrolidone; oils; moisturizers; thickeners; The angiogenesis inhibitor of the present invention may further contain other drugs (including traditional Chinese medicine) as necessary. The content of such other components and / or other drugs is not particularly limited, and an appropriate amount can be selected by those skilled in the art.

  Furthermore, the angiogenesis inhibitor of the present invention is not necessarily limited to in vivo or in vitro, and can be widely used in any application where it is desired to exert an inhibitory effect on angiogenesis. That is, the angiogenesis inhibitor of the present invention is not particularly limited, but may be used, for example, as a kind of additive added to food compositions such as health foods and used in the production field of livestock or farmed fish. The feed composition may be used as it is or in combination with other feed materials, or it may be used as it is or in combination with other pharmaceutical compositions as a pharmaceutical composition such as pharmaceuticals and quasi drugs. Also good.

  When the angiogenesis inhibitor of the present invention is used as a food composition, the form is not limited to a fixed food, but includes a beverage (for example, a liquid beverage). More specific examples include tea drinks, coffee drinks, soft drinks, milk drinks, confectionery, syrups, processed fruit products, processed vegetable products, pickles, and livestock meat in the form of liquids, pastes, solids, etc. Examples include, but are not limited to, products, fish products, delicacies, cans / bottles, instant food / drinks, oral liquids, liver oil drops, mouth fresheners, and jelly. Such a food composition containing the β-glucuronidase inhibitor of the present invention can be produced using techniques known to those skilled in the art.

  When the angiogenesis inhibitor of the present invention is used as a pharmaceutical composition, its dosage form is not particularly limited, and is processed into an appropriate dosage form according to the method described in the Japanese Pharmacopoeia. More specific examples of dosage forms include, for pharmaceutical compositions intended for oral administration, dosage forms such as capsules, tablets, powders, granules, fine granules, sustained release agents, and liquids. In the case of pharmaceutical compositions intended for parenteral administration, dosage forms such as injections, infusions, eye drops, ointments, creams, patches and the like can be mentioned. Since the dose can be easily varied depending on conditions such as the body weight of the subject person, it can be appropriately selected by those skilled in the art.

  The angiogenesis inhibitor of the present invention can be produced by a method usually used in the field depending on its use form, and can be appropriately ingested or applied by a method according to its form.

  Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited by these.

<Example 1: Production of arame extract>
100 g of lyophilized arame was placed in a mixer to prepare a dry powder. To this was added 2 L of distilled water, extraction was performed by heating at 100 ° C. for 10 minutes, and the extraction residue was removed by filtration to obtain a hot water crude extract (700 ml), to which ethanol was added Thus, a 90 (v / v)% ethanol solution was prepared.

  Thereafter, the obtained ethanol solution was filtered to remove only the supernatant. Subsequently, the supernatant was concentrated under reduced pressure to obtain 350 ml of an ethanol-soluble fraction. A column (φ3) containing 50 ml of a styrene-divinylbenzene adsorbent (Diaion HP20 manufactured by Mitsubishi Chemical Corporation) of the aqueous layer obtained by separating and extracting the ethanol soluble fraction with 350 ml of chloroform. (A) Distilled water (500 ml), (B) Distilled water (500 mL), (C) 30 (v / v)% ethanol aqueous solution (500 ml), (D) 60 Elution was successively performed using (v / v)% ethanol aqueous solution (500 ml) and (E) 100 (v / v)% ethanol (500 ml), and crude fractions (A), (B), (C), ( D) and (E) were collected in 500 ml portions. Next, after the solvents of the crude fractions (A), (B), (C), (D) and (E) were distilled off under reduced pressure, they were again dissolved or resuspended in 40 ml of distilled water and frozen. It dried and obtained the lamé fraction (A), (B), (C), (D) and (E).

<Example 2: Cell culture and luminal staining>
Using an angiogenesis kit (Cat No. KZ-1000 manufactured by Kurashiki Boseki Co., Ltd.), the following was performed according to the instruction manual attached to the kit: First, the cells in the kit were recovered from damage during transportation. The cells were allowed to stand for 3 hours at 37 ° C., 5% CO ₂ incubator. The culture medium in each well was mixed with the arame fraction (D) obtained in Example 1 so that the final concentration in the medium was 50 μg / ml, and VEGF (blood vessel so that the final concentration in the medium was 10 ng / ml. The medium was replaced with a fresh medium supplemented with endothelial growth factor (Vascular Endothelial Growth Factor), and cultured in a 37 ° C., 5% CO ₂ incubator. The medium was changed in the same manner on the fourth day, the seventh day, and the ninth day after the start of the culture.

  On day 11 after the start of culture, the wells were stained with a luminal staining kit (for CD31 staining) (Katashibo Industries, Ltd. Cat No. KZ-1225) as follows: First, after removing the medium from the wells and washing the cells with 1 ml of Dulbecco's phosphate buffer PBS (−), 1 ml of ice-cold 70% methanol was added to each well and left at room temperature for 30 minutes to leave the cells. Fixed. Next, after removing ethanol, 1 ml of PBS (-) containing 1% BSA (hereinafter referred to as a blocking agent) was added to each well to wash the cells. Further, 0.5 ml of a primary antibody diluted to 4000 times with a blocking agent was added to each well and incubated at 37 ° C. for 60 minutes. After removing the primary antibody and washing the cells three times with a blocking agent, 0.5 ml of a secondary antibody diluted 500-fold with the blocking agent was added to each well and incubated at 37 ° C. for 60 minutes. After incubation, the secondary antibody was removed and each well was washed 3 times with 1 ml of milliQ water.

  On the other hand, two tablets of BCIP / NBT (bromochloroindolyl phosphate / nitroblue tetrazolium) were dissolved in 20 ml of milli-Q water, and the filtered solution was used as a substrate solution. 0.5 ml of this substrate solution was added to the wells and incubated at 37 ° C. until the lumen became deep purple (about 10 minutes). The substrate solution was removed and each well was washed three times with 1 ml of milliQ water, and then the plate was left to stand and air dried. Using a inverted microscope equipped with a digital still camera (DIAPHOT-TMD manufactured by Nikon Corporation), three representative fields per well were photographed at a magnification of 40 times, and the images were analyzed using dedicated analysis software (Kurashiki Spinning Co., Ltd.) ) Angiogenesis quantification software), and the lumen length, lumen area, number of lumen joints (number of branch points) and number of lumen paths (obtained by branching) in the image The number of branches was analyzed. The obtained length, area, number of joints, and number of passes were recalculated with the result obtained in Comparative Example 1 (control) described later as 100%.

  A photograph showing an image obtained in this example is shown in FIG. In addition, the results of the lumen length, the lumen area, the number of lumen joints, and the number of lumen paths, which are obtained for the control and indicated by the image, are shown in FIGS. 4 to 7, respectively.

<Example 3: Cell culture and luminal staining>
For the medium in each well, add the lamé fraction (D) obtained in Example 1 so that the final concentration in the medium is 5 μg / ml, and VEGF so that the final concentration in the medium is 10 ng / ml. Incubation and luminal staining were performed in the same manner as in Example 2 except that each of the added media was used.

  A photomicrograph obtained in this example is shown in FIG. In addition, the results of the lumen length, the lumen area, the number of lumen joints, and the number of lumen paths, which are obtained for the control and indicated by the image, are shown in FIGS. 4 to 7, respectively.

<Comparative Example 1: Cell culture and luminal staining>
A medium (control) to which VEGF was added so that the final concentration in the medium was 10 ng / ml was used without containing the alame fraction (D) obtained in Example 1 for the medium in each well. Except that, incubation and luminal staining were performed in the same manner as in Example 2.

  A photograph showing an image obtained in this comparative example is shown in FIG. In addition, the results obtained in this comparative example (control) shown in the image, when the lumen length, the lumen area, the number of lumen joints, and the number of lumen paths are 100%, are shown in FIGS. 7 shows.

  As shown in FIGS. 1 to 3, in the photomicrograph of Comparative Example 1 using a control medium, blood vessels (branches in a complicated branch in the figure) were formed in the well by performing a series of incubations. However, according to the micrographs of Example 2 (FIG. 1) and Example 3 (FIG. 2), such a branch is hardly formed. From this, it can be seen that the alame fraction (D) used in this example has an effect of suppressing the neovascularization. In particular, in Example 2 set to a higher concentration (final medium concentration 50 μg / ml) than in Example 3 (final medium concentration 5 μg / ml), almost no angiogenesis occurred, and angiogenesis was suppressed. It turns out that the effect was improved more.

  Moreover, as shown in FIGS. 4 to 7, the alame fraction (D) used in Examples 2 and 3 was compared to the control (Comparative Example 1), the lumen length of the blood vessel, the lumen area, Both the number of luminal joints and the number of luminal paths show low values, which indicates that they have a sufficient anti-angiogenic effect. Further, as in the above micrograph, the higher the concentration of the lamé fraction (D), the lower the value in any of the lumen length, lumen area, number of lumen joints and number of lumen paths. You can also see that

<Example 4: Production of arame extract>
100 g of lyophilized arame was placed in a mixer to prepare a dry powder. To this was added 2 L of distilled water, extraction was performed by heating at 100 ° C. for 10 minutes, and the extraction residue was removed by filtration to obtain a crude hot water extract (700 ml). Next, among the obtained hot water crude extract, ethanol was added to 100 ml of the extract extracted in a beaker to prepare a 90 (v / v)% ethanol solution.

  Thereafter, the obtained ethanol solution was filtered to remove only the supernatant. Then, the supernatant was concentrated under reduced pressure and lyophilized to obtain 0.9 g of an ethanol-soluble fraction (F).

Using an angiogenesis kit (Cat No. KZ-1000 manufactured by Kurashiki Boseki Co., Ltd.), the following was performed according to the instruction manual attached to the kit: First, the cells in the kit were recovered from damage during transportation. The cells were allowed to stand for 3 hours at 37 ° C., 5% CO ₂ incubator. The medium in each well was divided into the ethanol fraction (F) obtained above so that the final concentration in the medium was 50 μg / ml, and VEGF (vascular endothelial growth) so that the final concentration in the medium was 10 ng / ml. The medium was replaced with a fresh medium supplemented with each factor (Vascular Endothelial Growth Factor), and cultured in a 37 ° C., 5% CO ₂ incubator. The medium was changed in the same manner on the fourth day, the seventh day, and the ninth day after the start of the culture.

  On day 11 after the start of culture, the wells were stained with a luminal staining kit (for CD31 staining) (Katashibo Industries, Ltd. Cat No. KZ-1225) as follows: First, after removing the medium from the wells and washing the cells with 1 ml of Dulbecco's phosphate buffer PBS (−), 1 ml of ice-cold 70% methanol was added to each well and left at room temperature for 30 minutes to leave the cells. Fixed. Next, after removing ethanol, 1 ml of PBS (-) containing 1% BSA (hereinafter referred to as a blocking agent) was added to each well to wash the cells. Further, 0.5 ml of a primary antibody diluted to 4000 times with a blocking agent was added to each well and incubated at 37 ° C. for 60 minutes. After removing the primary antibody and washing the cells three times with a blocking agent, 0.5 ml of a secondary antibody diluted 500-fold with the blocking agent was added to each well and incubated at 37 ° C. for 60 minutes. After incubation, the secondary antibody was removed and each well was washed 3 times with 1 ml of milliQ water.

  On the other hand, two tablets of BCIP / NBT (bromochloroindolyl phosphate / nitroblue tetrazolium) were dissolved in 20 ml of milliQ water, and the filtered solution was used as a substrate solution. 0.5 ml of this substrate solution was added to the wells and incubated at 37 ° C. until the lumen became deep purple (about 10 minutes). The substrate solution was removed and each well was washed three times with 1 ml of milliQ water, and then the plate was left to stand and air dried. Using a inverted microscope equipped with a digital still camera (DIAPHOT-TMD manufactured by Nikon Corporation), three representative fields per well were photographed at a magnification of 40 times, and the images were analyzed using dedicated analysis software (Kurashiki Spinning Co., Ltd.) ) Angiogenesis quantification software), and the lumen length, lumen area, number of lumen joints (number of branch points) and number of lumen paths (obtained by branching) in the image The number of branches was analyzed. The obtained length, area, number of joints, and number of passes were recalculated with the result obtained in Comparative Example 1 (control) as 100%.

  The results of the lumen length, the lumen area, the number of lumen joints, and the number of lumen paths shown in the images obtained in this example with respect to the control are shown in FIGS.

<Example 5: Production of arame extract>
100 g of lyophilized arame was placed in a mixer to prepare a dry powder. To this was added 2 L of distilled water, extraction was performed by heating at 100 ° C. for 10 minutes, and the extraction residue was removed by filtration to obtain a crude hot water extract (700 ml). Next, of the obtained hot water crude extract, 100 ml of the extracted solution separated in a beaker, and ethanol was added thereto to prepare a 60 (v / v)% ethanol solution.

  Thereafter, the obtained ethanol solution was filtered to remove only the supernatant. Subsequently, the supernatant was concentrated under reduced pressure and freeze-dried to obtain 1.2 g of an ethanol-soluble fraction (G).

  This ethanol soluble fraction (G) is used instead of the ethanol soluble fraction (F), and the ethanol soluble fraction is adjusted so that the final concentration in the medium is 50 μg / ml with respect to the medium in each well. Incubation and luminal staining were performed in the same manner as in Example 4 except that (G) and a medium to which VEGF was added so that the final concentration in the medium was 10 ng / ml were used.

  The results of the lumen length, the lumen area, the number of lumen joints, and the number of lumen paths shown in the images obtained in this example with respect to the control are shown in FIGS.

  As shown in FIGS. 8 to 11, the ethanol-soluble fractions (F) and (G) used in Examples 4 and 5 were compared to the control (Comparative Example 1), the lumen length of the blood vessel, A low value is shown in any of the luminal area, the luminal joint number, and the luminal path number, and it can be seen that it has a sufficient anti-angiogenic effect.

<Example 6: Quantification of fluorotannin>
Alame fractions (A) to (E) obtained in Example 1 and ethanol soluble fractions (F) and (G) obtained in Examples 4 and 5 were accurately weighed in 100 mg each, The total amount of phlorotannins in each fraction was measured by the Folin-Denis method using phloroglucinol as a standard substance (Journal of Japan Society for Food Science and Technology, Vol. 49, pp. 507-511). Table 1 and Table 2 show the total amount (%) of each obtained fluorotannin.

  As shown in Table 1, all of the lamée fractions (A) to (E) obtained in Example 1 contain phlorotannins, but their contents differ markedly depending on the fractions. I understand. In particular, the results of this alame fraction (D) and the micrographs obtained in Example 2 (FIGS. 1 and 2), as well as the lumen length, lumen area, number of lumen joints and number of lumen passes. Considering the results, it is considered that the angiogenesis inhibitory effect shown above may be largely contributed by fluorotannin contained as a main component in the alame fraction (D). Moreover, as shown in Table 2, in order to obtain an ethanol-soluble fraction, an ethanol solution prepared by adding ethanol to the hot water crude extract has a preparation concentration of particularly 60 (v / v). % To 98 (v / v)% (Example 4 prepared to 90 (v / v)%, Example 5 prepared to 60 (v / v)%) It can be seen that it is already contained at a high rate of nearly 20% by weight based on the weight of the melt fraction.

<Example 7: Preparation of food composition>
A food composition having the following composition was prepared using the alame fraction (D) obtained in Example 1 above.

Ingredient Weight (g)
Alame fraction (D) 0.6
Soy Saponin 2.0
Black vinegar extract 2.0
Apple fiber 2.0
Lecithin 1.0
Fructooligosaccharide 2.0
Fructose 1.0
Powdered vinegar 0.1
Cyclodextrin 1.0
Honey 1.0
Bone meal 1.0
Dextrin 4.9.

  Each component was mixed in a fluid granulator, granulated by spraying water, and dried at an inlet temperature of 80 ° C. to obtain a granular food product.

  Since the angiogenesis inhibitor of the present invention contains an extract derived from seaweed that can be widely used as food as an active ingredient, it is highly safe and can be used on a daily basis. The versatility in various fields can be improved. Since the angiogenesis inhibitor of the present invention also has an effect of suppressing angiogenesis in vivo, retinal diseases such as diabetic retinopathy, macular degeneration, retinal vein occlusion, retinal artery occlusion, and neovascular glaucoma Prevention of pathological conditions such as rheumatism, rheumatoid arthritis, various inflammatory diseases such as psoriasis, atherosclerosis, proliferation / metastasis of solid tumors (gastric cancer, colon cancer, lung cancer, pancreatic cancer, etc.), myocardial infarction It can be used for therapeutic purposes.

It is a microscope picture which shows the state of the angiogenesis expressed with the cell in the well which added the alame fraction obtained in Example 2. FIG. It is a microscope picture which shows the state of the angiogenesis expressed by the cell in the well which added the alame fraction obtained in Example 3. FIG. 2 is a photomicrograph showing the state of angiogenesis expressed by cells in a control well, obtained in Comparative Example 1. FIG. 5 is a graph for explaining the state of angiogenesis between a well to which an allame fraction has been added and a control well, and is obtained in Examples 2 and 3 based on the lumen length of the control. It is a graph which shows obtained lumen length (%). FIG. 5 is a graph for explaining the state of angiogenesis between a well to which an arame fraction has been added and a control well, obtained in Examples 2 and 3 based on the luminal area of the control. 5 is a graph showing the lumen area (%). FIG. 5 is a graph for explaining the state of angiogenesis between a well to which an alame fraction has been added and a control well, obtained in Examples 2 and 3 based on the number of luminal joints of the control. It is a graph which shows the obtained number of lumen joints (%). FIG. 5 is a graph for explaining the state of angiogenesis between a well to which an alame fraction has been added and a control well, obtained in Examples 2 and 3 on the basis of the number of luminal paths of the control. It is a graph which shows the obtained luminal pass number (%). FIG. 6 is a graph for explaining the state of angiogenesis between a well to which an ethanol-soluble fraction has been added and a control well, and Examples 4 and 5 based on the lumen length of the control. It is a graph which shows the luminal length (%) obtained by (1). FIG. 5 is a graph for explaining the state of angiogenesis between a well to which an ethanol-soluble fraction has been added and a control well, and in Examples 4 and 5 based on the luminal area of the control. It is a graph which shows the obtained luminal area (%). FIG. 4 is a graph for explaining the state of angiogenesis between a well to which an ethanol-soluble fraction has been added and a control well, and Examples 4 and 5 based on the number of control luminal joints. It is a graph which shows the number of luminal joints (%) obtained by (1). FIG. 4 is a graph for explaining the state of angiogenesis between a well to which an ethanol-soluble fraction has been added and a control well, and Examples 4 and 5 based on the number of luminal paths of the control It is a graph which shows the luminal pass number (%) obtained by (1).

Claims (10)

  Brown algae (Epidonia varneur), brown algae (Eisenia biloclis), brown turtle (Eisenia arborea) , Seaweed (Undaria pinnatifida), green sea turtle (Undaria peterseniana), hirome (Undaria undarioides), brown algae (Alaria promagida) (Hizikia fusiforme), and red moss (Sargassum horneri); brown algae (Coleoptera, Nemacystus decipieus) and Okinawa mosquito (Cladosifon okamuranus); An angiogenesis inhibitor comprising, as an active ingredient, an extract derived from at least one seaweed selected from the group.   The blood vessel according to claim 1, wherein the extract is an extract obtained from a supernatant of a C1-C3 alcohol solution of 50 (v / v)% or more containing the crude hydrothermal extract derived from the seaweed. Neonatal inhibitor.   The extract is derived from a fraction obtained by subjecting the supernatant concentrate to column chromatography using 40 (v / v)% to 90 (v / v)% aqueous C1-C3 alcohol. The angiogenesis inhibitor according to claim 2, which is an extract.   The angiogenesis inhibitor of Claim 3 whose adsorbent used for the said column chromatography is a styrene- divinylbenzene type adsorbent.   An angiogenesis inhibitor comprising a seaweed-derived extract as an active ingredient, wherein the extract contains fluortannin in a proportion of 15% by weight or more based on the weight of solids contained in the extract Neonatal inhibitor.   An angiogenesis inhibitor comprising phlorotannin as an active ingredient.   The angiogenesis inhibitor according to claim 6, wherein the fluorotannin is derived from seaweed. A method for producing an angiogenesis inhibitor,
Brown algae (Epidonia varneur), brown algae (Eisenia biloclis), brown turtle (Eisenia arborea) , Sea turtles (Undaria pinnatifida), green sea turtles (Undaria peterseniana), white turtles (Undaria unisariidae), brown turtles Ikia fusiforme, and the red seaweed (Sargassum horneri); the brown alga (Megatus decipius) and the red seaweed (Cladosifon okamuranus); Extracting at least one selected seaweed from hot water at 60 ° C. to 100 ° C. to obtain a hot water crude extract;
Combining the C1 to C3 alcohol or the C1 to C3 alcohol aqueous solution with the hot water crude extract to obtain a C1 to C3 alcohol liquid containing 50% (v / v)% or more of the alcohol; and the C1 to C3 Separating the supernatant of the alcohol solution;
Including the method.   A step of concentrating the supernatant to obtain a concentrate of the supernatant; and 9. A method according to claim 8, comprising the step of passing through a column using The method according to claim 9, wherein the column contains a styrene-divinylbenzene adsorbent.

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