WO2017061559A1 - Blood-vessel-wall strengthening drug, method for strengthening blood vessel wall, and vasoganglion-formation suppressing drug - Google Patents

Abstract

The purpose of the present invention is to provide drugs and a method with which it is possible to normalize abnormal blood vessels. The present inventors have found that arctigenin has a normalizing effect on abnormal blood vessels on the basis of findings that administering an Arctium lappa extract containing arctigenin to an abnormal-blood-vessel formation model increased the amount of pericyte-covered blood vessels, caused formation of a normal vasoganglion, improved the blood-flow state, and considerably decreased the area of low-oxygen regions. A blood-vessel-wall strengthening drug, a vasoganglion-formation suppressing drug, and a blood-vessel normalizing drug according to the present invention contain arctigenin as an active ingredient.

Description

Vessel wall reinforcing agent, method for reinforcing vessel wall, and vessel network formation inhibitor

The present invention relates to a vascular wall reinforcing agent and a vascular network for reconstituting normal blood flow in a tissue by suppressing the formation of an abnormal vascular network that randomly grows and reinforcing immature and fragile blood vessels. The present invention relates to a formation inhibitor and a blood vessel normalizing agent.

Angiogenesis is a process in which blood vessels are newly formed from existing blood vessels to build a blood vessel network. The formation of mature blood vessels is mainly caused by the stabilization of the blood vessel structure by adhesion of blood vessel wall cells such as pericite or vascular smooth muscle cells to the vascular endothelial cells that cover the inner lumen of the blood vessel. Generally, in a normoxic state, vascular wall cells line vascular endothelial cells and the vascular structure is stabilized. However, when a blood vessel is needed in a tissue in response to changes in environmental factors inside and outside the bloodstream, such as hypoxia and strong inflammatory stimuli, the new blood vessel rapidly expands from the existing blood vessel, resulting in an immature decline in strength. Blood vessel formation is induced.

Normal angiogenesis is physiologically related to the development of body tissues in the early stages of pregnancy (placentation and fetal development), wound healing processes (after surgery and injury), and formation of collateral circulation around the ischemic site ( It is known to play an important role in myocardial infarction, obstructive arteriosclerosis, and the like. On the other hand, abnormalities in angiogenesis cause various diseases such as cardiovascular diseases, skin diseases, and malignant tumors. For example, tumor tissue forms a disordered vascular network as the tissue grows rapidly, and the blood vessels themselves are fragile and frequently bleed, creating a highly stressed environment, creating a chronic hypoxic environment and a low hypoxic environment. Nutritional status is caused. Cancer cells that survived in such harsh environments have been reported to have higher malignancy and resistance to treatment.

In such a cancer tissue having immature tumor blood vessels, blood vessels are born but hypoxia persists due to circulatory failure. In the hypoxic state, the invasion and metastasis ability of cancer cells is enhanced, and immature tumor blood vessels provide a path for cancer cells to enter the blood vessels, thereby promoting cancer metastasis.

It has been pointed out that circulatory insufficiency of tumor tissues not only results in hypoxia and nutrition, but also affects the delivery of anticancer drugs to tumor tissues. Therefore, systems and compositions for delivering anticancer agents to tumor tissues have been proposed. For example, vascular endothelial growth factor (VEGF), which has an action of promoting angiogenesis, is induced by hypoxia. It has been pointed out that this anti-VEGF antibody (bevacizumab) targeting VEGF improves blood flow by inhibiting the formation of disordered vascular networks, which will improve the delivery of anticancer drugs to cancer tissues (Non-Patent Document 1). However, the effect of the anti-VEGF antibody (bevacizumab) is temporary, and mature tumor blood vessels regress with time, and the tumor tissue becomes hypoxic. Moreover, when the dose is increased, side effects based on the damage of normal vascular endothelial cells are enhanced. Therefore, it is difficult to adjust an appropriate administration period and dosage for the combined use of an anti-VEGF antibody (bevacizumab) and an anticancer agent (Non-patent Document 2).

Also, a method for targeting APJ (7-transmembrane G protein coupled receptor) as a drug delivery system is disclosed. However, this method does not deliver the drug to the mature blood vessels of normal tissues, but to the blood vessels of the tumor tissue containing mature blood vessels, and increases hypoxia and hypotrophic areas. There is a possibility of further enhancing treatment resistance (Patent Document 1).

In addition to cancer, tissue cells are placed under chronic hypoxia and nutritional conditions such as myocardial infarction, cerebral infarction and diabetic blood flow disorder. In such a tissue, it is known that rapid angiogenesis is performed to recover the tissue, and an immature and fragile blood vessel is formed. Therefore, it is thought to cause cerebral hemorrhage, thrombus formation and infarct recurrence, as well as diseases such as diabetic retinopathy and neovascular glaucoma. Chronic inflammation may also induce ectopic and unregulated angiogenesis due to the delivery of immune cells to tissues, causing causes such as wet age-related macular degeneration, rheumatoid arthritis and psoriasis It also becomes. Such disordered angiogenesis is also thought to exacerbate inflammation itself.

From recent studies, it has been pointed out that SASP (senescence-associated secretory phenotype) associated with cellular aging may be the true form of chronic inflammation associated with aging. This is the idea that senescent cells that accumulate in body tissues with aging contribute to increased inflammation. As side effects of this cellular aging, it is considered that carcinogenesis may be accelerated and chronic inflammatory diseases may be caused. DNA damage associated with aging, accumulation of reactive oxygen species and shortening of telomeres activate p53, RAS and p16 ^INK4a signaling pathways and induce cellular senescence. As a result, inflammatory cytokines such as IL-1β, IL-6 and IL-8, vascular endothelial growth factor (VEGF) and the like are secreted. This is SASP, and this has led to the spread of further cellular aging and chronic inflammation of surrounding cells, and it is thought that it will eventually lead to death of the individual through geriatric syndrome.

As described above, SASP causes chronic inflammation, but in the lesion area, intracellular energy consumption accompanying cell proliferation and hypermetabolism causes a hypoxic environment. This hypoxic environment is thought to contribute to the worsening of the disease state. When hypoxia occurs in tissue, the adhesion between vascular endothelial cells and connective tissue, extracellular matrix and basement membrane is weakened, and vascular endothelial cells can move easily. This causes branching and elongation of new blood vessels, causing (vascular remodeling) vascular damage. As a result of cytotoxicity, various intracellular substances are released, and these cytotoxicity-related molecular patterns (DAMPs) elicit inflammatory responses via their receptors, causing vascular cellular senescence. Aged blood vessels are formed from immature blood vessels, and blood circulation is poor due to increased interstitial pressure due to leakage of blood components. Therefore, vicious cycles where chronic diseases further progress due to persistent hypoxia and nutrition of tissues. It is thought that is causing. It is considered that a blood vessel normalizing agent having an effect of enhancing the strength of blood vessels and maintaining a blood vessel network can exert an effect on chronic diseases associated with aging due to aging.

Development of a method for normalizing abnormal blood vessels is desired in order to prevent and treat such cancer, diabetes, inflammatory diseases (including inflammation associated with aging) and cardiovascular diseases. In addition, immature angiogenesis, such as ulcerative colitis and inflammatory bowel diseases such as Crohn's disease (IBD), atherosclerosis and endometriosis, is considered to be an important contributing factor. There are many diseases. Therefore, the development of a method for arranging abnormal blood vessels into a mature normal blood vessel network is considered to make a great contribution to the prevention, treatment and improvement of these diseases.

International Publication No.2012 / 102363

The formation of an abnormal vascular network that grows randomly can cause various diseases as described above. In addition, in tissues with abnormal vascular networks, the blood vessels are immature and fragile, and a circulatory insufficiency creates a special microenvironment of hypoxia and nutrition that can affect the delivery of therapeutic agents. It has become a big problem. Therefore, development of a method for suppressing the formation of abnormal blood vessel networks, strengthening the blood vessel wall, and normalizing abnormal blood vessels is required.

An object of the present invention is to provide a drug and a method capable of normalizing abnormal blood vessels.

The present inventors have found that when a gobo cow extract containing arctigenin is administered to a mouse transplanted with human pancreatic cancer cells, which is a model of abnormal angiogenesis, the blood flow state in the tumor tissue is improved. Furthermore, the present inventors have found that administration of burdock extract containing arctigenin significantly reduces the hypoxic region in the tumor, increases the number of cells in the growth phase, and increases the number of pericite-coated blood vessels. Furthermore, the present inventors have found that administration of a gobo cow extract containing arctigenin forms a normal vascular network without the blood vessels inside the tumor being randomly packed, meandering, or entangled with adjacent blood vessels. It was. From these results, the present inventors have found that arctigenin has an action of strengthening the blood vessel wall, suppressing abnormal vascular network formation, and normalizing abnormal blood vessels, and completed the present invention.

That is, the present invention provides a vascular wall reinforcing agent for increasing blood vessels covered with vascular wall cells, which contains archigenin as an active ingredient.

In addition, the present invention provides the above-mentioned vascular wall reinforcing agent, wherein arctigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The present invention also provides a vascular network formation inhibitor comprising archigenin as an active ingredient.

The present invention also provides the vascular network formation inhibitor, wherein arctigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The present invention also provides a method for strengthening a blood vessel wall, comprising the step of ingesting architigenin and / or arctiin into a subject in need of strengthening the blood vessel wall.

In addition, the present invention provides a method for reinforcing the above-mentioned blood vessel wall, wherein arctigenin and / or arcticin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The present invention also provides a method for suppressing vascular network formation, comprising the step of ingesting archigenin and / or arctiin into a subject in need of suppression of vascular network formation.

In addition, the present invention provides a method for suppressing the formation of the above vascular network, wherein arctigenin and / or arcuin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention improve the blood flow state by normalizing abnormal blood vessels, prevent and improve various diseases caused by abnormal blood vessels, and Can contribute to treatment.

The figure which shows the blood-flow state in the tumor after administering the arguchigenin-rich burdock extract. The figure which shows the ratio (%) of the positive area | region of the pimonidazole in a tumor after administering an arctivenin high content gobo cow extract. The figure which shows the ratio (%) of an intratumoral BrdU positive cell after administering the architigenin-rich burdock extract. The figure which shows the ratio (%) of the pericyte covering blood vessel in a tumor after administering architigenin-rich burdock extract. The figure which shows the tumor blood-vessel structure after administering archigenin-rich burdock extract. The figure which shows the density | concentration of CPT-11 in the blood after administration of the arguchigenin-rich burdock extract. The figure which shows the appearance frequency of the ulcer tissue in a tumor after administering together with an arctiggenin-rich burdock extract and bevacizumab. The figure which shows the survival rate after administering together with the arguchigenin high content gobo cow extract and bevacizumab. The figure which shows the survival rate after administering together arcticogenin high content gobo cow extract and gemcitabine to a pancreatic cancer orthotopic transplant mouse | mouth. The figure which shows the fluctuation | variation of the tumor size by the existing chemotherapeutic agent with respect to a pancreatic cancer model, an arguchigenin-rich burdock extract, and these combination.

The present invention provides a vascular wall strengthening agent containing arctigenin as an active ingredient. The blood vessel wall reinforcing agent of the present invention reinforces the blood vessel wall by increasing blood vessels covered with blood vessel wall cells. The vascular wall reinforcing agent of the present invention can enhance the vascular wall and improve the vascular strength by increasing the frequency with which newly formed blood vessels or existing immature blood vessels are covered with vascular wall cells. Moreover, the vascular wall reinforcing agent of the present invention can suppress the formation of abnormal vascular network by reinforcing the vascular wall, and can be used as a vascular network formation inhibitor. The vascular network formation inhibitor of the present invention can induce a significant decrease in the sprouting of the first random microvascular that creates an abnormal vascular network.

Also, the vascular wall reinforcing agent and vascular network formation inhibitor of the present invention can normalize blood vessels by reinforcing the vascular wall and suppressing the formation of abnormal vascular networks. Therefore, the vascular wall reinforcing agent and vascular network formation inhibitor of the present invention can be used as a vascular normalizing agent.

In the present specification, “strengthening the blood vessel wall” means increasing blood vessels covered with blood vessel wall cells. In the present specification, “blood vessel wall cells” include pericytes (pericytes) and smooth muscle cells. As used herein, “suppressing vascular network formation” refers to reducing the vascular network, particularly abnormal vascular network, and reducing the first random microvascular sprouting that creates an abnormal vascular network. Including that.

In the present specification, “normalizing a blood vessel” means normalizing or maturing an abnormal blood vessel or blood vessel network to form a normal blood vessel or blood vessel network. In the present specification, “abnormal blood vessel” or “immature blood vessel” refers to an immature and fragile blood vessel that is not covered with blood vessel wall cells, a deformed blood vessel, a blood vessel that is excessively and abnormally meandering, and an adjacent blood vessel. Blood vessels, blood vessels with excessive branching, and blood vessels that do not circulate because the end of the blood vessel is not connected to other blood vessels. In the present specification, the term “abnormal vascular network” or “disordered vascular network” refers to a vascular network including the above-described abnormal blood vessels, a dense and complicated vascular network, and adjacent blood vessels. A blood vessel network and a blood vessel network in which blood vessels are not connected and do not circulate. As used herein, “normal blood vessel” or “mature blood vessel” refers to a blood vessel that is connected without interruption, a blood vessel that is covered with vascular wall cells, a blood vessel that is not excessively meandered or branched, and other ends. A blood vessel that is connected to other blood vessels and circulates sufficiently. In the present specification, the term “normal vascular network” refers to a vascular network composed of the above-described normal blood vessels, a vascular network having a regular and hierarchical structure (hierarchy), a vascular network that spreads uniformly without being dense, and a terminal network Refers to a vascular network that is connected to each other and circulates sufficiently.

The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention are used for complex branched and disordered vascular network caused by abnormal meandering or excessive vascular sprouting caused by ischemic or inflammatory conditions. Inhibits formation or reinforces immature and fragile blood vessels formed in this state into blood vessels that maintain normal strength. The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention suppress the formation of an abnormal vascular network and strengthen the vascular wall, thereby finally having a normal circulatory function. Can be formed.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can improve the blood flow state of the tissue and reduce the hypoxic region by normalizing the blood vessels. Further, the vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention suppress the formation of disordered vascular network, and have normal strength coated with vascular wall cells from immature blood vessels. By maturation into blood vessels, increase in vascular permeability (including suppression of bleeding tendency) is suppressed, and normal blood flow in the tissue can be reconstructed by preparing the vascular network.

Further, the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can improve the repair and development of damaged tissue by improving the blood flow state, Delivery of therapeutic agents can be improved. In addition, when the cause of abnormal vascular network formation is a tumor or infected tissue, improving the blood flow state improves the delivery of immune cells that exclude diseased cells and pathogens to the diseased tissue. be able to. In cancer, it is known that a hypoxic environment triggers a change in the cell state called epithelial-mesenchymal transformation in cancer cells, which changes into cells with high therapeutic resistance. By improving the above, malignant transformation of such cancer tissue can be suppressed. Moreover, improvement in the sensitivity of radiation therapy can be expected by improving the oxygen state of the tumor.

Arctinigenin used in the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention may be derived from a plant containing archigenin. Plants containing arctigenin are not particularly limited. Thistle, Ayoko Forsythia, Ginkgo biloba, Forsythia, Sylar Forsythia, Sesame, Momijigaigao, Shinchiku Himehagi, Datura Kizura, Teika Kazura, Mine Teika Kazura, Hime Teika Kazura, Tokyo Oyster Crane, Otari Kazura , Oats, spelled wheat, soft wheat, Mexican cypress and kaya. Of these, burdock, burdock, burdock sprout, young burdock, and forsythia are preferred because of their high arctigenin content. Arctigenin may be contained as an extract from these plants.

In the present invention, when arctigenin is derived from burdock, a burdock extract obtained using a method for producing a burdock extract described later can be used. Therefore, productivity at the time of manufacture can be improved, and a vascular wall reinforcing agent, a vascular network formation inhibitor, and a vascular normalizing agent can be prepared inexpensively and easily. Moreover, also when using plants other than burdock, it is possible to easily obtain an extract containing arctigenin by using the production method described later.

The burdock contains about 7% arctiin, which is classified as a lignan glycoside, and about 0.6% arctigenin, which is an aglycon of arctiin. Since the method for producing a burdock extract to be described later can significantly increase the content of arctigenin relative to arctiin, the extract obtained by this production method contains a high content of arctigenin. Therefore, if the extract obtained by the method for producing this burdock extract is used, compared with the conventional burdock extract, the vascular wall strengthening agent, the vascular network formation inhibitor and the vascular normalizing agent which have superior effects can do.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention may further contain alktiin. Arctiin may be derived from a plant containing arctiin, and may be derived from, for example, burdock, forsythia, or forsythia. The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention are contained in this burdock extract by containing an extract from a plant containing arctigenin, for example, a burdock extract obtained from burdock. The alktiin may be further contained.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention may contain archigenin and archtiin so that the weight ratio of archigenin / arctiin is 0.7 or more. The weight ratio of archigenin / arctiin is not particularly limited, but may be 1.3 or less. The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention comprise an extract of a plant containing archigenin and archtiin in a weight ratio of archigenin / arctiin = 0.7 to 1.3, for example, burdock extract May be. In addition, the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention may contain a burdock extract containing 3% or more archigenin. Such a burdock extract can be obtained by a method for producing a burdock extract described later. When the vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention contain a burdock extract obtained by a method for producing a burdock extract described later, the conventional burdock extract is contained. Higher vascular normalization effect.

For the vascular wall reinforcing agent, vascular network formation inhibitor, and vascular normalizing agent of the present invention, the extract powder obtained by the method for producing a burdock extract described later can be used as it is.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can further contain optional components. For example, the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention are pharmaceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants, coloring agents, and the like. Can be provided.

Examples of carriers and excipients used for vascular wall strengthening agents, vascular network formation inhibitors and vascular normalizing agents include lactose, glucose, sucrose, mannitol, dextrin, potato starch, corn starch, calcium carbonate, calcium phosphate, sulfuric acid Includes calcium and crystalline cellulose.

Examples of the binder include starch, gelatin, syrup, tragacanth gum, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like.

Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginate, sodium carboxymethylcellulose, and carboxymethylcellulose calcium.

Also, examples of lubricants include magnesium stearate, hydrogenated vegetable oil, talc and macrogol. As the colorant, any colorant allowed to be added to a pharmaceutical product can be used.

In addition, a vascular wall reinforcing agent, a vascular network formation inhibitor and a vascular normalizing agent are sucrose, gelatin, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, It may be coated with one or more layers of cellulose phthalate acetate, hydroxypropyl methylcellulose phthalate, methyl methacrylate, methacrylic acid polymer, and the like.

In addition, a pH regulator, a buffer, a stabilizer, a solubilizer, and the like may be added to the blood vessel wall reinforcing agent, the blood vessel network formation inhibitor, and the blood vessel normalizing agent as necessary.

Also, the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent can be provided as a preparation of any form. For example, vascular wall strengthening agents, vascular network formation inhibitors and vascular normalizing agents are capsules including tablets, troches, pills, powders and soft capsules such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets as oral preparations. , Syrups including granules, suspensions, emulsions, dry syrups, and liquids such as elixirs.

In addition, vascular wall reinforcing agents, vascular network formation inhibitors and vascular normalizing agents are for parenteral administration, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, transpulmonary administration. It can be a preparation for administration such as administration, enteral administration, buccal administration and transmucosal administration. For example, it can be an injection, a transdermal absorption tape, an aerosol, a suppository and the like. In addition, when an extract from a plant or the like is used, since the extract has a peculiar taste, it can be used as a preparation for masking the extract or a film coating agent coated with a coating agent. .

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can be administered to, for example, mammals. Mammals include, for example, humans, mice, rats, rabbits, cats, dogs, cows, horses and monkeys.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can contain archigenin so that the daily dose is 10 to 2000 mg per adult. Further, archigenin is not particularly limited, but may be administered 1 to 7 days a week. For example, arctigenin may be administered daily or 5 or 6 times a week.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention may be administered in combination with any drug. An arbitrary drug is, for example, a drug for preventing, ameliorating or treating any disease, for example, an anticancer drug. The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention normalize the blood vessels at the site to be treated, thereby enhancing the drug delivery of the drugs administered in combination to the site to be treated. be able to.

For example, in an environment where many immature blood vessels are formed, such as tumors and chronic inflammation, it is considered that circulatory insufficiency is likely to occur, drug delivery becomes poor, and the drug is less effective. Therefore, if the immature blood vessels are normalized by the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention to improve the blood flow state, it is considered that the combined drugs are more effective. In Test Examples 5 to 8 to be described later, when architigenin and an existing anticancer agent are administered in combination, the inhibition of ulcer formation is enhanced and the tumor growth is suppressed, compared with the case where the anticancer agent is administered alone, It has been shown to reduce the intratumoral ratio and prolong survival. As described above, the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can increase the drug delivery by normalizing the blood vessels and enhance the effect of the drug.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention take the form of, for example, pharmaceuticals, foods for patients, health foods, functional foods, foods for specified health use, dietary supplements and supplements. be able to. The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention also have an indication that it has the effect of strengthening the vascular wall, an indication that it has the effect of suppressing vascular network formation, and normalizes the blood vessel. It may be in the form of a functionally labeled food with a label indicating that there is an effect of the effect.

The present invention also provides a food composition for reinforcing a blood vessel wall, which contains arctigenin as an active ingredient and increases blood vessels covered with blood vessel wall cells. The present invention also provides a food composition for inhibiting vascular network formation, which contains arctigenin as an active ingredient. The present invention also provides a food composition for normalizing blood vessels, which contains arctigenin as an active ingredient. The food composition for strengthening blood vessel wall, the food composition for suppressing vascular network formation, and the food composition for normalizing blood vessel according to the present invention are configured in the same manner as the above-described vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent. can do.

In the present specification, the “food composition” includes not only general foods and drinks but also foods for patients, health foods, functional foods, foods for specified health use, dietary supplements and supplements. Common foods and drinks include, for example, various beverages, various foods, processed foods, liquid foods (soups, etc.), seasonings, energy drinks, and confectionery. In the present specification, the “processed food” refers to a product obtained by processing and / or cooking natural ingredients (animals, plants, etc.), such as processed meat products, processed vegetable products, processed fruit products, frozen foods, Includes retort foods, canned foods, bottled foods and instant foods. The food composition of the present invention may be a food with an indication that the blood vessel wall is strengthened, an indication that the vascular network formation is suppressed, or an indication that the blood vessels are normalized. Moreover, the food composition of the present invention may be provided in a form enclosed in a bag, a container or the like. The bags and containers used in the present invention can be any bags and containers normally used for food.

The present invention also provides a method for strengthening a blood vessel wall, which includes the step of ingesting archigenin and / or arctiin into a subject in need of strengthening the blood vessel wall. The present invention also provides a method for inhibiting vascular network formation, comprising the step of ingesting archigenin and / or arctiin to a subject in need of inhibition of vascular network formation. The present invention also provides a method of normalizing a blood vessel, comprising the step of ingesting archigenin and / or arctiin into a subject in need of blood vessel normalization.

The subjects to which the method of the present invention is applied include mammals such as humans, mice, rats, rabbits, cats, dogs, cows, horses and monkeys. Subjects who need vascular wall reinforcement, subjects who need to suppress vascular network formation, and subjects who need normalization of blood vessels are those who develop abnormal angiogenesis and abnormal angiogenesis Such as a subject suffering from a disease considered to be. Target diseases include, for example, cancer, cardiovascular diseases, various diseases with chronic inflammation, such as diabetic complications (nephropathy, retinopathy, neurosis), age-related macular degeneration, and neovascular glaucoma Inflammatory bowel disease (IBD) such as internal neovascular disease, ulcerative colitis and Crohn's disease, atherosclerosis, endometriosis, arteriosclerosis, psoriasis, brain and myocardial infarction, chronic kidney disease, non-alcoholic Examples include steatohepatitis, Alzheimer's disease, Parkinson's disease, obesity and lifestyle-related diseases, and skin photoaging.

Arctiin is a precursor of arctigenin and is known to be metabolized in vivo to become arctigenin. In the method of the present invention, only one of arctigenin or arcutin may be ingested, or both arctigenin and arcuinin may be ingested.

In the method of the present invention, arctigenin and / or arctiin may be derived from a plant containing arctigenin and / or arctiin, and may be derived from, for example, burdock, burdock, burdock sprout, young burdock and forsythia. Further, arctigenin and / or arctiin may be ingested as a burdock extract obtained using a method for producing a burdock extract described later. Moreover, you may be made to ingest arbitrary components with archigenin and / or archtiin. Optional ingredients include, for example, pharmaceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants, colorants, and the like.

Arctigenin and / or arctiin may be taken orally or parenterally (injection, application, patch, suppository, eye drops, nasal drop, inhalant, mouthwash, sublingual absorbent, implant, etc.) Good. In addition, archigenin and / or archtiin may be ingested so that the daily intake is 10 to 2000 mg per adult. Further, archigenin and / or archtiin is not particularly limited, but may be ingested 1 to 7 days a week. For example, archigenin and / or archtiin may be taken daily or 5 or 6 times a week.

The method of the present invention includes an embodiment in which archigenin and / or archtiin is ingested not only pharmaceutically but also non-pharmaceutically. For example, the method of the present invention includes embodiments in which archigenin and / or archtiin is ingested by a subject in the form of a food for a sick person, a health food, a functional food, a food for specified health use, a dietary supplement, and a supplement.

[Production method of burdock extract]
The burdock extract suitable for the present invention is produced through a herbal medicine cutting process, an extraction process (enzyme conversion process and extraction process using an organic solvent), a solid-liquid separation process, a concentration process, and a drying process.

(Herbal medicine cutting process)
In the crude drug cutting process, the raw burdock is cut into a size suitable for extraction. Herbal medicines that are raw materials have various sizes, shapes, and hardness such as various parts of plants, minerals, and animals, and cutting according to their characteristics is necessary. The burdock can be cut using any means known to those skilled in the art. For example, a commercially available cutting machine can be used.

In the method for producing a burdock extract suitable for the present invention, the activity of β-glucosidase, which is an enzyme inherent in the burdock, is measured in advance, and a suitable burdock can be selected. As a method for measuring the activity of β-glucosidase, for example, p-nitrophenyl-β-D-glucopyranoside (C12H15NO8: molecular weight 301.25) (manufactured by SIGMA-ALDRICH) is used as a substrate, and it is generated by acting a burdock product. By measuring the change in absorbance of p-nitrophenol at 400 nm, the enzyme activity can be measured. The amount of enzyme that produces 1 micromole of p-nitrophenol per minute can be expressed as 1 unit (U).

In order to obtain a burdock extract suitable for the present invention, a burdock having a β-glucosidase activity inherent in the burdock, for example, 0.4 U / g or more, preferably 1 U / g or more can be used. When it is less than 0.4 U / g, hydrolysis becomes insufficient, the weight ratio of archigenin decreases, and a desired burdock extract cannot be obtained efficiently.

Moreover, in the method for producing a burdock extract suitable for the present invention, a burdock cut to an arbitrary particle size can be used. It is considered that the smaller the particle size of the cut burdock, the more the enzyme conversion is promoted and the extract yield is increased. On the other hand, if the particle size is too small, the enzyme conversion may be too fast, making process control difficult, and hindering accurate solid-liquid separation in subsequent steps.

In order to obtain a burdock extract suitable for the present invention, as shown in the examples below, the burdock is cut to a particle size of 9.5 mm or less, for example, through a 9.5 mm sieve. . Further, in order to obtain a burdock extract suitable for the present invention, it is more preferable that the particle size of the burdock passes through the whole 9.5 mm sieve, for example, 60 to 100% is distributed on the 0.85 mm sieve. It is desirable to cut so that 65 to 80% is distributed on a screen of mm.

(Extraction process)
The extraction process is the most important process in terms of quality in the crude drug extract powder manufacturing process. This extraction step determines the quality of the herbal extract powder. In the method for producing a burdock extract suitable for the present invention, in order to extract the burdock extract, extraction is carried out in two stages, an enzyme conversion step and an extraction step using an organic solvent.

(Enzyme conversion process)
The enzyme conversion step is an important step in the method for producing a burdock extract suitable for the present invention. The enzyme conversion step is a step of converting arctiin contained in burdock into argtigenin by β-glucosidase, which is an enzyme inherent in burdock.

Specifically, the burdock cut material prepared in the above step is maintained at an appropriate temperature to cause β-glucosidase to act, thereby causing the reaction from arctiin to arctigenin. For example, an arbitrary solution such as water is added to the cut burdock and stirred at a temperature of about 30 ° C., so that the burdock can be maintained at an arbitrary temperature.

In order to obtain a burdock extract suitable for the present invention, the cut burdock is kept at a temperature around 30 ° C., for example, at a temperature between 20 and 50 ° C. When the temperature is lower than 20 ° C., hydrolysis becomes insufficient, the weight ratio of arctigenin decreases, and a desired burdock extract cannot be efficiently obtained. On the other hand, when the temperature is higher than 50 ° C., the enzyme is deactivated, the weight ratio of archigenin decreases, and a desired burdock extract cannot be efficiently obtained.

Further, the holding time is not particularly limited as long as it is held at the above temperature, and for example, it can be held for about 30 minutes. By maintaining the temperature between 20 ° C. and 50 ° C., regardless of the holding time, an appropriate amount of arctiin can be enzymatically converted to arctigenin, and a burdock extract suitable for the present invention can be obtained.

(Extraction process with organic solvent)
The extraction step with an organic solvent is a step of extracting arctigenin and arctiin from burdock using any appropriate organic solvent. That is, it is a step of extracting a burdock extract by adding an appropriate solvent in a state where the content of archigenin is increased by the enzyme conversion step. For example, a suitable solvent is added to the burdock extract, and the burdock extract is extracted by heating and stirring for a suitable time. In addition to heating and stirring, the burdock extract can be extracted using any extraction method known to those skilled in the art, such as heating reflux, drip extraction, immersion extraction, or pressure extraction.

Since arctigenin is sparingly soluble in water, the yield of arctigenin can be improved by adding an organic solvent. Any organic solvent can be used as the organic solvent. For example, alcohols such as methanol, ethanol and propanol, and acetone can be used. In view of safety, it is preferable to use ethanol as the organic solvent in the method for producing a burdock extract suitable for the present invention.

When the burdock extract is extracted by heating and stirring, the heating and stirring can be performed at an arbitrary temperature. However, in order to obtain a burdock extract suitable for the present invention, a temperature of 80 ° C. or higher, for example, 80 to 90 is used. Hold at a temperature between ° C. In addition, the time for heating and stirring is not particularly limited as long as heating and stirring is performed at the above temperature. By stirring and heating for about 30 minutes, for example, 30 to 60 minutes, architigenin and arctiin can be extracted from burdock into the solvent. .

The yield of arctigenin and arcthiin increases as the heating and stirring time increases. However, if the heating and stirring time is long, a lot of unnecessary oils and fats are dissolved, and the load of the concentration process is increased. Therefore, what is necessary is just to determine the time of heat stirring suitably according to a condition.

Further, the yield of arctigenin and arctiin increases as the amount of ethanol increases, so that the solubility of arctigenin and arctiin increases. However, if the amount of ethanol is large, a lot of unnecessary fats and oils are dissolved, and the load of the concentration process is increased. Therefore, the input amount may be appropriately determined according to the situation. In addition, the burdock extract can be simultaneously sterilized and sterilized by heating and stirring in this step.

(Solid-liquid separation process)
The solid-liquid separation step is a step of separating the burdock that has been extracted from the extract. Solid-liquid separation can be performed using any method known to those skilled in the art. Examples of the solid-liquid separation method include a filtration method, a sedimentation method, and a centrifugal separation method. Industrially, a centrifugal separation method is desirable.

(Concentration process)
The concentration step is a step of removing the solvent from the burdock extract prior to drying. Removal of the solvent from the burdock extract can be performed using any method known to those skilled in the art. However, it is preferable that the extract from the burdock obtained by the above process is not exposed to a higher temperature for a longer time. For example, by using the vacuum concentration method, it is possible to concentrate the burdock extract without being exposed to a high temperature for a long time.

The concentration of the burdock extract can be concentrated to a concentration at which a burdock extract having a desired concentration can be obtained. For example, it is desirable to concentrate to the extent that drying can be appropriately performed in the following drying process. In addition, when the burdock extract is dried into a powder formulation in the following steps, it is desirable to concentrate to a concentration at which appropriate formulation characteristics can be obtained.

Since arctigenin and arctiin are sparingly soluble in water, the amount of arctigenin and arctiin adhering to the production apparatus in the following drying process is large, and the final yield is greatly reduced. Therefore, dextrin can be added to the burdock extract obtained in this concentration step in order to prevent arctigenin and arctiin from adhering to the production apparatus. The amount of dextrin added is preferably about 15 to 30% with respect to the solid content of the concentrate, for example.

(Drying process)
It is a step of finishing the burdock extract obtained by the above step into a powder form. Drying can be performed using any method known to those skilled in the art. For example, freeze drying and spray drying are known as drying methods. The former is generally used at the laboratory level and the latter is used at the mass production level.

By the above manufacturing process, a burdock extract containing a high content of arctigenin can be obtained. This method for producing burdock extract must include a step of performing enzyme conversion at a temperature of 20 ° C. to 50 ° C., but does not need to include all of the other steps.

In addition, the above-described production process makes it possible to obtain a burdock extract with a high concentration of arctigenin at low cost and in a simple manner. Therefore, by using the burdock extract obtained by this method, the vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can be produced inexpensively and easily.

In addition, since the arguchigenin concentration of the burdock extract obtained by the above production process is high, it is one of the vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent compared with the case of using the conventional burdock extract. The total amount per day can be reduced. Therefore, the burden on the patient can be reduced.

Example 1
A burdock extract containing arctigenin was prepared by the following method.

The burdock (enzyme activity: 7.82 U / g) was cut and the whole 9.5 mm sieve was passed through a 0.85 mm sieve to confirm that 75% remained. 80 kg of this burdock slice was added to 560 L of water kept at 30 to 32 ° C. and stirred for 30 minutes, and then 253 L of ethanol was added, the temperature was raised to 85 ° C., and the mixture was further heated to reflux for 40 minutes. This liquid was centrifuged to obtain the resulting extract. Extracts obtained by repeating this operation twice were combined, concentrated under reduced pressure, and 25% dextrin was added to the extract solids, followed by spray drying. Arctigenin and arcthiin contents were 6.4% and 7.2%, respectively, and burdock extract powder (containing 25% dextrin) having an arctigenin / arctiin (weight ratio) = 0.89 was obtained.

[Test Example 1]
As an abnormal blood vessel model, a mouse transplanted with tumor cells was used. To functionally analyze the changes in the tumor microenvironment caused by repeated administration of arguchigenin-containing gobobull extract, the blood flow in the tumor was evaluated.

1 × 10 ⁶ cells / 200 μl of human pancreatic cancer cell Suit2 was transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan). On the 21st day of transplantation, the transplanted mice were divided into 4 groups: (a) untreated group, (b) gobo cow extract administered group, (c) bevacizumab administered group, and (d) gemcitabine administered group, and treatment was continued for 4 weeks. did. As for the burdock extract, mice were fed with 0.5% (w / w) of the burdock extract of Example 1 in the diet. When the daily food intake of the mouse is calculated as 3 to 5 g, arctigenin is administered at 15 to 25 mg / animal / day. Bevacizumab was administered intraperitoneally at a dose of 5 mg / kg once a week using 100 mg / 4 ml (Chugai Pharmaceutical) for intravenous infusion. Gemcitabine was intraperitoneally administered 100 mg / kg twice a week using a gemcitabine preparation for infusion (Eli Lilly and Company).

(Tumor size)
The mean tumor size in each group 4 weeks after the start of treatment was (a) 228 mm ^{2 in} the untreated group, (b) 189 mm ^{2 in} the gobobull extract administration group, (c) 56 mm ^{2 in} the bevacizumab administration group, and (d) The gemcitabine administration group was 68 mm ² .

(Incorporation of gadolinium DTPA in tumor tissue)
Four weeks after the start of treatment, uptake of gadolinium DTPA in each group of mouse tumor tissues was evaluated. Using a 9.4 Tesla MRI system (BIOSPEC 94 / 20USR), a dynamic contrast T1-weighted image of the tumor (time resolution 1.8-5.3 seconds) was taken up to 120 seconds after rapid intravenous injection of gadolinium DTPA. From this image, the signal intensity of gadolinium DTPA in the tumor tissue was measured. FIG. 1 is a graph showing the average value of the signal intensity every 30 seconds when the signal intensity at 0 seconds after gadolinium DTPA intravenous injection is taken as 100. An increase in the signal intensity of gadolinium DTPA within the tumor tissue means uptake of gadolinium DTPA into the tumor tissue. The degree of uptake of gadolinium DTPA into the tumor tissue is an indicator of the blood flow state in the tumor tissue.

(Functional analysis of changes in tumor microenvironment)
As shown in FIG. 1, in 90 to 120 seconds after intravenous injection of gadolinium DTPA, the signal intensity at the center of the tumor tissue after treatment in the group administered with argotigenin-containing burdock extract increased to 118.1 ± 13.7%. On the other hand, in the untreated group, it was 102.6 ± 3.4% in 90 to 120 seconds, and did not increase. Therefore, the blood flow tended to be better in the burdock extract administration group than in the untreated group (P <0.05, t-test). Moreover, in the gemcitabine treatment group in which the antitumor effect was observed, the signal intensity at the center of the tumor tissue did not increase as in the untreated group (not shown). On the other hand, in the bevacizumab treatment group having angiogenesis inhibitory action, the signal intensity tended to increase (not shown).

From the above results, it was suggested that the poor blood flow region of the tumor tissue may be reduced by the administration of archigenin-containing burdock extract and the rich blood flow region may remain.

[Test Example 2]
In order to analyze pathologically the changes in the tumor microenvironment due to repeated administration of argotigenin-containing gobo-bovine extract, hypoxia (Pimonidazole) and cell proliferation (BrdU) were evaluated by immunostaining. In addition, the ratio of blood vessels covered with perisite among blood vessels in the tumor was evaluated by double immunostaining of CD31 and αSMA.

Human pancreatic cancer cells Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl were transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan). On the 16th day after transplantation, the transplanted mice were divided into 4 groups: (a) untreated group, (b) gobo cow extract administered group, (c) bevacizumab administered group, and (d) gemcitabine administered group, and treatment was continued for 4 weeks. did. The burdock extract administration group gave mice a diet containing 2.0% (w / w) of burdock extract. When the daily intake of the mouse food is calculated to be 3 to 5 g, arctigenin is administered at 60 to 100 mg / animal / day. Bevacizumab was administered intraperitoneally at a dose of 5 mg / kg once a week using 100 mg / 4 ml (Chugai Pharmaceutical) for intravenous infusion. Gemcitabine was intraperitoneally administered 100 mg / kg twice a week using a gemcitabine preparation for infusion (Eli Lilly and Company).

(Tumor size)
Mean tumor size of each group after treatment after 4 weeks, (a) untreated group 1851mm ^2, (b) Goboushiekisu administered group 1035 mm ^2, (c) bevacizumab group 911 mm ^2, and (d) The gemcitabine administration group was 949 mm ² .

(Pathological analysis of changes in tumor microenvironment 4 weeks after treatment)
Four weeks after the start of treatment, 100 μL of BrdU (Sigma-Aldrich) 20 mg / mL and 100 μL of pimonidazole 16 mg / mL were administered intraperitoneally, and tumors were collected 1 hour later. In the collected tumor sections of each group, the hypoxic region (pimonidazole) and cell proliferation (BrdU) were evaluated by immunostaining in order to pathologically analyze changes in the tumor microenvironment. In addition, the ratio of blood vessels covered with perisite among blood vessels in the tumor was evaluated by double immunostaining of CD31 and αSMA.

Pimonidazole is a 2-nitroimidazole compound that is reduced under strong hypoxia with an oxygen partial pressure of 10 mmHg or less, and binds to intracellular proteins to form adducts. It is an oxygen probe.

BrdU is a thymidine analog and is incorporated into newly synthesized DNA in the S phase of the cell cycle. Since BrdU-labeled DNA can be detected with an anti-BrdU antibody, the anti-BrdU antibody is a proliferating cell detection tool that performs DNA replication.

The blood can flow without leaking because the outside of the blood vessel is lined with perisite. However, since many weak blood vessels composed only of endothelial cells are formed in the blood vessels inside the tumor, blood leaks and the delivery of the anticancer drug into the tumor is inhibited. An angiogenesis inhibitor typified by bevacizumab is considered to be able to enhance the effect of the anticancer agent used in combination by lowering the proportion of blood vessels not covered with perisite.

(Changes in the tumor microenvironment after treatment)
The percentage (%) of the positive area of pimonidazole in the tumor after each treatment is shown in FIG. The percentage of positive tumor pimonidazole areas was 40% in the untreated group, 18% in the bevacizumab group, 19% in the gobobull extract group, and 30% in the gemcitabine group. From this result, it became clear that the hypoxic region was remarkably reduced by bevacizumab treatment and gobobull extract treatment. This is thought to be due to the normalization of blood vessels.

Fig. 3 shows the ratio (%) of BrdU positive cells in the tumor after each treatment. The percentage (%) of BrdU positive cells in the tumor was 7% in the untreated group, 15% in the bevacizumab group, 11% in the gobobull extract group, and 6% in the gemcitabine group. From this result, it was clarified that the number of proliferating cells was increased by the treatment with bevacizumab and the treatment of gobo-bovine extract. This is thought to be due to the normalization of blood vessels.

Figure 4 shows the percentage (%) of pericyte-coated blood vessels within each tumor after each treatment. The percentage of perisite was determined by dividing the CD31 and αSMA double positive area by the CD31 positive area. The percentage of pericyte-coated blood vessels was 23.3% in the untreated group and 35.2% in the burdock extract group. Therefore, it is suggested that administration of burdock extract normalized blood vessels and increased pericyte-coated blood vessels. This result indicates that arctigenin can be used as a vascular wall strengthening agent to increase blood vessels covered with vascular wall cells such as pericytes.

Therefore, it is suggested that administration of arctigenin normalized the blood vessels in the tumor tissue. In addition, it is suggested that normalization of blood vessels provides an environment in which drugs can be easily delivered. Therefore, it is suggested that by administering the anticancer agent together with archigenin, drug delivery of the anticancer agent to the tumor tissue is enhanced, and as a result, the efficacy of the anticancer agent is enhanced.

[Test Example 3]
Using the vascular template method, changes in the vascular structure in the tumor by repeated administration of argotigenin-containing burdock extract were examined.

Human pancreatic cancer cells Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl were transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan), and the tumor size was about 200 mm ³ The mice were selected and treated. The prepared transplanted mice were divided into an untreated group and a burdock extract administration group, and the treatment was continued for 4 weeks. The burdock extract administration group was orally administered 750 mg / kg extract containing 10% arctigenin 5 times a week.

(Create blood vessel mold)
Four weeks after the start of treatment, the mouse was deeply anesthetized, and then thoracotomy was performed, and saline was perfused from the heart to remove blood. After the blood was removed, 10 mL of Mercox (Ladd research industry) containing 40% methyl methacrylate (Nisshin EM Co., Ltd.) with an accelerator (Ladd research industry) added was perfused from the heart. Thereafter, the mice were immersed in warm water at 60 ° C. for 60 minutes to cure the resin. The tumor was cut off, the tissue was corroded with 20% NaOH, the blood vessel template specimen was taken out, and the structural change of the blood vessel in the tumor was examined using a scanning electron microscope.

(Changes in tumor vasculature after treatment)
The blood vessels inside the tumor after each treatment are shown in FIG. In the untreated group of tumors, the blood vessels were dense and formed a complicated structure, and the end portions of the blood vessels also meandered and entangled with adjacent blood vessels. On the other hand, in the tumor treated with architigenin-containing burdock extract, the dense structure of blood vessels as seen in the untreated group was not seen, and there was little meandering of the blood vessels at the end part of the blood vessels. No intertwined structure was found. Therefore, it is suggested that the blood vessels were normalized by administration of the architigenin-containing burdock extract and a normal vascular network was formed. This result indicates that arctigenin can be used as a vascular network formation inhibitor that suppresses the formation of vascular networks, particularly abnormal vascular networks.

(Test Example 4)
In order to analyze the pharmacokinetic changes of anticancer drugs in tumors due to repeated administration of argotigenin-containing gobobull extract, blood and intratumor CPT-11 concentrations were evaluated.

Human pancreatic cancer cells Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl were transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan), and the tumor size was about 200 mm ³ The mice were selected and treated. The prepared transplanted mice were divided into an untreated group and a burdock extract administration group, and the treatment was continued for 4 weeks. The burdock extract administration group was orally administered 750 mg / kg extract containing 10% arctigenin 5 times a week.

Four weeks after the start of treatment, 50 mg / kg of campto intravenous infusion (Yakult) was intraperitoneally administered to the untreated group and the burdock extract group, and the tumor and blood were collected under deep anesthesia 30 minutes, 1 hour, and 3 hours, respectively. did. The collected blood was centrifuged at 1200 × g for 20 minutes at 4 ° C. to collect plasma. 200 μL of plasma was collected, an equal volume of acetonitrile was added, and centrifugation was performed at 12,000 rpm, 4 ° C. for 10 minutes. About 100 mg of the tumor was measured after collection, homogenized with 100 mg / 200 μL MilliQ, added with 300 μL of acetonitrile, and centrifuged at 12,000 rpm at 4 ° C. for 10 minutes. Each supernatant was collected and 10 μL was injected and subjected to HPLC analysis. For the sample, 10 mg of CPT-11 was dissolved in 5 mL of methanol, and 10 μL each was injected to perform HPLC analysis.

(Changes in pharmacokinetics of anticancer drugs in blood and tumor after treatment)
FIG. 6 shows the concentrations of CPT-11 in blood and in the tumor at each time after intravenous administration of campto instillation to each treatment group. The CPT-11 blood concentration in each time zone of the untreated group and the burdock extract administration group did not change clearly in either group. On the other hand, the CPT-11 concentration in the tumor did not change in both groups after 30 minutes and 1 hour, but the tumor CPT-11 concentration 3 hours after administration was 4 μg / g tumor in the untreated group. 8.7 μg / g tumor was observed in the arctigenin treatment group. It has been clarified that treatment with arctigenin can increase the concentration of CPT-11 in the tumor without affecting the blood CPT-11 concentration.
(Test Example 5)
In the process of tumor growth, nutrients and oxygen must be supplied through blood vessels throughout the enlarged tumor tissue. However, in many tumors, they are not sufficiently supplied due to immature blood vessel formation, and partial tissue necrosis is caused, and it is considered that a site where the tissue becomes ulcerous is generated. Therefore, in order to verify the blood vessel normalization effect of the above-mentioned burdock extract from the tissue state, ulcer formation in the tumor tissue after each treatment was observed.

Human colon cancer cells LS174T (ATCC CL-188) 5 × 10 ⁵ cells / 200 μl were transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan). On the 14th day after transplantation, the transplanted mice were divided into 4 groups: (a) untreated group, (b) gobo cow extract administered group, (c) bevacizumab administered group, and (d) gobo cow extract and bevacizumab combined administration group. The treatment was continued for 5 weeks. The burdock extract administration group was orally administered 250 mg / kg of an extract containing 10% architigenin five times a week. In the bevacizumab administration group, 5 mg / kg was administered intraperitoneally once a week using 100 mg / 4 ml (Chugai Pharmaceutical) for intravenous infusion.

FIG. 7 shows the ratio (%) of mice that produced ulcers in the tumors in each treatment group. The 50% ulcer formation rate was 8 days in the untreated group, 20 days in the gobo-bovine extract administration group, 10 days in the bevacizumab administration group, and 24 days in the combination administration group. Therefore, in the burdock extract administration group and the combination administration group, an inhibitory effect on ulcer formation in the tumor was observed compared with the untreated group and the bevacizumab administration group. In the bevacizumab administration group, the effect of suppressing ulcer formation was observed compared to the untreated group, but it was transient. This effect is thought to be due to the angiogenesis inhibitory action of bevacizumab. On the other hand, when a gobo cow extract was administered in combination with bevacizumab, inhibition of ulcer formation was enhanced. This is considered to be because the blood flow improving effect by the blood vessel normalizing effect by the treatment with gobo cow extract was enhanced in combination with the blood flow improving effect on the tumor with bevacizumab alone. Thus, the combined administration with arctigenin can remarkably reduce the hypoxia and hypotrophic regions due to blood vessel normalization, and is expected to suppress cancer malignancy.

(Life extension effect by combined use)
After the above treatment was continued for 5 weeks, the treatment was stopped and the survival was followed. The survival rate in each group is shown in FIG. As shown in FIG. 8, the 50% survival period was 103 days for the untreated group, 114 days for the burdock extract administration group, 107 days for the bevacizumab administration group, and 137 days for the combination administration group. Therefore, the combined administration group showed 33% and 20-28% survival-prolonging effects as compared to the untreated group and the single administration group, respectively.

(Test Example 6)
The antitumor effect of combined administration of burdock extract and gemcitabine was evaluated.

Human pancreatic cancer cell Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200μl transplanted under the armpit of a BALB / cAJcl nu / nu mouse (CLEA Japan), resulting in a tumor size of approximately 100mm ³ Were selected and treated as mice to be treated.

There are four groups of mice to be treated: (a) untreated group, (b) gemcitabine administration group, (c) burdock extract administration group, and (d) burdock extract and gemcitabine administration group (combination administration group) The treatment was continued for 4 weeks. As for the burdock extract, the burdock extract of Example 1 250 mg / kg (25 mg / kg or more as archigenin) was orally administered every day (5 times a week). Gemcitabine was administered intraperitoneally twice weekly at 150 mg / kg using an infusion gemcitabine preparation (Eli Lilly).

(Tumor weight)
After the treatment test, the tumor weight was measured for each administration group. As a result, the tumor weight after the treatment test was 2.05 g in the untreated group, 1.24 g in the gemcitabine administration group, 0.98 g in the burdock extract administration group, and 0.82 g in the combination administration group. Therefore, in all of the gemcitabine administration group, the burdock extract administration group and the combination administration group, suppression of tumor weight of about 40 to 60% was observed as compared with the untreated group.

From these results, it was shown that the combined use of burdock extract and gemcitabine suppresses tumor growth. This is presumably because gemcitabine became easier to act on tumors due to normalization of blood vessels by burdock extract. Therefore, it was shown that the combination agent of burdock bovine extract and gemcitabine has significantly higher anticancer activity than expected from the effect when each of these is administered alone.

(Test Example 7)
The 5-year survival rate after diagnosis of pancreatic cancer is less than 5% in 2013, and the average survival time is only 4-6 months. This is an extremely low value compared to other cancers, and effective measures are required. In Example 6 described above, the combined treatment of architigenin-rich burdock extract and gemcitabine for the pancreatic tumor transplantation model showed a tendency to suppress tumor growth compared to single agent treatment. Therefore, a model of orthotopic transplantation of pancreatic cancer to mouse pancreas was created as a model closer to the actual lesion, and the effect of combined treatment with gemcitabine and gobo-bovine extract on the survival rate was evaluated.

Human pancreatic cancer cells Miapaca-2 cells were suspended in high-concentration Matrigel solution (BD bioscience) at 5 × 10 ⁶ cells / 50 μl. An 8-week-old female BALB / cAJcl-nu / nu (Claire Japan) was opened under anesthesia, the cell suspension was injected into the pancreatic tissue and solidified, and then sutured and closed.

The transplanted mice were divided into 7 mice each in 4 groups: an untreated group, a burdock extract-administered group, a gemcitabine-administered group, and a group administered with these, and treatment was continued until all the mice died. The burdock extract administration group orally administered 250 mg / kg of an extract containing 10% arctigenin five times a week. In the gemcitabine administration group, 150 mg / kg was administered intraperitoneally twice a week using an infusion gemcitabine preparation (Eli Lilly and Company).

(Analysis of survival rate)
Survival was calculated by calculating and rendering survival curves and median survival MST using the Kaplan-Mayer method (GraphPad-PRISM 6.2, MDF). Significant differences were evaluated by Logrank (Mantel-COX) and Gehan-Breslow-Wilcoxon tests.

The change in survival rate (%) of each treatment group is shown in the upper graph of FIG. Median survival (MST) was 65 days in the untreated group (Non treat), 86 days (32.3% increase) in the gemcitabine group (Gemcitabine), and 88 days (35.4% increase) in the gobobull extract group (GBS-01) ), 117 days (80% increase) in combination treatment. Moreover, a significant difference was observed between the treated group and the untreated group regardless of whether the Gehan-Breslow-Wilcoxon test or the Log-RANK (Mantel-Cox) test was used.

As a result, in the pancreatic cancer orthotopic transplantation model, as shown in FIG. 9, the gobo-bovine extract administration group and the gemcitabine administration group showed substantially the same life-prolonging effect as compared with the untreated group. On the other hand, the combination administration group using these drugs in combination showed a significant life-prolonging effect compared to the untreated group and the single administration group. Therefore, it has been shown that the combined use of an anticancer drug and archigenin can greatly increase the survival rate even in pancreatic cancer, whose survival time is difficult to progress.

(Test Example 8)
Using a pancreatic cancer transplantation model, treatment with various existing anticancer agents, archigenin-rich burdock extract and combinations thereof was performed, and changes in tumor size were evaluated.

Miapaca-2 (ATCC CRL 1420) of human pancreatic cancer cells 5 × 10 ⁶ Cells / 200 μl are transplanted subcutaneously into the armpit of BALB / cAJc1-nu / nu mice (Claire Japan), raised for about 2 weeks, then about 100 mm ³ Alternatively, mice having a size of about 600 mm ³ were used as treatment target mice.

The prepared transplanted mice are divided into untreated groups, existing anticancer drug administration groups that are various anti-cancer components, burdock extract administration groups, and these combination administration groups, and each group has 5 to 6 tumors. The treatment was continued for about 4 weeks. As existing anticancer agents, carboplatin (CBDCA, Bristol-Myers, 60 mg / kg once abdominal administration), doxorubicin (Doxorubicin, Kyowa Hakko, 10 mg / kg abdominal administration twice a week), oxaliplatin (Oxaliplatin, Yakult, 8 mg) / kg once a week), Everolimus (Everolimus, Novartis, 5 mg / kg orally 5 times a week), Bortezomib (Bortezomib, Janssen Pharma, 1 mg / kg twice a week), Irinotecan, Yakult , 25 mg / kg twice a week by intraperitoneal administration) and hydroxychloroquine (Sanophy, 100 mg / kg by intraperitoneal administration five times a week). As for the burdock cow extract, 750 mg / kg of an extract containing 10% arctigenin was orally administered 5 times a week.

(Tumor size measurement)
Every 3-4 days after the start of treatment, the mice are held and the major axis (L), minor axis (W) and height (H) of the tumor are measured with precision digital calipers, and the tumor size = 4/3 * pi * The size was calculated using the formula L / 2 * H / 2 * W / 2 (pi = 3.14). In addition, when the height could not be measured, tumor size = 4/3 * pi * L / 2 * (W / 2) 2 was simply used.

Figure 10 shows the change in tumor size in each treatment group. As a result of combined treatment with existing anticancer drugs and burdock extract, carboplatin and oxaliplatin are 2.3 to 2.1 times (P <0.05) compared to single drug and 1.8 to 1.9 times (P <0.05) compared to single drug. A decrease in tumor size was observed. In addition, with irinotecan, the tumor size was reduced 2.1 times (P <0.05) compared to the burdock bovine extract alone and 1.5 times the tumor size compared to the anticancer agent alone. In doxorubicin, the tumor size was reduced by about 1.4 times compared to the gobo-bovine extract alone and 1.5 times (P <0.05) compared with the anticancer agent alone. Everolimus showed a 2.1-fold reduction in tumor size (P> 0.05) compared to the burdock extract alone and a 1.4-fold reduction in tumor size compared to the anticancer agent alone. Bortezomib was found to have a 1.8-fold (P <0.05) reduction in tumor size compared to the gobo-bovine extract alone and 1.4-fold compared to the anticancer agent alone. Hydrochloroquine, a therapeutic agent for malaria and autoimmune diseases, showed a 1.9-fold decrease in tumor size compared to single-drug bovine extract and 2.5-fold (P <0.05) compared to single anticancer agent.

From the above results, it was shown that argotigenin-rich burdock extract is a drug that can be expected to have a strong antitumor effect when combined with existing anticancer drugs having various medicinal mechanisms.

The vascular wall reinforcing agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can be used for the prevention, treatment and improvement of various diseases caused by abnormal blood vessel formation.

Claims (8)

1. A blood vessel wall reinforcing agent containing archigenin as an active ingredient to increase blood vessels covered with blood vessel wall cells.
2. 2. The vascular wall reinforcing agent according to claim 1, wherein the arctigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.
3. Vascular network formation inhibitor containing arctigenin as an active ingredient.
4. 4. The vascular network formation inhibitor according to claim 3, wherein the arctigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.
5. A method for strengthening a blood vessel wall, comprising a step of ingesting arctigenin and / or arctiin to a subject that needs to strengthen the blood vessel wall.
6. 6. The method for reinforcing a blood vessel wall according to claim 5, wherein the arctigenin and / or arcticin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.
7. A method for suppressing vascular network formation, comprising a step of ingesting archigenin and / or arctiin into a subject that needs to suppress vascular network formation.
8. 8. The method of suppressing vascular network formation according to claim 7, wherein the arctigenin and / or arcutin is derived from burdock, burdock, burdock sprout, juvenile burdock or forsythia.
   
   
   

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