KR20160129809A - Pharmaceutical composition having angiogenesis inhibition activity comprising cold water based extract of Cinnamomum cassia - Google Patents

Abstract

The present invention relates to a pharmaceutical composition having an angiogenesis inhibition activity comprising a cold water extract of a cinnamomum cassia. The cold water extract of a cinnamomum cassia represents an angiogenesis inhibition activity by inhibiting proliferation of vascular cells derived by a vascular endothelial growth factor (VEGF), a movement, invasive properties, a tubular formation, phosphorylation of an intracell signaling protein, an activation of a matrix metalloproteinase, and aortic sprouting. The present invention can be usefully applied to preventing and treating an eye syndrome such as a diabetogenous retinopathy, a glaucoma, etc. as well as cancer syndromes due to the formation of angiogenesis cells.

Description

[0001] The present invention relates to a pharmaceutical composition having an angiogenesis inhibitory activity comprising a cold water extract of broiler chickens as an active ingredient,

The present invention relates to a pharmaceutical composition having an activity of inhibiting angiogenesis, comprising Cinnamomum cassia cold water extract as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition having inhibitory activity against vascular endothelial growth factor (VEGF) Inhibits the phosphorylation and substrate metalloproteinase activity of extracellular signal regulated kinase (ERK), p38, and vascular endothelial growth factor receptor 2 and inhibits cell proliferation, cell migration, invasion, tube formation, and sprouting ) To inhibit the formation of angiogenesis.

Chemotherapy, chemotherapy, and radiation therapy are the first cancer treatments for cancer. Although cancer survival has been prolonged by many efforts to overcome cancer, conventional cancer therapy is still a source of fear because it can not be expected to have a smooth therapeutic effect in many cases due to side effects and pain. Recently, new research methods that can lessen damage to normal cells have been attempted. Research on biological therapeutic agents such as immunotherapy, gene therapy, resistance inhibition, neovascularization inhibitor, and chemical cancer prevention have been actively carried out. Studies to develop new anticancer substances from natural products with low side effects are actively being carried out in various fields.

Recently developed cancer drugs have brought new hope to cancer patients. Target anticancer drug is a new concept of anticancer drug that selectively acts on cancer cells and does not affect normal cells. It is a signal transduction inhibitor that suppresses the signal transduction pathways involved in the production and proliferation of cancer cells, Angiogenesis inhibitor, which blocks the formation of new blood vessels required for growth, and apoptosis inducer, which blocks the cell proliferation by inducing predetermined cell apoptosis of cancer cells. have. On the other hand, since cancer develops into a very complicated pathway, selective inhibition of the target factors alone can not prevent the growth of cancer cells efficiently. Therefore, a new multi-targeted anticancer agents ) Has been developed variously. In particular, the development of a multi-target anticancer agent which can block the key cancer pathway and block the angiogenesis of the cancer cell simultaneously and thus can reduce side effects with excellent efficacy in cancer patients who have failed the conventional anticancer drugs is actively under development. To develop such anticancer drugs, the United States established the National Cancer Institute (NCI) to develop a large-scale anti-cancer screening method from natural products, and has collected various natural resources from all over the world and has conducted anti-cancer search. In particular, Japan has invested in the development and discovery of new angiogenesis inhibitors, and now, several drugs developed in the past have been conducting clinical trials to see the effects of angiogenesis-related diseases. In Korea, much efforts have been made to develop useful anti-cancer substances from natural products since the 80's, but it is still a rudimentary stage. Today, research on the development of pharmacologically active substances from domestic natural resources is desperate in the global trend where the protection of intellectual property including material patent is strongly demanded. In order to enhance international competitiveness with advanced countries, development of new pharmacologically active substances It is important to secure its own technology.

Angiogenesis refers to the creation of new capillaries from existing blood vessels. Strictly controlled phenomena that rarely occur under normal physiological conditions, such as when the embryo develops during the development of the embryo, in the case of wound healing in adults, and in changes in the genital system in the female reproductive cycle. In adults, capillary endothelial cells do not divide relatively well, and the rate of cleavage is usually from months to years. Angiogenesis is a complicated process by the interaction of various kinds of cells with water-soluble factors and extracellular matrix components, and their functional groups are not yet fully characterized. First, angiogenesis is essential in primary or metastatic tumors. Cancerous tissues can grow over 1 ~ 2 mm ³ without nutrients and oxygen supply by neovascularization. (Folkman J, Semin. Cancer Biol. 1992, 3: 65-71). During neovascularization, primary cancer cells enter the blood vessels and migrate to other locations to form metastatic cancer. Therefore, neovascularization inhibitors have the potential to be commonly used in all solid tumors. Second, conventional chemotherapy treats cancer cells with rapid growth characteristics, so it is toxic to bone marrow cells and gastrointestinal cells, which have relatively high cell cycle, while neovascularization inhibitor has relatively few side effects It is possible. In addition, although chemotherapy with chemotherapy can be used as a therapeutic target for transformed cancer cells, new angiogenesis inhibitors are less likely to show tolerance in that normal vascular cells are treated. Third, because one blood vessel cell supplies nutrients and oxygen to hundreds of cancer cells, it can be an effective treatment for inhibiting many cancer cells through inhibition of one blood vessel cell. In the case of conventional anticancer chemotherapy, anticancer drugs flow out of blood vessels and affect cancer cells. However, blood vessel growth inhibitors directly contact vascular endothelial cells and thus drug delivery is easy. Anticancer drugs, which showed inhibitory effects in animal experiments, were not often effective in clinical trials. This is probably due to the difference in expression pattern of vascular endothelial cells and the distribution of different vascular growth factors in the microenvironment around each tumor. Therefore, expression of vascular growth factors, vascular endothelial cell diversity, and microenvironment surrounding cancer tissues have an important effect on the treatment of cancer through an angiostatic agent.

The general angiogenesis process that has been found so far in cancer tissues is as follows. First, angiogenesis is initiated by an angiogenic factor secreted from cancer cells. This inducer stimulates endothelial cells by binding to receptors of vascular endothelial cells, and stimulated endothelial cells have a variety of complex and complex processes including proliferation, invasion, migration and differentiation, and capillary formation Start a series of processes. The penetration and migration of endothelial cells require activation of histolytic enzymes, which is very similar to the penetration of cancer cells. Vascular Endothelial Growth Factor (VEGF) is an angiostatin promoting factor, and angiostatin (O'Reilly MS, Holmgren L, Shing Y et al., Cells 1994,21; 79: 315-328) and endostatin (O'Reilly MS et al., Cells 1997,24; 88 (2): 277-285) Most known as a factor.

Vascular endothelial growth factor (VEGF) is secreted in almost all cells and is highly correlated with tumor permeability (Takahashi Y et al., Cancer Res. 1995, 55 (18): 3964-3968) . Although vascular endothelial growth factor receptor (VEGFR) is generally found in vascular endothelial cells, it is expressed in nerve cells, Kaposi's sarcoma, hematopoietic stem cells, and vascular endothelial growth factor receptor-1 (VEGFR (VEGFR-2 (Kdr / flk-1)) and vascular endothelial growth factor receptor-3 (VEGFR-3 (flt-4)) There is a kind. When the vascular endothelial growth factor is incorporated, signal transduction is mediated by receptor dimerization as well as phosphorylation of the receptor tyrosine residues. Vascular endothelial growth factor receptor-1 (VEGFR-1) is involved in endothelial cell migration and vascular endothelial growth factor receptor-2 (VEGFR-2) is involved in endothelial cell growth and blood flow efficacy. In addition to direct angiogenic factors, there are nonspecific factors such as acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), transformation growth factor (TGF) The present invention relates to a pharmaceutical composition for the treatment and prophylaxis of diabetic nephropathy such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor, angiogenin, Related macrophage inflammatory protein (MIP), platelet factor 4 (PF4), and growth-related oncogene (GRO) (Moore BB et al., J Lab Clin Med 1998, 132 (2): 97-103). In addition, studies on the expression of vascular endothelial cell growth factor and its action on the neuronal vasculature are actively conducted by inhibiting the signal transduction process that regulates the expression and function of vascular endothelial growth factor. Antibody or receptor phosphorylation inhibitors to VEGF receptors that decrease the activity of angiogenesis promoting factors have been shown to inhibit metastasis of colorectal cancer, which is attributed to the reduction of blood vessels in the cancer tissue formed in the liver (Shaheen RM , et al., Cancer Res 1999, 59 (21): 5412-5416). In addition, studies on the mechanism of induction of endothelial cell death, the inhibition of the action of indirect factors that regulate angiogenesis promoter or survival factors of endothelial cells, and the methods of increasing the activity of neovascularization inhibitors present in the body actively It is progressing.

Diabetic retinopathy is a disease caused by retinal hypoxia and ischemia-induced neovascularization, and various factors are involved. Especially, neovascularization factor (VEGF), which is known as angiogenic factor and vascular permeability factor, It plays an important role. Although there is no drug that has been shown to prevent diabetic retinopathy or inhibit the progression of diabetic retinopathy, the development of inhibitors for various growth factors involved in angiogenesis in retinopathy has attracted attention. Recently, anti-angiogenic factors such as Abastin (bevacizu Mab) have been reported to reduce diabetic retinopathy (Avery RL, 2006, Ophthalmology, 113: 363-72; Spaide RF, 2006, Retina, 26: 275 -8; Iturralde D., 2006, Retina, 26: 279-84). However, many studies have been conducted to develop such substances as anti-angiogenic factors from natural products which have side effects and which have no side effects.

Cinnamomum cassia is an evergreen broad-leaved arboreous tree, mainly of the dicotyledonous plant. It is native to China and distributed in Sri Lanka, Indochina, Korea (Jeju) ) Eggplant, is the medicinal name of the shell.

Studies on the components and physiological activity of broiler chickens have shown that anti-inflammatory effects (Hong CH et al., J Ethnopharmacol. 2002, 83 (1-2): 153-9; Yu T et al., J Ethnopharmacol. (Boaduo NK et al., Pharm Biol., 2014, 52 (6): 286-7) (Yeh CF et al., J Ethnopharmacol. 2013, 147 (2): 321-6), and anti- Antioxidant activity (Hwa JS et al., J Ethnopharmacol. 2012, 139 (2): 605-15), anti-gastritis effect (Jung J et al., Yakugaku Zasshi. 2011, 131 (7): 1103-10) 2008, 31 (5): 946-8). Inhibition of melanin synthesis (Lim, CS et al., Biosci Biotechnol Biochem. 2010, 2006, 34 (3): 511), bone formation promoting action (Lee KH et al., Phytother Res 2006, 20 (11): 952-60), antibiotic action (Ooi LS et al. -22).

The extracts of dimethyl sulfoxide (DMSO) from Cinnamomum cassia were incubated with protein kinase C (PKC), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 VEGFR2), inhibits mitogen-activated protein kinase (MAPK (ERK1 / 2)) and phosphorylation of AKT, and that the extract inhibits the expression of zebrafish embryo spine (Rishipal R. Bansode et al., Food Science & Nutrition. 2013, 1 (2) (1983), pp. 177-186 1): 74-82) are known.

In addition, Cinnamomum zeylanicum extract inhibits the phosphorylation activity of purified vascular endothelial growth factor receptor 2 (VEGFR2) and inhibits mitogen-activated protein kinase (MAPK) and Stat3-mediated signaling pathways (Jianming Lu et al., Carcinogenesis 2010, 31 (3): 481-488).

In Korea Patent No. 213 897 discloses a broiler (Cinnamomum cassia) cancer metastasis because it inhibits the 2-hydroxy cinnamic point and neovascularization induced by the higher derivatives of the active ingredients that inhibits the aldehyde component is induced neovascularization of the organic solvent extract, It is also disclosed that this inhibitor can also be used as a drug for preventing and treating blindness due to neovascularization occurring during diabetic retinopathy or corneal transplantation.

However, in these documents, since DMSO and organic solvent extract are used, there is a problem that the solvent remains harmful to the human body and the industrialization is restricted due to an increase in manufacturing cost.

The cinnamaldehyde derivative was extracted with a mixture of chloroform and acetone. The extract was concentrated and extracted with a sodium hydroxide solution. The base layer was acidified and re-extracted with methylene chloride. The methylene chloride extract was concentrated and purified by silica gel chromatography There is a problem in that the separation process is very complicated due to the separation of the components through a step of purification by high pressure liquid chromatography and high performance liquid chromatography.

Korean Patent No. 213,897

Folkman, Semin. Cancer Biol. 1992, 3: 65-71 O'Reilly MS et al., Cell. 1994,21; 79: 315-328. O'Reilly MS et al., Cell. 1997, 24; 88 (2): 277-285. Takahashi Y et al., Cancer Res. 1995, 55 (18): 3964-3968. Moore BB et al., J Lab Clin Med. 1998, 132 (2): 97-103. Shaheen RM et al., Cancer Res. 1999, 59 (21): 5412-5416. Avery R. L., 2006, Ophthalmology, 113: 363-72. Spaide R. F., 2006, Retina, 26: 275-8. Iturralde D., 2006, Retina, 26: 279-84 Yeh CF et al., J Ethnopharmacol. 2013, 147 (2): 321-6 Hwa JS et al., J Ethnopharmacol. 2012, 139 (2): 605-15 Jung J et al., Yakugaku Zasshi. 2011, 131 (7): 1103-10 Lim CS et al., Biosci Biotechnol Biochem. 2010, 74 (3): 477-83 Kong YH et al., Biol Pharm Bull. 2008, 31 (5): 946-8 Lee KH et al., Phytother Res. 2006, 20 (11): 952-60 Ooi LS et al., Am J Chin Med. 2006, 34 (3): 511-22 Rishipal R. Bansode et al., Food Science & Nutrition. 2013, 1 (1): 74-82 Jianming Lu et al., Carcinogenesis. 2010, 31 (3): 481? 488

It is an object of the present invention to provide a pharmaceutical composition, a food composition, and a method for producing a broiler chilled cold water extract containing a broiler chilled cold water extract as an active ingredient by demonstrating the activity of inhibiting angiogenesis of a cinnamomum cassia cold water extract.

In order to achieve the above object, the present invention provides a pharmaceutical composition, a food composition and a preparation method thereof containing Cinnamomum cassia cold water extract as an active ingredient, wherein the composition is a vascular endothelial growth factor (VEGF ) Effectively inhibits angiogenesis by inhibiting proliferation, migration, invasion, and tube formation of vascular endothelial cells, phosphorylation of intracellular signaling proteins, activity of matrix metalloproteinase, and aortic germination can do.

The present invention relates to a pharmaceutical composition containing Cinnamomum cassia cold water extract as an active ingredient, and the broth cold water extract according to the present invention has an effect of inhibiting angiogenesis. Therefore, it is very useful as a therapeutic agent for a disease associated with abnormally proliferated cells requiring neovascularization in vivo, for example, a therapeutic agent for diabetic retinopathy or various tumors due to excessive neovascularization, and is useful as a pharmaceutical composition for inhibiting angiogenesis, Can be used as a food composition.

FIG. 1 shows results of MTT analysis as an experiment on cytotoxicity of broiler chilled cold water extract as an experimental example of the present invention.
FIG. 2 shows experimental results on the effect of chilled cold water extract on cell proliferation by vascular endothelial growth factor (VEGF) as an experimental example of the present invention.
FIG. 3 is a graph showing the effect of extracellular signal regulated kinase (ERK) on the vascular endothelial growth factor (VEGF) of poultry cold water extract (p38, And phosphorylation of vascular endothelial growth factor receptor 2) by Western blot analysis.
FIG. 4 is a graph showing experimental results on the effect of chilled cold water extract on cell migration by vascular endothelial growth factor (VEGF) as an experimental example of the present invention.
FIG. 5 shows experimental results on the effect of chilled cold water extract on cell invasion by vascular endothelial growth factor (VEGF) as an experimental example of the present invention.
FIG. 6 is a graph showing an experimental result on the effect of chilled cold water extract on the activity of matrix metalloproteinase by vascular endothelial growth factor (VEGF) as an experimental example of the present invention will be.
FIG. 7 shows experimental results on the effect of chilled cold water extract on tube formation by vascular endothelial growth factor (VEGF) as an experimental example of the present invention.
FIG. 8 shows experimental results on the effect of chilled cold water extract on rat aortic sprouting by vascular endothelial growth factor (VEGF) as an experimental example of the present invention.

The present invention relates to a pharmaceutical composition for inhibiting neovascularization comprising Cinnamomum cassia cold water extract as an active ingredient.

In one embodiment of the present invention, the concentration of the chilled cold water extract in the pharmaceutical composition may be less than 100 ug / ml, more specifically 0.5 to 50 ug / ml.

In one aspect of the present invention, the pharmaceutical composition is useful for the treatment of diabetic retinopathy, neovascular glaucoma, posterior capsular hyperplasia, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, corneal ulcer, cone diaphragm, macular degeneration, Sjogren's syndrome, Ophthalmopathy, hemangiomas, angiofibroma, psoriasis, ophthalmopathy, ophthalmic tumor, corneal transplant rejection, abnormal wounds, trachoma, bone disease, rheumatoid arthritis, osteoarthritis, , Pyogenic granuloma, proteinuria, abdominal aortic aneurysm disease, degenerative cartilage loss due to traumatic joint injury, nervous system herniated warts disease, cirrhosis, gynecological diseases, immature platelets of germinal membranes, inflammatory bowel disease, , Restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging, thyroid hyperplasia, Grave's disease, cancer incidence (ca ncer development, cancer invasion and cancer metastasis. The present invention is not limited thereto.

In one aspect of the present invention, the pharmaceutical composition may more particularly be used for the prevention or treatment of cancer development, cancer invasion or cancer metastasis due to diabetic retinopathy or neovascularization.

In one aspect of the present invention, the cancer may be selected from the group consisting of breast cancer, ovarian cancer, colon cancer, pancreatic cancer, kidney cancer, sarcoma, mesothelioma, teratoma, astrocytoma, melanoma, hemangioma and glioblastoma It is not limited.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , The sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. 1 to 200 mg / kg, preferably 10 to 100 mg / kg per day.

The composition of the present invention may be administered in the form of an individual therapeutic agent or in combination with another therapeutic agent exhibiting an effect of inhibiting angiogenesis, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

In the present invention, the term "individual " means all animals including humans who have already developed or are capable of developing a disease that can be prevented or treated through inhibition of neovascularization, and the composition comprising the extract of the present invention is administered to a subject The disease can be effectively prevented and treated.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, or rectally, though it is not intended to be limited thereto. The composition may also be administered by any device capable of transferring the active agent to the target cell.

In one embodiment of the present invention, the broiler chilled cold water extract is selected from the group consisting of extracellular signal regulated kinase (ERK) by vascular endothelial growth factor (VEGF), p38 or vascular endothelial growth factor receptor VEGFR) and inhibit the formation of angiogenesis.

In one embodiment of the present invention, the broiler chilled cold water extract inhibits vascular endothelial growth factor (VEGF) -induced vascular endothelial cell proliferation, inhibits vascular endothelial cell migration, inhibits tube formation, or And inhibits aortic sprouting, thereby exhibiting neovascularization-inhibiting activity.

The present invention relates to a food composition for inhibiting neovascularization comprising Cinnamomum cassia cold water extract as an active ingredient.

In one embodiment of the present invention, the concentration of the chilled cold water extract in the food may be less than 100 ug / ml, more specifically 0.5 to 50 ug / ml.

In one aspect of the invention, the food composition is selected from the group consisting of diabetic retinopathy, neovascular glaucoma, posterior capsular hyperplasia, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, corneal ulcer, cone diaphragm, macular degeneration, Sjogren's syndrome, Ophthalmopathy, hemangiomas, angiofibroma, psoriasis, ophthalmopathy, ophthalmic tumor, corneal transplant rejection, abnormal wounds, trachoma, bone disease, rheumatoid arthritis, osteoarthritis, , Pyogenic granuloma, proteinuria, abdominal aortic aneurysm disease, degenerative cartilage loss due to traumatic joint injury, nervous system herniated warts disease, cirrhosis, gynecological diseases, immature platelets of germinal membranes, inflammatory bowel disease, , Restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging, thyroid hyperplasia, Grave's disease, cancer incidence (ca ncer development, cancer invasion and cancer metastasis. The present invention is not limited thereto.

In one aspect of the present invention, the food composition may more particularly be used for preventing or alleviating cancer development, cancer invasion or cancer metastasis due to diabetic retinopathy or angiogenesis.

In one aspect of the present invention, the cancer may be selected from the group consisting of breast cancer, ovarian cancer, colon cancer, pancreatic cancer, kidney cancer, sarcoma, mesothelioma, teratoma, astrocytoma, melanoma, hemangioma and glioblastoma It is not limited.

In one embodiment of the present invention, the broiler chilled cold water extract is selected from the group consisting of extracellular signal regulated kinase (ERK) by vascular endothelial growth factor (VEGF), p38 or vascular endothelial growth factor receptor VEGFR) and inhibit the formation of angiogenesis.

In one embodiment of the present invention, the broiler chilled cold water extract inhibits vascular endothelial growth factor (VEGF) -induced vascular endothelial cell proliferation, inhibits vascular endothelial cell migration, inhibits tube formation, or And inhibits aortic sprouting, thereby exhibiting neovascularization-inhibiting activity.

The food according to the present invention may include a broiler chilled cold water extract, and suitable food supplementary additives.

The term "food-aid additive " in the present invention means a component which can be added to foods in a supplementary manner, and it can be appropriately selected and used by those skilled in the art as being added to produce health functional foods of each formulation. A coloring agent and a filler, a pectic acid and a salt thereof, an alginic acid and a salt thereof, an organic acid, a protective colloid thickener, a pH adjuster, a stabilizer, a stabilizer and a stabilizer such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, A preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, etc. However, the types of the food-aid additive of the present invention are not limited by the above examples.

In one aspect of the invention, the food may be a health functional food.

The term "health functional food " in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and rings by using raw materials and components having useful functions in the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the health functional food of the present invention can be manufactured by the method Unlike general medicines, there is an advantage that there are no side effects that may occur when a drug is taken for a long time by using a food as a raw material, and the portability In addition, the health functional food of the present invention can be ingested as an adjuvant for enhancing the effect of inhibiting angiogenesis.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the broiler chilled cold water extract according to the present invention is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight, of the raw material composition when the food is prepared. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary foods. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tau Martin and stevia extract, synthetic sweetening agents such as saccharine and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.

(A) adding Cinnamomum cassia to chicken broth at least twice the weight of broilers and extracting and filtering; And (b) drying the extract. The present invention also relates to a method for producing the cold water extract.

In one embodiment of the present invention, the extraction and filtration process may be repeated at least twice in the step (a).

In one embodiment of the present invention, the temperature of the cold water in the step (a) may be lower than 20 占 폚.

In one embodiment of the present invention, in step (a), the broiler chickens may be dried for 24 hours or more.

[ Application example ]

Application example  1: Treatment of diabetic retinopathy due to excessive neovascularization

Diabetes mellitus with a prevalence rate of 10% in Korea is one of the most important adult diseases in the world. It is now over 240 million people and will be increased to 380 million by 2025, according to the 2009 American Medical Association (JAMA). Blindness due to diabetes is the most common cause of blindness that occurs after age 25. Eye diseases commonly seen in diabetic patients include complications in various tissues such as conjunctivitis, cataract, glaucoma, ocular paralysis, optic neuropathy, but the most important cause of blindness is also retinopathy. Diabetic retinopathy progresses to proliferative diabetic retinopathy by inducing vitreous hemorrhage resulting in chronic retinal hypoxia, ischemia, and vascular permeability (Aiello LP, 1998, Diabetes Care. : 143-156). Several factors are implicated in this process, most notably vascular endothelial growth factor (VEGF), a neovascularizing factor and a vascular permeability factor. Initially, light vein expansions and blood vessels lose their elasticity, causing microvascular infiltration like acanthosis. As it progresses further, the vascular permeability increases and the blood components escape, resulting in swelling of the retina, bleeding and exudates. If the capillaries become clogged, the area where the blood circulation does not work is increased and new blood vessels begin to grow inside the retina. When these changes invade the central part of the retina, the vision deteriorates. Progression leads to proliferative retinopathy, which leads to neovascularization of the retina, optic disc, and iris, resulting in sudden vitreous hemorrhage or traction retinal detachment, resulting in severe visual loss. If early or midterm changes do not involve the central retina, visual acuity is not deteriorated at all and may be found to progress to the late stage. Therefore, the effect of the cold water extract of the present invention to inhibit angiogenesis is effective for the treatment of diabetic retinopathy.

Application example  2: tumor (cancer)

Tumor growth occurs only when cancer cells strengthen their ability to form new blood vessels that proliferate small blood vessels in their tissue masses. Cancer cells promote the growth of small blood vessels by secreting vascular endothelial growth factor (VEGF), an element that promotes angiogenesis or proliferates endothelial cells that form capillaries, do. If the elements necessary for these neovascularization are suppressed or blocked, the tumor becomes oxygen and malnutrition, becomes necrotic in the toxic material produced by cancer cells, and can not be transferred to other organs. Therefore, the effect of the broiler chick extract of the present invention on the inhibition of angiogenesis is effective for the cancer treatment by inhibiting the growth and metastasis of cancer cells.

Hereinafter, the present invention will be described in more detail with reference to the following examples, experimental examples and preparation examples. It should be understood, however, that the same is by way of illustration and example only and is not intended to limit the scope of the present invention.

[ Example ]

Example  1: Preparation of chilled cold water extract

After 24 hours, 5 times of cold water was added to the weight of the dried broiler chickens, and shaking extraction and filtration were repeated three times. The extracts were collected, concentrated with an evaporator at 40 ° C, and lyophilized to obtain chilled cold water extract.

[ Experimental Example ]

Experimental Example  1: Cytotoxicity test of chilled cold water extract

(MTT) cell viability assay was performed to determine whether the cold water extract (CCBE) obtained in Example 1 was toxic to human umbilical vein endothelial cells (HUVEC).

To this end, HUVECs were incubated with 20% fetal bovine serum (FBS) (Gibco), heparin (5 units / ml), basic fibroblast growth factor (bFGF) ) (3 ng / ml), 100 unit / ml penicillin, 100 μg / ml streptomycin and incubated at 37 ° C in a 5% CO ₂ incubator. Cell viability was maintained above 95% in all experiments.

The cultured vascular endothelial cells (HUVECs) were dispensed into 12-well plates at a concentration of 5 × 10 ⁴ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. The cells were treated with various concentrations of cold water extracts (1, 5, 10, 25, 50, 100 μg / ml) in various wells and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours.

After the cultivation was completed, 5 mg / ml MTT reagent was added to each well at a concentration of 100 μl / ml in the presence of the medium, and cultured in an incubator for 4 hours. After 4 hours, the culture medium was removed, and 150 쨉 l of dimethyl sulfoxide (DMSO) was added to each well. Cell viability was analyzed by measuring the absorbance at 595 nm using a spectrophotometer.

At this time, the cell viability was expressed as a relative value with 100% of the cell viability of the control group not treated with the broth cold water extract.

As a result, the cell viability was decreased as the treatment concentration of the cold water extract of broiler chickens increased (FIG. 1).

Thus, all of the following experiments were performed at concentrations that are not cytotoxic.

Experimental Example 2: Influence of broiler cold water extract on cell proliferation by vascular endothelial growth factor ( VEGF)

The vascular endothelial cells cultured in the same manner as in Experimental Example 1 were divided into 12-well plates at a concentration of ⁴ x 10 ⁴ cells / well. After 6 hours of serum starvation, broiler cold water extract was pretreated and treated with 20 ng / ml of vascular endothelial growth factor.

Twenty-four hours after the treatment, the number of cells was measured using a cell counter.

The results of the experiment showed that cold water extract of broiler chickens inhibited vascular endothelial cell proliferation by vascular endothelial growth factor (VEGF) (FIG. 2).

Experimental Example 3: Cell signaling to vascular endothelial growth factor (VEGF) of a cold water extract of broiler chick (extracellular signal regulated kinase (ERK), p38, and vascular endothelial growth factor receptor 2 phosphorylation) experiments

Vascular endothelial cells were plated on 60 mm plates and serum starvation was given for 6 hours on the next day. The broiler chilled cold water extract was treated 40 min before treatment with 20 ng / ml vascular endothelial growth factor.

The cells were treated with radioimmunoprecipitation assay (RIPA) buffer (12 mM EDTA, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0)) for 10 minutes after the vascular endothelial growth factor (20 ng / , 1 mM Na ₃ VO ₄ (Sigma-Aldrich, USA) 10 mM NaF (Sigma), 1 mM Phenylmethylsulfonyl fluoride (Sigma), 1% Triton X-100, 10% glycerol, Roche, Germany) was used to lyse the cells.

The dissolved cells were placed in a 1.5 ml tube, pipetted, and left on ice for 20 minutes. After centrifugation at 14,000 rpm for 20 minutes, the supernatant was recovered to separate proteins. Separated proteins were determined for each sample concentration using a Bicinchoninic Acid (BCA) protein assay kit (Pierce, Rockford, Ill.) And electrophoresed (SDS-PAGE).

Western blot analysis was performed using the separated proteins. After electrophoresis of the sample was completed, the protein was transferred from the gel to a polyvinylidene fluoride membrane (Pall Corporation), and the membrane was incubated with 3% bovine serum albumin (BSA ) Solution (Amresco, USA), and the primary antibody was treated and allowed to react at room temperature for 2 hours. The primary antibodies include anti-ERK antibodies, anti-p-ERK antibodies, anti-p38 antibodies, anti-p-p38 antibodies, anti-VEGF receptor 2 antibodies, and anti- -VEGF receptor 2 antibody was purchased from Cell Signaling Technology, Beverly, Mass. After completion of the reaction with the primary antibody, the cells were washed for about 15 minutes and reacted for 45 minutes with the secondary antibody.

After the reaction was sufficiently washed for at least 30 minutes, the band was confirmed using an enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology).

Experimental results showed that the phosphorylation of extracellular signal regulated kinase (ERK), p38, and vascular endothelial growth factor receptor 2 induced by vascular endothelial growth factor (VEGF) (Fig. 3).

Experimental Example 4: Influence of the effect of chilled cold water extract on cell migration by vascular endothelial growth factor ( VEGF)

First, the lower surface of the transwell insert was coated with gelatin, and 1 × 10 ⁵ vascular endothelial cells and chilled cold water extract were placed therein. In the lower chamber, 20 ng / ml, and incubated for 4 hours. After completion of the incubation, the inner surface of the well was wiped with a cotton swab to remove unmoved cells.

The number of cells that migrated downward was compared with the untreated group by staining with hematoxylin and eosin and counting in the 200x microscopic field. The cell migration of the untreated control group was defined as 100% and expressed as a relative value.

Experimental results showed that cold water extract of broiler chickens inhibited vascular endothelial cell migration by vascular endothelial growth factor (VEGF) (FIG. 4).

Experimental Example 5: Influence of the effect of chilled cold water extract on invasion of vascular endothelial growth factor ( VEGF)

The upper surface of the transwell insert was coated with matrigel, and 1 × 10 ⁵ vascular endothelial cells and chilled cold water extract were placed therein. In the lower chamber, 20 ng / ml of vascular endothelial growth factor And incubated for 24 hours. After completion of the incubation, the inner surface of the well was wiped with a swab or the like to remove unmoved cells.

The number of cells that migrated downward was compared with the untreated group by staining with hematoxylin and eosin and counting in the 200x microscopic field. The cell migration of the untreated control group was defined as 100% and expressed as a relative value.

Experimental results showed that cold water extract of broiler chickens inhibited vascular endothelial cell invasion by blood and vascular endothelial growth factor (VEGF) (FIG. 5).

Experimental Example 6: Influence of broiler cold water extract on matrix metalloprotease (MMP) by vascular endothelial growth factor ( VEGF)

The cell culture broth treated with broiler extract and vascular endothelial cell growth factor and the untreated cell culture broth were centrifuged at 1500 rpm for 5 minutes at 4 ° C and electrophoresed (SDS-PAGE) using 0.2% gelatin- Respectively. Thereafter, the cells were washed with 2.5% Triton X-100 solution for 30 minutes twice and incubated in an incubation buffer for 3 hours or 12 hours. Then, 0.25% Coomassie Brilliant Blue R250 solution The activity of substrate metalloproteinase was observed. Phorbol myristate acetate (PMA) (40 ng / ml) was used for positive control.

The results showed that the cold water extract of broiler chickens inhibited the activity of matrix metalloprotease (MMP) of vascular endothelial cells by vascular endothelial growth factor (VEGF) (FIG. 6).

Experimental Example 7: Influence of cold water extract of broiler chickens on tube formation by vascular endothelial growth factor ( VEGF)

A 24-well plate was coated with 200 쨉 l matrigel and incubated at 37 째 C for 30 minutes. The cultured endothelial cells 2 were dispensed to a 24-well plate coated at a concentration of × 10 ⁵ cells / well. Cells were pretreated with vascular endothelial growth factor (20 ng / ml) and chilled cold water extracts, and tube formation was observed microscopically 20 hours after dispensing. This experiment was carried out using 1% fetal bovine serum (FBS) M199 medium.

Experimental results showed that tube formation induced by vascular endothelial growth factor (VEGF) was inhibited by broiler cold water extract (Fig. 7).

Experimental Example 8: Effect of chilled cold water extract on the sprouting of rat aorta by vascular endothelial growth factor ( VEGF)

A 24-well plate was coated with 120 쨉 l matrigel and incubated at 37 째 C for 30 minutes. Aortas of 7-week-old rats (SD rats) were removed and the blood vessels were cleaned with phosphate buffered saline (PBS), and all of the fine blood vessels attached to the aorta were removed. The blood and thrombosed aorta were cut to a thickness of 1 mm, placed on a 24-well plate coated with matrigel, and once again 50 쨉 l matrigel was added thereto, Min. The vascular endothelial growth factor (20 ng / ml) and broth containing chilled cold water extract were placed on the hardened arteries surrounded by matrigel. After one week, the number of sprouting was observed under a microscope.

The results showed that the cold water extract of broiler chickens inhibited the sprouting of the rat aorta by vascular endothelial growth factor (VEGF) (Fig. 8).

[ Formulation example ]

Formulation example  One. Sanje  Produce

Cold water extract of broiler chickens ................ 10 mg

Sucrose ........................ 100 mg

Talc ............................ 10 mg

The above components are powdered and mixed, and filled in an airtight container to prepare a powder.

Formulation Example 2 . Manufacture of tablets

Chilled cold water extract ..................... 20 mg

Lactose ................................. 100 mg

Starch ................................. 100 mg

Magnesium stearate .................. Quantitative

The above components are mixed and tablets are prepared by tableting according to a conventional method for producing tablets.

Formulation example  3. Manufacture of capsules

Chilled cold water extract ..................... 10 mg

Lactose ................................. 50 mg

Starch ................................. 50 mg

Talc ................................. 2 mg

Magnesium stearate .................... Amount

The above components are mixed and filled in a gelatin capsule according to a conventional method for manufacturing a cambule to prepare a capsule preparation.

Formulation example  4. Preparation of granules

Chilled cold water extract ...................... 10 mg

Soybean extract ........................... 50 mg

Glucose ................................ 200 mg

Starch .................................. 500 mg

After mixing the above components, 100 mL of 30% ethanol is added, and the mixture is dried at 60 ° C to form granules, which are filled in a capsule to prepare granules.

Formulation example  5. Pennant  Produce

Chilled cold water extract ....................... 20 mg

Lactose ................................... 1,500 mg

Glycerin ............................... 1,500 mg

Starch ................................... 980 mg

After mixing the above components, they are prepared so as to be 4 g per one ring according to a conventional method for producing a pellet.

Formulation example  6. Liquid  Produce

Chilled cold water extract ........................ 100 mg

Sugar .................................... 20 g

Isomerization sugar ................................ 20 g

Lemon flavor .................................. Suitable amount

Purified water is added to the whole .................... 100 ml

The above components are mixed according to a conventional method for preparing a liquid preparation, filled in a 100 ml brown bottle, and sterilized to prepare a liquid preparation.

Formulation example  7. Preparation of injection preparation

Chilled cold water extract ........................ 1.0 mg

Sodium Metabisulfite ................... 0.5 mg

Methine paraben .............................. 0.8 mg

Propylparaben ............................ 0.1 mg

Sterilized distilled water for injection

The above ingredients are mixed and made into 3 ml by a conventional method, and then filled into a 3 ml capacity ampoule and sterilized to prepare an injection.

Formulation example  8. Manufacture of health functional foods

10 mg of vitamin C, 10 mg of vitamin C, 10 mg of vitamin C, 10 mg of vitamin C, 10 mg of vitamin C, 20 mg of vitamin A, Nicotinic acid amide 1.7 mg, folic acid 50,, calcium pantothenate 0.5 mg, inorganic mixture proper amount, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monobasic 15 mg, 90 mg of potassium, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride are mixed and prepared according to a conventional method for producing health food.

Formulation example  9. Manufacture of health drinks

20 mg of cold water extract of broiler chickens, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, 1 g of taurine, and purified water are mixed and prepared according to a conventional method for manufacturing health beverages.

Manufacturing example  10. Manufacture of confectionery

10 mL of the broth cold water extract of the present invention is added to 5 g of butter and 10 mL of milk to 100 g of wheat flour and cookies, crackers and snacks are prepared using this mixture.

Claims (6)

The cold water extract of Cinnamomum cassia is contained as an active ingredient and the cold water extract of the broth is contained in the pharmaceutical composition at a concentration of less than 100 ug / ml, and the vascular endothelial growth factor (VEGF) The present invention relates to a pharmaceutical composition for inhibiting angiogenesis, which inhibits angiogenesis by inhibiting phosphorylation of p38 or vascular endothelial growth factor receptor (VEGFR).
The method according to claim 1,
The pharmaceutical composition may be used for the treatment of diabetic retinopathy, neovascular glaucoma, posterior capsular hyperplasia, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, corneal ulcer, cone diaphragm, macular degeneration, Osteoarthritis, septic arthritis, hemangiomas, angiofibroma, psoriasis, pyogenic granuloma, osteoarthritis, osteoarthritis, osteoarthritis, rheumatoid arthritis, osteoarthritis, , Degenerative cartilage loss due to traumatic joint injury, neurodegenerative diseases of the nervous system, cirrhosis, gynecological diseases, immature platelets of inflammation, inflammatory bowel disease, dermatomyositis, arteriosclerosis, restenosis, central nervous system Inflammatory diseases, Alzheimer's disease, skin aging, thyroid hyperplasia, Grave's disease, cancer development, cancer invasion and cancer Wherein the composition is for the prevention or treatment of a disease selected from the group consisting of cancer metastasis.
The method according to claim 1,
The pharmaceutical composition may be used for the treatment of diabetic retinopathy; or
Wherein the composition is for prevention or treatment of cancer development, cancer invasion or cancer metastasis due to neovascularization.
The method of claim 3,
Wherein said cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, pancreatic cancer, kidney cancer, sarcoma, mesothelioma, teratoma, astrocytoma, melanoma, hemangioma and glioma. Composition.
The method according to claim 1,
The cold water extract of the broth suppresses vascular endothelial cell proliferation by vascular endothelial growth factor (VEGF), inhibits vascular endothelial cell migration, inhibits tube formation, or inhibits aortic germination (sprouting) And inhibiting angiogenesis. The present invention also provides a pharmaceutical composition for inhibiting angiogenesis.
The cold water extract of Cinnamomum cassia is contained in the food composition at a concentration of less than 100 ug / ml, and the extract of extracellular (ERK) by vascular endothelial growth factor (VEGF) The present invention relates to a novel angiogenesis inhibiting food composition which inhibits angiogenesis by inhibiting the phosphorylation of p38 or vascular endothelial growth factor receptor (VEGFR).

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