KR100547366B1 - Composition for the prevention or treatment of diseases associated with angiogenesis comprising the extract of Cnidium officinale Makino or the fraction therefrom - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of diseases caused by angiogenesis, and more particularly, a composition for the prevention or treatment of diseases caused by angiogenesis, including a fraction obtained by extracting and purifying the uterus as an active ingredient. It is about. Since the composition of the present invention has an activity of inhibiting angiogenesis, it can be usefully used for the prevention or treatment of diseases caused by angiogenesis such as arthritis, diabetic retinopathy, psoriasis and cancer.

Angiogenesis, Archery, Arthritis, Diabetic Retinopathy, Psoriasis, Cancer

Description

Composition for the prevention or treatment of diseases associated with angiogenesis comprising the extract of Cnidium officinale Makino or the fraction therefrom}

Figure 1 shows the extraction and fractionation method of the archery

Figure 2 is a photograph showing the inhibition of HUVEC tube formation by the extract and fractions.

Figure 3 illustrates the angiogenesis inhibitory effect in animal experiments by crude extract of the uterus (AS44-TW),

Figure 4 illustrates the angiogenesis inhibitory effect in animal experiments by the uterine fraction (AS44-C-2).

The present invention relates to a composition for preventing or treating diseases caused by angiogenesis, and more particularly, due to angiogenesis comprising an extract or fraction obtained by extracting and purifying Cnidium officinale Makino as an active ingredient. It relates to a composition for preventing or treating a disease.

Angiogenesis is the process of forming new capillaries from existing microvessels. Angiogenesis normally occurs during embryonic development, during tissue regeneration and wound healing, and during the development of the corpus luteum, a change in the genital system of women in a regular manner. In this case, it is strictly controlled [Folkman and Cotran, Int. . Rev. Exp. Pathol. 16 , pp 207-248, 1976.

In adults, vascular endothelial cells grow very slowly and do not divide relatively well compared to other types of cells.

The angiogenesis process is generally caused by stimulation of angiogenesis factors, resulting in the formation of lumen due to the degradation of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated.

However, there are diseases caused by angiogenesis that is not controlled autonomously and grows pathologically. Diseases related to angiogenesis in pathological conditions include hemangioma, angiofibroma, angioplasty and cardiovascular diseases such as atherosclerosis, angiogenesis, and edema sclerosis. Eye diseases caused by angiogenesis include corneal graft angiogenesis and angiogenesis. Glaucoma, diabetic retinopathy, corneal disease caused by neovascularization, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis and the like. Chronic inflammatory diseases such as arthritis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, blood-imposed diseases such as acne, Alzheimer's and obesity are also associated with angiogenesis, and cancer growth and metastasis necessarily depend on angiogenesis [D ' Amato RJ and Adamis AP, Ophthalmol., 102 , pp 1261-1262, 1995; Arbiser JL, J. Am. Acad. Derm., 34 (3) , pp 486-497, 1996; O'Brien KD et al. Circulation 93 (4) , pp 672-682, 1996; Hanahan D and Folkman J, Cell, 86, pp 353-364, 1996].

Angiogenesis, in particular, plays an important role in the growth and metastasis of cancer cells. Tumors are supplied with nutrients and oxygen for growth and proliferation through neovascularization, and new blood vessels that penetrate the tumor give metastasis by allowing metastatic cancer cells to enter the blood circulation [Folkman and Tyler, Cancer Invasion and metastasis , Biologic mechanisms and Therapy (SB Day ed.) Raven press, New York, pp 94-103, 1977; Polverini PJ, Critical Reviews in Oral Biology , 6 (3) , pp 230-247, 1995].

The main cause of death of cancer patients is metastasis, and the current metastasis of cancer does not contribute to the survival of cancer patients.

Arthritis, a representative disease of inflammatory diseases, is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial cavity between joints induces angiogenesis and destroys cartilage. In other words, synovial cells and vascular endothelial cells proliferate in the synovial cavity with the help of inflammation-inducing cytokines, forming articular pannus, a connective tissue layer that develops in the cartilage as the angiogenesis progresses, destroying cartilage that acts as a cushion. [Kocb AE, Polverini PJ and Lcibovich SJ, Arth. Rheum., 29 , pp 471-479, 1986; Stupack DG, Storgard CM and Cheresh DA, Braz J. Med. Biol. Rcs., 32 , pp 578-581, 1999, Koch AE, Atrh. Rheum., 41 , pp 951-962, 1998].

Many ocular diseases, which cause millions of blindness worldwide each year, are also caused by angiogenesis [Jeffrey MI and Takayuki A, J. Clin. Invest., 103 , pp 1231-1236, 1999]. For example, diseases such as macular degeneration, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma and corneal diseases caused by neovascularization are the diseases caused by neovascularization [Adamis AP]. , Aiello LP and D'Amato RA, Angiogenesis, 3 , pp 9-14, 1999]. Among them, diabetic retinopathy is a complication of diabetes, in which capillaries in the retina invade the vitreous body and eventually become blind.

Psoriasis, which is characterized by red spots and skin, is also a chronic proliferative disease of the skin. It does not heal and involves pain and malformations. In normal cases, keratinocytes proliferate once a month, whereas psoriasis patients proliferate at least once a week. This rapid proliferation requires a lot of blood supply, so angiogenesis is bound to occur actively [Folkman J, J. Invest. Dermatol., 59 , pp 40-48, 1972].

Obesity and adipose tissue can also be controlled by the lower vasculature, and treatment with anti-angiogenic inhibitors results in dose-dependent weight loss and adipose tissue loss [Rupnick MA et al. al., Proc. Natl. Acad. Sci. USA , 99 (16) , pp 10730-5, 2002].

In recent years, blood loss and inflammation in the Alzheimer's brain result in angiogenesis-inducing factors and angiogenesis, which suggest that beta-amyloid deposition and neurotoxic peptides are released. Drugs that inhibit angiogenesis, which can target WM, may prevent and treat Alzheimer's disease (Vagnucci AH et al., Lancet 361 , pp605-608, 2003).

Since angiogenesis inhibitors can be applied as a therapeutic agent for such various angiogenesis-related diseases, studies are being actively conducted to treat the above diseases by inhibiting angiogenesis.

Such angiogenesis inhibitors usually require long-term administration to patients, so they should be of low toxicity and be orally available for use as the ideal therapeutic. Therefore, the development of drugs and functional foods with low toxicity as angiogenesis inhibitors is required.

On the other hand, Cnidium officinale MAKINO is a perennial herb belonging to the umbelliferae, and in oriental medicine, its root stem is dried and used mainly for gynecological diseases such as headache, infertility, menstrual irregularities, anemia, tonic, and coldness. . The chemical components of the horoscope include phthalide-based compounds such as cnidilide, neocnidilide, ligustilide, butylphthalide and senkyunolide. It is known to contain (Fuzita M. and Kobayashi M . ; Chem. Pharm. Bull., 35 (4), pp1427-1433, 1987).

Korean Patent Publication No. 2002-82390 relates to a composition for inhibiting angiogenesis comprising glutathione and gardenia as an active ingredient, and discloses an angiogenesis inhibitory effect of glutathione and gardenia extract and mixtures thereof through in vivo experiments. In addition, Korean Patent Publication No. 2002-35912 discloses the use of horse chestnut extract as an angiogenesis inhibitor, and Korean Patent Publication No. 2003-51176 discloses an anti-ulcer therapeutic agent including a uterine extract.

However, none of these documents teaches or discloses that the extract of the uterus is used in the treatment of diseases caused by angiogenesis.

Accordingly, the present inventors have found that the uterus has the effect of inhibiting angiogenesis, and by extracting and purifying the active ingredient from the uterus, the present invention was completed by revealing that it can be used to inhibit angiogenesis.

It is an object of the present invention to provide a composition for the prevention or treatment of diseases caused by angiogenesis, including the fraction extracted from the uterus as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of diseases caused by angiogenesis, including crude extract of Cnidium officinale Makino.

The crude extracts comprise water, C ₁ to C ₄ lower alcohols, and mixtures thereof, preferably extracts soluble in water or methanol.

The present invention also provides a composition for the prevention or treatment of diseases caused by angiogenesis, including a non-polar solvent soluble extract or fraction of the uterus.

The non-polar solvent soluble extract includes extracts soluble in non-polar solvents such as hexane, chloroform, methylene chloride, ethyl acetate, preferably chloroform.

The purified fraction includes a purified product obtained by performing column chromatography of a chloroform soluble extract of Cheongung with a chloroform and methanol mixed solvent or an ethyl acetate and hexane mixed solvent with a developing solvent.

In addition, according to the present invention, the second fraction of the four fractions obtained by performing silica gel column chromatography at 100 ml per hour using a chloroform soluble fraction of Cheongung as a developing solvent using a chloroform: methanol (20: 1) mixed solvent as ethyl acetate: Silica gel column chromatography was performed using a hexane (1: 2) mixed solvent as a developing solvent, and an active third fraction was obtained from the four fractions obtained therefrom, which was then mixed with an ethyl acetate: hexane (1: 2) mixed solvent. The second fraction of the four fractions obtained by performing silica gel column chromatography using as a developing solvent.

Hereinafter, the present invention will be described in detail.

Crude crude extract of the present invention is about 1 to 20 times the weight (kg) by cutting the root or ground portion (leaf, stem) of the dried cheonunggi, preferably about 3 to 10 times water, C ₁ to Lower alcohol of C ₄ or a mixed solvent thereof, preferably water or methanol, at an extraction temperature of 20 ° C. to 100 ° C., preferably 50 ° C. to 100 ° C., for about 1 hour to 10 days, preferably about 2 hours to The crude extract obtained by filtration, concentrated under reduced pressure, or dried using a cold soak, hot water extraction, ultrasonic extraction, reflux cooling extraction method for 5 hours can be obtained as a polar solvent soluble extract.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the crude extract in distilled water, the suspension is about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, chloroform, methylene chloride, ethyl acetate, preferably Can be obtained by adding a nonpolar solvent such as chloroform to extract and separating the nonpolar solvent soluble layer 1 to 10 times, preferably 2 to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis. 3rd Ed. Pp 6-7,1998).

In a preferred embodiment of the present invention, the uterine fraction of the present invention is extracted with the organic solvent from the uterine arch. Specifically, the dried Cheongung was extracted with methanol to obtain a methanol extract (AS44-M), from which a hexane soluble fraction (AS44-H), a chloroform soluble fraction (AS44-C), a butanol soluble fraction (AS44-B) and Obtain the final aqueous fraction (AS44-W). The chloroform soluble layer (AS44-C) of Cheongung, whose angiogenesis inhibitory activity was confirmed, was subjected to silica gel column chromatography using chloroform: methanol mixed solvent, preferably 15: 1 to 25: 1 volume ratio of chloroform: methanol mixed solvent. Small fractions (AS44-C-1, -2, -3, -4). The second active fraction (AS44-C-2) was used as an ethyl acetate: hexane mixed solvent, preferably a 1: 1 to 1: 5 volume ratio of an ethyl acetate: hexane mixed solvent using silica gel column chromatography. Four small fractions were obtained again (AS44-C-2-A, -B, -C, -D), and the active fraction C was again mixed with an ethyl acetate: hexane solvent, preferably 1: 1. To 1: 5 volume ratio ethylacetate: hexane mixed solvent was subjected to silica gel column chromatography with a developing solvent to obtain three fractions (AS44-C-2-Ca, -b, -c).

In the embodiment of the present invention, the ability to inhibit angiogenesis of the fraction of the active ingredient isolated from the uterus was confirmed in various ways. As a result, crude water extract, chloroform soluble extract and fractions (AS44-C, AS44-C-2, AS44-C-2-C, AS44-C-2-Ca) inhibited HUVEC tube formation and inhibited HUVEC growth. In animal experiments, it was shown to be effective in treating diseases caused by angiogenesis by exhibiting the effects of inhibition of angiogenesis.

The present invention provides a composition for the prophylaxis or treatment of diseases caused by angiogenesis, which is isolated from the uterus by the above-described manufacturing method, the cheonunggi extract or fraction having angiogenesis inhibitory efficacy as an active ingredient.

Compositions for the prevention or treatment of diseases caused by angiogenesis of the present invention are hemangiomas, hemangiofibromas, angioplasty, arteriosclerosis, angiogenesis, edema sclerosis, corneal graft angiogenesis, neovascular glaucoma, diabetic retinopathy, corneas caused by neovascularization Diseases, degeneration of spots, pterygium, retinal degeneration, posterior capsular fibrosis, granuloconjunctivitis, senile degeneration, prematurity retinopathy, chronic inflammatory diseases such as arthritis, psoriasis, capillary dilatation, purulent granulomas, Alzheimer's disease, obesity, seborrheic dermatitis It may be applied to a disease selected from the group consisting of cancer diseases including acne and solid cancer, but is not necessarily limited thereto.

The cancer diseases include lung cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, Fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma Can be used to treat cancer diseases and metastases due to angiogenesis.

The composition of the present invention preferably contains from 0.01 to 99.9%, more preferably from 0.1 to 90%, of the extract or purified fraction.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general formulation.

That is, the composition containing the extract or fraction extracted from the cheongungung of the present invention can be administered in a variety of oral and parenteral formulations during the actual clinical administration, when formulated, commonly used fillers, extenders, binders, wetting agents And diluents or excipients such as disintegrants and surfactants.

Compositions comprising extracts or fractions according to the present invention are oral formulations, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods, respectively. Formulated in the form of can be used.

Such solid preparations are prepared by mixing at least one excipient such as dextrin, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the active ingredient fraction of the uterus. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or fractions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a dietary supplement for the prevention, improvement and alleviation of diseases caused by angiogenesis, including crude extracts and food supplements of food supplements.

The present invention also provides a health functional food for preventing and ameliorating diseases due to angiogenesis, which contains a ungmul nonpolar solvent soluble extract or a fraction separated therefrom and a food acceptable additive.

Examples of the food to which the extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing, ameliorating and alleviating diseases caused by angiogenesis. At this time, the amount of the extract in the food or beverage, in general, in the case of the health functional food composition of the present invention can be added to 0.01 to 90% by weight, preferably 0.1 to 80% by weight of the total food weight, the health beverage composition It can be added at a ratio of 0.01 to 90 g, preferably 0.1 to 50 g based on 100 ml.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Preparation of Crude Crude Extract 1

200 g of dried Cheongung purchased from Gyeongdong Market was placed in 1 liter of water, and extracted by heating twice at 90-100 ° C. for 6 hours. The extract was filtered and concentrated under reduced pressure to give 56.6 g of extract (AS44-TW).

Example 2 Preparation of Crude Crude Extract 2

800 g of dried Cheongung purchased from Gyeongdong Market was placed in 6 liters of 80% methanol, and extracted twice with 3 hours in a water bath using a reflux condenser. The extract was filtered and concentrated under reduced pressure to give 49.1 g (AS44-M) of methanol extract.

Example 3 Preparation of Soluble Fractions of Non-polar Solvents

3-1. Preparation of Soluble Hexane Fraction

49.1 g of the cheongung methanol extract of Example 2 was suspended in 1 liter of distilled water, and 200 ml of n -hexane ( n -hexane) was added thereto, and the mixture was shaken and left to be a fraction. Extraction and fractionation with hexane three times, the hexane soluble layers were combined and the solvent was evaporated by rotary evaporator to give 10 g of hexane soluble fraction (AS44-H).

3-2. Preparation of Soluble Fractions of Cheongung Chloroform

200 ml of chloroform was added to the aqueous layer separated from the hexane soluble layer of Example 3-1, shaken vigorously, and left to be fractionated. Extracted and fractionated three times with chloroform, the chloroform soluble layers were combined and the solvent was evaporated with a rotary evaporator to give 4.5 g of the chloroform soluble fraction (AS44-C).

3-3. Preparation of Soluble Fractions and Soluble Fractions of Cheongung Butanol

200 ml of butanol was added to the aqueous layer separated from the chloroform soluble layer of Example 3-2, shaken vigorously, and left to be fractionated. Extraction and fractionation with butanol three times, the butanol soluble layers were combined and the solvent was evaporated with a rotary evaporator to give 10.8 g of butanol soluble fraction (AS44-B).

At this time, the remaining aqueous layer was lyophilized to give 53.1 g of the aqueous fraction (AS44-W).

Each fraction obtained above was prepared at a concentration of 10 mg / ml, and used as a sample in the following experimental example.

Example 4. Isolation and Purification of the Active Ingredient from the Cheongung Extract

4.5 g of the chloroform soluble fraction (AS44-C) of Example 3-2 was adsorbed onto the same amount of silica gel, followed by silica gel column chromatography (7 × 70 cm, Merck). In this case, using a chloroform: methanol (20: 1, v / v) mixed solvent as a developing solvent, 100 mL per hour to give four fractions, namely small fraction 1 (1.11 g), small fraction 2 (0.39 g), Small fraction 3 (0.24 g) and small fraction 4 (1 g) were obtained and named AS44-C-1, AS44-C-2, AS44-C-3, AS44-C-4, respectively.

Among the active As44-C-2, silica gel column chromatography was again performed with an ethyl acetate: hexane (1: 2, v / v) solvent to obtain small fraction A (39 mg), small fraction B (23.4 mg), Small fraction C (167.3 mg) and small fraction D (100.5 g) were obtained, respectively AS44-C-2-A, AS44-C-2-B, AS44-C-2-C, AS44-C-2- Named D

The active AS44-C-2-C was subjected to silica gel column chromatography with ethyl acetate: hexane (1: 2, v / v) solvent to give a small fraction a (36.7 mg) and a small fraction b (61.8 mg). , Small fraction c (60.1 mg) was obtained and named AS44-C-2-Ca, AS44-C-2-Cb, AS44-C-2-Cc, respectively.

Reference Example 5. Isolation and Culture of HUVECs

Human umbilical vein endothelial cells (hereinafter abbreviated as 'HUVEC') were isolated from the umbilical vein of the newborn and primary cultured. Specifically, HUVECs were isolated according to the methods described in Grant et al . (Grant, DS et al ., Cell , 58 , pp933-943, 1989), and the isolated HUVECs were M-199 (Gibco BRL, 31100-035). , 10% FBS, 50 ㎍ / ㎖ ECGS (Endothelial cell growth supplement, Sigma E2759), 50 ㎍ / ㎖ heparin (Heparin, Sigma H3149) culture medium.

After the incubation of HUVECs was completed, it was confirmed that HUVECs were successfully isolated by immunostaining with an antibody of factor VIII (DAKO, Code No. M0616) (Valen, G. et al ., Free Radic. Biol. Med. , 26 , pp 1480-1488, 1999; Rhim, JS et al ., Carcinogenesis, 19 , pp673-681, 1998).

Experimental Example 1. Angiogenesis inhibitory effect of uterus

In order to determine the efficacy of angiogenesis inhibitory fractions and fractions, angiogenesis inhibition efficacy was measured at the cellular level through HUVEC tube formation inhibition assay using HUVEC.

1-1. HUVEC tube formation inhibition assay

The HUVEC angiogenesis inhibition assay is the most correlated with in vivo experiments in in vitro assays measuring angiogenesis.

Specifically, after passage of the HUVEC isolated and cultured in Reference Example 1 within 5 times (passage 5), the medium containing the basement membrane component Matrigel (BD Biosciences, Bedford, MA) was used 48 After dispensing 20,000-50,000 cells per well of a well plate, the cells were cultured under the primary culture medium of Reference Example 1.

At this time, the crude extracts, non-polar solvent soluble fractions, and purified products prepared in Examples 1 to 4 were respectively added to the medium at a concentration of 50 μg / ml, and DMSO was added to the control group.

After 18 hours of incubation, tube formation was observed under a microscope, and the results are summarized in Table 1 and Table 2 (-: no inhibition of tube formation, +/-: almost no inhibition, +: tube formation). With a slight suppression of the tube breaking, ++: the tube is broken a lot, +++: the tube is not formed at all).

As shown in Table 1, tube formation was strongly inhibited in the chloroform soluble fraction AS44-C. As mentioned in Example 4, four fractions were separated and purified with the AS44-C fraction to determine the effect of inhibiting HUVEC tube formation. As a result, the AS44-C-2 fraction most inhibited the tube formation of HUVEC. The AS44-C-2 fraction was re-fractionated to measure the activity. The AS44-C-2-C fraction was active. Among the four subfraction fractions, AS44-C-2-C-a was active. The results are summarized in Table 2 and FIG.

sample Inhibition of tube formation  AS44-TW +  AS44-M +  AS44-H +/-  AS44-C ++  AS44-B +/-  AS44-W +/-

sample Inhibition of tube formation  AS44-C +  AS44-C-1 +  AS44-C-2 +++  AS44-C-3 +  AS44-C-4 +  AS44-C-2-A -  AS44-C-2-B +  AS44-C-2-C +++  AS44-C-2-D +/-  AS44-C-2-C-a +++  AS44-C-2-C-b +/-  AS44-C-2-C-c +

Experimental Example 2. Cytotoxicity Assay

2-1. Cytotoxicity Test on HUVEC Cells

The cytotoxicity test on HUVEC cells isolated and cultured in Reference Example 1 was performed on the uterine fraction showing the tube forming inhibitory effect of HUVEC. Specifically, HUVECs were dispensed to contain 5,000 cells per well of a 96 well plate, and then each well was treated with a uterine fraction and observed changes in cell numbers after one day. Survival rate of HUVEC cells was determined by cell proliferation kit II (XTT: 2,3-bis- (2-methoxy-4-nitro-5- sulfophenyl) -2H-tetrazolium-5-carboxanilide disodium salt) , Cat. No. 1465015).

Table 3 shows the results of testing the toxicity of HUVECs of soluble chloroform fractions, indicating that HUVECs were more than 73% alive when chloroform fractions were added at concentrations that inhibit tube formation.

Toxicity Tests for HUVECs in the Cervix Fractions sample Survival rate (%) 50 μg / ml 5 μg / ml AS44-C 95.0 119.4  AS44-C-1 74.6 109.6  AS44-C-2 73.2 124.7

Experimental Example 3. Angiogenesis inhibitory effect experiment of crude extract of the uterus (AS44-TW) using mouse-Matrigel model

The mouse-Matrigel model was used to examine the effect of crude extracts of AS on the in vivo angiogenesis.

C57BL / 6 mice aged 6 to 8 weeks were injected subcutaneously with 0.4 ml of Matrigel, 50 ng / ml of basic Fibroblast Growth Factor (FGF) and 50 units / ml of heparin. After 3-5 days, the epidermis was removed, the matrigel was carefully removed to recover the gel, and then the amount of hemoglobin was obtained using a Dragkin's reagent (commercially available from Sigma), a reagent for measuring the total amount of hemoglobin in the blood. Measured and used as a control.

0.4 mg of the crude extract (AS44-TW) of Example 1 extracted from the uterus was dissolved in 1% DMSO, mixed with the above-mentioned basic FGF and heparin-containing Matrigel, and subjected to subcutaneous injection in the same manner as the control group, followed by hemoglobin content. Was measured and compared with the control.

The results are shown in Table 4 and FIG. 2, and the hemoglobin content was very low in the case of containing the crude extract (AS44-TW) compared to the control group that did not contain the crude extract (AS44-TW). 33% of the suppression was confirmed.

Hemoglobin content (g / dL) % Inhibition Control  613 0 Cheonungjojo Extract (AS44-TW)  407 33

Experimental Example 4. Experiment of angiogenesis inhibitory effect of AS44-C-2 using mouse-Matrigel model

In the same manner as in Experiment 3, C57BL / 6 mice were injected subcutaneously with 0.4 ml of Matrigel and 50 ng / ml of basic fibroblast growth factor (FGF) and 50 units / ml of heparin in combination with angiogenesis inducer. 2 mice orally administered 1.5 mg twice a day twice daily for 4 days, and then confirmed the angiogenesis inhibitory effect in animal experiments, it showed that about 80% inhibition of angiogenesis (see Table 5 and Figure 3).

Hemoglobin content (g / dL) % Inhibition Control 788 0 AS44-C-2 157 80

Experimental Example 5. Acute Toxicity Test

1. Oral administration

ICR-based mice and Sprague-Dawley rats were divided into four groups of 10 rats each, followed by oral administration of the cheonmgung methanol extract of the present invention at doses of 50, 100 and 500 mg / kg, respectively. No deaths occurred in all groups and no symptoms were apparent from the control group.

2. Intraperitoneal administration

ICR-based mice (weight 25 ± 5 g) and Sprague dooli rats were divided into four groups of 10 rats each, and the abdominal administration of the uterine water extract (AS44-TW) of the present invention at doses of 25, 50 and 75 mg / kg, respectively. After 24 hours, no one died in all three groups, and no symptoms were observed. As a result of the experiment, it was confirmed that the uterine water extract of the present invention has little acute toxicity.

Extracts and fractions of the present invention can be administered in the following formulations, the formulation examples below are merely to illustrate the invention, whereby the content of the invention is not limited.

Formulation Example 1 Preparation of Pills

Archery fraction from Example 3. 120 mg

Corn starch ............... 100 mg

Sterilized Distilled Water ...

The above components are mixed and refilled in a 0.3 cm diameter by a conventional pill manufacturing method to prepare pills.

Formulation Example 2 Preparation of Tablet

Archery fraction from Example 3 ....... 200 mg

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

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

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 3 Preparation of Capsule

Archery fraction from Example 3 ...... 100 mg

Lactose ... 50 mg

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

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

Magnesium Stearate .........

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

Crude crude extract of Example 1 ........ 1000 mg

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

Isomerized sugar ............... 20g

Lemon Flavor ......................

Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 5 Preparation of Healthy Food

Cruciform Crude Extract of Example 2 ............. 1000 mg

Vitamin Blend ...............

Vitamin A Acetate ............... 70 μg

Vitamin E ........... 1.0 mg

Vitamin B1 ......... 0.13 mg

Vitamin B2 ........... 0.15 mg

Vitamin B6 ............ 0.5 mg

Vitamin B12 ........................ 0.2 μg

Vitamin C ......................................... 10 mg

Biotin ............... 10 μg

Nicotinic Acid Amide ... 1.7 mg

Folic acid ............... 50 ㎍

Calcium Pantothenate ..................... 0.5 mg

Mineral mixture ........................

Ferrous Sulfate ............... 1.75 mg

Zinc Oxide ........... 0.82 mg

Magnesium Carbonate ......... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dicalcium Phosphate ............... 55 mg

Potassium citrate ... 90 mg

Calcium Carbonate ... 100 mg

Magnesium Chloride ............... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 6 Preparation of Healthy Drink

Crude crude extract of Example 1 ..... 1000 mg

Citric Acid ........................................ 1000 mg

Oligosaccharide ......................... 100 g

Plum concentrate ........................... 2 g

Taurine ......................................... 1 g

Purified water is added ............................................. 900 ml

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is mixed with a relatively suitable component for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the fraction containing the active ingredient extracted from the uterus of the present invention can be usefully used as a composition for the prevention or treatment of diseases caused by angiogenesis, such as arthritis, diabetic retinopathy, psoriasis and cancer.

Claims (12)

delete delete Chloroform soluble extract or TLC Rf: 0.20 ~ 0.49 (4 fractions) obtained by applying the extract to silica gel column chromatography using chloroform: methanol (20: 1) mixed solvent as a developing solvent at 100 ml / hr. Development solvent conditions; Hemangioma mediated by angiogenesis, angioma, angioma, angiosclerosis, angiogenesis, edema sclerosis, corneal graft Angiogenesis, neovascular glaucoma, diabetic retinopathy, corneal disease caused by neovascularization, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis, degenerative plaque, retinopathy of premature infants, arthritis, psoriasis, capillary dilator, purulent Selected from the group consisting of granulomas, Alzheimer's disease, obesity, seborrheic dermatitis, cancer diseases including acne and solid cancer More blood composition for the prevention or treatment of diseases caused by start-ups. delete delete delete delete delete delete delete delete delete

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