KR100533777B1 - Composition for inhibiting angiogenesis containing an extract of horse chestnut - Google Patents

Abstract

The present invention relates to a composition comprising the horse chestnut extract having angiogenic inhibitory activity as an active ingredient, and to a pharmaceutical composition for preventing and treating diseases caused by angiogenesis containing the same, which is a human umbilical vascular endothelial cell (HUVEC) Inhibitory experiments on the formation of capillaries such as capillaries and in vivo experiments using the mouse Matrigel model confirmed that the horse chestnut extract had angiogenesis inhibitory effects, and the horse chestnut extract was a matrix metalloproteinase (Matix metalloproteinase). As a result of confirming the inhibitory activity against MMP) enzymes, the composition comprising the horse chestnut extract of the present invention is effective in treating various diseases caused by angiogenesis such as tumor suppression, metastasis suppression, ocular disease caused by angiogenesis, psoriasis and arthritis. And diseases associated with excessive MMP activity.

Description

Composition for inhibiting angiogenesis comprising horse chestnut extract as an active ingredient {COMPOSITION FOR INHIBITING ANGIOGENESIS CONTAINING AN EXTRACT OF HORSE CHESTNUT}

The present invention relates to an angiogenesis inhibiting composition comprising the horse chestnut extract as an active ingredient, in particular horse chestnut extract found that the horse chestnut extract has the effect of inhibiting angiogenesis, can be used as a preventive and therapeutic agent for diseases related thereto It relates to a pharmaceutical composition containing an extract.

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 periodic manner, and in this case, it is strictly controlled (Folkman and Cotran; Int). Rev. Exp. Pathol., 16 , pp 207-248, 1976).

The formation of new blood vessels is precisely divided into vasculogenesis and angiogenesis. Angiogenesis is defined as angiogenesis of endothelial cells resulting from differentiation of blood in mesoderm early in embryonic development. Embryo vasculature begins when simple diffusion does not meet the needs of nutrition for rapidly improving cell proliferation for complex forms. Angiogenesis is a process of growing out of endothelial cells from existing blood vessels, and typically forms terminal veins and posterior capillaries of microvascular vessels. In adults, vascular endothelial cells do not divide relatively well, and the rate of endothelial division is usually months to years. However, angiogenesis in adults is the result of hormonal signaling, pro-angiogenesis in inflammatory cells, the release of cytokines, the activity of hydrolytic enzymes that release angiogenesis mediators isolated from extracellular matrix, or blood vessels Stimulation of angiogenesis factors is triggered as needed.

Angiogenesis usually involves reconstruction of blood vessels due to protease degradation of the vascular basement membrane, migration of vascular endothelial cells, proliferation, and formation of lumen by vascular endothelial differentiation, resulting in the formation of new capillaries. 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, neovascularized corneal disease, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granulomatous conjunctivitis, and chronic inflammatory diseases such as arthritis, psoriasis, capillary dilemma, purulent granulomas, and seborrheic There are dermatological diseases such as dermatitis and acne, and the growth and metastasis of cancer necessarily depends on angiogenesis (D'Amato RJ and Adamis AP; Ophthalmol., 102 , pp1261-1262, 1995; Arbiser JL; J Am Acad Derm , 34 (3) , pp486-497, 1996; O'Brien KD et al .; Circulation, 93 (4) , pp672-682, 1996; Hanahan D and Folkman J; Cell, 86 , pp353-364, 1996).

 Diseases caused by abnormal angiogenesis are deeply involved in the development or progression of the various diseases, and studies are actively being conducted to find substances that inhibit angiogenesis for the prevention or treatment of these diseases.

Especially in the case of cancer, angiogenesis plays two important roles. Firstly, it provides nutrition and oxygen for the growth and proliferation of primary and metastasized secondary tumors. Second, cancer cells to which metastatic capillaries penetrate the tumor. It gives the chance to enter the blood circulation system, causing cancer cells to metastasize throughout the body.

Tumors remain dormant for a period of time if angiogenesis does not occur and can no longer grow (Folkman and Tyler; Cancer Invasion and metastasis , Biologic mechanisms and Therapy (SB Day ed.) Raven press, New York, pp94 -103, 1977). Metastasis is also the main cause of death of cancer patients, and cancer cells are isolated from the primary tumor mass and grow on anatomically distant tissues or organs (Sugarbaker, Weingard and Rosoman; Cancer Invasion and Metastasis (LA Liotta) and IR Hart ed.) Boston, Nijhoff, pp 427-465, 1982).

Currently, the clinic uses chemotherapy or immunotherapy to improve the therapeutic performance of cancer. Although these treatments have some effects in some tumors, they do not contribute to improving the survival rate of cancer patients. Because of the transition.

Since cartilage destruction in the joints, blindness caused by various ophthalmic diseases, and hyperplasia of skin cells are also caused by angiogenesis, it is possible to inhibit the angiogenesis and progress and fundamentally cure diseases such as arthritis, various ophthalmic diseases, and psoriasis. Kocb AE, Polverini PJ and Lcibovich SJ; Arth.Rheum ., 29 , pp471-479, 1986; Stupack DG, Storgard CM and Cheresh DA, Braz; J Med Biol Rcs, 32 , pp578-581, 1999, Koch AE; Atrhritis Rheum, 41 , pp951-962, 1998; Jeffrey MI and Takayuki A; J Clin Invest, 103 , pp1231-1236, 1999; Folkman J, J Invest Dermatol, 59 , pp40-48, 1972.

Angiogenesis involves the breakdown of the basement membrane, and the enzymes belonging to the MMP family are the most representative enzymes that break down the basement membrane components. About 20 enzymes belonging to the MMP family are known to date. MMPs are a class of endopeptidase containing zinc capable of breaking down large biomolecules such as collagen, proteoglycans and gelatin, and are broadly classified into collagenase, gelatinase, stromelysin and membrane type MMP. They are all expressed in the form of proenzymes and then activated by cutting off a portion of itself (Bond, JS, et al . ; Int. J. Biochem., 75 , pp565-574, 1985; Chen, JM, Chen, WT; Cell , 48 , pp 193-203, 1987).

Collagenase acts on triple helix interstitial collagen and gelatin, and three kinds of fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8) and collagenase-3 (MMP-13) (Goldberg, GI, et al . ; J. Biol. Chem. , 261 , pp6600-6605, 1986; Fini, ME, et al . ; Biochemistry , 26 , pp6156-6165, 1987; Hasty, KA, et al .;. J. Biol Chem, 265 , pp11421-11424, 1990;.. Freije et al;... J. Biol Chem, 269, pp16766-16773, 1994).

Gelatinase is an enzyme that degrades denatured collagen and collagen IV, V, VII and X. It is a 72kDa gelatinase released by fibroblasts (gelatinase A, MMP-2) and a 92kDa gel released by monocytes. There are two types of tinases (gelatinase B; MMP-9), and are known to specifically act on collagen type IV, the main component of the basement membrane (Murphy, G., et al . ; Biochem. J., 258 , pp 463-472, 1989; Stetler-Stevenson, WG, et al . ; J. Biol. Chem. , 264 , pp1353-1356, 1989).

Types of stromelysin having a broad spectrum of proteolytic degradation include stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), stromelysin-3 (MMP-11), and matrylysine (MMP). -7), MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), etc. belonging to the metalloelase (MMP-12) and the membrane-type MMP Known.

MMPs are not only involved in angiogenesis, but also atherosclerosis, Alzheimer's disease, bone disease, proteinuria, abdominal aortic aneurysm, myelin demyelination, cirrhosis, renal glomeruloneum, immature rupture of the embryonic membrane, inflammatory bowel disease, and fasciitis Overexpression in various diseases such as macular degeneration (Wossener Jr .; Annals NY Acad. Sci., 732 , pp11-21, 1994; Warner et al . ; Am. J. Pathol., 158 , pp 2139-44, 2001; Stetler-Stevenson; Surg. Oncol. Clin. N. Am. , 10 , pp 383-92, 2001).

Therefore, the inhibitor of the activity of the MMP enzyme acts as an angiogenesis inhibitor and can be used for various MMP mediated diseases, and thus the development of new drugs is expected.

 In recent years, efforts to treat angiogenesis-related diseases by suppressing angiogenesis and there is no therapeutic agent to date, but there are no drugs used to date. Angiogenesis inhibitors usually require long-term administration to patients, so they should be of low toxicity and be orally available for the ideal treatment. Therefore, there is a demand for the development of a drug having low toxicity as an angiogenesis inhibitor.

In order to solve the problems described above, as a result of searching for an angiogenesis inhibitory effect on a plant extract having a relatively less toxicity than a chemical, the present inventors have found that the horse chestnut extract has an excellent angiogenesis inhibitory effect and completed the present invention.

Horse chestnut (Horse chestnut) belongs to the Hippocastanaceae, the horse chestnut extract of the present invention is obtained from the leaves or seeds (Saraz).

Horse chestnut trees include Japanese horse chestnut ( Aesculus turbinta BLUME), Chinese horse chestnut ( Aesculus chinensis Bge and Aesculus wilculus Rehd ), and European horse chestnut ( Aesculus hippocastanum L.), and the like.

Japanese lacquer tree is a deciduous tree growing in the suburbs of Gyeonggi-do, Japan and Korea. Its seed is Saraja (裟 羅 子) and has been used for the treatment of gastric pain, malaria, dysentery, etc. Its components include saponin, oleic acid and The same fatty oils, starches, etc. are known (Shin Min-kyo, information subsidy, Dohae Hyangje metabolism, published in 1998, Yeonglimsa pp. 384-385).

European horse chestnut is native to the north-eastern region of eastern Europe, but also grows in the south-central part of Korea, and its leaves contain triterpene saponins and the hydroxy coumarin-based esculin (Esculin: Esculetin 6-glucoside). It contains lacxin (fraxin) and scopolin and contains rutin, quercitrin and isoquercitrin. The active ingredient in the horse chestnut seed contains 3 to 5% of aescin or escin, which are diacylated tetra- and pentahydroxy-beta-amirin compounds with triterpene saponins. In particular, the seed is widely used as a raw material of pharmaceuticals as a medicine that is well-received from bruises and inflammation, and its component is known to be caused by aescin, an anti-inflammatory saponin. In addition, it contains quercetin's biosides and triosides flavonoids, proanthocyanidines and concentrated tannins, and contains about 50% starch (Hubner G). , et al .; 44th Ann. Congress of GA. , 139, 1966)

In Japan, the seeds are treated with water or lye, which removes all the tannins.

Normally, horse chestnut dry extract is prepared from horse chestnut seeds, and triterpene glycosides (16-20% of anhydrous aesin) are used as a standard. Horse chestnut dry or horse chestnut dry extract produced from horse chestnut is a medicine currently used for the prevention and treatment of swelling after surgery and post-traumatic, relieve hemorrhoid symptoms.

An object of the present invention is to provide an angiogenesis inhibitor comprising the horse chestnut extract as an active ingredient.

It is another object of the present invention to provide an angiogenesis inhibitor having low toxicity and oral administration using the compounds isolated from the horse chestnut extract as an active ingredient.

The present invention relates to a new use of horse chestnut (Horse chestnut) extract as an angiogenesis inhibitor, and provides an angiogenesis inhibiting composition comprising a horse chestnut extract having an angiogenic activity as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis, using the horse chestnut extract having angiogenesis inhibitory activity as an active ingredient.

In another aspect, the present invention provides a composition for inhibiting MMP activity comprising the horse chestnut extract as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by excessive MMP activity using the horse chestnut extract having MMP inhibitory activity as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The horse chestnut extract of the present invention can be extracted or purchased by various methods. Horse chestnut extract can be prepared according to a conventional method of extracting the active ingredient from the herbal medicine or the leaves or seeds of the plant, for example, conventional hot water extraction. The extract is concentrated or dried to give a horse chestnut extract. It is also preferable to use a horse chestnut dry extract already on the market.

The horse chestnut extract of the present invention may use the European horse chestnut extract which is currently sold as an anti-inflammatory agent, and extracts of Japanese and Chinese horse chestnut trees may be used.

In order to test the angiogenesis inhibitory effect of the horse chestnut extract, for example, at room temperature or daylight dried leaves of the horse chestnut is finely cut, the seed is rapidly dried at 30 ~ 40 ℃ or not dried and used to crush the mortar or crusher From about 3 to about 10 times, preferably about 5 to 7 times, the lower alcohol solvent, such as butanol, ethanol, methanol, distilled water or Mixtures of these, preferably water and methanol mixtures, and even more preferably, 80% methanol is added to the ultrasonic extraction method, reflux extraction method or conventional extraction method at a temperature of about 30 to 100 ℃, preferably about 50 to 80 ℃ It is extracted for 1 hour to 48 hours, preferably 3 hours to 10 hours to obtain a crude extract, which is used for angiogenesis inhibition experiments.

The horse chestnut extract of the present invention can also use the horse chestnut seed drying X which is currently commercially available.

The present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis, using as an active ingredient compounds such as aesin, esculin, esculin or quercitrin having angiogenesis inhibitory activity isolated from horse chestnut extract. .

In addition, the present invention provides a pharmaceutical composition for inhibiting MMP activity comprising horse chestnut extract as an active ingredient.

The pharmaceutical composition can be used for the prevention and treatment of diseases caused by angiogenesis due to excessive MMP activity.

The pharmaceutical composition of the present invention is for the treatment of diseases caused by angiogenesis, for example, rheumatoid arthritis, osteoarthritis, sepsis arthritis, arteriosclerosis, restenosis, MMP-mediated osteopenia, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging , Corneal ulcer, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, inflammatory bowel disease, and fascia membrane disease Treatment of age-related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, cone cornea, Sjogren's syndrome, myopia ophthalmic tumor, corneal graft rejection, angiogenesis, cancer infiltration and metastasis Can be used for

In the present invention, in order to see the angiogenesis inhibitory effect of the horse chestnut extract was confirmed through a tube formation assay (Tube formation assay) and mouse matrigel assay (Mouse matrigel assay) using vascular endothelial cells.

The composition comprising the horse chestnut extract of the present invention or an active ingredient thereof may be used as a single agent or in combination with other active ingredients that can enhance the efficacy.

In this case, a pharmaceutically acceptable diluent may be used, and the diluent includes, but is not limited to, saline solution, buffered saline solution, dextrose, maltodextrin, water, glycerol, ethanol and combinations thereof.

The horse chestnut extract of the present invention may be administered in a pharmaceutical composition with one or more pharmaceutically acceptable excipients.

The specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general health, sex and diet of the patient, and the time of administration. It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including the route of administration and the rate of release of the composition, the duration of treatment, and the drug (eg, chemotherapeutic agent) used or co-used with the specific composition.

The unit dose of the horse chestnut extract of the present invention is preferably 5 mg to 2 g, most preferably 10 mg to 1 g.

In addition, depending on the age and weight of the patient as well as the type of disease and the extent of the disease caused by angiogenesis, the dosage and the method of administration vary, but can generally be administered at 0.05 to 200 ㎎ / ㎏ body weight 1 to 3 times a day It is preferable to administer.

The pharmaceutical compositions of the present invention may be administered in a conventional manner via the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes.

Parenteral administration means administration modes including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion. Parenteral administration of the pharmaceutical compositions of the present invention may be carried out in unit dosage form in combination with a pharmaceutically acceptable carrier, i.e., nontoxic to the receptor and compatible with other agent components at the concentrations and dosages employed, under the desired purity. It is preferable to prepare.

In addition, the formulation of the pharmaceutical composition of the present invention can be applied to any formulation, and the prepared formulation can be used for oral, injection, and application. The formulation may be prepared in an injectable form (solution, suspension or emulsion), most preferably for oral use such as tablets, capsules, soft capsules, infusions, granules, pills and the like.

The composition is prepared by filling the horse chestnut extract with the capsule without excipients or uniformly and sufficiently in contact with the solid or liquid carrier or both. The product is then shaped into the desired formulation if necessary.

Examples of such carrier excipients include starch, water, saline, Ringer's solution and dextrose solution. Suitable formulations known in the art are preferably used as disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples and Experimental Examples.

Example 1  : Preparation of Horse Chestnut Extract

Leaves of Japanese horse chestnut purchased from Daelim horticulture seedlings were dried at room temperature, and the seeds were crushed with a grinder. After drying the pulverized leaves and seeds at room temperature, 500 g of horse chestnut leaves and 200 g of horse chestnut seeds were respectively ultrasonically extracted at 80 ° C. with 2 L of 80% methanol for 12 hours, and the extract was filtered. 120 g of crude extract and 53 g of horse chestnut seed crude extract were obtained.

Example 2  : Identification of horse chestnut

The crude extract obtained in Example 1 was suspended in distilled water and then extracted with 1 L of ethyl acetate. The ethyl acetate extract was evaporated to dryness in a vacuum dryer, and then dissolved in 50 ml of ethanol, followed by two-dimensional paper chromatography (developing solvent: BuOH: HAc: H ₂ O = 4: 1: 5 and 2% ethyl acetate). .

From the above, it could be confirmed by analyzing various analyzers such as TLC and ¹ H-NMR that aescin, quercitrin, esculletin and esculin were detected as main components.

Experimental Example 1  : Effect of Horse Chestnut Extract on Tube Formation of Human Umbilical Vessel Endothelial Cells

In order to conduct an angiogenesis experiment, a first experiment was performed to obtain a human Umbilical Vein Endothelial cell (HUVEC). (Grant DS, et al . ; Cell, 58 , pp933-943, 1989) In other words, the endothelium of the vein was isolated from the umbilical cord of fresh humans obtained by caesarean section. It was confirmed that the vascular endothelial cells by the cell immunological staining using the antibody of. The vascular endothelial cells identified above were cultured on gelled Matrigel (Matrigel, BD Biosciences, Bedford, USA) for 16 hours at a temperature of 37 ℃.

Vascular endothelial cells cultured by the above method was used as a control, and the horse chestnut extract was treated to Matrigel-like vascular endothelial cells at a concentration of 100 μg / ml or 10 μg / ml of commercial horse chestnut seed dry extract (Hwail Pharmaceutical, Korea). Also, the active ingredients of horse chestnut extract, esculin (Sigma E-8250, USA), esculletin (Sigma E-2631, USA), quercitrin (Sigma Q-3001, USA), aesin ( Sigma E-1378, USA) was treated at 50 μM concentrations, and compared with vascular endothelial cells treated with DMSO or water only as a control.

Experimental results are shown in FIGS. 1 to 9.

Figure 1 shows that the vascular endothelial cells cultured on Matrigel to form a reticular tubular structure can be seen that the reticular tubular structure is thought to be a process of angiogenesis. 2 and 4 are treated with European and Japanese horse chestnut extracts respectively to observe the vascular endothelial cells cultured on Matrigel. When the European horse chestnut extract was treated at a concentration of 100 µg / ml, it could be seen in FIG. 2 that the vascular endothelial cells did not form a tube at all, and in FIG. 3, the network connection was broken by the extract of 10 µg / ml. Could see. Figure 4 shows that when the Japanese horse chestnut extract was treated at a concentration of 100 ㎍ / ㎖, vascular endothelial cells do not form a tube.

 The following experiment was conducted to determine whether the main component of the horse chestnut extract has angiogenesis inhibitory activity. FIG. 5 is a tubular structure generated when DMSO alone is treated to vascular endothelial cells cultured on Matrigel in order to exclude the effect of DMSO used as a solvent of each component, and FIGS. 6 to 9 are active ingredients contained in horse chestnut extract. The results of observing the effect on the tube formation of vascular endothelial cells when 50 μM each was treated. When treated with aesin, vascular endothelial cells did not produce tubes at all (FIG. 9), and when treated with esculin, 83% inhibited tube formation (FIG. 7), and quercitrin suppressed 64%. It was seen that the reticular tubular connections were broken by vascular endothelial cells cultured on matrigel (FIG. 8). In contrast, esculin with sugar attached to esculinet inhibited tubular formation by 15% when treated at the same concentration (FIG. 6).

When the area of the tube formed on the Matrigel was analyzed by Image-Pro Plus (Media Cybernetics, Inc.), as shown in Table 1 and Table 2 below, the experimental group treated with the horse chestnut extract and its active ingredient, respectively, was not treated. It was confirmed that tube formation was inhibited in a concentration-dependent manner compared to the control group.

Sample and Concentration Pipe forming area Inhibition Rate (%) Control group (water treatment) 10.55 0 European horse chestnut (100 µg / ml) 0 100 European horse chestnut (10 µg / ml) 8.89 16 Japanese horse chestnut (100 µg / ml) 0 100

Sample and Concentration Pipe formation area (%) Inhibition Rate (%) Control group (DMSO treatment) 100 0 Esculin 85 100 Esculetin 17 16 Quercitrin 36 100 Aesin 0 100

Experimental Example 2  : Animal experiment to measure angiogenesis (mouse Matrigel model)

Subcutaneous injection of C57BL / 6 mice at 6 to 8 weeks of age (Korean Experimental Animal Center, Korea) with 0.4 ml of Matrigel, 50ng / ml of basic fibroblast growth factor (FGF) and 50 unit / ml of heparin Methanol extract obtained from Example 1 and commercially available European lactose extract (Hwail Pharmaceutical Co., Ltd., Korea) were orally administered 1 mg per mouse twice daily for 4 days, and the control group was orally administered with the same amount of water. On the 5th day after oral administration was started, the skin of the injection site was incised to recover the Matrigel, and the amount of hemoglobin was measured using a Drabkin's reagent (Sigma Company No. 525-2, USA). As shown in Table 10 and Table 3, the experimental group orally administered with the horse chestnut extract showed only 90% of the hemoglobin contained in the Matrigel compared to the control group administered with water, so the angiogenesis was inhibited by the horse chestnut extract. .

Hemoglobin content (g / ㎗) Control 635 ± 50 Horse chestnut extract administration group 62 ± 43

Experimental Example  3: Experiment of Inhibiting MMP Activity Using Fluorescence Substrate

MMP enzyme activity inhibition was measured using a spectrofluorometer (Perkin-Elmer LS50B) to determine whether the horse chestnut extract has an effect of inhibiting MMP. Among collagen enzymes of MMP series, MMP-13 was used as collagenase and MMP-2 was used as gelatinase.

MMP enzyme was genetically prepared in insect cells (Sf21 insect cells) using the baculovirus system. Recombinant virus comprising MMP-2 cDNA (GENEBANK No. XM_048244) was run using a Bac-N-Blue linear transfection kit (Invitrogen, product number K855-01, The Netherlands). Sf21 insect cells (Invitrogen, product number B821-01, The Netherlands) were used as host cells. Infected Sf21 cells were grown at 27 ° C. for 72 hours and the medium was harvested to purify MMP from the medium. MMP-2 was gelatin-sepharose affinity column (SIGMA, product number: G-5384) and eluted using 5% DMSO.

MMP-13 (GENEBANK No. NM_002427) was produced in the same manner and purified using SP-Sepharose (Amersham Pharmacia biotech, product no .: 17-0729-01). As an elution buffer, 20 mM Tris-Cl, pH7.0 , 5 mM calcium chloride and 2M sodium chloride were used. The prepared MMP enzymes were used after activation with 1 mM APMA (p-aminophenyl mercuric acetate) before use in activity measurement.

As a substrate of MMP-2, 2,4-dinitrophenyl-Pro-Leu-Gly-Met-Trp-Ser-Arg-OH (SEQ ID NO: 1, manufactured by Calbiochem) was used. MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH ₂ (SEQ ID NO: 2, manufactured by Calbiochem) was used. [MCA = methyl couaryl amide; Cha = L-cyclohexylalanine; Dpa = 3- (2,4-dinitrophenyl) -L-2,3-diaminopropanoic acid; Nva = L-Norvaline]

2 ml of a cuvette, 2 ml of a buffer solution containing 10 μM of substrate (50 mM Tricine (pH 7.5), 10 mM CaCl ₂ , 200 mM NaCl), and 10 nM of MMP enzyme, were added at room temperature using a spectrophotometer. Fluorescence change was measured for 5-10 minutes.

The final concentration of the European horse chestnut extract purchased in the buffer solution containing the substrate was 25 ㎍ / ㎖ and MMP was added, and the fluorescence over time was measured using a spectrofluorometer, and the enzyme alone did not contain an inhibitor. The fluorescence intensity by the same method was measured over time as a control.

As shown in Figures 11 and 12, when treated with horse chestnut extract, it was confirmed that 77% MMP-2 activity, 85% MMP-13 activity was inhibited, respectively.

Hereinafter, an example of the preparation of the pharmaceutical composition for inhibiting angiogenesis is described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Preparation Example 1 Manufacturing Method of Syrup

Syrup containing the horse chestnut extract of the present invention as an active ingredient 2% (dry weight / volume) is prepared by the following method.

Horse chestnut extract dry powder, saccharin and sugar were dissolved in 80 g of warm water. After cooling the solution, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to this mixture to make 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Horse chestnut extract dry powder ········· 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ······················

Preparation Example 2 Preparation of Tablet

Tablets containing the active ingredient are prepared by the following method.

25 g of horse chestnut extract dry powder was mixed with 17.5 g lactose, 18 g potato starch and 3.2 g colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 16 g of potato starch, 5 g of talc and 0.5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Horse chestnut extract dry powder ········ 25 g

Lactose ······················· 17.5 g

Potato starch 18 g

Colloidal silicic acid 3.2 g

10% gelatin solution ····················· 10 ml

Potato starch 16 g

Talc · 5 g

0.5 g of magnesium stearate

Preparation Example 3 Manufacturing Method of Injection Solution

Injection solution containing the active ingredient was prepared by the following method.

1 g of dry chestnut extract powder, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Horse chestnut extract dry powder ············ 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ·················

As described above, the horse chestnut extract of the present invention and / or aescin, esculletin, esculin, quercitrin is excellent in inhibiting angiogenesis, tumor suppressor, metastasis inhibitor, ocular disease caused by angiogenesis, psoriasis It can be usefully used for the treatment of various diseases related to angiogenesis such as.

1 is a control vascular endothelial cell photograph for observing the effect on the vascular formation of human Umbilical Vein Endothelial cells (HUVEC) of horse chestnut extract,

Figure 2 is a photograph of the tube formation of vascular endothelial cells cultured on Matrigel after treatment with horse chestnut extract at a concentration of 100 ㎍ / ㎖,

Figure 3 is a photograph of the tube formation of vascular endothelial cells cultured on Matrigel after treatment with horse chestnut extract at a concentration of 10 ㎍ / ㎖,

Figure 4 is a photograph of the tube formation of vascular endothelial cells cultured on Matrigel after treatment of the horse chestnut (Japan) extract at a concentration of 100 ㎍ / ㎖,

5 is a photograph taken of a control group treated with DMSO only in vascular endothelial cells cultured on Matrigel,

Figure 6 is a photograph observed after treatment with vascular endothelial cells cultured on Matrigel 50 μM of the active ingredient contained in the horse chestnut extract,

Figure 7 is a photograph observed after treatment of vascular endothelial cells cultured on Matrigel 50 μM of the active ingredient contained in the horse chestnut extract,

FIG. 8 is a photograph observed after treatment of quercitrin 50 μM, the active ingredient contained in the horse chestnut extract, to vascular endothelial cells cultured on Matrigel,

9 is a photograph observed after treatment of vascular endothelial cells cultured on Matrigel with 50 μM of aesin, an active ingredient contained in the horse chestnut extract,

Figure 10 shows the effect of horse chestnut extract to inhibit angiogenesis in the mouse Matrigel model,

11 is a graph showing that horse chestnut extract inhibits MMP-2 enzymatic activity,

12 is a graph showing that horse chestnut extract inhibits MMP-13 enzyme activity.

<110> AngioLab, Inc. <120> PHARMACEUTICAL COMPOSITION FOR INHIBITING ANGIGENESIS CONTAINING AN EXTRACT OF HORSE CHESTNUT <130> FPD / 200109-0034 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> substrate of MMP-2 <220> <221> MOD_RES <222> (1) 2223-dinitrophenyl <220> <221> MOD_RES <222> (9) <223> OH <400> 1 Xaa Pro Leu Gly Met Trp Ser Arg Xaa 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> substrate of MMP-13 <220> <221> MOD_RES <222> (1) <223> methyl coumaryl amide <220> <221> MOD_RES <222> (3) L-cyclohexylalanine <220> <221> MOD_RES <222> (5) <223> L-norvaline <220> <221> MOD_RES <222> (8) 3- (2,4-dinitrophenyl) -L-2,3-diaminopropanoic acid <220> <221> MOD_RES <222> (9) <223> NH2 <400> 2 Xaa Pro Xaa Gly Xaa His Ala Xaa Xaa 1 5

Claims (10)

delete Cancer, rheumatoid arthritis, osteoarthritis, sepsis arthritis, arteriosclerosis, restenosis, osteopenia, inflammatory diseases of the central nervous system, skin aging, abnormal wound union, traumatic joint damage Pharmaceutical composition for the prevention and treatment of degenerative cartilage loss, diabetic retinopathy, corneal ulcer, proliferative vitreoretinopathy, immature retinopathy, corneal transplant rejection, ophthalmic inflammation, cone cornea, Sjogren's syndrome, myopic ocular tumor. The composition according to claim 2, wherein the horse chestnut is Japanese horse chestnut, Chinese horse chestnut or European horse chestnut. Restenosis, aberrant wound union, degenerative cartilage loss due to traumatic joint injury, diabetic retinopathy, corneal ulcer, proliferative vitreoretinopathy, immature retinopathy, corneal transplant rejection Pharmaceutical composition for the prevention and treatment of conical cornea, myopia ophthalmic tumor, neovascularization, cancer infiltration and metastasis. Restenosis with esculin or esculin with angiogenesis inhibitory activity, abnormal wound union, degenerative cartilage loss due to traumatic joint injury, diabetic retinopathy, corneal ulcer, proliferative vitreoretinopathy, immature retinopathy Pharmaceutical composition for the prevention and treatment of corneal transplantation, keratoconus. Restenosis with quercitrin with angiogenic inhibitory activity, aberrant wound union, degenerative cartilage loss due to traumatic joint injury, diabetic retinopathy, corneal ulcer, proliferative vitreoretinopathy, immature retinopathy, rejection of corneal transplant , Pharmaceutical compositions for the prevention and treatment of cone cornea. delete Alzheimer's disease, bone disease, proteinuria, abdominal aortic aneurysm, nervous myelin sheath disease, liver cirrhosis, renal glomerular disease, which are active extracts of horse chestnut extract having matrix metalloproteinase (MMP) -based enzyme inhibitory activity A pharmaceutical composition for the prevention and treatment of immature rupture of the embryonic membrane, inflammatory bowel disease, macular degeneration associated with aging, and periodontal membrane disease. delete The method according to any one of claims 2, 4, 5, 6 and 8, A pharmaceutical composition in which the formulation is in the form of an oral preparation, an injection or a coating.