WO2007066928A1

WO2007066928A1 - Use of the extract of caesalpinia sappan l. and compounds therefrom

Info

Publication number: WO2007066928A1
Application number: PCT/KR2006/005084
Authority: WO
Inventors: Hyun Ok Yang; Min Young Kim; Young-Guen Kwon; Yeon Hee Choi
Original assignee: Korea Institute Of Science And Technology
Priority date: 2005-12-09
Filing date: 2006-11-29
Publication date: 2007-06-14
Also published as: KR100750657B1; KR20070060715A

Abstract

The present invention relates to an extract of Caesalpinia sappan L. having an angiogenesis inhibition activity and a use of the compound isolated therefrom, more precisely an extract of Caesalpinia sappan L. extracted by using water, alcohol or a mixture thereof , and a use of brazilin, sappanchalcone and brazilein isolated from the extract as an angiogenesis inhibitor. The extract of Caesalpinia sappan L. of the invention and a compound isolated therefrom have angiogenesis inhibition activity, so that they can be effectively used for the prevention and treatment of angiogenesis associated diseases such as vascular diseases, cardiovascular diseases, ophthalmic diseases, chronic inflammatory diseases, dermatological diseases, Alzheimer's disease, obesity and cancer.

Description

USE OF THE EXTRACT OF CAESALPINIA SAPPAN L. AND

COMPOUNDS THEREFROM

Technical Field

The present invention relates to an extract of Caesalpinϊa sappan L. having an angiogenesis inhibition activity and a use of the compounds isolated therefrom, more precisely an extract of Caesalpinia sappan L. extracted by using water, alcohol or a mixture thereof, and a use of brazilin, sappanchalcone and brazilein isolated from the extract as an angiogenesis inhibitor.

Background Art

Angiogenesis indicates the process of forming new capillary vessels from old capillary vessels. Normal angiogenesis is observed during embryonic development, tissue regeneration and wound healing and luteal phase, one of the female reproductive system cycles. But, even in this normal angiogenesis, the progress is strictly regulated (Folkman and Cotran, Int. Rev. Exp. Pathol. 16, pp207-248, 1976) .

In adults, vascular endothelial cells are growing very slowly and not differentiated very well, compared with other cells. Angiogenesis is the process of a capillary vessel generation, that is blood vessels are re-constructed by the decomposition of vascular basement membrane by proteinase activated by the stimulation of angiogenic factors, migration, proliferation and differentiation of vascular endothelial cells, and thus the formation of lumen. However, once angiogenesis is not spontaneously regulated and abnormally developed, various diseases are developed. Pathological aspects of angiogenesis associated diseases are as follows;

1) Vascular disease: This disease is exemplified by hemangioma, angiofibroma, vascular malformation, etc. (Tille JC and Pepper MS, Artherioscler. Thromb. Vase. Biol. 24(9), ppl578-1590, 2004; Nagai M.A. et al . , Laryngoscope. 106, ppl90-195, 1996) . This disease is a kind of vascular disorder caused by excessive angiogenesis. The involvements of angiogenic factors and their expressions in the development of such diseases have been well known;

2) Cardiovascular disease: This disease is exemplified by arteriosclerosis, vascular adhesion, scleroedema, etc. (O'Brien K. D. et al . , Circulation 93(4), pp672-682, 1996). In the case of arteriosclerosis, interruption of blood circulation is observed and thus angiogenesis is developed to maintain homeostasis; 3) Ophthalmic disease: This disease includes keratoplastic angiogenesis, angiogenic glaucoma, macular degeneration, diabetic retinopathy, retinosis of a precocious child, angiogenesis mediated cornea disease, pterygium, retinal degeneration, retrolental fibroplasia, granular conjunctivitis, etc. (D'Amato R. J. et al . , Ophthalmol., 102, ppl261-1262, 1995, Adamis A. P. et al . , Angiogenesis, 3, pp9-14, 1999). Diabetic retinopathy is a complication arisen by diabetes, which eventually deprives one's sight because of the invasion of capillary vessel in hyaloid;

4) Chronic inflammatory disease: The most representative chronic inflammatory disease is arthritis, which is caused by autoimmune disorder. The inflammation inducing cytokine mediates the proliferation of synovial cells and vascular endothelial cells in synovial cavity, resulting in angiogenesis. Thus, joint pannus, which is a connective tissue layer, is formed in cartilaginous part to destroy cartilage functioning like an impact absorbing pillow (Kocb AE et al . , Arth. Rheum., 29, pp471-479, 1986; Stupack DG, Storgard CM and Cheresh DA, Braz J. Med. Biol. Res., 32, pp578-581, 1999, Koch AE, Atrh. Rheum., 41, pp951-962, 1998) ;

5) Dermatological disease: This disease is exemplified by psoriasis, capillarectasia, purulent granuloma, dermatitis seborrheica, acne, etc. (Arbiser JL, J. Am. Acad. Derm., 34(3), pp486-497, 1996). Normal cycle of proliferation of keratinocytes is once a month but psoriasis patients exhibit once a week-proliferation cycle. For the frequent proliferation cycles, blood has to be supplied enough, leading to angiogenesis (Folkman J, J. Invest. Dermatol., 59, pp40-48, 1972);

6) Alzheimer's disease: Angiogenic factors have been found in the brain with Alzheimer's disease, which seems to be resulted from the decrease of blood flow rate and inflammation. As a result, β-amyloid is accumulated and neurotoxic peptide is secreted. So, angiogenesis inhibitors targeting endothelial cells of the abnormal brain can be used for the prevention and treatment of Alzheimer's disease (Vagnucci AH et al . , Lancet 361, pp605- 608, 2003) ;

7) Obesity: Obesity mediated angiogenesis increases fat accumulation. This accumulation can be regulated by vasculature in the lower part of adipose tissue and weight loss along with the decrease of adipose tissue can be achieved by treating an angiogenesis inhibitor dose- dependently (Rupnick MA et al., Proc. Natl. Acad. Sci . U.S.A., 99(16), ppl0730-5, 2002);

8) Cancer: Cancer herein includes lung cancer, nonsmall cell lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or cervix cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, breast cancer, tubal cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, bladder, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS (central nervous system) tumor, primary CNS lymphoma, spinal cord tumor, brainstem giloma, pituitary adenoma, etc. (Hanahan D et al . , Cell, 86, pp353- 364, 1996) . Cancer cells are supplied with nutrition and oxygen for their growth by newly generated blood vessels which are at the same time the pathways for metastasis through blood circulation system (Folkman and Tyler, Cancer Invasion and metastasis, Biologic mechanisms and Therapy (S. B. Day ed.) Raven press, New York, pp94-103, 1977; Polverini PJ, Critical Reviews in Oral Biology, 6(3), pp230-247, 1995) . Such metastasis is the leading cause of death of cancer patients and makes cancer treatments including chemo-therapy and immuno-therapy unsuccessful.

An angiogenesis inhibitor can be used as a therapeutic agent for angiogenesis-associated diseases. Thus, studies have been actively undergoing to treat such diseases by inhibiting angiogenesis .

In general, an angiogenesis inhibitor is supposed to be administered to a patient for a long term, so that it has to be very safe substance without or with very low toxicity and can be prepared in an appropriate form for oral administration. Therefore, a pharmaceutical compound with less toxicity or functional food can be a target for angiogenesis inhibitor.

Caesalpinia sappan L. is a member of Leguminosae, which is also called as 'SoBangMok", "SoBang" , "JeokMok" , and "HongJa" . This plant does not grow naturally in Korea, but is widely distributed in Fujiansheng, Guangdongsheng, Guangxisheng, Guizhiusheng, Yunnansheng and Hainansheng of China and in Taiwan. The heartwoods of the stems and the thick branches are called sappan wood, which have been known to have cardiovascular system associated activity and inhibitory activity to central nervous system as well as antimicrobial activity (Kim, et al . , "JungYakDaeSaJeon" , pp.3130, 1998) .

It has already been reported that Caesalpinia sappan

L. has cytotoxic effect on stomach cancer cells (Park, K. J., et al., Kor. J. Pharmacogn. 28(4), pp233-238, 1997), antioxidant effect (Lim, D. K., et al . , Kor. J. Food Sci. Technol. 28(1), pp77, 1996 ; Badami Shrishailappa et al . , Bio. Pharm. Bull., 26(11), ppl534-1537, 2003), antiinflammatory effect, antibacterial and deodorant effect (Kim, Y. S. et al . , Kor. J. Pharmacogn. 26(3), pp265-272, 1995 ; Hikino H Tet al . , Planta Med., 31(3), pp214-220, 1977 ; Kim, K. J. et al . , J. ethnopharmacology, 91(1), pp81-87, 2004 ; Xu, H. X. and Lee, S. F, phytotheraphy Research, 18(8), pp647-651, 2004), anticonvulsant effect (Baek, N. I. et al., Arch. Pharm. Res. 23(4), pp344-348, 2004), anticomplementary activity (Oh, S. R. et al . , Planta Med., 64(5), pp456-458, 1998) and topoisomerase-I inhibitory activity (Jeon, W. K. et al., Kor. J. Pharmacogn.

r

30(1), ppl-6, 1999), etc. However, no explanations on angiogenesis inhibitory activity have been made, yet.

The major ingredient of Caesalpinia sappan L. is alleged to be the colorless brazilin having the hematoxylin/flavonoid structure. The brazilin is oxidized in the air to be brazilein (Moon, C. K. et al . , Arch. Pharm. Res. 11(2), ppl49-154, 1988). Previous reports said that the major ingredient of Caesalpinia sappan L., the brazilin, is effective for the treatment of hypertension (Moon, C. K. et al., Drug Chem. Toxicol. 15(1), ppδl, 1992), regulates calcium content in platelets (Hwang, G. S. et al., Arch. Pharm. Res. 21(6), pp774-778, 1998), and lowers blood sugar (Kim, S. G. et al . , Arch. Pharm. Res. 21(2), ppl40-146, 1998) . Another ingredient of Caesalpinia sappan L. is sappanchalcone, which inhibits platelet coagulation (Morota, T. et al., Jpn. Kokai Tokkyo Koho, pp6, 1990). However, angiogenesis inhibition activity of Caesalpinia sappan L. has not been reported.

The present inventors investigated and confirmed that the extract of Caesalpinia sappan L. and compounds isolated therefrom have the angiogenesis inhibition effect. And the inventors further completed this invention by confirming that the extract of Caesalpinia sappan L. and compounds isolated therefrom can be effectively used for the prevention and treatment of angiogenesis associated diseases .

Disclosure

Technical Problem

It is an object of the present invention to provide an angiogenesis inhibitor containing the extract of Caesalpinia sappan L., brazilin, sappanchalcone and brazilein isolated therefrom as effective ingredients. Technical Solution To achieve the above object, the present invention provides an extract of Caesalpinia sappan L. containing brazilin, sappanchalcone and brazilein as effective ingredients and having angiogenesis inhibition activity and extracted by using water, alcohol and a mixture thereof.

The present invention also^' provides an angiogenesis inhibitor containing the extract of Caesalpinia sappan L.

The present invention further provides an angiogenesis inhibitor containing one or more compounds selected from a group consisting of brazilin, sappanchalcone and brazilein and pharmaceutically acceptable salts thereof.

The present invention also provides a composition for the prevention and treatment of angiogenesis associated diseases containing the angiogenesis inhibitor as an effective ingredient.

The present invention also provides health food containing the angiogenesis inhibitor for the prevention of cancer.

The present invention also provides a composition for the inhibition of metastasis containing the angiogenesis inhibitor as an effective ingredient.

The present invention also provides a method for inhibiting metastasis including the step of co- administering the effective dose of the metastasis inhibiting composition with cisplatin.

Hereinafter, the present invention is described in detail.

The present invention provides an extract of Caesalpinia sappan L. which is extracted by using water, alcohol and a mixed solvent thereof, contains brazilin, sappanchalcone and brazilein as effective ingredients and has angiogenesis inhibition activity. To obtain the extract of Caesalpinia sappan L., dried Caesalpinia sappan L. is cut into small pieces, to which water, Ci ~ C₃ lower alcohol or a mixture thereof, more preferably methanol, was added by 1 ~ 20 times the weight of the sample, more preferably 3 - 10 times the weight of the sample. The extraction was performed preferably at 20^°C ~ 100^°C for 1 ~ 10 days more preferably 2 - 72 hours by hot water extraction, ultrasonic extraction, reflux condensing extraction or enfleurage. It is most preferred to perform the extraction by enfleurage for 48 hours, but not always limited thereto.

In the preferred embodiment of the present invention, the extract of Caesalpinia sappan L. was prepared by the steps of drying Caesalpinia sappan L., cutting the dried Caesalpinia sappan L. into small pieces, enfleuraging the samples in methanol for 2 days, and filtering the solution to give an extract (see Fig. 1) . In the meantime, the water extract of Caesalpinia sappan L. was prepared by the steps of drying Caesalpinia sappan L., cutting the dried Caesalpinia sappan L. into small pieces, extracting the extract by reflux-condensation in water bath and freeze- drying the extract for further use .

The present invention also provides an angiogenesis inhibitor containing the extract of Caesalpinia sappan L.

The extract of Caesalpinia sappan L of the invention effectively inhibits HUVEC tube formation (see Table 1) and angiogenesis in an animal model (see Table 3 and Fig. 13), indicating that the extract can be effectively used as an angiogenesis inhibitor.

The present invention further provides an angiogenesis inhibitor containing one or more compounds selected from a group consisting of brazilin, sappanchalcone and brazilein or pharmaceutically acceptable salts thereof.

To isolate an effective ingredient from the extract of Caesalpinia sappan L., a nonpolar solvent selected from a group consisting of ether, hexane, methylene chloride, ethyl acetate and/or a mixture thereof can be used and more preferably hexane, methylene chloride or ethyl acetate can be selected.

A target compound having angiogenesis inhibition effect can be separated and purified by column chromatography using a filler selected from a group consisting of silica gel, sephadex, RP-18, polyamide and

XAD resin from the nonpolar solvent soluble extract

(Harborne J. B. Phytochemical methods: A guide to modern technique of plant analysis. 3rd Ed. ppβ-7, 1998) .

The isolation processes of the compounds of the invention from the extract of Caesalpinia sappan L. are as follows.

1) Isolation of brazilein

The process for isolating brazilein from the methanol extract of Caesalpinia sappan L. is summarized in Fig. 2.

Particularly, the methanol extract of Caesalpinia sappan L. was concentrated under the reduced pressure, during which a dark red crystal was educed. The dark red crystal product was dissolved in hot methanol at 20 ~ 100^°C, more preferably 50 ~ 100 ^°C and then stood at room temperature, leading to re-crystallization to give brazilein (see Fig.

2) . 2) Isolation of sappanchalcone The methanol extract of Caesalpinia sappan L. was concentrated under the reduced pressure and as a result a methanol soluble extract was obtained. The methanol soluble extract was isolated by using a mixed solvent of hexane: distilled water. To the separated distilled water layer was added another mixed solvent of methylene chloride: distilled water and the resultant methylene chloride layer proceeded to silica gel column chromatography and sephadex LH-20 chromatography for fractionation. An effective ingredient was separated, and then identified as sappanchalcone (see Fig. 3) .

3) Isolation of brazilin

Except the methylene chloride soluble layer obtained from the mixed solvent of methylene chloride : distilled water used for the isolation of sappanchalcone, the remaining distilled water layer was added with a mixed solvent of ethyl acetate : distilled water to separate ethyl acetate soluble layer, which proceeded to sephadex LH-20 chromatography and silica gel column chromatography for fractionation. From the fraction, an effective ingredient was isolated which was confirmed to be brazilin (see Fig. 4) . The compound of the present invention can be used to form a pharmaceutically acceptable salt, which is exemplified by such inorganic salts as alkali metal hydroxides (ex: sodium hydroxide, potassium hydroxide) , alkali metal bicarbonate (ex: sodium bicarbonate, potassium bicarbonate) , and alkali metal carbonate (ex: sodium carbonate, potassium carbonate, calcium carbonate) , such organic salts as primary, secondary and tertiary amine amino acids. The compound of the present invention can be prepared in the form of a solvate particularly a hydrate. The hydration can occur during the isolation of the compound or as time passes because of the hygroscopicity of the compound.

The angiogenesis associated diseases are exemplified by vascular diseases, cardiovascular diseases, ophthalmic diseases, chronic inflammatory diseases, dermatological diseases, Alzheimer's disease, obesity or cancer.

Vascular disease herein is selected from a group consisting of hemangioma, angiofibroma, vascular malformation, etc., and cardiovascular disease is selected from a group consisting of arteriosclerosis, vascular adhesion, scleroedema, etc. Ophthalmic disease is selected from a group consisting of keratoplastic angiogenesis, angiogenic glaucoma, macular degeneration, diabetic retinopathy, retinosis of a precocious child, angiogenesis mediated cornea disease, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasia, granular conjunctivitis, etc. Chronic inflammatory disease of the invention is arthritis, and dermatological disease herein is selected from a group consisting of psoriasis, capillarectasia, purulent granuloma, dermatitis seborrheica, acne, etc., but not always limited thereto.

In addition, cancer disease of the present invention is selected from a group consisting of lung cancer, nonsmall cell lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or cervix cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, breast cancer, tubal cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, bladder, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS (central nervous system) tumor, primary CNS lymphoma, spinal cord tumor, brainstem giloma, pituitary adenoma, etc. The composition of the present invention can be used for the treatment of angiogenesis associated cancer and metastasis thereof. The composition of the present invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation.

The composition of the invention can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients.

The formulations for the composition of the invention are powders, granules, coated tablets, pills, capsules, suppositories, syrups, juice, suspensions, emulsions, drops or injectable solutions and sustained released formulations.

These solid formulations are prepared by mixing the extract of Caesalpinia sappan L. or one or more compounds such as brazilin, sappanchalcone and brazilein with one or more suitable excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc., can be used.

Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the abovementioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin.

Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, and suppositories. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.

The effective dosage of the composition of the present invention containing the angiogenesis inhibitor as an effective ingredient can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage of the composition is 0.01 mg/kg ~ 10 g/kg per day, and preferably 1 mg/kg ~ 1 g/kg per day. Administration frequency is once a day or preferably a few times a day. The present invention also provides functional health food for the prevention of cancer which contains the angiogenesis inhibitor.

The angiogenesis inhibitor of the invention is addable to such food as beverages, gums, tea, vitamin complex, and other functional foods.

At this time, the content of the angiogenesis inhibitor in a functional food composition of the invention is generally 0.01 ~ 90 weight% for the total weight of the food product and more preferably 0.1 ~ 80 weight%, and the content of the angiogenesis inhibitor in a health improving beverage composition of the invention is 0.01 ~ 90 g/100 m# and more preferably 0.1 ~ 50 g /100 ml.

The health beverage composition of the present invention is not limited to a liquid type but includes various types of food as long as they contain the extract of the invention as an effective ingredient by the amount indicated above. The composition^'' of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages.

The natural carbohydrates can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol . As a sweetener, either natural sweetener such as thaumatin and stevia extract (ex: rebaudioside A and glycyrrhizin) or artificial sweetener such as saccharin and aspartame can be used. The ratio of natural carbohydrate to the composition of the present invention is preferably 1 ~ 20 g to 100 mi, more preferably 5 ~ 12 g to 100 mi.

In addition to the ingredients mentioned above, the composition of the present invention can include in variety of nutrients, vitamins, minerals (electrolytes), flavoring agents, coloring agents, pectic acid and its salts, arginic acid and its salts, organic acid, protective colloidal viscosifiers, pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The composition of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together. The present invention provides a composition for inhibiting metastasis which contains the angiogenesis inhibitor as an effective ingredient.

Cancer cells are supplied with nutrition and oxygen through newly generated blood vessels for the growth and proliferation. The new blood vessel happens to be the pathway of metastasis, that is cancer cells are transferred to other regions by way of the blood circulation system. Thus, the inhibition of angiogenesis might result in the inhibition of metastasis.

The present invention also provides a method for inhibiting metastasis including the step of coadministering the effective dose of the metastasis inhibiting composition of the invention with cisplatin.

Description of Drawings

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Fig. 1 illustrates the process of preparing the methanol extract of Caesalpinia sappan L. and isolating the effective ingredient from the extract.

Fig. 2 illustrates the purification process of brazilein from Caesalpinia sappan L.

Fig. 3 illustrates the purification process of sappanchalcone from Caesalpinia sappan L.

Fig. 4 illustrates the purification process of brazilin from Caesalpinia sappan L.

Fig. 5 is a set of photographs illustrating the inhibition of HUVEC tube formation according to the treatment of 50 βg/ml of each methanol extract and water extract of Caesalpinia sappan L. and effective ingredients such as brazilin, sappanchalcone, and brazilein isolated and purified from Caesalpinia sappan L.

Fig. 6 is a set of photographs illustrating the cytotoxic effects of brazilin, sappanchalcone and brazilein on HUVECs .

Fig. 7 is a graph illustrating the inhibition of VEGF (vascular endothelial growth factor) mediated HUVEC proliferation by brazilin.

Fig. 8 is a graph illustrating the inhibition of VEGF (vascular endothelial growth factor) mediated HUVEC proliferation by sappanchalcone.

Fig. 9 is a graph illustrating the inhibition of VEGF (vascular endothelial growth factor) mediated HUVEC proliferation by brazilein.

Fig. 10 is a set of photographs and a graph illustrating the inhibition of VEGF mediated endothelial cell migration by brazilein.

Fig. 11 is a set of photographs and a graph illustrating the inhibition of VEGF mediated endothelial cell sprouting by brazilein in the mouse artery.

Fig. 12 is a set of photographs illustrating the results of ERK Western blotting with brazilin, sappanchalcone and brazilein.

Fig. 13 is a graph illustrating the angiogenesis inhibition effect of the methanol extract of Caesalpinia sappan L. in an animal model.

Fig. 14 is a graph illustrating the angiogenesis inhibition effects of sappanchalcone and brazilein in an animal model .

Fig. 15 is a graph illustrating the anticancer effect of brazilein observed in the animal xenograft model injected with HeLa cell line.

Fig. 16 is a graph illustrating the anticancer effect of brazilein observed in the animal xenograft model injected with HCT116 cell line. Mode for Invention

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1; Preparation of the extract of Caesalpinia sappan L^ <!-!> Preparation of the methanol extract of Caesalpinia sappan L .

9 kg of the dried Caesalpinia sappan L. , purchased from Kyungdong herb market (Seoul, Korea), was enfleuraged in 18 ! of 100% methanol for 2 days. This enfleurage was repeated three times. The methanol enfleuraged solution was filtered with a filter paper under normal pressure to give an extract (Fig. 1) . <l-2> Preparation of the water extract of Caesalpinia sappan L .

200 g of the dried Caesalpinia sappan L., purchased from Kyungdong herb market (Seoul, Korea), was dipped in 1 I of water, followed by heat-extraction at 90^°C ~ 100^°C for 6 hours, which was repeated two times. The extraction solution was filtered with a filter paper under normal pressure and freeze-dried to give 51.5 g of the water extract of Caesalpinia sappan L. Example 2 : Isolation, purification and identification of the effective ingredients

<2-l> Isolation of the extract of Caesalpinia sappan L. by using a nonpolar solvent

To isolate effective ingredients of Caesalpinia sappan L., the extract of Caesalpinia sappan L. prepared in Example 1 was concentrated under the reduced pressure until it became thick. The educed crystal was filtered with a glass filter to give 21 g of dark red crystal. The methanol soluble substance remaining in the methanol solution was concentrated again under the reduced pressure, resulting in 450 g of methanol soluble substance.

230 g of the methanol soluble substance was suspended in 1 I of distilled water, to which π-hexane was added 1 t by 1 ( with vortexing vigorously. The mixture was left as it was to prepare fractions. Extraction with hexane was performed three times to separated hexane soluble layer and distilled water layer. 1 ( of methylene chloride was added to the distilled water layer and fractionation was performed. As a result, 41.1 g of methylene chloride soluble layer was obtained (Fig. IB) .

To the remaining distilled water layer was added 1 I of ethyl acetate, followed by fractionation. As a result, 150 g of ethyl acetate soluble layer was obtained (Fig. 1C) . <2-2> Isolation, purification and identification of brazilein

900 mg out of 21 g of the dark red crystal, educed from the concentration process of the methanol extract of

Caesalpinia sappan L. under ^' the reduced pressure, was dissolved in 300 Vλl of hot methanol (50 "C) and then left at room temperature for 24 hours, leading to re- crystallization. As a result, 16 g of pure single Compound 1 was obtained, which was then identified as brazilein from the following data (Fig. 2) .

Compound 1: reddish-brown crystals (MeOH-H₂O), [a] D ,25

-700 (DMSO; C 1.00), UV λ_π MeOH nm: 445, IRv : 3409, 2924,

2855, 1739, 1599, 1498 cm^"1, mp. 260-265^°C, EI-MS (m/z) : 284 [M]⁺ Ci₆Hi₂O₅, ¹H-NMR (500 MHz, DMSO-d₆) : δ 7.80 (IH, d, J=8.7 Hz, H-I), 7.10 (IH, s, H-Il), 6.56 (IH, d, .7=8.5 Hz, H-2) , 6.35 (IH, s, H-4), 6.32 (IH, S, H-8), 4.45 (IH, d, J=Il.7 Hz, H-6a) , 4.00 (IH, d, J=Il.7 Hz, H-6b) , 2.85 (2H, d, J=3.7 Hz, H-7) ¹³C-NMR (125 MHz, DMSO-d₆) : δ 39 (C-7) , 72 (C-6) , 74 (C-6a) , 102 (C-4) , 104 (C-Il), 110 (C-Ia), 110 (C-2), 117 (C-8) , 126 (C-Ha), 130 (C-I), 151 (C-12), 152 (C-IO), 157 (C-4a) , 158 (C-7a) , 162 (C-3), 179 (C-9)

compound 1 (brazilein) <2-3> Isolation, purification and identification of sappanchalcone

4.1 g of the methylene chloride soluble layer obtained in Example <2-l> proceeded to silica gel (70 - 230 mesh, 350 g) column (φ=3.0 x 50 cm) chromatography using the mixed solvent of methylene chloride/methanol as an elution solvent by stepwise gradient density elution (20:1 → 1:1). As a result, 6 fractions, AS30-M1 (1.3 mg), AS30- M2(870 mg) , AS30-M3(560 mg) , AS30-M4 (270 mg) , AS30-M5(300 mg) and AS30-M6(800 mg), were obtained. The 4^th fraction (AS30- M4) proceeded to sephadex LH-20 column (φ=3.0 x 50 cm) chromatography with 50% methanol to give three fractions. From the 2^nd fraction (AS30-M42) , 60 ing of Compound 2 was isolated and identified as sappanchalcone from the following data (Fig. 3) .

Compound 2: Yellow needles, [a] D²⁵ +15.2 (MeOH; c 1.00), UV λ_max ^Me0H nm: 366, IRv : 3370, 2928, 1602, 1514, 1445

198-199^°C, EI-MS (m/z) : 286 [M]⁺ Ci₆Hi₄O₅, ¹H- NMR (500 MHz, DMSO-d₆) : δ 7.57 (IH, d, J=8.5 Hz, H-6¹), 7.49 (IH, d, J=15.6 Hz, H-b) , 7.36 (IH, d, J= 15.6 Hz, H-a) , 7.11 (IH, br s, H-2), 6.98 (IH, d, .7=8.1 Hz, H-6), 6.79 (IH, d, J=8.1 Hz, H-5) , 6.51 (IH, br s, H-3^■ ) , 6.45 (IH, d, J=8.5 Hz, H-5¹), 3.88 (3H, -OMe) ¹³C-NMR (125 MHz, DMSO- d₆) : δ 56 (C-OMe), 100 (C-3^■ ) , 109 (C-5¹), 115 (C-2) , 116

(C-5), 121 (C-I') , 123 (C-6) , 125 (C-6¹), 128 (C-I), 133

(C-a) , 144 (C-b) , 146 (C-3), 149 (C-4) , 162 (C-2 ' ) , 164 (C- 4^■ ) , 193 (C=O)

compound 2(sappanchalcone)

<2-4> Isolation, purification and identification of brazilin

18 g out of 150 g of ethyl acetate soluble layer obtained in Example <2-l> proceeded to sephadex LH-20 column (φ=5.0 x 50 cm) chromatography using the mixed solvent of methylene chloride :methanol (15:1). The 4^th fraction proceeded again to sephadex LH-20 column (φ=5.0 x 50 cm) chromatography using the mixed solvent of methylene chloride :methanol (20:1), resulting in 4 fractions. Among these four fractions, the 3^rd fraction (AS30-E43, 1.67 g) proceeded to silica gel (70 ~ 230 mesh, 350 g) column (cp=3.0 x 50 cm) chromatography using the mixed solvent of methylene chloride : ethyl acetate as an elution solvent by stepwise gradient elution (7 : 3→5 : 5->3 : 7 ethyl acetate). The

2^nd fraction of them proceeded to silica gel (70 ~ 230 mesh, 150 g) column (φ=2.5 x 30 cm) chromatography using the mixed solvent of methylene chloride/ethyl acetate as an elution solvent by stepwise gradient elution (7:3→5:5 ethyl acetate) for purification and thereby 160 fflg of Compound 3 was isolated from the first fraction (AS30-E4321) and identified as brazilein from the following data (Fig. 4) .

Compound 3: [a] D²⁵ +93.7 (MeOH; c 1.44), UV λ_max ^Me0H nm: 274 , IRv : 3393, 2926, 2852, 1622, 1509 cm^"1, EI-MS (m/z) : 286 [M]⁺ Ci₆Hi₄O₅, ¹H-NMR (500 MHz, DMSO-d₆) : δ 7.18 (IH, d, J=8.4 Hz, H-I), 6.70 (IH, s, H-8) , 6.59 (IH, s, H-Il), 6.46 (IH, dd, J=8.3, 2.4 Hz, H-2) , 6.29 (IH, d, .7=2.4 Hz, H-4) , 3.96 (IH, br s, H-12) , 3.92 (IH, dd, J=I.2, 11.3 Hz, H-6a) , 3.69 (IH, d, J=Il.2 Hz, H-6b) , 3.02 (IH, d, .7=15.6 Hz, H- 7a), 2.77 (IH, d, J=15.6 Hz, H-7b) ¹³C-NMR (125 MHz, DMSO- d₆) : δ 43 (C-4a) , 51 (C-3) , 71 (C-9) , 78 (C-10), 104 (C- Ha), HO (C-I), 112 (C-7a) , 113 (C-Ia), 115 (C-8) , 131 (C- 11), 132 (C-2), 137 (C-4), 145 (C-6a) , 145 (C-6) , 155 (C- 12) , 158 (C-7)

compound 3(brazilin)

Experimental Example 1: Isolation and culture of HUVECs

Human umbilical vein endothelial cells (referred as ¹HUVEC¹ hereinafter) were isolated from umbilical vein of an infant, followed by culture. Particularly, HUVECs were isolated by the method of Grant, et al . (Grant, D. S. et al . , Cell, 58, pp933-943, 1989), and the isolated HUVECs were cultured in a culture medium comprising M- 199 (Gibco BRL, 31100-035), 10% FBS, 50 μg/vA ECGS (endothelial cell growth supplement, Sigma E2759) and 50 //g/mfc heparin (Sigma H3149) .

Upon completion of the culture, HUVECs were immuno- stained by using the antibody (DAKO, Code No. MO616) of factor VM, confirming that HUVECs were successfully isolated (Valen, G. et al . , Free Radic. Biol. Med., 26, ppl480-1488, 1999; Rhim, J. S. et al . , Carcinogenesis, 19, pp673-681, 1998) . Experimental Example 2 : Inhibition effect on HUVEC tube formation

To investigate whether the effective ingredients brazilin, sappanchalcone, and brazilein isolated from

Caesalpinia sappan L. and the methanol extract and water extract of Caesalpinia sappan L. had angiogenesis inhibition activity, HUVEC tube formation inhibition assay was performed to measure angiogenesis inhibition effect at cellular level (Fig. 5) .

HUVEC tube formation inhibition assay stands closest to in vivo experiment among many in vitro assays.

Particularly, HUVECs separated and cultured in Experimental Example 1 were sub-cultured less than 5 times (passage 5) and distributed to a 48 well plate using a medium containing matrigel (BD Biosciences, Bedford, MA) , the basement membrane component, at the concentration of 20,000 ~ 50,000 cells per well, followed by further culture with the same medium composition as described in the primary culture medium of Experimental Example 1.

The extract of Caesalpinia sappan L. of Example 1 and brazilin, sappanchalcone and brazilein isolated and purified in Example 2 were added to the medium respectively at the concentration of 10 μg/ml and 50 μg/ml. A control group was treated with DMSO. 18 hours later, tube formation was observed under microscope and the results are shown in Table 1 (-: tube formation was not inhibited at all, +/-: tube formation was hardly inhibited, +: tube formation was slightly inhibited, tube cutoff was detected, ++: tube cutoff was detected a lot, +++ : tube formation was inhibited, no tube formation was observed) .

As shown in Fig. 5 and Table 1, the methanol extract and water extract of Caesalpinia sappan L. and brazilin, sappanchalcone and brazilein exhibited HUVEC tube formation inhibition effect with the concentration of 50 μg/ml.

Experimental Example 3 : Cytotoxicity test

To measure the cytotoxicity of brazilin, sappanchalcone and brazilein, the effective ingredients isolated from Caesalpinia sappan L., HUVEC survival rate was measured (Fig. 6) .

The 24-well plate was coated with 2% gelatin, to which HUVECs were seeded at the concentration of 1 x 10⁵ cells per well. On the next day, the cells were cultured for 6 hours in 1% M199 medium to make starvation condition. After starvation, brazilin, sappanchalcone and brazilein were treated thereto at different concentrations of 1 ng/m£, 10 ng/mi, 100 ng/mi, 1 βg/vd and 10 βg/vλl. 48 hours later, OD₅₇₀ was measured by MTT assay (Ealey PA, et al . , Growth Regul, 5: 36-44 (1995)) to quantify live cells.

Fig. 6 is a set of photographs (40X) illustrating HUVECs 48 hours after treating brazilin, sappanchalcone and brazilein. As shown in these pictures, cytotoxicity was not observed at any concentration. As shown in Table 2, when HUVECs were treated with the extract of Caesalpinia sappan L. and its' effective ingredients brazilin, sappanchalcone and brazilein at the concentrations of 50 βg/ml and 10 βg/ml, at which tube formation was inhibited, for 24 hours, HUVEC survival rate was not changed.

Experimental Example 4: Inhibition of HUVEC proliferation

To investigate the cell growth inhibition activity of brazilin, sappanchalcone and brazilein, VEGF was added to the samples treated with the extract and the effective ingredients of Caesalpinia sappan L. at the concentration of 10 ng/ml, and then HUVEC growth rate was measured (Fig. 7 ~ Fig. 9) .

The 24-well plate was coated with 2% gelatin, to which HUVECs were seeded at the concentration of 2xlO⁴ cells per well. On the next day, the cells were cultured for 6 hours in 1% M199 medium to make starvation condition. After starvation, brazilin, sappanchalcone and brazilein were treated thereto at different concentrations of 1 ng/in#, 10 nq/ml, 100 ng/m£, 1 μg/ml and 10 μg/ml. 30 minutes later, VEGF (10 ng/ml) was added. 40 hours later, OD₅₇₀ was measured by MTT assay to quantify proliferated cells.

Fig. 7 ~ Fig. 9 are graphs illustrating the quantified growth rates of HUVECs. As shown in these graphs, after treating with brazilin, sappanchalcone and brazilein for 40 hours, VEGF mediated HUVEC proliferation was significantly inhibited by brazilin and brazilein at 10 μg/vd and by sappanchalcone at 1 μg/ml. Experimental Example 5: Inhibition of VEGF mediated endothelial cell migration by brazilein

<5-l> Chemotactic migration test

To investigate the inhibition of HUVEC migration by brazilein, chemotactic migration test was performed (Fig. 10) .

The lower part of the polycarbonate filter (6.5 mm in diameter, 8 IM pore size) in the insert chamber (Corning Costa, USA) was coated with 10 μg of gelatin. A growth medium supplemented with 1% (v/v) FBS containing VEGF was loaded in the lower part wells. HUVECs (IxIO⁶ cells/m#) were pre-treated with brazilein at different concentrations of 0.001 μM, 0.01 μM, 0.1 μM and 1 μM at room temperature for 30 minutes and then distributed in the upper part wells by 100 μβ/well, followed by culture for 4 hours in a 37 ^°C incubator. The polycarbonate filter of the insert chamber was taken off and cells were fixed. The fixed cells were stained with hematoxylin (Sigma-Aldrich Co., USA) and eosin

(Sigma-Aldrich Co., USA) (Fig. 10A). The number of HUVECs of each experimental group that had passed through the filter was counted (Fig. 10B) .

As a result, the number of -HUVECs passed through the filter was reduced brazilein content dependently, suggesting that VEGF mediated HUVEC migration was inhibited by brazilein. <5-2> Wounding migration assay- To investigate the inhibition effect of brazilein on non-chemotactic cell migration of HUVECs, wounding migration assay was performed (Fig. 11) .

HUVECs were loaded in a 6 well plate coated with gelatin at the concentration of 3xlO⁵ cells per well, followed by culture. 200 βi tip was used to make a line wound in a certain area. The cells were treated with 0.1 μg/m£ and 1 μg/vai of brazilein for 30 minutes, followed by- treatment of 20 ng of VEGF. 18 hours later, the cell migration to the wounded area was observed under a microscope (Fig. HA) .

As a result, wound healing caused by VEGF mediated HUVEC migration was reduced brazilein content dependently (Fig. HB) .

Experimental Example 6: Inhibition of VEGF mediated ERK phosphorylation

To investigate the effect of brazilein on VEGF mediated ERK phosphorylation, Western blotting was performed (Fig. 12) .

A 60 mm plate was coated with 2% gelatin, to which

HUVECs were inoculated by 80 ~ 90%. On the next day, the cells were cultured for 6 hours in 1% M199 medium to make starvation condition. After starvation, brazilin, sappanchalcone and brazilein were treated thereto at different concentrations (1 ng/mi, 10 ng/wi, 100 ng/m#, 1 βg/ml and 10 μg/ml) for 30 minutes, followed by the treatment of VEGF (10 ng/ml) for 10 minutes. Then, the cells were lysed in 2xSDS PAGE sample buffer (24 mM Tris-HCl, pH 6.8, 5 % glycerol, 0.8 % SDS, 6 mM 2-mercaptoethanol , 0.04% bromophenol blue) . The lysate was heated for 10 minutes, followed by SDS-PAGE and Western blotting using an anti- phospho-ERK antibody.

Fig. 12 is a set of photographs illustrating the level of ERK phosphorylation according to the concentrations of brazilin, sappanchalcone and brazilein. As shown in these photographs, brazilin, sappanchalcone and brazilein did not inhibit VEGF mediated ERK phosphorylation.

This result indicates that the extract of Caesalpinia sappan L. and the effective ingredients isolated therefrom target not VEGF mediated ERK phosphorylation pathway but another pathway for angiogenesis inhibition.

Experimental Example 7: Inhibition of angiogenesis in an animal model (Mouse-Matrigel model)

To investigate the inhibition of angiogenesis by the methanol extract of Caesalpinia sappan L. and the effective ingredients such as sappanchalcone and brazilein prepared in the above Examples, a mouse-matrigel model was used (Fig. 13 and Fig. 14) .

0.4 mi of matrigel (BD biosciences, USA), 50 ng/rod. of FGF (fibroblast growth factor) , and 50 unit/m-C of heparin (Sigma-Aldrich Co., USA) were subcutaneously injected to a C57BL/6 mouse (Japan SLC, Inc., Japan) at 6 ~ 8 weeks old. 3 ~ 5 days later, epidermis was removed and matrigel was carefully separated to recover the gel. Hemoglobin was quantified by using Drakin's reagent (Sigma), which used as a control .

The methanol extract of Caesalpinia sappan L. was dissolved in 10% ethanol, which was orally administered by 1.5 mg, twice a day. The 10% ethanol was orally administered to a control by the same amount and with the same administration frequency. In the meantime, brazilein (0.5 mg, 1 mg) and sappanchalcone (0.2 mg, 0.5 mg) were also dissolved in 10% ethanol, which were orally administered twice a day. After 4 days of the oral- administration, hemoglobin of each group was quantified and compared .

The experimental group treated with the methanol extract of Caesalpinia sappan L. exhibited angiogenesis inhibiting effect approximately 33.3% compared with the control group (Table 3, Fig. 13). The experimental group treated with sappanchalcone also exhibited angiogenesis inhibiting effect up to 70% with the content of 0.2 mg and 41.7% with the content of 0.5 mg. The experimental group treated with brazilein exhibited angiogenesis inhibiting effect up to 58.6% with the content of 0.5 mg and 96.4% with the content of 1.0 mg, compared with the control group (Table 4, Fig. 14) .

As a result, the administration of the methanol extract of Caesalpinia sappan L., sappanchalcone and brazilein lowered hemoglobin content in blood, in particular, brazilein was confirmed to inhibit angiogenesis significantly.

Experimental Example 8 : Cytotoxicity to various cancer cells

To investigate the cytotoxicity of brazilein to various cancer cells, the following 7 cancer cell lines were tested for survival rate.

IXlO⁴ cells of each cancer cells were inoculated in a 96 well plate. On the next day, DU145, HepG2, and MCF7 cells were treated with brazilein diluted in EMEM, AGS and A549 cells were treated with brazilein diluted in Ham's F12K medium, HCT116 cells were treated with brazilein diluted in McCoy' s 5 α medium, and HeLa cells were treated with brazilein diluted in DMEM medium at different concentrations of 100, 30, 10, 3, 1, 0.3, 0.1 uM. 24 hours later, the media were washed with PBS and added with MTT, followed by measuring the cancer cell survival rate. The concentration that was able to inhibit cancer cell survival rate upto 50% was determined and shown in Table 5. IC₅₀ for those 7 cancer cell lines ranged 0.99 ~ 30.22 uM. Therefore, it was confirmed that brazilein had anticancer activity.

Cytotoxicity to various cancer cell lines (IC₅₀=50% survival/growth inhibition concentration)

Experimental Example 9: Anticancer activity in the animal xenograft model

HeLa cell line, a cervical cancer cell line, was subcutaneously injected under the side of a female nude mouse at 6 weeks old by IxIO⁷. When the volume of a generated solid tumor reached 100 mm³, brazilein was orally administered to the mouse 2 ~ 3 times a week by 0.1 mg/kg per administration. In the meantime, the conventional anticancer agent cDDP [cis-Diammineplatium (II) dichloride; cisplatin] was intravenously injected through the tail vein once a week, for three weeks, by 4 mg/kg. Another group was orally treated with cDDP (4 mg/kg) and brazilein (0.1 mg/kg) together 2 - 3 times a week. A control group was treated with a solvent only. The volume of the solid tumor was calculated by the following formula. Solid tumor volume = the longest diameter of a tumor x the shortest diameter x the shortest diameter /2

AS shown in Fig. 15, on the 35^th day from the cancer cell implantation, the volume of a solid tumor was 100 mm³. Thereafter, samples were treated respectively. As a result, on the 70^th day from the implantation, the volume of the solid tumor was reduced about 68.7% in cDDP treating group, while the volume of the solid tumor was reduced 56.5% in brazilein treating group. The volume of the solid tumor in cDDN and brazilein co-treating group was also reduced 77.6%, indicating that brazilein exhibited excellent anticancer activity when it is treated to a sample both separately and together with cDDP (Fig. 15) .

Colon cancer cell line HCT116 was intravenously injected through the tail vein of a female nude mouse at 6 weeks, and anticancer activity of brazilein was investigated in analogy to the procedure as described above. Brazilein was orally administered every other day.

As a result, the brazilein treating group exhibited 37.3% tumor suppressing effect on the 54^th day from the implantation, compared with a control (Fig. 16) .

Experimental Example 10 : Acute toxicity test in animals via oral administration Male ICR mice and SD rats were used (10 animals per group) in the tests for acute toxicity. As a result, LD₅₀ was at least 2 g/kg. Concentrations of brazilein used in the toxicity test and the number of dead animals are shown in Table 6. From the test with those different concentrations for 14 days, it was confirmed that no death or abnormal symptoms were detected even at the highest concentration of 2 g/kg.

Acute toxicity test in animals via oral administration

Manufacturing Example 1: Preparation of pills

Brazilein of Example <2-2> 120 mg

Corn starch 100 mg

Sterilized distilled water Proper amount The above components are mixed and prepared in the form of pill of 0.3 cm in diameter according to the conventional pill preparation method. Manufacturing Example 2 : Preparation of tablets

Sappanchalcone of Example <2-3> 200 mg

Lactose 100 mg

Starch 100 mg

Magnesium stearate Proper amount The above components are mixed and prepared in the form of tablet according to the conventional tablet preparation method.

Manufacturing Example 3 : Preparation of liquid formulation Extract of Caesalpinia sappan L. of Example 1 1000 mg Sugar 2O g

Isomerized sugar 2O g

Lemon flavor Proper amount

Refined water was added to the above components to make the total volume to 1000 mi. The above components were mixed according to the conventional production method for liquid formulation, filled in a brown bottle and sterilized to prepare a liquid formulation. Manufacturing Example 4 : Preparation of health beverages

Extract of Caesalpinia sappan L. of Example 1 1000 mg Citric acid 1000 mg

Oligosaccharide 100 g Plum extract 2 g

Taurine 1 g

Refined water was added to the above components to make the total volume to 900 ϊd. The above components were mixed according to the conventional health beverage production method, and the mixture was heated with stirring at 85 ^°C for one hour. The solution was filtered and stored in a sterilized 2 i container with sealed and sterilized. The container was then stored in a refrigerator for further use as a health beverage composition. The composition of the beverage followed the preferable combination of each component but not always limited thereto and can be modified considering demand class, demand country, purpose of use, local and national preference, etc. Industrial Applicability

As explained hereinbefore, the extract of Caesalpinia sappan L. and the compounds isolated therefrom have angiogenesis inhibiting effect, so that they can be effectively used for the prevention and treatment of angiogenesis associated diseases such as vascular diseases, cardiovascular diseases, ophthalmic diseases, chronic inflammatory diseases, dermatological diseases, Alzheimer's disease, obesity and cancer.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[Claim l]

An extract of Caesalpinia sappan L. which is extracted by using water, alcohol or a mixture of the two and contains brazilin, sappanchalcone and brazilein as active ingredients and has angiogenesis inhibiting activity.

[Claim 2]

The extract of Caesalpinia sappan L. according to claim 1, wherein the alcohol is Ci ~ C₃ lower alcohol.

[Claim 3]

An angiogenesis inhibitor containing the extract of Caesalpinia sappan L. of claim 1.

[Claim 4]

An angiogenesis inhibitor containing one or more compounds selected from a group^" consisting of brazilin, brazilein and sappanchalcone, or their pharmaceutically acceptable salts.

[Claim 5]

A composition for the prevention and treatment of angiogenesis associated disease containing the angiogenesis inhibitor of claim 3 or claim 4 as an effective ingredient.

[Claim 6]

The composition for the prevention and treatment of angiogenesis associated disease according to claim 5, wherein the angiogenesis associated disease is vascular diseases, cardiovascular diseases, ophthalmic diseases, chronic inflammatory diseases, dermatological diseases, Alzheimer's disease, obesity or cancer.

[Claim 7]

The composition for the prevention and treatment of angiogenesis associated disease according to claim 6, wherein the vascular disease is selected from a group consisting of hemangioma, angiofibroma and vascular malformation .

[Claim 8]

The composition for the prevention and treatment of angiogenesis associated disease according to claim 6 wherein the cardiovascular disease is selected from a group consisting of arteriosclerosis, vascular adhesion and scleroedema. [Claim 9] The composition for the prevention and treatment of angiogenesis associated disease according to claim 6, wherein the ophthalmic disease is selected from a group consisting of keratoplastic angiogenesis, angiogenic glaucoma, macular degeneration, diabetic retinopathy, retinosis of a precocious child, angiogenesis mediated cornea disease, pterygium, retinal degeneration, retrolental fibroplasias and granular conjunctivitis. [Claim lθ]

The composition for the prevention and treatment of angiogenesis associated disease according to claim 6, wherein the chronic inflammatory disease is arthritis. [Claim ll]

The composition for the prevention and treatment of angiogenesis associated disease according to claim 6, wherein the dermatological disease is selected from a group consisting of psoriasis, capillarectasia, purulent granuloma, dermatitis seborrheica and acne.

[Claim 12]

The composition for the prevention and treatment of angiogenesis associated disease according to claim 6, wherein the cancer is selected from a group consisting of lung cancer, nonsmall cell lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or cervix cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, breast cancer, tubal cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, bladder, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS (central nervous system) tumor, primary CNS lymphoma, spinal cord tumor, brainstem giloma and pituitary adenoma.

[Claim 13]

A functional health food containing the angiogenesis inhibitor of claim 3 or claim 4.

[Claim 14]

A metastasis inhibiting composition containing the angiogenesis inhibitor of claim 3 or claim 4.

[Claim 15] A method for inhibiting metastasis including the step of co-administering the effective dose of the metastasis inhibiting composition of claim 14 with cisplatin.