KR20100051303A - Composition comprising the extract of sponge as an active ingredient for treating and preventing the disease caused by overexpressed mmp-9 or mmp-2 - Google Patents

Abstract

PURPOSE: A composition containing an extract of a sea sponge is provided to prevent and treat diseased occurring from matrix metalloproteinase(MMP)-9 OR MMP-2 which directly take parts in vascularization. CONSTITUTION: A composition for inhibiting MMP-9 or MMP-2 contains an extract of a sea sponge as an active ingredient. The sea sponge is selected from the group consisting of a cliona celata, a callyspongia elegans, a calcarea, a glass sponges, a demospongiae, a euspongia officinalis, a keratosa, a halichondria japonica, or a h. panacea. The extract is a polar or a non-polar solvent extraction of the sea sponge. The formulation of a health food containing the composition includes powder, granule, capsules, tablets, or liquid.

Description

Composition comprising the extract of sponge as an active ingredient for treating and preventing the disease caused by overexpressed MMP -9 or MMP-2}

The present invention relates to a pharmaceutical composition and health functional food for the prevention and treatment of diseases caused by overexpression of MMP-9 or MMP-2 containing the sponge extract fraction as an active ingredient.

[1] tetler-Stevenson et al., Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu. Rev. Cell. Biol. 9 , pp. 541-573, 1993

Matrisian, LM Metalloproteinases and their inhibitors in matrix remodeling. Trends. Genet. 6 , pp. 121-125, 1990

Beeley NRA et al., Nature, 362 , pp. 839-841, 1993

Backstrom JR et al., J Neurosci, 16 (24) , pp. 7910-9, 1996

[Reference 5] Woessner JG, Jr. FASEB J, 5 , pp. 2145-2154, 1991

[Document 6] Republic of Korea Patent Publication No. 2002-71674

[7] Newby AC et al., J Pathol, 190 , pp. 300-9, 2000

[Reference 8] Bendeck MP et al., Circ Res, 75 , pp. 539-45, 1994

[Reference 9] Cho A. et al., Circ Res, 91 , pp. 845-51, 2002

10 Galis ZSet al., Circ Res, 1 , pp.852-9, 2002

[11] Senior RM et al., J Biol Chem, 266 , pp.7870-5, 1991

12 Okada Y. et al., J Biol Chem, 267 , pp.21712-9, 1992

[13] Galis ZS et al., Circ Res, 75 , pp. 181-9, 1994

[14] Fabunmi RP et al., Biochem J, 315 , pp. 335-42, 1996

15. Cho A. et al., Arterioscler Thromb Vasc Biol, 20 , pp. 2527-32, 2000

16. Faisal Khan KM et al., J Biol Chem, 277 , pp. 2353-9, 2002

Moon SK et al., Biochem Biophys Res Commun, 301 , pp. 1069-78, 2003

Moon SK et al., J Cell Physiol, 198 , pp. 417-27, 2004

[19] Bond M. et al., FEBS Lett, 435 , pp. 29-34, 1998

Menno PJ et al., 25 , pp. 904-914, 2005

Document 21 Sennett S.H. In: Marine Chemical Ecology, McClintock JB, Baker BJ, eds. (Boca Raton, FL: CRC Press), pp. 523-542, 2001

22. Page MJ et al., Aquaculture, 250 , pp.256-269, 2005

23. Ettinger-Epstein P. et al., J Nat Prod, 70 , pp.648-651, 2007

[24] Gunasekera SP et al., J Nat Prod, 65 , pp. 1643-8 2002

Suh, UH et al., Ochnaflavone inhibits TNF-alpha-induced human VSMC proliferation via regulation of cell cycle, ERK1 / 2, and MMP-9. J Cell Biochem , 99 , pp. 1298-1307, 2006

UH Jin et al., Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha-induced human aortic smooth muscle cells. Vascular Pharmacol , 44 , pp.345-53, 2006

SJ Suh et al., Raphanus sativus and its isothiocyanatesinhibit vascular smooth muscle cells proliferation and induce G (1) cell cycle arrest. Int Immunopharmacol , 6 , pp.854-61, 2006

Matrix metalloproteinases (MMPs) play an important role in the degradation of various components of the extracellular matrix (ECM). Diseases mediated by MMPs include atherosclerosis, restenosis, MMP-mediated osteopenia, inflammatory diseases of the central nervous system, skin aging, rheumatoid arthritis, pulmonary arthritis, corneal ulcers, abnormal wound unions, bone diseases, proteinuria, Abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, myelin deprivation disease, cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, inflammatory bowel disease, periodontal disease, macular degeneration associated with aging, diabetic retinal degeneration, proliferative Vitreoretinopathy, immature retinopathy, ocular inflammation, cone cornea, Sjogren's syndrome, myopia, ocular tumor, inflammation, dry eye, corneal graft rejection, angiogenesis, cancer infiltration and metastasis (tetler-Stevenson et al., Tumor cell interactions with the extracellular matrix during invasion and metastasis.Annu . Rev. Cell.Biol . 9 , pp.541-573, 1993).

Of these MMPs, in particular, MMP-2 and MMP-9 act specifically on type IV collagen, resulting in atherosclerosis, restenosis, rheumatoid arthritis, pulmonary arthritis, corneal ulcer, abnormal wound union, abdominal aneurysm disease, traumatic joint injury. Degenerative cartilage loss, demyelination of the nervous system, cirrhosis, immature rupture of the embryonic membrane, inflammatory bowel disease, periodontal disease, macular degeneration associated with aging, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation, Corneal, Sjogren's syndrome, myopia, ocular tumors, inflammation, dry eye, corneal rejection, angiogenesis, obesity, cancer invasion and metastasis, stroke, tumor growth disease (Matrisian, LM Metalloproteinases and their inhibitors in matrix remodeling.Trends.Genet . 6 , pp . 121-125, 1990).

In addition, the level of MMP-9 in spinal fluid is associated with multiple sclerosis and other neurological disorders (Beeley NRA et al., Nature, 362 , pp. 839-841, 1993), and also contributes to the degradation and accumulation of amyloid beta protein. (Backstrom JR et al., J Neurosci, 16 (24) , pp. 7910-9, 1996).

In addition, MMP-9 is known to play an important role in human tumors, angiogenesis-related diseases (obesity, dry eye, cataracts), cardiovascular disease and inflammation, cancer infiltration and metastasis. Cancer metastasis and infiltration are basic characteristics of malignant cancer cells. Cancer initially causes metastasis as well as invasion into blood vessels. Cancer cells go through several important steps in the process of infiltration and metastasis. Among these processes, the extracellular matrix surrounding the cancer tissue and the basement membrane are regarded as walls in cancer invasion, so that the breakdown of the extracellular matrix constituting the basement membrane is an essential early stage. Several proteases are involved in this process and reach the final stage through the degradation of the extracellular matrix (ECM) and the basal membrane, which are external environmental barriers (Woessner JG, Jr. FASEB J, 5 , pp. 2145-2154, 1991). . However, it is the inactive pro MMP-9 that is secreted from the cell, which is the production of an active form of plasminogen by the urokinase-type plasminogen activator (uPA). It is activated through a series of enzyme activations. Activated MMP-9 enhances the invasive capacity of cancer cells (Korean Patent Publication No. 2002-71674).

On the other hand, the expression of MMP-2 and MMP-9 is known to be related to arteriosclerosis, and the relationship between the expression of MMP protein and the proliferation of vascular smooth muscle cells and the migration to endometrium has been studied. Increased proteolytic activity by damage to the vascular wall catalyzes the breakdown of the extracellular matrix (ECM) surrounding vascular smooth muscle cells, which is also a necessary process for vascular smooth muscle cells to migrate into the endothelium (Newby AC et al. , J Pathol, 190 , pp. 300-9, 2000). In response to vascular damage, MMP-2 (72 kDa) and MMP-9 (92 kDa) gelatinases are involved as mediators of the development of these disorders. In rat vascular injury model, MMP-9 was expressed within 6 hours after rat carotid artery injury and continued expression for 6 days. On the other hand, the activity of MMP-2 increased after 4 days of injury (Bendeck MP et al., Circ Res, 75 , pp. 539-45, 1994). The facts from recent knockout studies of genes have been reported to be critical for the development of vascular wall damage due to MMP-9's ability to regulate the proliferation and migration of vascular smooth muscle cells (Cho A. et al., Circ Res, 91 , pp. 845-51, 2002; Galis Z Set al., Circ Res, 1 , pp.852-9, 2002). It has also been found that MMP-2 and MMP-9 have similar substrate specificities but differences in their expression (Senior RM et al., J Biol Chem, 266 , pp.7870-5, 1991; Okada Y. et al. , J Biol Chem, 267 , pp. 21712-9, 1992). MMP-2 is constitutively expressed in several cell types, including smooth muscle cells, and its expression is not induced by cytokines or growth factors (Galis ZS et al., Circ Res, 75 , pp. 181-9, 1994; Fabunmi RP et al., Biochem J, 315 , pp. 335-42, 1996). MMP-9 expression, in contrast, has been shown in several different laboratories, indicating that MMP-9 expression is fundamentally low and induced by TNF-α treatment (Cho A. et al., Arterioscler). Thromb Vasc Biol, 20 , pp. 252732, 2000). In addition, studies of signal transduction pathways regulating MMP-9 have shown that extracellular signal-regulated kinase 1/2 (ERK1 / 2), p38 and PI-3, which are mitogen-activated protein kinases (MAPK) when treated with several different inducers Kinase and other pathways have been shown to be important for the expression of MMP-9. In particular, TNF-α and Nerve growth factor (NGF) regulate the expression of MMP-9 through the ERK1 / 2 pathway in vascular smooth muscle cells (VSMC). (Cho A. et al., Arterioscler Thromb Vasc Biol, 20 , pp. 2527-32, 2000; Faisal Khan KM et al., J Biol Chem, 277 , pp. 2353-9, 2002). As shown in previous studies, promoter activity of MMP-9 induced by TNF-α in vascular smooth muscle cells is mediated by AP-1 and NF-κB (Moon SK et al., Biochem Biophys Res Commun, 301). , pp. 1069-78, 2003; Moon SK et al., J Cell Physiol, 198 , pp.417-27, 2004; Bond M. et al., FEBS Lett, 435 , pp.29-34, 1998). In addition, NF-κB is a key transcription factor involved in the activation of inflammatory cytokine genes such as TNF-α and IL-1β. Thus, various studies have been conducted to inhibit atherosclerosis through the study of the signaling pathways involved in NF-κB activation and the behavior of various proteins depending on NF-κB activity (Menno PJ et al., 25 , pp.904). -914, 2005).

Sponge is a fishery raw material that needs to create new added value. It contains a large amount of various powerful biologically active ingredients. Therefore, it is expected to create added value by developing these active ingredients. It is necessary to develop new functional food materialization technology and foster related industries in order to utilize the high value of sea sponges.

The sponge (marine sponge, porifera) is the lowest in the animal classification and the simplest among the welfare animals and does not have sensory cells or neurons. Mediterranean sea sponges are especially well known and Euspongia officinalis is a sponge used in bathrooms. Many marine natural substances have been separated from the sea surface and provide biologically active compounds with abundant structural diversity from various metabolic processes. The identification of natural products from sponges is one of the important resources that play a leading role in the medical and industrial sectors, but these materials have the disadvantage of being difficult to research and industrialize because they are present in very small amounts (Sennett SH In: Marine Chemical Ecology). , McClintock JB, Baker BJ, eds. (Boca Raton, FL: CRC Press), pp. 523-542, 2001; Page MJ et al., Aquaculture, 250 , pp.256-269, 2005). Thus, studies on interspecific variations of natural species of sponges and their understanding of their mechanisms can be a prerequisite for the optimization of seeding and culture of sponges. One of them, Luffariella variabilis, produces a manoalide family of substances with potent anti-inflammatory properties, but their supply is by collecting (Ettinger-Epstein P. et al., J Nat Prod, 70 , pp. 648-651, 2007). Therefore, research on the chemical diversity and growth environment of these materials is being done. In addition, (+) discodermolide identified from Discoderma species collected at the beach of Bahamas in the 1990s is known to act as an immunosuppressive and cytostatic inhibitor, and in addition to the incidence of cancer cells resistant to pacitaxol in vitro. It has been shown to inhibit proliferation (Gunasekera SP et al., J Nat Prod, 65 , pp. 1643-8 2002).

In Korea, there is an urgent need for researches on screening biologically active substances from marine organisms, which are reports of various resources, and to reveal their function and structure. In particular, in order to increase the possibility of developing and utilizing sea sponges as one of marine resources, The present invention has been accomplished by discovering physiological activity with antagonism against MMP-9 and MMP-2, which are known as important pathological targets in the metastasis process.

In the present invention, in order to develop and study the utilization of marine waste resources and new materials of bioactive substances, in human tumors, angiogenesis-related diseases (obesity, dry eye, cataracts), cardiovascular disease and inflammation, arteriosclerosis and cancer development mechanism Targeting matrix metalloproteinase-9 (MMP-9) and MMP-2, known to play an important role, we have studied the extract of sea sponges with antagonistic activity. There has been no report on the prophylactic therapeutic activity of diseases (obesity, dry eye, cataracts), cardiovascular disease, inflammation and artherosclerosis.

In order to achieve the above object, the present invention provides an inhibitor for MMP-9 or MMP-2 containing the sponge extract fraction as an active ingredient.

 The present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by overexpression of MMP-9 or MMP-2 containing the sponge extract fraction as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of diseases caused by overexpression of MMP-9 or MMP-2 containing the sponge extract fraction as an active ingredient.

The sponges defined herein are Cliona celata, Callyspongia elegans, Calcarea, Glass sponge, Demospongiae, Bath sponge (Euspongia officinalis) Keratinous sponges (Keratosa), orange beach sponges (Halichondria japonica) or gray beach sponges (H. panicea), preferably amber sponges (Cliona celata).

The extract fractions defined herein are characterized as being polar solvent soluble extraction fraction or nonpolar solvent soluble extraction fraction of sponge.

The polar solvent soluble extract fraction as defined herein is an extract fraction soluble in water, methanol, ethanol, butanol or a mixed solvent thereof, preferably water, methanol, butanol or a mixed solvent thereof, more preferably Butanol soluble extract fraction.

The non-polar solvent soluble extraction fraction as defined herein comprises an extraction fraction soluble in hexane, chloroform, methylene chloride or ethyl acetate, preferably hexane or ethyl acetate.

Diseases caused by overexpression of MMP-9 or MMP-2 as defined herein include acute and chronic inflammatory diseases, cardiovascular disease, arteriosclerosis, restenosis, rheumatoid arthritis, pulmonary arthritis, corneal ulcers, abnormal wound union, abdominal pain Aneurysms, degenerative cartilage loss due to traumatic joint injuries, demyelination of the nervous system, cirrhosis, immature rupture of the embryonic membrane, periodontal disease, macular degeneration associated with aging, airway inflammation, retinal degeneration, proliferative vitreoretinopathy, immature retinopathy , Cornea, Sjogren's syndrome, myopia, ophthalmic tumor, dry eye, corneal transplant rejection, angiogenesis, obesity, cancer infiltration and metastasis, stroke or tumor growth disease, etc., preferably acute, chronic inflammatory disease, cardiovascular disease, Atherosclerosis, rheumatoid arthritis, abdominal aortic aneurysm, airway inflammation, retinal degeneration, proliferative vitreoretinopathy, immature retinopathy, cancer infiltration and metastasis, stroke or Curing bowel disease, and more preferably grade. Including chronic inflammatory diseases, cardiovascular diseases, atherosclerosis, rheumatoid arthritis, airway inflammation, retinal degeneration or cancer invasion and metastasis.

Hereinafter, the method for obtaining the extract fraction of the present invention will be described in detail.

The extract fraction of the present invention is washed with fine sponges, and then washed with water, water, C ₁ to C ₄ lower alcohols or a mixed solvent thereof, preferably methanol, about 1 to 5 times the weight of dried sponges (w / v), preferably 2 to 4 times, cold extraction at 5 to 100 ° C., preferably at 20 to 90 ° C., more preferably at 60 ° C. for 20 to 30 hours, preferably 24 hours, Hot water extraction, ultrasonic extraction, reflux cooling extraction, heating extraction, etc., preferably extracted by cold needle extraction or hot water extraction method, filtered and concentrated under reduced pressure to obtain the crude sponge extract of the present invention.

The crude sponge extract obtained in the above step, preferably the sponge methanol extract is suspended in 30 to 50 times, preferably 40 to 45 times the volume (w / v) of water by weight, and then hexane, chloroform, ethyl acetate and A non-polar solvent soluble extract fraction which is fractionated 1 to 5 times, preferably 2 to 4 times by sequentially adding the same volume with water, and then soluble in non-polar solvents such as hexane, chloroform and ethyl acetate; And polar solvent soluble extract fractions soluble in polar solvents such as butanol, water and the like.

The present invention provides an inhibitor for MMP-9 or MMP-2 containing the sponge extract fraction obtained by the above production method as an active ingredient.

The inhibitor as defined herein is characterized by inhibiting the enzymatic activity or enzyme production of MMP-9 or MMP-2.

The inhibitor as defined herein is characterized by inhibiting transcriptional or promoter activity for MMP-9 or MMP-2 expression.

The present invention further relates to the use of the sponge extract fraction as an inhibitor against MMP-9 or MMP-2.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases caused by the overexpression of MMP-9 or MMP-2 containing the sponge extract fraction obtained by the above production method as an active ingredient.

The pharmaceutical composition containing the extract fraction of the present invention comprises the extract fraction in an amount of 0.1 to 50% by weight based on the total weight of the composition.

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 extract fraction of the present invention is a drug that can be used with confidence even for long-term use for the purpose of prevention because there is little toxicity and side effects.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the extract fractions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, 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.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.5 g / kg to 5 g / kg, preferably 1 g / kg to 3 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in 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 also provides a health functional food for the prevention and improvement of diseases caused by overexpression of MMP-9 or MMP-2 containing the sponge extract fraction obtained by the above production method as an active ingredient.

The composition comprising the extract fraction of the present invention can be used in a variety of drugs, foods and beverages for the prevention and improvement of diseases caused by overexpression of MMP-9 or MMP-2. Foods to which the extract fraction of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. Can be.

The amount of the extract fraction in the food or beverage of the present invention can generally be added in an amount of 1 to 5% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 10 g based on 100 ml. Preferably at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention contains the spongy extract fraction as an essential ingredient in the indicated proportions, and there is no particular limitation on the liquid component, and it may contain various flavors or natural carbohydrates as additional ingredients, like ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described 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 said 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 adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices 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 in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The sponge extract fraction of the present invention inhibits matrix metalloproteinase (MMP) -9 and MMP-2 directly involved in angiogenesis, inhibits p-ERK signal mediating expression of MMP-9, By confirming the efficacy of inhibiting NF-κB nuclear migration, it can be used in pharmaceutical compositions and dietary supplements useful for the prevention and treatment of diseases caused by overexpression of MMP-9 or MMP-2.

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

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

Example 1 Preparation of Sponge Methanol Crude Extract

260 g of fresh pumpkin sponge (Cliona celata) collected from the east coast was washed with water, added 1 liter of 100% methanol, immersed at 60 ° C for 24 hours, extracted 3 times at room temperature for 2 hours, and then decompressed, concentrated and dried 13 g (yield: 5%, hereinafter referred to as "PM").

Example 2. Preparation of Spongy Nonpolar Solvent Soluble Extract Fraction

2-1. Preparation of Sponge Hexane Extract Fraction

12 g of P-M of Example 1 was suspended in 500 mL of distilled water, poured into a 2000 mL fraction funnel, and then shaken to add an equal amount of hexane to separate the water layer and the hexane layer. The process was repeated three times. Concentration under reduced pressure, concentration, and drying yielded 1.2 g of a hexane extracted fraction of sea sponge (yield: 10% or less, referred to as "P-H") and a water-soluble fraction, which were used as samples in the following experimental example.

2-2. Preparation of Sponge Ethyl Acetate Extract Fraction

0.25 g of ethyl acetate extract fraction (yield: 2.31% or less) was performed by the same process as Example 2-1, except that 500 mL of ethyl acetate was added to the water-soluble fraction of Example 2-1 and extracted three times. , "PE") and water-soluble fractions were obtained and used as samples in the following experimental example.

Example 3. Preparation of Sponge Polar Solvent Soluble Extract Fraction

Except that 500 mL of butanol was added to the water-soluble fraction of Example 2-2, and extracted three times, the same process as in Example 2-1 was carried out, followed by 2.0 g of a sponge butanol extract fraction (yield: 18.96% or less, " PB ") and 7.4 g of water-soluble fraction (yield: 86.55% or less," PW ") were obtained and used as a sample of the following experimental example.

The extracted fractions obtained in the above example were dissolved in DMSO (dimetylsulfoxide) and used for activity assay.

Reference Example 1. Experimental Cell Culture

Human liver cancer cells (2.4 × 10 ³ cells, American Type Culture Collection, ATCC, MD, USA) and HASMC (2.2 × 10 ³ cells, Human Aortic Smooth Muscle Cells; Bio-Whittaker Co., San Diego, CA, USA) DMEM medium (Gibco) containing 10% heat-treated fetal bovine serum (FBS; Gibco-BRL, USA), penicillin (100 units / ml), streptomycin (100 μg / ml), and sodium bicarbonate (2.2 g / l) -BRL, USA) was incubated for 24 hours at 37 ℃, 5% CO ₂ incubator (CO2 incubator-2, Astec Co., Japan).

Experimental Example 1. Identification of Glycolipide-like Substances in the Sponge Extract Fraction

In order to confirm the active ingredient of the sponge extract fraction obtained in the above embodiment, thin layer chromatography (TLC) was performed as follows. Whatman's HP-K silica gel (silica gel: size 10 cm x 10 cm: thickness 200μ) was used, and the extracted sponge fraction was dissolved in a developing solvent (chloroform (30): methanol (60): water (8)) and dipped. After the development, color development was performed with sulfuric acid diluent.

Experimental results, as shown in Figure 2, the extraction fraction was developed in a solvent of chloroform (30): methanol (60): water (8) to the standard ganglioside glycolipids (GM3, GM1, GM2, GD3, lactosyl ceramide (LC)) As a result, it was confirmed that the development was performed in a similar position, and that a substance or a similar substance such as glycolipid GM1, GM2, GM3, and LC used as a standard material existed.

Experimental Example 2. Cytotoxicity Test of Sponge Extract Fraction

To determine the effect of sponge extract fractions on human liver cancer cells and HASMC, the XTT method (Boehringer Mannheim, Mannheim, Germany) (Suh, UH et al., Ochnaflavone inhibits TNF-alpha-induced human VSMC) proliferation via regulation of cell cycle, ERK1 / 2, and MMP-9.J Cell Biochem , 99 , pp.1298-1307, 2006).

Cell proliferation assays were determined by purchasing a commercial proliferation assay kit-II (XTT, Boehringer Mannhein, Germany). Human liver cancer cells and HASMC cells cultured in Reference Example 1 were inoculated with 10 ³ cells per 100 μl of DMEM medium in a 96 well culture plate (T-2, NUNC CO., Sweden) and cultured for 24 hours. The medium on the plate was discarded and replaced with 100 μl of fresh medium added with different concentrations (0, 125, 250, 500 and 1000 μg / ml) of spongy extract fractions. Plates were maintained at 5% CO ₂ and incubated in a humidified incubator (CO ₂ incubator-2, Astec Co., Japan) at 37 ° C. for 24 hours. After incubation, discard the medium and wash the cells with PBS saline solution (Boehringer Mannheim, Mannheim, Germany) (sodium 3 ′-[1- (phenyl-aminocarbonyl) -3,4-tetrazolium]- 50 μl of XTT reagent prepared by mixing bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate and N- methyl dibenzopyrazine methyl sulfate (50: 1 mixture)) and 100 μl of electron coupling reagent were prepared. Was added. This was again maintained at 37 ° C., 5% CO ₂ incubator for 4 hours, and the absorbance was measured at 490 nm with an ELISA meter (Molecular Devices, USA).

As a result, as shown in Fig. 3 and 4, the graph shows the percentage of cell proliferation using the group without the sponge extract fraction as a control, each data was carried out three independent experiments to average ± SD Indicated. Thus, the sponge extract fractions were confirmed to show no cytotoxicity to human liver cancer cells and HASMC.

Experimental Example 3 Measurement of MMP-9 and MMP-2 Inhibitory Effects of Sponge Extract Fractions on Human Liver Cancer Cells and Human Smooth Muscle Cells (HASMC)

In order to confirm the effect of the sponge extract fraction obtained in the above example on the activity of MMP-9 and MMP-2, various concentrations of sponge extract fractions were treated on TNF-α-induced human liver cancer cells and HASMC. UH Jin et al., Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha-induced human aortic smooth muscle cells.Vascular Pharmacol , 44 , pp.345-53, 2006) was measured as follows.

Human liver cancer cells (1 × 10 ⁵ cells / well) and HASMC cells (1 × 10 ⁵ cells / well) cultured in Reference Example 1 were prepared using PBS saline solution (NaCl 137mM, KCl 27mM, Na ₂ HPO ₄ 10mM, KH _2). PO ₄ 2 mM; Sigma, St. Louis, USA) and then treated with sponge extract fractions (500, 1000 μg / ml) in the presence of 100 ng / ml TNF-α and incubated in serum-free DMEM medium for 24 hours. . Then, 20 μl of the medium obtained from each human liver cancer cell and HASMC cell culture medium (DMEM medium) was added to 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS and 0.00625% (w / v) bromo. Rediluted in 5 μl of buffer containing bromophenol blue. 25 μl of this solution was subjected to electrophoresis (Amersham Bioscience Co., NJ, USA, Might Small II) for 2 hours without heating on 10% polyacrylamide gel containing 0.1% (w / v) gelatin. The gel was then immersed in 2.5% Triton X-100 (2 X 30 min) at room temperature, washed with NanoPure distilled water and the gel was washed in reaction buffer (50 mM Tris-HCl, pH 7.5), 200 mM NaCl, 2.5 mM CaCl ₂ ). It was immersed and kept at 37 ° C. for 24 hours, and stained with Coomassie brilliant blue (Sigma, St. Louis, USA) R-250 to identify a white band corresponding to enzymatic activity. The activity of MMP-9 and MMP-2 was measured by the brightness of the white band corresponding to the active part, and the molecular weight was estimated by comparison with the pre-stained SDS-PAGE markers (see FIG. 5).

As a result, as shown in FIG. 5, the intensity of the band obtained in gelatin zymography decreased as the concentration of the sponge extract fraction increased. In particular, when the cells were directly treated with PH, the IC ₅₀ for MMP-9 was 375 μg / ml and the IC ₅₀ for MMP-2 was 652 μg / ml. In addition, when the PE was directly treated, the IC ₅₀ for MMP-9 was 232 μg / ml and the IC ₅₀ for MMP-2 was 615 μg / ml.

Experimental Example 4. Effect of Sponge Extract Fractions on MMP-9 and MMP-2 Enzyme Activities

In order to assay the inhibitory effect on the gelatin degradation activity of the MMP-9 and MMP-2 of the sponge extract fraction obtained in the above example, the experiment was carried out as follows.

The conditioned medium (DMEM medium) of Experimental Example 3 obtained from TNF-α-treated human liver cancer cells and HASMC cell culture medium was 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS and 0.00625%. (w / v) Rediluted in the same buffer containing bromophenol blue. The solution was then electrophoresed without heating on a 10% polyacrylamide gel containing 0.1% (w / v) gelatin. After electrophoresis, the gel was immersed in 2.5% Triton X-100 (2 X 30min) at room temperature, then washed with NanoPure distilled water, the gel was sliced into slices and spongy extract fractions of various concentrations (0, 500 and 1000 μg / ml). It was immersed in the reaction buffer containing (50mM Tris-HCl (pH 7.5), 200mM NaCl, 2.5mM CaCl ₂ ) and kept at 37 ℃ for 24 hours. Staining with Coomassie Brilliant Blue R-250 identified white bands corresponding to enzymatic activity.

As shown in FIG. 6 and FIG. 7, it was confirmed that the intensity of the band obtained in the gelatin zymography decreased with the addition of the sponge extract fraction. In particular, the IC ₅₀ for the MMP-9 of PH was 276 µg / ml. The IC ₅₀ for MMP-2 was 889 μg / ml. The IC ₅₀ of MMP-9 of PE was found to be 198 ㎍ / ml, and the IC ₅₀ of MMP-2 was found to be 412 ㎍ / ml.

Experimental Example 5. Inhibition of p-ERK Signal Mediating Expression of MMP-9 Induced by TNF-α from Sponge Extract Fractions

Method described in the literature to determine the effect of the sponge extract fraction obtained in the above example on the phosphorylation inhibition of ERK protein, the major signal molecule that regulates the expression of MMP-9 gene of human liver cancer cells and HASMC Wes button blot (SK Moon et al according to, ERK1 / 2 mediates TNF-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-B and AP-1:.. Involvement of the ras dependent pathway J Cell Physiol , 198 , pp. 417-27, 2004) was measured as follows.

Human liver cancer cells (1 × 10 ⁵ cells / well) and HASMC (1 × 10 ⁵ cells / well) cultured in Reference Example 1 were washed with PBS physiological saline, and serum-free medium of Experiment 3 (DMEM medium) In the presence of 100 ng / ml TNF-α, 1000 μg / ml of the spongy extract fractions were treated at time intervals of 5 minutes, 15 minutes, and 30 minutes. Sample treated cells were washed twice with PBS saline and Lipa buffer (Tris-HCl: 50 mM, pH 7.4; NP-40: 1%; Na-deoxycholate): 0.25 for protein extraction. %; NaCl: 150 mM; EDTA: 1 mM; PMSF: 1 mM; Aprotinin, leupeptin, pepstatin: 1 μg / ml each; Na3VO4: 1 mM; NaF: 1 mM , 5 mM), and then mixed with 30 μl of Reagent A and 2 μl of the lysate using a Dc protein assay kit (Bio-rad, Calif.) On a 96well Elisa microplate. After the addition, 200 μl of reagent B was added and mixed again. After leaving for 15 minutes, the absorbance was measured at 750 nm, and the amount of protein was measured by comparing the absorbance of the standard protein BSA (bovine serum albumin). Quantified samples were blotted onto Nitrocellulose membrane (Amersham co.) And confirmed by Western blot using p-ERK and ERK antibodies.

As a result, 1000 ㎍ / ㎖ sponge ethyl acetate as shown in Figure 8 The extracted fractions were found to completely inhibit the phosphorylation of ERK induced at 5 and 15 minutes after TNF-α treatment.

Experimental Example 6. Measurement of the inhibitory effect of migration of sponge extracts in NF-κB nuclei

The method introduced by Schreiber et al. (1989) to determine the effect of the sponge extract fraction obtained in the above example on the inhibition of nuclear migration of p65, a large subunit of NF-κB (SJ Suh et al. , Raphanus sativus and its isothiocyanatesinhibit vascular smooth muscle cells proliferation and induce G (1) cell cycle arrest.Int Immunopharmacol , 6 , pp.854-61, 2006).

Cells cultured in Reference Example 1 (1 × 10 ⁵ cells / well) were washed with PBS physiological saline, and 300, 700 and 1000 μg in the presence of 100 ng / ml TNF-α in the serum-free medium of Experimental Example 3 After pretreatment of the sponge extract fraction at a concentration of / ml for 6 hours, treatment with 100 ng / ml of TNF-α for 30 minutes, the fully grown cells were washed twice with 1 ml of PBS buffer, and then treated with 0.5 ml of PBS and scraper ( scraper and transfer to EP tube. 400 μl of solution A (HEPES: 10 mM, pH7.9; KCl: 10 mM; EDTA: 0.1 mM; EGTA: 0.1 mM), immediately before use, 1 mM of dithiotreitol (DTT), 0.5 mM of phenylmethylsulfonyl fluoride (PMSF), A protease cocktail (apropriate concentration) was added and used, and mixed with 0.5% Nonidet NP40 (Sigma) to dissolve the cell membrane, and centrifuged at 6,000 rpm for 15 minutes to separate nuclei. 50 μl of solution C (HEPES: 20mM, pH7.9; NaCl: 0.4M; EDTA: 1mM; EGTA: 1mM) was used to dissolve the protein in the isolated nucleus, and DTT 1mM, PMSF 1mM, protease inhibitor cocktail ( Nucleoproteins were extracted by addition of apropriate concentration and stored at -70 ° C.

As a result, as shown in Figure 9, the analysis of the isolated nuclear protein by Western blot, p65 was treated with TNF-α increased the mobility into the nucleus, sponge ethyl acetate In the sample treated with the extracted fraction, it was confirmed that the migration in the nucleus of p65 was inhibited depending on the throughput.

Hereinafter, the preparation examples of the composition including the sponge extract fraction of the present invention, but the present invention is not intended to limit it, but to explain in detail only.

Formulation Example 1 Preparation of Powder

P-M 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

P-H 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components and tableting according to the conventional tablet manufacturing method to prepare a tablet.

Formulation Example 3 Preparation of Capsule

P-E 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

P-B 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na2HPO4,12H2O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

P-M 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

P-H 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture quantity

Ferrous Sulfate 1.75 mg

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7 Preparation of Healthy Drink

P-E 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

[National R & D project supporting this invention]

[Task unique number]

20070008

[Name of Buddha]

Korea Maritime Fisheries Research and Development Institute

[Name of research project]

Fisheries Specific Development Project

[Name of Research Project]

Development of industrialization technology for the treatment of chronic diseases from sponges and echinoderm

[Host]

Yeungnam University Industry-Academic Cooperation Foundation

[Research period]

September 20, 2007 ~ September 19, 2010

1 is a diagram illustrating a process of fractionating sponge extracts using various solvents,

FIG. 2 is a result of fractionation of sea sponge extract fractions using thin layer chromatography (TLC),

3 is a diagram showing the effect of inhibiting the cell proliferation in human liver cancer cells and arterial smooth muscle cells of the sponge hexane extract fractions by XTT assay,

Figure 4 is a diagram measuring the effect of inhibiting cell proliferation in human liver cancer cells and arterial smooth muscle cells of the sponge ethyl acetate extract fractions by XTT assay,

5 is a Zymograph result showing that the inhibition of metalloproteinase-9 (MMP-9) production of hepatic cancer cells and arterial smooth muscle cells induced by Tumor necrosis factor-α (TNF-α) with spongy extract fractions ,

Figure 6 is a diagram measuring the enzyme inhibitory activity against MMP-9 and MMP-2 in human hepatocellular carcinoma cells and arterial smooth muscle cells of the sponge hexane extract fractions by using zymography,

7 is a diagram showing the enzyme inhibitory activity against MMP-9 and MMP-2 in human liver cancer cells and arterial smooth muscle cells of the sponge ethyl acetate extract fractions using zymography,

FIG. 8 is a Western Blot result showing that sponge extract fractions inhibited p-extracellular signal-regulated kinase (p-ERK) of TNF-α-induced human liver cancer cells and arterial smooth muscle cells.

Figure 9 is a schematic diagram of the promoter location of the MMP-9 gene (A) and Westton blot results (B) showing that the sponge extract fraction inhibits the migration of NF-κB in the nucleus.

Claims (9)

Inhibitor against MMP-9 or MMP-2 containing sponge extract fraction as active ingredient. A pharmaceutical composition for the prevention and treatment of diseases caused by overexpression of MMP-9 or MMP-2 containing a sponge extract fraction as an active ingredient. According to claim 2, wherein the sponges (Cliona celata), pretty sponges (Callyspongia elegans), lime sponges (Calcarea), hexagon sponges (Glass sponge), common sponges (Demospongiae), bath sponges ( A pharmaceutical composition comprising Euspongia officinalis), keratinous sponges (Keratosa), orange beach sponges (Halichondria japonica) or gray beach sponges (H. panicea). According to claim 2, wherein the disease caused by overexpression of MMP-9 or MMP-2 is acute chronic inflammatory disease, cardiovascular disease, arteriosclerosis, restenosis, rheumatoid arthritis, pulmonary arthritis, corneal ulcer, abnormal wound Fusion, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, myelin deprivation disease, cirrhosis, immature rupture of the embryonic membrane, myocardial disease, macular degeneration associated with aging, airway inflammation, retinal degeneration, proliferative vitreoretinopathy, A pharmaceutical composition characterized by immature retinopathy, cone cornea, Sjogren's syndrome, myopia, ophthalmic tumor, dry eye, corneal rejection, angiogenesis, obesity, cancer infiltration and metastasis, stroke or tumor growth disease. The pharmaceutical composition according to claim 2, wherein the extract fraction is a polar solvent soluble extract fraction or a nonpolar solvent soluble extract fraction of the sponge. A dietary supplement for the prevention and improvement of diseases caused by overexpression of MMP-9 or MMP-2 containing the sponge extract fraction as an active ingredient. The method according to claim 6, wherein the sponges are Cliona celata, Callyspongia elegans, Calarea, Glass sponge, Despongiae, Bath sponges A dietary supplement comprising Euspongia officinalis), keratinous sponges (Keratosa), orange beach sponges (Halichondria japonica) or gray beach sponges (H. panicea). According to claim 6, wherein the disease caused by overexpression of MMP-9 or MMP-2 is acute chronic inflammatory disease, cardiovascular disease, arteriosclerosis, restenosis, rheumatoid arthritis, pulmonary arthritis, corneal ulcer, abnormal wound Union, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injuries, myelin deprivation disease, cirrhosis, immature rupture of the embryonic membrane, periodontal membrane disease, age-related macular degeneration, airway inflammation, retinal degeneration, proliferative vitreoretinopathy, immature Health functional food characterized by retinopathy, cone cornea, Sjogren's syndrome, myopia, ophthalmic tumor, dry eye, corneal rejection, angiogenesis, obesity, cancer infiltration and metastasis, stroke or tumor growth disease. 7. The dietary supplement of claim 6 which is a powder, granule, tablet, capsule or beverage.

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