WO2006115321A1

WO2006115321A1 - Composition comprising the extract of salvia miltiorrhiza bge showing enhancing activity for the prevention or treatment of blood circulatory disease.

Info

Publication number: WO2006115321A1
Application number: PCT/KR2006/000026
Authority: WO
Inventors: Hyun Wook Chang; Jong-Keun Son; Seung Ho Lee; Kun Ho Son; Kyung Soo Nam; Cheorl Ho Kim
Original assignee: Industry Academic Cooperation Foundation of Yeungnam University
Current assignee: Industry Academic Cooperation Foundation of Yeungnam University
Priority date: 2005-04-25
Filing date: 2006-01-04
Publication date: 2006-11-02
Anticipated expiration: 2007-10-25
Also published as: KR20060111857A; KR100767238B1

Abstract

The present invention relates to a composition comprising a polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza for the prevention and treatment of blood circulatory system disorder. The extracts from Salvia miltiorrhiza have potent inhibiting effect on inhibition of proteolytic activity of MMP-9 and on migration of VSMC cell therefore it can be used as the therapeutics or health food for treating and preventingblood circulatory system disorder caused by MMP hyper- activation.

Description

COMPOSITION COMPRISING THE EXTRACT OF SALVIA

MILTIORRHIZA BGE SHOWING ENHANCING ACTIVITY

FOR THE PREVENTION OR TREATMENT OF BLOOD

CIRCULATORY DISEASE.

Technical Field

[1] The present invention relates to an extract of Salvia miltiorrhiza B GE. showing potent inhibiting effect onmatrix metalloproteinase-9 activity and migration of TNF- α-induced human aortic smooth muscle cells and a composition comprising the same having preventing and treating arteriosclerosis.

[2]

Background Art

[3] The proliferation and migration of vascular smooth muscle cells (VSMC) may play a key role in the development of intimal thickening after arterial wall injury or in atherosclerosis (Ross, R., New England Journal of Medicine 314. pp488-500. 1986). VSMC in the media has low mitogenic activity. During the early stages of arterial wall injury or atherosclerosis, aortic smooth muscle cells may undergo transition from a contractile to a synthetic phenotype and begin proliferating in response to various growth factors, causing intimal thickening of the arterial walls (Chamley-Campbell, et al., Physiological Review 59_, ppl-61. 1979; Schwartz et al., Journal of American College of Cardiology 20, ppl284-1293. 1992; Ross, Nature 362, pp801-809. 1992). However, in addition to growth factor stimulation, the replication and migration of VSMC may require the degradation or remodeling of extracellular matrix surrounding the cells (Matrisian, L.M., Trends in Genetics 6, ppl21-125. 1990; Sasaguri et al., Laboratory Investigation 71, pp261-269.1994). VSMC synthesizes important components of the extracellular matrix, including collagens, elastin, and proteoglycans (Galis et al., Circulation Research 75, ppl81-189. 1994). An imbalance between the accumulation and degradation of extracellular matrix may be crucial in the development of intimal thickening that forms after vascular wall interventions (Strauss et al., Circulation Research 75, pp650-658. 1994).

[4] An understanding of the mechanisms of SMC invasion and migration may be important to devise therapies aimed at preventing artherosclerosis mediated smooth muscle cell's migration and intimal thickening. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases whose enzymatic activity is directed against components of the extracellular matrix (ECM) (Kleiner et al., Cancer chemotherapy and pharmacology 43, pp42-51. 1999). MMPs comprise three main groups: the interstitial collagenases (MMP-I), the type IV collagenases or gelatinases (MMP-2 and -9), and the stromelysins (MMP-3) (Woessner, 1991 Woessner, J.F.Jr., FASEB Journal 5, pp2145-2154. 1991; Chung et al., FASEB Journal 18QOl ppl 123-1125. 2004a). Among these MMPs, gelatinases degrade denatured interstitial collagens, native basement-membrane collagens; the expression of MMP-9 has been implicated in the progression of atherosclerotic lesions (Newby and Zaltsman, Journal of Pathology 190. pp300-309. 2000). Recent reports from an in vivo study concluded that MMP-9 is critical for the development of arterial lesions by regulating both VSMC migration and proliferation. On the basis of in depth reports from several different laboratories, it has been generally concluded that the basal levels of MMP-9 are usually low, and that its expression can be induced by treatment of vascular smooth muscle cells with tumor necrosis factor-α (TNF-α) (Cho et al., Thrombosis and Vascular Biology 20, pp2527-2532. 2000). Recent results from our laboratory demonstrated that ERK1/2 mediates TNF-αinduced MMP-9 expression in VSMC via the regulation of NF-kB and AP-I (Moon, S. K., et al., Biochemical and Biophysical Research Communication 301. ppl069-1078. 2003; Moon, S.K., et al., Archives of Biochemistry and Biophysics 418, pp39-48. 2003b).

[5] The endogenous MMP inhibitors, known as tissue inhibitors of metalloproteinase

(TIMPs), maintain a balance between matrix formation and destruction but have short half-lives (Brew et al., Biochimica et Biophysica Acta 1477. pp267-283, 2000). Therefore, synthetic inhibitors of MMPs, such as BB94 (batimastat) and BB2516 (marimastat) have been developed. BB94 has a structure that mimics collagen and facilitates chelation of the zinc ion in the active site of the MMP molecule, thereby causing an inactive protease. During the last few years, substances of this type have been used to study the importance of extracellular matrix remodeling in various types of diseases, including rheumatoid arthritis, cancer invasion and cardiovascular diseases. Thus, synthetic MMP inhibitors have been demonstrated to affect the wound- healing response in injured rat carotid arteries (Zempo et al., Thrombosis and Vascular Biology 16, pp28-33.1996; Bendeck et al., Circulation Research 78, pp38-43. 1996). To reduce migration of VSMC from aortic medial explants (Kenagy et al., Thrombosis and Vascular Biology 16, ppl373-1382.1996), and to inhibit the macrophage modulation of phenotypic change and DNA synthesis in cultured rabbit aortic SMC MMP-9 inhibitors have been designed (Fitzgerald et al., Atherosclerosis 145. pp97-106.1999).

[6] Due to the critical role of anti-migration in vascular disease development, the modulation of migration has become an interesting target in both therapeutic and preventive approaches in atherosclerosis (Chung et al., FASEB Journal 18(14^*). ppl670-1681, 2004b; Ha et al. Toxicology and Applied Pharmacology 200Q^*). ppl-6, 2004a; Ha et al., Pharmacological Research 50Gl pp279-285. 2004c).

[7] Several natural products have been used for vascular diseases, and some traditional herbal prescriptions have been also employed for treatment of atherosclerosis (Yoshie et al., Pharmacological Research 43, pp481-488.2001; Kim et al., International Im- munopharmacology 3, pp723-734.2003).

[8] There have been reported that the crude extract of Salvia miltiorrhiza Bunge

(Labiatae) (SM) contains phenolic compounds that are effective in protecting liver microsomes, hepatocytes, and erythrocytes against oxidative damage (Li et al., Journal of Asian Natural Products Research 414), pp271-280, 2002; Liu et al., Liver 21 f 6^*). pp384-390, 2001) and several active components such as tanshinones, D(+)3,4-dihydroxyphenol lactic acid, protocatechuic aldehyde, salvianolic acids (A, B, C, D, E, F) and rosmarinic acid have been isolated and identified therefrom (Li, L.N., Journal of Chinese Pharmaceutical Science 6, pp57-64.1997).

[9] However, there has been not reported or disclosed about therapeutic effect for atherosclerosis disease of the crude extract of Salvia miltiorrhiza Bunge (Labiatae) in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[10]

[11] To investigate an inhibiting effect of Salvia miltiorrhiza Bunge (Labiatae) on blood circulation disorder through several biochemical experiments and to confirm whether the crude extract and non-polar solvent soluble extract play an important role in inhibiting blood circulation disorder, the inventors of the present invention have intensively carried out several biological experiments such as MMP-9 inhibition activity, the cytotoxicity of HASMC cell etc, and finally completed present invention by confirming that the crude extract and non-polar solvent soluble extract inhibit MMP-9 activity, and migration of TNF-α-induced human aortic smooth muscle cells.

[12] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter.

[13]

Disclosure of Invention Technical Problem

[14] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE as an active ingredients for the treatment and prevention of blood circulatory system disorder caused by MMP hyper-activation.

[15] Technical Solution

[16] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE as an active ingredient for the treatment and prevention of blood circulatory system disorder caused by MMP hyper-activation.

[17]

[18] Above described polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE can be fractionated from crude extract prepared by the steps consisting of: extracting the rhizoma, root, or herb of plant material with water, lower alcohols such as methanol, ethanol, preferably methanol and the like, or the mixtures thereof.

[19] Above described polar solvent soluble extract can be prepared by extracting above crude extract with polar solvent, for example, water, lower alcohol such as methanol, ethanol, butanol preferably butanol and the like, or the mixtures thereof.

[20] Above described non-polar solvent soluble extract can be prepared by extracting above crude extract with non-polar solvent, for example, hexane, ethyl acetate or dichloromethane, preferably ethyl acetate.

[21] It is an object of the present invention to provide a use of a polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE for the manufacture of therapeutic agent for the treatment and prevention of blood circulatory system disorder caused by MMP hyper-activation in human or mammal.

[22]

[23] It is an object of the present invention to provide a method of treating or preventing blood circulatory system disorder in a mammal comprising administering to said mammal an effective amount of polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE, together with a pharmaceutically acceptable carrier thereof.

[24]

[25] It is another object of the present invention to provide a health food or food additives comprising above polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE, together with a sitologically acceptable additive for the prevention and improvement of blood circulatory system disorder caused by MMP hyper-activation.

[26]

[27] Above described blood circulatory system disorder comprises atherosclerosis, ischemic myocardial infarction, angina pectoris, hypertension, stroke, hyperlipemia, anemia, migraine, arrhythmia, apoplexy, angioma, hemangiofibroma, vascular deformity, angiostenosis and the like, preferably, atherosclerosis.

[28]

[29] Above described extracts may be extracted from any of Salvia genus plants such as

Salvia japonica or Salvia plebia.

[30]

[31] The pharmaceutical composition of the present invention can contain about 0.01 ~

50 % by weight of the above extract based on the total weight of the composition.

[32]

[33] The health food of the present invention comprises above extracts as 0.01 to 80 %, preferably 1 to 50 % by weight based on the total weight of the composition.

[34]

[35] Above health food can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc.

[36]

[37] An inventive extracts isolated from of Salvia miltiorrhiza BGE may be prepared in accordance with the following preferred embodiment.

[38]

[39] Hereinafter, the present invention is described in detail.

[40]

[41] An inventive extract of Salvia miltiorrhiza BGE can be prepared in detail by following procedures,

[42]

[43] The inventive crude extract of Salvia miltiorrhiza BGE can be prepared by follows;

For example, the rhizoma of Salvia miltiorrhiza BGE is dried, cut, crushed and mixed with 5 to 25-fold, preferably, approximately 10 fold volume of distilled water, lower alcohols such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably methanol; the solution is treated with hot water at the temperature ranging from 20 to 100°C, preferably from 60 to 100°C, for the period ranging from 15mins to 24 hours with extraction method by the extraction with hot water, cold water, reflux extraction, or ultra-sonication extraction with 1 to 5 times, preferably 2 to 3 times, consecutively; the residue is filtered to obtain the supernatant to be concentrated with rotary evaporator, at the temperature ranging from 20 to 100°C, preferably from 50 to 70°C and then dried by vacuum freeze-drying, hot air-drying or spray drying to obtain dried crude extract powder of Salvia miltiorrhiza BGE which can be soluble in water, lower alcohols, or the mixtures thereof.

[44]

[45] Additionally, polar solvent soluble and non-polar solvent soluble extract of present invention can be prepared by following procedure; the crude extract prepared by above step, is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5-fold volume of non polar solvent such as ethyl acetate, chloroform, hexane and the like; the non-polar solvent soluble layer is collected to obtain non-polar solvent soluble extract of the present invention and remaining polar solvent soluble layer is collected to obtain polar solvent soluble extract of the present invention which is soluble in water, lower alcohols, or the mixtures thereof. Also, above described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well-known in the art, for example, the procedure disclosed in the literature(Harborne J. B. Phytochemical methods: A guide to modern techniques of plant analysis, 3^rd Ed. pp6-7, 1998).

[46]

[47] To investigate the effect of Salvia miltiorrhiza BGE effect on blood circulation disorder through several biochemical experiments and to confirm whether the crude extract and non-polar solvent soluble extract play an important role in inhibiting blood circulation disorder, the inventors of the present invention have intensively carried out several biological experiments such as MMP-9 inhibition activity, the cytotoxicity of HASMC cell etc, and we have confirmed that the polar solvent soluble extract as well as non-polar solvent soluble extract inhibit MMP-9 activity, and migration of TNF- α-induced human aortic smooth muscle cells.

[48] The extract of Salvia miltiorrhiza BGE have been widely used long years agoas herbal medicine. Accordingly, it shows unique properties and lots of advantages including: (a), no known adverse effect; (b), no difficulty for oral consumption; (c), low cost; and (d), a long history of use by the human population (Change H.M. and But, P.P., 1986. Pharmacology and Applications of Chinese Material Medica, World Scientific Publication, Singapore.1986), all of which are indicative of its potential application as an anti-atherosclerotic agent.

[49]

[50] In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising the polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza prepared by above preparation method for the treatment and prevention of blood circulatory disease caused by MMP hyper-activation in human or mammal as active ingredients.

[51]

[52] It is another of the present invention to provide a treating method and preventing method comprising administering a pharmaceutical composition comprising said extract prepared by above preparation method to the mammals including human.

[53]

[54] The inventive composition for treating and preventing blood circulatory disease may comprises above extracts as 0.01 ~ 50 % by weight based on the total weight of the composition.

[55]

[56] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

[57]

[58] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

[59]

[60] The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[61]

[62] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

[63]

[64] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

[65]

[66] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[67]

[68] The desirable dose of the inventive extract or composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 10 g/kg, preferably, 1 to 3 g/kg by weight/day of the inventive extract or compounds of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the amount of inventive extract should be present between 0.01 to 50% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.

[69]

[70] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.

[71]

[72] Also, the present invention provide a composition of the health food beverage for the prevention and improvement of blood circulatory disease adding above described extracts 0.01 to 80 % by weight, amino acids 0.001 to 5 % by weight, vitamins 0.001 to 2 % by weight, sugars 0.001 to 20 % by weight, organic acids 0.001 to 10 % by weight, sweetener and flavors of proper amount.

[73]

[74] Above described extract of Salvia miltiorrhiza can be added to food and beverage for the prevention and improvement of blood circulatory disease.

[75]

[76] To develop for health food, examples of addable food comprising above extracts of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as power, granule, tablet, chewing tablet, capsule or beverage etc.

[77]

[78] Also, the extract of the present invention will be able to prevent and improve blood circulatory disease by way of adding to child and infant food, such as modified milk powder, modified milk powder for growth period, modified food for growth period.

[79] [80] Above described composition therein can be added to food, additive or beverage, wherein, the amount of above described extract in food or beverage may generally range from about 0.1 to 80w/w %, preferably 1 to 50 w/w % of total weight of food for the health food composition and 1 to 30 g, preferably 3 to 10 g on the ratio of IOOD of the health beverage composition.

[81]

[82] Providing that the health beverage composition of present invention contains above described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cy- clodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 D of present beverage composition.

[83]

[84] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforement ioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[85]

[86] The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc. [87]

[88] The above extract of Salvia miltiorrhiza BGE may be 20 to 90 % high concentrated liquid, power, or granule type.

[89]

[90] Similarly, the above extract of Salvia miltiorrhiza BGE can comprise additionally one or more than one of lactose, casein, dextrose, glucose, sucrose and sorbitol.

[91]

[92] Inventive extract of the present invention have no toxicity and adverse effect therefore; they can be used with safe.

[93]

[94] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[95]

Advantageous Effects

[96] The extract of Salvia miltiorrhiza BGE have been widely used long years agoas herbal medicine. Accordingly, it shows unique properties and lots of advantages including: (a), no known adverse effect; (b), no difficulty for oral consumption; (c), low cost; and (d), a long history of use by the human population (Change H.M. and But, P.P., 1986. Pharmacology and Applications of Chinese Material Medica, World Scientific Publication, Singapore.1986), all of which are indicative of its potential application as an anti-atherosclerotic agent.

[97]

[98] In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising the polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza prepared by above preparation method for the treatment and prevention of blood circulatory disease caused by MMP hyper-activation in human or mammal as active ingredients.

[99]

Brief Description of the Drawings

[100] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[101]

[102] Fig. 1 shows the effect of the extract of Salvia miltiorrhiza BGE on cell proliferation; [104] Fig. 2 shows the photographs of HASMC cells treated with control group (A), 100

D/ml and 500 D/ml of ethylacetate soluble extract (B and C) and the relative inhibition effect on cell proliferation of ethylacetate soluble extract (D);

[105]

[106] Fig. 3 represents the MMP-9 gelatinolytic effect on the cultured conditioned media of HASMC treated with TNF-α(100 ng/ml); lane 1 treated with control group, lane 2 with buffer and 50 D of 70% EtOH soluble extract, lane 3 with buffer and 50 D of chloroform soluble extract, lane 4 with buffer and 50 D of ethylacetate soluble extract, lane 5 with buffer and 50 D of butanol soluble extract, lane 6 with buffer and 50 D of water soluble extract;

[107]

[108] Fig. 4 represents the inhibitory effects on the MMP-9 gelatinolytic activity of various concentration of EtOAc soluble extract (0, 10, 20, 30, 40, 50, 70, 100 and 500 D/ml) and zymographs performed by controlled medium obtained from HASMC cells in the presence of TNF-α or not;

[109]

[110] Fig. 5 presents the inhibitory effect of ethylacetate soluble extract on the migration of VSMC cells, Fig. A shows the migrated cells excluded TNF-α or inventive extract (a), the cells treated with 100 ng/ml of TNF-α (b), the cells treated with TNF-α and 100 D/ml of inventive extract (c), the cells treated with TNF-α and 200 D/ml of inventive extract (d), Fig. B shows total number of migrated cells stained with hematoxylin and eosin;

[111]

[112] Fig 6 depicts the inhibitory effect of the inventive extract compared with previously known MMP inhibitors on MMP-9; (A) cultured medium treated with DMSO, EDC and 50 D/ml of inventive extract, (B) the intensity of the bands estimated with Gel-Print System transformed into expressed mean+SE value in zymogram studies.

[113]

Best Mode for Carrying Out the Invention

[114] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[115]

[116] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner. Mode for the Invention

[118] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

[119]

[120] Example 1. Preparation of the polar and nonpolar solvent soluble extract of

Salvia miltiorrhiza

[121]

[122] 1-1. Preparation of water soluble extract

[123] lkg of dried of Salvia miltiorrhiza BGE purchased from Kyung-dong Market located in Seoul was cut into small pieces, mixed with 5OmL of distilled water and extracted with hot water for 15 mins at 100°C. The residue is filtered to obtain the supernatant to obtain 35g of water extract.

[124]

[125] 1-2. Preparation of methanol soluble extract

[126] Remaining residue obtained from Example 1-1 was extracted with 70% ethanol three times for 5 hours at 70°C. The residue was filtered with 0.45 um of filter paper and polled supernatant was concentrated, dried with freezing dryer (Speed Spec 3000, Bio-Rad Co. U.S.A.) to obtain 55g of 70% ethanol soluble extract, (yield: 5.5%).

[127]

[128] 1-3. Preparation of chloroform soluble extract

[129] 50g of 70% ethanol soluble extract obtained from Example 1-2 was dissolved in

500 ml of distilled water and fractionated with chloroform three times. The polled chloroform soluble layer was concentrated with vaccuo to obtain 15.4g of chloroform soluble extract (yield: 30.8%).

[130]

[131] 1-4. Preparation of ethylacetate soluble extract

[132] Remaining residue excluding chloroform soluble extract obtained from Example

1-3 was fractionated with ethylacetate three times. The polled ethylacetate soluble layer was concentrated with vaccuo to obtain 11.6g of ethylacetate soluble extract (yield: 23.2%).

[133]

[134] 1-5. Preparation of butanol soluble extract

[135] Remaining residue excluding ethylacetate soluble extract obtained from Example

1-4 was fractionated with butanol three times. The polled butanol soluble layer was concentrated with vaccuo to obtain 3.8g of butanol soluble extract (yield: 7.5%).

[136]

[137] 1-6. Preparation of ethanol soluble extract [138] Remaining residue excluding butanol soluble extract obtained from Example 1-5 was fractionated with ethanol three times. The polled ethanol soluble layer was concentrated with vaccuo to obtain 17.5g of ethanol soluble extract (yield: 3.5%).

[139]

[140] For the bioassay test, all the samples prepared from Example 1 were dissolved in

DMSO and further diluted in culture media.

[141]

[142] Experimental Example 1. The cytotoxic effect on HASMC

[143] Materials

[144] (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) were obtained from Sigma Chemical Co. (MO, USA). The drugs were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 40 mM. Recombinant human TNF-α and BB94 was obtained from R&D systems (MN, USA) and British Biotechnology (Oxford, U.K.), respectively.

[145]

[146] Cell Culture

[147] Human aortic smooth muscle cells (MASMC) were prepared by the procedure disclosed in the literature (Moon, S. K., et al., Biochemical and Biophysical Research Communication 301, ppl069-1078. 2003).

[148] HASMC were cultured in smooth muscle cell growth medium-2 containing 10%

FBS, 2 ng/ml human basic fibroblast growth factor, 0.5 ng/ml human epidermal growth factor, 50 D/ml gentamicin, 50 D/ml amphotericin-B, and 5 D/ml bovine insulin. For all experiments, early passage HASMC were grown to 80-90% confluence and made quiescent by serum starvation (0.1% FBS) for at least 24 hrs. The serum-free medium contained secreted proteins such as MMP-9. The amount of secreted proteins in the conditioned media were estimated and quantified by cell numbers.

[149]

[150] Cell Viability AssayfXTT proliferation assay)

[151] The cytotoxicity of the inventive extracts prepared by Example 1 was performed by the procedure disclosed in the literature (Ha et al., Pharmacological Research 50(3). pp279-285. 2004c).

[152] The cytotoxic effect of the extracts from SM and its ingredient herbs on HASMC was investigated using a commercially available proliferation kit (XTT II, Boehringer Mannheim, Mannheim, Germany) (Ha et al., 2004c). Briefly, the cells were plated in 96- well culture plates at a density of IxIO⁴ cells per well in DMEM culture medium and allowed to attach for 2 hrs. The extracts were added to various final concentrations (0, 10, 50, 100, 250, 500 and 1000 D/ml) in triplicates. After 72 hrs of culture, 50 ml of XTT reaction solution (sodium 3'-[l-(phenyl-aminocarbonyl)-3,4-tetrazolium] - bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate; mixed in proportion 50:1) was added to the wells. The optical density was read at 490 nm wavelength in an ELISA plate reader after 4 hrs incubation of the plates with XTT in an incubator (37°C and 5% CO + 95% air). All determinations were confirmed using replication in at least three identical experiments. The data were shown mean+SE as percent of control.

[153] [154] Result [155] As shown in Fig.l, the inventive extract prepared in Example 1 showed weak cytotoxic effect on HASMC cells in a dose dependent manner (IC > 500 D/ml), which is not so harmful to human body. The CH 2 Cl 2 fraction, water extract and 70% EtOH extract exhibited weak cytotoxic activity (IC of > 100 D/ml, 90 D/ml, and >100 D/ml, re-

50 spectively). However, the EtOAc and BuOH fraction exhibited higher cytotoxic activity (IC of 70 and 80 D/ml, respectively) <£ee Table 1). Although all fractions and the 70% EtOH extract had weak cytotoxic activity, typical concentration dependent cytotoxic effect of EtOAc fraction against VSMC is shown in Fig. 2.

[156] Table 1 Cell Cytotoxicity (IC 50 , D/ml)

[157] [158] Experimental Example 2. Gelatin zymography assay [159] Methods [160] Substrate gel zymography of the activity of MMP-9 was performed with a Mini- Protein II apparatus from Bio-Rad, according to a method disclosed in the literature (Demeule et al., Biochimica et Biophysica Acta 1478. pp51-60.2000).

[161] To examine the inhibitory activity of SM against MMP-9 activity, the cultured conditioned media of HASMC, which was treated with TNF-α (100 ng/ml), have been subjected to the gelatin zymography. Then, we investigated

[162] The controls using either DMSO or EtOH showed no effect on proteolytic activity of MMP-9 (Data not shown). Proteolytic activity is indicated by the presence of clear bands on a dark background and the intensity of the band is proportional to the enzyme expression.

[163] Cells were grown to sub-confluence and were rinsed with phosphate-buffered saline

(PBS) and then incubated in serum-free medium for 24 h. The amount of gelatinase in the conditioned media were estimated and quantified by cell numbers. Conditioned medium were resuspended in a sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.00625% (w/v) bromophenol blue, then were agitated for 20 min at room temperature and loaded without boiling onto 7.5% acrylamide/ bisacrylamide (29.2:0.8) separating gel containing 0.1% (w/v) gelatin. Electrophoresis was carried out at a constant voltage of 100 V. After electrophoresis, the gels were soaked in 0.25% Triton X-100 (2x30 min) at room temperature and rinsed in NanoPure water. For gelatinase inhibition assays, the methanol extract and its fractions were freshly solubilized in the Tris buffer used for developing the zymogram; the gel slab was cut into slices corresponding to the lanes and then put in different tanks containing the stated concentrations of fractions. The gel containing gelatin was incubated at 37°C for 20 h in the incubation buffer containing 50 mM Tris-HCl (pH 7.6), 20 mM NaCl, 5 mM CaCl₂ and 0.02% Brij-58 with or without 0, 1, 10, 50, 100, 500, 1000 D/ml of fraction or extract. The gel was then stained for 15-30 min in 0.1% (w/v) Coomassie blue R-250 in 30% methanol and 10% acetic acid, and destained in the same solution without the Coomassie blue dye.

[164]

[165] Densitometric and statistical analysis

[166] The intensity of the bands obtained from zymogram studies was estimated with

Gel-Print System (Core Bio Corp., Seoul, KOREA). The values are expressed as means+SE.

[167]

[168] Result

[169] The effects on the MMP-9 gelatinolytic activity of the 70% EtOH extract, CH Cl fraction, EtOAc fraction, BuOH fraction and water extract at 50 D/ml each as shown in Fig. 3. The control line that conditioned medium was incubated in tris-CaCl -buffer only showed presence of MMP-9. Also, the 70% EtOH extract, CH Cl fraction and water extract showed low inhibitory activities. The EtOAc extract and BuOH fraction showed strong inhibition of proteolytic activity of MMP-9, EtOAc fraction being the strongest inhibitory activity.

[170] When the inhibitory effects on the MMP-9 gelatinolytic activity by each fraction and extract including 70% EtOH extract, CH Cl fraction, BuOH fraction, and water extract were examined, 70% EtOH extract, CH 2 Cl 2 fraction and water extract showed weak activity (IC > 100 D/ml), while BuOH fraction showed weak inhibition of MMP- 9 proteolytic activity (IC = 63 D/ml) (gee Table 2). [171] Especially, as shown in Fig. 4, EtOAc fraction inhibited MMP-9 in a dose- dependent manner (lowest registered values of IC : 24 D/ml). Although treatment of VSMC with up to 50 D/ml of EtOAc fraction neither inhibited their growth nor indeed morphological changes (Fig. 2). But EtOAc fraction showed the strongest inhibitory effect on proteolytic activity of MMP-9.

[172] [173] Table 2 MMP-9 inhibitory activity (IC , D/ml)

[174] [175] Relative MMP-9 inhibitory activity of SM compared with established MMP-9 inhibitors

[176] From the above results, it is suggested that SM is a strong inhibitor of MMP-9. The inhibitory effect against MMP-9 activity of SM was compared with established MMP inhibitors such as BB 94, catechin and its derivates, which are present abundantly in green tea. As shown in Fig. 6, the inhibitory effect of SM against gelatinolytic activity of MMP-9 was higher than that of EGC, but lower than that of EGCG and BB94.

[177] [178] Experimental Example 3. Migration assays [179] [180] Methods [181] Matrigel Migration assay was performed by the procedure disclosed in the literature (Chung et al., FASEB Journal IS(UX ppl670-1681, 2004b).

[182] Briefly, Matrigel-coated filter inserts (8 D pore size) that fit into 24-well invasion chambers were obtained from Becton-Dickinson (NJ, USA). HASMC (5 X 10 cells/ well) to be tested for invasion were detached from the tissue culture plates, washed, re- suspended in conditioned medium collected from TNF-α treated HASMC for 24 h, and then added to the upper compartment of the invasion chamber in various concentration of SM (0, 50, 100, 250 and 500 D/ml). Five hundred ml of same conditioned medium was added to the lower compartment of the invasion chamber. Cells without TNF-α treated conditioned medium served as control. The Matrigel invasion chambers were incubated at 37°C for 24 hrs in 5% CO 2. After incubation, the filter inserts were removed from the wells, and the cells on the upper side of the filter were removed using cotton swabs. The filters were fixed, mounted, and stained according to the manufacturer's instructions. The cells that invaded through the Matrigel and were located on the underside of the filter were counted. Three to five invasion chambers were used per condition. The values obtained were calculated by averaging the total number of cells from three filters.

[183]

[184] Inhibitory effect of EtOAc fraction on migration of VSMC

[185] VSMC were resuspended in conditioned medium (5x10⁴ cells/200 ml), added to the upper components of the matrigel invasion chamber supplemented with various concentration of EtOAc fraction (0, 50, 100, 250 and 500 D/ml) and incubated for 24 h at 37°C and 5% CO 2.

[186] As shown in Fig. 5, the total number of cells that invaded to the underside of the filters was significantly decreased by EtOAc fraction treatment, in a dose-dependent manner (IC = 15 D/ml). The results showed that SM effectively inhibited the invasion of VSMC.

[187]

[188] These results suggested that SM could be used as a potential anti-atherosclerotic agent and that EtOAc fraction is an anti-atherosclerotic agent with low cytotoxicity acting on MMP-9 and may serve as a lead compound with development of anti- atherosclerotic drugs.

[189]

[190] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[191]

[192] Preparation of powder

[193] Dried powder of Example 1-4 50mg

[194] Lactose lOOmg

[195] Talc lOmg

[196] Powder preparation was prepared by mixing above components and filling sealed package.

[197]

[198] Preparation of tablet

[199] Dried powder of Example 1-4 50mg

[200] Corn Starch lOOmg

[201] Lactose lOOmg [202] Magnesium Stearate 2mg

[203] Tablet preparation was prepared by mixing above components and entabletting.

[204] Preparation of capsule

[205] Dried powder of Example 1-5 50mg

[206] Corn starch lOOmg

[207] Lactose lOOmg

[208] Magnesium Stearate 2mg

[209] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

[210]

[211] Preparation of injection

[212] Dried powder of Example 1-4 50mg

[213] Distilled water for injection optimum amount

[214] PH controller optimum amount

[215] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2ml ample and sterilizing by conventional injection preparation method.

[216]

[217] Preparation of liquid

[218] Dried powder of Example 1-5 0.1~80g

[219] Sugar 5~10g

[220] Citric acid 0.05-0.3%

[221] Caramel 0.005-0.02%

[222] Vitamin C 0.1-1%

[223] Distilled water 79-94%

[224] CO gas 0.5-0.82%

[225] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

[226]

[227] Preparation of health food

[228] Extract of Example 1-6 lOOOmg

[229] Vitamin mixture optimum amount

[230] Vitamin A acetate 70mg

[231] Vitamin E l.Omg

[232] Vitamin B₁ 0.13mg

[233] Vitamin B₂ 0.15mg

[234] Vitamin B 0.5mg

[235] Vitamin B 0.2mg [236] Vitamin C lOmg

[237] Biotin lOmg

[238] Amide nicotinic acid 1.7mg

[239] Folic acid 50mg

[240] Calcium pantothenic acid 0.5mg

[241] Mineral mixture optimum amount

[242] Ferrous sulfate ,.1.75mg

[243] Zinc oxide 0.82mg

[244] Magnesium carbonate 25.3mg

[245] Monopotassium phosphate 15mg

[246] Dicalcium phosphate 55mg

[247] Potassium citrate 90mg

[248] Calcium carbonate lOOmg

[249] Magnesium chloride 24.8mg

[250] The above-mentioned vitamin and mineral mixture may be varied in may ways.

Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

[251]

[252] Preparation of health beverage

[253] Extract of Example 1-6 lOOOmg

[254] Citric acid lOOOmg

[255] Oligosaccharide lOOg

[256] Apricot concentration 2g

[257] Taurine Ig

[258] Distilled water 900D

[259]

[260] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85°C for 1 hour, filtered and then filling all the components in IOOOD ample and sterilizing by conventional health beverage preparation method.

[261]

[262] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[263]

Industrial Applicability

[264] As described in the present invention, the polar solvent soluble extract and non- polar solvent soluble extract inhibit MMP-9 inhibition activity, the cytotoxicity of HASMC cell etc,therefore, it can be used as the therapeutics or health food for treating and preventing blood circulatory disorder such as atherosclerosis without adverse action.

Claims

[1] A pharmaceutical composition comprising polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE as an active ingredients for the treatment and prevention of blood circulatory system disorder caused by MMP hyper-activation.

[2] The pharmaceutical composition according to claim 1 wherein said polar soluble extract is extracted with the solvent selected from the group consisting of water, lower alcohol and the mixture thereof.

[3] The pharmaceutical composition according to claim 2 wherein said lower alcohol is butanol.

[4] The pharmaceutical composition according to claim 1 wherein said non-polar solvent is selected at least one from the group consisting of hexane, carbon tetrachloride, chloroform, methylene chloride, ethyl ether and ethyl acetate.

[5] The pharmaceutical composition according to claim 4 wherein said non-polar solvent is ethyl acetate.

[6] The pharmaceutical composition according to claim 1 wherein said blood circulatory system disorder comprises atherosclerosis, ischemic myocardial infarction, angina pectoris, hypertension, stroke, hyperlipemia, anemia, migraine, arrhythmia, apoplexy, angioma, hemangiofibroma, vascular deformity and angi ostenosis.

[7] The pharmaceutical composition according to claim 6 wherein said blood circulatory system disorder is atherosclerosis.

[8] A use of a crude extract, polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE for the preparation of therapeutic agent for the treatment and prevention of blood circulatory system disorder caused by MMP hyper-activation.

[9] A health food comprising polar solvent soluble or non-polar solvent soluble extract of Salvia miltiorrhiza BGE, together with a sitologically acceptable additive for the prevention and improvement of blood circulatory system disorder caused by MMP hyper-activation.

[10] The health food according to claim 9 wherein said health food is provided as powder, granule, tablet, chewing tablet, capsule or beverage type.