KR100891881B1 - Composition for preventing and treating hyperlipidemia and vascular disease due to highly activated MMP comprising 3,4,5-trihydroxybenzaldehyde as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing and treating vascular diseases caused by hyperlipidemia and MMP overactivity including 3,4,5-trihydroxybenzaldehyde as an active ingredient. It relates to a composition for the prevention and treatment of vascular diseases caused by hyperlipidemia and MMP hyperactivity comprising 4,5-trihydroxybenzaldehyde as an active ingredient. The composition of the present invention, without cytotoxicity, increases blood cholesterol and decreases the amount of triglycerides, and inhibits MMP activity of arterial smooth muscle cells, resulting in MMP hyperactivity such as hyperlipidemia and arteriosclerosis caused by abnormal lipid metabolism. It has the effect of preventing and treating vascular diseases. Therefore, the composition of the present invention can be used for the purpose of preventing and treating vascular diseases such as hyperlipidemia and arteriosclerosis.

Bamboo, hyperlipidemia, arteriosclerosis, MMP, prevention, treatment

Description

Composition for preventing and treating hyperlipidemia and vascular disease due to highly activated MMP comprising 3,4,5-trihydroxybenzaldehyde as an active ingredient 5-trihydroxybenzaldehyde as an active ingredient}

The present invention relates to a composition for preventing and treating vascular diseases caused by hyperlipidemia and MMP overactivity including 3,4,5-trihydroxybenzaldehyde as an active ingredient. It relates to a composition for the prevention and treatment of vascular diseases caused by hyperlipidemia and MMP hyperactivity comprising 4,5-trihydroxybenzaldehyde as an active ingredient.

Modern people have a variety of metabolic diseases such as hyperlipidemia due to high intake of instant foods and meat-based diets and high content of cholesterol and triglycerides in blood, resulting in many vascular diseases such as arteriosclerosis. Among them, arteriosclerosis is a disease in which fat accumulates in the walls of blood vessels and thickens the walls of blood vessels over a long period. Hyperlipidemia, especially hypercholesterolemia, is a major risk factor, and hypertension and diabetes are also known as risk factors. have. In the progression of arteriosclerosis, damage to vascular endothelial cells constituting the inner surface of blood vessels occurs due to oxidative lipids, high blood pressure and rapid blood flow, and fat gradually accumulates in the walls of blood vessels. Inflammation proceeds and the walls of blood vessels thicken due to the proliferation of vascular smooth muscle cells. As a result, blood vessels become narrow and blood flow is not smooth. If the coronary arteries of the heart is narrowed due to excessive exercise, the heart is suffering from angina due to insufficient blood supply to the heart. On the other hand, if the activity of the protease produced by the macrophages in the thickened blood vessel wall (called arteriosclerosis plaque) is strong, a part of the tissue covering the plaque will burst. At this time, the fat and peripheral proteins in the plaque come out into blood vessels and form a blood clot, which often blocks the coronary arteries or blood vessels of the brain, causing myocardial infarction or stroke.

A study by the Lipid Research Clinica Program (LRCP) reported that a 1% reduction in blood cholesterol levels reduced the risk of coronary artery disease by about 2%, and high-density cholesterol, which transports and removes cholesterol from the blood vessel walls to the liver. Increased HDL-cholestrol is known to inhibit the progression of coronary stenosis and prevent atherosclerosis.

Conventional arteriosclerosis treatment is a method of taking hyperlipidemia-lowering drugs for a long time, or if the symptoms are severe, surgical removal of plaque and insertion of a metal stent to prevent the narrowing of blood vessels. Despite the need for continuous management such as drug therapy and diet, due to the nature of the disease, the incidence of metabolic-related diseases in modern people is not significantly lowered due to the difficult management.

On the other hand, Geum japonicum THUNBERG is a perennial herb belonging to the Rosaceae family native to Ulleungdo, southern and mid-central mountain meadows and roadside hills of Korea. It is used for edible, ornamental and medicinal purposes, and young leaves are eaten as herbs, and it is used as a medicine for gastric ulcers, hypertension, hemorrhoids in Kwanchocho and oriental medicine and folk medicine, and it is 3,4,5-trihydroxybenzaldehyde isolated from the snakeroot extract. It is known that has an excellent antioxidant effect, but it is not known about the control of triglyceride content and inhibitory activity against MMP.

Therefore, the present inventors were studying the physiological effects of 3,4,5-trihydroxybenzaldehyde isolated from bamboo radish extract, and 3,4,5-trihydroxybenzaldehyde was used to determine the concentration of high density cholesterol (HDL-cholesterol) and triglyceride in the blood. The present invention was completed by finding out that there is an effect of preventing and treating vascular diseases caused by MMP overactivity such as hyperlipidemia and arteriosclerosis by inhibiting concentration control and matrix metalloproteinase (MMP) activity.

Accordingly, it is an object of the present invention to provide new uses of 3,4,5-trihydroxybenzaldehyde.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating vascular diseases caused by hyperlipidemia or MMP overexpression comprising 3,4,5-trihydricbenzaldehyde or a pharmaceutically acceptable salt thereof as an active ingredient. To provide a composition.

In order to achieve the another object of the present invention, the present invention provides a food composition for preventing and improving vascular disease by hyperlipidemia or MMP overexpression comprising 3,4,5-trihydroxybenzaldehyde or salts thereof as an active ingredient. .

In order to achieve the another object of the present invention, the present invention provides a food composition for preventing and improving vascular disease by hyperlipidemia or MMP overexpression comprising the extract as an active ingredient.

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

The composition of the present invention is characterized in that it comprises 3,4,5-trihydroxybenzaldehyde (3,4,5-trihydroxybenzaldehyde) or a salt thereof as an active ingredient.

The 3,4,5-trihydroxybenzaldehyde of the present invention is represented by the following formula (I).

The 3,4,5-trihydroxybenzaldehydes according to the invention can be used on their own or in the form of salts. In this case, the salt is preferably used in the form of a pharmaceutically acceptable salt when used as a pharmaceutical composition. As the pharmaceutically acceptable salt, an acid addition salt formed by a pharmaceutically acceptable free acid is preferable. The free acid may be an organic acid or an inorganic acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutaric acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The 3,4,5-trihydroxybenzaldehyde of the present invention can be isolated and purified from nature, commercially available and used or prepared by chemical synthesis methods known in the art.

Preferably, the 3,4,5-trihydroxyhexibenzaldehyde of the present invention may be used separately from nature according to methods known in the art. More preferably Geum japonicum THUNBERG) can be separated by conventional extraction and chromatography.

Extraction, separation of 3,4,5-trihydroxybenzobenzaldehyde from snakeskin is, for example, water, methanol, ethanol, propanol, pentanol, butanol, pentane, cyclopentane, cyclohexane, 1-pentene, diisobutyl Ethylene, xylene, dichloromethane, 1,2-dichloroethane, 1-chlorobutane, 1-chloropentane, 1-chloropropane, 2-chloropropane, bromoethane, methanol, ethanol, 1-propanol, 2-propanol , 1-pentanol, 2-butoxyethanol, benzene, toluene, chlorobenzene, ether, ethyl ether, diethyl ether, diisopropyl ether, chloroform, methyl acetate, ethyl acetate, acetone, carbon tetrachloride, carbon disulfide ), Diethyl sulfide, 4-methyl-2-propanone, tetrahydrofuran, 2-butanone, 1-nitropropane, 1,4-dioxane, dimethyl sulfoxide, aniline, In organic solvents such as diethylamine, acetonitrile, pyridine, ethylene glycol Selected may be any one or extracted with a mixture of these solvents.

Preferably, the extraction for the preparation of the snakeskin extract of the present invention can be carried out by the following method. That is, the dried bamboo radish powder sample was extracted with 80% methanol, extracted with a mixed solvent of n-hexane: water (1: 1), the aqueous layer was separated, and then mixed with a mixed solvent of methylene chloride: water (1: 1). Extract and separate the aqueous layer again. The aqueous layer may be prepared by extracting a mixed solvent of ethyl acetate: water (1: 1) and separating the ethyl acetate layer.

The ratio of the solvent such as methanol and methanol during extraction is not particularly limited, but the solvent may be added in 1 to 20 times (by weight) to the powder or fraction of the snake. Preferably, the powder of snakewood to increase the extraction efficiency Alternatively, the solvent may be added in 2 to 5 times the fractions.

The temperature during extraction is preferably carried out at room temperature under atmospheric pressure. In addition, the extraction efficiency can be further increased when stirring with a shaker (shaker) during extraction.

Bamboo radish used for extraction can be harvested, washed and used as is or dried. As a drying method, both dry, shade, hot air drying and natural drying can be used. In addition, in order to increase the extraction efficiency can be used by grinding the bamboo or its dry matter with a grinder.

Separation of 3,4,5-trihydroxybenzaldehyde from the extract may be performed by chromatography using a method known in the art, for example, silica gel column chromatography. The 3,4,5-trihedizaldehyde may be separated by reverse phase column chromatography and high performance liquid chromatography (HPLC).

Preferably, the 3,4,5-trihydroxybenzaldehyde of the present invention is finely pulverized dried bamboo radish and then added three times 80% methanol to the bamboo radish powder and extracted at room temperature for 24 hours. The extract is centrifuged to remove precipitates and the supernatant is recovered to prepare a serpentine extract containing 3,4,5-trihydroxybenzaldehyde. The extract was sequentially extracted with n-hexane, methylene chloride, ethyl acetate and fractionated, wherein an ethyl acetate fraction was passed through a silica gel column to obtain a fraction eluted with an ethyl acetate-methanol solvent. After passing this through a silica gel column, a fraction prepared by eluting with a chloroform-methanol solvent was prepared, and then passed through a Sephadex LH-20 column with 80% methanol solvent to separate 3,4,5-trihydricbenzaldehyde. have.

3,4,5-trihydroxybenzaldehyde of the present invention is effective in hyperlipidemia by increasing the content of HDL cholesterol, lowering the content of triglycerides in the blood, inhibits the activity of MMP-9 of arterial smooth muscle cells, the migration It was found to be effective in vascular diseases caused by MMP overactivity by decreasing its ability. These results indicate that the 3,4,5-trihydroxybenzaldehyde of the present invention is effective for the prevention and treatment of vascular diseases caused by hyperlipidemia and MMP hyperactivity, or improvement of symptoms, respectively. First revealed by

Intimal thickening (stenosis) after atherosclerosis and arterial wall injury is known to be due to the proliferation and migration of vascular smooth muscle cells (VSMC) (Ross R., J. Med. 314, pp 488-500 , 1986). VSMC in the medial vascular media has low cell division, but at the onset of atherosclerosis or vascular wall injury, aortic smooth muscle cells enter the proliferation phase in cooperation with a variety of growth factors to enlarge the vessel (Chamley-Campbell J. et al., Physiol. Rev. 59, pp 1-61, 1979; Schwartz RS et al., J. Am. Coll. Cardio 20, pp 1284-1293, 1992). However, the replication and migration of VSMC requires remodeling and degradation of extracellular matrix around cells (Matrisian LM, Trends. Genet. 6, pp121-125, 1990), and MMPs (Matrix metalloproteinases) play a major role in the degradation enzymes. (Dollery CM et al., Circ.Res. 77, pp863-868, 1995; Pauly RR et al., Circ.Res. 75, pp 41-54, 1994; Cho A. et al., Circ.Res. 91, pp 845-851, 2002).

MMPs are a type of zinc-dependent endoproteinase that degrades extracellular matrix (Kleiner, D. E. et al., Cancer chemotherapy and pharmacology 43, pp 42-51, 1999). MMPs include interstitial collagenases (MMP-1), type IV collagenases or gelatinases (MMP-2, -9), and stromelysins (MMP-3). (Woessner J. et al., FASEB J. 5, pp 2145-2154, 1991; Chung, TW et al., FASEB Journal 18 (10), pp 1123-1125, 2004a), among these MMPs, gelatinases. Collagen (gelatinase, collagen) is known to degrade the natural basement membrane, and the expression of MMP-2 / -9 induces atherosclerotic lesions (Newby AC et al., J. Pathol. 190, pp 300-309, 2000). .

Recent in vivo ( in In vivo studies have shown that MMP-9 is critical for inducing arterial lesions through VSMC migration and proliferation (Galis, ZS et al., Circ.Res. 91, pp852-859, 2002; Cho, A. et al. , Arterioscler. Thromb. Vasc. Biol. 20, pp2527-2532, 2000). The activity of MMPs is inhibited and regulated by endogenous MMP inhibitors (tissue inhibitors of metalloproteinases), and the regulation of MMPs and endogenous MMP inhibitors balances matrix formation and destruction (Brew K. et al., Biochim). Biophys.Acta. 1477, pp 267-283, 2000).

Therefore, synthetic MMP inhibitors (eg, BB94 batimastatR and BB2516 marimastatR, etc.) have been developed for the treatment of atherosclerosis using this mechanism. These MMP inhibitors have been developed over the last few years and are being studied for rheumatoid arthritis, cancer and cardiovascular diseases. Therefore, a substance showing inhibitory activity against MMP can be used as a material for preventing and treating atherosclerosis.

The present inventors have confirmed that the 3,4,5-trihydroxybenzaldehyde of the present invention is effective against hyperlipidemia and vascular disease due to MMP overactivity, and the 3,4,5-trihydroxybenzaldehyde or a pharmaceutical thereof To prepare a pharmaceutical composition for preventing and treating hyperlipidemia containing an acceptable salt as an active ingredient and a pharmaceutical composition for preventing and treating vascular diseases due to MMP overactivity.

The pharmaceutical composition according to the present invention may contain 3,4,5-trihydroxybenzaldehyde alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, `` pharmaceutically acceptable '' refers to a composition that is physiologically acceptable and does not normally cause an allergic or similar reaction when administered to a human.

Vascular disease due to MMP overactivity refers to vascular diseases such as arteriosclerosis, angina pectoris, myocardial infarction, hypertension, anemia, migraine, stroke and hemangioma, in which increased MMP activity is the cause of disease development or characteristics of disease progression. May be atherosclerosis.

The pharmaceutical composition for the prevention and treatment of vascular diseases caused by hyperlipidemia and MMP hyperactivity of the present invention can be administered to a mammal by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, transdermal administration, subcutaneous, intravenous, intramuscular or intraperitoneal administration. Preferably the pharmaceutical composition of the present invention may be administered orally. In the above, 'oral administration' refers to administering the pharmaceutical composition of the present invention through the oral cavity so that the active ingredient contained in the pharmaceutical composition of the present invention is delivered into the individual.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredients with solid excipients, milling them, adding suitable auxiliaries and then processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

The total effective amount of the 3,4,5-trihydroxybenzaldehyde of the present invention may be administered to a patient in a single dose, and the fractionated treatment protocol administered for a long time in multiple doses. May be administered). The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably, the preferred total dose of 3,4,5-trihageoxybenzaldehyde of the present invention may be from about 0.01 μg to 1,000 mg, most preferably 0.1 μg to 100 mg per kg of patient body weight per day. However, the concentration of 3,4,5-trihydroxybenzaldehyde is not only dependent on the route of administration and frequency of treatment, but also on various factors such as the age, weight, health status, sex, severity of the disease, diet and excretion rate. Since the effective dosage for the patient is determined in consideration of this, a person of ordinary skill in the art should consider the prevention of vascular disease due to hyperlipidemia and MMP overactivity of 3,4,5-trihydricbenzaldehyde. And appropriate effective dosages depending upon the particular use as therapeutic agent. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown.

On the other hand, the present invention provides a food composition for preventing and improving vascular disease due to hyperlipidemia and MMP overactivity, including 3,4,5-trihydroxybenzaldehyde and salts thereof.

The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, the health food can be ingested by granulating, encapsulating and powdering the 3,4,5-trihydroxybenzaldehyde itself of the present invention. In addition, it can be prepared in the form of a composition by mixing with 3,4,5-trihydroxybenzaldehyde of the present invention and known active ingredients known to have the effect of preventing and improving vascular diseases caused by hyperlipidemia or MMP overactivity.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage corned beef) Breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese), edible vegetable oils, margarine, vegetable protein , Retort foods, frozen foods, various seasonings (e.g., miso, soy sauce, sauce, etc.), etc., can be prepared by adding 3,4,5-trihydricbenzaldehyde of the invention.

The preferred content of the 3,4,5-trihydroxybenzaldehyde of the present invention in the food composition of the present invention may include from about 0.01 to 10% by weight relative to the total weight of the food.

On the other hand, the food composition of the present invention can be prepared with known food additives. The food additive may preferably use one or two or more additives of sugars such as white sugar, mannitol, glucose, sorbitol, xylitol, inositol, lactose and fructose or amino acids such as citric acid, ascorbic acid and amino acids.

On the other hand, the present invention provides a food composition for preventing and improving vascular disease due to hyperlipidemia and MMP overactivity, including the snakewood extract extracted with an organic solvent having 1 to 6 carbon atoms as an active ingredient, the extraction solvent and method of the snakeskin extract is As described above.

In one embodiment of the present invention, after extracting the bamboo radish powder with 80% methanol, the extract is sequentially extracted with n-hexane, methylene chloride, ethyl acetate and fractions, wherein the ethyl acetate fractions are first silica gel column chromatography, The 3,4,5-trihydroxybenzaldehyde was isolated via secondary silica gel column and Sephadex LH-20 column chromatography.

On the other hand, in another embodiment of the present invention, the experimental animal ingesting a high fat diet using the ethyl acetate fraction containing 3,4,5-trihydroxysibenzaldehyde of the present invention and 3,4,5-trihydroxysibenzaldehyde The total cholesterol content, HDL cholesterol content and blood triglyceride content in were investigated. As a result, there was no difference in the total cholesterol content among the experimental groups, but it was found that the HDL cholesterol content was high and the blood triglyceride content was low, which was effective for hyperlipidemia.

In another embodiment of the present invention, the effect of 3,4,5-trihydroxybenzaldehyde of the present invention on MMP-9 inhibitory activity and cell migration ability was investigated. As a result, MMP-9 activity was different depending on the cell type, but almost disappeared when 3,4,5-trihydroxybenzaldehyde was treated at a concentration of 50 to 500 μg / ml, and 3,4,5-trihydroxybenzaldehyde was treated. Was found to effectively inhibit the invasion-migration activity of human arterial smooth muscle cells.

Accordingly, the present invention provides a composition for the prevention and treatment of vascular diseases due to hyperlipidemia and MMP hyperactivity. The composition of the present invention can be used for the purpose of preventing and treating vascular diseases such as hyperlipidemia and arteriosclerosis.

Below. The present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

From bamboo  3,4,5- Of trihydroxybenzaldehyde  detach

<1-1> Bamboo  Preparation of Extract

The snakewood used in the present invention was harvested and used in the packaging of the Gyeongnam Agricultural Research and Development Institute Herbal Herbal Testing Team (FIG. 1), and was used while keeping the ground portion of the harvested snakewood in the shade.

The dried bamboo radish sample was completely dried at 50 ° C. in a dry oven, and then finely ground to obtain bamboo powder. Then, 3 liters of 80% methanol was added to 1 kg of the powder sample, and then shaken occasionally at room temperature for 24 hours. It was left. The extracted supernatant was filtered with Whatman No. 2 filter paper, and 2 liters of 80% methanol was added to the remaining snakeskin sample and extracted again for 24 hours and filtered in the same manner as described above. I collected the liquid. The filtrate was concentrated under reduced pressure at 45 ° C. with a rotary vacuum evaporator to obtain 80% methanol extract, which completely blown off the solvent.

After lyophilizing the 80% methanol extract, it was dissolved in a solvent mixed with n-hexane: water in a volume ratio of 1: 1, transferred to a separatory filter, and then shaken with a separatory filter shaker for about 1 hour, and then left to separate a hexane layer. Then, hexane was added to the remaining water layer in the same volume ratio and extracted in the same manner as above. This process was carried out once and a total of three times to obtain an n-hexane layer and an aqueous layer, and the n-hexane layer was concentrated under reduced pressure with a rotary vacuum evaporator to obtain an n-hexane fraction. Next, the aqueous layer was partitioned three times in a volume ratio of methylene chloride: water in a volume ratio of 1: 1 to obtain a methylene chloride layer and an aqueous layer (ii), and the methylene chloride layer was concentrated under reduced pressure with a rotary vacuum evaporator to fractionate methylene chloride. Got. Next, the aqueous layer (ii) was partitioned ethyl acetate: water three times in a volume ratio of 1: 1 to obtain an ethyl acetate layer and an aqueous layer, and the ethyl acetate layer was concentrated under reduced pressure with a rotary vacuum evaporator to fractionate ethyl chloride. Got. Finally, water was evaporated from the aqueous layer to obtain a water fraction.

<1-2> 3,4,5- Of trihydroxybenzaldehyde  detach

The ethyl acetate (EtOAc) fraction (49.5 g) in the solvent fraction of Geum japonicum THUNBERG isolated in Example <1-1> was silica gel column with EtOAc-MeOH solvent (100: 0 → 0: 100; gradient). Chromatography (silica gel column chromatography) was performed and divided into five fractions (Fraction I to V). Four fractions (fractions I, II, III, V) were subjected to secondary silica gel column chromatography using CHCl ₃ -MeOH solvent system (100: 0 → 0: 100; gradient) to obtain active fraction (antioxidant activity). The active fractions were separated from the active material by Sephadex LH-20 column chromatography under MeOH-H ₂ O solvent conditions (8: 2). The active substances separated here were finally separated by preparative chromatography (Shimadzu) to separate five single substances. The separated compounds were analyzed by LC / MS / MS, 1H-NMR, and 13C-NMR to confirm three types of structures. The structure of Compound 2, which has the highest antioxidant activity and the highest content, is represented by Chemical Formula 1 below. It was identified as 3,4,5-trihydroxygebenzaldehyde (3,4,5-trihydroxybenzaldehyde, THBA).

<Formula 1>

< Example  2>

Investigation of the effect on the blood cholesterol and triglyceride content of the composition of the present invention

Male Sprague-Dawley three-week-old male rats were purchased from a central laboratory animal and divided into two groups to have a normal diet and a high fat diet. THBA (3,4,5-) isolated from 1% and 5% (w / w) of dietary intake, respectively, from 10 to 4 weeks after induction of obesity, and 1% and 5% (w / w) of dietary intake, respectively. trihydroxybenzaldehyde) was orally administered at a concentration similar to that of the ethyl acetate fraction (3 mg / ml) to make a high fat control group, 1% GJ group, 5% GJ group, and THBA group. The normal control group was fed AIN-76A diet # 100000 (Dytes Inc., Bethleham, PA, USA) during the 10-week experimental period from 4 weeks of age, and the high-fat diet group used beef tallow as a source of fat for AIN-76A diet. -76A high fat diet # 100496 (Dytes Inc., Bethleham, PA, USA) was fed 40% of the total calories to fat. Each experimental group was eight.

The test animals were separated and bred one by one, and water and diet were supplied without restriction. Dietary intake and body weight were measured twice a week during the experiment. The dietary efficiency (FER) was calculated by dividing the weight gain during the experiment period by the dietary intake during the experiment period from the experimental diet supply day to the sacrifice day.

Experimental animals were sacrificed at 14 weeks of age to collect blood and organs. Blood was collected by sacrificial heart puncture and centrifuged at 3000 rpm for 15 minutes to obtain serum, and these samples were stored at -70 ° C until analysis.

The total cholesterol, HDL cholesterol, and triglyceride contents of the serum were measured using a kit of Sigma Chemical Co., Ltd. (St. Louis. MO). Differences between the groups were tested by one-way ANOVA and Duncan's multiple range test was performed at the α = 0.05 level. Significance was tested at the α = 0.05 and α = 0.01 levels, and all statistical analyzes were processed using SAS program. The results are expressed as mean ± standard deviation (SD).

As a result, as shown in Table 1 below, the total cholesterol content of blood was not different among the experimental groups, but the HDL cholesterol was lower in the high fat control group than the normal control group, and the bamboo radish extract group was significantly increased than the high fat control group. Serum triglyceride was 79.62 ± 23.12mg / dl in the high fat control group, which was significantly increased in comparison with 50.49 ± 8.11mg / dl in the normal control group, while it was significantly decreased in the beet fed group. The ± 10.81mg / dl and THBA (3,4,5-trihydroxybenzaldehyde) group showed 47.11 ± 8.37mg / dl significantly decreased compared to the high fat control group.

Total cholesterol (mg / dl) HDL cholesterol (mg / dl) Triglyceride (mg / dl) Normal Control (General Dietary) 64.33 ± 9.54 38.82 ± 7.42 50.49 ± 8.11 High fat control group (high fat diet) 67.51 ± 6.71 22.86 ± 3.90 79.62 ± 23.12 1% GJ group (High Fat Diet + 1% Bamboo Radish Ethyl Acetate Extract) 63.44 ± 10.12 27.50 ± 10.94 58.50 ± 9.82 5% GJ group (High Fat Diet + 5% Bamboo Radish Ethyl Acetate Extract) 65.93 ± 11.67 32.59 ± 10.05 44.26 ± 10.81 THBA group (high fat diet + THBA) 64.22 ± 9.41 30.34 ± 9.15 47.11 ± 8.37

< Example  3>

Bamboo  Extract of human arterial smooth muscle cells ( HASMC Toxicity to)

<3-1> Cell Culture

Human arterial smooth muscle cells (HASMC) were cultured (Moon, et al., Biochem. Biophys. Res. Commun. 301, pp 1069-1078, 2003) by the method described in the literature. The culture medium contained smooth muscle cell growth medium-2 (10% FBS, 2 ng / ml human primary fibroblast growth factor, 0.5 ng / ml human endothelial growth factor, 50 μg / ml gentamicin (50 μg / ml amphotericin-B). , 5 μg / ml bovine insulin) were used, and all experiments were cultured with 80-90% confluence of primary human HASMC (passage) and serum starvation. , 0.1% FBS).

<3-2> Cytotoxicity Assay

Cytotoxicity was measured by XTT kit (XTT II, Boehringer Mannheim, Mannheim, Germany) in HASMC according to the methods described in the literature (Ha, et al, pharmacological Research 50 (3), pp 279-285, 2004). The cells were grown in 96-well culture plates with 1 × 10 ⁴ cells / well DMEM for 2 hours and THBA (3,4,5-trihydroxybenzaldehyde) isolated from the serpentine extract at various concentrations (0, 10, 50). , 200, and 500 μg / ml) and divided into three experimental groups. After 20 hours 50 μl of XTT reducing solution (sodium 3 ′-[1- (phenyl-aminocarbonyl) -3, 4-tetrazolium] -bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate and N- Methyl dibenzopyrazine methyl sulfate; mixed at 50: 1) was added. After 24 hours, it was measured in an incubator with XTT at 490 nm with an ELISA plate reader (37 ° C., 5% CO ₂ + 95% air). The experiment was conducted three times independently, and the results were expressed as mean (SEAN ± SE).

As a result, as shown in Figure 3, the toxicity of THBA (3,4,5-trihydroxybenzaldehyde) to human arterial smooth muscle cells (HASMC) was examined at various THBA concentrations (0, 10, 50, 200, 500 ㎍ / ml) As you can see, it was weak. In particular, THBA is unlikely to cause any harm to the human arterial smooth muscle cells (HASMC) as the proliferation rate is 65.6% compared to normal cells even at a concentration of 500 ㎍ / ml is not harmful to the human body.

<Example 4>

MMP-9 Inhibitory Activity and Cell Migration

<4-1> Inhibitory assay of MMP-9 produced by THBA human arterial smooth muscle cells (HASMC)

In order to examine the MMP-9 inhibitory activity of the snakeskin extract and THBA of the present invention, MMP-9 activity was carried out with a slight modification of the method described in the literature (Demeule, et al., Biochimica et Biophysica Acta 1478, pp51-60, 2000; Chung, et al., FASEB Journal 18 (14), pp 1670-1681, 2004b; Ha, et al., Toxicology and Applied Pharmacology 200 (1), pp 1-6, 2004a).

Cultures of human arterial smooth muscle cells (HASMC) treated with TNF-α (100 ng / ml) (untreated for comparison) were treated with 62.5 mM Tris-HCl (TrisHCl, pH 6.8), 10% glycerol ( glycerol), 7.5% of gelatin 0.1% (w / v) without suspension and suspended in a buffer containing 2% SDS and 0.00625% (w / v) bromophenol blue. Acrylamide / bisacrylamide (29.2: 0.8) electrophoresis was performed at a constant voltage of 100 volts in a separation gel for electrophoresis. After electrophoresis, the gel was infiltrated twice with Triton X-100 0.25% solution for 30 minutes at room temperature and then washed with pure water.

To test the inhibitory effects of THBA on TNF-α-induced MMP-9 expression, THAS was treated by HASMC in concentrations (0, 20, 50, 100, 200, 500 μg / ml). zymography) was performed as follows. HASMC cells were cultured in 10% FBS / DMEM medium, washed with phosphated buffed saline (PBS), and incubated with THBA in TMEM-treated serum-free DMEM medium for 24 hours. After the normal growth cells were collected and suspended in a polyacrylamide 10% gel containing 0.1% gelatin and electrophoresed. The electrophoresis gel was washed with Triton X-100 2.5% (v / v) solution for 1 hour to remove SDS, and then incubated at 37 ° C for 24 hours to decompose gelatin protein. The gel was then stained with Coomassie Brilliant blue R-250 (Bio-Rad, USA) to determine the degree of enzyme activity. In contrast, we examined the inhibitory effects of THBA on MMP-9 in LPS-treated raw 264.7 cells and Hep3B cells in which MMP-9 is constantly expressed.

As a result, as shown in Figure 4, MBA-9 inhibitory activity by THBA is HASMC treated with TNF-α, raw 264.7 cells treated with LPS, Hep3B cells constantly expressing MMP-9, the difference by cell type It was found that the activity almost disappeared at a concentration of 50 to 500 μg / ml.

<4-2> Experimental migration of arterial smooth muscle cells

In order to test the migration ability of TNF-α-induced arterial smooth muscle cells, the Matrigel migration assay described in the literature was used (Chung, et al., FASEB Journal 18 (14), pp 1670-1681, 2004b; Ha et al., Pharmacological Research 50 (3), pp 279-285, 2004c). HASMC (5 × 10 ⁴ cells / well) cells after fixation of Matrigel-coated filtration inserts (8 m empty size) in 24-well invasion chambers from Becton-Dickinson, USA Was incubated for 24 hours. It was treated with TNF-α (100 ng / ml) followed by various concentrations of THBA (0, 20, 50, and 100 μg / ml) after 24 hours of incubation. As a control group, 80 μg / ml of EGCG (epigallocatechin-3-gallate), which is known to prevent TNF-α treatment and atherosclerosis prevention and treatment, was used. The Matrigel invasion chamber was kept at 37 ° C., 24 hours, 5% CO ₂ .

As a result, as shown in FIG. 5, the number of migrated cells (cells infiltrated into the filter paper) was greatly reduced in concentration-dependent manner by the addition of THBA, and this result was comparable to EGCG used as a control. These results show that THBA isolated from snakeskin extract effectively inhibits the invasion-migration activity of human arterial smooth muscle cells.

< Production Example  1>

THBA Hyperlipidemia with or MMP Due to overactivity  Preparation of drugs for the treatment or prevention of vascular diseases

<1-1> Preparation of Tablet

25 mg of THBA was added to a U-type mixer with 26 mg of lactose for excipients, 3.5 mg of Avicel (microcrystalline cellulose), 1.5 mg of sodium starch glyconate as a disintegration aid, and 8 mg of low-hydrosyprophylcellulose (L-HPC) for binders. Mix for 20 minutes. After mixing was completed, 1 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a humidity test.

<1-2> Preparation of Syrup

2 g THBA, 0.8 g saccharin and 25.4 g sugar were dissolved in 80 g of warm water. After cooling the solution, 8.0 g of glycerin, 0.04 g of flavor, 4.0 g of ethanol, 0.4 g of sorbic acid, and an appropriate amount of distilled water were mixed thereto. Water was added to the mixture to make 100 ml, thereby preparing a syrup containing acteoside as an active ingredient of 2% (W / V).

<1-3> Preparation of Capsule

A capsule was prepared by mixing THBA 50 mg, lactose 50 mg, starch 46.5 mg, talc 1 mg and an appropriate amount of magnesium stearate and filling it into hard gelatin capsules.

<1-4> Preparation of Injection Solution

100 g was prepared by dissolving 1 g of THBA, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid in distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

As described above, the composition of the present invention, while not cytotoxic, reduces the increase of high-density cholesterol and triglycerides in the blood, and inhibits the MMP activity of arterial smooth muscle cells, resulting in hyperlipidemia and atherosclerosis caused by abnormal lipid metabolism. It has the effect of preventing and treating vascular diseases caused by the same MMP overactivity. Therefore, the composition of the present invention can be used for the purpose of preventing and treating vascular diseases such as hyperlipidemia and arteriosclerosis.

Figure 1 shows the growth of the ground portion of the snake.

Figure 2 shows the extraction process of the organic solvent of the separation process of the 3,4,5-trihydroxybenzaldehyde (THBA) of the present invention from the serpentine.

Figure 3 shows the effect on the cell proliferation of 3,4,5-trihydroxy sibenzaldehyde of the present invention.

Figure 4 shows the effect of inhibiting the MMP-9 activity of 3,4,5-trihydroxybenzaldehyde of the present invention.

Figure 5 shows the inhibitory effect on the migration ability of human arterial smooth muscle cells of the 3,4,5-trihydroxybenzaldehyde of the present invention.

Claims (12)

A pharmaceutical composition for preventing and treating hyperlipidemia comprising 3,4,5-trihydroxygebenzaldehyde (3,4,5-trihydroxybenzaldehyde) or a pharmaceutically acceptable salt thereof as an active ingredient represented by the following formula (I). <Formula I>

The composition of claim 1, wherein the 3,4,5-trihydroxybenzaldehyde is isolated from Geum japonicum THUNBERG. A pharmaceutical composition for preventing and treating vascular diseases due to MMP overactivity, which comprises 3,4,5-trihydroxybenzaldehyde and a pharmaceutically acceptable salt thereof represented by the following formula (I): <Formula I>

The composition of claim 3, wherein the vascular disease due to MMP overactivity is selected from the group consisting of atherosclerosis, angina pectoris, myocardial infarction, hypertension, anemia, migraine, stroke and hemangioma. A food composition for preventing and improving hyperlipidemia, comprising 3,4,5-trihydroxybenzaldehyde and salts thereof represented by the following Formula (I): <Formula I>

Food composition for preventing and improving vascular diseases due to MMP overactivity, including 3,4,5-trihydroxybenzaldehyde and salts thereof represented by the following formula (I): <Formula I>

7. The composition of claim 6, wherein the vascular disease due to MMP overactivity is selected from the group consisting of atherosclerosis, angina pectoris, myocardial infarction, hypertension, anemia, migraine, stroke and hemangioma. The composition of claim 5 or 6, further comprising at least one additive selected from the group consisting of sugars and organic acids. The composition of claim 8, wherein the sugar is at least one selected from the group consisting of sucrose, mannitol, glucose, sorbitol, xylitol, inositol, lactose and fructose. The composition of claim 8, wherein the organic acid is at least one selected from the group consisting of citric acid, ascorbic acid and amino acids. delete delete

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