KR100707708B1 - Health care composition comprising compound SCE25­20 isolated from Saururus chinensis for the prevention of inflammatory, allergy or blood circulatory diseases - Google Patents

Abstract

The present invention relates to a composition for the prophylaxis or treatment of inflammation, allergic diseases and vascular diseases, specifically, inflammatory, allergic diseases and blood vessels containing the compound SCE25-20 isolated from the extract of Saururus chinensis as an active ingredient It relates to a composition for preventing or treating a disease. The compound of the present invention inhibits the activity of lipoxygenase (5-LO, 5-lipoxygenase), which causes various allergic and asthmatic diseases, and is dependent on cyclooxygenase-2 (COX-2, cyclooxygenase-2) inhibits production of (prostaglandin D ₂ , PGD ₂ ), inhibits degranulation in mast cells, inhibits nitric oxide radical production, and inhibits inducible nitric oxide synthase (iNOS) protein expression, By inhibiting MMP-9 activity related to sclerosis or vascular disease, it can be usefully used for the prevention or treatment of inflammation, allergic diseases and vascular diseases.

Triticale extract, anti-inflammatory, anti-allergic, anti-asthma, vascular disease

Description

Health care composition containing compound SC2502 isolated from Saururus chinensis for the prevention of inflammatory, allergy or blood circulatory diseases}

1 is a view showing the entire process of separating the compound SCE25-20 from the three hundred seconds extract of the present invention,

FIG. 2 is a diagram showing the effect of inhibiting cyclooxygenase-2 dependent prostaglandin D ₂ production in mouse bone marrow-derived mast cells (BMMC) against compound SCE25-20 isolated from three hundred sec extract ,

3 is a diagram showing the inhibitory activity test results of 5-lipoxygenase (5-lipoxygenase) involved in (leukotriene C ₄₎ Synthesis of leukotrienes C ₄ to cause bronchial asthma and allergic reactions,

Figure 4 is a diagram showing the experimental results of the free inhibitory response of beta-hexosaminidase (β-hexosaminidase, β-HEX) as an indicator of degranulation in mast cells causing various inflammatory diseases, bronchial asthma and allergic reactions ,

FIG. 5 is a diagram showing the inhibition of nitric oxide (NO) radical product causing various inflammatory diseases (A) and protein expression test results (B) of nitric oxide synthase (iNOS) producing the same.

6 is a diagram showing the results of experiments with Zymography of MMP-9 inhibitory activity involved in vascular smooth muscle migration.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammation, allergic diseases or vascular diseases containing SCE25-20 as an active ingredient, which is a compound isolated from the three hundred seconds extract having the effect of preventing inflammation, allergic diseases and vascular diseases. It is about.

Inflammation is the defense of biological tissue against local injuries. In other words, in response to various harmful stressors, the biodefense reaction to remove the injury caused by the stimulus and restore the original state is an inflammatory response. Inflammatory stimuli include infection or chemical or physical stimuli. Bio constituents involved in the inflammatory response include low-molecular or high-molecular chemicals such as free radicals, proteins, sugars, lipids, plasma, blood cells, blood vessels, and connective tissue. The process of inflammation is usually divided into two types, which can be divided into acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are associated with the removal of foreign bodies by posting a microcirculatory system. Chronic inflammation is long in duration, and tissue growth is seen.

Allergic diseases are most often caused by chemical mediators (primarily histamine, prostaglandins, leukotrienes, TNFα, cytokines, etc.) that are released from granules of mast cells in tissues activated by antigen-antibody responses. Since the use of allergy therapeutic drugs currently used remains symptomatic, there is an urgent need for the development of more fundamental therapeutic drugs.

Key mediators to induce inflammatory and allergic diseases include prostaglandindes, leukotriens, and platelet activators (PAFs), including phospholipase A2 and cyclooxygenase ( cyclooxygenase) and lipoxygenase to form the precursor arachidonic acid (arachidonic acid). In addition, enzymes related to nitric oxide synthase (NOS) biosynthesis, an enzyme that generates nitric oxide (NO), are also known to play an important role in mediating the inflammatory response. NOS, an enzyme that produces NO from arginine (L-Arginine), is also a major goal in blocking inflammation. According to recent research, there are several types of NOS, such as brain NOS (brain NOS) in the brain, nNOS (neuronal NOS) in the nervous system, and endothelial NOS (eNOS) in the vascular system. The small amount of NO produced by them plays an important role in maintaining normal homeostasis, such as inducing neurotransmission and vasodilation, whereas various inflammatory cytokines and lipopolysaccharides (lipopolyssacharide, LPS) NO, which is rapidly produced by iNOS (induced NOS) induced by external stimulants, is known to cause cytotoxicity and various inflammatory reactions. MJ et al., Mediators of inflammation, 4 , pp 387-396, 1995: Appleton L. et al., Adv. Pharmacol ., 35 , pp 27-28, 1996).

In order to treat the above inflammatory diseases, various types of inflammatory disease treatments have been developed. The inflammatory disease therapeutic substances reported to date are induced by damaged tissue cells, cells involved in inflammation, or chemotactic factors in acute inflammation. It is a drug that inhibits the production of eicosanoids from the cell membrane of the white blood cells. Allergy agents are also drugs that inhibit degranulation inhibition (histamine free) inhibition, histamine receptor blockade, leukotriene production inhibition and leukotriene receptor blockade, which are free during antigen-antibody reactions.

On the other hand, drugs that have recently attracted attention as a therapeutic agent for inflammation and allergic asthma are drugs that simultaneously inhibit degranulation, inhibit leukotriene C ₄ production, inhibit prostaglandin D ₂ production and inhibit nitric oxide (NO) radical production.

Proliferation and migration of vascular smooth muscle cells is a major factor in the development of stenosis after arteriosclerosis and arterial wall injury (Ross R., J. Med. 314 , pp 488-500, 1986). VSMC present 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 and 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) Replication and migration of VSMCs requires remodeling and degradation of extracellular matrix around cells (Matrisian LM, Trends. Genet . 6 , pp 121-125, 1990). Among the degrading enzymes, MMPs are important (Dollery CM et al., Circ.Res . 77 , pp 863-868, 1995; Pauly RR et al., Circ.Res . 75 , pp 41-54, 1994; Cho A. et al., Circ.Res. 91 , pp 845-851, 2002). Many studies have reported that MMP-9 is important for the proliferation and migration of arterial smooth muscle cells (Newby AC et al., J. Pathol. 190 , pp 300-309, 2000).

On the other hand, three hundred seconds is a perennial herb belonging to the tree three hundred and more, 0.5-1m in height. Underground stem long stretched sideways, leaves alternate, egg-shaped, basal-shaped. 2-3 leaves under the inflorescence are white. In late June to July, water inflorescences face down and fall down. Inflorescences stand up straight, with small white flowers growing slowly from the base to the end. A flower chin leaf is not on the flower rotary axis, and is attached to the end of a small peduncle of 2-3 mm in length. Flowers are bisexual, without cover. There are 6 surgeries facing the carpel, and the base is attached to the carpel. The pistil consists of four carpels, and the bases stick together. 2 seeds per 1 carpel, but 1 seed per carpel. The leaves, flowers, and roots are white, and two to three leaves on the upper part are called white because it is white. It lives in low wetland and is distributed in Korea, Japan, and Southeast Asia. There are two species of genus 300, of which S. cernuus is distributed in eastern North America.

It is also called horse riding or red riding horse. It improves blood circulation, releases blood, lowers fever, releases poison, stops cramps, and stops pain, resulting in overworked illness, sore muscles and bone nodes, soreness and bruises. , Arthralgia, pain after surgery, snake bite poison (Bae Gi Hwan, Korean medicinal plant, Kyohaksa, p 204, 2000).

The present inventors have found that at the same time, leukotriene C ₄ and inhibited against a strong inhibitory ability is particularly COX-2 in the production of prostaglandin D ₂ (PGD ₂₎ from the mast cell is a compound SCE25-20 separating the Houttuynia cordata extract from rat bone marrow (LTC ₄₎ It has been shown to possess the potent inhibitory effect on production and to have the free inhibitory ability of beta-hexosaminidase (β-HEX), an indicator of degranulation in mast cells. In addition, we strongly inhibited the expression of nitric oxide (NO) radicals and the expression of iNOS protein, a NO synthetase, in RAW264.6, a cell line derived from phagocytes, and MMP-9 inhibitory activity, which plays an important role in vascular smooth muscle migration. The present invention was completed.

It is an object of the present invention to provide a pharmaceutical composition and food composition for the prevention and treatment of inflammation, allergic diseases and vascular diseases containing the compound SCE25-20 isolated from the extract of three hundred seconds effective for inflammation, allergic diseases and vascular diseases .

In order to achieve the above object, the present invention is an inflammatory, allergic disease comprising a compound SCE25-20 represented by the following structural formula (1) isolated from three hundred seconds extract having the effect of preventing and treating inflammatory, allergic diseases and vascular diseases And it provides a pharmaceutical composition for the prevention and treatment of vascular diseases.

The inflammation-related diseases include arteriosclerosis, rheumatoid arthritis, asthma, acute pain, chronic pain, neuropathic pain, postoperative pain, pains such as migraine and joint pain, neuropathy, nerve damage, irritable bowel syndrome, shock caused by endotoxin , Inflammatory bowel disease and cancer, preferably atherosclerosis, rheumatoid arthritis.

The allergic diseases include bronchial asthma, allergic rhinitis, allergic asthma or allergic dermatitis, preferably allergic rhinitis, allergic dermatitis.

The vascular disease is coronary artery disease such as ischemic myocardial infarction, angina pectoris, myocardial infarction, hypertension, stroke, hyperlipidemia, anemia, migraine, arteriosclerosis, arrhythmia, stroke, hemangioma, hemangiofioma, vascular malformation, vascular adhesion, preferably ischemic Myocardial infarction, stroke.

Hereinafter, the process of separating the compound of the present invention will be described in more detail.

The trichophytium crude extract of the present invention, by cutting the dried root or ground portion (leaves, stems) of three hundred seconds, about 3 to 20 times the weight (kg), preferably about 5 to 10 times of water, C ₁ to Lower alcohol of C ₄ or a mixed solvent thereof, preferably methanol, for about 1 to 10 days, preferably about 3 to 8 days, at 20 to 50 ° C., preferably at 20 to 30 ° C. extraction temperature, The extract obtained by ultrasonic extraction, reflux cooling extraction, preferably cold needle extraction method is filtered and concentrated under reduced pressure to obtain a crude extract that is a polar solvent soluble extract.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the crude extract in distilled water, the suspension is about 1 to 100 times, preferably about 1 to 5 times the volume of non-polar solvents such as hexane, ethyl acetate, chlorform It can be obtained by extracting and separating 1 to 10 times, preferably 2 to 5 times, non-polar solvent soluble layer. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods : A guide to modern techniques of plant analysis. 3rd Ed. Pp 6-7, 1998).

The compound of the present invention is subjected to adsorption chromatography, such as silica gel column chromatography with a non-polar solvent soluble extract obtained in the above process, hexane / ethyl acetate mixed solvent or ethyl acetate: methanol mixture, 1: 0 to 10 After developing a solvent having a: 1 (w / w) ratio on the column and fractionating the same, reverse phase chromatography such as silica gel column chromatography or Rp-18 column chromatography was repeatedly performed to isolate Sauserneol F. can do.

Dichloromethane, chloroform, hexane, ethyl acetate, methanol, distilled water or a mixed solvent thereof may be used as the developing solvent.

In a preferred embodiment of the present invention, the crude methanol extract of three hundred seconds is suspended in distilled water, and then fractionated in order of n-hexane, ethyl acetate, n-butanol, and water to prepare each solvent fraction. Sauserneol F can be obtained using an ethyl acetate layer using a column.

The present invention contains a compound SCE25-20 isolated from the extract of the three hundred seconds obtained by the above method as an active ingredient, and provides a pharmaceutical composition effective for the prevention or treatment of inflammation, allergic diseases, vascular diseases.

The pharmaceutical composition containing the compound SCE25-20 isolated from the tritical extract of the present invention preferably contains 0.01 to 99.9%, and more preferably 0.1 to 90% of the compound 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.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising the compounds of the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, according to conventional methods. Carriers, excipients and diluents which may be used in the composition comprising the extract include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, 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 the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . 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.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The present invention provides a health functional food comprising a compound SCE25-20 isolated from three hundred seconds showing the effect of preventing inflammation, allergic diseases and vascular diseases, and food supplements additives.

The compounds of the present invention can be used in various ways, such as drugs, foods and beverages for the prevention and treatment of inflammation, allergic diseases and vascular diseases. Examples of the food to which the compound of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

The compound of the present invention may be added to food or beverage for the purpose of preventing and treating inflammation, allergic diseases or vascular diseases. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably It can be added at a ratio of 0.3 to 10 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are conventional 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. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of 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 compounds of the present invention, 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 salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages 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 from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

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

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

Example 1. Preparation of 300 seconds crude extract

9.7 kg of the dried three hundred sec roots purchased from Daegu Yangnyeong-si were chopped and immersed in 13 l of 70% methanol, and then extracted at room temperature. Then, each extract was further extracted twice by adding 10 L of methanol. , The filtrate was mixed. The methanol extract was depressurized, concentrated and dried to give 1 kg of total extract.

1.0 kg of dried 300 parts of the ground portion (stem, leaf) cultivated in the herbal medicine college of Yeungnam University College of Pharmacy extract was prepared in the same manner as the preparation method to obtain a total extract 100g.

Example 2. Preparation of Fractions of Three hundred Second Extract

2-1. Preparation of 300 seconds hexane fraction

950 g of the crude extract of the three hundred sec root of Example 1 was suspended in 1.4 L of distilled water, and then hexane-soluble fractions and water-soluble fractions were obtained, respectively, under reduced pressure, concentration and drying to obtain 136 g of hexane-soluble fractions and 850 g of water-soluble fractions. It was.

2-2. Preparation of ethyl acetate fraction of 300 seconds

Suspended in 1.4 L of distilled water in the three hundred seconds water-soluble fraction of Example 2-1, and extracted three times by adding 1.4 L of ethyl acetate, to obtain an ethyl acetate soluble fraction and a water-soluble fraction, respectively, under reduced pressure, concentration and drying This gave 182 g of ethyl acetate soluble fraction and 680 g of water soluble fraction.

2-3. Preparation of 300 seconds butanol fraction

Suspended in 1.4 L of distilled water in the three hundred seconds water-soluble fraction of Example 2-2, and then extracted three times by adding 1.4 L of butanol, to obtain a butanol-soluble fraction and a water-soluble fraction, each of them was decompressed and concentrated to obtain a butanol-soluble fraction. 42 g and 620 g of a water soluble fraction were obtained.

Example 3. Isolation of SCE25-20 Compounds

130 g of the 300 sec ethyl acetate soluble part of Example 2-2 was adsorbed onto 250 g of silica gel (No.7734, 70-230 mesh, Merck) and silica gel column chromatography (12 × 60 cm) was carried out at 1000 ml per hour. It was. At this time, as the developing solvent, n-hexane as the initial solvent, gradually increasing polarity (n-hexane: ethyl acetate (n-hexane 100% → ethyl acetate 100%) → ethyl acetate: methanol (ethyl acetate 100% → methanol 100%) Finally, the polar fractions were washed with methanol, and PGD ₂ , LTC ₄ , degranulation inhibition, NO production inhibition through NO iNOS protein expression inhibition, and MMP-9 inhibitory substance were eluted and divided into 39 fractions.

For each fraction, silica gel chromatography and Rp-18 (ODS) column chromatography were repeatedly performed to obtain the following compound. The separation process is shown in FIG.

Compound SCE25-20 was prepared by silica gel column chromatography (12 x 600 cm, developing solvent; n-hexane: ethyl acetate (n-hexane 100% → ethyl acetate 100%) → ethyl acetate fraction of ethyl acetate fraction of Example 2-2: Methanol (ethyl acetate 100% → methanol 100%) was carried out at 1000 ml / hr to obtain 39 fractions. The 25th fraction (1.3 g) was subjected to Rp-18 (ODS) column chromatography (4 × 50 cm, developing solvent; methanol: water (50:50) → methanol 100%) at 200 ml / h 24 Fractions were obtained. 0.23 g of the C compound SCE25-20 was obtained in the 20 th thereof, and the compound was analyzed by optical rotation and NMR analyzer.

60.6˚ (c 0.2, CHCl₃)

60.6˚ ( c 0.2, CHCl ₃ )

¹ H-NMR (CDCl ₃ , 250 MHz) δ 6.74-6.93 (9H, m, aromatic protons), 5.93 (2H, s, -OCH ₂ O-), 5.92 (2H, s, -OCH ₂ O-), 5.40 (2H, d, J = 6.0 Hz, H-7, H-7 ′), 4.61 (1H, d, J = 8.4 Hz, H-7 Hz), 4.09 (1H, m, H-8 Hz), 3.90 (3H, s, -OCH ₃ ), 2.20 to 2.30 (2H, m, H-8, 8 '), 1.14 (3H, d, J = 6.2 Hz, H-9 ˝), 0.69 (6H, d, J = 6.3 Hz, H-9, 9´)

¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ 150.9 (C- _{3 ′} ), 148.1 (C-3 ′), 147.9 (C-4 ′), 147.8 (C-3), 146.8 (C-4), 146.7 (C-4 '), 137.1 (C-1'), 135.8 (C-1), 134.4 (C-1 '), 121.5 (C-6'), 119.7 (C-6), 119.3 (C- 6 '), 119.1 (C-5'), 110.5 (C-5 '), 108.5 (C-2'), 108.2 (C-2 '), 108.0 (C-5), 107.3 (C-2), 101.4 (-OCH ₂ O-), 101.3 (-OCH ₂ O-), 84.5 (C-8 '), 84.1 (C-7), 83.9 (C-7'), 78.8 (C-7 '), 56.2 (-OCH ₃ ), 44.3 (C-8), 44.2 (C-8´), 17.4 (C-9˝), 15.1 (C-9, 9´)

Experimental Example 1. Prostaglandin D ₂  Investigate the impact on biosynthesis

The following experiment was carried out to examine the effect on the production of prostaglandin D ₂ (PGD ₂ ) of Compound SCE25-20 purified in three hundred seconds prepared in the above Example.

Mouse bone marrow-derived mast cells (BMMC) were derived from BALB / C mice by Murakami et al. [Murakami et al .; J. Biol. Chem. 269 , pp 22269-22275 (1994)], medium under 50% WEHI-3 conditions containing culture supernatant of WEHI-3 cells (provided by Professor Kudo Ichiro, Pharmacy, Showa University, Japan), IL-3 producing cells (conditioned medium, containing 10% FCS).

After 3 weeks of culture, mast cells were treated with SCF / LPS / IL-10 mixed stimulant (Sigma) for 30 minutes. PGD ₂ quantification of the supernatant after cell stimulation was measured by Enzyme linked immunoassay (EIA) using the PGD ₂ assay kit (Cayman). At this time, in order to suppress the production of PGD ₂ produced by COX-1, BMMC was treated with aspirin (aspirin, 10 ug / ml) 1 hour beforehand in BMMC. Anthricin was pre-treated for 15 minutes in advance, and the amount of PGD ₂ produced by adding a stimulant was measured. The production of PGD ₂ by cyclooxygenase-1 (COX-1) was measured after 1 hour after treatment with SCF / LPS / IL-10 mixed stimulant (Sigma).

As a result, the inhibition rate of the PGD ₂ production of each compound of SCE25-20 is shown in FIG. 2, and the compound SCE25-20 inhibited the production of PGD ₂ in a dose-dependent manner, and the concentration required for 50% inhibition was COX-2. 7.1 μΜ.

Experimental Example 2. Analysis of the effect on the production of leukotriene

In order to examine the effect on the production of leukotriene C ₄ (LTC ₄ ) of the compound SCE25-20 prepared in the above example was carried out.

Mast cells derived from murine bone marrow were harvested from BALB / C mice by Murakami et al. [Murakami et al .; J. Biol. Chem . 269 , pp 22269-22275, 1994], isolated from the bone marrow, medium under 50% WEHI-3 condition containing culture supernatant of WEHI-3 cells (provided by Kudo Ichiro, Faculty of Pharmacy, Showa University, Japan). conditioned medium, containing 10% FCS).

After 3 weeks of culture, mast cells were treated with SCF (Sigma) for 30 minutes. LTC ₄ quantification of the supernatant after cell stimulation was measured by EIA using the LTC ₄ assay kit (Cayman). Compound SCE25-20 was pretreated for 15 minutes in advance, and then the amount of LTC ₄ produced was measured by adding a stimulant.

As a result, Figure 3 shows the inhibition of LTC ₄ production of each of the SCE25-20 compounds, compound SCE25-20 inhibited the production of LTC ₄ dose-dependently, the concentration required to inhibit 50% was 1.34 μM.

Experimental Example 3. Effect of compound SCE25-20 on mast cell degranulation (β-Hexosaminidase, β-HEX)

The mouse bone marrow-derived mast cells (BMMC) were treated with compound SCE25-20 by concentration (0.1-10 μM) in 2Ｘ105 cells / ml, and then preincubated for 30 minutes at 37 ° C. and 5% CO _2. After stimulation with SCF (KL, c-kit ligand, 100 ng / ml) was incubated for 15 minutes and centrifuged for 5 minutes at 3000rpm, 4 ℃. The supernatant was mixed 1: 2 with β-hex (substrate [100 mM citrate buffer (citric acid 0.955%, sodium citrate dihydrate 1.478%, pH 4.5), 1.3 mg / ml p-nitrophenyl-N-acetyl-bD-glucosaminide) and 37 After the reaction was carried out at 1 ° C. for 1 hour, the reaction was stopped with 0.2 M glycine (pH 10.7) to measure absorbance at 405 nm using ELASA, and the measured value was converted into% release granules.

Experimental results, it is shown in Figure 4 for the β-HEX (β-Hexosaminidase) glass inhibitory effect.

Experimental Example 4 Removal Effect of NO Radicals Released from LPS-Induced Phagocytes

In order to confirm the radical scavenging effect of the compound of the present invention SCE25-20, the following experiment was performed.

Compound SCE25-20 dissolved in the included RPMI 1640 medium (pH 7.6) was pretreated for 30 minutes with a concentration of 1.6-50 μM in advance, followed by incubation for 24 hours with LPS (200 ng / ml). After incubation, the concentration of NO was measured using the Gries method.

The amount of nitric oxide was confirmed by analyzing nitrite in the cultured supernatant. Raw 264.7 cells were dispensed into 5 × 105 cells in a 24-well plate and stabilized for 24 hours. The culture medium was replaced, pretreated with SCE25-20 for 30 minutes by concentration, and activated by treatment with LPS (200 ng / ml). After incubation at 37 ℃ 5% CO ₂ for 24 hours, the supernatant was taken by centrifugation for 5 minutes at 120 ℃, 4 ℃. The culture supernatant was mixed 1: 1 with 100 ml of grease reagent (Griess reagent, 1% sulfanilamide, 0.1% naphylethylenedimine dihydrochloride, 2.5% phosphoric acid) for 10 minutes at room temperature. The absorbance developed after the reaction was measured at 570 nm wavelength using an ELISA reader. Fresh culture medium was used as the blank in all experiments and the concentration of nitrite was converted from the standard curve obtained from the sodium nitrate (NaNO ₂ ) reaction.

As shown in FIG. 5, compound SCE25-20 significantly inhibited NO radicals in a dose-dependent manner, and the concentration required for 50% inhibition was 1.66 μM. In addition, compound SCE25-20 dose-dependently inhibits protein expression of iNOS involved in NO radical generation in a dose-dependent manner, indicating that inhibition of NO radical is due to iNOS protein expression.

Experimental Example 5. MMP-9 Inhibitory Activity Experiment

MMP-9 activity was performed by the methods described in the literature (Demeule, et al., Biochimica et Biophysica Acta 1478 , pp 51-60, 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). Compound SCE25-20 was run by concentration (0, 3.2, 6.4, 12.5, 25, 50 μΜ).

As a result, compound SCE25-20 inhibited the activity of MMP-9 in a dose dependent manner as shown in FIG. 6.

Formulation Example 1 Preparation of Injection

Compound SCE25-20 ...................................... 10 mg

Mannitol ............... 180 mg

Sterile Distilled Water for Injection ............... 2974 mg

Na ₂ HPO _{4 12} H ₂ O ... 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 2 Preparation of Tablet

Compound SCE25-20 of Example 2-1 ... 10 mg

Lactose ........................... 100 mg

Starch ........................... 100 mg

Magnesium Stearate ...............

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 3 Preparation of Capsule

Compound SCE25-20 ......................................... 10 mg

Crystalline Cellulose ............... 3 mg

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 Liquid

Compound SCE25-20 ............... 20 mg

Isomerized sugar ......................................... 10 g

Mannitol ......................................... 5 g

Purified water ...............

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation Example 5 Preparation of Healthy Drink

Compound SCE25-20 ......................................... 100 mg

Vitamin C ............... 15 g

Vitamin E (powder) ........................ 100 g

Iron Lactate ............... 19.75 g

Zinc Oxide ........................... 3.5 g

Nicotinic acid amide ......................... 3.5 g

Vitamin A ......................... 0.2 g

Vitamin B1 ...................... 0.25 g

Vitamin B2 ........................ 0.3g

Water .........................................

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the manufacture of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

As mentioned above, SCE25-20, a novel compound of the present invention, is isolated from 300 seconds and inhibits the production of prostaglandins D ₂ and leukotriene C ₄ involved in inflammation and allergic reactions, inhibits degranulation, and nitric oxide (NO By inhibiting radical products, it has anti-inflammatory and anti-allergic effects, and it can also be used as pharmaceutical composition and health functional food for preventing and treating vascular disease by inhibiting MMP-9.

Claims (2)

A health functional food for the prevention of arteriosclerosis, rheumatoid arthritis, allergic rhinitis, allergic dermatitis, ischemic myocardial infarction or stroke, comprising the compound SCE25-20 of the formula (1) and a food supplement acceptable food supplement.

The dietary supplement of claim 1 which is a tablet, capsule, pill or liquid. .

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