KR101831438B1 - Composition comprising uldavioside A compound for preventing or treating of inflammatory disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an inflammatory disease containing an uldavioside A compound isolated from Ulmus davidiana Planch extract. The above-mentioned urodiboside A compound can be easily used as a therapeutic agent for inflammatory diseases or a health functional food by inhibiting the expression of inflammatory cytokines.

Description

[0001] The present invention relates to a composition for preventing or treating an inflammatory disease containing an urodiboside A compound,

The present invention relates to a composition for preventing or treating an inflammatory disease containing an uldavioside A compound isolated from Ulmus davidiana Planch extract.

Inflammation is a cause of various diseases such as rhinitis, bronchitis, hepatitis and arthritis, and is closely related to human health. In particular, in the case of a degenerative disease that is aggravated by the aging of the population due to the extension of the life expectancy, the researchers report that the pathophysiology is closely related to the inflammation and immune response. And there is an increasing interest in the development of therapeutic agents for the treatment of inflammation.

Inflammatory responses are the most important pathological mechanism of host defense mechanisms against wounds, microbial infections, but sustained and excessive inflammatory responses impair tissue. The macrophage is the most representative immune cell that regulates this inflammatory response. The activated macrophages are TNF-α, IL-6 (interleukin-6), PGE ₂ (prostaglandin E ₂ ) nitric oxide (ROS), reactive oxygen species (ROS), and the like (Laskin, DL et al., 2011). On the other hand, overexpression of such inflammation-related factors leads to rheumatoid arthritis, osteoporosis, sepsis, vascular disease, cancer and the like (Lawrence, T. et al., 2002).

Ulmus davidiana Planch is a dried cork layer of bark and flesh of Ulmus davidiana var. Japonica Nakai belonging to the Ulmaceae (Ji Hyung Jun, et al., 1988). In the oriental medicine, it has been reported that it has an excellent effect on germane, anticancer, wound healing medicine and inflammation (Duke JA, 1985) because it is tasteful, nontoxic, swelling, arthritis, gastric ulcer and gastrointestinal disease. There are natural products such as β-sitosterol, phytosterol, stimasterol, tannin, starch, and polysaccharide. (Matsuzaki T., et al., 1985) and the chemical composition (Duke JA et al., 1985) and the separation of friedelin, epifriendelalol and taraxerol, , 1985). However, attempts to study diverse physiological activities of the carrageenan have not been conducted yet.

Accordingly, the present inventors have completed the present invention by confirming that the cryoprecipitate A compound, which is isolated from the extract of the corynebacterium rhizoma extract, has anti-inflammatory activity in the course of studying an anti-inflammatory composition containing the extract of Cryptomeria japonica.

Korean Patent Laid-Open Publication No. 2000-0041190 discloses prevention and treatment of diseases such as wounds of pharmaceutical compositions containing milky blood, Korean Patent Publication No. 2006-0078305 discloses herbal medicine extracts containing yugun , But the composition of the present invention is different from that of the present invention because there is no description of the compound of the present invention.

In addition, as a non-patent document, Son B. W., et al. Describes a cryobioxide A compound isolated from elm, but has no anti-inflammatory activity.

Korean Patent Laid-Open No. 2000-0041190, Pharmaceutical composition, 2000. 07. 15. Disclosure. Korean Patent Laid-Open No. 2006-0078305, cosmetic composition having skin elasticity enhancement and anti-inflammatory effect containing extracts of yugaengpei, lily, bananas, white wax and white algae, 2006. 07. 05. disclosed.

Jae Hyun Lee, et al., Korean Journal of Medicinal Herbs, Korean Medical Index, 295, 1988. Duke J. A., Handbook of medicinal herbs, CRC press, 495, 1985. Laskin, D. L. et al., Macrophages and tissue injury: agents of defense or destruction ?, Annu. Rev. Pharmacol. Toxicol., 51, 267-288, 2011. Lawrence, T. et al., Anti-inflammatory lipid mediators and insights into the resolution of inflammation, Nat. Rev. Immunol., 2, 787-795, 2002. Matsuzaki T., et al., Antioxidative of tea leaf catechins, Nippon Nogeikaga Kogyo Kaishi, 59, 129-134, 1985. Son B. W., et al., Catechin glycoside from Ulmus davidiana, Arch. Pharm. Res., 12 (3), 219-222, 1989.

It is an object of the present invention to provide a composition for the prevention or treatment of inflammatory diseases containing uldavioside A compound isolated from Ulmus davidiana Planch extract.

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases, which comprises, as an active ingredient, a cryovioside A compound represented by the following formula (1).

[Chemical Formula 1]

Said inflammatory disease is selected from the group consisting of allergic diseases, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, Sepsis, bed sores, degenerative chronic inflammatory diseases and nephritis.

The cryobiocid A compound may be added in an amount of 0.001 wt% to 50 wt% based on the total weight of the composition.

The present invention also relates to a health functional food for the improvement of inflammatory diseases, which comprises, as an active ingredient, a cryoprecipitate A compound represented by the following formula (1).

The health functional food may be selected from the group consisting of a drink, a meat, a sausage, a bread, a candy, a snack, a noodle, an ice cream, a dairy product, a soup, an ionic drink, a beverage, an alcoholic beverage, a gum, a tea and a vitamin complex.

The cryobiocid A compound may be added in an amount of 0.001% by weight to 50% by weight based on the total weight of the health functional food.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases containing the compound of the present invention as an active ingredient.

The above-mentioned urodibioside A compound can be synthesized according to a conventional method, and can be prepared as a pharmaceutically acceptable salt, or can be isolated and purified from an extract of Leucocyte.

The above-mentioned yukigi is Ulmus davidiana var. Japonica Nakai belonging to Ulmaceae, dried cork layer and bark.

The extract can be extracted with water, C1 to C4 lower alcohols or a mixed solution thereof as a solvent. The C1 to C4 alcohols may be selected from the group consisting of methanol, propanol, isopropanol, butanol, and isobutanol. Preferably, it is extracted from water.

The compound isolated from the carrot extract can be purified by column chromatography. The chromatography was carried out by silica gel column chromatography, HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography LH-20 column chromatography, and high-performance liquid chromatography.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the compound of formula 1 and an excipient. The compound may be added in an amount of preferably 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 40% by weight, and most preferably 0.001% by weight to 30% by weight, based on the total weight of the composition.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may be formulated with the compound of the present invention in combination with at least one excipient such as starch, calcium carbonate, sucrose, , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween-61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, body weight and the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the compound of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.

In addition, the present invention provides a health functional food for improving inflammatory diseases, which comprises a compound of the formula (I) of the present invention and a pharmaceutically acceptable food-aid additive.

The compound may be added in an amount of preferably 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 30% by weight, and most preferably 0.001% by weight to 10% by weight based on the total weight of the whole food.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods.

The present invention relates to a composition for preventing or treating an inflammatory disease containing an uldavioside A compound isolated from Ulmus davidiana Planch extract. The above-mentioned urodibioside A compound inhibits the expression of an inflammatory cytokine, thereby inhibiting the expression of an inflammatory cytokine, thereby inhibiting the expression of an inflammatory cytokine, thereby inhibiting the expression of an inflammatory cytokine, thereby preventing allergic diseases, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, , Inflammatory diseases such as tonsillitis, throat, bronchitis, pneumonia, pancreatitis, sepsis, bedsores, degenerative chronic inflammatory diseases and nephritis, and health functional foods.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the correlation between ¹ H- ¹ H COZY (bold lines) and ¹ H- ¹³ C HMBC for an uldavioside A compound of the present invention (arrow).
2 is a graph showing changes in the expression of TNF-α (A), IL-1β (B) and NO (C) as inflammation-related factors in Raw264.7, C2C12 and NIH3T3 cells following the treatment with the urodiboside A compound of the present invention Respectively.
Figure 3 shows the cytotoxicity results of Raw264.7 (A), C2C12 (B) and NIH3T3 (C) cells of the cryovioside A compound of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Isolation of cryptobioside A compound >

Yugun pei was purchased from a farm and dried. After adding 1 L of purified water to 10 g dried rhizome, the mixture was treated with a semi-continuous low-temperature vacuum extractor at 25 ° C for 12 hours. The Rhizopus japonicus extract was extracted three times, and impurities were removed using a filter paper to obtain hot water extract . The diaion HP-20 column chromatography (Diaion HP-20 column chromatography) was performed on the isocratic elution conditions with 30% methanol, 50% methanol, 70% methanol and 100% Fractions were obtained (Fr. E-1 to E-3). Of these, Fr. E-1 was subjected to sephadex LH-20 column chromatography according to the elution conditions of 70% methanol and the like to separate the urodibioside A compound (9 mg).

Example 2: Identification of the physico-chemical structure of the urodavioside A compound [

Uldaviosidea;

Light brown powder;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 1 below;

ESI-MS [M + H] ⁺ m / z 423.1, C ₂₀ H ₂₂ O ₁₀ .

Position Cuda Bioside A [delta] _C (ppm) ^a [delta] _H (ppm) ^b 2 82.9 4.59 (1H, d, J = 6.8 Hz) 3 68.6 3.98 (1 H, m) 4 28.4 2.84 (1H, dd, J = 16.5, 5.5 Hz)
2.53 (1H, dd, J = 16.5,8.3 Hz)  4a 103.2 5 158.2 6 97.2 6.12 (1H, d, J = 2.0 Hz) 7 157.6 8 96.8 6.06 (1H, d, J = 2.0 Hz)  8a 156.9 9 132.1 10 115.2 6.82 (1H, d, J = 2.0 Hz) 11 146.2 12 146.3 13 116.1 6.75 (1H, dd, J = 8.2 Hz) 14 119.9 6.70 (1H, dd, J = 8.2, 2.0 Hz)   One' 108.7 5.47 (1H, d, J = 3.4 Hz)   2' 78.2 4.13 (1H, d, J = 2.7 Hz)   3 ' 80.3   4' 75.4 4.07 (1H, d, J = 9.6 Hz)
3.84 (1H, d, J = 9.6 Hz)   5 ' 64.9 3.60 (2H, m) ^{a 13 C NMR (150 MHz in} CD 3 OD) spectroscopy data
^{b 1} H NMR (600 MHz in CD ₃ OD) spectroscopy data

< Example  3. Culture of animal cells>

In order to confirm the activity of the compound of the present invention, the macrophage cell line Raw264.7, the myocardial cell C2C12, and the fibroblast cell line NIH3T3 were inoculated into 10% FBS (fetal bovine serum), 100 U / ml penicillin, / Ml streptomycin in DMEM (Dulbecco's Modified Medium) medium and cultured at 37 ° C in a 5% CO ₂ incubator.

Example 4. Confirmation of Expression of Inflammation-Related Factor of Cryoprecipitate A Compound [

Expression of NO (nitric oxide), TNF-alpha (tumor necrosis factor-alpha), and IL-1 beta (interleukin-1 beta), which are inflammatory factors, in order to confirm the anti- Were measured using an enzyme-linked immunosorbent assay (ELISA) kit.

The Raw264.7, C2C12, and NIH3T3 cells cultured in the above Example 3 were seeded in a 48 well plate at 1 x 10 &lt; ⁵ &gt; cells Cells were cultured for 24 hours. Then, the medium was completely removed, DMEM medium (containing 100 U / ml of penicillin and 100 μg / ml of streptomycin) containing 0.5% FBS was added And cultured for 18 hours. After culturing for 18 hours, the cells were treated with 1 μM and 10 μM of the urodibioside A compound, followed by pre-reaction for 1 hour, followed by treatment with 2 μg / ml of LPS (lipopolysaccharide) and culturing for 24 hours. At this time, Raw264.7, C2C12, and NIH3T3 cells, which were not treated with anything, were used as a control group.

The expression levels of NO, TNF-α and IL-1β secreted in cell culture media were measured using ELISA kits (ENZO, USA). At this time, the experimental method was carried out based on the experimental method provided in the kit, and the results are shown in FIG.

As shown in FIG. 2, in the cells of Raw264.7, C2C12 and NIH3T3, inflammatory factors such as TNF-α (2A), IL-1β (2B) and NO (2C) Indicating that the expression is decreased in dependence on the treatment concentration of the cryobiocide A compound.

As a result, it was found that the compound of the present invention had anti-inflammatory activity.

Example 5: Cytotoxicity of cryovioside A compound < RTI ID = 0.0 >

The cytotoxicity of the urobiosid A compound of the present invention was confirmed by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay.

The Raw264.7, C2C12, and NIH3T3 cells cultured in the above Example 3 were seeded in a 48 well plate at 1 x 10 &lt; ⁵ &gt; cells Cells were cultured for 24 hours, and then cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 0.5% FBS (fetal bovine serum), 100 U / ml penicillin and 100 μg / ml streptomycin ) Medium and incubated for 18 hours. After 18 hours of incubation, the cryovioside A compound was treated with 1 [mu] M and 10 [mu] M and cultured for 24 hours. At this time, Raw264.7, C2C12, and NIH3T3 cells, which were not treated with anything, were used as a control group. After 24 hours, the cells were treated with MTT solution at a concentration of 100 μg / ml and reacted at 37 ° C. for 3 hours. The culture solution was removed, and the formazan precipitate formed was dissolved in 200 μl of DMSO (dimethyl sulfoxide) And the results are shown in Fig.

As shown in Fig. 3, survival rates of Raw264.7 (3A), C2C12 (3B) and NIH3T3 (3C) cells treated with the cucumber biologic A compound were increased compared to the control. As a result, it was found that the cryovioside A compound of the present invention did not show cytotoxicity.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of the compound of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. A 10% gelatin solution was added to the mixture, followed by pulverization and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of activity and 5 g of magnesium stearate was made into tablets.

Formulation example  1-2. Injection preparation

1 g of the compound of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The cryobioside A compound of the present invention was added to the cooking seasoning at 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

A food for health promotion was prepared by adding 0.1% by weight of the compound of the present invention to wheat flour, and preparing bread, cake, cookie, cracker and noodle by using this mixture.

Preparation Example 2-3. Manufacture of soups and gravies

The health-enhancing soup and juice were prepared by adding 0.1% by weight of the compound of the present invention to the soup and the juice.

Formulation Example 2-4. Manufacture of dairy products

The dulabioside A compound of the present invention was added to milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Manufacture of vegetable juices

0.5 g of the compound of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation Example 2-6. Manufacture of fruit juices

0.1 g of the compound of the present invention was added to 1,000 ml of apple juice or grape juice to prepare vegetable juice for health promotion.

Claims (7)

Inflammatory bowel disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, atherosclerosis, inflammatory bowel disease, glomerulonephritis, inflammatory bowel disease, , Inflammatory diseases selected from the group consisting of tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, degenerative chronic inflammatory diseases and nephritis.
[Chemical Formula 1]

delete The method according to claim 1,
Wherein the compound is added in an amount of 0.001% by weight to 50% by weight based on the total weight of the composition. Inflammatory bowel disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, atherosclerosis, inflammatory bowel disease, glomerulonephritis, inflammatory bowel disease, , Inflammatory diseases selected from the group consisting of tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, degenerative chronic inflammatory diseases and nephritis.
[Chemical Formula 1]

delete 5. The method of claim 4,
The health functional food is selected from the group consisting of a drink, a meat, a sausage, a bread, a candy, a snack, a noodle, an ice cream, a dairy product, a soup, an ionic drink, a drink, an alcoholic beverage, a gum, Health functional food for improvement of disease. 5. The method of claim 4,
Wherein said compound is added in an amount of 0.001% by weight to 50% by weight based on the total weight of the health functional food.

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