KR20070044602A - Composition for inhibiting inflammatory vascular injury and atherogenesis comprising dry-roasted glycyrrhiza extract - Google Patents

Abstract

The present invention relates to an anti-inflammatory or anti-thrombotic composition containing a roasted licorice extract as an active ingredient, and more particularly to inhibiting inflammatory vessel damage and vascular sclerosis containing a roasted licorice methylene chloride fraction extract or lyocacalcon A as an active ingredient. It relates to a baked licorice composition of improved blood circulation. Baked licorice extract or purified ricokalcon A used as an active ingredient in the present invention can prevent the adhesion of monocytes to vascular endothelial cells early to block the development of inflammatory vascular sclerosis, thereby inhibiting inflammatory vascular damage or blood circulation Has an improvement effect.

Baked licorice, Baked licorice methylene chloride fraction extract, Ricokalcon A, Anti-inflammatory, Vasclerosis, Anti-thrombosis.

Description

COMPOSITION FOR INHIBITING INFLAMMATORY VASCULAR INJURY AND ATHEROGENESIS COMPRISING DRY-ROASTED GLYCYRRHIZA EXTRACT}

1 is a chemical formula of ricochalcone A identified in Examples 1-3.

2 is a calibration curve of lyocacalcon A obtained in Example 1-4.

Figure 3 shows the inhibitory effect of ethylene extract and methylene chloride fraction extract of baked licorice on THP-1 monocyte adhesion to vascular endothelial cells of Experimental Example 1.

Figure 4 shows the inhibitory effect of purified ricokalcon A in baked licorice extract on THP-1 monocyte adhesion to vascular endothelial cells of Experimental Example 1.

FIG. 5A shows adhesion molecules-1 in vascular endothelial cells treated with Tumor Necrosis Factor (hereinafter referred to as 'TNF')-α in Experimental Example 2 (vascular cell adhesion molecure-1, hereinafter referred to as 'VCAM-1') Figure 5B is a photograph showing the inhibitory effect of the roasted licorice methylene chloride fraction extract on protein expression, Figure 5B shows the effect of inhibiting the expression of VCAM-1 according to the concentration of purified ricokalcon A in the methylene chloride fraction extract in Experimental Example 2 It is shown.

[Industrial use]

The present invention relates to a composition for anti-inflammatory or hematopoietic improvement comprising baked licorice extract as an active ingredient, and more particularly, to inflammatory vascular damage, vascular sclerosis, blood circulation, containing baked licorice extract or ricokalcon A as an active ingredient. Baked licorice composition for improving or preventing or treating thrombosis.

[Private Technology]

Inflammatory chain reaction is caused by the reaction of inflammatory cytokines in the artery wall. Secretion of cytokines, such as tumor necrosis factor-α, affects the hardening of arterial walls (Desfaits AC, Serri O, Renier G. Normalization of lipid peroxides, monocytes adhesion, and tumor necrosis factor-alpha production in NIDDM patients after gliclazide treatment Diabetes Care 21: 487-493, 1998; Fazio S, Linton MF.The inflamed plaque: Cytokine production and cholesterol balance in the vessel wall.Am J Cardiol 88: 12E-15E, 2001).

The expression of cell adhesion molecules is a common phenomenon in the outbreak of inflammation, which occurs in the early stages of vascular hardening (Schonbeck U, Mach F, Libby P. CD154 (CD40 ligand). Int J Biochem Cell Biol 32: 687-693 , 2000). Adhesion molecule proteins are observed in lesions of atherosclerosis, and appear at the site where human atherosclerotic plaques are formed (Lessner SM, Prado HL, Waller EK, Galis ZS.Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model. Am J Pathol 160: 2145-2155, 2002).

Flavonoids are known to act as anti-inflammatory substances that inhibit the expression of cell adhesion molecules, including VCAM-1 (Bito T, Roy S, Sen CK, Shirakawa T, Gotoh A, Ueda M, Ichihashi M, Packer L. Flavonoids differentially regulated IFN gamma-induced ICAM-1 expression in human keratinocytes: molecular mechanisms of action.FEBS Lett 520: 145-152, 2002; Choi JS, Choi YJ, Park SH, Kang JS, Kang YH. Flavones mitigate tumor necrosis factor a-induced adhesion molecule upregulation in cultured human endothelial cells: Role of nuclear factor-kB. J Nutr 134: 1013-1019, 2004).

Flavonoids are pigments present in plants such as vegetables and fruits such as green tea, legumes, grapes, garlic and onions. Flavonoids exist in various forms and are classified into subgroups of flavonols, flavones, isoflavones, flavonones, flavan-3-ols, and anthocyanidins. However, not all flavonoids have the same bioactivity. Flavonoids act as a natural antioxidant that removes free radicals from cells and tissues, and have been reported as anti-inflammatory substances that inhibit cell adhesion molecules and matrix protease (Bito T, Roy S, Sen). CK, Shirakawa T, Gotoh A, Ueda M, Ichihashi M, Packer L. Flavonoids differentially regulate IFN gamma-induced ICAM-1 expression in human keratinocytes: molecular mechanisms of action.FEBS Lett 520: 145-152, 2002; Sartor L, Pezzato E, Dell Aica I, Canito R, Biggin S, Garbisa S. Inhibition of matrix-proteases by polyphenols: chemical insights for anti-inflammatory and anti-invasion drug design. Biochem Pharmacol 64: 229-237, 2002). In addition, the flavonoids are proanthocyanidins, and phenolic compounds such as polyphenols, acid has been suggested that utilization for the prevention or treatment of cardiovascular disease are (Cotelle N. Role of flavonoids in oxidative stress Curr Top Med Chem 1.: 569-590, 2001).

Licorice is a perennial herb of rosemary legumes and used as a sweetener for sweet roots, and has been used traditionally in Chinese medicine as a medicine for lung and inflammatory diseases in Tibet and India (Demizu S, Kajiyama K). , Takahashi K, Hiraga Y, Yamamoto S, Tamura Y, Okada K, Kinoshita T. Antioxidant and antimicrobial constituents of licorice:. isolation and structure elucidation of a new benzofuran derivative Chem Pharm Bull 36: 3474-3479, 1988). Licorice extract has also been added to enhance this effect or to make the medicinal herbs less bitter (Vaya J, Belinky P, Aviram M. Antioxidant constituents from licorice root: isolation, structure elucidation and antioxidative capacity toward LDL oxidation.Free Radic Biol Med 23: 302-313, 1997). However, no clear mechanism of licorice's inflammatory effects has been identified.

Ricokalcon A, one of the flavonoids found in licorice, has recently been identified as a compound having various functionalities, including antibacterial and anticancer activity. Recently, it has been found by the present inventors that nonpolar compounds including licochalcone A are increased in the process of baking licorice under certain conditions. Korean Patent).

Disclosure of Invention An object of the present invention is to provide a baked licorice extract containing a high activity of licochalcone and a baked licorice extract that inhibits adhesion of monocytes to vascular endothelial cells activated with inflammatory cytokines to maximize anti-inflammatory effects.

In order to achieve the above object, the present invention provides a composition for treating inflammation containing the roasted licorice extract as an active ingredient. The present invention also provides an anti-vascular damage, anti-vascular sclerosis or anti-thrombosis composition comprising purified ricokalcon-A as an active ingredient in methylene chloride fraction extract of baked licorice.

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

Plants produce several types of antioxidants that prevent the damage of molecules from reactive reactive oxygen species. Phenolic compounds are the major compounds in antioxidants produced by plants.

The present inventors inhibited the expression of cell adhesion molecules of VCAM-1 induced by tumor necrosis factor-a and prevented the aggregation of cell adhesion molecules during the screening of natural plant materials having anti-inflammatory activity. Confirmation of inhibition was completed the present invention. In addition, lycokalcon A isolated from the roasted licorice extract and the roasted licorice extract of the present invention inhibited the cell adhesion molecule protein expression induced by the tumor necrosis factor-α which is an inflammatory tumor necrosis factor.

In Experimental Example 1 of the present invention, the tumor necrosis factor tumor necrosis factor-α-treated vascular endothelial cells significantly increased THP-1 monocyte adhesion compared to the untreated group, unlike the ethanol extract, methylene chloride fraction of baked licorice When the extract was pretreated, it was confirmed that the adhesion of monocytes to vascular endothelial tissue was suppressed. This monocyte adhesion inhibition activity of the methylene chloride fraction extract of baked licorice was achieved by inhibiting the expression of the cell adhesion molecule protein VCAM-1 induced by tumor necrosis factor-α.

In addition, the expression of VCAM-1 protein was inhibited and the adhesion of THP-1 monocytes to vascular endothelial cells was remarkably suppressed when treated with purified ricokalcon A in methylene chloride fraction extract of baked licorice.

Baked licorice methylene chloride fraction extract in the present invention inhibits the expression of cell adhesion molecule proteins, such as VCAM-1, and shows an effect on blood circulation improvement due to inhibition of aggregation of blood cells, so as to prevent inflammatory cardiovascular diseases such as vascular sclerosis and thrombosis. It can be used for prevention and treatment. Preferably, the fraction extract of the non-polar solvent in the baked licorice extract and the purified ricokalcon A can be said to have the ability to prevent the initial process of blood cell aggregation and vascular curing associated with inducible VCAM-1 expression. Accordingly, the present invention provides a composition for treating inflammation of baked licorice extract comprising methylene chloride fraction extract of roasted licorice or ricokalcon A as an active ingredient.

Licorice extract may be prepared according to the conventional method for producing a plant extract. Conventional extraction equipment, ultrasonic grinding extractors or fractionators can be used. That is, some or all of licorice may be baked, and then extracted or fractionated with water or an organic solvent to prepare an extract. For example, the leaves, branches, roots, fruits, stems, flowers, shells, etc. of licorice are baked, and a solvent is added to the pulverized product thereof to obtain an extract. The solvent may be water, alcohol having 1 to 5 carbon atoms or alcohol dilution water. The alcohol dilution water may be a dilution of alcohol in water at 50 to 99% by volume.

Baked licorice extract of the present invention is first roasted licorice root, and then extracted with the solvent. The extract thus obtained may then be fractionated with one or more solvents selected from the group consisting of methylene chloride, hexane, acetone, ethyl acetate, chloroform and mixtures thereof, in which case it is preferable to carry out with methylene chloride. Two or more solvents may be used in the fractionation, and each solvent extract may be prepared by using the solvent sequentially according to the polarity of the solvent. According to one embodiment of the present invention, the extracted licorice is preferably extracted with ethanol, followed by extraction with methylene chloride. The methylene chloride fraction extract of the baked licorice may then be filtered under reduced pressure and / or lyophilized to remove the solvent. It is also possible to purify ricokalcon A from the methylene chloride fraction of the baked licorice.

Baked licorice methylene chloride fraction extract of the present invention comprises lycochalcone A that inhibits inflammatory monocyte activity and expression of cell adhesion molecule proteins. Lycocalcon A may be included as an active ingredient in a fraction extract using a methylene chloride nonpolar solvent when extracted as a nonpolar material.

The composition for inflammatory vascular damage, vascular hardening or blood circulation improvement of the present invention as described above may include a licorice methylene chloride fraction extract or an active ingredient of ricokalcon A alone, and includes a pharmacologically acceptable carrier. can do. The carrier includes saline, buffered saline, water, glycerol and ethanol, and the like, but is not limited thereto, and any suitable pharmacologically acceptable agent known in the art may be used. In addition, conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.

Inflammatory vascular injury of the present invention and the composition for inhibiting vascular sclerosis and blood circulation improvement may include an active ingredient alone or two or more, the content of the active ingredient is preferably adjusted appropriately. When the active ingredient is included alone, the licorice extract is preferably 30 to 99% by weight, and the content of ricokalcon A is preferably 5 to 50% by weight based on the total composition. In addition, when it contains two or more active ingredients, it is preferable to include 10 to 90% by weight, more preferably 15 to 70% by weight based on the whole composition. When the content is less than 10% by weight, the effects of anti-inflammatory, anti-vascular sclerosis or blood cell anti-aggregation and anti-thrombosis may be insignificant, and when the content exceeds 90% by weight, the effect on the dose may be reduced It is economical.

Inflammatory vascular damage of the present invention and the composition for inhibiting vascular sclerosis and blood circulation can be used as a pharmaceutical or food additive. The formulation of the composition may be a warning, granule, powder, syrup, liquid fluid extract, emulsion, suspension, acupuncture, tablet, injection, capsule, cream, troche or pasta and may be used orally or parenterally. .

The dosage of the composition of the present invention is a conventional dosage of a medicament, for example, 300 to 3,000 mg for daily licorice extract, and 300 to 1,000 mg for ricokalcon A. The dosage is not limited to this, and it is preferable to be applied differently according to the age, sex, condition of the patient and the drug used in combination.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only preferred embodiments of the present invention, and the present invention is not limited by the following examples.

Example  1: Preparation of Baked Licorice Extract

Example  1-1: Purchase of Materials

M199 medium, RPMI 1640 medium, 3- (4,5-dimethylthiazolyl) -diphenyl tetrazolium bromide (MTT) were all purchased from Sigma-Aldrich (St. Louis, Mo.). Also other reagents required for this experiment were purchased from Sigma, unless otherwise noted. Fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, bovine brain extract, human epidermal growth factor and hydrocortisone were purchased from East Rutherford (NJ).

Example  1-2: Preparation of Ethanol and Methylene Chloride Extract

Glycyrhiza among licorice varieties baked purchased roots from inflata Bat Daekwang yakeopsa (Seoul, Korea). 0.5 kg of roasted licorice root is mixed with 3 liters of ethanol solution (95: 5, v / v), and the ultrasonic extraction process is repeated three times for 30 minutes using a PowerSonic 420 Ultrasonic Cleaning Apparatus (manufactured by Hsinshin Instrument Co., Ltd.) It was carried out by. The extract was obtained to give 125 g of residue in vacuo (hereinafter 'ethanol extract'). 1 g of distilled water was added to 50 g of the ethanol extract and suspended, followed by extracting by adding 1 liter of methylene chloride three times at room temperature using a separating funnel to obtain 6.5 g of methylene chloride soluble fraction extract (hereinafter, 'methylene chloride fraction extract').

Example  1-3: Ricokalcon  Sympathy of A

6.2 g of the methylene chloride fraction extract obtained in Example 1-2 was eluted with n-hexane: ethyl acetate (100: 0-0: 100, v / v) and fractionated by silica gel column chromatography. Fractions extract (1.2g) repeating HPLC (JAL analytical Co., Japan) SiO 2 : n-hexane-ethyl acetate, 3: 2, v / v) gave 80 mg of compound X. The compound X was found to be ricokalcon A by FR-IR (KBr), λ max (nm), UV, ¹ H NMR spectra, ¹³ C NMR data, and electron ionization mass spectrometry.

Analysis results of ricokalcon A using the measuring equipment is as follows, the chemical formula of ricokalcon A is as described in FIG. IR (KBr) (cm- ¹ ): 3437 (OH), 1637, 1603, 1560 (aromatic ring C = C), 1215, 1166 (CO); EI-MS m / z : 338 ([M] ⁺ , 70), 308 (60), 307 (100), 121 (95), 93 (20%); UV lmax (nm) 378, 305, 263 (CH ₃ OH), 451, 348, 252 (CH ₃ ONa), 39, 313, 261 (AICI ₃ ), 381, 311, 260 (NaOAc), 380, 315, 264 (NaOAc / H ₃ BO ₃ ); ¹ H-NMR d _H (ppm): d6.59 (1H, s, H-3), 9.52 (OH), 7.56 (1H, s, H-6), 7.64 (1H, d, a), 8.02 ( 1H, d, b), 7.97 (1H, d, H-2 '), 6.93 (1H, d, H-3'), 9.52 (OH), 6.93 (1H, d, H-5 '), 7.97 ( 1H, d, H-6 '), 6.27 (1H, dd.H-2``), 5.12 (1H, dd, H-3''), 5.07 (1H, dd, H-3''), 1.45 (3H, s, H-4 "), 1.45 (3H, s, H-5 "), 3.85 (OCH3); ¹³ C-NMR d _C (ppm): 115.8 (C-1), 159.7 (C-2), 101.0 (C-3), 160.0 (C-4), 126.8 (C-5), 129.3 (C-6 ), 119.4 (α), 140.0 (β), 131.5 (C-1 '), 131.7 (C-2'), 116.0 (C-3 '), 162.3 (C-4'), 116.0 (C-5 ' ), 131.7 (C-6 '), 40.6 (C-1''), 148.6 (C-2''), 110.8 (C-3''), 27.4 (C-4''), 27.4 (C- 5 ''), 55.9 (OCH3), 188.5 (C = O)

For cell culture, ethanol extract of roasted licorice, methylene chloride fraction extract of roasted licorice and purified and isolated lyocacalcon A were dissolved in dimethyl sulfoxide. The final culture concentration of dimethyl sulfoxide was 0.5% or less.

Example  1-4: licorice and baked licorice Ricokalcon  Comparison of content of A

In order to determine the calibration curve of lyocacalcon A, the lyocacalcon A sample obtained in Example 1-3 was prepared in HPLC methanol at concentrations of 1.0 mg / ml, 0.1 mg / ml, 0.01 mg / ml, and 0.001 mg / ml, respectively. After obtaining the chromatogram under the HPLC conditions shown above, the linear equation between the peak area and the concentration is obtained. The first equation obtained here was obtained as the area (Y) = 197.93 X concentration (X) + 0.0799, and the proportional equation of the area and the concentration that completely satisfies the first equation with the R value of linearity was 1. Accordingly, the content of lyocacalcon A contained in the sample is obtained by substituting the area values of the peaks obtained from the samples, respectively, and the calibration curve is shown in FIG. 2. Each point shown here is a comparative product lyocacalcon. It means that the area value of each peak obtained at each density | concentration of A, and the point where that density | concentration meet.

According to Figure 2, the content of ricokalcon A extracted from licorice was found to increase about 4.9 times in baked licorice (13.8mg / 100g baked licorice) than non-baked licorice (2.8mg / 100g licorice).

Experimental Example  One: Mononuclear  Measurement of adhesion inhibition effect

Experimental Example  1-1: Blood vessel  Incubation and Preparation

Human umbilical vein endothelial cells were isolated from the umbilical cord using collagenase enzymes (Worthington Biochem. Corp., Lakewood, NJ) (Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial) cells derived from umbilical veins: Identification by morphologic and immunologic criteria Clin Invest 52:. 2745-2756, 1973), were primarily cultured in 37 ℃, 5% CO ₂ incubator. Cells in 25 mM HEPES-buffer M199 containing 10% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.75 mg / ml human envelope growth factor and 0.075 mg / ml hydrocortisone Incubated. The cells were passaged and used within 10 passages. The identification of vascular endothelial cells was characterized by acetylation labeled with a fluorescent substance of DiI (1.1'-dioctadecyl-, 3,3,3-tetramethylindocarbocyanine perchlorate) (Molecular Probes Co., Eugene, OR, USA) while maintaining a pebble shape. This was done by investigating the low density protein (Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetyl-low density lipoprotein. J Cell Biol 99: 2034-2040, 1984).

The vascular endothelial cells were applied at 90-95%, and the cells were cultured overnight with ethanol extract, methylene chloride fraction extract, and purified lyocacalcon A of roasted licorice of Example 1, and tumor necrosis factor-α (Roche Molecular Biochemicals, Mannheim, Germany) was treated with 10 ng / ml for 6 hours. In addition, cells treated with tumor necrosis factor-α alone without treatment with fraction extract or lyocacalcon A were also prepared.

Experimental Example  1-2: In vitro  Adhesion test

Human leukemia mononuclear cell line THP-1 was obtained from American type culture collection (Rockville, MD), and vascular endothelial cells were applied in 4-well glass at a concentration of 1.0 x 10 ⁵ and cultured in 25 mM HEPES-buffered M199 medium. . Before treating the cells with tumor necrosis factor-α 10ng / ml for 6 hours (Park SH, Park JHY, Kang JS, Kang YH.Involvement of transcription factors in plasma HDL protection against TNF-α-induced vascular cell adhesion molecule-1 Int J Biochem Cell Biol 35: 168-182, 2003), and the cells that were pretreated with the baked licorice ethanol extract and methylene chloride fraction extract of Example 1 and the cells that did not undergo the pretreatment step were prepared. The THP-1 cells were labeled with 5 μM calcein AM (Molecular Probes Inc., Eugene, OR) in RPMI 1640 medium containing 10% FBS, each of the three types of THP-1 cells treated 5.0 × 10 ^5. To the cell monolayer of vascular endothelial cells treated with tumor necrosis factor-α was incubated for 2 hours. At this time, the control group was observed vascular endothelial cells not treated with tumor necrosis factor-α.

Clean co-cultured cells and measure adhesion to vascular endothelial cells of THP-1 monocytes at 485 nm excitation wavelength and 538 nm emission wavelength on a fluorescence microscope (Diagnostic Instrument, Livingston, Scotland) with a SPOT II digital camera. 3 is shown.

In addition, each cultured with vascular endothelial cells in an environment pretreated with purified 25 μM lyocacalcon A and an environment not pretreated with lycocalcon A, obtained in Examples 1-3, followed by 10 ng / ml tumor necrosis for 6 hours. It was activated by treatment with factor-α. The vascular endothelial cells were cocultured with THP-1 mononuclear cells labeled with Calcein AM for 2 hours and observed by fluorescence microscopy to measure the degree of adhesion to vascular endothelial cells of THP-1 monocytes.

Experimental Example  1-3: Experiment Result

As shown in FIG. 3, mononuclear cell adhesion was observed in a small amount in control vascular endothelial cells that were not treated with tumor necrosis factor-α. In addition, THP-1 adhesion to vascular endothelial cells exposed only to tumor necrosis factor-α was significantly increased. However, monocyte adhesion was significantly inhibited in vascular endothelial cells exposed to tumor necrosis factor-α pretreated with 25 μg / ml methylene chloride fraction extract. In contrast, ethanol extracts of baked licorice did not show this effect. Therefore, it can be seen that the methylene chloride fraction extract containing the non-polar substance of the baked licorice extract has an inhibitory effect on the aggregation of mononuclear leukocytes in cytokine-stimulated vascular endothelial cells.

In FIG. 4, mononuclear cell adhesion was small in cells not treated with tumor necrosis factor-α, and adhesion was markedly increased when tumor necrosis factor-α was treated. However, the pretreatment with 25 μM lyocacalcon A showed significantly weaker staining, indicating that monocyte adhesion was inhibited. This suggests that the cells co-exposed to non-polar material, lycochalcone A and tumor necrosis factor-α, contained in the methylene chloride fraction extract substantially inhibit the adhesion of THP-1 induced by tumor necrosis factor-α.

Experimental Example  2: VCAM Effect of Baked Licorice Extract on Inhibition of -1 Expression

Experimental Example  2-1: Weston Blot  Experiment method

After pretreatment of cells not treated with tumor necrosis factor-α, cells treated with tumor necrosis factor-α only 10 ng / ml, 25 μg / ml of ethanol extract of Example 1-2 or 25 μg / ml of methylene chloride fraction extract Cultured cells were prepared by treating cell tumor necrosis factor-α 10 ng / ml.

Cell lysates were 10% SDS, 1% β-glycerophosphate from cultured vascular endothelial cells. Obtained in 1M Tris-HCl lysis buffer containing 0.1 M Na ₃ VO ₄ , 0.5 M NaF and protease inhibition cocktail.

To measure VCAM-1 expression in the cells, cell lysates containing the same amount of total protein were fractionated by 8% SDS-PAGE and transferred to nitrocellulose membrane. 8% SDS-PAGE was performed on the protein of cell lysate. Electrophoretic protein bands were transferred to nitrocellulose membranes and TBS-T buffer containing 5% skim milk for 3 hours to prevent nonspecific binding [0.5M Tris-HCl (pH 7.5), 1.5M NaCl, 0.1% Tween 20] was used. The membrane was incubated for 3 hours with a 1,000-fold dilution of primary antibody (polyclonal rabbit acti-human antibody, Santa Cruz Biotechnologies, Santa Cruz, CA) of VCAM-1 protein, and then washed three times with TBS-T buffer. It was then reacted with a secondary antibody of goat anti-rabbit IgG horseradish peroxidase (1: 10,000, Jackson Immuno Reseach Laboratories, West Grove, Pa., USA) for 1 hour.

The amount of VCAM-1 protein was detected by a Supersignal West pico chemiluminescene (Pierce Biotech. Inc., Rockford, Ill.) And photographed on a Konica X-ray film as shown in FIG. 5A. At this time, treatment with monoclonal mouse β-actin antibody (1: 5,000) was measured as a control.

Experimental Example  2-2: Ricokalcon  According to A concentration VCAM -1 Experimental method for expression suppression effect

The Western blot analysis method of Experimental Example 2-1 was also used here. The prepared vascular endothelial cells were pretreated with 10, 20, 30, and 50 μM of ricokalcon A and tumor necrosis factor-α 10 ng / ml was added. In the cells prepared here, the expression level of VCAM-1 was measured in the same manner as in Experimental Example 2-1. The measurement results are shown in Fig. 5B.

Experimental Example  2-3: Results

As shown in FIG. 5A, VCAM-1 expression was weak in control vascular endothelial cells not treated with tumor necrosis factor-α, and VCAM-1 expression was significantly increased upon tumor necrosis factor-α treatment. When the tumor necrosis factor-α treated cells were treated with 25 μg / ml ethanol extract of Example 1, the expression of VCAM-1 was not inhibited, but the methylene chloride fraction extract of Example 1 was treated with 25 μg / ml. Was found to significantly inhibit VCAM-1 expression.

5B, VCAM-1 expression was significantly increased in cells treated with tumor necrosis factor-α only. In the case of cells pretreated with ricochalcone A, the expression of VCAM-1 was significantly suppressed depending on the concentration. Although the degree of expression was slightly inhibited when 10 μM of lyocacalcon A was treated, the expression of this VCAM-1 was completely inhibited by the treatment of at least 20 μM of lyocacalcon A.

VCAM-1 is mainly responsible for the aggregation of monocytes in vascular endothelial tissue, causing damage to vascular hardening vascular endothelial tissue, and is known to be involved in the generation of atherosclerotic plugs. The present invention investigated whether the inhibitory effect of baked licorice methylene chloride fraction extract or lycakalcon A on monocyte adhesion of vascular endothelial cells by tumor necrosis factor-α was due to the inhibition of VCAM-1 expression of vascular endothelial cells. To this end, Western blot analysis was attempted using an antibody specific for the VCAM-1 protein.

As shown in FIG. 5, VCAM-1 expression was relatively low in control cells not treated with tumor necrosis factor-α, and increased significantly in cells stimulated with tumor necrosis factor-α. Vascular endothelial cells pretreated with 25 μg / ml ethanol extract and exposed to tumor necrosis factor-α were not significantly inhibited in VCAM-1 protein expression, but pretreated with 25 μg / ml methylene chloride fraction extract. Cells exposed to -α almost completely inhibited the expression of the VCAM-1 protein. In addition, cells treated with lycochalcone A and tumor necrosis factor-α inhibited VCAM-1 expression depending on the concentration of lyocacalcon A, and the expression of VCAM-1 was decreased when lyocacalcon A was pretreated 20 μM or more. Completely suppressed.

Therefore, it was confirmed that methylene chloride fraction extract or ricokalcon A component of baked licorice inhibits the adhesion of monocytes to vascular endothelial tissue by inhibiting the expression of VCAM-1 protein in vascular endothelial cells. The baked licorice composition comprising the substance as an active ingredient can be provided for the purpose of preventing and treating vascular sclerosis and coronary artery disease while blocking the initial process of vascular hardening, which begins with the adhesion of monocytes, and blood, including circulating platelets. It can be used for pharmacological use such as thrombosis due to blood improvement by inhibiting aggregation of cells.

Baked licorice extract of the present invention, in particular, methylene chloride fraction extract and purified ricokalcon A prevents the adhesion of monocytes to vascular endothelial cells can block the development of vascular sclerosis and thrombosis early, this anti-inflammatory vessel It can be used as a composition for injury, antivascularization and antithrombosis.

Claims (6)

Inflammation treatment composition comprising baked licorice extract. The composition of claim 1, wherein the composition for treating inflammation is inflammatory vascular injury, vascular sclerosis or thrombosis. The method of claim 1, wherein the roasted licorice extract is a composition for treating inflammation that is extracted from the licorice, the alcohol having 1 to 5 carbon atoms or a mixture thereof with a solvent. The composition for treating inflammation according to claim 3, wherein the roasted licorice extract is further extracted using at least one solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate and chloroform. According to claim 1, The baked licorice extract is an inflammation treatment composition is extracted by sequentially adding ethanol and methylene chloride. The composition for treating inflammation according to claim 1, wherein the baked licorice extract contains ricokalcon A.

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