KR20050080882A - A antiinflammatory composition containing stylopine isolated from leaf of chelidonium majus - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising stylopine isolated from the leaves of celandine (Papaveraceae, Chelidonium majus L. (Papaveraceae)) as an active ingredient. In more detail, the composition contains stilpine as an active ingredient, nitric oxide (NO), prostaglandin E ₂ (progetaglandin E ₂ , PGE ₂₎ , tumor necrosis factor-α (tumor necrosis factor-α, TNF-α ), Interleukin-1β (IL-1β) and IL-6 production, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) It suppresses expression and shows anti-inflammatory response.

Description

A antiinflammatory composition containing stylopine isolated from leaf of Chelidonium majus}

The present invention relates to an anti-inflammatory composition comprising stylopine isolated from the leaves of celandine (Papaveraceae, Chelidonium majus L. (Papaveraceae)) as an active ingredient.

Generally, cytokines and tumor necrosis factor-α (tumor) such as nitric oxide (NO), prostaglandin E ₂ (PGE ₂ ), interleukin-1β, IL-1β, IL-6, etc. It is known that mediators such as necrosis factor-α and TNF-α induce inflammation (Lee et al., 1992; Moncada et al., 1991; Nathan, 1997; Sautebin, 2000; Wheeler and Bernard, 1999). . In addition, macrophage lines play an important role in inflammatory diseases by producing the inflammatory mediators, and the production of the mediators from macrophage lines increases lipopolysaccharide (LPS) and lipothycosaccharides as mRNA expression is increased in various inflammatory tissues. Immune stimulants, including extracellular cytotoxicity, such as acid (lipoteichoic acid) were found (Penglis et al., 2000; Yamashita et al., 2000).

No and pro-inflammatory cytokine play a major role in the defense mechanism, but the overproduction of these inflammatory mediators is known as sepsis, rheumatoid arthritis, atherosclerosis, lung It affects the pathogenesis of several diseases such as pulmonary fibrosis and chronic hepatitis (Coker and Laurent, 1998; Isomaki and Punnonen, 1997).

On the other hand, celandine (Papaveraceae, Chelidonium majus L. (Papaveraceae)) is a plant doeon used in a variety of diseases in the European and Chinese herbal medicines. Natural extracts from various parts, such as roots, germination and leaves, can be found in many alkaloids such as stilpin, sanguinarine, chelidonine, chelerythrine, berberine and coptisisine. Have been reported to have (Colombo and Bosisio, 1996). In addition, both natural extracts of C. majus and purified components derived from them have been reported to have antiviral, anti-inflammatory, anti-tumor and antimicrobial properties in vitro and in vivo (Lenfeld et al., 1981; Colombo and Bosisio, 1996; Biswas and Khuda-Bukhsh, 2002). In particular, stilpin is a major component of various phytomedicinal plants, including C. majus , which are known to exhibit GABAA receptors, detoxification of xenobiotics and other allosteric modulation of anti-inflammatory (Kubo, et al., 1994; Haberlein, et al., 1996; Satou, et al., 2002; Ikezawa, et al., 2003). However, the effect of stilpin isolated from C. majus on NO, PGE ₂ and pro-inflammatory cytokine production induced by stimulation of LPS remains unidentified.

Therefore, the present inventors have conducted a number of studies to find a substance that can suppress the production and expression of inflammatory mediators induced by stimulation of LPS in RAW 264.7 macrophage line, stilpine (stylopine) isolated from the leaves of celandine And inhibits the production of inflammatory mediators NO, PGE ₂ and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and the expression of iNOS and COX-2. .

Accordingly, it is an object of the present invention to provide an anti-inflammatory composition containing stilpin as an active ingredient.

In order to achieve the above object, the anti-inflammatory composition according to the present invention is characterized in that it contains a stilpine (stylopine) as an active ingredient.

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

Chelidonium majus L. is a biennial plant of the dicotyledonous poppy genus Poppyaceae. It is usually distributed in Korea, Japan, northeast China, Sakhalin, Mongolia and Siberia. The Papaveraceae plant C. majus contains abundant specific alkaloids and flavonoid components, which have been reported to have antiviral, anti-inflammatory, anti-tumor and antimicrobial properties (Colombo and Bosisio, 1996). In addition, in oriental medicine, the whole plant is called baekryechae (白 屈 菜), is used as an analgesic for abdominal pain due to gastroenteritis and stomach ulcers, dysentery, jaundice hepatitis, skin ulcers, tuberculosis, scabies, ringworm.

In the present invention, it has been found that the stilpine contained in celandine grass has an anti-inflammatory effect, and it is clear that what mechanism of action of stilpin has in macrophages involved in the inflammatory response. Therefore, the stilpine isolated in the present invention can be used more advantageously in the treatment of patients with diseases caused by excessive inflammatory response by establishing a more specific prescription amount in the treatment of inflammation.

On the other hand, the stilpin component may be used after extraction from the leaves of celandine by the methods commonly known in the art. An example of the extraction method of styropine is a method using methanol, which is described in more detail in the following examples.

 Meanwhile, according to the present invention, diseases which may occur due to excessive inflammatory reactions are commonly known in the art, and effects may be obtained by administering a composition according to the present invention to treat such diseases. Examples of diseases that can be treated by the compositions of the present invention, namely diseases caused by excessive inflammatory reactions, include sepsis, rheumatoid arthritis, atherosclerosis, and pulmonary fibrosis. ), Chronic hepatitis, and the like, but the present invention is not limited thereto, and inflammatory response diseases commonly known in the art are also included in the scope of the present invention.

Styropine isolated from the leaves of celandine of the present invention can significantly inhibit the production and COX-2 expression of NO, PGE ₂ TNF-α, IL-1β and IL-6 in RAW 264.7 macrophage lines. , Which exhibits anti-inflammatory effects with down regulation of iNOS and COX-2.

In the anti-inflammatory composition of the present invention, the effective amount of the active ingredient stropin may be appropriately selected depending on the administration method, the preparation form, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease, but is not particularly limited thereto. In general, it can be formulated to be administered in a dose of 0.001 to 500 mg / kg body weight. Of course, the dose of the active ingredient may be an amount outside the above range.

The formulation for administering the stilpine within the above range may have a conventional form, and may be used, for example, in the form of a pill, a capsule, a drink, a medicine, and the like. They can be administered orally or various parenteral routes of administration for the treatment of diseases caused by hyperinflammatory reactions, which are suitable for the dosage form and are well known to those skilled in the art and can be easily selected by those skilled in the art. Excipients, carriers, or diluents, and the like.

Example

Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to these examples.

In the following, all values are expressed as mean ± SD of three independent determiantion. In addition, all experiments were performed at least three times, and each trial was performed at least three times as independent observations. Statistical analysis was performed by analysis of variance (ANOVA) and Student's t- test.

On the other hand, the reagents used in the present invention are summarized as follows.

DMEM (Dulbecco's Modified Eagle's Medium), Fetal bovine serum (FBS) and antibiotics (antibiotics) were purchased from GIBCO BRL (Grand Islang, NY).

Rabbit anti-iNos, rabbit anti-COX-1 and COX-2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

LPS ( Salmonella entritidis phenol extraction), Tween 20, bovine serum albumin (BSA), dimethyl sulfoside (DMSO), 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium Bromide (MTT), sodium dodecyl sulfate (SDS), N ^G -nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma Chemical Company (ST Louis, MO).

TNF-α, IL-1β, IL-6 and PGE ₂ immune kit (Quantikine ™) were purchased from the R & D system (Minneapolis, MN, USA).

96 well tissue culture plates and other tissue culture products were purchased from Life Technologies (Gaithersburg, MD).

All reagents were subjected to a colorimetric Limulus amoebocyte lysate assay (limit of measurement, 10 pg / ml; Whittaker Bioproducts, Walkersville, MD) to determine the LPS content.

Reference Example 1 Extraction and Separation of Styropine

Leaf of celandine (Papaveraceae, Chelidonium majus L. (Papaveraceae)) used in the experiments was purchased in May 2003 in a herbal pharmaceutical partners Jeonbuk. The proof of the specimen was kept in the herbarium of Oriental Medicine Center of Wonkwang University. The proof number is LGF777.

First, dried leaves (500 g) of C. majus were extracted with MeOH for 12 hours. The MeOH extract was then concentrated and suspended in water and separated successively with n-hexane, EtOAc and BuOH.

The n-hexane aqueous portion (5.2 g) was extracted with a MeOH-CHCl ₃ 1: 1 solution on a Sephadex LH-20 column to give six subfractions (AF). Then, fraction B (1.3 g) was separated by reverse-phase HPLC in water using a gradient elution of CH ₃ CN 40-70% for 50 minutes in water, followed by separation in 100% CH ₃ CN for 10 minutes. The component (332 mg) was obtained.

REFERENCE EXAMPLE 2 RAW 264.7 Macrophage Line and Its Culture

RAW 264.7 macrophage lines were purchased from ATCC, USA (American Type Culture Collection, TIB71, Maryland, USA).

The macrophage line rodent macrophage RAW 264.7 was 1 × in DMEM supplemented with 10% heat inactivated FBS, feticillin G (100 IU / mL), streptomycin (100 μg / mL), and L-glutamine (2 mM). Incubated at 37 ° C. in a humid atmosphere containing 5% CO ₂ and 95% air while maintaining 10 ⁶ cell numbers / ml. The next day, the medium was replaced with fresh DMEM, and the cells were stimulated with LPS with or without stilpine from celandine for a period of time. Styropine was dissolved in DMSSO and then diluted with medium to final concentration.

Reference Example 3 MTT Analysis for Cell Viability

Cells were placed in a 96 well plate containing 100 μl of DMEM medium of 10% FBS at a density of 2.5 × 10 ⁵ cells / ml and incubated overnight. After 24 hours following seeding, 100 μl fresh medium or stilpin (1-20 μg / ml) was added and the plates were incubated for 24 hours.

The cells were washed once before adding 50 μl of FBS-free medium containing 50 μg / ml MTT. After incubation at 37 ° C. for 4 hours, it was dissolved in 50 μl DMSO. Optical density at 570 nm was determined with a microplate reader. At this time, the optical density of formazan (formazan) formed in the control (untreated) cells was 100% survival.

Reference Example 4 Nitrite Analysis

Accumulated nitrite (NO) and oxidized NO were measured in the culture medium by Griess reaction.

First, 100 μl cell culture medium was mixed with 100 μl Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydorchloride / 2.5% phosphoric acid) and then at room temperature for 10 minutes. After incubation, the absorbance was measured at 540 nm with a microreader. At this time, fresh culture medium was used as the base group in all experiments, the level of nitrite in the sample was calculated from the standard nitrite curve prepared in the culture medium.

Reference Example 5 PGE ₂ , TNF-α, IL-1β, and IL-6 Analysis

Cells (1 × 10 ⁶ / mL) were preincubated for 2 hours with stilpin in 24-well plates and then incubated with LPS (1 μg / mL) for 6 or 18 hours. After the suspensions were removed, PGE ₂ , TNF-α, IL-1β and IL-6 levels were quantified with an immunoassay kit according to the manufacturer's protocol.

Reference Example 6 Western Blot

Cytoproteins were extracted from control and stilpine-treated RAW 264.7 cells. Washed cell pellets in cold lysis buffer (10 mM Tris-base, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 5 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 10 μg / ml) leupeptin, 25 μg / ml aprotinin), and resuspended at 4 ° C. for 30 minutes. Nuclei and cell debris were removed by microcentrifugation, and the suspensions were frozen. 30 μg of cellular proteins in the treated and untreated cell extracts were electroblotted into nitrocellulose thin films and separated by 10% sodium dodecyl sulfate (SDS) -polyacylamide gel electrophoresis. The immunoblot was then removed. Incubate overnight at 4 ° C. with blocking solution (5% skim milk) and incubate for 4 hours with a suitable dilution of the first antibody (for rabbit anti-iNos, rabbit anti-COX-1 and COX-2) It was. The blot was washed twice with PBS and incubated for 1 hour at room temperature with a 1: 5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG. The stain was again washed three times with Tween 20 / Tris-buffered saline (TTBS), developed in 10 ml of 1: 1 mixed solution of the ECL observation system for 1 minute, dried quickly and exposed to film for 2-20 minutes. Protein concentration was determined by Bio-Rad protein assay reagent according to the manufacturer's instructions.

Example 1 Effects of Styropine on iNOS Expression in NO Production and LPS-Stimulated RAW 264.7 Macrophage Lines

The effect of stropin on NO production and iNOS protein expression was measured in LPS stimulated RAW 264.7 macrophage line.

First, the treatment concentration of stilpin used in the present invention had no effect on NO production and iNOS protein expression in RAW 264.7 macrophage line unstimulated with LPS. In contrast, the production of NO was significantly reduced in RAW 264.7 macrophage lines treated with LPS (1 μg / ml). In addition, the expression of iNOS protein was measured by western blotting analysis to assess whether the reduction in NO production by stilpin was related to iNOS.

Consistent with the decrease in NO production in FIG. 2A, stilpin inhibited the expression of iNOS protein in a concentration dependent manner (FIG. 2B) in RAW 264.7 macrophage line stimulated with LPS. NO inhibitor L-NAME (10 μM), positive control also inhibited NO production in activated RAW 264.7 (FIG. 2A). As a result, it was evident that Styropine inhibits NO production and iNOS expression concentration dependently on LPS without cytotoxicity.

Example 2 PGE in LPS-stimulated RAW 264.7 Macrophage Line ₂  Effect of Styropine on Secretion

The effect of stropin on PGE ₂ production was measured in RAW 264.7 macrophage line activated with LPS.

As shown in FIG. 3, in the case of LPS alone treatment, PGE ₂ synthesis was secreted in large amounts in RAW 264.7 macrophage line. On the other hand, in the group treated with different concentrations of stilpin, PGE ₂ secretion induced by stimulation of LPS in a concentration dependent manner was inhibited (FIG. 3).

Example 3 Effect of Stilpin on COX-2 Activity and Expression in LWA-stimulated RWA 264.7 Macrophage Line

To study the mechanism of inhibition of PGE ₂ secretion by storopin, COX-2 activity and expression in LPS stimulated RWA 264.7 macrophage lines were measured.

As shown in FIG. 4, LPS alone induced high levels of COX-2 production in LPS stimulated RWA 264.7 macrophage line. On the other hand, COX-2 activity was inhibited in a concentration-dependent manner in the group treated with different concentrations of stilpin. Styropine in the range of drugs used showed a concentration dependent decrease in LPS-induced COX-1 and COX-2 protein expression (FIG. 4B). In addition, it was confirmed that COX-2 activity and protein expression were more inhibited than COX-1 in the same amount of stilpin. This demonstrates that inhibition of PGE ₂ production is concentration dependent by stifling the LPS-stimulated RAW 264.7 macrophage line by inhibition of COX activity and protein expression.

Example 4 Effects of Styropine on TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW 264.7 Macrophage Lines

To determine if stilpin regulates the production of pro-inflammatory cytokine, the TNF-α, IL-1β and IL-6 The production was measured.

Styrene alone treatment did not affect the secretion of TNF-α, IL-1β and IL-6 in unstimulated RAW 264.7 macrophage lines. On the other hand, when treated with different concentrations of stilpine, it was confirmed that the concentration-dependent inhibition of the secretion of TNF-α, IL-1β and IL-6 (Fig. 5).

This indicates that cataposides were not reduced in cell viability in the range of drugs used in the present invention compared to cells treated with LPS alone, so the observed effect was not due to the cytotoxicity of stilpin ( 5D).

From the above results, stiloff from C.majus as an anti-inflammatory drug in traditional herbal medicines showed NO, PGE ₂ , COX-2, TNF-α, IL-1β, and LPS-stimulated RAW 264.7 macrophage lines. It has been shown to inhibit the production of IL-6 in a concentration dependent manner.

It has been described that the stilpin of the present invention is a major inhibitor of protein expression of iNOS and COX-2, and inhibits PGE ₂ production like NO in LPS-stimulated RAW 264.7 macrophage lines (FIGS. 2-4).

It has also been shown that stilpin of the present invention inhibits the production of pro-inflammatory satokines (TNF-α, IL-1β and IL-6) induced in macrophages in LPS-stimulated RAW 264.7 macrophages.

As described above, stilpin can inhibit the activity of various mediators involved in the inflammatory response, namely NO, PGE ₂ and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Thus, furthermore, the production of NO, PGE ₂ and pro-inflammatory cytokines, respectively, inhibits the nuclear factor-κB (NF-κB) response components essential for the expression of iNOS, COX-2 and pro-inflammatory cytokine genes involved in the inflammatory process and It may be confirmed that the component induces NF-κB activity.

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1 is a view showing the chemical structure of the stiloff pin isolated from the leaves of celandine.

FIG. 2 is a diagram showing the effect of stilpin on NO production (A) and iNOS protein expression (B) in RWA 264.7 macrophage lines.

FIG. 3 shows the effect of stilpine on PGE ₂ production in RWA 264.7 macrophage lines.

FIG. 4 is a diagram showing the effect of stilpin on COX activity (A) and COX protein expression (B) in RWA 264.7 macrophage line.

FIG. 5 shows the effects of stilpin on TNF-α (A), IL-1β (B) and IL-6 (C) in RWA 264.7 macrophage lines.

Claims (6)

Anti-inflammatory composition characterized in that it contains a stilpine (stylopin) as an active ingredient in a dose of 0.001 ~ 500mg / kg body weight for the treatment of diseases caused by excessive inflammatory reactions. The anti-inflammatory composition according to claim 1, wherein the stilpin is isolated from the leaves of celandine. The anti-inflammatory composition of claim 1, wherein the anti-inflammatory composition inhibits the production of pro-inflammatory cytokines of TNF-α, IL-1β and IL-6. The anti-inflammatory composition according to claim 1, wherein the anti-inflammatory composition inhibits PGE ₂ secretion. The anti-inflammatory composition of claim 1, wherein the anti-inflammatory composition inhibits the production of nitric oxide (NO). The anti-inflammatory composition of claim 5, wherein the anti-inflammatory composition inhibits inducible nitric oxide synthase (iNOS) expression.