KR20090036238A - Ishige okamurae extract having anti-inflammatory effect - Google Patents

Abstract

An Ishige okamurae extract having anti-inflammatory effects is provided to inhibit transcriptional activity of the NF-kappaB(nuclear factor kappa B) in an RAW 246.7 cell and suppress generation of TNF-alpha(tumor necrosis factor alpha), IL-1beta(interleukin 1beta), IL-6 and PGE2(prostaglandin E2). An Ishige okamurae extract comprises 1-1000mg/mL of Ishige okamurae extracts as an active ingredients. The Ishige okamurae extracts is obtained by marine algae having anti-inflammatory effects in screened macrophage from marine supply source. The Ishige okamurae extracts does not show cytotoxin against macrophage. The Ishige okamurae extracts is extracted by ethanol. A unit active ingredient from the Ishige okamurae lowers transcriptional activity of NF-kappaB.

Description

Ishige okamurae extract having anti-inflammatory effect

The present invention relates to an extract of Ishige Okamura, which has anti-inflammatory activity, and more particularly, inhibits transcriptional activity of NF-κB in RAW 246.7 cells (Abelson murine leukemia virus-induced tumor, a macrophage cell line) cells. The present invention relates to an extract of Ishige okamurae , which has anti-inflammatory activity by inhibiting the production of inflammatory mediators TNF-α, IL-1β, IL-6 and PGE ₂ .

Inflammation is the organism's response to the upper body associated with biological, chemical or physical stimuli. In acute inflammation, this reaction is to inactivate or destroy invading organisms, remove irritants, and finally put them in a state for tissue reconstruction. However, in chronic inflammation, this response is periodontal disease, colitis, arthritis. It is associated with multiple diseases such as atherosclerosis, Alzheimer's disease, asthma, multiple sclerosis and inflammatory bowel disease (Balkwill et al., 2004).

NF-κB is known to play the most important role in the immune system (Li and Verma, 2002). NF-κB is an inhibitor of inflammatory mediators and effector enzymes such as cytokines involved in inflammation, including tumor necrosis factor-α, interleukin, cyclooxygenase, and nitric oxide synthase. It has been reported to regulate expression (Bonizzi et al., 2004). Prior literature shows that such cytokines in inflammatory mediators induce matrix metalloproteinases (MMPs) that degrade extracellular matrices such as collagen and fibronectin (Lark et al., 1997).

In addition, NF-κB regulates the expression of genes outside the immune system, thereby affecting various aspects of normal physiology and physiology during disease. NF-κB is active in the nucleus and is inhibited by sequestration in the cytoplasm by a κB inhibitor (IκB). NF-κB is activated once released from IκB. Phosphorylation of IκB by IκB kinase (IKK) causes ubiquitrylation and degeneration by proteasomes. Cytokines such as interleukin-1 and tumor necrosis factor-α activate NF-κB by phosphorylation of IκB.

The vitrified NF-κB dimer is translocated into the nucleus and binds to a specific sequence in a promoter or enhancer of the gene of interest that plays an important role in inflammation (Karin et al., 2000).

Recently, mouse models of two inflammation-related cancers have been associated with cancer progression with inflammatory mediators known as gene-transcription factor NF-κB and tumor necrosis factor-α (Pikarsky et al., 2004: Greten et al. , 2004). In the inflammation-related model of liver cancer in this document, it has been found that activation of NF-κB is not critical at the early stage of tumor development but fatal at the stage of transition to malignancy. As such, any anti-inflammatory therapy may be most effective during the early stages of cancer development.

In addition, epidemiological studies that have identified chronic infectious diseases and inflammation as major risk factors for various forms of cancer have reported that there is some association between inflammation and cancer (Balkwill et al., 2001; Coussens et al. ., 2007). It is well known that NF-κB transcription factors play a major role in inflammation and innate immunity. Previous studies have reported that in inflammation, NF-κB finally acts as a growth signal, as well as affects the avoidance of apoptosis, unlimited replication potential and metastasis (Garten et al., 2004; Hanaha et al., 2000; Garber et al., 2004).

Therefore, if a therapeutic agent is developed that can inhibit the activation of the NF-κB transcription factor, it is expected that such therapeutic agent can inhibit inflammation as well as cancer development. In recent years, it has been reported that various natural compounds can be used as potential anti-inflammatory agents (Calzado et al., 2007).

Ishige okamurae (IO; L) has a narrow leaf, thick outer layer, pointed vertices and rough surface arrangement. These seaweeds are brown algae, stearic acid, methyl myristate and palmitic acid, (2S) -O-palmitoyl-2-O-myristoyl-3-O- (6-sulfo-alpha-D-quinobyra Nosyl) glycerol and its derivatives (Zhongguo et al., 2002). However, no information on the effect of IO extracts on anti-inflammatory has been reported to date.

Therefore, the present invention is focused on the fact that an agent capable of inhibiting the activation of NF-κB can prevent the development of cancer. The study of the effect of the extract in detail has led to the present invention, through which the screening of marine algae that can inhibit the activation of NF-κB, Ishige Ishige ( Ishige okamurae , IO) has been shown to inhibit the activation of NF-κB, resulting in an anti-inflammatory effect.

In order to achieve the above object, the present invention inhibits the transcriptional activity of NF-κB in RAW 246.7 cells, and inhibits the production of inflammatory mediators TNF-α, IL-1β, IL-6 and PGE ₂ to prevent inflammation. Ishige Okamura with activity okamurae ) extract (hereinafter referred to as IO extract in the present invention).

The Ishige Okamura extract is preferably an ethanol extract.

The present invention also provides an anti-inflammatory composition containing an extract of Okamura as an active ingredient.

The present invention screens marine algae with anti-inflammatory effects in macrophages from marine sources and found for the first time that IO extracts can inhibit the production of inflammatory mediators through inactivation of NF-κB transcription factors in macrophages.

In the present invention, the inhibitory effect of IO extract on cell growth in RAW 246.7 cells was evaluated. The IO extract did not show any cytotoxicity against macrophages. This is supported by the fact that IO has long been used as a traditional food in Korea. It has also been reported that the ethyl acetate fraction of IO markedly inhibited the enzymatic activity of vial enzymes (Ahn et al., 2002).

Previous studies have shown that IO strongly inhibits algal tissue growth, spore fixation, zygote formation and germline growth, also increases foot repulsion, and inhibits immature settlement of muscles. (Cho et al., 2001). Therefore, IO has not only been used as an edible seaweed in Korea but also has an anti-inflammatory effect in macrophages and thus may have therapeutic potential in preventing or treating chronic inflammation.

In the present invention, in order to investigate whether IO can suppress chronic inflammation, the level of the main inflammatory mediators such as cytokines and PGE ₂ synthesized by cyclooxygenase-2 is determined as a major player in the chronic inflammatory response. Measurements were made using immunoassay on in macrophages.

In addition, in the present invention, the IO extract reduced the expression levels of TNF-α, IL-1β and PGE ₂ in lipopolysaccharide-promoted RAW 246.7 cells, which indicates that chronic inflammation in macrophages will be inhibited by the IO extract. Instructs the fact that it can. This is a previous report that pre-inflammatory cytokines such as TNF-α, IL-1β and IL-6 were the strongest derivatives of COX-2 associated with reducing the mortality rate of rectal cancer below 50% (Marnett et al., 2002). Is supported by.

NK-κB plays an absolute role in regulating apoptotic cell death of normal and malignant cells as well as regulating various inflammations. Thus, this is an attractive target for anticancer and anti-inflammatory strategies.

In particular, it is often described as the central mediator of the immune response because a wide variety of bacteria and viruses can activate it. Activation of NF-κB expresses inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. Further evidence supporting the role of inflammation in the development and growth of gastrointestinal malignancies is the fact that constitutive expression of NF-κB has been identified in many gastrointestinal malignancies, including hepatocellular carinoma and colorectal cancel. (Hoffmann et al., 2007).

Therefore, in order to investigate whether IO extract could affect the transcriptional activity of NF-κB in RAW 246.7 cells, reporter gene analysis using a reporter construct comprising NF-κB binding site-luciferase was performed.

This revealed that IO extract-treated cells inhibited the transcriptional activity of NF-κB enhanced by TNF-α treatment. These results suggest that the inhibitory effect of IO extract on the production of inflammatory mediators is obtained by inactivation of NF-κB transcription factor, which plays a critical role in inflammation in macrophages. This is according to a study (Oakley et al., 2005) that the correlation between chronic inflammation and NF-κB was observed in chronic inflammation. This observation can be further confirmed by the discovery that IO extracts can reduce the expression level of NF-κB transcription factor involved in chronic inflammation.

The expression level of NF-κB in the nucleus was significantly reduced in a concentration dependent manner. This result was due to the reduction of inflammatory mediator levels due to inactivation of NF-κB in macrophages. In addition, previous studies have shown that activation of NF-κB plays an important role in colon cancer (Yu et al., 2004) as well as chronic inflammation caused by the production of inflammatory mediators (Sebban et al., 2006).

In chronic inflammation, such as osteoarthritis, preinflammatory cytokines induce matrix metalloproteinases (MMPs), which release a cartilage extracellular matrix (Dinarello and Moldawer, 1999). Therefore, the inhibitory effect of IO extract on MMP expression and activity was further analyzed in media regulated from HTT1080 cells. It has been reported that fibrosarcoma HT1080 cells secrete type IV collagenase, MMP-2 and MMP-9, and that these enzymes play a major role in cancer metastasis (Nagase et al., 1998). Yoon et al., 2001).

IO extract showed a dose dependent inhibitory effect on MMP-2 and MMP-9 activity, respectively. In contrast to the gelatinase activity of MMP-9, MMP-2 can break down gelatin and fibrillar collagen, respectively, making MMP-2 more important for complete digestion of collagen during cellular invasion. (Aimes et al., 1995). Therefore, it can be expected that the IO extract has an excellent effect of inhibiting metastasis of cancer cells.

The results of the present invention provide the first experimental evidence that IO can confer anti-inflammatory effects through inactivation of NF-κB transcription factors in macrophages. Further studies have shown that a single active ingredient purified from IO can inhibit the activation of NF-κB transcription factor at lower concentrations and reduce the incidence of metastasis in in vivo models as well as chronic inflammation. This result is valuable.

Hereinafter, a preferred embodiment for the practice of the present invention will be described in more detail. However, the scope of the present invention is not limited by these examples.

<Example 1>

IO  Preparation of Extract

Brown algae Ishige okamurae was collected from October 2004 to March 2005 on the coast of Jeju Island, South Korea. Fresh IO was washed three times with tap water to remove salts, creeping plants, and sand adhering to the surface of the sample and stored at -20 ° C. The frozen sample was lyophilized, homogenized with a grinder and then extracted. 1 kg of dried IO powder was extracted with 95% EtOH (1:10 w: v) and evaporated in vacuo. The concentrated IO extract was used after dissolving in DMSO.

<Example 2>

Cell culture

Cell lines were transferred to T-75 tissue culture flasks (Nunc, Denmark) in a monolayer using appropriate medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 10 μg / mL penicillin-streptomycin in a 5% CO ₂ and 37 ° C. wet environment. Individual cultures were made. DMEM was used as culture medium for RAW 264.7 cells. Cells were treated with a scraper and passed three times per week, five times before being used for the experiment.

<Example 3>

MTT  analysis

Toxicity levels of IO extracts to RAW 246.7 cells were determined according to the method described in Hansen et al., 1989, MTT [3- (4,5-dimethyl-2-yl) -2,5-diphenyltetrazolium bromide ] Using the method.

Cells were incubated at a density of 5 × 10 ³ cell / well in 96-well plates. After 24 hours, cells were washed with fresh medium and treated with different concentrations of IO extract. After 48 hours of incubation, the cells were rewashed and 20 μl of MTT (5 mg / mL) was added and incubated for 4 hours. Finally, formazan salt formed by dissolving DMSO (150 μl) was dissolved, and the amount of formazan salt was determined by measuring OD at 540 nm using a GENios ™ microplate reader (Tecan Austria GmbH, Austria). Relative cell viability was measured by the amount of MTT converted to formazan salt. The viability of the cells was quantified in% relative to the control (OD of blank treated OD / OD of control OD-blank of 100) and a dose response curve was generated. Data is presented as mean from three or more experiments, with P <0.05 considered to be significant.

<Example 4>

TNF -α, IL -1β and IL -6 Enzyme Immunoassay

Solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) according to the manufacturer's protocol without prior extraction and purification of inflammatory mediators such as TNF-α, IL-1β and IL-6 (Code RPN) 2751, RPN 2718 and RPN 2708, Amersham Pharmacia Oiosciences, NJ, USA). Standard curves from 10.24-400, 50-2450 and 51-2000 pg / mL were used to determine the concentrations of TNF-α, IL-1β and IL-6, respectively. The results were calculated using linear regression of a four-parameters logistic model.

Example 5

PGE ₂ Enzyme immunoassay

PGE ₂ was measured by enzyme immunoassay without prior extraction and purification according to the manufacturer's protocol (Code RPN 222, Amersham Pharmacia Oiosciences, NJ, USA). Standard curves were generated using concentrations ranging from 2.5 to 320 pg / well. PGE ₂ concentrations in the samples were calculated using linear regression of the 4-parameter logistic model.

<Example 6>

Transfection  And reporter gene analysis

RAW 246.7 cells were planted in 24-well plates and incubated at 37 ° C. At about 70-80% adhesion, cells were washed with DMEM and incubated for 5 hours with DMEM without serum and antibiotics.

The cells were then subjected to pcDNA 3.1 300 where the total amount of DNA per well was controlled by 1 μg of the reporter construct containing the NF-κB binding site sequence and the Lipofectamine ™ reagent (Invitrogen, USA) according to the manufacturer's instructions. The DNA mixture containing ng was transfected.

After incubation for 72 hours, the cells were eluted and luciferase activity was measured using a luminometer (Tecan Austria GmbH, Austria). Luciferase activity was normalized to transfection efficiency by β-galactosidase activity using o-nitrophenyl β-galactopyranoside as substrate. Β-galactosidase staining was performed by a method as described in Lin et al. Data are shown as mean ± standard deviation (n = 3).

<Example 7>

Extraction of nuclear proteins

Cells were treated with IO extracts in the absence or presence of TNF-α and nuclear proteins were extracted. Briefly, cells were harvested with 1 mL of ice cold PBS and centrifuged at 4 ° C., 5000 rpm for 1 minute. Cell pellets were 0.4 mL buffer A containing 10 mM HEPES, ph 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Eluted on ice for 15 minutes.

Then 25 mL of 10% nonidet P-40 solution was added and the sample was vortexed for 15 seconds and then centrifuged at 4 ° C. for 5 minutes at 15000 rpm. The pellet was washed again with 0.5 mL Buffer A and resuspended in 5 mL Buffer B containing 20 mM HEPES, ph 7.9, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF.

The eluted nuclei were allowed to stand on ice for 30 minutes and then centrifuged at 4 ° C. for 5 minutes at 15000 rpm. Nuclear protein concentration was determined by DC protein analysis (Bio-Rad, Hercules, CA, U.S.A.). Nuclear extracts were stored at -80 ° C until use.

<Example 8>

Weston Blot  analysis

Western blot analysis was performed according to standard procedures. Briefly, RAW 264.7 cells were treated with 50 mM Tris-HCl, pH 7.5 0.4% Nonidet P-40, 120 mM NaCl. Elution was performed in the elution buffer containing 2 mM phenylmethylsulfonyl fluoride, 80 μg / mL leupetin, 3 mM NaF and 1 mM DTT.

Cell eluate (10 μg) was dissolved on a 4-20% Novex® gradient gel (Invitrogen, USA), electrophoresed with nitrocellulose membrane and blocked with 10% skim milk. . Using p50 monoclonal antibody (Cat. No. sc-8414, Santa cruz Biotechnology, Inc., USA) with a chemiluminescent ECL kit (Amersham Pharmacia Biosciences, NJ, USA) according to the manufacturer's instructions p50 was detected. Western blots were quantified with ImageMaster software (Amersham Pharmacia Biosciences, NJ, USA).

Example 9

gelatin Zymography

MMP-2 and MMP-9 activity was measured by zymography in the absence or presence of IO extracts as described above. Cells were exposed to various concentrations of IO extracts for 1 hour before each HT1080 cells were treated with 10 ng / mL PMA, followed by incubation for 3 days in FB free medium. Sample buffer (125 mL Tris-HCl, pH 6.8 3% SDS, 40% glycerol, without boiling the control medium containing 50 μg of total protein (or the reaction product of the active MMP-2 with varying concentrations of the IO extract) 0.02% bromophenol blue) and electrophoresed on 10% polyacrylamide gels containing 1.5 mg / mL gelatin under non-reducing conditions.

After electrophoresis, the gel was washed twice with 50 mM Tris-HCl (pH 7.5) containing 2.5% Triton X-100, 37 in development buffer containing 10 mL CaCl ₂ , 50 mL Tris-HCl and 150 mL NaCl. Incubate overnight at ° C. Gels were stained with 0.25% Coomassie Blue R-250 in 30% methanol and 10% acetic acid and bleached without Coomassie blue dye in the same solution. Gelatinolytic bands were observed as clear bands against a blue background and the intensity of the bands was measured using Imagemaster software (Amersham Pharmacia Biotect, Sweden).

<Example 10>

Statistical analysis

All data were compared using a two-tailed independent Student's t-test. P values less than 0.05 were considered statistically significant. Data are presented as mean ± SE.

<Example 11>

RAW  246.7 on proliferation of cells IO  Effect of Extract

Since the purpose of the present invention is to test the inhibitory effect of IO extract on inflammation, the effect of IOE dissolved in DMSO on the viability of RAW 246.7 cells was first measured by MTT assay.

1 is a diagram showing the effect of IO extract on the viability of RAW 246.7 cells in the presence of FBS, where cells were treated with the indicated concentration of IO extract, cell viability was measured by MTT assay after 24 hours, and data Mean ± SD from three experiments.

As shown in FIG. 1, the IO extract showed no cytotoxic effect on cell viability even when treated for 24 hours at the highest test concentration (100 μg / mL). Therefore, a study of anti-inflammatory effects was conducted by selecting IO extract concentrations ranging from 1 to 10 μg / mL.

<Example 12>

For the production of inflammatory mediators IO  Inhibitory Effect of Extracts

In order to elucidate the inhibitory effect of IO extract on the production of inflammatory mediators, immunoassays for TNF-α, IL-1β, IL-6 and PGE ₂ were performed. To this end, RAW 246.7 cells were treated with different concentrations of IO extract for 1 hour followed by lipopolysaccharide 1 μg / mL to promote inflammation. After incubation for 1 day, control medium was collected and thus the level of inflammatory mediators was measured.

Figure 2 shows the inhibitory effect of the IO extract on the production of inflammatory mediators in RAW 246.7 cells, after treatment of cells with different concentrations of IO extract, then treated with 1 μg / mL polyliposaccharide to promote inflammation. I was. After incubation for 1 day, the conditioned medium was collected and subjected to immunoassay for TNF-α (Pennel A), IL-1β (Pennel B), IL-6 (Panel C) and PGE ₂ (Panel D). It was. 1 μM of dexamethasone and 10 μM of aspirin were used as controls in cytokines and PGE ₂ , respectively. Data are shown as mean ± SD from three experiments.

As shown in FIG. 2, the IO extract showed a dose dependent inhibitory effect on the production of all inflammatory mediators. Treatment with 10 μg / mL IO extract reduced TNF-α levels by 30% compared to the blank group (see FIG. 2A). No significant difference was found in the inhibitory effect on the production of TNF-α between the group treated with 1 μM dexamethasone and the group treated with IO extracts of about 5 μg / mL.

As shown in FIG. 2B, the inhibitory effect of the IO extract on the production of IL-1β was remarkable compared to the inhibitory effect on the production of other cytokines. 10 μM of IO extract showed 80% inhibitory effect on the production of IL-1β. The level of IL-6 in the presence of 10 μM of IO extract was similar to the level in the presence of 1 μM of dexamethasone (FIG. 2C). The inhibitory effect of the IO extract on the production of PGE ₂ was higher than those for other cytokines (FIG. 2D).

In particular, treatment with 1 μg / mL of IO extract reduced the level of PGE ₂ by 60%, similar to the level when treated with 10 μM of aspirin used as a positive control. Although the inhibitory effect of IO extract on PGE ₂ levels was somewhat different according to the levels of cytokines, the trend of inhibition effect was similar among treatment groups as the concentration of IO extract increased.

Example 13

RAW  In 246.7 cells NF for transcriptional activity of -κB IO  Effect of Extract

Whether transcriptional activity of NF-κB in RAW 246.7 cells is affected by IO extracts was further studied using a reporter construct comprising the NF-κB binding DNA sequence-luciferase gene.

3 is a diagram showing the effect of IO extract on the transcriptional activity of NF-κB in RAW 246.7 cells, the cells were treated with different concentrations of IO extract for 1 hour and then treated with 10 ng / mL TNF-α NF-κB was activated.

(A) PCDNA 3.1 vectors were co-infected to normalize luciferase activity for all treatment groups, and infected cells were stained with β-galactosidase. (B) The transcriptional activity of NF-κB was measured by luciferase analysis after co-infection with the NF-κB reporter vector and the pcDNA 3.1 vector. Data are presented as mean ± S.E. In three or more experiments. Student's t-test was used to confirm the level of saliency (P <0.01).

As shown in FIG. 3A, RAW 246.7 cells were co-infected with a reporter vector of NF-κB and a pcDNA 3.1 vector representing about 40% SA-β gal positive cells. As shown in FIG. 3B, the transcriptional activity of NF-κB in cells treated with IO extracts was markedly reduced in a dose dependent manner compared to the untreated blank group (P <0.01).

Transcriptional activity of NF-κB by 10 ng / mL TNF-α treatment was enhanced by 500% compared to the untreated blank group. Treatment with 1 and 5 μg / mL IO extracts reduced transcriptional activity of NF-κB by about 5 and 2 fold as compared to the untreated blank group, respectively. These results show that IO extracts can inhibit the production of inflammatory mediators such as TNF-α, IL-1β, IL-6 and PGE ₂ by inhibiting the transcriptional activity of NF-κB in RAW 246.7 cells.

<Example 14>

RAW  In 246.7 cells p50 For protein expression IO  Effect of Extract

To directly confirm the inhibitory effect of the IO extract on the activation of NF-κB transcription factor involved in inflammation, Western blot analysis was used to test the expression level of p50, which is part of the NF-κB transcription factor, in the presence of IO extract. .

FIG. 4 shows Western blot analysis of p50 protein onset in RAW 246.7 cells after treatment with IO extract. As indicated, Western blot analysis of cell elution was performed using p50 monoclonal antibody. Lower panel shows individual relative protein expression%. Values are expressed as relative protein expression using the following formula. % Relative protein expression = (strength of band / intensity of blank band) X 100. Expression of β-actin protein was used as a control for standardization of p50 protein.

As shown in FIG. 4, there was no significant difference between the TNF-α promoting group and the group treated with the 1 μg / mL IO extract. However, when treated with an IO extract of about 5 μg / mL, the expression level of p50 in the nucleus was significantly decreased (P <0.01) compared to the TNF-α promoting group. Moreover, the expression level of p50 increased with increasing concentration of IO extract. These results show that the inhibitory effect of IO extract on the production of inflammatory mediators may be due to the inactivation of NF-κB transcription factor in RAW 246.7 cells.

<Example 15>

HT1080  In the cell MMP For IO  Inhibitory Effect of Extracts

Based on these results, further studies were conducted on whether the IO extracts could inhibit the expression and activity of MMPs involved in chronic inflammation. Regulatory medium of HT1080 cells treated with IO extract was used for gelatin zymography.

Figure 5 shows the effect of IO extract on the activity of MMP-2 and MMP-9 from HT1080 cells, cells were treated with 10 ng / mL PMA under serum-free conditions to induce MMP expression. After incubation for 3 days, the control medium was reacted with various concentrations of the IO extract for 1 hour, followed by gelatin zymography. The bottom panel shows the relative enzyme activity as% of each blank group.

As shown in FIG. 5, treatment with PMA increased the expression level and activity of MMP-2 and MMP-9 compared to the blank group. IO extracts of 5 μg / mL or more showed a dose dependent inhibitory effect on the expression level and activity of MMP-2 and MMP-9 (P <0.01). The expression level of MMP-9 in the treatment group treated with 10 μg / mL of the IO extract was 8-fold reduced compared to the group treated with 1 ng / mL of phorbol 12-myristate 13-acetate (PMA). There was no significant difference between the 1 μg / mL IO extract treated group and the PMA treated group. However, significant inhibitory effects on MMP-2 and MMP-9 expression, respectively, were observed at IO extract concentrations above 5 μg / mL (P <0.01).

Ishige Okamura extract according to the present invention inhibits the transcriptional activity of NF-κB in RAW 246.7 cells and anti-inflammatory activity by inhibiting the production of inflammatory mediators TNF-α, IL-1β, IL-6 and PGE ₂ It is a very useful invention in the development of natural anti-inflammatory agents in the pharmaceutical industry.

1 is a diagram showing the effect of IO extract on the viability of RAW 246.7 cells in the presence of FBS.

Figure 2 shows the inhibitory effect of IO extract on the production of inflammatory mediators in RAW 246.7 cells.

Figure 3 shows the effect of IO extract on the transcriptional activity of NF-κB in RAW 246.7 cells.

Figure 4 shows Western blot analysis of protein onset of p50 in RAW 246.7 cells after treatment with IO extract.

5 is a diagram showing the effect of the IO extract on the activity of MMP-2 and MMP-9 from HT1080 cells.

Claims (4)

(Ishige to Ishikawa's Okamura having inflammatory activity-suppressed transcriptional activity of NF-κB in RAW 246.7 cells and inflammatory mediators of TNF-α, IL-1β, IL-6 and inhibit the production of PGE _2, wherein okamurae ) extract. The Ishige Okamura extract according to claim 1, wherein the extract is an ethanol extract. An anti-inflammatory pharmaceutical composition comprising the extract of Ishige Okamura of claim 1 or 2 as an active ingredient. The anti-inflammatory pharmaceutical composition according to claim 3, wherein the Ishige Okamura extract is contained at 1 to 1000 mg / mL.

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