KR20140072464A - Composition for prevention or treatment of inflammatory diseases comprising hexane fraction from orostachys japonicus - Google Patents

Abstract

The present invention relates to an use of a nucleic acid fraction derived from Orostachys japonicas having anti-inflammatory activity, and more particularly to a composition and a health functional food comprising a nucleic acid fraction of Orostachys japonicas as an active ingredient for preventing or treating (alleviating) inflammatory diseases. The nucleic acid fraction of Orostachys japonicas of the present invention has an effect of inhibiting nitric oxide (NO) generation through inhibition of iNOS expression. Also, the present invention inhibits NF-κB activation and reduces Akt phosphorylation by inhibiting transportation of NF-κB p65 from cytosol into nucleus and by inhibiting IκBα phosphorylation. Moreover, the present invention has an outstanding anti-inflammatory effect through a mechanism of repressing a cell surface expression of CD14 and TLR4 proteins, which are LPS receptors. Thus, the composition of the present invention comprising the nucleic acid fraction of Orostachys japonicas as an active ingredient can effectively inhibit inflammation to thereby being usefully applied to inflammation-associated diseases.

Description

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

The present invention relates to a method of treating osteoarthritis japonicus) it relates to the use of nucleic acid-derived fractions, and more particularly to prevent or treat inflammatory conditions (composition and health functional food for improving) comprising a nucleic acid fraction of wasong as an active ingredient.

Inflammation reactions are bio-defense reactions caused by various factors such as infection by pathogens or tissue damage, and they perform an initial protective action to confine the damage only to the infected area or damaged area. In most cases, this inflammatory response leads to the elimination of pathogenic factors and the induction of specific adaptive immunity using components of endogenous immunity (Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm. Immunologic Research. 23, pp.99-109, 2001). The rubor, tumor, calor, and dolor, which are known to be associated with inflammation, are caused by inflammatory mediators and cytokines at the site of inflammation, And increased localization of blood vessels, increase of extracellular flux of plasma components due to increased permeability of blood vessels, increase of extracellular flux of immune cells due to increase of adhesion to circulating immune cells of vascular endothelial cells, (Gallo RL, Murakami M, Takaaki O, Zaiou M. Biology and clinical relevance of naturally occurring antimicrobial peptides, J. Allergy. Clin. Immunol. 110, pp. 823 2002, Graeme B. Ryan, MB, and Guido M., Acute Inflammation, American Journal of Pathology, 86 (1), pp. 185-274, 1977). Histamine produced in some cells as a response to tissue damage, and low molecular peptide kinin, which is inactivated in the blood and activated by tissue damage, also contributes to increased inflammation-related vasodilation and vascular permeability (Yamaki K, Thorlacius H, Xie X, Lindbom L, Hedqvist P, Raud J., Characteristics of histamineinduced leukocyte rolling in the undisturbed microcirculation of the rat mesentry. British J. Pharmacol. 123, pp. 390-399, 1998; Brocklehurst WE, Role of Kinins and Prostaglandins in Inflammation, Proc. Roy. Soc. Med., 64, pp. 4-6, 1971).

In general, the acute inflammatory reaction is rapid and lasts for a short period of time. Acute inflammation is accompanied by a systemic reaction called acute phase reaction. On the other hand, chronic inflammation can be induced as a result of persistent immune activation in connection with some diseases such as infections or autoimmune diseases, and the accumulation and activation of macrophages is evidence of a chronic inflammatory response (Huang AL, Vita JA , Effects of Systemic Inflammation on Endothelium-Dependent Vasodilation. Trends, Cardiovasc. Med. 16, p. However, persistent chronic inflammatory reactions can cause serious damage to host cells or tissues.

The inflammatory response at the site of infection is initiated by the response of macrophages to the pathogen. Inflammatory mediators such as reactive oxygen species and active nitrogen species (e.g., NO), prostaglandins, rucotrienes, etc. produced by macrophages activated by pathogens, and inflammatory mediators such as TNF-a, IL-6 and IL-8 Inflammatory cytokines are known to be involved in the inflammatory response (Renauld JC, New insights into the role of cytokines in asthma, J. Clin. Pathol 54, pp.577-589, 2001; Blake GJ, Ridker PM , Tumor necrosis factor-alpha, inflammatory biomarkers, and atherogenesis. Eur. Heart J. 23, pp.345347, 2002). Activation of NF-κB, a transcription factor of genes involved in the production of these inflammatory mediators, is crucial to the inflammatory action of macrophages. Inflammatory genes such as inducible nitric oxide synthase (iNOS2), cyclooxygenase (COX-2), TNF-α, IL-6 and IL- lt; / RTI >

Nitric oxide (NO) is produced from L-arginine by nitric oxide synthetase (NOS) in the human macrophage. The NOS of the human body includes three types of endothelial constitutive NOS (hereinafter referred to as ecNOS), neuronal constitutive NOS (hereinafter referred to as ncNOS) and inducible NOS (hereinafter referred to as iNOS) There is an isomerism. Both ecNOS and ncNOS are expressed in endothelial cells and neuroendocrine cells, and are dependent on calcium and calmodulin, while iNOS is a lipopolysaccharide (LPS) -1, TNF-α, and immunostimulants such as radiation alone, it is expressed in many cells and is independent of calcium and calmodulin. NO plays a protective role against tumor cells and pathogens at high concentrations, and low levels of NO produced by vascular endothelial cells regulate blood pressure, while NO generated from neurons is a neurotransmitter, And performs various related physiological responses. Non-inducible constitutive NOS (cNOS) plays an important role in maintaining the homeostasis of human body. NO produced by ecNOS acts on the vasculature to inhibit vasodilation, platelet adhesion and aggregation, and NO produced by ncNOS It acts on the nervous system to increase the long-term memory ability important for memory ability, or to cause depression as a neurotransmitter, and it is also involved in digestive organ motility and penile erection.

On the other hand, NO produced by iNOS expression induced by a specific cytokine or LPS acts on inflammation, host defense, and the like. In addition, activation of transcription factor NF-κB in macrophages by stimulation of endotoxin induces iNOS expression, which leads to increased production of NO (Butler, AR, Chemistry & Industry , 16: 828, 1995). When iNOS expression is induced by external stimuli such as LPS, inflammatory factors, and irradiation, a large amount of NO is continuously produced for 4 to 6 hours, which is known to cause inflammation in the human body. In addition, in rats, a large amount of iNOS mRNA and protein is expressed in macrophages by external stimulation of LPS, and synthesized NO performs microbial sterilization and antitumor action. However, it is known that excessive NO production is unnecessarily caused to cause inflammatory diseases such as arthritis and sepsis, immune diseases such as tissue graft rejection reaction, autoimmune disease diabetes, and nerve cell death.

Therefore, the development of an iNOS activity inhibitor is highly likely to be developed as a therapeutic agent for such diseases. From this viewpoint, a compound that inhibits NO production by iNOS can be used as a therapeutic agent for various inflammatory diseases of the human body. Studies on the inhibition of NO production have been mainly focused on the development of substances that specifically inhibit the enzyme activity of iNOS, including precursors of derivatives of L-arginine, derivatives of L-citrulline, Aminoguanidine derivatives, and isothiourea derivatives (Babu, BRB and Griffith OW, Current Opinion in Chemical Biology, 2: 491, 1998).

On the other hand, NF-κB is activated by various external stimuli such as inflammation-induced cytokines, toxic compounds, bacterial infections, viral infections, radiation, UV, and active oxygen It is known that it acts to regulate the expression of proteins involved in various cell responses such as apoptosis, immune response and inflammatory response. Such NF-κB is a transcription factor composed of homodimers or heterodimers of five proteins including p50 and p65 (RelA) proteins. In the cytoplasm, NF- Lt; RTI ID = 0.0 > inactivated < / RTI > However, IκB kinase (IκB kinase, IKK) is activated through a variety of external stimuli such as inflammatory cytokines, toxic compounds, bacterial infections, viral infections, radiation, UV and active oxygen to phosphorylate IκB bound to NF- , Resulting in the release of NF-κB from IκB. The liberated NF-κB enters the nucleus and functions as a transcription factor for a variety of inflammatory or immune responses, cell proliferation-related genes.

Although many anti-inflammatory drugs that can act on such mechanism have been developed to date, long-term use due to side effects has been a problem. Efforts to find the active ingredient in natural products have been actively pursued as a solution to such problems.

Meanwhile Orostachys japonicus is a perennial herbaceous plant of the stones and has been widely used in anti-inflammation, antipyretic, detoxification and hemostasis in Korea. Friedelin, epi-friedlanol, grutinone, glutinol, triterpenoid, β-sitosterol, campesterol, sterol, fatty acid ester, kaempferol, quercetin, flavonoid, 4-hydroxybenzoic acid, 3,4 and aromatic acids such as dihydroxybenzoic acid and gallic acid. However, single substances related to some foods have been reported, but there has been no research on physiological activity and molecular biology.

Thus the inventors of the eopeumyeonseo adverse effect on the living body was excellent anticancer action is seeking to find a natural substance, wasong (Orostachys japonicus ), and their pharmacological effects were examined. As a result, Orostachys japonicus- derived nucleic acid fractions reduce intracellular NO concentration through inhibition of iNOS protein expression; Inhibit the activation of NF-κB by inhibiting the migration of NF-κB p65 from the cytoplasm to the nucleus and inhibiting IκBα phosphorylation; Weaken Akt phosphorylation; The present inventors have completed the present invention by confirming that it is possible to effectively prevent or treat inflammation-related diseases, since they can exhibit anti-inflammatory activity through the mechanism of inhibiting the cell surface expression of LPS receptors CD14 and TLR4 protein.

Korean Patent Publication No. 10-2010-0033573 Korean Patent Publication No. 10-2005-0058635

Accordingly, it is an object of the present invention to provide a composition which is free from side effects in vivo and has excellent anti-inflammatory activity, thus effectively preventing or treating inflammatory diseases.

Another object of the present invention is to provide a health functional food which is free from adverse effects on living body and has excellent anti-inflammatory activity and can effectively improve inflammatory diseases.

In order to achieve the object of the present invention, the present invention provides a composition for preventing or treating an inflammatory disease comprising an active ingredient of a nucleic acid fraction of Orostachys japonicas .

In one embodiment of the present invention, the nucleic acid fraction of the above-mentioned source can be a fraction obtained by adding a nucleic acid to an ethanol extract obtained by adding 95% ethanol to a feed powder.

In one embodiment of the invention, the nucleic acid fraction of the over-the-wash may be included in the composition at a concentration of 10-100 μg / ml.

In one embodiment of the present invention, the nucleic acid fraction of the over-secretase reduces intracellular NO concentration through inhibition of iNOS protein expression; Inhibit the activation of NF-κB by inhibiting the migration of NF-κB p65 from the cytoplasm to the nucleus and inhibiting IκBα phosphorylation; Weaken Akt phosphorylation; It may have anti-inflammatory activity through mechanisms that inhibit the cell surface expression of LPS receptors CD14 and TLR4 protein.

In one embodiment of the present invention, the inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, rheumatoid multiple muscular pain, arteriosclerosis, inflammatory visceral disease, ulcerative colitis, osteoporosis, Crohn's disease, encephalomyelitis, meningitis, pancreatitis, Inflammatory bowel disease, osteomyelitis, encephalitis, meningitis, rhinitis, acute bronchitis, chronic bronchitis, osteoarthritis, gout, spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, vasculitis, lymphocytic chorioamnionitis, glomerulonephritis, uveitis, Anaphylactic shock, burns, infections, bacterial infections, viral infections, fungal infections and congenital infections, psoriasis, atopic infections, psoriasis, acute respiratory distress syndrome, asthma, pain, septic shock, ischemia, ulceration, multiple sclerosis, Alzheimer's disease, Alzheimer ' s disease, Alzheimer ' s disease, Alzheimer ' s disease, Parkinson ' s disease, autoimmune diseases and autoimmune diseases.

The present invention also provides a health functional food for improving inflammatory diseases comprising an active ingredient of a nucleic acid fraction of Orostachys japonicas .

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ram noodles, gums, candy, ice cream, alcoholic beverages, .

The nucleic acid fractions of Orostachys japonicas of the present invention inhibit NO production through inhibition of iNOS expression as well as NF-κB p65 through inhibition of NF-κB p65 migration from the cytoplasm to the nucleus and inhibition of IκBα phosphorylation Inhibits Akt phosphorylation and has an excellent antiinflammatory effect through mechanisms that inhibit the cell surface expression of LPS receptors CD14 and TLR4 proteins. Therefore, the composition of the present invention, which comprises it as an active ingredient, And can be effectively used for inflammation-related diseases.

Figure 1 graphically shows the cell viability by treatment with mouse macrophages at different concentrations (12.5, 25, 50, 75, 100 μg / ml) of the fresh nucleic acid fractions of the present invention (OJH: fraction of fresh nucleic acids).
FIG. 2 is a graph showing the amount of nitric oxide produced by pretreatment of mouse macrophages according to the concentration of the carrier nucleic acid fraction (12.5, 25, 50, 75, 100 μg / ml) of the present invention (OJH;
FIG. 3 shows Western blot analysis of the expression levels of iNOS and COX-2 protein in mouse macrophages according to the pretreatment of the concentration of the source nucleic acid fractions (25, 50, 75, 100 μg / ml) of the present invention (OJH: Nucleic acid fractions).
FIG. 4 shows RT-PCR analysis of the expression levels of iNOS and COX-2 gene in mouse macrophages according to the pretreatment of the concentration of the nucleic acid fractions of the present invention (25, 50, 75, 100 μg / ml) Waxy nucleic acid fractions).
FIG. 5 shows Western blot analysis of the amount of phosphorylated NF-κB p65 and IκBα protein expression in mouse macrophages after pretreatment of the fresh nucleic acid fractions of the present invention over time (OJH: fraction of nucleic acid fractions of wastes).
Figure 6 shows the Western blot analysis of the inhibition of NF-κB p65 migration from the cytoplasm to the nucleus by pretreatment of mouse macrophages with the concentration of the source nucleic acid fraction (25, 50, 75, 100 μg / ml) of the present invention (OJH: fraction of wasteweed nucleic acid).
FIG. 7 is a Western blot analysis of the degree of degradation of p-IκBα according to the pretreatment of mouse macrophages (25, 50, 75, 100 μg / ml) according to the concentration of the nucleic acid fractions of the present invention.
8 is a graph showing the degree of migration of NF-κB p65 to the nucleus according to the pretreatment of the nucleic acid fractions of the present invention by immunofluorescence using a confocal microscope (control group; LPS; LPS (1 μg / ml), LPS + OJH, OJH (100 占 퐂 / mL) and LPS treatment group.
FIG. 9 is a graph showing the changes in the amount of phosphorylated ERK, JNK, p-38 and Akt protein expression in the mouse macrophages according to the pretreatment of the fractions of the nucleic acid fractions of the present invention (25, 50, 75 and 100 μg / (OJH: nucleic acid fractions of Wassong).
FIG. 10 shows Western blot analysis of the amount of phosphorylated Akt protein expressed over time after pretreatment of the nucleic acid fractions of the present invention with mouse macrophages (OJH: fraction of wasteweed nucleic acid).
FIG. 11 is a graph in which cells expressing CD14 after pretreatment of the wild-type nucleic acid fractions of the present invention with mouse macrophages were analyzed using a flow cytometer (curves of gray areas show basic signals from an isotype-matched IgG control group, The graph represents the untreated group, the dotted line represents the LPS-treated group, and the bolded line graph represents the LPS-treated group after the transfection of nucleic acid fractions.
12 shows Western blot analysis of the expression level of TLR4 protein expressed on the cell surface by pretreatment of mouse macrophages with the concentration of the source nucleic acid fractions of the present invention (25, 50, 75, 100 μg / ml) (OJH: Waxy nucleic acid fractions).

The present invention relates to the use of anti-inflammation of the nucleic acid fraction of Wassong, and is characterized by providing a composition for preventing or treating an inflammatory disease containing the nucleic acid fraction of Wassong as an active ingredient.

In the present invention, " Orostachys " japonicus ) is a perennial herbaceous plant with a dicotyledonous rosemary crassacea called a rocksol, and it is said that a herbal book has a special effect on fever, geothermal, hepatitis, eczema, burn and the like. In particular, it is known that diabetes, paralysis, arthritis, gastrointestinal diseases, sore legs, limp, constipation, vomiting, various diseases and various diseases.

Friedelin, epi-friedlanol, grutinone, glutinol, triterpenoid, β-sitosterol, campesterol, sterols, fatty acid esters, kaempferol, quercetin, flavonoids, 4-hydroxybenzoic acid, 3 , 4-dihydroxybenzoic acid, and gallic acid. However, single substances related to some foods have been reported, but there has been no research on physiological activity and molecular biology.

The present inventors have found that the nucleic acid fractions of the present invention having the above characteristics can reduce the intracellular NO concentration through inhibition of iNOS protein expression; Inhibit the activation of NF-κB by inhibiting the migration of NF-κB p65 from the cytoplasm to the nucleus and inhibiting IκBα phosphorylation; Weaken Akt phosphorylation; It has been confirmed that it has anti-inflammatory activity through the mechanism of inhibiting the cell surface expression of LPS receptors CD14 and TLR4 protein, thereby proving that it can prevent or treat inflammatory diseases.

As a result of examining the inhibitory effect of intracellular nitric oxide in the fraction of wastes nucleic acid in Experimental Example 2 of the present invention, it was found that the amount of nitric oxide produced by LPS alone treatment in mouse macrophages was dependent on the treatment concentration of the fraction As shown in Fig. In particular, at a concentration of 100 μg / ml, it appeared to be similar to that of the control group in which nothing was treated.

In addition, in the following Experimental Example <3-1> of the present invention, the effect of the fractions of the transgene nucleic acid on the expression of iNOS and COX-2 protein was examined when LPS alone was administered to mouse macrophages, Whereas it was confirmed that the expression of iNOS protein was inhibited in the test group pretreated with the fractions of the present invention. However, the pretreatment of the fresh nucleic acid fractions of the present invention did not affect COX-2 protein expression (see FIG. 3).

In the following Experimental Example <3-2 of the present invention, the effect of the fraction of wasted nucleic acid on the expression of iNOS and COX-2 gene was examined. As a result, -2 gene expression (see Fig. 4).

As a result of examining the effect of the nucleic acid fractions of the present invention on NF-B activity, the NF-B p65 and IκBα phosphorylation proceeded from 15 minutes after treatment with LPS alone in mouse macrophages, In contrast, the NF-B p65 and IκBα phosphorylation occurred in 15 minutes and continued until 30 minutes and was suppressed after 60 minutes in the pretreatment group of the fresh nucleic acid fractions of the present invention 5). In addition, the present inventors investigated the migration of NF-κB p65 from the cytoplasm to the nucleus through western blot analysis. As a result, as shown in FIG. 6, the migration of NF-κB p65 from the cytoplasm into the nucleus was enhanced in the case of treatment with LPS only, whereas in the pretreatment group of the fresh nucleic acid fraction of the present invention, NF- it was confirmed that the migration of p65 to the nucleus was inhibited (see Fig. 6).

Further, as a result of examining the effect of the fresh nucleic acid fraction on MAPKs and PI3K / Akt activity in Experimental Example 5 of the present invention, LPS stimulation in mouse macrophages induces phosphorylation of MAPKs and Akt, In the pretreatment group, the phosphorylation of Akt was inhibited depending on the concentration of the fraction treated. However, pretreatment of the fresh nucleic acid fractions of the present invention did not inhibit the phosphorylation of ERK1 / 2, JNK, and p38 (see FIG. 9).

In the following Experimental Example 6 of the present invention, the effect of the fractions of the nucleic acid fragments on the cell surface expression of CD14 and TLR4 proteins was examined. As a result, when LPS alone was administered to mouse macrophages, CD14 expression was significantly increased on the cell surface, It was confirmed that CD14 expression was weakened in the experimental group pretreated with the fresh nucleic acid fraction of the present invention (see FIG. 11). It was also confirmed that TLR4 expression was effectively inhibited depending on the treatment concentration in the test group pretreated with the nucleic acid fractions of the present invention (see FIG. 12).

From these results, the present inventors have experimentally proved that the fractions of Waste nucleic acid have excellent anti-inflammatory activity.

Therefore, the composition of the present invention comprising the source nucleic acid fraction as an active ingredient can effectively prevent or treat an inflammatory disease.

The term "inflammatory disease" in the present invention means a tumor necrosis factor-alpha (TNF-alpha) secreted by macrophage-like immune cells, IL-1 (inflammatory cytokines) such as interleukin-1, IL-6, prostagladin, luecotriene or nitric oxide (NO).

In the present invention, "treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term treatment as used herein refers to the act of treating when "treating" is defined as above. Thus, "treatment" or "treatment regimen" of an immune disorder in a mammal may include one or more of the following:

(1) inhibiting the development of inflammatory diseases,

(2) prevent the spread of inflammatory diseases,

(3) relieving inflammatory diseases,

(4) preventing recurrence of inflammatory diseases and

(5) relieving the symptoms of inflammatory diseases (palliating)

In the present invention, the above-mentioned nucleic acid fractions may be fractions obtained by extracting a natural extract from a natural source using an extractive isolation method known in the art, followed by addition of a nucleic acid solvent.

The 'extract' as defined in the present invention is extracted from the wort using an appropriate solvent, and includes, for example, a crude extract of Wassong, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

As an appropriate solvent for extracting the extract from the feed, any organic solvent that is pharmaceutically acceptable may be used, and water or an organic solvent may be used, and for example, purified water, methanol acetone, ether, benzene, chloroform (such as methanol, ethanol, propanol, isopropanol, and butanol) chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane may be used alone or in combination. It is preferable to use ethanol (alcohol), more preferably 95% ethanol.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the method for producing the worts extract of the present invention, and any known method can be used.

For example, the extract of Wakoshi contained in the composition of the present invention can be prepared into a powdery state by an additional process such as vacuum distillation, freeze-drying, or spray drying, .

The extract of WakSong obtained through the above process can be further fractionated into fractions of Waste nucleic acid of the present invention by fractionation using an organic solvent.

The method for producing the nucleic acid fractions of the present invention according to one embodiment of the present invention will be described in more detail as follows.

First, 200 g of the warsh powder was extracted three times with 95% ethanol for 3 hours, and the ethanol extract was dried using a rotary evaporator to obtain a dry powder in which ethanol was evaporated, and then the above-mentioned waxy ethanol extract dried After the water is added to the powder to dissolve it, the added water and the same amount of hexane are mixed, stirred vigorously, and then allowed to stand for 30 minutes to separate into a nucleic acid fraction and a water fraction. The fractions of the fresh source nucleic acid fractions after the completion of the fractionation can be dried to finally produce the fresh source nucleic acid fractions of the present invention.

The composition of the present invention containing the above-mentioned source nucleic acid fraction as an active ingredient may be a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the source nucleic acid fraction of the present invention may be contained in the composition at a concentration of 10 to 100 μg / ml, and the source nucleic acid fraction of the present invention may be contained in an amount of 0.1 to 95% .

Inflammatory diseases in which the pharmaceutical composition of the present invention may exhibit therapeutic effects include arthritis, rheumatoid arthritis, rheumatoid multiple muscular pain, arteriosclerosis, inflammatory visceral disease, ulcerative colitis, osteoporosis, Crohn's disease, Osteoarthritis, ankylosing spondylitis, psoriatic arthritis, vasculitis, lymphocytic chorioamnionitis, glomerulonephritis, uveitis, ileitis, liver, osteoarthritis, osteoarthritis, osteoarthritis, meningitis, pancreatitis, peritonitis, osteomyelitis, encephalitis, meningitis, rhinitis, acute bronchitis, Inflammatory bowel disease, inflammation, kidney inflammation, asthma, pain, septic shock, ischemia, ulcer, multiple sclerosis, scleroderma, hemorrhagic shock, anaphylactic shock, burn, infection, bacterial infection, , Psoriasis, atopic dermatitis, Reishmaniasis, schistosomiasis, hemodialysis, seizures, cardiovascular transplantation, ischemia, recurrent disease, hemochromatosis, hemochromatosis, diabetes mellitus Alzheimer's (Alzheimer) disease, Parkinson's (Parkinson) itdoe include bottles, Allografts geobujeung and autoimmune diseases such as, but not limited thereto. The composition of the present invention may also be a food composition, which may contain, as an additional component, various flavors or natural carbohydrates, such as conventional food compositions, in addition to containing the active ingredient, the source nucleic acid fraction.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the food composition may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate, etc.), pectic acid, Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The active ingredient of the present invention is a natural substance, which has few side effects such as chemical agents. Therefore, it can be safely used for long-term administration for the purpose of prevention or treatment of inflammatory diseases.

The present invention also provides a health functional food for the improvement of inflammatory diseases, which comprises the nucleic acid fraction of the present invention as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of improving inflammatory diseases.

In the present invention, the term &quot; health functional food &quot; refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture of the carrier nucleic acid fraction, which is an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives by a conventional method, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a normal hard capsule with a mixture of the nucleic acid fractions of the present invention, the active ingredient of the present invention, with additives such as excipients, etc. The soft capsule is prepared by dissolving the nucleic acid fractions And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding the mixture of the active ingredient of the present invention, the carrier nucleic acid fraction, the excipient, the binder and the disintegrant in a conventionally known method, and if necessary, Or it may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of the active ingredient of the present invention and the carrier nucleic acid fraction with an excipient, a binder, a disintegrant, etc. by a known method, and if necessary, adding a flavoring agent, And the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

The present invention also provides the use of a composition comprising an effective amount of a nucleic acid fraction of the present invention for the manufacture of a medicament for the prevention or treatment of an inflammatory disease or a food. The composition of the present invention containing the above-mentioned Wong-Song nucleic acid fraction as an active ingredient can be used for the manufacture of medicines or foods for the prevention or treatment of inflammatory diseases.

The present invention also provides a method of preventing or treating an inflammatory disease comprising administering to a mammal a nucleic acid fraction of the invention.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

In the therapeutic method of the present invention, the composition comprising the nucleic acid fractions of the present invention as an active ingredient may be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intramuscularly, transdermally, topically, And can be administered in a conventional manner.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example >

reagent

Mouse macrophage line RAW 264.7 was distributed from ATCC (American Type Culture Collection, Manassas, Va.). DMEM (Dulbecco's modified Eagle's medium), fetal bovine serum (FBS) and antibodies were purchased from Hyclone; Lipopolysaccharide (LPS) and grease reagents were purchased from Sigma (St. Louis, MO, USA); Antibodies to iNOS, COX-2, phospho-IκBα, NF-κB p65, phospho-ERK1 / 2, ERK1 / 2, phospho-p38, p38, phospho-JNK, JNK, Beverly, Mass., USA). Other compounds used analytical grade.

Statistical processing

All experiments were repeated at least 3 times and the results were expressed as mean + standard deviation. All experimental data were processed by the SPSS program (SPSS 12.0, SPSS Institute, and Chicago, IL). Statistical analysis was performed using ANOVA and Duncan's Multiple Range Test. Statistical significance was determined when the p-value was less than 0.05.

Cell culture

As mouse macrophage line, RAW264.7 cells were purchased from ATCC and cultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS and 1% 5,000 units / ml penicillin-5,000 μg / ml streptomycin at 37 ° C and 5 The cells were cultured in a% CO ₂ incubator, and the medium was changed at 2-day intervals. Subculture was performed 2 to 3 times a week.

< Example  1>

Welcome  Nucleic acid Fraction  Produce

First, 200 g of the warsh powder was extracted three times with 95% ethanol for 3 hours. The ethanol extract was dried on a rotary evaporator to obtain a dry powder, and then water was added to the dried powder of the wort ethanol extract The mixture was stirred vigorously and then allowed to stand for 30 minutes to separate the nucleic acid fraction (2.386 g) and the water fraction. After the fractionation, the fractions of the fresh nucleic acid fractions were dried using a rotary evaporator and used in the following experiments.

< Experimental Example  1>

The Welcome  Nucleic acid Fraction  Cytotoxicity measurement

In order to examine whether the fraction of the nucleic acid of the present invention prepared in Example 1 showed cytotoxicity, CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega Corporation, Madison, USA) Survival rate was measured.

Briefly, RAW 264.7 cells were seeded at a concentration of 5 × 10 ⁴ cells in a 96-well plate, treated with the concentration of the source nucleic acid fraction of the present invention at a concentration of 5% CO _{2 at} 37 ° C. for 24 hours, To the plate was added 20 μl of MTS [3- (4,5-dimethylthiaxol-2-yl) -5- (3carboxy-methoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt] CO ₂ 37 ° C. Absorbance was measured at 490 nm using a fluorescence multi-detection reader (Synergy HT, BioTek, USA).

As a result, as shown in FIG. 1, it was confirmed that there was no change in the cell survival rate according to the treatment concentration of the fresh nucleic acid fraction. From these results, the fresh nucleic acid fraction of the present invention can be safely used without toxicity even at 100 μg / ml And it was found.

< Experimental Example  2>

The Welcome  Nucleic acid Fraction  Nitric oxide ( NO ) Inhibitory effect

In order to confirm the nitric oxide suppression effect of the fresh nucleic acid fraction of the present invention prepared in Example 1, the amount of nitric oxide produced by the fraction treated with RAW 264.7 cells, a mouse macrophage cell line, was measured.

Nitric oxide (NO) is an important intracellular proinflammatory mediator, which reacts with a superoxide radical to produce peroxynitrite anion (ONOO-). The nitrite peroxide thus produced induces inflammatory activity and causes arthritis, ulcers It is known to cause a variety of pathological conditions such as chronic colitis and systemic lupus erythematosus. Therefore, the inflammatory reaction can be inhibited by a mechanism of inhibiting nitric oxide (NO) production.

To evaluate the production of nitric oxide (NO), RAW 264.7 cells were adjusted to 2 × 10 ⁵ cells / mL using DMEM medium supplemented with 10% FBS, and then attached to a 24-well plate. (1 μg / mL) was treated with lipopolysaccharide (LPS) (1 μg / mL) for 1 hour and then cultured at 37 ° C. for 15 hours in 5% CO ₂ . 50 μl of 1% sulfanilamide (in phosphoric acid) and 50 μl of 0.1% naphthylenediamine dihydrochloride were mixed with 100 μl of each cultured medium, and incubated at room temperature for 10 minutes. Then, the absorbance was measured at 560 nm. The amount of NO produced was measured using a sodium nitrite (NaNO 2) standard curve.

As a result, as shown in FIG. 2, it was confirmed that the amount of nitric oxide produced by LPS-only treatment was reduced depending on the treatment concentration of the source nucleic acid fraction of the present invention. Especially at 100 μg / ml concentration, it appeared to be similar to that of the control without any treatment.

< Experimental Example  3>

The Welcome  Nucleic acid The fraction iNOS  And COX -2 protein and gene expression Mitch Influence

In order to confirm the anti-inflammatory effect of the fresh nucleic acid fractions of the present invention prepared in Example 1, iNOS and COX-2 proteins and gene expression inhibition by RAW264.7 macrophages pretreatment of the fractions of Wongsong nucleic acid fractions were measured by Western blot And RT-PCR analysis.

iNOS is an abbreviation for inducible nitric oxide synthase. It is called inducible NOS. It is induced by immunostimulants such as lipopolysaccharide (LPS), cytokine such as IL-1 and TNF-α, Is activated only when a large amount is expressed in many cells to induce nitric oxide (NO) production. It is known that nitric oxide produced in such a manner acts on inflammation expression, host defense, and the like. In addition, COX-2 is an enzyme that synthesizes prostaglandin, an inflammatory mediator gene, which is one of the original cancer genes expressed in vascular endothelial cells, smooth muscle cells constituting blood vessel wall, subcutaneous subepithelial cells and epithelial cells. It is known to convert to nodal to exacerbate fever, fatigue, epithelial rupture, inflammation and various allergic symptoms.

<3-1> Welcome  Nucleic acid Fraction iNOS  And COX -2 protein expression

In Experimental Example 2, it was confirmed that the fresh nucleic acid fraction of the present invention can effectively reduce the production of LPS-induced nitric oxide (NO) in RAW264.7 macrophages. In this experiment, Lt; RTI ID = 0.0 &gt; expression-inhibition &lt; / RTI &gt; In addition, Western blot analysis was also conducted to determine whether the fraction of Wong's nucleic acid can inhibit the expression of COX-2, an enzyme that synthesizes prostaglandin, an inflammatory mediator.

RAW264.7 cells were pre-treated for 1 hour with LPS (1 μg / ml) for 12 hours, and then the culture medium was removed. Cells were washed twice with PBS. The cells were then scraped with a cell scraper by adding a cell elution buffer while placing the plate on ice. The supernatant was centrifuged at 12,000 rpm for 10 min at 4 ° C in ice. The supernatant was collected and transferred to a new tube. The supernatant was mixed with the loading buffer, and then loaded on 10% SDS-PAGE for electrophoresis. After electrophoresis, the gel was transferred to PVDF (polyvinylidene fluoride membrane). The transferred PVDF membrane was reacted with PBST buffer containing 5% non-fat dry milk for 2 hours in order to block nonspecific proteins, and the primary antibody (anti-iNOS, anti-COX-2 ) At 4 ° C for 12 hours and washed three times with PBST buffer every 10 minutes. After washing, the cells were reacted with a secondary antibody (HRP conjugated goat anti rabbit IgG) at 4 ° C for 2 hours and then washed 3 times with PBST. Blot was detected using ECL detection kits (Santa Cruz, CA, U.S.A.) ECL detection kit (Santa Cruz, Calif., USA) and developed on X-ray film. The intensity of the bands was measured by densitometry analysis using PDQuest software (version 7.0, Bio-Rad, USA).

As a result, as shown in FIG. 3, when RAW264.7 cells were treated with LPS alone, the expression of iNOS and COX-2 protein was significantly increased, whereas the expression of iNOS protein in the experimental group pretreated with the fresh nucleic acid fractions of the present invention was inhibited . However, pretreatment of the fresh nucleic acid fractions of the present invention did not affect COX-2 protein expression.

<3-2> Welcome  Nucleic acid Fraction iNOS  And COX -2 gene expression

In this experiment, the amount of mRNA of the gene was analyzed by RT-PCR in order to confirm whether the inhibition of NO production by the fractions of the transgene nucleic acid is achieved through inhibition of iNOS and COX-2 gene expression.

RAW264.7 cells were pretreated with the Waste nucleic acid fractions of the present invention at a concentration of 25, 50 and 100? / Ml for 1 hour, and then treated with LPS (1? / Ml) for 12 hours. The cells were then lysed by adding 1 ml of TRI-regent (Bio science), treated with 200 μl of chloroform (Sigma), and then subjected to vortexing for 15 seconds, followed by centrifugation at 5,000 rpm for 3 minutes. (vortexing) and incubated at room temperature for 5 minutes. Then, the RNA, DNA, and protein layers were separated by centrifugation at 14,000 rpm for 20 minutes. RNA was transferred to a new microfuge tube and 500 μl of isopropanol (MERCK) was added. After vortexing, it was left at 4 ° C for 5 minutes, then rinsed at 14000 rpm for 15 minutes. After removing the supernatant, TRIzol reagent was added to the RNA Melted. The extracted RNA was measured with a spectrophotometer.

2 μg of total RNA isolated by the above method was used for cDNA synthesis using SuperScript ™ II reverse transcriptase system kit according to the manufacturer's instructions. A PCR mixture (final volume 20 μl) consisting of 2 μl of cDNA, 2 μl of 10 × PCR buffer, 0.2 μl of Taq DNA polymerase, 0.2 μl of forward / reverse primer (see Table 1 below) and 1% diethyl pyrocarbonate water PCR was performed according to the conditions shown in Table 2. The amplified cDNA products were separated by 2% agarose gel electrophoresis, stained with ethidium bromide and visualized using Gel Doc ^TM XR Image Analyzer. And quantified using PDQuest software (version 7.0, Bio-Rad. USA).

As a result, as shown in Fig. 4, it was confirmed that the pretreatment of the fresh nucleic acid fractions of the present invention did not affect the expression of the iNOS and COX-2 gene due to stimulation by LPS.

Primer set used in PCR gene Primer sequence iNOS Forward 5'-CCTTGTTCAGCTACGCCTTC-3 ' Reverse 5'-AAGGCCAAACACAGCATACC-3 ' COX-2 Forward 5'-TCCTCCTGGAACATGGACTC-3 ' Reverse 5'-GCTCGGCTTCCAGTATTGAG-3 ' GAPDH Forward 5'-TGTTCCTACCCCCAATGTGT-3 ' Reverse 5'-GTGAGGGAGATGCTCAGTG-3 '

PCR conditions denaturation 95 5min / 95 30sec annealing 58 1min extension 72 1mim final extension 72 10min

< Experimental Example  4>

The Welcome  Nucleic acid The fraction NF -KB activity

In order to confirm the effect of the fractions of the present invention on the NF-κB signal pathway prepared in Example 1, the degree of inhibition of NF-κB and IκBα phosphorylation by RAW264.7 macrophage pretreatment of the transfection nucleic acid fractions was evaluated Respectively. Phosphorylated NF-κB and IκBα proteins were identified by Western blot analysis.

NF-κB is activated by various external stimuli such as inflammation-inducing cytokines, toxic compounds, bacterial infections, viral infections, radiation, UV, and active oxygen, , Immunoreactivity and inflammatory response, and the like. Such NF-κB is a transcription factor composed of homodimers or heterodimers of five proteins including p50 and p65 (RelA) proteins. In the cytoplasm, NF- Lt; RTI ID = 0.0 &gt; inactivated &lt; / RTI &gt; However, IκB kinase (IκB kinase, IKK) is activated through a variety of external stimuli such as inflammation-inducing cytokines, toxic compounds, bacterial infections, viral infections, radiation, UV and active oxygen to phosphorylate IκB bound to NF- , Resulting in the release of NF-κB from IκB. The liberated NF-κB enters the nucleus and functions as a transcription factor for a variety of inflammatory or immune responses, cell proliferation-related genes.

<4-1> Western Blat  analysis

In this experiment, the amount of phosphorylated NF-κB p65 and IκBα protein was measured by Western blot analysis, and the same method as in Experimental Example <3-1> was performed. However, the nucleic acid fractions of the present invention were pretreated for 2 hours, and anti-phospho-p65 and anti-phospho-IκBα were used as primary antibodies.

As a result, as shown in FIG. 5, phosphorylation of p65 and IκBα proceeded from 15 minutes after treatment with LPS alone, and the humanization was not inhibited with time, whereas in the pretreatment group of the waste nucleic acid fractions of the present invention, p65 and IκBα phosphorylation Was observed at 15 minutes and continued until 30 minutes, and it was confirmed that it was inhibited at 60 minutes.

In addition, the present inventors investigated the migration of cytoplasmic nucleus of NF-κB p65 and the degradation of p-IκBα through western blot analysis. As a result, as shown in FIG. 6, the migration of NF-κB p65 from the cytoplasm into the nucleus was enhanced in the case of treatment with LPS only, whereas in the pretreatment of the fresh nucleic acid fraction of the present invention, migration of NF- Was suppressed.

In addition, the present inventors examined whether the migration of NF-κB p65 nuclei due to pretreatment of the fresh nucleic acid fraction to pNKBα was due to p-IκBα degradation. As a result, as shown in FIG. 7, Dependent phosphorylation of IκBα in the cytoplasm.

<4-2> Confocal  Using a microscope Immunofluorescence

In this experiment, immunofluorescence was performed using a confocal microscope to visually observe the degree of migration of NF-κB p65 to the nucleus.

The RAW264.7 macrophages were pretreated with the nucleic acid fractions of the present invention for 1 hour, treated with LPS and reacted for 1 hour. Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature and increased in permeability with 100% methanol for 10 min at -20 ° C. To examine the location of NF-κB p65 in cells, the cells were treated with a polyclonal antibody (1: 200) against NF-κB p65. The cells were treated with a second-order rhodamine-conjugated goat anti-rabbit IgG antibody and reacted at room temperature for 2 hours. After washing with PBS, the nuclei were stained with 1 μg / mL of 4'-6-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy using a Zeiss LSM 510 Meta microscope.

As a result, as shown in FIG. 8, when NF-κB p65 was transferred to the nucleus by LPS alone treatment, the level of NF-κB p65 in the nucleus was inhibited in the pretreatment group of the fresh nucleic acid fraction of the present invention LPS (1 μg / ml), LPS + OJH, OJH (100 μg / mL) and LPS treatment groups.

< Experimental Example  5>

The Welcome  Nucleic acid The fraction MAPKs  And PI3K / Akt  Effect on activity

In Example 4, it was confirmed that the nucleic acid fractions of the present invention directly inhibited NF-κB activity. In order to further investigate the molecular mechanism of inhibition of NF-κB activity, Western blot analysis of MAPKs (ERK1 / 2, JNK, p38) and PI3K / Akt activity in RAW264.7 macrophages by pretreatment of the fractions of the nucleic acid fractions of Wongsong was confirmed.

The molecular signaling mechanism that controls the induction of iNOS or COX-2 expression through the activation of NF-κB transcription factors by various external stimuli is the three enzymes belonging to the major MAPK superfamily, including extracellular-regulated protein kinase (ERK ), c-Jun NH2-protein kinase (JNK) / stress-activated pretein kinase (SAPK), and serine / threonine protein kinase p38 MAPK.

Western blot analysis was carried out in the same manner as in Experimental Example < 3-1 >. 2), anti-ERK1 / 2, anti-phospho-p38, anti-phospho-p38, anti-phospho-p38, and anti-phospho-p38 as the primary antibodies were prepared by pretreating the fresh nucleic acid fractions of the present invention for 2 hours and then stimulated with LPS p38, anti-phospho-JNK, anti-JNK, anti-phospho-Akt were used.

As a result, as shown in FIG. 9, it was confirmed that LPS stimulation induced phosphorylation of MAPKs and Akt, while phosphorylation of Akt was dependent on concentration of fraction treated in the pretreatment of the fresh nucleic acid fraction of the present invention. However, pretreatment of the fresh nucleic acid fractions of the present invention did not inhibit phosphorylation of ERK1 / 2, JNK, and p38. As shown in Fig. 10, phosphorylation of Akt began to take place at 15 minutes and continued until 60 minutes, but phosphorylation was almost inhibited at 90 minutes.

< Experimental Example  6>

The Welcome  Nucleic acid The fraction CD14  And TLR4  Effect of protein on cell surface expression

In order to investigate the effect of the fractions of the transgene nucleic acid on the cell surface proteins, CD14 and TLR4, which are known as LPS receptors, RAW264.7 macrophages were pre-treated with the fresh nucleic acid fractions of the present invention and stimulated with LPS, And TLR4 expression were confirmed using flow cytometry and Western blot analysis.

CD14 is a glycosylphosphatidylinositol-linked protein that circulates in the blood or on the cell surface and has no direct signaling ability because it lacks an intracellular domain, And it plays an important role in signal transduction by binding to the LPS receptor. TLR4 is also a pathogen-recognizing receptor that recognizes LPS, a cell wall component of Gram-negative bacteria. Activation of TLR4 significantly promotes the production of pro-inflammatory proteins such as iNOS, COX-2 and TNF-a.

First, to evaluate cell surface CD14 protein expression, the cells were divided into 12-well plates at a concentration of 5 × 10 ⁵ cells / well, treated with a 100 μg / ml concentration of the fresh nucleic acid fraction of the present invention, , And treated with LPS (1 μg / ml) for reaction for 12 hours. The treated cells were collected and reacted with FITC-rabbit anti-mouse CD14 or isotype-matched rabbit IgG antibody on ice. Then, the cells were finally washed, fixed with 1% paraformaldehyde, and analyzed for fluorescence intensity using a Becton Dickinson FACS caliber flow cytometer and CELL-Quest Pro software (BD Bioscience, San Jose, Calif.).

As a result, as shown in FIG. 11, the expression of cell surface CD14 was markedly increased in the case of treatment with LPS only, whereas in the pretreatment group of the fresh nucleic acid fraction of the present invention, CD14 expression was attenuated (curve of gray area The graph shows the baseline signal from the isotype-matched IgG control, the linear graph shows the untreated group, the dotted graph shows the LPS-treated group, and the bolded line graph shows the LPS-treated group after the transfection of nucleic acid fractions.

In addition, as shown in FIG. 12, it was confirmed that TLR4 expression was effectively inhibited depending on the treatment concentration in the experimental group pretreated with the fresh nucleic acid fraction of the present invention. (Whole protein lysate was analyzed by Western blotting. GAPDH). &Lt; / RTI &gt;

Consequently, the present invention's nucleic acid fractions of the present invention reduce the intracellular NO concentration in a concentration-dependent manner; inhibit iNOS protein expression; Activation of NF-κB (nuclear factor-kappa B) through inhibition of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation; Weaken Akt phosphorylation; It was confirmed that LPS receptors have anti-inflammatory activity through the mechanism of inhibiting the cell surface expression of CD14 and TLR4 proteins.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

OJH: Waste nucleic acid fraction of the present invention
LPS: lipopolysaccharide
NO: Nitric oxide
iNOS: inducible nitric oxide synthase
NF-κB: nuclear factor-kappa B
IκBα: inhibitory factor kappa B alpha

Claims (7)

A composition for preventing or treating an inflammatory disease comprising an active ingredient of a nucleic acid fraction of Orostachys japonicas . The method according to claim 1,
Wherein the nucleic acid fraction is a fraction obtained by adding a nucleic acid to an ethanol extract obtained by adding 95% ethanol to a feed powder. The method according to claim 1,
Wherein the nucleic acid fraction of the source is contained in the composition at a concentration of 10 to 100 μg / ml. The method according to claim 1,
Wherein the nucleic acid fraction of the over-secretion reduces intracellular NO concentration through inhibition of iNOS protein expression; Inhibit the activation of NF-κB by inhibiting the migration of NF-κB p65 from the cytoplasm to the nucleus and inhibiting IκBα phosphorylation; Weaken Akt phosphorylation; A composition for preventing or treating an inflammatory disease, which has an anti-inflammatory activity through a mechanism of inhibiting cell surface expression of LPS receptor CD14 and TLR4 protein. 5. The method according to any one of claims 1 to 4,
Wherein the inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, rheumatoid multiple muscular pain, arteriosclerosis, inflammatory visceral disease, ulcerative colitis, osteoporosis, Crohn's disease, encephalitis, meningitis, pancreatitis, peritonitis, osteomyelitis, encephalitis, Acute bronchitis, chronic bronchitis, osteoarthritis, gout, spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, vasculitis, lymphocytic chorioamnionitis, glomerulonephritis, uveitis, ileitis, liver inflammation, kidney inflammation, asthma, pain, septic shock, Anemia, anaphylactic shock, burns, infection, bacterial infections, viral infections, fungal infections and congenital infections, psoriasis, atopic dermatitis, leishmaniasis, schistosomiasis, acute myelogenous leukemia, anemia, ulcer, multiple sclerosis, sclerosis, hemorrhagic shock, , Hemodialysis, seizures, cardiovascular transplantation, ischemia, recurrent disease, hemochromatosis, hemochromatosis, diabetes, Alzheimer's disease, Parkinson's disease, An autoimmune disease, an autoimmune disease, an autoimmune disease, an autoimmune disease, and an autoimmune disease. A health functional food for improving inflammatory diseases comprising the nucleic acid fraction of Orostachys japonicas as an active ingredient. The method according to claim 6,
Wherein the food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods Health functional foods.

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