KR20140125137A

KR20140125137A - Compound comprising extracts or fractions of Potentilla genus having anti-inflammation activity

Info

Publication number: KR20140125137A
Application number: KR20130042928A
Authority: KR
Inventors: 남정환; 정진철; 홍수영; 김수정; 유동림; 권오근; 이경태; 안소영; 김현삼; 조광수; 손황배
Original assignee: 대한민국(농촌진흥청장)
Priority date: 2013-04-18
Filing date: 2013-04-18
Publication date: 2014-10-28

Abstract

The present invention is applied to a composition having an anti-inflammatory activity comprising a plant extract of the genus Escherichia or a fraction thereof. More specifically, the present invention relates to a composition having anti-inflammatory activity, which comprises a safflower seed extract and a canola extract, or fractions thereof, which are plants of the genus Escherichia.
The present invention relates to a method for the production of nitric oxide (NO), prostaglandin (prostaglandin E2, PGE2), which is secreted from a macrophage by lipopolysaccharide (LPS) Generation or the like, thereby being useful for preventing or treating various inflammation.

Description

[0001] The present invention relates to a composition having an anti-inflammatory activity comprising a plant extract of the genus Escherichia or a fraction thereof,

The present invention relates to a composition having an anti-inflammatory activity comprising a plant extract of the genus Escherichia or a fraction thereof. More specifically, the present invention relates to a composition having anti-inflammatory activity, which comprises a safflower seed extract and a canola extract, or fractions thereof, which are plants of the genus Escherichia.

Inflammation is one of the damage of tissue, external stimulation or defense of biological tissues against various infectious agents. Enzyme activation, inflammation mediator secretion, cell infiltration due to organic interactions of various inflammatory mediators and various immune cells in blood vessels and body fluids And a series of complex pathologies such as fluid exudation, circulatory disturbances, tissue degeneration and hyperplasia. In the process of inflammatory reaction, macrophages are collected at the wound area at the initial stage and attack the invading bacteria. After that, plasma accumulates on the injured area and blood flow is increased, resulting in external symptoms such as fever, erythema, edema and pain . If the inflammatory reaction continues or occurs excessively, it progresses to a major pathological condition (irritable allergic disease, chronic inflammatory disease) of the disease, resulting in serious abnormal disorder.

Nonsteroidal anti-inflammatory drugs (NSAIDS), a widely used agent for the treatment of most inflammatory diseases, are produced from arachidonic acid called prostaglandin, called cyclooxygenase (COX) Inhibition of the enzymatic activity involved in the biosynthesis of the glycosaminoglycans, exhibits an anti-inflammatory action. In addition to the main therapeutic effect, it causes severe adverse effects such as gastrointestinal disorders, hepatic disorders, renal diseases and the like. Therefore, the development of a novel anti-inflammatory analgesic agent that is excellent in anti-inflammatory efficacy is widely demanded because there is no side effect and it is not difficult to use for a long period of time. This is why research on material development through the verification of efficacy from natural resources is being activated recently.

Various biochemical phenomena are involved in the cause of inflammation in living body. Macrophage is a cell with various functions. It produces various cytokines and NO by chemical stimulation and plays an important role in the inflammation reaction. In particular, inducible nitric oxide synthase (iNOS) expressed by macrophage-stimulated cytokines such as lipopolysaccharide (LPS) and interferon gamma and TNF-α produces large amounts of nitrogen oxides (NO) over a long period of time. These oxidative stresses are known to promote NFκB activity, a transcription factor of inflammatory responses inhibited by IκB. Activated NFκB is known to migrate to the nucleus and promote gene expression that induces inflammatory responses such as iNOS, COX-2, and several cytokines such as IL-1β and TNF-α. When these factors are inhibited, (Baeuerle et al., Annu. Rev. Immunol., 12: 141-179, 1994).

Nitrogen oxides (NO) are produced from L-arginine by three major nitrogen oxide synthase (NOS) isomers, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). While nNOS and eNOS are regulated by Ca2 + / calmodulin, iNOS is regulated at the transcriptional level by inflammatory stimuli such as interleukin, interferon, and LPS. NO produced by nNOS or eNOS plays a normal physiological function such as vasodilation, neurotransmission, cell destruction to pathogen, etc. However, overexpressed NO produced by macrophages in iNOS is involved in various pathophysiological And reacts with superoxide to form peroxynitrite, which acts as a powerful oxidant to damage cells, activates NFκB in macrophages activated by inflammatory stimuli, , Cancer, atherosclerosis, and the like.

Prostaglandins are unsaturated fatty acid derivatives containing 20 carbons with a cyclic structure. They are chemical mediators mainly involved in chronic inflammatory diseases and are also involved in autoimmune diseases such as asthma. Prostaglandins are biosynthesized by COX, which has two isomers, COX-1 and COX-2. COX-1 is a constant enzyme present in tissues such as the stomach or kidney, and is involved in maintaining normal homeostasis, whereas COX-2 is a transient and rapidly expressed expression in cells by cytokines or cytokines in inflammation or other immune responses do. NSAIDs used in the treatment of chronic inflammatory diseases such as acute or rheumatoid arthritis are known to exhibit various side effects such as gastrointestinal disorders as mentioned above by inhibiting COX-1 enzyme as well as inhibiting COX-2 enzyme.

Therefore, when a substance that inhibits the production of tumor necrosis factor (TNF-α) and nitrogen oxide (NO), inhibits prostaglandin production, or inhibits the production of IL-1β is searched, will be.

On the other hand, the plants of the wing of the Yangmyeong family are the perennial plants belonging to the primrose family. They are mainly grown in sunny mountainous areas, grasslands and sometimes high mountains. They are distributed in various parts of Korea, including Samhwacho, , Watery grasses, and three-leafed rice blossoms. Twenty-four from Salicornia (Potentilla kleiniana , orchid ) grows in a humid place, height is 20 ~ 60cm, and the lower half grows at an angle. The roots are palm-like compound leaves with long petiole, three leaves are running on the stem, and petiole becomes shorter as it goes up. It is used for the juice of juvenile food. It is called as a snake (snakes), and it is prescribed for fever, cold, sore throat, boil, eczema etc. Potentilla supina ) is also known as a spotted gabi , grows in the field and is about 50cm in height. The stem is gathered tightly and the lower side grows at an angle to the side, straight up, the branches are staggered, the leaves are staggered and there are petiole. Fruits are round oaks, ripened in September-October, no hairs, young stems and leaves are used for food.

However, there has been no study or report on the effect of the above-described extracts or fractions thereof on the inflammation. Accordingly, the present inventors have completed the present invention by confirming that Saffron ginseng and Ganoderma lucidum extract, or fractions thereof, of the plants of the genus Escherichia can inhibit the production of nitrogen oxides or inhibit the production of prostaglandins.

Korean Patent No. 10-0240477

The present invention provides a composition having an anti-inflammatory activity comprising a plant extract of the genus Escherichia or a fraction thereof.

The present invention relates to a composition having an anti-inflammatory activity comprising a plant extract of the genus Escherichia or a fraction thereof.

The present invention also relates to a pharmaceutical composition for preventing or treating an inflammatory disease comprising the above composition as an active ingredient.

The present invention also relates to a cosmetic composition and a food composition for preventing or improving an inflammatory disease containing the composition as an active ingredient.

The water extracts of the Japanese radish and the extracts or fractions thereof of the present invention inhibit mRNA expression of TNF-α, IL-1β which are inflammatory cytokines secreted from macrophages by lipopolysaccharide (LPS) (NO), prostaglandin (prostaglandin E2, PGE2), and the like, thereby preventing or treating various inflammation.

Figure 1 relates to the efficacy of ethanol fractions for LPS-induced iNOS and COX-2 protein expression.
Fig. 2 relates to the efficacy of the SARS ethanol fraction on LPS-induced inflammatory mRNA expression.
Figure 3 relates to the efficacy of the L. angustifolia ethanol fraction for LPS-induced inflammatory mRNA expression.
Figure 4 relates to the efficacy of the ethanol fraction for LPS-induced NF-kB nuclear translocation.

The plants of the Saccharomyces cerevisiae include, but are not limited to, alfalfa or canola seedlings.

In order to prepare the extract of Sorghum ganoderma L. and Canola ganoderma L. extract of the present invention, water, C1-C3 alcohol or a mixture thereof may be used as an extraction solvent, and extraction with methanol or ethanol may be used. It is not. The amount of the extraction solvent may be 2 to 20 times by weight, and may be 5 to 10 times by weight, relative to the dry weight of the plant, but is not limited thereto.

Examples of the method for extracting the plant include a method using an extraction device such as hot water extraction, immersion extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, reflux cooling extraction and ultrasonic extraction, or adsorption resin including XAD and HP- A method using a conventional extraction method of the art can be used. It may be heated, refluxed or extracted at room temperature, but is not limited thereto. The extraction number is preferably 1 to 5 times, more preferably 3 times, but is not limited thereto. It may be extracted once to five times by an ultrasonic extraction method. The temperature for extraction is preferably from 10 to 100 ° C, more preferably room temperature, but is not limited thereto. The extraction time may be from 1 day to 7 days, and may be from 3 days to 7 days, but is not limited thereto.

The extract can be concentrated under reduced pressure, but is not limited thereto. The vacuum concentration may be performed using a vacuum rotary evaporator, but the present invention is not limited thereto. The drying may be carried out under reduced pressure, vacuum drying, boiling, spray drying, room temperature drying or freeze drying, but is not limited thereto.

The syrups of the present invention can be obtained by suspending a water-soluble extract of Sambacho or Phellinus linteus in water, and then sequentially fractionating the extract with a solvent having a different polarity. The solvent may be any solvent selected from the group consisting of ethanol, EtOH, hexane, chloroform, ethyl acetate, EtOAc, and mixtures thereof. In one embodiment of the present invention, all the above fractions exhibited activity, but the ethanol fraction showed the highest activity.

The term "anti-inflammatory activity" as used herein refers to inhibiting inflammation, and the term " inflammation " as used herein means the pathological condition of abscess formed by the invasion of external bacteria. Inflammation reaction is a biological defense reaction process for repairing and regenerating damage caused by invasion that brings about a fundamental change in the cell or tissue of a living body. It is a process of damaging tissues (cells) or infecting external sources (bacteria, fungi, Allergen-inducing substances), various inflammatory mediators and immune cells are involved in local blood vessels and body fluids, and a series of complex physiological responses such as enzyme activation, inflammatory mediator secretion, body fluid infiltration, cell migration, and tissue destruction, And fever pain. In normal cases, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, if the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, the major pathological condition of the disease (irritable disease, Chronic inflammation), and become obstacles in the treatment process such as transfusion, drug administration, or organ transplantation.

The extract of the present invention or the extract of Ganoderma lucidum extract or its fraction is not an artificially synthesized compound but has advantages of safety, toxicity and side effects because it is based on the components obtained from natural extracts, so that it can be taken for a long time. The composition may be used not only for humans but also for animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs,

Accordingly, the present invention can be used as a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises as an active ingredient an extract of Samcheonchon or P. ganoderma extract or a fraction thereof, and the composition can be treated to prevent or treat an inflammatory disease.

The " inflammatory disease " of the present invention is defined as any condition specified by a local or systemic bio-defense response to an external physical or chemical stimulus or infection of an external infectious agent such as a bacterium, a fungus, or a virus. This reaction is associated with activation of various inflammatory mediators and enzymes associated with immune cells (such as iNOS, COX-2, etc.), secretion of inflammatory mediators such as NO, TNF-a, IL-6, IL- , Body fluid infiltration, cell migration, tissue destruction, and is manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of specific function of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as above, it may be acute, chronic, ulcerative, allergic, Whether it is necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, Inflammatory bowel syndrome, inflammatory pain, migraine headache, headache, back pain, fibromyalgia, fascia disease, viral infection (e.g., C type infection), bacterial infection, fungal infection, burn, wound due to surgical or dental surgery, Prostaglandin E Overdose Syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, eczema, atopic inflammation, acne, multiple sclerosis .

The term "prophylactic" of the present invention refers to any action that inhibits or delays the onset of the disease by the administration of a composition comprising the samphanuum or canine ganoderma extract or fractions thereof according to the present invention.

The term "treatment" of the present invention refers to any action that improves or alleviates the symptoms of the disease by administration of a composition containing the extract or the extract of Panax notoginsenoside or extract thereof according to the present invention.

In the present invention, RAW 264.7 macrophage cells were tested for the inhibitory effect on the production of nitrogen oxides (NO), prostaglandin E2 (PGE2) and inflammatory mRNA expression , And cytotoxicity evaluation experiments were performed.

As a result of the measurement, the extract of Radix Salvia miltiorrhizae and Ganoderma lucidum extract or its fractions were not cytotoxic (see Table 1). In addition, it was confirmed that production and secretion of inflammatory cytokine and chemokines NO and PGE2 were markedly inhibited (see Tables 2 to 3).

In the present invention, the inflammatory factor involved in the inflammatory reaction is nitric oxide (NO). NO is produced by nitric oxide synthase (NOS) from L-arginine through the intermediate metabolite hydroxyarginine and NO and citrulline. NO has a biological activity and acts on the vascular system in vivo to mediate vasodilation, platelet adhesion and aggregation, neurotransmission, and digestive organ exercise. In addition, NO has been shown to play a role in several metabolic regulatory and physiological actions (neurotransmission, blood clotting, blood pressure control, immunity against cancer cells, etc.) and is also produced in inflammatory cells as well as nonimmune cells, .

In general, NO is present in the form of gas in the atmospheric state, and because it is a free radical structure, it is not only poisonous but also has a half-life of 6 to 10 seconds, which is very unstable and is known to be rapidly converted into stabilized end products NO ₃ and NO ₂ have.

On the other hand, NO synthase involved in the NO synthesis (NOS) is divided into calcium (Ca ² ⁺⁾ or calmodulin (camodulin) significantly cNOS (constitutive NOS) and iNOS (inducible NOS) in accordance with whether or not dependence. cNOS is dependent on calcium or calmodulin, mainly in the brain, epithelial cells, neutrophils, and gastric mucosal cells. iNOS is independent of calcium or calmodulin, and iNOS is present in macrophages, hepatocytes, cancer cells, IFN-γ, TNF-α, and the like, and bacterial LPS (lipopolysaccharide), an endotoxin. Because of the activation of cyclooxygenase-2 (COX-2) by the stimulation, prostaglandin (PGs), which is an inflammatory mediator, is produced. Therefore, iNOS expression and COX-2 expression are closely related to each other. It also affects COX-2 expression. Inflammatory diseases caused by these inflammatory reactions include, in addition to arteriosclerosis, gastritis, colitis, arthritis, nephritis, hepatitis, cancer or degenerative diseases.

The pharmaceutical composition for prevention or treatment of inflammatory diseases, including the sphagnum ganoderma or the extract of Aspergillus oryzae, or fractions thereof, according to the present invention may be administered to mammals such as rats, mice, livestock and humans in various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intracerebral or intracerebroventricular injection, Lt; / RTI >

The amount of the pharmaceutical composition for preventing or treating inflammatory diseases used in the present invention should be a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The effective amount may vary depending on the route of treatment, the use of excipients and the likelihood of use with other medicaments, as will be appreciated by those skilled in the art.

The pharmaceutical treatment forms of the composition for preventing or treating inflammatory diseases of the present invention may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmaceutically active compounds.

The pharmaceutical compositions for the prevention or treatment of inflammatory diseases of the present invention may be prepared into pharmaceutical formulations using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal . In the preparation of the formulations, the active ingredient may be mixed with or diluted with the carrier, or enclosed in a carrier in the form of a container.

Accordingly, the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention can be administered orally or parenterally in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral preparation, And may further comprise suitable carriers, excipients and diluents conventionally used in the production of the composition.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweetening agents, have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In another aspect, the present invention relates to a cosmetic composition comprising, as an active ingredient, a composition comprising samphanaceae or extract of Aspergillus oryzae or fractions thereof. The cosmetic composition according to the present invention may be formulated into a cosmetic, a gel, a water-soluble powder, a fat-soluble powder, a water-soluble liquid, and an essence in a cream by adding a pH adjusting agent, a flavoring agent, an emulsifier, have.

In addition, in another aspect, the present invention provides a food composition for improving inflammatory diseases, which comprises as an active ingredient an extract of Sam Roiot bean or L. anthracis extract or a fraction thereof.

The food composition of the present invention includes forms such as pills, powders, granules, infusions, tablets, capsules or liquid preparations. Foods to which the composition of the present invention can be added include, for example, beverages, gums, tea, vitamins And health supplement foods.

There are no particular restrictions on other components other than the composition containing the above-described Sam-Nam-gwang or Ganoderma lucidum extract or its fractions as an essential ingredient that can be contained in the food composition of the present invention. The composition of the present invention is not limited to a variety of herbal medicine extracts, Natural carbohydrates and the like as additional components.

The food composition may further include one or more herbal medicine extracts selected from the group consisting of Sanchosan, Gongsuanggang, Pungyoung, Huangbai, Seokchangpo,

The food supplementary additives may further include flavors, flavors, colorants, fillers, stabilizers and the like which are commonly used in the art.

Examples of such 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. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors of synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These people can be used independently or in combination.

In addition, the food composition of the present invention may be any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, Lt; / RTI >

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example 1> Experimental method

1. Cell culture

RAW 264.7 cells were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco, New York, USA) containing 10% FBS and penicillin (100 μg / ml) and streptomycin And cultured under the condition. RAW 264.7 cells were pretreated with various concentrations (10, 20, and 40 μM) of the sample solution for 1 hour, treated with LPS and cultured for 24 hours.

2. MTT assay Cytotoxicity test

To determine the degree of toxicity of the cells, MTT (dimethyl thiazolyl diphenyl tetrazolium salt, a type of tetrazole, Sigma Chemical Co., St. Louis, MO, USA) was used. Cells were equally divided into 96-well plates (353077, falcon ™, USA) at 1 × 10 ⁵ cells / ml and cultured for 24 hours. After washing with PBS (Phosphate buffer saline, Bioneer Co., Deajeon, Korea), 100 μl of H ₂ O ₂ (Hydrogen peroxide solution, BBC Biochemical, Washington, USA) After incubation for 45 minutes, the supernatant was removed, washed twice with PBS buffer, and then 200 μl of the medium was injected for another 24 hours. After addition of 50 μl of 5 mg / ml MTT reagent, the supernatant was removed. 100 μl of DMSO (dimethyl sulfoxide, Sigma Chemical Co., St. Louis, Mo., USA) was added and incubated at 540 nm in an ELISA microplate reader The survival rate was calculated by measuring the absorbance with a Power Wave 340, Bio-Tek Instruments Co., USA).

3. Nitric oxide ( NO ) Quantity measurement

The amount of NO produced from RAW 264.7 cells was measured by using Griess reagent [1% (w / v) sulfanilamide in 5% (v / v) phosphoric acid and 0.1% (w / v) naphtylethylenediamine- As the form of NO ₂ ^- . Griess reagent was mixed with 100 쨉 l of each well and reacted on a 96-well plate for 10 minutes, and the absorbance was measured at 540 nm.

4. PGE ₂ Measurement of quantity

Cells from the cells treated with the sample and control were taken and the amount of PGE ₂ was quantified according to the instructions of the assay design kit (Amersham pharmacia Biotech Co., USA).

5. Western blot exam

Cells treated with the sample and the control group were lysed with lysis buffer PRO-PREP (Intron Biotechnology Co., Sungnam, Korea), centrifuged and the supernatant was taken to extract the proteins. The protein concentration of the supernatant was quantified using a Bradford reagent to obtain 30 μg of the protein. The extracted protein was electrophoresed on a 10% SDS-polyacrylamide gel and the gel protein was blotted with PVDF membrane. (Santa Cruz, CA, USA), COX-2 (Santa Cruz, Calif., USA) and P65 (Santa Cruz, CA, USA) at a ratio of 1: 1000 after 90 minutes of blotting with 5% USA), PARP-1 (Santa Cruz, CA, USA), α-tubulin (Santa Cruz, CA, USA) and β-actin And washed three times at intervals of 10 minutes. The second antibody (iNOS-Rabbit, COX-2-Goat, P65-Rabbit, PARP-1-Rabbit and α-tubulin-mouse, β-actin-mouse) was left at room temperature for 1 hour at 1: -TBS three times at 10-min intervals and then developed with chemiluminescence (Gel logic 2200 PRO, Carestream Molecular Imaging Co., USA).

6. RT - PCR exam

Total cellular RNA was extracted according to the kit protocol using Easy Blue ^® kits (IntronBiotechnology). 1 μg of RNA was reverse transcribed using MuLV reverse transcriptase, 1 mM dNTP and 0.5 μg / μL of oligo (dT ₁₂ _-18 ) in each sample to obtain cDNA. A total volume of 25 μL samples containing 1 unit of Taq DNA polymerase, 0.2 mM dNTP, X10 reaction buffer and 100 pmol of 5 'and 3' primers was added to the cDNA using thermal cycler (Perkin Elmer Cetus, Foster City, CA, USA) And then subjected to PCR analysis [Takara thermal cycler dice (Takara Bio Inc., Japan)].

The primer information is as follows:

1. iNOS (55 C, 50 CYCLE)

Forward primer: 5'-CATGCTACTGGAGGTGGG TG-3 '

Reverse primer: 5'-CATTGATCTCCGTGACAGCC-3 '

2. COX-2 (55 C, 50 CYCLE)

Forward primer: 5'-GGAGAGACTATCAAGATAGT-3 '

Reverse primer: 5'-ATGGTCAGTAGACTTTTACA-3 '

3. TNF-α (55 ° C, 50 CYCLE)

Forward primer: 5'-AGCACAGAAAGCATGATCCG-3 '

Reverse primer: 5'-CTGATGAGAGGGAGGCCATT-3 '

4. IL-6 (55 C, 50 CYCLE)

Forward primer: 5'-GAGGATACCACTCCCAACAGACC-3 '

Reverse primer: 5'-AAGTGCATCATCGTTGTTCATACA-3 '

5. IL-1 [beta] (55 [deg.] C, 50 CYCLE)

Forward primer: 5'-TGCAGAGTTCCCCAACTGGTACATC-3 '

Reverse primer: 5'-GTGCTGCCTAATGTCCCCTTGAATC-3 '

[Beta] -actin (55 [deg.] C, 50 CYCLE)

Forward primer: 5'-ATCACTATTGGCAACGAGCG-3 '

Reverse primer: 5'-TCAGCAATGCCTGGGTACAT-3 '

7. Statistical analysis

The values of the test values were expressed as mean ± SD, and the analysis was significant with Student's t- test.

< Example 2> Experimental results

One. RAW 264.7 Cytotoxicity of macrophages

Prior to identifying the anti - inflammatory effects of the samples, the MTT assay was performed to exclude the possibility of inhibiting the inflammatory mediators from toxic effects on RAW264.7 cells. As shown in Table 1, all samples showed no toxicity at a concentration of 100 μg / ml. The following Table 1 relates to cytotoxicity in various fractions.

2. RAW In 264.7 macrophages Nitric oxide Generation inhibitory effect

The amount of NO produced in the culture of RAW264.7 cells activated by LPS at a concentration that does not show cytotoxicity as a result of MTT assay was measured using a grease reagent. When the viablity of Lactobacillus crassiflora and Lactobacillus farina was decreased to less than 80% at 10 μg / ml or more, the experiment was carried out at 10 μg / ml or less. The inhibition of NO formation was observed in all samples except chloroform fraction. The inhibition effect of NO production by Samcheoncho ethanol, canola gabi ethanol and canine gabi ethyl acetate fraction was good, but it can not be judged to be remarkably superior to other samples To confirm the inhibitory effect of other inflammatory metabolites. The following Table 2 relates to the efficacy of the various fractions on LPS-induced NO production.

3. RAW In 264.7 macrophages PGE ₂ Generation inhibitory effect

Samples were confirmed to inhibit the production of PGE ₂ , another inflammatory metabolite induced by LPS, in the extract fraction. Ethanol from Samcheoncho and Ganbang ethanol from dogs were effective. In the previous NO assay and the results, considering the stability as a health functional food, the experiment was carried out by choosing Samchoanchoa ethanol and Canola gabi ethanol. Table 3 below shows LPS-induced PGE ₂ RTI ID = 0.0 > production. &Lt; / RTI >

4. RAW In 264.7 macrophages iNOS And COX -2 protein Expression inhibition effect

The inhibitory effects of iNOS and COX-2, which are enzymes involved in the production of NO and PGE ₂ , were inhibited by ethanol and dextrin ethanol. LPS-induced iNOS and COX-2 protein expression was inhibited by the sample. β-actin was used as an internal control (see FIG. 1).

5. RAW 264.7 Inflammation in macrophages mRNA Expression inhibition effect

Inhibition of mRNA expression of iNOS, COX-2 and TNF-α, IL-1β, and IL-6, which are inflammatory cytokines, was confirmed by ethanolamine. It was confirmed that LPS-induced inflammatory mRNA expression was inhibited by the sample in a concentration-dependent manner (see FIG. 2).

Inhibition of mRNA expression of iNOS, COX-2 and the inflammatory cytokines TNF-α, IL-1β, and IL-6 was observed in canine gabi ethanol. It was confirmed that the remaining mRNA expression of LPS-induced inflammatory mRNA was inhibited by the sample in a concentration-dependent manner except for IL-6 (see FIG. 3).

6. RAW In 264.7 macrophages NF -κB translocation into the nucleus

The inhibition of translocation of p65, a subunit of NF-κB into the nucleus, was confirmed by ethanol and deoxyribonucleic acid. The translocation of p65 into the nucleus induced by LPS was inhibited by the sample in a concentration-dependent manner. PARP and α-tubulin were used as internal controls for nuclear and cytoplasm, respectively (see FIG. 4).

Claims

A composition having an anti-inflammatory activity comprising a plant extract of the genus Escherichia or a fraction thereof.

The method according to claim 1,
Wherein the plant of the genus Escherichia is a green tea or a canola.

The method according to claim 1,
Wherein the extract is a water extract or a methanol extract of a plant of the genus Escherichia.

The method according to claim 1,
Wherein the fraction is a fraction using a solvent selected from the group consisting of ethanol, chloroform, hexane, ethyl acetate, and a mixed solvent thereof.

A pharmaceutical composition for preventing or treating an inflammatory disease comprising the composition of any one of claims 1 to 4 as an active ingredient.

6. The method of claim 5,
Wherein the plant extract in the genus Paracetamus or a fraction thereof suppresses the production of nitrogen oxides (NO).

6. The method of claim 5,
Wherein said extract of the genus Escherichia or a fraction thereof inhibits the production of prostaglandin E2 (PGE2).

A cosmetic composition for preventing or ameliorating an inflammatory disease comprising the composition of any one of claims 1 to 4 as an active ingredient.

A food composition for preventing or ameliorating an inflammatory disease comprising the composition of any one of claims 1 to 4 as an active ingredient.