KR20200020354A - Pharmaceutical composition for preventing or treating inflammatory disease comprising extract of Pilea martinii as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases containing Pileamartinii extract, and more specifically, to a food composition for preventing or improving inflammatory disease containing Pileamartiniiextracts, a pharmaceutical composition for preventing or treating inflammatory diseases containing Pileamartinii extracts, a method for preventing or treating inflammatory diseases containing a step of injecting the pharmaceutical composition to an entity except a human, a quasi-drug composition for preventing or treating inflammatory diseases containing the extract, and a cosmetic composition for alleviating or improving inflammation containing the extract. The Pileamartinii extract provided in the present invention inhibits nitrogen oxide (NO) induced by lipopolysaccharide from generating and IL-6, TNF-α, PGE_2 secretion; has an mRNA expression inhibition effect of iNOS and COX-2 proteins induced by lipopolysaccharide and inhibition of the same; and inhibits inflammatory responses through an NF- κB signal transmission process inhibition, thereby being useful as a composition for preventing or improving inflammatory diseases.

Description

Pharmacy composition for preventing or treating inflammatory disease comprising extract of Pilea martinii as effective component

The present invention relates to a composition for preventing or treating an inflammatory disease comprising a pilea martinii extract, and more particularly, a food composition for preventing or improving an inflammatory disease including a pilate martini extract, an inflammatory agent comprising a pilate martini extract. A pharmaceutical composition for preventing or treating a disease, a method for preventing or treating an inflammatory disease comprising administering the pharmaceutical composition to an individual except a human, an quasi-drug composition for preventing or improving an inflammatory disease including the extract, and the extract It relates to a cosmetic composition for reducing or improving inflammation.

Inflammation is a locally occurring immune response that minimizes damage and restores damaged areas when cells or tissues are damaged or destroyed by various factors such as harmful substances or organisms from outside. And a defense mechanism that is useful for removing products produced by tissue damage. As a result of this defense mechanism, pain, edema, redness or fever may occur, resulting in dysfunction. The various factors causing the inflammation may include physical factors such as trauma, burns, frostbite, radioactivity, chemical factors such as acids, and immunological factors due to antibody reactions. It is also caused by.

Inflammation normally acts to restore the normal structure and function by neutralizing or removing the onset factors and regenerating the upper tissues in vivo through the inflammatory response.However, the degree of inflammation becomes more than a certain level or becomes chronic. This is a problem if you progress to the same disease state. In addition to observing the inflammatory response in almost all diseases, it is known that enzymes related to inflammatory reactions play an important role in carcinogenesis.

Recently, the progress of the inflammatory response in the body is known to be associated with the activity of the cyclooxygenase (COX) enzyme. The COX enzyme is a major enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), and two kinds of isozymes, COX-1 and COX -2 is known to exist. The COX-1 is constantly present in tissues such as the stomach and kidneys, and is involved in maintaining normal homeostasis, while the COX-2 is involved in mitogen or cytokines during inflammation and other immune responses. Is an enzyme that is transiently and rapidly expressed in cells.

Another powerful anti-inflammatory mediator, Nitric Oxide (NO), is produced from L-arginine by NO synthetase (NOS), and can be produced by many types of substances such as endotoxins or cytokines such as UV or external stress. Produced in cells. Such inflammatory stimuli can increase the expression of inducible NOS (iNOS) in the cell, thereby inducing NO production in the cell, thereby inducing a inflammatory response by activating macrophages.

Therefore, in recent years, in order to effectively alleviate inflammation, studies have been conducted on substances capable of inhibiting NO production. However, some side effects are problematic for anti-inflammatory substances developed by these studies. For example, nonsteroidal anti-inflammatory drugs used in the treatment of chronic inflammatory diseases such as acute or rheumatoid arthritis are known to exhibit side effects such as gastrointestinal disorders by inhibiting COX-2 enzyme as well as COX-1 enzyme. (Fries J., 1996; Singh G., 1998). Therefore, there is a need for a study on the anti-inflammatory effects of natural products with few side effects.

On the other hand, Philea Martini is a plant belonging to the nettle ( urticales ), which occurs extensively in China and grows mainly in the shaded and wet areas of the forest. The antimicrobial activity is known as a pharmacological activity on the filet of martini, but other effects are not known (African Journal of Microbiology Research 6.19 (2012): 4132-4137.).

Accordingly, the present inventors have conducted research to develop a natural-derived material having anti-inflammatory effects and low side effects, and as a result, Philea martini extract inhibits the expression of COX-2 and iNOS and inhibits the signaling of NF-κB, The present invention was completed by confirming that it inhibits the secretion of nitric oxide and inflammatory factors.

One object of the present invention is to provide a food composition for preventing or ameliorating an inflammatory disease comprising a extract of Pilea martinii .

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including the extract of Philea Martini.

Yet another object of the present invention is to provide a method for preventing or treating an inflammatory disease, which comprises administering to the subject other than humans the Philea martini extract.

Yet another object of the present invention is to provide an quasi-drug composition for preventing or improving inflammatory diseases, including the extract of Philea martini.

Yet another object of the present invention is to provide a cosmetic composition for alleviating or improving inflammation comprising a Philea martini extract.

One aspect of the present invention for achieving the above object, provides a food composition for the prevention or amelioration of inflammatory diseases, including pilea martinii extract.

The term "pilreah Martini (Pilea martinii)" in the present invention can be confirmed that a plant belonging to the nettles neck (urticales), occurring widely in China is mainly the shade of the forest that grows in wet areas. Antimicrobial activity is known as a pharmacological activity against the filet of martini, but other effects are not known.

The term "extract" in the present invention may refer to a liquid component obtained by immersing in a solvent and then extracted for a predetermined time at room temperature or warmed state, a solid component obtained by removing the solvent from the liquid component.

The phyllea martini extract with anti-inflammatory response can be extracted from various organs of natural, hybrid, and mutant plants, for example, root, stem, leaf, flower, trunk of fruit, bark of fruit, as well as plant tissue culture. Extractable from

In the present invention, the fila martini extract may be extracted from the root, stem, leaf, flower, or fruit of the fila martini, but is not particularly limited thereto.

Specifically, the Philea Martini extract may be an outpost extract of Philea Martini.

In the present invention, the fila martini extract includes, but is not limited to, extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms and mixed solvents thereof.

In the present invention, the extract may include an extract obtained by an extraction treatment, a diluent or concentrate of the extract, a dried product obtained by drying the extract, or any one of these modifiers or purified products.

The fila martini extract can be prepared using common extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, and specifically, methods such as hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction may be used.

As used herein, the term "inflammatory disease" is a general term for diseases of which inflammation is the main lesion, for example, inflammatory skin disease, inflammatory bowel disease such as Crohn's desease and ulcerative colitis, peritonitis , Osteomyelitis, cellulitis, meningitis, encephalitis, pancreatitis, trauma-induced shock, bronchial asthma, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondyloarthritis, ankylosing spondylitis, lighter syndrome, Psoriatic arthrosis, enteropathic spondylitis, juvenile arthrosis, juvenile ankylosing spondylitis, reactive arthrosis, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculosis arthritis, viral arthritis, fungal arthritis, syphilis arthritis, Lyme Disease, arthritis associated with 'angiitis syndrome', nodular polyarteritis, irritable vasculitis, Lou Gehrig's granulomatosis, rheumatoid Sexual muscle pain, articular cell arteritis, calcium crystalline arthritis, pseudogout, non-articular rheumatoid arthritis, bursitis, hay fever, epicondylitis (tennis elbow), neuropathic joint disease (charco and joint), hemarthrosic arthritis Noch-Scholine purpura, hypertrophic osteoarthritis, multiple psychotic retinopathy, surcoilosis, hemochromatosis, sickle cell disease and other hemochromatosis, hyperlipoproteinemia, hypomagglobulinemia, familial Mediterranean fever, Behart's disease Systemic lupus erythematosus, recurrent fever, psoriasis, multiple sclerosis, sepsis, septic shock, multiple organ dysfunction syndrome, acute respiratory distress syndrome, chronic obstructive pulmonary disease, acute lung injury and It may be any one selected from the group consisting of broncho-pulmonary dysplasia, specifically, inflammatory skin disease, ulcerative colitis, chronic phase May be bronchitis, osteoarthritis, sepsis. This is not restrictive.

The term "prophylaxis or improvement of inflammatory diseases" of the present invention means the action of inhibiting inflammation, the regulation of the inflammatory response is known to be very complicated, which is known to reduce the enhancement and damage of the repair system in vivo . However, if the inflammatory response persists due to repeated tissue damage or regeneration, overproduction of ROS and RNS in inflammation-related cells results in permanent genetic modification. As such, ROS and RNS are deeply involved in the inflammatory response that regulates the actions of various cells in vivo. During the inflammatory process, high levels of inflammatory cytokines, nitric oxides (NO), and prostaglandin E2 (PGE ₂ ) are induced by inducible nitric oxide synthase (iNOS) and cyclooxygena. Produced by an enzyme (cyclooxygenase-2, COX-2). Inflammation is a cause of various inflammatory diseases, the composition comprising the filament martini extract of the present invention may have a prophylactic and ameliorating effect against various inflammatory diseases through the inhibitory action of the nitric oxide, inflammatory cytokines or transcription factors, etc. have.

Prevention or improvement of the inflammatory disease may be achieved by inhibiting the production of nitric oxide.

In a specific embodiment of the present invention, the treatment of the Philea martini extract to macrophages induced by the LPS-induced inflammatory response and measuring the amount of nitric oxide produced, the amount of nitric oxide increased by the LPS stimulation to the concentration of the Philea martini extract It was confirmed that the decrease according to (Fig. 2).

Prevention or improvement of the inflammatory disease may be achieved by inhibiting the expression of iNOS protein, COX-2 protein or mRNA encoding the same.

In another specific embodiment of the present invention, after treating the cholera martini extract in macrophages induced by the LPS-induced inflammatory response, the protein levels of iNOS and COX-2 were checked and mRNA activation was analyzed. Induced by the inflammatory response was confirmed that the protein level of iNOS and COX-2 increased and the phosphorylation activity of mRNA was increased, but the protein level and phosphorylation activity was reduced according to the concentration of the Philea Martini extract (Fig. 3).

Prevention or improvement of the inflammatory disease may be achieved by inhibiting the activity of NF-κB.

In another specific embodiment of the present invention, after treating the cholera martini extract in macrophages induced by LPS-induced inflammatory response and analyzed the binding activity of NF-κB and AP-1, increased by LPS stimulation NF-κB and AP-1 activity was found to decrease when treated with more than 30μg / ml of Philea Martini extract (Fig. 6a).

Prevention or treatment of the inflammatory disease may be achieved by inhibition of secretion of IL-6, TNF-α or PGE ₂ .

In another specific embodiment of the present invention, after treating the Philea martini extract to macrophages induced by LPS-induced inflammatory response, the amount of inflammatory cytokines IL-6, TNF-α and PGE ₂ were measured. , It was confirmed that the amount of cytokine secretion increased by the LPS stimulation significantly decreased with the concentration of the Philea martini (FIGS. 4 and 7).

The term "prevention" in the present invention refers to any action of inhibiting or delaying an inflammatory disease by administration of a composition comprising a Philea martini extract according to the present invention.

The term "improvement" in the present invention means any action in which the symptoms of an inflammatory disease are ameliorated or beneficially altered by administering the composition.

The food composition of the present invention provides various types of food additives or functional foods. Specifically, it may be processed into leaching tea, liquid tea, beverage, fermented milk, cheese, yogurt, juice, probiotic or health supplement containing the composition, and may be used in the form of various other food additives.

The food composition of the present invention may further comprise a food acceptable carrier.

There is no restriction | limiting in particular in the kind of food which can add the composition containing the Philea martini extract of this invention, For example, there are various drinks, gum, tea, a vitamin complex, health supplements, etc. The food composition may be added to other ingredients that do not interfere with the therapeutic effect of menopausal disorders, the kind is not particularly limited. For example, various herbal extracts, food acceptable additives, natural carbohydrates, and the like may be contained as additional ingredients, such as conventional foods.

The food supplement additive is added to prepare the food composition of each formulation can be used by those skilled in the art as appropriate. For example, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters , Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in the carbonated beverage, and the like, but the kind is not limited by the above examples.

At this time, the content of the extract included in the food is not particularly limited, but may be included in 0.01 to 100% by weight, specifically 1 to 80% by weight relative to the total weight of the food composition.

When food is a beverage, it may be included in a ratio of 1 to 30 g, specifically 3 to 20 g, based on 100 ml. In addition, the composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. Also, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included. It may also contain amino acids such as lysine, tryptophan, cysteine, valine and the like. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), butylhydroxytoluene ( BHT), etc.), colorants (such as tar pigments), colorants (such as sodium nitrite, sodium nitrite), bleach (sodium sulfite), seasonings (such as MSG glutamate), sweeteners (ducin, cyclate, saccharin, sodium, etc.) , Food additives such as flavorings (vanillin, lactones, etc.), swelling agents (alum, aluminium, D-potassium hydrogenate, etc.), reinforcing agents, emulsifiers, thickeners (pigments), coatings, gum herbicides, foam inhibitors, solvents, improvers, etc. ) Can be added. The additive is selected according to the type of food and used in an appropriate amount.

The food composition of the present invention may be prepared by a method commonly used in the art, and may be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material, and can be excellent in portability.

Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises Philea martini extract.

The term "treatment" in the present invention refers to any action in which the inflammatory disease is improved or advantageously modified by administration of a composition comprising the extract of Philea martini according to the present invention.

As used herein, the term "pharmaceutical composition" means that is prepared for the purpose of preventing or treating a disease, and can be used by formulating in various forms according to each conventional method. For example, it may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like, and may be used in the form of external preparations, suppositories, and sterile injectable solutions. In particular, it may be used in a form suitable for topical administration, for example, eye drops, creams, ointments, gels or lotions.

The composition of the present invention may be prepared in the form of a pharmaceutical composition for the prevention or treatment of inflammatory diseases, further comprising a suitable carrier, excipient or diluent commonly used in the manufacture of a pharmaceutical composition, the carrier is an unnatural carrier (non-naturally occuring carrier).

In the present invention, carriers, excipients and diluents which may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, and the like in the above extracts and fractions thereof. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solvents, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, etc. according to the desired method, and the active ingredient of the pharmaceutical composition may be administered to the age of the subject to be administered. , Sex, weight, pathology and severity, route of administration or prescriber's judgment. Dosage determination based on these factors is within the level of one of skill in the art and its daily dosage may be, for example, from 0.1 mg / g / day to 100 mg / g / day, more specifically from 5 mg / g / day to 50 mg / day. g / day, and the frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or several times in divided doses. The dosage does not limit the scope of the invention in any aspect.

Another embodiment of the present invention provides a method for preventing or treating an inflammatory disease, comprising administering to a subject other than humans, Philea martini extract.

The above, Philea martini extract, inflammatory disease, prevention is as described above.

As used herein, the term "individual" may mean any animal, including a human, who has or is likely to develop an inflammatory disease. The animal may be a mammal such as, but not limited to, a human, a cow, a horse, a sheep, a pig, a goat, a camel, a antelope, a dog, a cat, and the like, which require treatment of similar symptoms.

The method for preventing or treating the present invention may specifically include administering the composition in a pharmaceutically effective amount to an individual having or at risk of developing an inflammatory disease.

The term "administration" in the present invention means introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered through various routes as long as it can reach the desired tissue. If desired, administration may be by administration of instillation, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, pulmonary administration, rectal administration, etc. And may specifically be administered via a route of oral administration.

Yet another aspect of the present invention provides an quasi-drug composition for preventing or improving inflammatory disease, comprising a Philea martini extract.

As used herein, the term "quasi drug" refers to articles that have a lesser action than pharmaceuticals among articles used for the purpose of diagnosing, treating, ameliorating, alleviating, treating, or preventing a disease in humans or animals. For example, quasi-drug products under the Pharmaceutical Affairs Law exclude products used for the purpose of medicines, and include products used for the treatment or prevention of diseases of humans / animals, and products with slight or no direct action on the human body.

Specifically, the quasi-drug may include external skin preparations and personal hygiene products. More specifically, it may be, but is not limited to, a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, or an ointment.

When the composition according to the present invention is used as an quasi-drug additive, the composition may be added as it is or used with other quasi-drugs or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use.

Another aspect of the present invention provides a cosmetic composition for alleviating or improving inflammation comprising a Philea martini extract.

In the present invention, the cosmetic composition is a solution, external ointment, cream, foam, nourishing lotion, flexible lotion, pack, flexible water, latex, makeup base, essence, soap, liquid cleaning agent, bath, sunscreen cream, sun oil, It may be prepared in a formulation selected from the group consisting of suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays, but It is not limited.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as conventional components, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , Chelating agents, pigments, preservatives, fragrances and the like may be appropriately blended, but is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and the like. May be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as the carrier component, and particularly in the case of a spray, additionally chlorofluorohydro Propellants such as carbon, propane / butane or dimethyl ether may be included, but are not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsion may be used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil and the like can be used, and in particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester May be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxyde, bentonite, agar or tracant may be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters may be used, but are not limited thereto. These may be used alone or in combination of two or more thereof.

In addition to the said essential component, you may mix | blend with the cosmetic composition of this invention the other component normally mix | blended with cosmetics as needed. Specifically, as a compounding component which may be added, an oil-fat component, a humectant, an emollient, surfactant, organic and inorganic pigment, organic powder, a ultraviolet absorber, a preservative, a fungicide, antioxidant, a plant extract, a pH adjuster, alcohol, a pigment, Fragrances, blood circulation accelerators, cooling agents, restriction agents, purified water, and the like, but are not limited thereto.

Philea martini extract provided by the present invention inhibits the production of nitric oxide (NO) induced by lipopolysaccharide and secretion of IL-6, TNF-α, PGE ₂ , induced by lipopolysaccharide (LPS) iNOS and COX-2 proteins and their mRNA expression inhibitory effect, and can inhibit the inflammatory response by inhibiting the NF-κB signaling process, it can be useful as a composition for the prevention or improvement of inflammatory diseases.

1 is a graph showing the cytotoxicity analysis results of the Philea Martini extract.
Figure 2 is a graph showing the results of the analysis of nitric oxide production according to the treatment of Philea Martini extract in LPS-stimulated macrophages.
Figure 3 is a graph analyzing the protein level and mRNA activity of iNOS and COX-2 according to the treatment of the filla martini extract in LPS-stimulated macrophages.
Figure 4 is a graph analyzing the amount of PGE ₂ secretion according to the treatment of the Philea Martini extract in LPS-stimulated macrophages.
Figure 5 is a graph analyzing the phosphorylation activity of MAPK signaling system transcription factor (p38, ERK, JNK) according to the treatment of the Philea martini extract in LPS-stimulated macrophages.
Figure 6 is a graph showing the phosphorylation activity and protein level analysis results of NF-κB signaling system transcription factor according to the treatment of the Philea martini extract in LPS-stimulated macrophages.
Figure 7 is a graph showing the results of the analysis of the secretion of inflammatory cytokines according to the treatment with Philea martini extract in LPS-stimulated macrophages.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1 Preparation of Philea Martini Extract

400 ml of 99.9% (v / v) methanol was added to the whole dried and purified Pilea martinii plant (37 g), followed by extraction for 3 hours followed by 2 hours of sonication for 15 minutes. Proceed. The obtained extract was filtered through a non-fluorescent cotton and concentrated under reduced pressure at 45 ° C. with a rotary evaporator (N-1000SWD, EYELA). Finally, the concentrate was lyophilized to obtain 2.52 g of Pilea martinii extract.

Experimental Example 1. Cytotoxicity Analysis of Philea Martini Extract

Pilreah Martini (Pilea martinii) the survival of the cells for pilreah Martini extract the lipid polysaccharide is treated in macrophages RAW 264.7 versus with the extract with different concentrations (0, 10, 30, 50 μg / ml) the lipid polysaccharide (LPS) with MTT Measured by analytical method. At least three repeated experiments were performed, and the results of the independent experiments were averaged and expressed as percentages compared to the viability of control cells.

As a result, as shown in Figure 1, it could be confirmed that the fillia martini extract does not exhibit cytotoxicity even up to 50μg / ml.

This suggests that the Philea martini extract is not toxic and therefore has high safety.

Experimental Example 2 Analysis of Nitric Oxide Production Inhibitory Activity of Extracts from Philea Martini

Since nitric oxide is a very unstable molecule and is rapidly converted into nitrite after generation, the nitric oxide production inhibitory effect was confirmed by analyzing the nitrite production level using the Griess reaction assay. RAW 264.7 macrophages were pretreated for 2 hours with different concentrations of Philea Martini extract and stimulated for 22 hours with LPS (1 μg / ml). The cell culture was then treated with Greries reagent and the discoloration level measured to confirm the level of nitrite production.

As a result, as shown in Figure 2, compared with the normal group, LPS treated group showed a very high level of nitrite production, while the filla martini treated group showed a concentration-dependent decrease in nitrite production.

This suggests that the Philea Martini extract can inhibit the production of nitric oxide and thereby reduce the inflammatory diseases caused by it.

Experimental Example 3. Inhibitory activity analysis of i.e.

3-1. Analysis of Protein Production Inhibitory Activity of iNOS and COX-2

Inhibitory activity of phyllae martini extract against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which are indicative of inflammatory reactions, was analyzed. RAW 264.7 macrophages were pretreated for 2 hours with different concentrations of Philea Martini extract and stimulated for 22 hours with LPS (1 μg / ml). Equal amounts of total protein (10 μg / lane) were isolated by 10% SDS-PAGE and expression of iNOS and COX-2 proteins were detected using anti-iNOS and anti-COX-2 protein specific antibodies. β-actin was used as loading control. The results are shown as representatives of three independent experiments.

As a result, as shown in Figure 3a, it was confirmed that the protein level of iNOS and COX-2 decreased concentration-dependently of the Philea Martini extract.

This suggests that the protein levels of iNOS and COX-2 are reduced by the Philea martini extract, which may reduce the symptoms of inflammatory diseases.

3-2. mRNA production inhibitory activity of iNOS and COX-2

After pretreatment for 2 hours with different concentrations of Philea martini extract was confirmed whether the inhibition of iNOS and COX-2 mRNA expression in LPS-stimulated cells. RAW 264.7 cells pretreated for 2 hours at the indicated concentrations were stimulated with LPS (1 μg / ml) for 22 hours. Total RNA was isolated from these cells and expression of iNOS and COX-2 mRNA was detected by RT-PCR analysis. As a control for the initial similar content of cDNA of the samples, PCR of GAPDH was performed. The results are shown as representatives of three independent experiments.

As a result, as shown in Figure 3b, in the case of iNOS, it can be seen that the level of mRNA decreases as the concentration of the filla martini increases, COX-2 also can be seen that the level of mRNA decreases from 30μg / ml .

This suggests that Philea Martini can reduce the expression levels of iNOS and COX-2, thereby reducing the symptoms of inflammatory diseases.

Experimental Example 4 PGE of Philea Martini Extract ₂  Secretion inhibitory activity assay

PGE ₂ secretion inhibitory activity was measured by using a mouse PGE ₂ antibody-coated ELISA kit in a culture of LPS-stimulated RAW 264.7 cells after 2 hours of pretreatment with different concentrations of Philea martini extracts to measure the amount of PGE ₂ secreted. It was. Data were obtained from three independent experiments and expressed as mean ± standard deviation.

As a result, as shown in Figure 4, it was confirmed that the amount of PGE ₂ significantly decreases with the concentration of the Philea Martini.

This suggests that the Philea martini extract may have an effect of improving the inflammatory disease by reducing the secretion of PGE ₂ , which is one of the factors involved in the inflammatory response.

Experimental Example 5 Analysis of MAPK Signaling Inhibitory Activity of the Extract of Philea Martini

In order to confirm the inhibitory activity of the fila martini extract against the MAPK signaling system, one of the higher signaling systems of the inflammatory response, the phosphorylation activity of p38, ERK, JNK proteins used in the MAPK signaling system was analyzed. Mouse RAW 264.7 macrophages were pretreated with 30 μg / ml Philea Martini extract for 2 hours and stimulated with LPS (1 μg / ml) for a designated time. Equal amounts of total protein (15 μg / lane) were isolated from whole cell extracts by 10% SDS-PAGE and then electrically transferred to nitro cellulose membranes. The transferred proteins were analyzed using phosphate specific anti-protein antibodies against p38, ERK, and JNK, respectively, and then blotted back to the respective anti-protein antibodies to analyze the results. All experiments were repeated at least three times and representative values of the results of three independent experiments are shown in the figure.

As a result, as shown in Figure 5, all p38, ERK, JNK protein can be confirmed that the LPS-treated group induces the inflammatory response to increase the phosphorylation activity, while in the group treated with LPS and Philea martini and LPS-treated group In comparison, it was confirmed that the phosphorylation activity is greatly reduced.

This suggests that the Philea martini extract can improve inflammatory diseases by inhibiting the phosphorylation of proteins required for MAPK signaling mechanisms, thereby preventing the signaling and reducing the inflammatory response.

Experimental Example 6 Analysis of NF-κB Signaling Inhibitory Activity of the Extract of Philea Martini

6-1. Binding Activity Analysis of NF-κB and AP-1

In order to confirm the inhibitory activity of the Philea martini extract on the NF-κB signaling system, which is one of the higher signaling systems of the inflammatory response, the transcription factors NF-κB and AP-1, which are used in the NF-κB signaling system, are identified. Binding activity was analyzed. Mouse RAW 264.7 macrophages were pretreated for 2 hours with different concentrations of Philea martini extract and prepared nuclear extract (5 μg / lane) obtained by stimulation with LPS (1 μg / ml). The binding activity of NF-κB and AP-1 was performed by electrophoretic mobility shift assay (EMSA). All experiments were conducted at least three times independently and representative values were shown in the figure.

As a result, as shown in Figures 6a and 6b, it is possible to confirm the binding activity of NF-κB and AP-1 induced by LPS, the activity of NF-κB and AP-1 as the concentration of the Philea Martini extract increases This decrease can be seen.

6-2. IκB-α Proteolytic Activity Assay

The degradation activity against well known IκB-α in the mechanism of inflammatory response was analyzed. Macrophages were pretreated with 30 μg / ml of Philea Martini extract for 2 hours and stimulated with LPS (1 μg / ml) for a designated time to prepare cytoplasmic extracts. Equal amounts of protein (10 μg / lane) were separated by 10% SDS-PAGE and then electrophoresed to nitro cellulose membranes. The membrane was analyzed by reacting anti-IκB-α antibodies. β-actin was used as loading control.

As a result, as shown in Figure 6c, when the inflammatory response is induced by LPS stimulation, it can be confirmed that IκB-α is degraded and decreased with time, whereas in the group of fila martini extract treatment group IκB-α over time Was able to confirm that it maintains a constant level.

6-3. p65, c-Jun, c-Fos Protein Analysis

Other transcription factors used in the NF-κB signaling system were further identified. Nuclear extracts (10 μg / lane) were prepared from cells stimulated for 2 hours after treatment with 30 μg / ml of Philea martini extract for a time designated by LPS (1 μg / ml). 10% SDS-PAGE was performed to isolate and transfer to nitrocellulose membrane and analyzed using phosphate-specific anti-p65, anti-p65, anti-c-Jun and anti-c-Fos. Lamin B was used as a loading control. All experiments were performed at least three times independently and representative values were shown in the figure.

As a result, as shown in Figure 6d, it can be seen that the level of p65 and phosphorylated p65 is increased by LPS induction, the level was reduced in the group treated with Philea martini extract. In addition, c-Jun and c-Fos also can be seen that the level is reduced in the Philea martini extract treatment group compared to the LPS treatment group.

Taken together, Philea Martini extract inhibits the binding activity of the transcription factors NF-κB and AP-1, inhibits the activity of p65, and decreases the levels of c-Jun and c-Fos, which is the upper signaling system of inflammatory response. Inhibition of NF-κB signaling suggests that inflammatory diseases can be treated.

Experimental Example 7 Analysis of Inflammatory Cytokine Secretion Activity of Philea Martini Extract

In order to confirm the effect of the Philea martini extract on inflammatory cytokines directly involved in the inflammatory response, the secretion of lipopolysaccharide-induced IL-6 and TNF-α in mouse RAW 264.7 macrophages was analyzed. Different concentrations of Philea Martini extract were pretreated for 2 hours and then stimulated with LPS (1 μg / ml) for 22 hours. The amount of secreted IL-6 and TNF-α was measured using mouse IL-6 or TNF-α kits. Data were obtained from three independent experiments and expressed as mean ± standard deviation.

As a result, as shown in Figure 7, it can be seen that the secretion of IL-6 and TNF-α significantly increased when only LPS treatment, and the concentration-dependent secretion decreases when treated with Philea martini. .

This suggests that the Philea martini extract can reduce the secretion of inflammatory cytokines that are directly involved in the inflammatory response and thus have a therapeutic effect on inflammatory diseases.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (8)

A food composition for preventing or ameliorating an inflammatory disease comprising a extract of Pilea martinii.
The method of claim 1,
The extract is a food composition that is extracted with water, alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
The method of claim 1,
The prevention or improvement of the inflammatory disease is achieved by inhibiting the expression of iNOS protein, COX-2 protein or mRNA encoding it.
The method of claim 1,
The prevention or improvement of the inflammatory disease is achieved by inhibiting the activity of NF-κB, food composition.
Pharmaceutical composition for preventing or treating inflammatory diseases, including the extract of Philea Martini.
A method for preventing or treating an inflammatory disease, comprising administering to a subject other than humans, Philea martini extract.
A quasi-drug composition for preventing or ameliorating an inflammatory disease comprising a Philea martini extract.
Inflammation alleviation or improvement cosmetic composition comprising a Philea martini extract.

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