KR102010691B1 - Composition for Anti-inflammation Using Pratol - Google Patents

Abstract

The present invention is directed to an activity that inhibits the production of NO, PGE ₂ and inflammatory mediator cytokines in LPS-induced mouse macrophage lines (RAW 264.7 cells), which is a substance derived from Trifolium pretense . An anti-inflammatory composition comprising pratol as an active ingredient is disclosed.

Description

Composition for anti-inflammatory using pratol {Composition for Anti-inflammation Using Pratol}

The present invention relates to a composition for anti-inflammatory using pratol.

Inflammation is a local biological defense response in response to physical trauma, harmful chemicals, bacteria, fungi, viruses, or pathological conditions caused by irritants in metabolites in vivo. Inflammation is triggered by various inflammatory mediators produced from damaged tissues and migrating cells. During the inflammatory reaction, plasma accumulates at the site of inflammation, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever. In normal cases, the organism restores normal structure and function by neutralizing or eliminating the pathogens through the inflammatory response and regenerating the upper tissue, but in other cases, the disease progresses to a disease state such as chronic inflammation.

Recent advances in molecular biology have led to a great deal of research into the inflammatory response at the molecular level.

Inflammatory reactions involve a variety of biochemical phenomena, but macrophages, in particular, are known to play an important role in the inflammatory response by producing nitric oxide (NO) and various inflammatory cytokines by chemical stimulation (Ito T., et. al., Curr Drug Traget Inflamm Allergy, 2 (3): 257-265, 2003). In particular, lipopolysaccharide (LPS), known as endotoxin of Gram-negative bacteria, stimulates macrophages and activates signaling pathways such as NF-κB (mitogen activated protein kinase (MAPK)) pathways and tumors. Secretion and nitric oxide (NO) of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL1-β) and IL-6; Prostaglandin E2 (PGE2) promotes the expression of inflammatory response factors. In various mammalian cells and tissues, NO is produced from L-arginine by nitric oxide synthase (NOS). NOS has several isoforms. Brain NOS (brain NOS) in the brain, nNOS (neuronal NOS) in the nervous system, endothelial NOS (eNOS) in the vascular endothelial system are always expressed in a certain level in the body, a small amount produced by these monoxide Nitrogen (NO) plays an important role in maintaining the homeostasis of the body by performing various physiological reactions related to blood pressure control, neurotransmitter, learning, memory, and the like. In contrast, iNOS (induced NOS), whose expression is induced by a stimulus, produces excess NO. Nitric oxide produced by iNOS reacts with superoxide to form peroxynitrite. It acts as a powerful oxidant and damages cells and is involved in various pathological processes, including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236: 658-671, 2011; Riehemann et al., FEBS Lett., 442: 89-94, 1999; Stammler et al., Science, 258: 1898-1902, 1992).

On the other hand, cyclooxygenase (COX) has hydroperoxidase (HOX) activity with COX function and synthesizes PGG ₂ and PGH ₂ intermediates from arachidonic acid, and these compounds are PGE ₂ and PGF ₂ , PGD ₂ , prostacyclin and thromboxane A ₂ (thromboxane A2, TxA2) are produced. There are two isoforms in COX. While COX-1 is always expressed in most tissues and synthesizes prostaglandins (PGs), which are required for cell protective action, COX-2 is rapidly induced during the inflammatory response to produce PGE ₂ and the like. Play an important role (Weisz A., Biochem. J., 316: 209-215, 1996; (Miller MJ et al., Mediators of inflammation, 4: 387-396, 1995: Appleton L. et al., Adv) Pharmacol., 35: 27-28, 1996).

Expression of iNOS and COX-2, which leads to overproduction of NO and PGs, is regulated by the nuclear transcription factor NF-κB. NF-κB is a nuclear protein of the Rel gene family. In the cytoplasm, NF-κB binds to I-κB and is inactive.However, if I-κB kinase is activated by any factor, It is activated by the release of I-κB. NF-κB, composed of p50 and p65 heterodimers, activates and induces gene expression of iNOS and COX-2 that migrate to the nucleus and induce an inflammatory response (Oh, GT et al., Atherosclerosis, 159 (1): 17-26, 2001).

Bacterial endotoxins such as LPS (lipopolysaccharide) activate NF-κB, which is a transcription factor by binding to toll-like receptor 4 (TLR4), and induce the expression of iNOS and COX-2 to induce NO, inflammatory cytokines, PGE _2, etc. Secrete several inflammatory modulators (Chen YC, et al, Biochem. Pharmacol., 61: 1417-1427, 2001; Dobrovolskaia MA, et al., J Immunol., 170: 508-19, 2003; Ji Y, et al., Cell Physiol Biochem., 25: 631-640, 2010). NO, Inflammatory cytokines such as TNF-α, IL-6, PGE ₂ , arthritis (Jang CH et al., Rheumatology, 2006, 45 (6): 703-710), fibromyalgia (Hernandez ME et. Al., BMC Res Notes., 2010, 3 (1): 156), Sjogren's syndrome (Baturone R. et. Al., Scand J Rheumatol., 2009, 38 (5): 386-389), etc. (Jang CH et al., Rheumatology, 45 (6): 703-710, 2006; Hernandez ME et. Al., BMC Res. Notes., 3 (1): 156, 2010; Baturone R. et. al., Scand J Rheumatol., 38 (5): 386-389, 2009).

These findings can either inhibit NO production Drugs that inhibit the production of inflammatory cytokines such as TNF-α and IL-6, and drugs that inhibit the expression of iNOS or COX-2, have the potential as effective anti-inflammatory agents (Karin M. et al., Cold Spring Harb Perspect Biol., 1, pp 1-14, 2009).

Non-steroidal anti-inflammatory drugs (NSAIDS), which are widely used as anti-inflammatory agents, are known to cause serious side effects such as gastrointestinal disorders, liver disorders and nephropathy (Rainsford KD., Subcell biochem., 42: 3). -27, 2007; Guruprasad P. Aithal., Rheumatology., 7: 139-150, 2011; Praveen PN Rao et al., Pharmaceuticals., 3: 1530-1549, 2010).

Therefore, there is still a need for the development of new drugs that have anti-inflammatory activity and have fewer side effects and lasting effects.

The present invention discloses the anti-inflammatory activity of pratol , a substance isolated from red clover ( Trifolium pretense L.).

An object of the present invention to provide an anti-inflammatory composition using pratol.

Other and specific objects of the present invention will be presented below.

In the present invention, Pratol (Pratol) showed NO and PGE ₂ inhibitory activity in a dose-dependent manner in rat macrophage (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide) and iNOS In addition to reducing the expression of and COX-2 as well as confirming the inhibitory effect of the production of inflammatory cytokines TNF-α, IL-6 and IL-1β was completed.

In view of the above, the present invention can be understood as an anti-inflammatory composition comprising pratol, a prodrug thereof, a hydrate thereof, or a solvate thereof represented by the following [Formula 1] as an active ingredient.

[Formula 1]

Pratol (Pratol; 7-hydroxy-2- (4-methoxyphenyl) chromen-4-one) of the present specification is a substance isolated from red clover (Trifolium pretense L.) as an O-methylated flavone. Red shamrock is a perennial plant of real rosemary legaceae, and is introduced in Europe for grasses and now spreads wild throughout Korea. It is a plant commonly found in various places such as ranches and roads in Jeju Island. Since ancient times, the flowers, leaves, and stems of red shamrocks have been used as medicines.In particular, petals are put into ointments or boiled in water to treat wounds, burns, gout and eye diseases, and boiled with tea to relieve fever, cough and asthma. have.

In addition, in the present specification, "prodrug" refers to a drug that modulates physical and chemical properties by chemically changing a drug, and itself does not show physiological activity, but it does not affect the action of chemicals or enzymes in the body after administration. By means of the original drug can change the drug means.

In the present specification, "hydrate" refers to a compound to which water is bound, and includes a compound containing no chemical bonding force between water and the compound.

In addition, in this specification, "solvate" means the compound which generate | occur | produced between the molecule | numerator or ion of a solute, and the molecule | numerator or ion of a solvent.

As used herein, the term "active ingredient" alone refers to a component that can exhibit the desired activity or exhibit itself with a carrier which is inactive.

In addition, in the present specification, "anti-inflammatory" is meant to include improvement (reduction of symptoms), treatment, inhibition of the onset or delay of the inflammatory disease as defined below.

In addition, in the present specification, the "inflammatory disease" refers to an inflammatory response specified as a local or systemic biological defense response against external physical and chemical stimuli or infection or autoimmunity of an external infectious agent such as bacteria, fungi, viruses, and various allergens. It can be defined as a pathological symptom that causes. These inflammatory responses include activation of various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, NO, TNF-α, IL-6, etc.), fluid infiltration, It is accompanied by a series of complex physiological reactions such as cell migration and tissue destruction, and is manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of an inflammatory disease as above, whether it is acute, chronic, ulcerative, allergic or Irrespective of necrosis 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, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, viral infection (eg, type C infection), bacterial infection, fungal infection, burn, wound by surgical or dental surgery, pro Prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis Will be included.

The anti-inflammatory composition of the present invention may include the active ingredient in any amount (effective amount) as long as it can exhibit the improvement activity of an inflammatory disease intended to be treated according to the use, formulation, formulation purpose, etc. Silver will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. Here, the "effective amount" refers to the intended medical and pharmacological effects, such as an improvement effect of an inflammatory disease, when the composition of the present invention is administered to a mammal, preferably a human, during the administration period as suggested by a medical expert. The amount of the active ingredient included in the composition of the present invention can be mentioned. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

In addition to the active ingredient, the anti-inflammatory composition of the present invention is administered or ingested for the purpose of increasing and enhancing the anti-inflammatory effect or through the addition of similar activities such as anti-allergic activity, skin protective activity (inhibition of UV damage to the skin, skin moisturizing, etc.). In order to enhance the convenience of the present invention, it may further include any compound or natural extract that has already been proven safe in the art and known to have the corresponding activity.

Such compounds or extracts include compounds, extracts, and medicines contained in procedures such as the Pharmacopoeia of each country ("Korea Pharmacopoeia" in Korea) and the Health Functional Foods Code of Korea ("Health Functional Food Standards and Standards" in Korea). Laws governing the manufacture and sale of compounds, extracts or health functional foods licensed under the laws of each country governing the manufacture and sale of pharmaceutical products in Korea ("Pharmaceutical Act on Health Functional Foods") And compounds or extracts which have been individually functionally recognized as such. For example, MSM (dimethylsulfonylmethane) with 'improving arthritis' functionality in Korea Health Functional Food Code, N-acetylglucosamine with 'improving arthritis' and 'skin moisturizing' functionality, and glucosamine with 'improving arthritis', etc. Compound extracts such as FAC (Fatty acid Complex) containing CMO, P. falciparum, Turmeric extract, Chicken breast cartilage powder, Rose hip powder, Boswellia extract, Complexes such as beeswax alcohol and whole ginseng extract, complexes such as fatty acid complex, perilla extract, complexes such as green lipped mussel extract oil, hop extract and golden extract, and Enterococcus faecalis individually recognized for its 'hypersensitivity immune response' function Heat treated dry powder, complex such as guava leaf extract, stalk extract, leaflet extract, picaopreto powder Compounds such as horses, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) and the like will correspond to such compounds or extracts.

One or more such compounds or natural extracts may be included in the anti-inflammatory composition of the present invention together with the active ingredients.

The anti-inflammatory composition of this invention can be grasped | ascertained as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, sweets, bread, sweets, noodles, and the like. Foodstuffs, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, and the like can be prepared as functional food preparations.

In addition, the food composition of the present invention can distinguish any product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in the legal and functional divisions. For example, it is a health functional food pursuant to the Act on Health Functional Foods, or confectionery, soybeans, teas, beverages, etc. according to each food type in the Food Code (KFDA Notification, Food Standards and Standards) of the Korean Food Sanitation Act. Special purpose foods, and the like.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives can generally be understood as substances which are added to the food, mixed or infiltrated in the manufacture, processing or preservation of the food, which must be ensured because of its daily and long-term intake with the food. In the Food Additive Code according to the laws of each country governing the manufacture and distribution of foods ("Food Sanitation Law" in Korea), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA notice and standards), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners and flavoring agents in terms of function. , Preservatives, emulsifiers, acidulants, thickeners and the like.

Sweeteners are used to impart a suitable sweetness to foods, either natural or synthetic, which can be used in the compositions of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing preferably. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. In addition, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo and the like can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

Sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used as a preservative, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin etc. can be mentioned, As acidic acid, acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used. The acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As the thickener, suspending implementers, sedimenting agents, gel formers, swelling agents and the like can be used.

In addition to the food additives described above, the food composition of the present invention may include a bioactive substance or minerals known in the art for the purpose of supplementing and reinforcing the functionality and nutritional properties and ensuring the stability as a food additive.

Examples of such physiologically active substances include catechins, vitamin B1, vitamin C, vitamin E, vitamin B12, tocopherol, dibenzoyl thiamine, and the like contained in green tea. Examples of the minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc and the like.

In the food composition of the present invention, the food additive as described above may be included in an amount that can achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to food or food additives.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical compositions of the present invention may be prepared in oral or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. "Pharmaceutically acceptable" here means that the subject of application (prescription) is not toxic as far as adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers It may be prepared in a formulation such as. Examples of suitable pharmaceutically acceptable carriers include lactose, glucose, sucrose, dextrose, sugars such as sorbitol, mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable Yu etc. can be mentioned. If formulated, it may be formulated to include diluents and / or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, if necessary.

When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories with suitable carriers according to methods known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof. Preferably, PBS (phosphate buffered saline) containing Ringer's solution or triethanol amine or sterilized for injection Water, isotonic solutions such as 5% dextrose, and the like. When formulated as a transdermal administration, it may be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, linen, aerosol and the like. Nasal inhalants can be formulated in the form of aerosol sprays using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like.

Regarding the formulation of pharmaceutical compositions, it is known in the art and specific reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. The document is considered part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention range from 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g, depending on the condition, body weight, sex, age, severity of the patient and route of administration. It can range from / kg. Administration can be done once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

The anti-inflammatory composition of this invention can be grasped | ascertained as a cosmetic composition in another specific aspect. When the anti-inflammatory composition of the present invention is identified as a cosmetic composition, its use may be understood as a relief of inflammatory skin irritation.

The cosmetic composition of the present invention may include, in addition to the active ingredient, components conventionally used in the cosmetic composition, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and pharmaceuticals, and carriers.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

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, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention can be prepared according to the manufacturing method of the cosmetic composition usually carried out in the art, except for including the active ingredient exhibiting anti-inflammatory activity.

As described above, according to the present invention can provide an anti-inflammatory composition using pratol.

The anti-inflammatory composition of the present invention may be commercialized as foods, cosmetics, drugs, etc. for the use of improvement of inflammatory diseases, the use of alleviating inflammatory skin irritation, and the like.

1 shows the results of cell viability when treated with the concentration of pratol.
2 is a result showing the NO and PGE ₂ production inhibitory activity of pratol.
Figure 3 shows the results of the expression inhibitory activity of pratol iNOS and COX-2.
4 is a result showing the inhibitory activity of inflammatory cytokine production of pratol.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

EXAMPLES Anti-inflammatory Activity of Pratol

Example 1 Experimental Method

Pratol used in the experiment was purchased from Extrasynthese (Genay CEDEX, France).

Example 1-1 Cell Culture

RAW264.7 cells used in the anti-inflammatory activity confirmation experiment were purchased through the Koeran Cell Line Bank. Cells were incubated in Dulbecco's Modified Eagle medium (DMEM) medium containing 1% penicillin and 10% fetal calf serum (FBS) in an incubator at 37 ° C and 5% CO2.

Example 1-2 Cytotoxicity Evaluation

MTT experiments were performed to determine the effect of the sample on cell survival. The MTT experiment was carried out by the dehydrogenase action of mitochondria in living cells, resulting in a yellow, water-soluble substrate, 3- (4,5-dimethylthiaxo-2-yl) -2,5-diphenyltetrazolium bromide (MTT). This test uses the ability of mitochondria to reduce to mazan.

RAW 264.7 cells were incubated for 24 hours in DMEM medium containing 1% penicillin and 10% FBS. 2.0 x 10 ⁴ cells / well per well was dispensed into 24 well plates and incubated for 48 hours before treatment of the samples by concentration. The supernatant was removed and diluted with DMEM medium to a concentration of MTT reagent (0.5 g / L) in 400 μL of each well and treated for 3 hours. Thereafter, the supernatant was removed, DMSO was added to the formazan crystals, and dissolved at room temperature for 1 hour, and then transferred to a 96 well plate, and the absorbance was measured at 550 nm using a microplate reader (Tecan, Mannedorf, Swizerland).

Example 1-3 Anti-inflammatory Activity Measurement

Experimental methods performed to confirm the anti-inflammatory activity of Pratol are as follows.

<1-3-1> NO production amount measurement

RAW 264.7 cells were dispensed at 1.5 × 10 ⁵ cells / well per well in 24 well plates using DMEM medium supplemented with 1% penicillin and 10% FBS and incubated for 24 hours. The samples were then incubated for 24 hours by treatment with concentrations (25, 50, 100 μM) LPS. As a control, DMEM medium without any treatment, a medium treated only with LPS (1 μg / mL), and a medium treated with LPS and 25 μM APDC were used. Then, 100 μL of the supernatant of each well and 100 μL of the Griess reagent were mixed in a 96-well plate, and reacted for 1 hour while blocking the light.

<1-3-2> Prostaglandin E ₂ (PGE ₂ ) Inhibition Measurement

50 μL of the medium diluted with the concentration (25, 50, 100 μM) and LPS (1 μg / mL) was treated in each well incubated with RAW 264.7 cells and incubated for 24 hours. As a control group, a group treated with nothing (DMEM medium only), a group treated with LPS (1 μg / mL) only, and a group treated with LPS and 25 μM APDC simultaneously. After 24 hours, the cultures were centrifuged at 10,000 rpm for 3 minutes, and then the supernatants were harvested and the PGE ₂ content at each concentration using a mouse ELISA (enzyme-linked immnunosorbent assay) kit (R & D Systems Inc., Minneapolis, MN, USA). Was measured.

<1-3-3> Determination of Inflammatory Mediated Cytokines (TNF-α, IL-6, IL-1β)

RAW 264.7 cells were aliquoted into each well of a 24 well plate (1.5 × 10 ⁵ cells / well) and incubated for 24 hours. Subsequently, the samples were incubated for 24 hours by co-treatment with (25, 50, 100 μM) LPS for each concentration. DMEM medium without treatment as a control, medium treated with LPS (1 μg / mL) only, LPS and 25 Medium treated with μM APDC at the same time was used. After 24 hours, the culture medium was centrifuged at 10,000 rpm for 3 minutes, and then the supernatant was collected, and the contents of inflammatory mediators of each concentration were measured using a mouse ELISA kit (R & D Systems Inc., Minneapolis, MN, USA). .

<1-3-4> Western blotting

RAW 264.7 cells were dispensed in 100 mm dish (1.0 × 10 ⁵ cells / well) and incubated for 24 hours. DMEM media treated with diluted samples (25, 50, 100 μM) by concentration and nothing treated as control, media treated with LPS (1 μg / mL) only, and media treated with LPS and 25 μM APDC simultaneously It was. The cells were detached, put in a RIPA buffer containing a protease inhibitor cocktail (1.0%), and lysed by vortexing every 10 minutes for 1 hour. After centrifugation at 1300 × g for 15 minutes, the supernatant was transferred to another e-tube, and the protein was quantified by BCA protein assay kit (Thermo Fisher Scientific, Waltham, Mass., USA). In order to make a western sample, 20 μg of protein and 2 × Laemmli sample buffer were mixed at a 1: 1 ratio and then boiled at 100 ° C. for 5 minutes. After the sample was cooled at 4 ° C., 20 μL of the sample was added to each line of SDS-polyacrylamide gel and loaded for 1 hour. The separated proteins were transferred to a polyvinylidene difluoride (PDVF) membrane and the proteins of the membrane were blocked for 1 hour with 5% nonfat skim milk dissolved in Tris buffered saline (TBST) containing 0.4% Tween 20. Thereafter, the membrane was reacted with skim milk in which primary antibody was dissolved for 24 hours. After washing 6 times with TBST every 10 minutes, the secondary antibody dissolved in TBST at 1: 3,000 ratio was reacted for 1 hour. After washing 6 times with TBST every 10 minutes, each protein band was detected using an enhanced chemiluminescence (ECL) kit (Biosesang, Korea).

Example 1-4 Statistical Processing

Student's t-test was used for all the experimental results and statistically significant as (*) for p-values <0.05 and (**) for p-values <0.01. Each result is expressed as mean ± SD of three independent experiments.

Example 2 Results of Confirming Anti-inflammatory Activity of Pratol

Example 2-1 Cell Viability

When Pratol was treated to RAW 264.7 cells, MTT experiments were performed to find the viable concentrations.

L-added medium was treated with Pratol at 25, 50, and 100 μM concentrations, DMEM medium without any treatment, LPS (1 μg / mL) -only medium, and LPS and 25 μM APDC at the same time. Cells were cultured and used as controls. After incubating the cells for 48 hours and performing the MTT experiment, the cell viability of pratol did not change significantly within the treated concentration [FIG. 1]. Thus, the treatment of pratol at a concentration of 25-100 μM showed no effect on cytotoxicity.

 Example 2-2 Anti-inflammatory Activity of Pratol through Inhibition of NO and PGE2 Production

<2-2-1> NO Production Inhibitory Activity of Pratol

Groups treated with Pratol in concentration ranges (25, 50, 100 μM) that were not toxic to RAW 264.7 cells, and none treated as controls, LPS (1 μg / mL) treated groups, and LPS and 25 μM The group treated with APDC at the same time was used. After 24 hours, NO production was induced when LPS alone was treated, and NO production was inhibited by more than 60% when treated with APDC inhibitor. Comparing the LPS-treated group with LPS-stimulated RAW 264.7 cells, Pratol was shown to inhibit more than 45% of NO production at high concentrations (100 μM), indicating that Pratol inhibited high NO production inhibitory activity. It can be seen that (Fig. 2a).

<2-2-2> Inhibitory Activity of Pratol on PGE ₂ Production

In the same conditions as in Example 2-2-1, PGE2 production inhibition experiment of pratol was performed using the PGE2 ELISA kit, and the concentration of PGE in the group treated with both LPS and pratol was compared with that of LPS alone. _{2 It} was confirmed that the production amount is reduced. In particular, high levels of Pratol (100 μM) treated PGE ₂ production decreased more than the inhibitor APDC treatment. These results show that pratol effectively reduces PGE ₂ production in LPS-stimulated RAW 264.7 cells within the range of no cytotoxicity [FIG. 2B].

<2-2-3> Inhibitory Activity of Pratol's iNOS and COX-2 Expression

The protein expression level was measured by Western blotting to determine whether the inhibition of NO and PGE ₂ production by pratol was related to the inhibition of iNOS and COX-2 expression.

RAW 264.7 cells stimulated with LPS (1 μg / mL) were treated with 25, 50 and 100 μM of Pratol at concentrations of 25, 50 and 100 μM and treated with DMEM medium, LPS-only medium and LPS and 25 μM APDC. Incubation was performed for 24 hours using the treated media. As a result, it was confirmed that significantly reduced protein expression of iNOS and COX-2 when Pratol was treated compared to the LPS alone treatment group [FIG. 3].

 Example 2-3 Inflammatory Cytokine Expression by Pratol

The effect of Pratol on the expression of inflammatory mediators TNF-α, IL-6, and IL-1β in RAW 264.7 cells was investigated using an ELISA kit.

RAW 264.7 cells stimulated with LPS (1 μg / mL) were treated with various concentrations of pratol (25, 50, 100 μM) to confirm the inhibitory activity of TNF-α, IL-6 and IL-1β production. 4 is shown. As a result, it was confirmed that pratol inhibits the expression of TNF-α, IL-6 and IL-1β in a concentration-dependent manner when compared with the LPS alone treatment group.

Claims (5)

Pratol (Pratol), or a hydrate thereof, or an anti-inflammatory composition comprising a solvate thereof as an active ingredient.
The method of claim 1,
The anti-inflammatory refers to amelioration, treatment, inhibition of onset or delayed on the inflammatory disease,
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, pulmonary fibrosis, irritability Bowel Syndrome, Inflammatory Pain, Migraine, Headache, Back Pain, Fibromyalgia, Fascia Disease, Viral Infection, Bacterial Infection, Fungal Infection, Burn, Surgical or Dental Surgery, Prostaglandin E Over Syndrome, Atherosclerosis Sclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema and multiple sclerosis.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the pharmaceutical composition.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the food composition.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the cosmetic composition.

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