KR20200069116A - Pharmaceutical composition for prevention or treatment inflammatory diseases comprising comprising root extract Hovenia dulcis, or fractions thereof, or compounds isolated from therefrom - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases, particularly to a pharmaceutical composition or a food composition for preventing or treating inflammatory diseases, including extract of Hovenia dulcis roots, a fraction thereof or a compound isolated therefrom, as an active ingredient. According to the present invention, extract of Hovenia dulcis roots, a fraction thereof or a compound isolated therefrom inhibits production of inflammatory cytokines, prostaglandin E2 and nitrogen oxide, which are inflammation-mediating factors, and thus can be used advantageously to prevent, treat or alleviate inflammatory diseases. In addition, extract of Hovenia dulcis roots, a fraction thereof or a compound isolated therefrom is derived from natural substances, and thus can be used safely for preventing or treating inflammatory diseases with no side-effects.

Description

Pharmaceutical composition for prevention or treatment of inflammatory diseases comprising root extract Hovenia dulcis, or fractions thereof, or compounds isolated from therefrom }

The present invention relates to a composition for the prevention or treatment of inflammatory diseases, which includes a hawthorn root extract, a fraction thereof or a compound isolated therefrom as an active ingredient, specifically, a hawthorn root extract, a fraction thereof, or a compound isolated therefrom. It relates to a pharmaceutical composition for preventing or treating and a food composition for preventing or improving.

Inflammatory response refers to the mechanism of restoring and regenerating the damaged area when an invasion that causes any organic changes such as physical action, chemical substances, or bacterial infection is applied to a living body or tissue.

Macrophages regulate the inflammatory response and immune function, and play an important role in maintaining homeostasis. Lipopolysaccharide (LPS), the outer membrane component of Gram-negative bacteria, responds to the early stage of macrophage infection and plays a central role in host defense, but macrophages activated by excessive lipopolysaccharide stimulation are tumor necrosis factor ( It secretes pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and secretes large amounts of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) (Jenog et al., 2014). NO is produced from inducible NO synthase (iNOS) when macrophages are activated, and excessive NO production causes inflammation and causes harmful effects such as pathological vasodilation, cytotoxicity, and tissue damage. Known (Forstermann and Sessa, 2012). Gene expression of pro-inflammatory cytokines and proteins such as TNF-α, IL-1b and IL-6 are mitotic-activated protein kinases (MAPKs) and nuclear factor kappa B (NF). -kB). When NF-kB is activated in the expression of genes related to immune and inflammatory reactions, IkBα (inhibitory kappa B α), which is bound to NF-kB, is decomposed, and NF-kB enters the nucleus from the cytoplasm, and then TNF-α, It plays an important role as a transcription factor for cytokine expression such as IL-6 (Jang et al., 2005, Majdalawieh and Ro, 2010).

HO-1, an enzyme that regulates the metabolism of heme oxygenase (HO), breaks down the cells' heme into carbon monoxide (CO), ferrous iron, and biliverdin. It is converted, and biliverdine is reduced back to bilirubin by a reductase (Elbirt and Bonkovsky, 1999). HO-1 and the produced byproducts are recognized as enzymes that protect cells and tissues from oxidative stress and effects on gene expression and enzyme activity related to anti-inflammatory and anti-apoptosis (Gozzelino et al., 2010, Tsan et. al., 1989). Bilirubin inhibits the expression of iNOS and COX-2 and the production of NO, and biliverdine has been reported to inhibit the production of the pro-inflammatory cytokines IL-6 and IL-1β. In particular, in the RAW 264.7 macrophages treated with lipopolysaccharide, studies have been reported that HO-1 suppresses cell damage through anti-inflammatory action by inhibiting NO production and iNOS gene expression.

Hovennia dulcis Thunb. is a broad-leaved broad-leaved tree of the buckthorn family. It is a tree with strong cold resistance and sound resistance and strong blindness. It grows mainly in Seoraksan, Odaesan, Jirisan and Hallasan in South Korea, and grows better in the southern region than in the middle and northern regions. do. Many of the chemical components of lush trees have been known, and the main active substances are saponin-based hovenoside I-VII, hovacerboside A1, hovenidulcioside A1, A2, B1, B2, Hoduloside III, etc., and flavonoid-based quercetin, camperol, (+) ampelosin, hovenitin I~III, hobenodulin The structure was studied by separating Hovenodulinol (Park et al., 2015). The pharmacological activity reported so far has been reported to suppress sweet taste, hepatocyte protection, muscle relaxation inhibition, diuretic, antioxidant, anti-cancer and diabetes treatment effects (Yand et al., 2013). In a recent study, literature on the anti-inflammatory efficacy and compositional analysis of oleaceae fruit extract was reported (Park et al., 2016).

Although there have been reports that the extract of hawthorn fruit has an anti-inflammatory effect, there are no studies on the anti-inflammatory effect of extracts by region (leaf, stem, root).

Therefore, it is necessary to develop a composition for the prevention or treatment of inflammatory diseases, which shows excellent efficacy in the treatment and prevention of inflammatory diseases by utilizing the root extract of hawthorn, and can reduce side effects and drug resistance by using natural raw materials.

Accordingly, in the present invention, in order to confirm the anti-inflammatory effect of the root extract, which is excellent in the NO production inhibitory effect of the extract of each stalk (leaf, stem, root), NO, prostaglandin E2 and pro-inflammatory cytokines (IL-1β, IL -6, TNF-α) secretion was measured, and HO-1, MAPKs and Ik-Ba were examined as the regulatory mechanism. In addition, the chemical structure was identified by spectroscopic methods by separating and purifying the active ingredient from the root extract of hawthorn.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including the hawthorn root extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a food composition for the prevention or improvement of inflammatory diseases, including the hawthorn root extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the compound of formula (I) as an active ingredient.

[Formula Ⅰ]

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases comprising the compound of formula (I) as an active ingredient.

[Formula Ⅰ]

One aspect of the present invention for achieving the above object is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including hawthorn root extract or a fraction thereof as an active ingredient.

In the present invention, by confirming that the root tree extract extract has the effect of inhibiting the production of inflammatory mediators such as inflammatory cytokines, prostaglandin E2 and nitric oxide, the extract can be used as an active ingredient in the treatment of inflammatory diseases Did.

In the present invention, hut trees may be purchased from the market or directly collected. The hut tree of the present invention is made of Korean (Hovenia dulcis var.Korean Nakai.), Japanese (Hovenia dulcis var. tomentella Makino) and Chinese (Hovenia acerba Lindl., Hoveniaacerba var. var. fulvotomemtosa) Includes all hut trees.

The term "extract" of the present invention refers to an extract obtained by extracting the root of the barley tree, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude product or a purified product of the extract, or a mixture thereof. It includes extracts of all formulations that can be formed using the extract itself and the extract.

The method of extracting the root of the hut tree tree is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method may include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, etc. These may be performed alone or in combination of two or more methods.

In the present invention, the type of the extraction solvent used for extracting the root of the hawthorn tree is not particularly limited, and any solvent known in the art may be used.

Non-limiting examples of the extraction solvent may include water, alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof, and these may be used alone or in combination of one or more. Specifically, ethanol may be used, but is not limited thereto, and the ethanol is 0.1 to 99.99% (v/v), specifically 30 to 90% (v/v), and more specifically 80% (v/v) methanol May be, but is not limited to.

In the present invention, the root tree root extract may be 80% (v/v) methanol extract or water extract of root tree root.

As an example of preparing the ethanol extract of the root of the lush tree, the crushed root of the lush tree is extracted with methanol, then concentrated under reduced pressure and freeze-dried to prepare the ethanol extract of the root of the tree.

The term "fraction" of the present invention means a result obtained by performing a fraction to separate a specific component or a specific component group from a mixture comprising various various constituents.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity) Or a chromatographic fractionation method for performing separation according to affinity), and combinations thereof. Specifically, a method of obtaining a fraction from the extract by treating a predetermined solvent to the extract obtained by extracting the root of the hut of the present invention.

In the present invention, the type of fractional solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionated solvent include polar solvents such as water and alcohols having 1 to 4 carbon atoms; Non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane; Or mixed solvents thereof. These may be used alone or in combination of one or more, but are not limited thereto. Specifically, hexane, ethyl acetate, butanol or water may be used alone or in combination of one or more, but are not limited thereto. It is not.

In addition, the extract or fraction may be prepared and used in a dry powder form after extraction, but is not limited thereto.

The hawthorn root extract and fractions thereof of the present invention can be effectively used for the prevention or treatment of inflammatory diseases.

The term "inflammatory disease" in the present invention is atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, Rheumatoid fever, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, and multiple sclerosis It includes, but is not limited to.

In the present invention, the term “prophylaxis” means any action that suppresses or delays the onset of inflammatory disease by administration of the composition.

In the present invention, the term "treatment" refers to all acts of relieving an inflammatory disease by administration of the composition, and refers to a disease, a symptom of a disease, a secondary disease of a disease or disease, or a predisposition therefor. A russetia root extract is administered to a subject (human or animal) having a disease, symptom of the disease, a secondary disease of the disease or condition, or a predisposition to it, with the purpose of treatment, alleviation, alleviation, remediation, or improvement. It is defined as the application or administration of the containing composition.

The pharmaceutical composition of the present invention may include the extract of the hawthorn root relative to the weight of the total composition in 0.0001 to 80% by weight, specifically 0.01 to 40% by weight, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the manufacture of the pharmaceutical composition, and the carrier includes a non-naturally occurring carrier. can do.

The pharmaceutical composition may be formulated and used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method. .

The term “pharmaceutically acceptable” means exhibiting properties that are not toxic to cells or humans exposed to the composition.

Specifically, the type of the carrier is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable carrier can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more. In addition, if necessary, other conventional additives such as antioxidants, buffers, and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to form a solution such as aqueous solutions, suspensions, emulsions, etc. It can be formulated and used in dosage forms, pills, capsules, granules or tablets.

The method of administration of the pharmaceutical composition for preventing or treating an inflammatory disease according to the present invention is not particularly limited, and may be in accordance with a method commonly used in the art. As a non-limiting example of the above administration method, the composition may be administered by oral administration or parenteral administration. In addition, the composition for preventing, improving or treating inflammatory diseases of the present invention may be prepared in various dosage forms according to a desired mode of administration.

Another aspect of the present invention provides a food composition for preventing or improving an inflammatory disease, which includes a hawthorn root extract or a fraction thereof as an active ingredient.

At this time, the definition of the roots, extracts, fractions, inflammatory diseases and prevention of the hut is as described above.

The term "improvement" of the present invention means any action that at least reduces the parameters associated with the condition being treated with the administration of the composition of the present invention, for example the severity of symptoms.

The term "food" of the present invention means meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , Vitamin complexes, dietary supplements and health foods, and includes all foods in the ordinary sense.

Since the food composition of the present invention can be consumed on a daily basis, a high inflammatory disease improvement effect can be expected, and thus it can be very useful for health promotion purposes.

The functional food (functional) food (food for special health use), in the same terms as food (food for special health use, FoSHU), in addition to nutrient supply, the medical and medical effects are processed so that the bio-regulatory function appears efficiently Means food. Here, the term'function (sex)' means obtaining a useful effect for health use such as adjusting nutrients or physiological effects on the structure and function of the human body. The food of the present invention can be manufactured by a method commonly used in the art, and in the case of the preparation, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the food can be prepared without limitation as long as the formulation is recognized as food. The food composition of the present invention can be manufactured in various types of formulations, and unlike general medicines, it has the advantage of not having side effects that may occur when taking medicines for a long time using natural substances as raw materials, and is excellent in portability. The food of the invention can be consumed as an adjuvant to enhance the effect of improving inflammatory diseases.

The health food refers to food having an active health maintenance or enhancement effect compared to general food, and health supplement food means food for health supplement purposes. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.

Specifically, the health functional food is a food prepared by adding the extract of the present invention to food materials such as beverages, teas, spices, gums, confectionery, or by encapsulation, powdering, suspension, etc. It means to bring about an effect, but unlike general medicines, it has the advantage that there is no side effect that can occur when taking medicines for a long time using food as a raw material.

The food composition may further include a physiologically acceptable carrier, and the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, and vision. For example, vitamin A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantotenic acid, and the like. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr); And amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition is a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleached and highly bleached, sodium hypochlorite, etc.), antioxidants (butyl hydroxyanisole (BHA), butyl hydride Roxytoluene (BHT, etc.), colorants (such as tar pigment), colorants (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (such as MSG sodium glutamate), sweeteners (dulcine, cyclate, saccharin) , Sodium, etc.), flavoring agents (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen sulphate, etc.), strengthening agents, emulsifying agents, thickeners (foaming agents), coating agents, gum starting agents, foam inhibitors, solvents, improvers, etc. Food additives. The additive may be selected according to the type of food and used in an appropriate amount.

As an example of the food composition of the present invention may be used as a health drink composition, in this case it may contain various flavoring agents or natural carbohydrates, etc. as additional ingredients, such as ordinary beverages. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be generally about 0.001 to 0.4 g, specifically about 0.002 to 0.3 g per 100 mL of the health drink composition of the present invention.

In addition to the above, the health drink composition includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acids, alginic acids, salts of alginic acids, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohol or carbonic acid. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients may be used independently or in combination. The proportion of these additives is not very important, but is generally selected from 0.001 to 0.10 parts by weight per 100 parts by weight of the health drink composition of the present invention.

Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the compound of formula (I) as an active ingredient.

[Formula Ⅰ]

At this time, the definition of the root tree root, extract, inflammatory disease, prevention, treatment and pharmaceutical composition is as described above.

In the present invention, "Formula I" means a compound represented by the following formula.

The compound represented by the formula (I) of the present invention is named 27-O-protocatechuoylbetulinic acid.

The 27-O-protocatechuyl betulinic acid may be specifically isolated from the roots of hawthorn, but is not particularly limited thereto, and may be purchased from a natural product or commercially available.

In the present invention, when the compound represented by the formula (I) is separated and purified from nature, it can be obtained from natural materials using a method of extracting and separating conventional substances, and the compound represented by the formula (I) is natural The extraction method for separation and purification from can be a solvent extraction method using water or an organic solvent. At this time, the organic solvent is not limited to this, but methanol (methanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), butanol (butanol), acetone (acetone), ether (ether), benzene ( Various solvents such as benzene), chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination.

In addition, the extract obtained from the above method can then obtain the active ingredient from the extract using a separation and purification method known in the art, in general, various synthetic resins such as silica gel (silica gel) or activated alumina (alumina) Column chromatography (filled) and high-speed liquid chromatography (HPLC) may be used alone or in combination. The method of extracting and separating the active ingredient is not necessarily limited to the above method.

Another aspect of the present invention provides a food composition for preventing or improving an inflammatory disease comprising the compound of Formula I as an active ingredient.

[Formula Ⅰ]

In this case, the definition of 27-O-protocatechuoyltooleric acid, which is the root tree, extract, separation and purification process, inflammatory disease, prevention, improvement, food composition, and compound represented by the formula (I), is as described above. .

The overlapping contents are omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the technical field to which the present invention pertains.

The root tree extract of the present invention, the fractions thereof and the compounds isolated therefrom can be used for the prevention, treatment, or improvement of inflammatory diseases by inhibiting the production of inflammatory mediators, inflammatory cytokines, prostaglandin E2 and nitric oxide. In addition, since the root tree root extract of the present invention, the fractions thereof and the compounds isolated therefrom are derived from natural products, they can be safely used without side effects in the prevention or treatment and improvement of inflammatory diseases.

1A, 1B, 1C, and 1D confirm the effect of hawthorn root extract on nitric oxide and prostaglandin E2 production, iNOS mRNA and COX-2 mRNA expression in RAW 264.7 cells stimulated with lipopolysaccharide The results show that RAW 264.7 cells were stimulated with RE at the indicated level for 24 hours in the presence or absence of LPS (1 μg/ml), and FIG. 1A determined the amount of NO using a grease reaction in the culture medium. Figure 1b is the detection of PGE2 levels in cell culture medium by ELISA analysis, Figures 1c and 1d are analyzed for iNOS or COX-2 protein levels, respectively, 6 hours after LPS stimulation.
2A, 2B, 2C, 2D, 2E, 2F, and 2G are root extracts and/or LPS to confirm the effect of lush tree root extract on the expression of pro-inflammatory cytokines in RAW 264.7 cells. (1 μg/ml) shows the results when treated at the indicated concentration for 24 hours, and FIGS. 2A, 2B, and 2C are respectively IL-6, IL-1β or TNF in the culture medium using ELISA assay, respectively. Protein levels of -α are measured, and FIGS. 2d, 2e, and 2f are mRNA levels of IL-6, IL-1β or TNF-α, respectively, by real-time PCR analysis using specific primers. Figure 2g shows the cell viability after treatment with the root extract for 24 hours with a cell counting kit-8 and the results are expressed as a percentage compared to the untreated control.
Figures 3a and 3b are lipopolysaccharide-stimulated RAW 264.7 macrophages in order to confirm the effect of the lush root extract on NF-kB pathway activation cells before lipopolysaccharide (1 mg/ml) treatment for 5 minutes It shows the results of pre-treatment for 30 minutes with the root extract of hawthorn at the indicated concentration, and FIG. 3A shows the results of Western blot analysis of the nuclear and cytosolic proteins with the indicated antibodies, and FIG. 3B shows the results of the experiment as a relative density graph. It is shown.
Figure 4a shows the phosphorylation and total protein expression of ERK, p38 and JNK as lipopolysaccharide-stimulated shed root extract cells to confirm the effect of shed root extract on activation of lipopolysaccharide-stimulated MAPKs in RAW 264.7 cells Extracted from and analyzed by Western blot, Figure 4b shows the results of the experiment in a relative density graph.
5a, 5b, and 5c are LPS-stimulated RAW 264.7 cells in order to confirm the effect of hut root extract on cytosolic Nrf2, nuclear Nrf2 and HO-1 expression in cells, various concentrations of hut root extract (10 to 40 μg/ml) for 1 hour pretreatment and lipopolysaccharide (1 μg/ml) for 24 hours, FIG. 5a shows the cytosolic Nrf2, nuclear Nrf2 and HO-1 protein expression by Western blot As a result of analysis, Figure 5b is a graph showing the relative density of the results of the experiment, Figure 5c is a graph showing the mRNA expression level of HO-1 according to the concentration of the root extract of hut tree.
Figures 6a and 6b is to determine the effect of lush root extract on the expression of iNOS, COX-2, HO-1 and Nrf2 in LPS-stimulated RAW 264.7 cells, cells of various concentrations of shed root extract (10-40 μg /ml) was first pretreated with 10 μM of tin protoporphyrin IX (SnPP) for 1 hour, and the result of stimulation with lipopolysaccharide (1 μg/ml) for 24 hours is shown.FIG. 6A shows NOS, COX-2, HO The expression of -1 and Nrf2 protein expression was analyzed by Western blot, and FIG. 6B shows the result of the experiment as a relative density graph.
Figure 7a shows a representative HPLC chromatogram of the extract (root, stem, leaf) according to the parts of the hut at 254nm, Figure 7b is the structure of the main compound of the hut root extract 27-O-protocatechuoyl betulinic acid It shows.

Hereinafter, examples and experimental examples will be described in detail to help understanding of the present invention. However, the following examples and experimental examples are only illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples and experimental examples. Examples and experimental examples of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only provided to more easily understand the present invention, and the scope of the present invention is not limited by the examples.

material

The Hovenia dulcis samples used in this experiment were collected from the wild mountains of Goseong-gun, Gyeongnam in August 2017, and then used for experiments after shade and refinement after accurate assessment, and the samples were sent to the National Bokdudae Arboretum Resource Plant Industry Division. Archived (E01708130066). 1.5 kg of dried stalk, stalk, leaf, and root samples were taken, 10 L methanol was added, extracted at 30° C. for 24 hours, and then centrifuged to recover the supernatant first, and again the precipitate was re-extracted and centrifuged to recover the supernatant 1 After mixing with the supernatant and concentrated under reduced pressure, it was used as a sample for anti-inflammatory effect and HPLC analysis while being stored in a freezer at -20℃.

reagent

Cell culture reagents such as Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Lipopolysaccharide (LPS) and 3'-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(3-(4,5-Dimethylthiazol-2-yl)-2 ,5-Diphenyltetrazolium Bromide, MTT) was purchased from Sigma (Louis, MO, USA). Falcon (Corning, NY, USA) was used for 96-well cell culture flasks and other tissue culture dishes. The primary antibodies iNOS, COX-2 and Lamin B used in the experiment were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA), p65, phosphor-p65, IκBα, HO-1, Nrf2, phospho- ERK, ERK, phospho-p38, p38, phosphor-JNK, JNK (Cell Signaling, Beverly, MA, USA). The secondary antibody, horseradish peroxidase (Horseradish peroxidase, HRP-conjugated anti-rabbit or goat antibodies), was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The Greases Reagent System was purchased from Promega (Madison, WI, USA), and the enzyme-linked immunosorbent assay kit was purchased from R&D System (Minneapolis, MN, USA).

Cell culture

RAW 264.7 cells, a mouse macrophage cell line, were purchased frozen from the American Type Culture Collection (ATCC, Manassas, VA, USA) with 10% fetal calf serum (FBS) and 1% antibiotic (100 U/ml penicillin G, 100 μg/ ml Streptomycin) was added and cultured in a DMEM medium at 37°C, 5% CO ₂ environment.

Cell viability measurement

To determine the presence or absence of cytotoxicity of the Root Extract (RE), it was measured using a cell counting kit (Cell counting kit)-8 (Dojindo, Japan). RAW 264.7 cells were dispensed into 96-well plates at 2x10 ⁴ cells/ml and cultured for 24 hours, and then the root extract (RE) was treated at different concentrations (0, 10, 20 and 40 μg/ml) and cultured for 24 hours. After adding 10 μl of CCK-8 solution per well, the mixture was reacted for 2 hours at 37° C. and 5% CO ₂ conditions, and absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

Measurement of nitric oxide (NO) and prostaglandin E2 (PGE2)

As part of measuring the anti-inflammatory effect of extracts in each region on the inflammatory response induced in RAW 264.7 cells by lipopolysaccharide stimulation, NO in the cell culture was quantified and compared. To measure NO scavenging activity, cells were divided into 2x10 ⁴ cells/ml in 96-well plates and cultured for 24 hours, followed by pre-treatment with root extracts at a concentration of 0, 10, 20, 40 μg/ml and cultured for 1 hour. Then, the lipopolysaccharide was treated at a concentration of 1 μg/ml and cultured for 24 hours. After absorbing 50 μl of the culture supernatant and the same amount of grease reagent (Promega, Madison, WI, USA), the absorbance value was measured using a microplate reader (BioTek, Winooski, VT, USA) at 540 nm. The NO value was calculated using a standard curve for each concentration of sodium nitrite (NaNO ₂ ). And the PGE2 level in the cell culture was measured using a PGE2 enzyme-linked immunosorbent assay kit (R&D System, Minneapolis, MN, USA).

Cytokine (IL-1β, IL-6, TNF-α) measurement

After culturing the cells in the same way as the NO measurement, the cell culture solution was recovered from each well. The concentration of TNF-α, IL-1β, and IL-6 in the cell culture supernatant was measured using an enzyme linked immunosorbent assay (Elisa) kit (R&D system, MN, USA).

Quantitative polymerase reaction Real-time reverse transcription polymerase chain reaction (RT-PCR)

RAW 264.7 cells were dispensed into 6-well plates at 6x10 ⁵ cells/ml, incubated for 24 hours, and then the root extract was treated for 1 hour, and then the lipopolysaccharide was reacted at a concentration of 1 μg/ml for 24 hours. Subsequently, all RNA was isolated using Trizol (Invitrogen, USA) reagent. Total RNA was quantified and each cDNA was synthesized from 1 μg of all RNAs using RT-PreMix (Promega, Madison, WI, USA). MRNA expression was confirmed using the template of each cDNA and primers of iNOS, COX-2, IL-6, IL-1β, TNF-α and β-actin. Table 1 shows the primer sequence of each gene.

gene Primer sequence iNOS Forward 5’-CCC TTC CGA AGT TTC TGG CAG CAG C Reverse 5’-GGC TGT CAG AGC CTC GTG GCT TTG COX-2 Forward 5’-ACT CAC TCA GTT TGT TGA GTC ATT C Reverse 5’-TTT GAT TAG TAC TGT AGG GTT AAT G IL-6 Forward 5’-CAA GAA AGA CAA AGC CAG AGT CCT Reverse 5’-TGG ATG GTC TTG GTC CTT AGC IL-1β Forward 5’-TGC AGA GTT CCC CAA CTG GTA CAT C Reverse 5’-GTG CTG CCT AAT GTC CCC TTG AAT C TNF-α Forward 5’-ACA AGC CTG TAG CCC ACG Reverse 5’-TCC AAA GTA GAC CTG CCC HO-1 Forward 5’-AAG ATT GCC CAG AAA GCC CTG GAC Reverse 5’-AAC TGT CGC CAC CAG AAA GCT GAG β-actin Forward 5’-AGT GTG ACG TTG ACA TCC GTA AAG A Reverse 5’-GGA CAG TGA GGC CAG GAT GG

Western blot analysis

RAW 264.7 cells were treated with root extract and lipopolysaccharide, and then HO-1 inhibitor (SnPP) was pretreated 1 hour ago. After washing twice with ice-cold phosphate buffered saline (PBS) with ice, radioimmunoprecipitation assay (RIPA buffer) (25 mM TrisHCl pH 7.6, 150 mM NaCl, 1 % NP-40, 1% sodium dioxycholate, 0.1% SDS, protease inhibitor) was reacted on ice for 30 minutes, whole cell lysate was prepared and centrifuged for 20 minutes to collect supernatant. Protein quantification of each sample was performed by measuring absorbance at 595 nm using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). The same amount of protein (30 μg) was separated with 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and converted using iBlot 2 blotting system (Thermo Fisher, Waltham, MA USA). The PVDF membrane was reacted in a 5% non-fat milk block solution for 1 hour, and then the primary antibody (1:1000 dilution) was maintained at 4°C overnight. After washing three times with TBST solution, the secondary antibody is horseradish peroxidase (HRP)-conjugated anti-rabbit, or anti-mouse IgG (1:10000 dilution) coupled with horseradish peroxidase. ) For 1 hour at room temperature. Subsequently, after washing three times, the protein was confirmed using a ChemiDoc™ imaging system (Bio-Rad, Hercules, CA, USA).

HPLC analysis of dulcis extract by region

The HPLC instrument used a pump, auto sample, column oven, DAD (Agilent 1260 infinity HPLC system, Agilent Technologies, USA), and all solvents used in the analysis were J.T. HPLC grade solvents purchased from Baker (Phillipsburg, NJ, USA) were used. Water (solvent A) and acetonitrile (solvent B) containing 0.1% formic acid were used as the mobile phase, and 10 μl of the sample was injected at a flow rate of 1 mL/min, and analyzed under gradient conditions at a wavelength of 254 nm. Table 2 shows the HPLC conditions for the separation of the extracts of different parts of the oleander extract and the HPLC analysis conditions for the separation of the main compounds.

Indicators Condition column ZORBAX Eclipse plus C ₁₈ (4.6 X 150 mm, 5 μm) Flow rate 1.0 ml/min Injection volume 10 μl UV detection 350 nm Operating time 50 minutes Gradient Hours (minutes) % A ^z % B ^y 0 95 5 6 95 5 30 30 70 35 30 70 45 0 100 50 0 100

^z Formic acid in 0.1% water.

^y Formic acid in 0.1% acetonitrile

Separation and structural identification of active ingredients of mulberry root extract

The root extract having the most excellent anti-inflammatory effect among the extracts by parts of the family tree was separated using a recirculating preparative HPLC system. 1 g of extract was injected into a JAIGEL-ODS AP column (20 x 500 mm), and a mixed solvent (water:acetonitrile=1:2, v/v) was eluted at 10 mL per minute, and absorbance was measured at 254 nm using a UV detector. And divided into 6 small fractions (HR1-HR6). Double HR4 was subjected to preparative TLC with a mixed solvent of EtOAc:MeOH (30:1) and purified by Sephadex LH-20 (95% MeOH) to obtain compound 1 (30 mg). The main components of the final purified root extract are the chemical structural hydrogen and carbon skeleton obtained through Q Exactive plus Orbitrap LC-MS/MS (Thermo Fisher Scientific, MA, USA) and 500 MHz Bruker AM NMR (Bruker, MA, USA). After obtaining the information, the structure of the purified major components was identified.

Statistics processing

All experiment results were performed three or more times, and were expressed as mean ± standard deviation (Mean ± SD) based on the average value. Statistical processing of the experiment results was performed by GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA) was used for two-way ANOVA and the p-value was less than 0.05.

Comparative Experimental Example 1. Confirmation of the effect of dulcis root extract on NO and PGE2 production and iNOX, COX-2 expression

In order to compare the anti-inflammatory effect of the extracts of different parts of the family tree, treatment of the extracts of each part in the RAW 264.7 macrophages, which induced the inflammatory response with lipopolysaccharide (LPS), simultaneously inhibited the production of nitric oxide (NO), an indicator of inflammation. As a result of the measurement, as shown in Table 3, it exhibited an excellent inhibitory effect on the root of the hawthorn extract (RE). Anti-inflammatory effects and regulatory mechanisms were observed using root extracts at concentrations of 10, 20, and 40 μg/ml that do not exhibit cytotoxicity.

Sample Cytotoxic IC ₅₀ (μg/ml) NO production suppression IC ₅₀ (μg/ml) Methanol extract of leaves >40 >40 Methanol extract of stem >40 37.64 ± 0.73 ^z Methanol extract of root >40 12.51 ± 0.48 27- O -protocatechuyl betulinic acid >40 5.42 ± 0.61

^z values are expressed as mean±standard deviation of 3 replicates.

At this time, GAPDH was used as an internal control for real-time PCR analysis (n=4), and data were expressed as mean±standard deviation for 3 experiments (*p<0.05, * compared to when used alone in LPS). *p<0.001) The results are shown in FIGS. 1A to 1D.

The initial inflammatory response is induced by inducible nitric oxide synthase (iNOS) and cyclooxygenase (cyclooxygenase-2, COX-2) and inflammatory mediators (NO, PGE2) by these proteins. In the present invention, in order to investigate the anti-inflammatory effect of the root extract, 10, 20, and 40 μg/ml of the root extract were pre-treated for 1 hour in RAW 264.7 cells, followed by treatment of 1 μg/ml lipopolysaccharide for 24 hours to obtain NO. As a result of examining the effect on the production amount in the same way, it was reduced by 53.1% at 10 μg/ml and 94.7% at 40 μg/ml (FIG. 1A ). In addition, as a result of investigating the effect on the production of PGE2 induced by lipopolysaccharide, it was reduced by 47.2% at 10 μg/ml and 90.1% at 40 μg/ml (FIG. 1B ).

In order to investigate the mechanism of action for the anti-inflammatory effect of the root extract, mRNA expression of inflammatory expression enzymes (iNOS, COX-2) in the body was investigated. NO produced by iNOS plays a useful role in the immune response, but continuous production of NO is an important factor causing chronic inflammatory diseases. Therefore, RT-PCR was performed to investigate the effect of root extract on the expression of iNOS, which produces excessive NO in the body. In the group treated with lipopolysaccharide, iNOS and COX-2 mRNA increased significantly, and in the group treated with root extract, the expression level of iNOS and COX-2 mRNA was significantly increased compared to the group treated with lipopolysaccharide. As it decreased, iNOS and COX-2 mRNA inhibitory effects showed a similar tendency of NO and PGE2 production inhibitory effects (FIGS. 1C and 1D ). Therefore, it was confirmed that the root extract has an inhibitory effect on inflammation by inhibiting iNOS and COX-2 expression.

Experimental Example 1. Confirmation of the effect of rotten tree root extract on pro-inflammatory cytokine production and mRNA expression

In the process of inducing an inflammatory reaction, the production of inflammatory mediators such as NO and PGE2 is accompanied by the production of inflammatory cytokines through an immune response, and is an important indicator of inflammation. In particular, when activated by various stimulating factors such as lipopolysaccharide in macrophages, pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) increase, and these induce iNOS expression. Therefore, in order to investigate the effect of the root extract on the production of TNF-α, IL-6, and IL-1β, protein levels were measured using an enzyme-linked immunosorbent assay. At this time, the data were expressed as the mean±standard deviation of 3 experiments (*p<0.05 and **p<0.001 when compared to the case where LPS was used alone).

Root extract was pretreated to RAW 264.7 cells at various concentrations for 1 hour and stimulated with lipopolysaccharide. The results of measuring TNF-α, IL-6 and IL-1β in the cell supernatant after 24 hours are shown in FIGS. 2A, 2B, or 2C, respectively. In the group treated with lipopolysaccharide, TNF-α, IL-6, and IL-1β expression was significantly increased, and in the group treated with root extract, inflammatory factors were concentration-dependently stronger than those treated with lipopolysaccharide group. It was confirmed to decrease. Based on the above results, it was decided to determine whether the level of mRNA also inhibits infectious factors. As a result of measuring the mRNA expression of TNF-α, IL-6, IL-1β by quantitative polymerase reaction method, lipopolysaccharide stimulation significantly increased the expression of mRNA, and TNF-α in the experimental group pre-treated with root extract, Both IL-6 and IL-1β mRNA expression was inhibited concentration-dependently (FIG. 2D, FIG. 2E or FIG. 2F, respectively). Through these results, it was confirmed that the root extract showed an anti-inflammatory effect of suppressing NO production by regulating the expression of iNOS by reducing the production of TNF-α, IL-1β and IL-6. The cytotoxicity of each root extract used in all experiments was determined by MTT analysis, and it was confirmed that the root extract did not show toxicity in RAW 264.7 cells by showing a survival rate of 90% or higher at all concentrations used (FIG. 2g). .

Experimental Example 2. Confirmation of the effect of dulcis root extract on Ik-B expression

When inflammation is caused by RAW 264.7 cells or lipopolysaccharide, inflammatory mediators are secreted by various signal transduction mechanisms. Nuclear factor kappa B (NF-kB, Nuclear Factor-kappa B) are representative pathways. NF-kB is an inducible transcription factor expressed in all cells, and is known to regulate the expression of various genes related to the inflammatory response of cells, inducing activation of immune cells and defense against extracellular stimulation (Ghosh et al., 2012). Lipopolysaccharide binds to toll-like receptor 4 (TLR4) present in the cell membrane, and when activated, the transcription factor NF-kB in the cytoplasm is activated, Ik-Ba (inhibitory kappa Ba) bound Is degraded, and accordingly, NF-kB moves from the cytoplasm into the nucleus to induce transcription such as COX-2, iNOS (Kim et al., 2013). In the present invention, results of analysis to investigate the relationship between NF-kB activity and inhibition of production of inflammation-mediated factors reduced by root extract treatment are shown in FIGS. 3A and 3B. β-actin was used as an internal protein for the cytosol and nuclear fractions, respectively, and the data are expressed as the mean±standard deviation of 3 experiments (*p<0.05, ** compared to when LPS was used alone). p<0.001).

In the group treated with lipopolysaccharide, the migration of NF-kB subunit p65 into the nucleus was significantly reduced compared to the untreated group, and in the group treated with root extract, the lipopolysaccharide treated group and In comparison, expression was decreased in a concentration-dependent manner. In addition, in the group treated with lipopolysaccharide, the degradation of Ik-Ba was apparent compared to the untreated group, and in the root extract, the degradation of Ik-Ba was inhibited compared to the lipopolysaccharide treated group. As a result, the root extract reduces the activation of the transcription factor NF-kB, inhibits the transcription of downstream signaling molecules iNOS and COX-2, and reduces the production of NO, thereby exhibiting anti-inflammatory effects. I could infer.

Experimental Example 3. Confirmation of the effect of dulcis root extract on MAPK activity

The signal transduction pathway of mitotic activated protein kinase (MAPK) plays an important role in the activation of the inflammatory response. In particular, it is known that p38 MAPK plays an important role in the secretion of cytokines induced by lipopolysaccharide, and p38, JNK, and ERK signaling pathways play an important role in lung-related inflammatory diseases (Yang et al., 2015). ). Particularly, in macrophages, lipopolysaccharide stimulates TLR4 on the surface to induce activation of the lower cell signaling pathway, MAPK, and the activated signaling pathway expresses various inflammatory mediators such as pro-inflammatory cytokines, NO, and PG. Induce (Kim et al., 2010). In the present invention, the results of examining whether the anti-inflammatory effect of the root extract is caused by MAPK molecular regulation are shown in FIGS. 4A and 4B. Data are expressed as the mean±standard deviation of 3 experiments (*p<0.05 and **p<0.001 compared to when LPS was used alone).

As a result of Western blot analysis of the effect on the expression of phosphorylated MAPKs in macrophages according to root extract treatment, it was found that all proteins belonging to MAPKs were activated in lipopolysaccharide-derived RAW 264.7 cells. It was confirmed that the expression of phosphorylated ERK, JNK, and p-38 decreases as the amount of the root extract increases, compared to the lipopolysaccharide-treated group. Therefore, the root extract showed the effect of inhibiting the expression of several inflammatory mediators such as anti-inflammatory cytokines, NO, and prostaglandins (PG) by inhibiting the activation of MAPK.

Experimental Example 4. Confirmation of the effect of lush root extract on HO-1 expression induction and the effect of lush root extract on Nrf2 activity

Force Oxygenase (Heme oxygenase-1, HO-1) is an enzyme that is involved in the production of wilberdine, carbon monoxide (CO), and free iron by oxidizing the heme, thereby protecting against oxidative stress. Weakens the inflammatory response. Expression of HO-1 or CO treatment in activated macrophages inhibits the production of cytokines that promote inflammation, and also suppresses the production of inflammatory mediators. Expression of nuclear factor-E2-related factor 2 (Nrf2), a transcriptional regulatory factor of HO-1 protein expression, into the nucleus acts as a very important factor. In order to investigate whether the root extract was transcribed to Nrf2, after processing the root extract on RAW 264.7 cells, HO-1 expression and Nrf2 activation were analyzed using Western blot. While Nfr2 gradually decreased according to the concentration of the root extract, Nrf2 inside the nucleus showed an increasing pattern, thereby confirming that transcription of Nrf2 into the nucleus was achieved (FIGS. 5A, 5B, and 5C). Data are expressed as the mean±standard deviation of 3 experiments (*p<0.05, **p<0.001 when compared to using LPS alone).

It was found that the expression of HO-1 increased when the lipopolysaccharide was treated by pre-treatment of the root extract than when the lipopolysaccharide was treated alone. In FIGS. 5A and 5B, the expression level of the transcription regulator Nrf2 in the cytoplasm and nucleus was confirmed, but decreased in the cytoplasm, but increased in the nucleus. Therefore, it can be concluded that the root extract induces HO-1 by increasing Nrf2 activity. To investigate the direct relationship between the anti-inflammatory effect of the root extract and the expression of HO-1, the experiment was performed using the HO-1 inhibitor Snpp. In an experiment in which SnPP was treated with root extract (40 μg/ml) for 12 hours, iNOS and COX-2, which were inhibited by the root extract, were partially increased by co-administration of SnPP (FIGS. 6A and 6B ). Data are expressed as the mean ± .standard deviation of 3 experiments (*p<0.05, **p<0.001 when compared to using LPS alone).

From these results, it was confirmed that HO-1 expression by the root extract inhibits the production of NO and PGE2.

Experimental Example 5. Analysis of the components of hut tree parts and identification of the structure of the main components

In order to compare the differences in the main components of the extract of oleander by region, it was analyzed using HPLC. As a result, a certain main peak (29.5 minutes) was found in the root extract compared to the leaf and stem extracts (FIGS. 7A and 7B ). In the present invention, the main component extracted from the root extract was separated and purified using a Recycling preparative HPLC system. Compound 1 (white powder) was subjected to obitrap/mass spectrometry analysis, and a molecular ion peak of m/z 607.3649 [M]+ was observed to determine a molecular weight of 608.8046.

Typical 3 oxymethine protons of triterpene, represented by four angular methyl groups at δ1.05, 0.91, 0.90, and 0.77 at ¹ H NMR and multiplets at δ3.16 Could confirm. Another methyl group bound to the sp ² carbon in δ1.74 was identified, and in δ4.77 and 4.63, two olefin protons represented by singlets were identified, and this compound had an isopropenyl group, and δ4 One oxygenate methylene group represented by a doublet at .72, 4.55 and a protocatechuoyl group containing a proton coupling of the ABX system at δ7.53, 6.92, 7.46 in the aromatic region It could be estimated that the lupane family of triterpenoids. ^{In the 13} C-NMR spectrum (in Acetone-d6, 125 MHz), a total of 37 signals were identified to confirm that it was a triterpene. Five methyl groups were identified at δ27.7, 15.3, 16.3, 16.1, and 18.8, three oxymethine carbons at δ77.7, and olefin carbons of isopropenyl groups at δ149.9 and 109.4, δ62.8 It can be confirmed once again that it is a typical signal of oxygenated methylene carbon, and δ 165.9 was able to confirm the carboxy carbon of the protocatechuoyl group. Therefore, it was found by NMR spectroscopy and MS analysis that the chemical structure of the major component of the root of the hawthorn tree was 27-O-protocatechuoyl betulinic acid (27- O- protocatechuoylbetulinic acid), which is a lupane-based triterpenoid. Kang et al ., 2017).

In the present invention, the anti-inflammatory effect of methanol extracts by leaves (stalks, stems, roots) was investigated. As a result of confirming the inhibitory effect of nitric oxide (NO) production, which is an inflammation-mediated substance, by simultaneously treating the extracts by regions in RAW 264.7 macrophages that induced inflammation with lipopolysaccharide, there was an excellent inhibitory effect on the root extract. The anti-inflammatory effects and related molecular mechanisms of the root extracts of the lush tree were confirmed. As a result, the root extract in the NF-kB and MAPK signaling pathways, which are the main pathways of the inflammatory response, inhibits nuclear migration of NF-kB induced by lipopolysaccharide, and inhibits phosphorylation of ERK, JNK, p38, iNOS, It was confirmed that the expression of COX-2 was reduced and the production of NO and pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) was suppressed. In addition, the root extract showed the anti-inflammatory effect by inducing HO-1, an anti-inflammatory protein, by activating Nrf2 in macrophages. In addition, a high anti-inflammatory effect was also confirmed in the 27-O-protocatechuyl betulinic acid compound, which identified the structure using NMR and MS devices after separating the main components from the root extract of hawthorn. Therefore, the root of the hawthorn root and its main components are likely to play an important role in the control of inflammatory diseases and indicate that they can be used as effective natural anti-inflammatory agents in preventing or treating the development of various inflammatory diseases such as arthritis and inflammatory bowel disease.

As described above, the present invention has been described through materials, examples, comparative experiments and experimental examples. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. Therefore, the above-described experimental examples are to be understood as illustrative and non-limiting in all respects. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (10)

Pharmaceutical composition for the prevention or treatment of inflammatory diseases, which includes the extract of oleaceae root or fractions thereof as an active ingredient. The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the extract is extracted with water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof. According to claim 1, wherein the inflammatory disease is atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis , Rheumatoid fever, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis Pharmaceutical composition for the prevention or treatment of inflammatory diseases that is abnormal. Food composition for the prevention or improvement of inflammatory diseases, including hawthorn root extract as an active ingredient. The food composition for preventing or improving inflammatory diseases according to claim 4, wherein the extract is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The method of claim 4, wherein the inflammatory disease is atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis. , Rheumatoid fever, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis Food composition for preventing or improving inflammatory diseases that is abnormal. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the compound of formula (I) as an active ingredient.
[Formula Ⅰ]

The method of claim 7, wherein the inflammatory disease is atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, liver Any selected from the group consisting of spondylitis, rheumatoid lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis A pharmaceutical composition for the prevention or treatment of inflammatory diseases that is at least one. Food composition for the prevention or improvement of inflammatory diseases comprising the compound of formula (I) as an active ingredient.
[Formula Ⅰ]

The method of claim 9, wherein the inflammatory disease is atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, liver Any selected from the group consisting of spondylitis, rheumatoid lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis Food composition for the prevention or improvement of inflammatory diseases that are more than one.

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