KR102369924B1 - Composition for prevention, improvement or treatment of inflammatory diseases comprising an extract of Campanula takesimana Nakai as and active ingredient - Google Patents

Abstract

It relates to a composition for preventing, improving, or treating inflammatory diseases, comprising the extract of oleracea as an active ingredient, and according to the composition according to one aspect, inflammatory mediators NO (Nitric Oxide), PGE ₂ (Prostaglandin E2), IL-6 By effectively reducing the secretion of (Interleukin-6) and TNF-α (Tumor necrosis factor-α) and inhibiting the enzymatic activity of XOD (Xanthine Oxidase), it has an effect that can be usefully used for the prevention, improvement or treatment of inflammatory diseases. there is.

Description

Composition for prevention, improvement or treatment of inflammatory diseases comprising an extract of Campanula takesimana Nakai as and active ingredient}

It relates to a composition for preventing, improving, or treating inflammatory diseases, comprising the extract of Sumcholong flower as an active ingredient.

Inflammatory reaction is a biological defense reaction caused by various factors such as infection by pathogens or tissue damage, and performs an initial protective action to limit damage only to the infected or damaged site. In general, acute inflammatory response proceeds rapidly and is maintained for a short period of time, and acute inflammation is accompanied by a systemic response called an acute phase reaction. On the other hand, chronic inflammation can be induced as a result of continuous immune activation in relation to some diseases, such as infections or autoimmune diseases, and the accumulation and activation of macrophages increases the chronic inflammatory response. However, in the case of a persistent chronic inflammatory response, it can cause serious damage to host cells or tissues.

Nitric oxide (NO) is produced by nitric oxide synthetase in human macrophages. NO produced by specific cytokines or LPS acts on inflammation and host defense mechanisms. However, excessive production of NO is known to cause inflammatory diseases such as arthritis and sepsis, autoimmune diseases, immune diseases such as type 1 diabetes, and neuronal cell death. Therefore, from this point of view, materials that inhibit NO production can be used as therapeutic agents for various inflammatory diseases in the human body.

Plant-derived materials have been used for a long time due to their excellent safety features.

Campanula takesimana Nakai is a species endemic to Ulleungdo in Korea in the Campanulaceae family. In the food ingredients announced by the Ministry of Food and Drug Safety, the leaf part is specified as an edible part. It grows up to 30 ~ 100cm, and there are white chrysanthemum flowers with dark spots on a white background and dark purple purpurea chrysanthemums. However, there is no direct mechanism study for the prevention, improvement or treatment of inflammatory diseases of the sagebrush.

Accordingly, the present inventors performed a direct efficacy study on the prevention, improvement or treatment of inflammatory diseases of the extract of oleraceae.

One aspect is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising an extract of Campanula takesimana Nakai as an active ingredient.

Another aspect is to provide a composition for topical skin application for preventing or treating inflammatory diseases comprising an extract of Campanula takesimana Nakai as an active ingredient.

Another aspect is to provide a cosmetic composition for relieving skin inflammation comprising an extract of Campanula takesimana Nakai as an active ingredient.

Another aspect is to provide a health functional food composition for preventing or improving inflammatory diseases comprising the extract of Campanula takesimana Nakai as an active ingredient.

One aspect provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising an extract of Campanula takesimana Nakai as an active ingredient.

The island chorong flower ( Campanula takesimana Nakai) may be one or more selected from the group consisting of whole, root, stem, branch, leaf, seed or fruit.

As used herein, the term “active ingredient” or “effective amount” refers to any amount of a composition used in the practice of the invention provided herein sufficient to alleviate, inhibit the progression or prevent a disease, disorder or condition, or one or more symptoms thereof. can mean

As used herein, the term "inflammatory disease" refers to nitric oxide (NO), IL-6 ( Interleukin-6), TNF-α (Tumor necrosis factor-α), or prostaglandin (Prostaglandin) may refer to a disease induced by an inflammatory substance (inflammatory cytokine).

Accordingly, in one embodiment, the extract is NO (Nitric oxide), PGE ₂ (Prostaglandin E2), IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor-α) and XOD (Xanthine Oxidase) It may be to reduce one or more activities selected from the group consisting of.

Without being limited by a particular theory, the extract reduces the secretion of inflammatory mediators Nitric oxide, Prostaglandin, Interleukin-6, and Tumor necrosis factor-α in macrophages (RAW264.7), thereby preventing or treating inflammatory diseases. It may be something that can work.

In one embodiment, 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, oleaginous at least one selected from the group consisting of spondylitis, rheumatoid lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, parotiditis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis. It could be a disease.

As used herein, the term “gout” may refer to a disease in which urate crystals are deposited in cartilage, tendons, and surrounding tissues of joints as the concentration of uric acid in the blood increases.

Since the uric acid is produced by oxidizing xanthine by xanthine oxidase, in one embodiment, by inhibiting the activity of xanthine oxidase by the extract, prevention of gout and related arthritis or It may be something that can be effective in treatment.

As used herein, the term “prevention” refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or concomitant symptom thereof, preventing the acquisition or reacquisition of the disease or disorder, or preventing the disease or disorder from occurring. How to reduce the risk of acquisition. For example, the prevention refers to any action of suppressing or delaying the occurrence of inflammation or inflammation-related diseases, disorders, or symptoms by administration of the composition according to the present invention.

As used herein, the term “improvement” may refer to any action that at least reduces a parameter related to alleviation or treatment of a condition, for example, the degree of a symptom.

As used herein, the term "treatment" refers to alleviation or amelioration of pathological symptoms, reduction of the site of disease, delay or alleviation of disease progression, amelioration, alleviation or stabilization of disease state or symptoms, partial or complete recovery, prolongation of survival, or other It is used in the sense of including all beneficial treatment results and the like.

In addition, the extract may be one extracted using water, alcohol, for example, C1-C6 alcohol, for example, C1-C4 alcohol, or a mixture thereof as a solvent. The C1-C6 alcohol may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, hexanol, or the like. The solvent may be, for example, a mixture of water and alcohol, that is, an aqueous alcohol solution. The alcohol concentration of the aqueous alcohol solution is 1 to 99.5 (v/v)%, for example, 10 to 99.5 (v/v)%, 1 to 70 (v/v)%, 1 to 40 (v/v)%, 5 to 25 (v/v)%, 7 to 20 (v/v)%, 5 to 25 (v/v)%, or 10 to 20 (v/v)%. The aqueous alcohol solution may be an aqueous ethanol solution.

The extraction is 3 to 10 (volume/weight) times, for example, 3 to 7 (volume/weight) times, 3 to 5 (volume/weight) times, 5 to 10 (volume) times, / weight) times, or may include adding 4 to 10 times. For example, it may include adding 3 to 10 L of the extraction solvent with respect to 1 kg of the material derived from the S.

The extract may be extracted by any extraction method such as hot water extraction, solvent extraction, distillation extraction, supercritical extraction, etc. conventionally used to extract natural plants, and preferably extracted with purified water, an organic solvent, or a mixed solvent thereof. can be The extraction is performed by warmed liquid extraction, pressurized liquid extraction (PLE), microwave assisted extraction (MAE), subcritical extraction (SE), or a combination thereof. can be The subcritical extraction may be subcritical water extraction (SWE). Subcritical water extraction is also referred to as superheated water extraction or pressurized hot water extraction (PHWE). The warmed liquid extraction may be reflux extraction.

The extraction is performed at 4 to 70 °C, for example, 4 to 50 °C, 4 to 40 °C, 4 to 30 °C, 10 to 70 °C, 15 to 70 °C, 20 to 70 °C, 4 to 50 °C, 10 to 50 It may be carried out at ℃, 4 to 40 ℃, 4 to 30 ℃, 10 to 40 ℃, 10 to 35 ℃, or 10 to 30 ℃. The extraction time may vary depending on the selected temperature, for example, from 1 hour to 2 months, for example, from 1 hour to 1 month, from 1 hour to 15 days, from 1 hour to 10 days, from 1 hour to 5 days, from 1 hour to 3 days. , 1 hour to 2 days, 1 hour to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 days, 5 hours to 1 day, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days, or 10 hours to 2 days. The extraction may include mixing the whole, a part thereof, or materials derived therefrom in the solvent and leaving it for a certain period of time. The standing may include moderate agitation. The extraction may be repeated one or more times, for example, 1 to 5 times.

The extraction may be performed by separating plant residues and extracts by known methods such as filtration. The extraction may also include removing the solvent from the obtained extract by a known method such as concentration under reduced pressure. The extraction may also include preparing a dry extract by drying the obtained extract, such as freeze-drying.

The extract is 0.0001 wt% to 99.0 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt% with respect to the total weight of the composition %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight.

The term “pharmaceutical composition” may refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Advantageous effects include enabling diagnostic decisions; amelioration of a disease, symptom, disorder or condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and responding to a disease, symptom, disorder or condition in general.

The pharmaceutical composition may be administered parenterally during clinical administration and may be used in the form of general pharmaceutical formulations. Parenteral administration may refer to administration via a route other than oral administration, such as rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. When the pharmaceutical composition of the present invention is used as a pharmaceutical, it may further contain one or more active ingredients exhibiting the same or similar function.

The pharmaceutical composition may be in the form of a solution, suspension, syrup, or emulsion in an aqueous or oily medium, or may be formulated in the form of a powder, powder, granule, tablet or capsule, and additionally a dispersing agent or stabilizer for formulation can do. When formulating the pharmaceutical composition, it may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc. usually used when formulating the pharmaceutical composition. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, liulinji, glycerogelatin, etc. may be used.

The pharmaceutical composition can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (Ascorbic acid) to increase stability or absorption Acid) or Glutathione, Antioxidants, Chelating agents, Small Molecular Proteins or other Stabilizers may be used as pharmaceuticals.

In addition, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or administration route of the pharmaceutical composition. In addition, the type and degree of response to be achieved by administration of the pharmaceutical composition, the type of subject to be administered, age, weight, general health condition, symptoms or degree of disease, sex, diet, excretion, simultaneous or This may vary depending on a number of factors, including drugs and other components of the composition used together, and similar factors well known in the medical field. A person of ordinary skill in the art can readily determine and prescribe an effective dosage for a desired treatment. Administration of the pharmaceutical composition according to the present invention may be administered once a day, may be administered divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way. The dosage of the pharmaceutical composition may be 1 ug/kg/day to 1,OOO mg/kg/day per day.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of treatment of atopic dermatitis.

Another aspect provides a composition for topical skin application for preventing or treating inflammatory diseases comprising an extract of Campanula takesimana Nakai as an active ingredient.

The external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof.

The external preparation for skin is a component used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorants, and various skin nutrients can be appropriately blended as needed.

The external preparation for skin includes metal-blocking agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

Another aspect provides a cosmetic composition for alleviating skin inflammation comprising an extract of Campanula takesimana Nakai as an active ingredient.

The cosmetic composition consists of a softening lotion, a nourishing lotion, a nourishing cream, a moisture cream, a massage cream, an essence, an ampoule, a gel, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, a powder, a gel, a lotion and an ointment. It may have a formulation selected from the group. The cosmetic composition may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelate Agents, colors, preservatives, fragrances, etc. may be appropriately mixed, but the present invention is not limited thereto.

Another aspect provides a health functional food composition for preventing or improving inflammatory diseases comprising an extract of Campanula takesimana Nakai as an active ingredient.

The health functional food is a food manufactured using raw materials or ingredients (hereinafter, 'functional raw materials') that are easily deficient in daily meals or have a useful function for the human body, to maintain health or prevent certain diseases or symptoms, and / or any food that helps to improve, there is no particular restriction on the form of the final product. For example, the health functional food may have a formulation selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leached teas, or health drinks.

The content of the active ingredient (that is, the extract of oleracea flower) contained in the health functional food is not particularly limited, suitably depending on the type of food, desired use, etc., for example, 0.0001 to 99% by weight of the total food weight, 0.0001 to 95 % by weight, 0.0001 to 90% by weight, 0.0001 to 80% by weight, 0.0001 to 50% by weight, 0.001 to 99% by weight, 0.001 to 95% by weight, 0.001 to 90% by weight, 0.001 to 80% by weight, 0.001 to 50% by weight , 0.01 to 99% by weight, 0.01 to 95% by weight, 0.01 to 90% by weight, 0.01 to 80% by weight, 0.01 to 50% by weight, 0.1 to 99% by weight, 0.1 to 95% by weight, 0.1 to 90% by weight, 0.1 to 80% by weight, 0.1 to 50% by weight, 0.1 to 30% by weight, 0.1 to 10% by weight, 1 to 99% by weight, 1 to 95% by weight, 1 to 90% by weight, 1 to 80% by weight, 1 to 50 wt%, 1-30 wt%, 1-10 wt%, 10-99 wt%, 10-95 wt%, 10-90 wt%, 10-80 wt%, 10-50 wt%, 10-30 wt% , 25 to 99% by weight, 25 to 95% by weight, 25 to 90% by weight, 25 to 80% by weight, 25 to 50% by weight, 25 to 30% by weight, 40 to 99% by weight, 40 to 95% by weight, 40 to 90% by weight, 40 to 80% by weight, 40 to 50% by weight, 50 to 99% by weight, 50 to 95% by weight, 50 to 90% by weight, 50 to 80% by weight, 60 to 99% by weight, 60 to 95 % by weight, 60 to 90% by weight, or 60 to 80% by weight, but is not limited thereto.

The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents or natural flavoring agents, coloring agents, thickeners (cheese, chocolate, etc.), pectic acid or salts thereof, alginic acid or salts thereof, It may further contain at least one selected from the group consisting of organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The proportion of these additives is generally selected from 0.001 to about 20 parts by weight per 100 parts by weight of the total health functional food, but is not limited thereto.

The terms and methods described for the above invention are equally applied between the respective inventions.

According to the composition comprising the extract of Seomcholong flower according to an aspect as an active ingredient, inflammatory mediators NO (Nitric Oxide), PGE ₂ (Prostaglandin E2), IL-6 (Interleukin-6) and TNF-α (Tumor necrosis factor) -α) secretion and inhibiting the enzymatic activity of XOD (Xanthine Oxidase), there is an effect that can be usefully used for the prevention, improvement or treatment of inflammatory diseases.

1 is a chromatogram analyzed by high-performance liquid chromatography on a hot-water extract of Seomchorong flower or a 30% alcohol extract of Seomchorong flower.
Figure 2 is a diagram showing the change in the amount of nitric oxide (NO) secretion changed when the macrophage (RAW264.7) is treated with the extract of Campanula takesimana Nakai.
Figure 3 is a picture showing the change in the amount of Prostaglandin E2 (PGE ₂ ) secretion, which is changed when the macrophage (RAW264.7) is treated with the extract of Campanula takesimana Nakai.
Figure 4 is a diagram showing the change in IL-6 (Interleukin-6) secretion amount changed when processing the extract of Campanula takesimana Nakai in macrophages (RAW264.7).
Figure 5 is a diagram showing the change in the amount of TNF-α (Tumor necrosis factor-α) secretion is changed when processing the extract of Campanula takesimana Nakai in macrophages (RAW264.7).
6 is a diagram showing the change in SOD (Superoxide dismutase) activity that is changed when processing the extract of Campanula takesimana Nakai.
Figure 7 is a diagram showing the change in xanthine oxidase (Xanthine Oxidase) activity that is changed when processing the extract of Campanula takesimana Nakai.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of wild greens extract

Of the composition of the present invention, the hot-water extract of oleracea is prepared by the following process. First, dry leaves of Campanula takesimana Nakai 2 kg and 40 kg of purified water were put in an extraction tank and extracted under reflux at 98°C for 5 hours. The extracted sample was filtered through a bag filter (55 um), then the thin film was concentrated under reduced pressure, and a water-soluble powder was obtained through freeze-drying.

In the composition of the present invention, the 30% alcohol extract of oleracea is prepared by the following process. First, dry leaves of Campanula takesimana Nakai 2 kg and 40 kg of 30% alcohol were placed in an extraction tank and extracted under reflux at 60° C. for 5 hours. The extracted sample was filtered through a bag filter (55 um) and concentrated under reduced pressure to obtain a water-soluble powder.

Experimental Example 1: High-performance liquid chromatography analysis of wild vegetable extract

The hot water extract and 30% alcohol extract obtained in Example 1 were analyzed by high-performance liquid chromatography (HPLC) and UV/Vis detector. As HPLC equipment, Waters e2695 Series system, Waters 2489 UV/Vis detector (Waters, Milford, MA, USA) was used, and all solvents used for analysis were J.T. An HPLC-grade solvent purchased from Baker (Phillipsburg, NJ, USA) was used.

(5 μm, 250 Х 4.6 mm, Waters, Milford, MA, USA) 컬럼을 사용하였으며, 컬럼의 온도는 30℃ 주입 용량은 20㎕, 측정 파장은 210nm로 설정하였다. 이동상은 아세토니트릴(ACN)과 3차 증류수(D.W)를 사용하였으며, 1㎖/분 의 속도로 ACN -D.W (1:9 - 10:0, v/v) 혼합 용액을 50분간 분석하였다. 분석 시료는 섬초롱꽃 열수추출물 또는 섬초롱꽃 30%주정추출물 분말 300mg을 정밀히 칭량하여 30% 메탄올 30㎖을 가한 후 20분간 초음파 진탕기에 넣어 용해하여 실온에서 방냉 후 상등액을 취해 0.45 μm 멤브레인 필터로 여과하여 사용하였다.XSelect HSS C18 100 was analyzed for the hot-water extract or 30% alcohol extract obtained from Example 1

A (5 μm, 250 Х 4.6 mm, Waters, Milford, MA, USA) column was used, the column temperature was set to 30°C, the injection volume was set to 20 μl, and the measurement wavelength was set to 210 nm. Acetonitrile (ACN) and tertiary distilled water (DW) were used as the mobile phase, and a mixed solution of ACN-DW (1:9 - 10:0, v/v) was analyzed for 50 minutes at a rate of 1 ml/min. For the sample to be analyzed, precisely weigh 300 mg of hot-water extract or 30% ethanol extract powder of Zinnia oleracea, add 30 ml of 30% methanol, dissolve in an ultrasonic shaker for 20 minutes, cool at room temperature, take the supernatant, and filter with a 0.45 μm membrane filter. was used.

As shown in FIG. 1 , the hot water extract of S. oleraceae or 30% alcohol extract of S. oleracea is a composition containing a number of components, and when comparing each chromatogram, there is a difference in the aspect and content of the composition depending on the extraction method. could confirm that there was

Experimental Example 2: Changes in Nitric oxide (NO) secretion during treatment with extract Confirm

Nitric oxide (NO) secretion amount with respect to the extract obtained in Example 1 Change was confirmed.

Specifically, macrophages, RAW264.7 cells, were obtained from the American Type Culture Collection (ATCC, MD, USA) and cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 5% CO2, 37 °C. . After attaching RAW264.7 cells to a 24-well plate at a concentration of 1X10 ⁵ cells/500 μl/well for 24 hours, 0.5 mg/ml LPS and the extract of Example 1 in phenol-red free DMEM 6.25, 12.5, 25, They were treated with concentrations of 50, 100, and 200 μg/ml and incubated for 24 hours. Then, transfer 100 μl of the culture supernatant to a 96-well plate, mix 100 μl of Griess (0.1% N-(1-naphtyl)ethylenediamine, 1% sulfanilamide) reagent, and react for 10 minutes. nm was measured with a microplate reader (BioTek, Vermont, USA). Nitric Oxide (NO) secretion was calculated by converting a standard curve from the dilution range of sodium nitrite to the amount of nitrite in each sample.

As a result, as shown in FIG. 2 , the amount of NO produced increased by LPS was reduced by the treatment with the oleracea extract, and both the hot water extract and the 30% ethanol extract showed an NO reduction effect.

Experimental Example 3: Cytokine (PGE) _2, Changes in IL-6, TNF-α) secretion

Cytokine (PGE _2, IL-6, TNF-α) secretion amount with respect to the extract obtained in Example 1 Changes were confirmed using ELISA (Enzyme-linked Immunosorbant Assay: ELISA).

Specifically, after attaching macrophages (RAW264.7) to a 24-well plate at a concentration of 2X10 ⁵ cells/well for 24 hours, 0.5 mg/ml LPS and the extract of Example 1 were 6.25, 12.5, 25, 50, 100 , treated at a concentration of 200 μg/ml and incubated for 24 hours. Then, after diluting the culture supernatant to an appropriate concentration, 50 μl of each was transferred to a 96-well plate coated with cytokine antibody and allowed to react for 24 hours. After washing 3 times with washing buffer, 100 μl of biotinylated antibody reagent was treated in each well and reacted at room temperature for 1 hour, washed 3 times, and then treated with 100 μl of sterptavidine-HRP solution in each well After reacting at room temperature for 1 hour, it was washed 3 times with a washing buffer again. Here, 100 μl of di(2-ethylhexyl)-2,4,5 trimethoxybenzalmalonate (TMB) substrate was treated and reacted for 5-30 minutes, 100 μl of stop solution was treated, and absorbance was measured at 550 nm.

As a result, as shown in FIGS. 3, 4 and 5, the amount of cytokines (PGE _2, IL-6, TNF-α) increased by LPS was decreased in a concentration-dependent manner by the treatment with the extract of Seomchoronchi, in particular, The reduction effect on PGE ₂ by the hot water extract was remarkable.

Experimental Example 4: Superoxide dismutase (SOD) activity test during treatment with extract

Superoxide dismutase (SOD) activity of the extract obtained in Example 1 was confirmed using an SOD Determination kit (Sigma, MO, USA). During the production of uric acid, a large amount of superoxide radicals are generated by xanthine oxidase. These reactive oxygen species (ROS) contribute to oxidative stress in the body, and many pathological processes such as inflammation, arteriosclerosis, cancer, and aging. get involved in Therefore, the inflammatory reaction is further aggravated by the active oxygen generated during the process of gout. SOD is an enzyme that catalyzes the disproportionation reaction that converts superoxide ions into oxygen and hydrogen peroxide, and is an antioxidant enzyme that removes excess free radicals accumulated in the body. Therefore, as the activity of SOD increases, when free radicals are removed, it helps to relieve gout inflammation.

Specifically, after diluting the extract of Example 1 to an appropriate concentration, 20 μl of each was dispensed into a 96-well plate, and 200 μl of a water-soluble tetrazolium salt (WST) solution and 20 μl of an enzyme reaction solution were added. After treatment at 37 °C for 20 minutes, absorbance was measured at 450 nm with a microplate reader (BioTek, VT, USA).

) 으로 계산하였다.SOD enzyme activity

) was calculated as

As a result, as shown in FIG. 6, SOD activity was increased in a concentration-dependent manner in both hot water and 30% alcohol extract.

Experimental Example 5: Xanthine oxidase inhibitory activity test during treatment with extract

The xanthine oxidase inhibitory activity of the extract obtained in Example 1 was confirmed using a Xanthine oxidase assay kit (AAT Bioquest, CA, USA). Xanthine oxidase promotes oxidation from purine base to hypoxanthine or xanthine to uric acid and peroxide, which is immediately decomposed into hydrogen peroxide. In this case, the degree of inhibition of the activity of xanthine oxidizing factor can be confirmed by measuring the absorbance.

로 XOD activity를 계산하였다. Specifically, the extract of Example 1 and xanthine oxidase were mixed with Amplite Red diluted solution, Horseradish Peroxidase diluted solution, and xanthine diluted solution, and then treated in a dark room at room temperature for 30 to 60 minutes. After that, absorbance was measured at 570/610 nm with a microplate reader.

to calculate the XOD activity.

As a result, as shown in FIG. 7 , both the hot water extract and 30% alcohol extract of S. oleracea significantly inhibited xanthine oxidase according to the concentration.

Formulation Example 1: Preparation of tablets

With respect to Example 1, according to a conventional tablet manufacturing method, the ingredients in Table 3 below were mixed and compressed to prepare tablets.

Raw material name unit weight (mg) Example 1 50 corn starch 100 lactose 100 stearic acid 2

Formulation Example 2: Preparation of capsules

With respect to Example 1, the ingredients in Table 4 below were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare soft capsules.

Raw material name unit weight (mg) Example 1 50 corn starch 100 lactose 100 stearic acid 2

Formulation Example 3: Preparation of liquid formulation

With respect to Example 1, the ingredients in Table 5 were mixed according to the beverage preparation method suitable for preference, and a liquid was prepared by filling in a vial or pouch.

Raw material name unit weight (g) Example 1 2.5050 Xanthan Gum 0.0075 fructooligosaccharide solution 0.7500 Coconut Flower Extract Powder 1.0500 Ssanghwa Concentrate 1.5000 red ginseng flavor 0.0450 Purified water 9.1425

Formulation Example 4: Preparation of jelly

With respect to Example 1, the ingredients in Table 6 below were mixed according to the jelly preparation method suitable for the taste, and the ingredients were filled in a three-sided cloth to prepare a jelly.

Raw material name unit weight (g) Example 1 2.0000 food gel 0.3600 carrageenan 0.0600 calcium lactate 0.1000 sodium citrate 0.0600 Complex Golden Extract 0.0200 Enzyme-treated Stevia 0.0440 fructooligosaccharide solution 5.0000 Red Grape Concentrate 2.4000 Purified water 13.9560

Formulation Example 5: Preparation of nutritional cream

With respect to Example 1, a nourishing cream was prepared with the composition of Table 7 below according to a conventional method.

Raw material content (%) Example 1 1.0 sitosterol 4.0 Polyglyceryl 2-oleate 3.0 3.0 Ceteares-4 2.0 cholesterol 3.0 dicetyl phosphate 0.4 concentrated glycerin 5.0 Sunflower Oil 22.0 Carboxyvinyl Polymer 0.5 triethanolamine 0.5 antiseptic a very small amount Spices a very small amount Purified water remaining amount

The above composition ratio is generally formulated as a formulation example by mixing suitable components, but the mixing ratio and raw materials may be arbitrarily changed as necessary.

Since the extract of the present invention is stable in all formulation example test conditions, there was no problem in formulation stability.

Claims (9)

A pharmaceutical composition for preventing or treating inflammatory diseases comprising a hot water extract of Campanula takesimana Nakai at an amount of 50ug/mL to 1,500ug/mL. The method according to claim 1, wherein the extract is NO (Nitric oxide), PGE ₂ (Prostaglandin E2), IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor-α) and XOD (Xanthine Oxidase) from the group consisting of A pharmaceutical composition that reduces one or more selected activities. The method 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, sacrificial spondylitis One or more diseases selected from the group consisting of , rheumatoid lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, parotiditis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis. A pharmaceutical composition that is. A composition for external application for skin for preventing or treating inflammatory diseases, comprising Campanula takesimana Nakai hot water extract at an amount of 50ug/mL to 1,500ug/mL. A cosmetic composition for relieving skin inflammation comprising a hot water extract of Campanula takesimana Nakai at an amount of 50ug/mL to 1,500ug/mL. A health functional food composition for preventing or improving inflammatory diseases comprising a hot water extract of Campanula takesimana Nakai at an amount of 50ug/mL to 1,500ug/mL. The method according to claim 6, wherein the extract is NO (Nitric oxide), PGE ₂ (Prostaglandin E2), IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor-α) and XOD (Xanthine Oxidase) from the group consisting of A health functional food composition that reduces one or more selected activities. The method according to claim 6, 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, sacrificial spondylitis One or more diseases selected from the group consisting of , rheumatoid lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, parotiditis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis. A health functional food composition that is phosphorus. The method according to claim 6, wherein the health functional food has a formulation selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leached teas, and health drinks. Health functional food composition.

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