KR101538743B1 - Composition for Preventing, Improving or Treating of Ｔｈ1-mediated Immune Disease or Ｔｈ2-mediated Immune Disease Comprising Extracts from Saussurea lappa Clarke and Biota orientalis (L.) Endl. as an Active Ingredients - Google Patents

Abstract

The present invention provides a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease, which comprises an extract of Camellia sinensis and an extract of Araliaceae as an active ingredient. The present inventive compositions of the present invention have shown that the secretion of IL-6 and TNF-α, the expression of PAR-2 and ICAM1, and the secretion of IgE, which are involved in the activation of NF-κB, Significantly inhibit and effectively reduce the number of mast cells and eosinophils, thereby exhibiting an excellent effect in the prevention and treatment of Th1-mediated immune diseases or Th2-mediated immune diseases. The present invention can provide an inexpensive production cost and ease of use by using a natural material as a material.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease which comprises an extract of Aspergillus oryzae as an active ingredient and a Th2-mediated Immune Disease Comprising Extracts from Saussurea lappa Clarke and Biota orientalis (L.) Endl. as an Active Ingredients}

The present invention relates to a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease, which comprises an extract of Camellia sinensis and an extract of Aspergillus oryzae.

Allergy is increasing day by day due to changes in eating habits, cleanliness of hygiene, intensification of pollution, etc. 20-25% of the whole people are suffering from atopic dermatitis (AD), asthma, rhinitis Lt; / RTI > This is a serious social problem by significantly reducing the quality of life. In addition, food allergies are identified in 6-8% of infants and 35% of children in AD are due to food allergies.

In addition, patients with atopic dermatitis are rapidly increasing due to the use of food additives, excessive use of chemicals, and pollution. Accordingly, there is an increasing demand for searching for natural materials effective for prevention and treatment of atopic dermatitis. Research into the polyol-based materials with improved moisturizing properties that improve immunity and inhibit inflammation (NF-κB mechanism) associated with atopic dermatitis and commercialize them in the form of nano-liposomes that are easy to penetrate into the skin It is progressing. The development of nano-liposome formation and stabilization methods for developing the natural materials in the form of atopic lotion or cream, and the development of lotions and creams effective for the prevention and treatment of atopic dermatitis are under way.

Ultimately, exploring PAR-2 (protease-activated receptor-2) and herbal medicines that inhibit the NF-κB mechanism involved in the progression of atopic disease is also meaningful in securing the original technology. It is important to make the form of nano liposome that can be made and stabilize it, which plays an important role in manufacturing cosmetics by securing original technology.

Atopy is a typical skin disease that causes skin dryness, which affects about 10-20% of the total population. The causes are genetic factors and environmental factors. When the genetic factors are not changed significantly, the environmental factors have a great influence on the increase of the prevalence rate. Among them, exposure to outdoor and indoor allergens such as house dust mites and ticks It is also related to increased food additives and fat intake due to reduced breastfeeding and increased intake of processed foods.

The symptoms of atopic dermatitis include dry skin, epidermal hyperplasia, inflammation, itching, etc., and the cause of atopic dermatitis has not been clarified yet. The current treatment methods focus on corticosteroids, antihistamine agents, inhibition of DNA synthesis through UV therapy, inhibition of cell hyperplasia, inflammation and itching, but these methods have the disadvantage that they can not be treated with the underlying side effects have. Since atopic dermatitis is especially present in infants, it is difficult to administer the medication, so it is important to develop it as an external medicine.

Atopic dermatitis is associated with inflammation, which is associated with the activation of T lymphocytes by atopic dermatitis reaction with immunoglobulin E by allergen-inducing substances, which secrete cytokines in the skin with atopic dermatitis. Ultimately, in order to alleviate symptoms of atopic dermatitis, it is necessary to search for prevention of inflammation and prevention of epidermal hyperplasia among herbal medicines, because moisturization is maintained while inflammation is reduced and epidermal hyperplasia is prevented. Although studies on the prevention and treatment of atopic dermatitis which can be used as an injectable or external agent have been continuously performed, functional products suitable for effective atopic prevention and treatment have not yet been developed. It is necessary to develop as a topical agent that can eliminate inflammation and eliminate factors that cause itching and atopic dermatitis along with moisturizing, since atopic dermatitis is especially prone to itching due to dry skin disease. Do.

The most problem of atopic dermatitis is the abnormal activation of immune function and the increase of cytokine due to the increase of inflammation reaction. Therefore, it is important to search for medicinal herbs that can suppress such inflammatory reaction and develop it as a preventive and therapeutic agent for atopy. Most of the studies to reduce the inflammation and immune function to normalize the development of functional products to prevent or treat atopic dermatitis, the development of many products that are mostly developed for treatment of atopic dermatitis is more effective than taking a product.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop natural products capable of preventing or treating diseases, diseases or conditions caused by Th1 or Th2 immune responses such as inflammatory diseases and allergies. As a result, the present inventors have completed the present invention by experimentally confirming that the combination of mulberry and Japanese cabbage extract has a prophylactic or therapeutic effect of immune disease which is remarkably improved as compared with a mulberry extract or a mulberry tree extract.

Accordingly, it is an object of the present invention to provide a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease, comprising a woody and opium tree extract.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, the present invention provides a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease, comprising a woody and opioid extract.

The present inventors have made efforts to develop natural products capable of preventing or treating diseases, diseases or conditions caused by Th1 or Th2 immune responses such as inflammatory diseases and allergies. As a result, And the present invention has the effect of preventing or treating immune diseases which have remarkably improved compared with those of the present invention.

Saussurea lappa Clarke (Saussurea lappa Clarke) used in the present invention is a plant of Asteraceae, which is used to trim roots and root roots in autumn and to dry thick roots. Known chemical components include alrentolactone, inulin, and saponin. It has been used as a placebo in oriental medicine and has been used in various respiratory diseases such as bronchitis and pulmonary tuberculosis. It is an extremely safe plant that has been used in herbal medicine for a long time and has already confirmed human stability due to ingestion and has no stimulation to the skin and mucous membranes.

Biota orientalis (L.) Endl.), Which is used in the present invention, is an evergreen tree belonging to the Cupressaceae family, also known as hardball, and is originated in China and widely distributed throughout Korea. It is also known to be effective against hemorrhage of the wife, hemorrhage of the puffer fish or rectum, and prevention of hypertension and paralysis. The antimicrobial activity, All of which have been reported to exhibit excellent effects. Leaves also contain flavonoids, essential oils, lead, tannins, and resins. Flavonoids include quercetin, chemperol, myristate, hinokiflavone, and a mentoflavone.

As used herein, the term " wicker extract " refers to a mixture obtained by extracting from the roots of the seeds using a suitable solvent.

In the present specification, the term " Cryptomeria japonica extract " means a mixture obtained by extracting from the leaves of the clavatus with the appropriate solvent.

In the present specification, the term " mallow and cinnabar and tree blend extract " means a mixture of the marihuana extract and cervical spruce extract, preferably 1: 1 mixture of municipal extract and cervix extract.

The method of isolating the extract of the present invention, which is an active ingredient in the composition of the present invention, comprises extracting the extract from a natural product according to a conventional method known in the art, that is, a conventional solvent under the conditions of ordinary temperature and pressure Can be separated by using.

Various extracting solvents can be used when the extracts of Chenopodiaceae and Chenopodiaceae which are used in the composition of the present invention are obtained by treating the extractive solvent in the seeds and the spruce trees. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, , Ethylene glycol or butylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

According to one embodiment of the present invention, the extract of the present invention is obtained by treating water, ethanol, butanol, butylene glycol, ethyl acetate,

According to a particular embodiment of the present invention, the extract of the present invention is obtained by treating butylene glycol in a woody and spruce tree.

As used herein, the term " extract " has the meaning conventionally used in the art as a crude extract, but broadly includes fractions obtained by further fractionation of the extract. That is to say, the extracts of Momordica keiskei and Cynomolgus koreensis are obtained not only by using the above-mentioned extraction solvent but also by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained through the purification method is also included in the horseradish perilla extract of the present invention.

The horseradish and herbal extracts used in the present invention may be prepared in powder form by an additional process such as vacuum distillation and lyophilization or spray drying.

As used herein, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve the efficacy or activity of the following herbs and spice extracts. Since the present invention is a composition extracted from natural and herbal plant material such as moss and spruce tree, there is no adverse effect on the human body even when administered in an excessive amount. Therefore, the quantitative upper limit in which the composition of the present invention is included in the composition of the present invention is selected by a person skilled in the art .

According to the present invention, the horseradish peroxidase and mixed leaves of the present invention inhibit the secretion of IL-6 and TNF-α and the expression of PAR-2 and ICAM1, which are involved in the activation of NF-κB, Ameliorating or treating a Th1-mediated immune disorder or a Th2-mediated immune disorder by effectively reducing the number of mast cells, eosinophils, and mast cells.

The compositions of the present invention can be used for the prevention or treatment of various Th1-mediated immune diseases, diseases or conditions.

As used herein, the term "Th1-mediated immune disorder" refers to a cytokine, such as IL-1β, IL-2, IL-12, IL-17, IFN- TNF-alpha, and the like.

As used herein, the term " Th1 cell " refers to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion, and functional activity. For example, Th1 cells synthesize IL-2 and IFN-y, but IL-4, IL-5, IL-10 and IL-13 do not synthesize cytokine expression patterns. Th1 cells are involved in cell-mediated immune responses, organ-specific autoimmune diseases and delayed hypersensitivity reactions to a variety of intracellular pathogens.

The Th1-mediated immune disease to which the composition of the present invention is applied is not particularly limited, and is preferably applied to transplant rejection, autoimmune disease or inflammatory disease. More preferably, the Th1-mediated immune disease to which the composition of the present invention is applied is an autoimmune disease or inflammatory disease, and even more preferably it is colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes and rheumatoid arthritis.

The Th1-mediated immune diseases to which the composition of the present invention is applied are selected from the group consisting of colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, Reactive Arthritis, osteoarthritis, psoriasis, , Myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves disease, leprosy, syphilis, Lyme disease, Borreliosis, neurogenic-borrelia, tuberculosis, sarcoidosis, lupus, Uveitis, irritable bowel syndrome, Crohn's disease, Shoggran's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, muscular dystrophy, amyotrophic syndrome, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis Socio-emphysema, Parkinson's disease, multiple sclerosis, autism spectrum disorders, attention deficit disorder, and attention deficit hyperactivity disorder. It is not.

The compositions of the present invention may be used for the prevention or treatment of various Th2-mediated immune diseases, diseases or conditions.

As used herein, the term " Th2-mediated immune disease " refers to a disease involving IgE and mast cells due to the production and activity of allergen-specific Th2 cells.

As used herein, the term " Th2 cell " refers to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion, and functional activity. For example, Th2 cells exhibit IL-4, IL-5, IL-10, and IL-13 cytokine expression patterns. Th2 cells are involved in the humoral immune response.

The Th2-mediated immune disease to which the composition of the present invention is applied is not particularly limited, and is preferably applied to an allergic disease, more preferably to atopic dermatitis.

Th2-mediated immune diseases to which the composition of the present invention is applied include, but are not limited to, atopic dermatitis, other skin diseases associated with atopy, allergic rhinitis (acute or chronic), alopecia and allergic bronchial asthma.

The compositions of the present invention may be prepared with pharmaceutical compositions.

According to one embodiment of the present invention, the composition of the present invention comprises: (a) a pharmaceutically effective amount of the above-described inventive horsetail and Quercus serrata extract; And (b) a pharmaceutically acceptable carrier. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve efficacy or activity of the herbs and herbs described above.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably parenterally administered.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of this invention are in the range of 0.001-100 mg / kg on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention can be provided as a food composition. When a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising an effective ingredient of the present invention and an effective ingredient of the present invention is prepared from a food composition, But includes ingredients that are typically added during the manufacture of the food product, including, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract and licorice extract may be further added in addition to the male extract of the present invention have.

A composition for preventing, ameliorating or treating a Th1-mediated disease or a Th2-mediated immune disease comprising the extract of the present invention as an active ingredient may be prepared from a functional food composition. When the composition of the present invention is prepared with a functional food composition, it includes components that are conventionally added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of being made with a drink, flavorings or natural carbohydrates may be added as an additional ingredient in addition to the extracts of Chenopodium album var. For example, natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (e.g., maltose, sucrose, etc.); oligosaccharide; Polysaccharides (e.g., dextrin, cyclodextrin and the like); And sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). Natural flavoring agents (e.g., tau marin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may be used as flavorings.

The composition of the present invention can be prepared with a cosmetic composition. When a composition for preventing, ameliorating or treating a Th1-mediated disease or a Th2-mediated immune disease comprising the extract of the present invention as an active ingredient is prepared from a cosmetic composition, in addition to the male extract as an active ingredient, Include commonly used ingredients and include customary adjuvants such as, for example, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising an extract of Chenopodium albumus and an extract of Chenopodium albumus as an active ingredient.

(Ii) The inventive compositions of the present invention have the following effects: secretion of IL-6 and TNF-α, PAR-2 and ICAM1 expression, activation of NF-κB, Suppresses the secretion more effectively, and effectively reduces the number of mast cells and eosinophils, thereby exhibiting an excellent effect in the prevention and treatment of Th1-mediated immune diseases or Th2-mediated immune diseases.

(Iii) The present invention can provide an inexpensive production cost and ease of use by using natural materials as materials.

FIG. 1 shows the results of measurement of the expression levels of PAR-2, ICAM-1 and IL-6 by house dust mite.
Expressed as relative mRNA levels for beta-actin expressing mRNA. ^{a, b} indicates that there is a statistically significant difference in the alphabet on the bar in one variable.
FIG. 2 shows the results of measurement of the effect of the mixed extracts of horseradish perennials, Bombyx mori, and horseradish perianths on TNF-α and IL-6 secretion levels.
^{a, b, and c} indicate that there is a statistically significant difference between the two variables on the bar.
FIG. 3 shows the results of measurement of the effect of the mixed extracts of horseradish perennials, Bombyx mori, and horseradish perianths on PAR-2, ICAM1, MCP-1 and IL-6 mRNA expression.
^{a, b, and c} indicate that there is a statistically significant difference between the two variables on the bar.
Fig. 4 is a picture of symptoms of a mouse or the like caused by atopic induction.
Fig. 5 is a graph showing the sensory test score according to the mixed treatment of horseradish, cinnabar and wood + cinnabar.
^{A, b, and c} indicate that there is a statistically significant difference between the different alphabets (P <0.05).
FIG. 6 shows the results of measurement of serum IgE and TNF-? Levels according to the mixed extracts of horseradish, cedarwood, and horseradish perianth.
^{A, b, and c} indicate that there is a statistically significant difference between the different alphabets (P <0.05).
FIG. 7 shows the results of measurement of the number of mast cells and neutrophils according to the mixed extract treatment of horseradish, cedarwood, and horseradish + cinnabar.
^{A, b, and c} indicate that there is a statistically significant difference between the different alphabets (P <0.05).
FIG. 8 shows the results of measuring the mRNA expression levels of IL-4, IL-13 and IFN-γ in skin tissues according to the mixed treatment with horseradish, cinnabar and wood +
^{A, b, and c} indicate that there is a statistically significant difference between the different alphabets (P <0.05).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

1. Materials

(With dried roots of Saussuerea lappa Clarke) and with Biota orientalis (L.) Endl. (Dried leaves of cinnabar and leaf) powder in 1,3-butylene glycol Were added at 70%, respectively, and sonicated at 60 ° C for 2 hours. That is, 300 g of 1,3-butylene glycol was added to 1 kg of each of the dried woody powder and the cowpea powder, and the mixture was ultrasonically extracted at 60 DEG C for 2 hours. After the extraction, the precipitate was removed by centrifugation and the supernatant was used for the experiment.

2. Cell culture

In human corneal HaCaT cells, PAR-2 expression and cytokine secretion

Human corneal HaCaT cells were cultured in RPMI medium containing 10% FBS, 2 mM L-glutamine, 100 IU / ml penicillin and 100 / ml streptomycin antibiotic. In order to measure the expression of PAR-2 (protease-activated receptor-2), a mite extract, known as an atopy inducer, was purchased from the Bank of Arthropods of Yonsei University and treated with 3 μg / mL of HaCat cells to induce atopy. 2 expression was increased. The extracts of mite, mungbean, or horseradish and mulberry were treated with mite extract and the expression level of PAR-2 was measured. Two days before the cytokine secretion experiment, 1 x 10 ⁶ HaCaT cells were transferred to a 12-well culture plate and cultured in RPMI, 10% FBS, 2 mM L-glutamine and antibiotics. On the day of the cytokine secretion experiment, KRB solution (pH 7.2) containing 0.2% (w / v) BSA and horseradish, cinnabar extract or horseradish and cinnabar and mixed extracts was changed to pH 7.2. After 2 hours, KRB solution The concentrations of TNF-α and IL-6, which are inflammatory cytokines secreted into the collected KRB solution, were measured by ELISA kit (eBioscience, San Diego, CA, USA) and the KRB solution was stored at 80 ° C. until measurement Respectively. Cells in the wells were scraped and lysed with homogenization buffer (1 M HCl containing 5% (v / v) HCOOH, 1% (v / v) trifluoroacetic acid and 1% (w / v) NaCl) The amount of TNF-α and IL-6, the remaining inflammatory cytokines, was measured by real-time PCR.

3. Animal experiments

Dermatitis induction and sample treatment

Ng / Nga mice at 5-6 weeks of age were cleaned and then allowed to stand for 24 hours to heal the skin micro-wounds. 200 μl of a 1% DNCB solution (acetone: olive oil = 3: 1) was applied to the back region, and after 4 days, 150 μl of 0.2% DNCB solution was applied 2-3 times a week to the back region. After 4 to 5 weeks of treatment, the dermatitis is sufficiently induced and the skin of the back area is completely peeled off (facilitating the permeation of the therapeutic drug). When a new dermatitis is formed in this area, After the evaluation, the blood was collected and the blood cells were settled, and serum was taken and stored at -70 ° C for freezing. Immediately before the completion of the DNCB treatment, the drug to be tested for its therapeutic effect was dissolved in a suitable solvent (a solvent in which the compound was completely dissolved and no skin irritation) at a desired concentration. (150 μl) twice a day twice a day, for 2 weeks, with 0.2% DNCB treatment. Sensory evaluation was conducted weekly. After the sensory evaluation, the blood was taken and the skin of the back was excised and stored in 10% formalin solution. Blood was collected by centrifuging the blood cells and stored frozen at -70 ° C.

4. Evaluation Method

Sensory evaluation

The dermatitis of NC / Nga mice used a clinical visual assessment method commonly used in atopic dermatitis. The visual evaluation results are expressed by the total sum of the scores evaluated for each of the following five items. Evaluation items are divided into erythema, pruritus & dry skin, edema & escoriation, erosion, and lichenification. Each item is scored as zero (0), weak (1), severity (2), and severe (3). So you get a score between 0 and 15. In general, when using DNCB to cause dermatitis, it can be judged that the dermatitis reaches its peak at about 12-13 points.

An ELISA (Pharmingen: manufacturer's recommendation)

To investigate the anti - atopic effect of mixed extracts of P. vannamei and P. vivax, the extracts were treated with atopic dermatitis - induced animal models and serum IgE levels were measured by enzyme immunoassay (ELISA). Specifically, in an animal model in which DNCB was treated to induce atopic dermatitis, 30% butylene glycol (1,3-butylene glycol) was added to the control group 10 days after the completion of the DNCB treatment, The extracts were mixed with 200 μl of the herbal extract twice daily for 8 weeks. The blood was collected from the capillaries of the mucous membranes at intervals of 4 weeks, and the blood cells were collected. The serum was collected and the serum IgE .

ELISA was performed by diluting the capture antibody (Capture Ab) (anti-mouse IgE capture mAB, BD Bioscience) 250 times with coating buffer and then adding 50 μl to the 96 well plate for ELISA and incubating overnight at 4 ° C After allowing the capture antibody to react with the plate, the antibody was washed 5 times with PBST to remove unreacted antibody. Each well was then blocked with 100 μl of the assay diluent at room temperature for 1 hour and incubated with standard samples (100/50/25 / 12.5 / 6.3 / 3.2 / 1.6 ng / ml), 4.15 [mu] l of IgE standard sample, and sample sample. At this time, blood samples were diluted 200-500 times as the blood IgE concentration exceeded 10 μg / ml when the disease was induced and the clinical score exceeded 6-7 points. 50 μl of the standard and sample samples were added to each well, covered with lap, allowed to react at room temperature for 2 hours, and then washed 5 times with PBST. 50 μl of a detection antibody (detection Ab + Avidin-HRP) (biotinylated anti-mouse IgE, BD Bioscience) was added to each well, followed by lapping and incubation at room temperature for 1 hour. After washing with PBST 5 times to remove unreacted antibody, 50 μl of TMB substrate was added to each well. After reacting at room temperature for 30 minutes, 25 μl of a stop solution was added to the reaction solution. The absorbance was measured at 450 nm and the concentration of IgE was measured. Serum levels of TNF-α were measured by the same method as for IgE, but the antibodies were used for TNF-α (eBioscience, San Diego, CA, USA) and IgE (mdbioproducts, St Paul, MN, USA).

5. Dyeing and observation of skin tissue

The skin was collected and stained with toluidine blue to observe mast cells. HE staining (Heamatoxylin-Eosin) was performed to observe the morphology of the cells. First, the experiment was completed for 6 weeks. The skin of the back area was resected to 1 × 1 cm ² and stored in 10% formalin solution. After one day, it was stored in PBS solution for about one week and embedded to make a paraffin block. The skin tissue prepared with paraffin block was fixed to a cutting device (Shandon finesse E +) and cut to a thickness of 5 μm. The cut tissue was placed in ethanol (40% ethanol) and sterilized before sterilization. Then, the skin tissue expanded by using a slide glass was placed in lukewarm water at 37 占 폚. This serves to spread the cut skin tissue. Finally, the skin tissue was removed by using a slide glass, dried at room temperature, and stored in a slide box.

Tissue was stained using HE staining and toluidine blue staining to observe the overall condition of the skin and observed at a ratio of 10 times on an optical microscope.

Before the toluidine blue staining, the toluidine solution was prepared by adding 1 g of toluidine blue + 1 g of sodium borate + 100 ml of dH ₂ O to the flask and mixing well. When used, filter on the filter paper. After cutting, 20 slide glasses stored in a slide box were placed on the iron pedestal, and then a functional process was performed. After the dehydration process, the skin tissue was covered with a cover glass using a microscope.

Before performing HE staining, make reagents necessary for staining. Meyer hematoxylin was applied to the filter paper every time it was used. Eosine was placed in a flask with 1.0 g of eosin yellow wax and 100 ml of distilled water on a stirrer, which was placed on a stirrer and filtered to filter paper. Acid alcohol 1% was prepared by mixing 99 ml of 70% alcohol and 1 ml of hydrochloric acid.

In order to perform the dyeing, 20 skin tissues were prepared by cutting the water after cutting, and then the functional process was performed. After drying, the skin tissue was placed in hematoxylin, which was once filtered through the filter paper, for 5 minutes, and then sufficiently washed with dH ₂ O (third distilled water). Then, it was placed in 1% acidic alcohol for a few seconds, washed in dH ₂ O (third distilled water) flowing once more, placed in a filtered eosin for 6 minutes, and then dH ₂ O (third distilled water) Washed and finally dehydrated. After dehydration, the skin tissue was covered with a cover glass and observed with a microscope.

6. Expression of IL-4, IL-13 and INF-r mRNA in skin tissue

After completion of the experiment, dorsal skin tissues were obtained from 5 mice of each experimental group. Total RNA of skin tissue was isolated using a monophasic solution of phenol and guanidine isothiocyanate (Trizol reagent, Gibco-BRL, Rockville, Md.) And then extracted and precipitated with isopropyl alcohol. Equal amount of total RNA was synthesized by using SuperScript III reverse transcriptase (Invitrogen, Grand Island, NY, USA) and PCR was performed using High fidelity Taq DNA polymerase (Invitrogen, Grand Island, NY, USA) Respectively. Equal amounts of cDNA were mixed with CyberGreenMix (Invitrogen, Grand Island, NY, USA) and analyzed using a real-time PCR machine (BioRad Laboratories, Hercules, Calif.). Expression levels of the gene of interest were evaluated based on the β-actin, a housekeeping gene. The following primers were used for the PCR reactions: mouse IFN-y, forward 5'-CGGCACAGTCATTGAAAGCCTA-3 'and reverse 5'-GTTGCTGATGGCCTGATTGTC-3'; IL-4, forward 5'-TCTCGAATGTACCAGGAGCCATATC-3 'and reverse 5'-AGCACCTTGGAAGCCCTACAGA-3'; Mouse IL-13, forward 5'-cagctccctggttctctcac-3 'and reverse 5'-ccacactccataccatgctg-3'; Mouse β-actin, forward 5'-CATCCGTAAAGACCTCTATGCCAAC-3 'and reverse 5'-ATGGAGCCACCGATCCACA-3'. The primers were designed to bind to at least one intron to distinguish products derived from mRNA and genomic DNA.

Experiment result

1. Anti-atopic effect in HaCaT, a skin cell

PAR-2, ICAM-1 and IL-6 expression by house dust mite

As shown in FIG. 1, the expression of PAR-2, ICAM-1 and IL-6 gene was increased by house dust mite. Up to 3 μg / mL, gene expression was increased by house dust mite, but at higher concentrations, there was no effect or decreased tendency. Based on the above results, in the present invention, 3 mcg / mL of dust mites were used (Fig. 1).

The concentration of TNF-α and IL-6 secreted by the medium

In preliminary experiments, when the extracts of various medicinal herbs were treated with 3 μg / mL of mite extract, the extracts of Mackerel and Cynobacterium tended to inhibit the expression of PAR-2, and these two extracts were tested for anti-atopic effect . The treatment with 50 μg / mL of Butylene Glycol Extract of Hibiscus japonicus, Cervus spp., And Mulberry + Cervus spp. (1: 1) was effective in the treatment of atopy. The treatment of mulberry and mulberry extracts reduced the secretion of TNF-α and IL-6, respectively, compared with the mite extract-treated control, but it was not statistically significant. The concentration of TNF-α and IL-6 secreted in the medium was significantly reduced when skin cells were treated with 1: 1 mixture of wheat germ and cinnabar (Fig. 2).

Amount of mRNA of PAR-2, IL-6 and ICAM-1 expressed in skin cells

The expression of PAR-2, which is known to increase when atopic symptoms appear in skin cells, and the adhesion molecule, ICAM-1, which increases expression when PAR-2 is enhanced by inflammation, and IL -6 expression was measured. Each of the waxy and cynomolgus extracts did not significantly reduce the expression of PAR-2, but were significantly reduced when treated with the horseraceous &amp; Expression of IL-6 and ICAM1 was similar to that of PAR-2.

2. Effect of mixed extracts of horseradish and cinnabar species in atopic animals

Sensory evaluation

After applying atorvic induction, the extracts of Mulberry, Quercus mongolica and Mulberry + Quercus variabilis were applied, and as a result, the changes of the state were scored (evaluation items were erythema, pruritus and dry skin ), Edema & escoriation, Erosion, and Lichenification. Each of these items was classified as No (0), Weak (1), Severity (2), Severity (3) The scores of at least 0 and up to 15 points were obtained.) The extracts of each of the mulberry and mulberry trees tended to improve the atopic state of the mulberry compared with the control group. However, when the mixed extract of Mulberry + (Fig. 4 and Fig. 5).

Serum IgE and TNF-a concentrations

The most serious problem of atopic dermatitis is the abnormal activation of immune function, which leads to an increase in the incidence of cytokines due to an inflammatory reaction. When atopic symptoms were exacerbated, abnormal immune function was observed. Therefore, serum IgE levels indicating immune function were measured in order to evaluate the relief and relief of atopic symptoms. In addition, TNF-α concentration, which indicates the degree of inflammation in the blood, was measured. The result showed a pattern similar to sensory symptoms. The concentration of TNF-α was decreased in the control group and the control group, and the number of control group was decreased more than that of control group. The IgE concentration, which indicates immunoreactivity, was significantly increased in the control group as compared with the normal control group. The IgE concentration was significantly decreased in the horseradish, the cabbage, This means that the mixed extract of Corynebacterium corydalis and Cervus nipponensis reduces immunity and inflammation of the immune system, and consequently, the combination of the Corynebacterium and Cervus elaphus extract effectively improves atopic symptoms (Fig. 6).

The presence of mast cells and eosinophils in the skin

The number of mast cells and eosinophils per unit area of skin tissue, which is an indicator of inflammation - induced immune response, were examined. As a result, the numbers of mast cells and eosinophils in the control group were significantly higher than those in the normal control group. As a result of daily application of the mixed extracts of horseradish peroxidase, horseradish peroxidase, and horseradish periplankton for 6 weeks on the mice that induced atopy, there was almost no difference in the horsetail, but the horseshoe perennial itself showed a higher amount of mast cells and eosinophils than the control group And the amount of mast cells and eosinophils was remarkably decreased in the mixture of the waxy and cinnabar extract extracts (Fig. 7).

IL-4, IL-13 and IFN-γ expression in skin

T cells that show immune function in the body are divided into two types, Th1 and Th2 cells. Th1 cells secrete IFN-γ and secrete Th2 cells by synthesizing IL-4 and IL-13. These two are in harmony with one another and control immune function to make immune function the best. Atopic dermatitis is characterized by over-stimulation of the function of atopic dermatitis. It is necessary to inhibit the activity of Th1 and Th2 cells in order to relieve and alleviate atopic symptoms.

Experimental results showed that IL-4, IL-13 and IFN-γ were significantly expressed in the skin tissues in the control group as compared with the normal control group. The hysterectomy did not significantly decrease the expression of these, but the basta tree was statistically significantly reduced. Furthermore, expression of IL-4, IL-13 and IFN-γ in skin tissues was markedly decreased in the hornblown and cynomolgus mixed extract treatment group as compared with the control group, but not decreased as in the normal control group (Fig. 8).

conclusion

DNCB treated mice were treated for 6 weeks with mixed extracts of horseradish, cinnabar, and horseradish. The results showed that the single extract of Quercus serrata inhibited the immune function and decreased the inflammation incidence. Significantly suppressed immune function and hyperactivity of inflammation as compared with unicellular or mulberry extracts, resulting in inhibition of atopic progression.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A pharmaceutical composition for preventing or treating atopic dermatitis comprising as an active ingredient, a mixture of Saussurea lappa Clarke and Biota orientalis (L.) Endl. Butylene glycol extract in a weight ratio of 1: 1.
delete delete delete 2. The composition of claim 1, wherein said composition inhibits the secretion of IL-6 (Interleukin-6) and TNF-a.
2. The composition of claim 1, wherein the composition inhibits expression of PAR-2 (Protease activated receptor-2) and ICAM1 (Intercellular Adhesion Molecule 1).
2. The composition of claim 1, wherein the composition inhibits the secretion of IgE.
A composition for preventing or ameliorating atopic dermatitis comprising as an active ingredient, a mixture of Saussurea lappa Clarke and Biota orientalis (L.) Endl. Butylene glycol extract in a weight ratio of 1: 1.
A cosmetic composition for prevention or improvement of atopic dermatitis comprising as an active ingredient, a mixture of Saussurea lappa Clarke and Biota orientalis (L.) Endl. Butylene glycol extract in a 1: 1 weight ratio.

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