KR102168765B1 - A composition for treating atopic dermatitis comprising a fermented product of balloon flower root by bioconversion - Google Patents

Abstract

The present invention provides a composition for preventing, ameliorating or treating atopic dermatitis, comprising a fermented product treated with a fibrinolytic enzyme after culturing bellflower roots in a liquid medium and inoculating Basidiomycota hyphae for culture fermentation as an active ingredient.

Description

A composition for treating atopic dermatitis comprising a fermented product of balloon flower root by bioconversion}

The present invention relates to a composition for the treatment of atopic dermatitis comprising a bioconverted bellflower ferment.

Atopic dermatitis is an intractable immune disease characterized by pruritus, dryness, and eczema erythematosus. Genetic factors and environmental influences are on the rise as the main pathogenic factors. According to a 2010 survey by the Korea Centers for Disease Control and Prevention, the prevalence of atopic dermatitis, which had itchy rashes in Korean children for the past 12 months, was 20.6% for elementary school students and 12.9% for middle school students, indicating that the incidence of atopic dermatitis tends to be higher with younger age. Compared to the results of the survey, 13.4% and 6.7%, the incidence of disease was significantly increased. Between 1990 and 2010, Africa increased from 9.9% to 20.9%, Europe from 18.9% to 19.6%, and Asia in Japan from 10.1% to 13.6%. It has a high prevalence rate in Western societies and migrant groups due to urbanization, and it is more frequent in Western Europe than Eastern Europe and in developed countries than in developing countries.

Steroids, immunosuppressants, antihistamines, skin moisturizers, skin emollients, etc. are all used to treat atopic dermatitis, but among the drugs used as pharmacological treatments, representative drugs used for mild patients are topical steroids, topical calcineurin inhibitors and antihistamines. Among them, antihistamines are used to relieve itching, but their efficacy is not so high that they are not very helpful. Accordingly, in the treatment of atopic dermatitis, T cell suppression immunomodulators such as steroids or calcineurin inhibitors are mainly used. Currently, topical steroids used in mild patients and most drugs used in patients with moderate symptoms have serious side effects, so the combination of drug therapy and phototherapy is trying to increase the therapeutic effect and reduce side effects.

As described above, as part of an effort to reduce side effects in the treatment of atopic dermatitis, the need to develop raw materials and products derived from natural products has emerged. Functional materials derived from natural products, which are known to be effective in improving atopic skin, mostly rely on mast cell degranulation inhibitory effects. Antigranulation inhibition in mast cells, which is expected to have the same effect as antihistamines, is expected to be partially effective in relieving symptoms of atopic dermatitis, but antihistamines are basically only used to relieve itching in atopic dermatitis, and this is also highly effective. Considering the fact that it is not very helpful, the effect of inhibiting degranulation in mast cells is not expected to be significant.

Until now, technologies such as pharmaceutical compositions for the treatment of atopic dermatitis using natural extracts as active ingredients (KR 2018-0138265 and KR 1898262) have been developed, but they are fermented with microorganisms (Basicillus mycelium) or bioconverted through additional enzyme treatment. Technology development for atopic dermatitis treatment or prevention agent using fermented bellflower as an active ingredient is insufficient.

Therefore, the present invention relates to a composition for the treatment of atopic dermatitis and its effect, including fermented with basidiomycete hyphae or fermented bellflower fermented by further enzyme treatment.

The present invention was conceived to solve the problems of the prior art as described above, in a composition or food composition for treating atopic dermatitis comprising fermented bellflower fermented by fermentation with basidiomycete hyphae or further enzymatic treatment About. However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned may be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the invention, various specific details, such as specific forms, compositions and processes, etc. are set forth. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the context of “in one embodiment” or “embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

Basidiomycete in the present invention is a fungus that becomes a cell called basidiomycete as a result of sexual reproduction as a subfamily of fungi to create spores. It cannot make nutrients by itself, so it is attached to other organisms and is parasitic, and many are known as mushrooms. Shiitake mushrooms, Pseudomonas mushrooms, Chaga mushrooms, Maitake mushrooms, Wood ear mushrooms, Snowflake mushrooms and skirt mushrooms are representative. Basidis are densely formed on the wrinkled side of mature mushrooms. Most of basidiomycete mushrooms live by-products in the fluids of higher plants, and play an indispensable role in the biological circulation of substances by decomposing cellulose and lignin in particular, but they also form mycorrhizal roots at the roots of trees to live in common. There are also many. There are other types that are considered important as edible bacteria or that need attention as poisonous mushrooms.

In the present invention, mycelium is a vegetative cell of a filamentous fungus (fungus). Functional differentiation, such as nutritional or physiological functions, can also be seen. Formally, the presence or absence of a partition wall is used for classification, and it develops in the germination tube of spores and elongates by apical growth. A group of branched hyphae is called mycelium. Depending on their physiological function, they are divided into basal hyphae that elongate as they enter the surface or inside of the medium or host and parasitic hyphae that elongate in the air. Parasitic hyphae depend on the transfer of nutrients from the basal hyphae, and some of them differentiate into spore-forming organs. If organic matter is present and environmental conditions such as suitable humidity and temperature are good, various mycelial structures are formed. The diaphragm of the mycelium of associa fungi is simple, but in basidiomyces, it can be seen that it forms a clamp connection. In fungi, it is branched into several branches and tangled like a thread. In mushrooms, it aggregates to form fruiting bodies. Because of the lack of chlorophyll, photosynthesis is not possible and it is attached to host organisms and absorbs nutrients. In the case of basidiomycete, which is a mushroom, a hyphae germinated from a spore has one nucleus, and when it fuses with another hyphae, one cell has two nuclei. A hypha with one nucleus is called a primary hyphae, and a hyphae with two nuclei is called a secondary hyphae. In the present specification, the basidiomycete hyphae can be specifically selected and used from hyphae such as medicinal and edible basidiomycete shiitake mushrooms, phylum mushrooms, chaga mushrooms, Maitake mushrooms, tree ear mushrooms, snowflake mushrooms, and skirt mushrooms, These basidiomycosis hyphae are the Korea Microbial Conservation Center (KCCM), the Korea Research Institute of Bioscience and Biotechnology Life Resource Center (KCTC), the Korea Cell Line Bank (KCLB), the Korea Agricultural Microbial Resource Center (KACC), and the American Standard Strain Culture Recorded Preservation Center (ATCC). The strain can be distributed and used by such institutions.

In the present invention, bellflower (Platycodon grandiflorum) is a perennial plant having a thick rootstock. The stem stands upright, grows to a height of 40~80cm, and rarely prunes branches. The leaves are alternately placed on each node, or 2-3 leaves may appear in one place. The leaves are shaped like an elongated egg or oval, and there is no such thing as a petiole. Both ends of the leaf are sharp and have sharp teeth at the edge, and the back of the leaf looks like white powder. Several large bell-like flowers bloom at the end of the stem. The end of the flower is divided into five and has five stamens. The diameter of the flower is about 4cm and the color is dark azure, but some flowers bloom in white. Rhizome is used as a medicinal material, and it is known that Platycodin and Platycodigenin, a kind of saponin contained in the rhizome, have an expectorant and antitussive action.

In the present specification, the bellflower or bellflower powder means composed of all or part of bellflower, and a part of bellflower may be one or more selected from the group consisting of bellflower root, root hair, stem, branch, leaf, bark, and bark, but limited to this no.

In the present invention, biotransformation refers to inducing a structural change of a substance added through a biological method and converting it into a chemically modified form to induce the generation of new components.

In the present invention, fermentation refers to a process in which microorganisms decompose organic substances using their own enzymes. Fermentation is a phenomenon in which organic substances are converted into simple compounds by enzyme action to release free energy, but in general, it refers to a phenomenon in which microorganisms accumulate metabolites through the decomposition process of organic substances. In other words, alcohol fermentation in which yeast decomposes sugar anaerobicly into ethyl alcohol and carbon dioxide, and lactic acid fermentation in which lactic acid bacteria anaerobicly decompose sugar into lactic acid, etc. are typical fermentations, but at present, acetic acid bacteria absorb oxygen in the air. A phenomenon in which alcohol is oxidized to acetic acid by use, and a phenomenon in which molds oxidize glucose to gluconic acid using oxygen in the air are also called acetic acid fermentation, gluconic acid fermentation, and the like. In addition, these are divided into anaerobic fermentation and aerobic fermentation (oxidative fermentation), and other anaerobic fermentations other than those mentioned above include glycerol fermentation, acetone butanol fermentation, 2, 3-butylene glycol fermentation, butyric acid fermentation, and propionic acid. Fermentation, methane fermentation, and the like, and aerobic fermentation include citric acid fermentation, itaconic acid fermentation, succinic acid fermentation, 2-ketoglutaric acid fermentation, oxalic acid fermentation, fumaric acid fermentation, and sorbose fermentation. In addition, the production of amino acids, vitamins and antibiotics by microorganisms is also called glutamic acid fermentation, riboflavin fermentation, penicillin fermentation, etc., but it is not a very good name. In principle, the name of the fermentation action is called the product, but sometimes the name of the substrate is given by cellulose fermentation or pectin fermentation.

In the present invention, atopic dermatitis refers to a refractory immune disease of skin diseases characterized by pruritus, dryness, and erythema eczema. Atopic dermatitis is known to have a combination of genetic and environmental factors, and skin barrier abnormalities and immunological abnormalities are increasing as major pathogens. The terms'atopic' and'allergy' began to be used in the early 20th century and are derived from Greek. 'atopy' is a term meaning out of place by prefixing'a', which is used in the opposite sense to the Greek word'topos', which means place. The word'allergy' is a combination of the Greek word'allos' meaning'different' and the word'ergon' meaning'to work'. When our body reacts abnormally to things that must be taken normally, that is, a word for'modified reaction', it means hypersensitivity. Currently, these two words are used interchangeably.

When foreign substances invade from the outside, our body removes foreign substances in order to protect itself from it and exhibits a reaction to protect the body, which is called an immune response. On the contrary, hypersensitivity reactions work in a harmful direction by destroying tissues by showing an immune response even to things that are not harmful to our body. Hypersensitivity reactions can be divided into four categories: type 1 hypersensitivity reaction, type 2 hypersensitivity reaction, type 3 hypersensitivity reaction, and type 4 hypersensitivity reaction, of which type 1 hypersensitivity reaction is commonly referred to as allergy. In type 1 hypersensitivity reactions, immunoglobulin-E (IgE) is produced against foreign substances (or'allergens') coming from the outside, and when this IgE is combined with the allergen, urticaria, cough, runny nose, sneezing, shortness of breath, etc. Symptoms appear. Type 1 hypersensitivity reactions are also referred to as immediate or IgE-mediated hypersensitivity reactions because hypersensitivity reactions are induced by IgE-mediated after exposure to allergens, and the period until hypersensitivity reactions occur is very short. On the other hand, type 4 hypersensitivity reactions do not immediately show symptoms after exposure to the allergen, but appear gradually, and are also called delayed or cell-mediated hypersensitivity reactions because there is no production of IgE. As it was recently revealed that the basic pathology of allergy is caused by inflammation, cases that appear irrespective of IgE are also included in allergy. After all, allergy can be defined as hypersensitivity reactions that form inflammation in target organs, whether or not related to IgE. That is, allergies include IgE-dependent allergies and IgE-independent allergies.

In the strict sense of the present invention, the term'atopic' refers to a tendency to abnormally generate IgE for foreign substances entering the body from the outside. Therefore, the term'atopic' is not medically identical to the term'allergy'. However, they are actually used interchangeably in the same meaning. In other words, when IgE-mediated hypersensitivity reaction is clinically expressed as a symptom, it is referred to as'atopic disease' and is called'allergic disease', and traditionally, atopic dermatitis, asthma, allergic rhinitis, and allergic conjunctivitis are classified as these diseases. have. Atopic diseases correspond to IgE-dependent (IgE mediated) allergic diseases, and IgE-independent allergic diseases do not correspond to atopic diseases. In addition, sometimes the term'atopic dermatitis' is too long to be replaced with the word'atopic' for short, but as mentioned above, the definitions of the terms'atopic dermatitis' and'atopy' are clearly different medically, so the exact term is used. I need it.

In addition, although atopic dermatitis, urticaria and allergic rhinitis are IgE-dependent (IgE-mediated) allergic diseases, there are distinct differences in the treatment of the same individual diseases.

In the case of atopic dermatitis, “2015 Korea Atopic Dermatitis Treatment Guidelines” published in 2015 suggests “antihistamines, local calcineurin inhibitors, and local steroids” for mild atopic dermatitis. In addition, for moderate and severe atopic dermatitis, systemic In treatment, "cyclosporine, short-term systemic steroids, other systemic immune modulators (AZA, MMF, MTX, 　IFN-γ, Alitretinoin), phototherapy, antigen-specific immune therapy, and biological agents" are suggested.

In the case of allergic rhinitis, according to the “Clinical Treatment Guidelines for Allergic Rhinitis” published in November 2005 by the Korean Society for Asthma and Allergy, “antihistamines, leukotriene receptor antagonists, steroids, blood vessel counts” Festival” is suggested, and steroids are accepted as immunosuppressants because their effects appear through suppression of almost all immune responses.

More specifically, antihistamines are used primarily in the treatment of allergic rhinitis. Oral antihistamines are effective for runny nose, sneezing, nasal itching, and eye symptoms through antagonism of the H1 receptor of target cells, but when there is a nasal congestion symptom, more effective nasal antihistamines are used. However, in patients who do not achieve sufficient effects with antihistamines, the administration of intranasal steroids, leukotriene receptor antagonists, and oral vasoconstrictors is controlled. Nasal steroids are the most effective treatment for nasal congestion.

Leukotriene receptor antagonists block the binding of leukotriene, an important inflammatory mediator of allergic rhinitis, to the receptor, helping to improve nasal and ocular symptoms in patients with allergic rhinitis, and are more effective in improving symptoms when administered with intranasal steroids. to be. By itself, it is similar in effect to antihistamines, and is less effective than intranasal steroids. Leukotriene receptor antagonists are also used in the treatment of asthma, so they are particularly useful in allergic rhinitis patients with asthma.

In the treatment of atopic dermatitis, antihistamines are basically only used to relieve itching of the skin, and this is also not very helpful because the efficacy is not high. This is because in atopic dermatitis, there are too many mediators that cause itching in addition to histamine, so antihistamines alone cannot effectively control itching. As IL-31 was recently reported as the main factor closely related to itching among several mediators that cause itching, a monoclonal antibody of IL-31 Receptor A was developed as a medicine and started to be used for the treatment of itching in atopic dermatitis.

On the other hand, in the treatment of urticaria, which is the same IgE-dependent allergic disease, clinical symptoms including itching are caused by a medium called histamine secreted from mast cells, so that major symptoms of urticaria, including itching, are effectively suppressed by antihistamines.

Although atopic dermatitis, urticaria and allergic rhinitis are the same IgE-dependent allergic diseases, the lesions are different from each other to the skin and respiratory system, and the treatments used are different, and even the same treatments have different purposes and effects, which are suitable for each disease. And treatment methods are being adopted.

In the present invention, atopic dermatitis is known as a representative refractory disease that occurs mainly in infancy, and generally refers to atopic eczema accompanied by itching, dry skin, and inflammation. Immunological mechanisms of atopic dermatitis include the enhancement of the Th2 immune response, the imbalance of the Th1 and Th2 immune responses among Th cells, the cytokine dysfunction, and the increase in the activity of Langelhans cells. Recently, it was reported that Th17 immune response also adversely affects atopic dermatitis by Nograles et al.

The common mechanism of IgE-dependent allergic reaction is ① Allergens (external antigens) that cause allergies enter our body, and antigen-specific Th2 cells are activated, ② B cells are activated by cytokines secreted by activated Th2 cells. When IgE antibody-producing B cells are converted to IgE antibody production and secreted, ③ the secreted IgE antibody binds to mast cells and sensitization of mast cells is induced. ④ At this time, the allergen from the outside is obese. Mast cells are activated while binding to IgE bound to the cell surface, secreting various chemical allergy mediators such as histamine, leading to allergic symptoms.

Of these, if the process from atopic dermatitis to the secretion of Th2 cytokines is described in more detail, it can be divided into 7 steps. TSLP is produced from keratinocytes, fibroblasts, mast cells, and dendritic cells of the skin caused by allergens or viruses (step 1), and TSLP activates immature dendritic cells (step 2), and immature dendritic cells activated by TSLP Cells secrete the chemokines IL-8 and eotaxin-2, which not only attract eosinophils and neutrophils, but also secrete thymus and activation-regulated chemokine (TAC) and macrophage-derived chemokine (MDC) to transport Th2 cells to the lesion site. Draw in (step 3). Myeloid dysphagia cells (mDCs) activated by TSLP mature and migrate to the lymph nodes (step 4). Mature dendritic cells activated by TSLP express OX40L on the surface, which promotes antigen-specific naive CD4+ T cells to become inflammatory Th2 cells in lymph nodes. Th2 cells produce IL-4, IL-5, IL-13 and TNF, but not IL-10 (step 5). Since TARC and MDC are generated at the inflammatory site of the lesion, the inflammatory Th2 cells move back to the site of inflammation, which is the lesion site (step 6). In the inflammatory site, Th2 cytokines IL-4, IL-5, IL-13 and TNF are produced by inflammatory Th2 cells, and these cytokines promote IgE production, eosinophil increase and mucus production (step 7) .

When atopic dermatitis patient scratches the skin due to itching or is exposed to allergens, TSLP is secreted from the keratinocytes of the skin, and this secreted TSLP affects Langerhans cells and as a result secretes Th2 cytokines from activated inflammatory Th2 cells. It is known to make.

From an immunological point of view, atopic dermatitis is a disease that occurs when the balance of specific immune responses called Th1/Th2 is broken along with the enhancement of the Th2 immune response. At this time, it is TSLP (Thymic stromal lymphopoietin) that plays an important role. TSLP is secreted from skin keratinocytes, etc. in the stage of atopic dermatitis development, causing Th2 immune response by differentiating naive Th0 cells into Th2 cells. When TSLP is secreted excessively, the Th2 immune response increases and the Th1/Th2 balance collapses, leading to atopic dermatitis progression or worsening. From an immunological point of view, treatment of atopic dermatitis needs to restore the balance of Th1/Th2 immune responses by suppressing the production of TSLP and reducing the Th2 immune response.

TSLP is mainly made from keratinocytes of the skin and epithelial cells of the airways and nasal mucosa, and stimulates immature myeloid dendritic cells (mDC), which is a heterodimeric TSLP receptor (receptor) in order for the immature dendritic cells to become mature dendritic cells. Express. TSLP-activated dendritic cells differentiate naive CD4+ T cells into Th2 phenotype, and promote the proliferation of Th2 memory cells by expressing costimulatory molecule OX40 ligand together with Th2 chemotactic cytokines TARC (CCL17) and MDC (CCL22). In addition, TSLP inhibits the IL-12 p40 receptor expressed in dendritic cells, suppresses the Th1 immune response, and further promotes the Th2 immune response. TSLP activates the transcription of the IL-4 gene in Th2 cells, promotes the production of IL-13 in mast cells, and increases the response of basophils to infiltrate eosinophils, thereby exacerbating the allergic inflammatory response. Therefore, TSLP plays a key role in the initiation of atopic dermatitis. TSLP is expressed in epithelial cells of atopic dermatitis patients. In addition, TSLP overexpression in skin keratinocytes in mice amplifies inflammatory responses to sensitized inhaled antigens.

Atopic dermatitis is the most characteristic symptom of severe itching. Relieving it is an effective way to improve the quality of life and prevent the worsening of skin lesions. Briefly summarizing the mechanism by which the itching of atopic dermatitis is amplified, when external irritation penetrates the damaged skin barrier and reaches the skin, an allergic inflammatory reaction is triggered, and the resulting itching is'itching → scratching → inflammation → (again) → itching. → scratching → inflammation →… … It is amplified beyond control through the vicious cycle of'.

The mechanism from skin inflammation to itching in atopic dermatitis has not yet been fully elucidated, but interleukin-31 (IL-31) is considered to be the main factor closely related to itching among several mediators that are produced by inflammatory reactions and cause itching. . IL-31 is mainly produced by Th2 cells, and sends a signal through binding to the IL-31 receptor expressed in the sensory nerve and skin keratinocytes. At this time, IL-31 is the sensory nerve. It binds to IL-31 receptor to induce itching, and increases expression of TARC(CCL17), MDC(CCL22), S100A7, β-defensin2 by binding to IL-31 receptor of keratinocytes, and expression of filaggrin, corneodesmosin By suppressing the skin lesions appearing in atopic dermatitis worsen.

It is known that S. aureus bacteria are proliferating on the skin of many atopic dermatitis patients (about 90%) including severe atopic dermatitis patients. Recently, it is known that Th2 cytokines create favorable conditions for proliferation of S. aureus, whereas S. aureus is known to promote the secretion of Th2 cytokines.

Th2 cytokine is known to promote the death of keratinocytes by S. aureus alphatoxin, and deltatoxin is known to promote IgE production and increase the effect of Th2 cytokine. In addition, Staphylococcal enterotoxin B, known as superantigen, is known to increase degranulation in mast cells and promote the secretion of IgE antibodies and aggravate lesions by promoting the secretion of Th2 cytokines. In addition, it is known that Peptidoglycan of S. aureus can induce Th2 immune response in mice. It is known that Peptidoglycan is a ligand of TLR2. It has also been reported that TSLP production in keratinocytes is induced through the pathways of TLR2 and TLR6 by the membrane of S. aureus and diacylated lipopeptide.

In conclusion, Th2 cytokine helps the proliferation of S. aureus, whereas S. aureus can induce TSLP and Th2 cytokine, so S. aureus may exacerbate atopic dermatitis.

To overcome this, the clinical guidelines recommend the use of antibiotics in the treatment of secondary infections. In particular, the materials presented in this patent are TLR4 agonist, and Th1 adjuvant of LPS competitive inhibitor. As it can be used as a Class II antibiotic adjuvant, it is expected that additional effects on atopic dermatitis can be expected.

The guidelines for treatment according to the Korean Atopic Dermatitis Treatment Guidelines (2015) are as follows.

Atopic dermatitis is treated with mild patients and moderate/severe patients. This is because in the case of mild patients, even if the symptoms worsen, it does not progress to more than moderate symptoms, but in the case of moderate patients, it progresses to severe.

Regardless of symptoms, atopic dermatitis patients should use moisturizers as basic treatment, avoid exacerbation factors, and receive patient education. For skin lesions of patients with mild atopic dermatitis, topical anti-inflammatory treatments such as topical steroids and local calcineurin inhibitors are used as active treatments to eliminate the lesion, and antihistamines are used when itching is complained of. For reference, unlike general inflammation, general nonsteroidal anti-inflammatory drugs are not used as a basic treatment.

For patients with moderate or higher severity, systemic treatments must be used as active treatment, including treatments for mild patients. In addition, in the case of moderate to severe atopic dermatitis, in systemic treatment, "cyclosporine, short-term systemic steroids, other systemic immunomodulators (AXA, MMF, MTX, IFN-γ, Alitretinoin), phototherapy, antigen-specific immune therapy, biological agents Use ". In addition, when moderate or severe dermatitis worsens, poultice therapy and antibiotics may be used.

After symptoms disappear, topical calcineurin inhibitors or topical steroids are applied 2-3 times a week for maintenance, and proactive treatment is recommended to prevent recurrence. Other than that, although the effect has not yet been clearly demonstrated, evening primrose oil, probiotics and prebiotics, and vitamin D can be used as an adjunct treatment.

Most of the symptoms of atopic dermatitis appear on the skin, so it can be misunderstood as a simple skin disease.However, atopic dermatitis is caused by a continuous recurrence of underlying inflammation due to not only abnormal skin barrier but also abnormally activated immune system function. Although they can be classified, since they are different from general inflammation, nonsteroidal anti-inflammatory drugs commonly used in inflammatory diseases are rarely used as therapeutic agents.

Therefore, in the treatment of atopic dermatitis disease, treatment of Th2 immune hypersensitivity rather than inflammation shows superior long-term effects. Therefore, the method for treating atopic dermatitis, strictly speaking, can be said to change the direction of the immune response such as suppressing the Th2 immune hypersensitivity reaction rather than suppressing inflammation.

In the present invention, atopic dermatitis is one of the skin diseases having the main symptom of itching, and itching, skin dryness, skin lesions, lichenification, atopic eczema, erythema and Th2 immune hypersensitivity inflammation, etc. are mainly initiated in infancy or childhood. Accompany. Symptoms derived from atopic dermatitis are characterized by chronic recurrence and are accompanied by persistent mental and physical pain to the patient. As treatments for atopic dermatitis, steroids, calcineurin inhibitors, antihistamines, immunomodulators, interferon-gamma, and the like are currently being used. Of these, the steroids are most widely used as treatments for atopic dermatitis due to their rapid improvement. If you reduce or quit, the symptoms rapidly worsen, and there is a disadvantage in that there is a risk of various serious side effects including Cushing's syndrome. Due to the shortcomings of such steroids, calcineurin inhibitors, immunomodulators, interferon-gamma, etc. are used in place of steroids, but the immunomodulators are at risk of side effects such as burning skin, high blood pressure, and renal dysfunction. In addition, the antihistamine is accompanied by symptoms such as drowsiness and dry mouth, and when the interferon-gamma is injected, the therapeutic effect appears several weeks later, and an immediate symptom relief effect cannot be expected. Atopic dermatitis symptom relief using natural substances extracted from nature (especially plants) is capable of effectively preventing, alleviating or treating atopic dermatitis symptoms without side effects even if used for a long period of time by supplementing the shortcomings of these conventional chemical compositions. Sexual skin disease treatments are in the spotlight.

In addition, in the case of atopic dermatitis, in addition to steroidal anti-inflammatory drugs, immunosuppressants, and antihistamines, secondary infection treatments are used as needed, but immune hypersensitivity itself is not improved and side effects are serious, making it difficult to use repeatedly for a long time. Therefore, in the reality that there is no suitable oral systemic treatment that can suppress the immune hypersensitivity reaction to Th2 that occurs in the body in mild treatment and maintenance treatment, which are mainly used for external skin, the treatment effect is increased and side effects are reduced by combining drug treatment and phototherapy. It is trying to reduce it, and also eating health functional foods such as probiotics (lactic acid bacteria), which are known to be helpful in improving atopic dermatitis, are being made.

Taken together, the difference between almost all products that have a mechanism to suppress the immune response up to now is that steroids suppress immunity most widely, whereas recent products are characterized by suppressing only a narrow range of immunity. However, it can be said that there is no product that solves the Th1/Th2 imbalance along with the enhanced Th2 immune response enough to change the disease progression.

The present invention contains a Th1 adjuvant capable of resolving the Th1/Th2 imbalance along with such an enhanced Th2 immune response, and fermented with basidiomycete hyphae or further comprising fermented bellflower bioconverted through enzymatic treatment. It relates to a composition for the treatment of atopic dermatitis and its effect.

In the present invention, IgE is an antibody protein also called immunoglobulin E. It is often referred to as "allergic antibodies" because it plays an important role in allergic reactions. When you are allergic to a specific substance (allergen), the immune system erroneously judges that generally harmless substances are harmful to the body. When exposed to these specific substances, the immune system begins to produce IgE to protect our body. The IgE antibody remains in the body and causes an allergic reaction the next time it comes into contact with the allergen. As a result, in people with allergies, blood IgE levels increase, and IgE exhibits specificity for each allergen.

In the present invention, the pharmaceutical composition refers to a composition administered for a specific purpose. For the purposes of the present invention, the pharmaceutical composition of the present invention is used for the inhibition of atopic dermatitis active substance, and may include a composition therefor and a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutical composition according to the present invention contains 0.1 to 100% by weight of an active ingredient corresponding to the pharmaceutical composition of the present invention based on the total weight of the composition. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

In the present invention, the food composition is variously used as a food composition for improving atopic dermatitis disease, and the food composition comprising the composition of the present invention as an active ingredient includes various foods such as beverages, gums, teas, vitamins It can be prepared in the form of a combination drug, powder, granule, tablet, capsule, confectionery, rice cake, bread, and the like. Since the food composition of the present invention is composed of bellflower, a natural food with little toxicity and side effects, and a bioconverted fermented product thereof, it can be safely used even when taken for a long time for prophylactic purposes. When the composition of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 100% of the total weight. Here, when the food composition is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and as an additional component, various flavoring agents or natural carbohydrates, etc. may be included as in ordinary beverages. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and conventional sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition is various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, It may contain stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. These ingredients may be used independently or in combination The proportion of these additives is not so important, but 100% by weight of the composition of the present invention It is generally selected from 0.1 to 100 parts by weight per part.

In the present invention, the cosmetic composition is a composition for improving atopic skin condition or atopic skin disease. Cosmetic composition comprising the composition of the present invention as an active ingredient is a lotion, nutritional lotion, nutritional essence, massage cream, beauty bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sun Screen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory {cosmetic}, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, Cosmetic cream, jasmine oil, bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap {non-medical use}, cream soap, facial wash, hair rinse, makeup soap, tooth whitening gel, toothpaste, etc. It can be manufactured in the form of. To this end, the composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in the manufacture of cosmetic compositions. As carriers, excipients or diluents that can be further added to the cosmetic composition of the present invention, purified water, oil, wax, fatty acid, fatty alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, kill Rating agents, buffers, lower alcohols, and the like are included, but are not limited thereto. In addition, whitening agents, moisturizing agents, vitamins, sunscreen agents, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed. Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm seed oil, jojoba oil, and avocado oil may be used as the oil. Can be used. Stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as fatty acids, and cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as fatty alcohols. And as fatty acid esters, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As surfactants, cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible. In addition, hygroscopic agents, thickeners, antioxidants, and the like, which are widely known in the cosmetic field, may be included, and the types and amounts thereof are as known in the art.

In the present invention, the amylase-based hydrolase is a family of enzymes that preferentially hydrolyze α-glycosidic bonds of polysaccharides to generate low molecular weight carbohydrates/sugar fragments, including α-amylase, β-amylase, and γ-amylase. Can be lifted. In the present invention, the cellulase-based hydrolase may include cellulase, hemicellulase, pectinase, or glucanase capable of decomposing bark, root skin, roots, leaves, and the like. As commercially available cellulase preparations, for example, Cellulase T "Amano", Cellulase A "Amano" (manufactured by Amano Enzyme Inc.), Tricerase KSM, Multi-Fact A40, Cellulase GC220 (Genene or higher) Koa Kyowa Co., Ltd.], Cellulase GODO-TCL, Cellulase GODO TCD-H, Vesselex, Cellulase GODO-ACD [manufactured by GODO SHUSEI CO., LTD.]), Cellulase [manufactured by GODO SHUSEI CO., LTD.] Toyobo Co., Ltd.], Cellizer, Cellulase XL-522 (manufactured by Nagase ChemteX Corporation), Cellsoft, Dennimax (manufactured by Novozymes) (Manufactured by Novozymes A/S)], Cellulose AC40, Cellulose AL, Cellulose T2 [manufactured by HBI enzymes Inc.], Cellulase "Onozuka" 3S, Cellulase Y-NC [Yakult Yakult Pharmaceutical Industry Co., Ltd. production], SumiTim AC, SumiTim C [Shinnihon Chemical Co., Ltd. (SHINNIHON CHEMICALS Corporation) production], Entiron CM , Entiron MCH, and bioheat (above made by Rakuto Kasei Industrial CO., LTD.), etc. are mentioned.

According to one embodiment of the present invention, the present invention comprises as an active ingredient a fermented product treated with fibrinolytic enzyme after cultured and fermenting bellflower in a liquid form and inoculating basidiomycete hyphae as an active ingredient, preventing atopic dermatitis, It provides a composition for improvement or treatment.

In the present invention, the culture medium may be made into a bellflower medium by powdering bellflower, treating the bellflower powder with a hydrolase in a liquid state, and sterilizing it.

In the present invention, the hydrolase may be at least one of an amylase-based hydrolase and a cellulase-based hydrolase.

In the present invention, the fibrinolytic enzyme may be a cellulase alone or a combination of one or more selected from the group consisting of cellulase, hemicellulase, pectinase, or glucanase.

In the present invention, the basidiomycete hyphae may be made of a basidiomycete selected from the group consisting of shiitake mushrooms, phylum mushrooms, chaga mushrooms, maitake mushrooms, wood ear mushrooms, snowflake mushrooms, and skirt mushrooms.

The composition of the present invention decreases the expression level of IL-4, IL-5, IL-13, TSLP (thymic stromal lymphopoietin), or IL-31 at the site of atopic dermatitis, or It is possible to increase the expression level of IL-2, IL-12, or IFN-γ in the serum, decrease the amount of IgE in serum, or increase the amount of galectin-9. In addition, the present inventors confirmed that the activity of the bioconverted bellflower fermentation according to the present invention is completely inhibited as a result of treatment with TAK242, a specific inhibitor of TLR4, and the bioconverted bellflower fermentation according to the present invention uses TLR4. It was confirmed to induce activation of macrophages through. It could be evaluated as a substance having a mechanism different from that of general anti-inflammatory agents that inhibit the production of PGE ₂ and NO in macrophages.

The composition provided by the present invention may be used in the form of a pharmaceutical composition, a cosmetic composition, or a food composition, but is not limited thereto.

According to another embodiment of the present invention, (a) the step of culturing bellflower in a liquid phase; (b) performing a biotransformation fermentation process of culture-fermenting by inoculating basidiomycete hyphae in the liquid bellflower medium cultured in step (a); And (c) a composition for preventing, improving or treating atopic dermatitis comprising the step of performing a bioconversion enzyme treatment process of treating a fibrinase from the fermented product produced by the biotransformation fermentation process of step (b). It provides a method of manufacturing.

In the present invention, the culture medium of the bellflower in the step (a) may be made into bellflower medium after the bellflower is pulverized and the bellflower powder is treated with a hydrolase in a liquid state. Here, after the hydrolytic enzyme is treated, sterilization or sterilization may be performed as necessary, but is not limited thereto.

In the present invention, the hydrolase of step (a) may be at least one of an amylase-based hydrolase and a cellulase-based hydrolase.

In the present invention, the basidiomycete hyphae of the step (b) may be made of basidiomycete selected from the group consisting of shiitake mushrooms, prickly pear mushrooms, chaga mushrooms, maitake mushrooms, wood ear mushrooms, snowflake mushrooms, and skirt mushrooms. In the present invention, the terminal fungus hyphae is 2% (v/v) to 18% (v/v), preferably 5% (v/v) to 15% (v/v), more preferably 7% ( v/v) to 13% (v/v), but is not limited thereto.

In addition, in the present invention, the temperature during the culture fermentation of step (b) is performed under conditions of 28 to 30° C. and pH 4.5 to 7, so that the concentrated liquid bellflower medium at the point when the concentration of the residual carbon source is depleted below a certain concentration is It is preferable to culture for 7 to 10 days in a fed-batch process to be added, but is not limited thereto.

In the present invention, the fibrinolytic enzyme of step (c) may be a cellulase alone, or a combination of one or more selected from the group consisting of cellulase, hemicellulase, pectinase, and glucanase, and a specific example is a fibrinolytic enzyme. Is commercially available enzymes, that is, fibrinolytic enzymes, Viscozyme (a complex derived from Aspergillus), cellulase (from Aspergillus niger), a piltrase (a complex derived from Tricoderma reesei), and a rapidase (Rapidase: Aspergillus niger) Derived complex) and Sumizyme (Sumizyme: Aspergillus niger-derived pectinase-containing complex) may be used one or more selected from the group consisting of, but is not limited thereto. In the present invention, the fibrinolytic enzyme may be added in an amount of 0.01% (v/v) to 1% (v/v), preferably 0.01% (v/v) to 0.1% (v/v), It is not limited thereto.

In the present invention, the bioconversion enzyme treatment process of step (c) may be performed while stirring for 1 to 3 hours under a temperature condition of 50 to 60°C, but is not limited thereto.

In the present invention, the bellflower biotransformation product of step (d) is IL-4, IL-5, IL-13, TSLP (thymic stromal lymphopoietin), or IL-31 at the lesion site of atopic dermatitis. Decrease the expression level of, increase the expression level of IL-2, IL-12, or IFN-γ in the spleen, decrease the amount of IgE in the serum, or the amount of galectin-9 Can increase

The bioconverted bellflower fermented product provided by the present invention is a material for improving, preventing, or treating atopic dermatitis as compared to the bellflower extract (bellflower raw material) that is not fermented with microorganisms (basidiomycete hyphae) or additionally fermented through enzyme treatment. Since it contains a lot, it is expected to have the effect of reducing the occurrence of atopic dermatitis when it is developed into food using this.

1 is a schematic diagram showing a process of obtaining a culture medium of bellflower, a bioconversion process, and a fermented powder according to an embodiment of the present invention.
2 is a result of treatment with Tak242, a selective inhibitor of TLR4 according to an embodiment of the present invention, that the activity of the bioconverted bellflower fermentation is completely inhibited, and that the immunologically active active ingredient in the bellflower fermentation is a polysaccharide fraction. It is a graph.
3 is a graph confirming that neutrophil infiltration by LPS is inhibited when simultaneously treated with LPS according to an embodiment of the present invention. Figure 3 (a) evaluated the ability to induce intraperitoneal neutrophil infiltration according to intraperitoneal administration of LPS. Figure 3 (b) evaluated the neutrophil infiltration induction ability and anti-LPS efficacy of the bioconverted bellflower fermented polysaccharide fraction.
4 is a graph showing the results of measuring the expression level of IL-4, IL-5, or IL-13 in lesion tissue in an animal model of atopic dermatitis according to an embodiment of the present invention.
5 is a graph showing the results of measuring the expression level of IL-2, IL-12, or IFN-γ in the spleen in an animal model of atopic dermatitis according to an embodiment of the present invention.
6 is a graph showing the results of measuring the IgE concentration in the blood, the ear thickness as the lesion site, and the concentration of TSLP and IL-31 in the ear tissue in an atopic dermatitis animal model according to an embodiment of the present invention.
7 is a graph showing the result of measuring the expression level of galectin-9 in serum in an animal model of atopic dermatitis according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Bioconversion process of fermentation and enzyme treatment

Example 1-1. Bellflower pretreatment work

For the pretreatment of the purchased bellflower from the raw material to powdering, the degree of contamination of the mold in the raw material was measured, and then washing was performed to remove foreign matter and contaminants from the bellflower raw material. Washing was carried out through a total of three steps: ① air washing to remove foreign matter and mold spores, ② water washing to remove residual pesticides, and ③ alcohol (Et-OH) to remove contamination of microorganisms. The raw material was washed with water through the washing step. After washing and cutting, the raw material was dried in a hot air dryer capable of mass drying, and then coarse and finely pulverized, and then the bellflower raw material was powdered.

Example 1-2. Obtaining fermented powder of bellflower

The bellflower powder from which foreign matter and mold contamination was removed was subjected to enzymatic treatment and heat treatment sterilization after adding water for liquid culture medium. As the enzyme, amylase-based hydrolase was used, and after the enzyme was added, the reaction was performed at 60° C. for 1 hour, and sterilized at high temperature for 30 minutes to perform culture medium of bellflower.

In this case, commercially available enzyme products can be used. In this case, it was carried out in an appropriate amount (optimum amount suggested in the product manual for each enzyme product) and appropriate conditions (appropriate temperature and pH suggested in the product manual for each enzyme product).

Thereafter, shiitake mushroom hyphae among the basidiomycete hyphae separately cultured in the culture (fermentation) medium were added. Basidiomycete mycelium seed culture solution was inoculated with 10% (v/v) and cultured under conditions of 28~30℃ and pH 4.5~7. In addition, it can be cultured for 7 to 10 days in a fed-batch culture process in which concentrated liquid bellflower medium is added when the concentration of the residual carbon source is depleted below a certain concentration. The basidiomycete hyphae described above can be specifically selected and used from mycelium such as medicinal and edible basidiomycete mushrooms, such as shiitake mushrooms, phylum mushrooms, chaga mushrooms, maitake mushrooms, wood ear mushrooms, snowflake mushrooms, and skirt mushrooms. Is preferable, and the effect was confirmed to be the same. In addition, these basidiomycete hyphae are known as the Korea Microbial Conservation Center (KCCM), the Korea Research Institute of Bioscience and Biotechnology Life Resources Center (KCTC), the Korea Cell Line Bank (KCLB), the Korea Agricultural Microbial Resource Center (KACC), and ATCC) and other institutions can obtain and use the strain.

Cellulase among fibrinolytic enzymes was used for enzymatic treatment in the bioconversion process for the fermented product. At this time, cellulase may be used alone, or commercially available enzyme products including various enzymes such as cellulase, hemicellulase, pectinase, and glucanase may be used. I can. In this case, an appropriate amount (optimal amount suggested in the product instructions for each enzyme agent) was added and carried out. As enzymes, commercially available enzymes, namely, fibrinolytic enzymes, Laminex (Laminex: a complex derived from Tricoderma reesei), Viscozyme (a complex derived from Aspergillus), Cellulase (from Aspergillus niger), Piltrase (Filtrase: Tricoderma reesei-derived complex), Rapidase (Rapidase: Aspergillus niger-derived complex) and Sumizyme (Sumizyme: Aspergillus niger-derived pectinase-containing fibrinolytic enzymes) can be used by selecting any one or multiple from fibrinolytic enzymes. Nex is preferred, and its effect was confirmed to be the same. The enzyme/substrate reaction was performed by adding the recommended concentration (0.05%) suggested in the product manual of Laminex enzyme preparation, and rotating at 250 rpm for 1 to 3 hours at 50 to 60°C.

The bellflower fermented product produced by the bioconversion process was lyophilized and powdered after enzymatic inactivation and sterilization at 90°C for 1 hour. In addition, a solution of fermented bellflower fermented product having the above immune activity is dissolved in 20 times of water, or a fermented bellflower fermented product is centrifuged at 10,000 rpm for 30 minutes to remove insoluble residues, and the supernatant is freeze-dried to obtain a water-soluble powder. Alternatively, 5 times the amount of ethanol was added to the supernatant to induce precipitation at 4° C. for more than 24 hours, and the precipitate was lyophilized to obtain a powder of a polymeric polysaccharide fraction having immunological activity. The culture medium of the bellflower, the bioconversion process, and the fermentation powder acquisition process are described in FIG. 1.

Example 2. In vitro efficacy evaluation of bioconverted bellflower fermentation

Cell culture.

The RAW264.7 cell line used in the experiment was purchased from ATCC (Manassas, VA). DMEM medium was purchased and used from Welgene (Gyeongsan, Korea), and both fetal bovine serum (FBS) and antibiotics added to the medium were purchased and used from Thermo Fisher Scientific (Waltham, MA), and contained 5% CO ₂ It was incubated in the air condition at 37°C saturated humidity.

Cytokine measurement in RAW264.7 cell line.

RAW264.7 cell line After inoculating a 96-well plate at 2×10 ⁵ cells/well, it was incubated overnight, and each sample was diluted according to the concentration and treated. After 24 hours, 100 µl of the supernatant was collected, the same amount of Griess solution was added, and left for 15 minutes, and the absorbance of 570 nm was measured.

result. Proof of active ingredient in bioconverted bellflower fermentation

In order to confirm the innate immune activity efficacy of fermented bellflower powder, the ability to secrete cytokines and secretion patterns in RAW264.7 cells, a macrophage cell line, were investigated, and the site of action (specific receptor) was inferred and confirmed through the secretion pattern. In order to do so, the site of action was confirmed by treating an inhibitor for the inferred specific receptor to check whether signal transduction was blocked.

As a result of examining the cytokine secretion pattern, TNF-α, IL-6, IL-10 and IFN-β were secreted in the same manner as LPS, and IL-1β, IL-4, IL-5, IL-12p70 and IFN-α Was not secreted. Since LPS is well known as a representative ligand recognized by TLR4, it could be inferred that fermented bellflower powder would be recognized by TLR4 and mediate signals.

As the treatment of fermented bellflower powder effectively induces the production of four cytokines in macrophage cell lines, it was attempted to confirm the active ingredient in fermented bellflower powder (see FIG. 2). The polysaccharide fraction in the fermented bellflower powder was isolated and purified and treated on a macrophage cell line to measure the expression levels of four cytokines, and treated at a concentration of 1/20 of the fermented bellflower powder in consideration of the recovery rate of the polysaccharide fraction. As a result, even when only the polysaccharide fraction was treated, cytokines similar to those treated with the bellflower fermented powder were produced, and the active ingredient of the bellflower fermented powder was confirmed to be a polysaccharide.

In addition, as a result of measuring the amount of cytokines by treatment with TAK242, a specific inhibitor of TLR4, it was confirmed that the production of all four cytokines induced by treatment of fermented bellflower powder and polysaccharide fraction during TAK242 treatment was completely inhibited. Therefore, the fermented bellflower powder and the polysaccharide fraction of the fermented bellflower powder were identified as TLR4 agonists that induce activation of macrophages by binding to TLR4.

As a result of investigating the cytokine secretion pattern related to immune activity in macrophages of fermented bellflower powder, it was confirmed that it induces very high expression of TNF-α, IL-6 and IFN-β, of which IFN-β is type I. Interferon has antibacterial and antiviral effects, and is a representative cytokine that induces Th1 immune response by contributing to the differentiation of Th1 cells. It is a cytokine with immunomodulatory function that suppresses Th2 and Th17 immune responses while inducing Th1 immune responses. .

Therefore, fermented bellflower powder is a Th1 adjuvant of TLR4 agonist that can specifically activate Th1 immune response, and it is a substance that has a mechanism different from general anti-inflammatory drugs that inhibit PGE ₂ and NO production in macrophages. Could be evaluated.

Example 3. In vivo efficacy evaluation of bioconverted bellflower fermentation in atopic dermatitis model

Experimental animals .

As for the mice used in the experiment, 6-week-old female BALB/c mice were purchased from Orient Bio (Seongnam, Korea), and they were reared by supplying sufficient water and feed while maintaining the room temperature at 22±2°C until use in the experiment. .

Intraperitoneal neutrophil invasion induction and anti-LPS efficacy evaluation Animal experiments.

To measure the LPS-induced neutrophil induction ability, mice were randomly separated into 10 mice per group, and LPS (SIGMA, St. Louis, MO) and the developed material were injected into the abdominal cavity after an adaptive diet for one week. After 6 hours, the mice were sacrificed, the abdominal cavity was washed with PBS to recover the cells infiltrated into the abdominal cavity, and the number of neutrophils was counted through Kimja staining.

Animal testing of anti-atopic dermatitis efficacy evaluation.

To induce atopic dermatitis, mice were randomly separated into 10 mice per group, and 1% DNCB (dinitrochlorobenzene) was applied to one ear after an adaptive diet for 1 week to sensitize. After 3 days, 10 mg/ml of mite extract was applied to the same ear and stimulated, and from 1 week after sensitization, the developed material was administered in a diet according to the daily intake. Afterwards, DNCB sensitization and mite extract stimulation were repeated once a week for 4 weeks to induce atopic dermatitis for a total of 5 weeks, and then the mice were sacrificed. After measuring the ear thickness, the ear tissue and spleen were removed and blood was collected from the heart.

ELISA.

Blood collected from the heart was treated at 4° C. for 30 minutes to induce blood coagulation, and then centrifuged at 2,000 g for 30 minutes to separate the serum. The amount of IgE in the isolated serum was measured using a mouse IgE ELISA kit (Abcam, Cambridge, Cambridgeshire, England), and the amount of IL-1β, IL-4, IL-6, and IFN-γ was determined by each ELISA kit (R&D systems, minneapolis, MN). The ear tissue was placed in TNT lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0), crushed using a homogenizer, and then centrifuged at 13,000 rpm for 30 minutes to recover the supernatant. The amount of TSLP (Thymic Stromal Lymphopoietin) in the supernatant was measured using a mouse TSLP quantikine ELISA kit (R&D systems, minneapolis, MN), and the amount of IL-31 was an IL-31 mouse ELISA kit (Thermo Fisher Scientific, Waltham, MA). It was measured using. The amounts of IL-4, IL-5, IL-13, and IFN-γ were measured using respective ELISA kits (R&D systems, minneapolis, MN).

Real-time PCR.

Total RNA in the extracted spleen was extracted using a tissue RNA preparation kit (QIAGEN, Hilden, Germany). The extracted RNA was synthesized as cDNA using reverse transcriptase (TAKARA, Kusatsu, Shiha, Japan), and amplified by real-time PCR (Bio-Rad, Hercules, CA) using each primer to confirm the expression level of mRNA.

Tissue staining.

The extracted ear tissue was fixed with 3.7% formaldehyde and then dehydrated and embedded in paraffin. The paraffin block containing the tissue was sliced to a thickness of 4 μm and attached to the slide glass. Then, through a hydration process, stained with hematoxylin and eosin Y, and observed under a microscope.

Results 1. Demonstration of bioconverted bellflower fermentation as a competitive inhibitor for LPS

As a result of confirming the effect of intraperitoneal administration of LPS inducing neutrophil infiltration, it was confirmed that when 200ng of LPS was injected, neutrophil invasion occurred to the maximum and remained constant thereafter. However, when the same amount of the bioconverted bellflower fermented polysaccharide fraction was injected, unlike LPS, it was confirmed that intraperitoneal neutrophil infiltration was hardly induced, and neutrophil infiltration by LPS was inhibited when simultaneously treated with LPS (Fig. 3). Reference). Unlike LPS, the bioconverted bellflower fermentation polysaccharide fraction does not induce intraperitoneal neutrophil infiltration, and rather, LPS-induced intraperitoneal neutrophil infiltration is inhibited by the bioconverted bellflower fermentation polysaccharide fraction, which is a competitive inhibitor for LPS. Proved. Therefore, the bioconverted bellflower fermentation product could be evaluated as a safe material that does not induce an inflammatory reaction in the body, but rather a material that can act competitively with LPS.

Results 2. Effect of Bioconverted Bellflower Fermentation on Atopic Dermatitis Prevention

Results 2-1. Confirmation of inhibition of Th2 cytokine, which is promoted as atopic dermatitis is induced

In order to evaluate the atopic dermatitis inhibitory activity of bioconverted bellflower fermentation, an animal model of atopic dermatitis induced with DNCB and mite extract was used. Ten 6-week-old female Balb/c mice per experimental group were subjected to acclimatization for 1 week and then sensitized by applying 20 µl of 1% DNCB to the right ear, and after 3 days, 20 µl of 10 mg/ml mite extract was applied to the same ear and stimulated. DNCB and mite extract were applied once a week for a total of 5 weeks to induce atopic dermatitis, and bioconverted bellflower fermentation was fed for a total of 4 weeks from 1 week after the induction of atopic dermatitis. Subsequently, the mice were sacrificed to evaluate the inhibitory effect of bioconverted bellflower fermentation on atopic dermatitis.

By measuring the amount of Th2 cytokine in the lesion tissue of atopic dermatitis-induced mice, the effect of reducing the enhanced Th2 immune response of the bioconverted bellflower fermentation and the effect of regulating the Th1/Th2 immune response was confirmed. As shown in Figure 4, as a result of confirming the amount of IL-4, IL-5, or IL-13 in the lesion tissue (herein the ear tissue) by the RT-PCR method, 5 compared to normal mice by induction of atopic dermatitis. It was confirmed that the amount of expression over-fold increased. On the other hand, in the case of mice administered with the bioconverted bellflower fermentation, it was found that the expression levels of IL-4, IL-5, and IL-13 were all decreased, and thus it was confirmed to have excellent inhibitory activity (see FIG. 4).

Results 2-2. Confirmation of recovery of decreased Th1 cytokine as atopic dermatitis is induced

Results In the same manner as in the evaluation method in 2-1, the amount of Th1 cytokine in the spleen of atopic dermatitis-induced mice was measured, along with the effect of reducing the enhanced Th2 immune response of the bioconverted bellflower fermentation and Th1/Th2. The effect of regulating the immune response was confirmed. As shown in Figure 5, as a result of confirming the expression level of IL-2, IL-12, or IFN-γ among Th1 cytokines using the RT-PCR method, as atopic dermatitis is induced, IL-2, IL-12 , Or it was confirmed that the expression level of IFN-γ was decreased. On the other hand, it was confirmed that the expression levels of IL-2, IL-12, or IFN-γ were recovered in the mice administered with the bioconverted bellflower fermentation (see FIG. 5). That is, the expression levels of IL-2, IL-12, and IFN-γ were all increased, indicating that they had excellent Th1/Th2 immune response regulating activity.

Results 3. Effect of bioconverted bellflower fermentation on atopic dermatitis treatment

Through the measurement of IgE in serum, ear thickness, TSLP in ear tissue, and IL-31, which induces itching in atopic dermatitis, the therapeutic effect of bioconverted bellflower fermentation against atopic dermatitis was confirmed. By inducing atopic dermatitis, serum IgE is about 7 times, ear thickness is about 2 times, TSLP protein expression in ear tissue is about 6 times, and IL-31 protein expression in ear tissue is about 6 compared to normal mice. It was confirmed that it increased more than twice. On the other hand, when 40 mg/kg bellflower raw material was administered, the amount of IgE in the serum was suppressed by 20.8%, the ear thickness was suppressed by 25.2% compared to the positive control, and the expression level of TSLP protein in the ear tissue generated from keratinocytes was 22.5. % Was suppressed, and it was confirmed that the amount of IL-31 protein in the ear tissue was suppressed by 16.0%. On the other hand, when bioconverted bellflower fermentation was administered at 40 mg/kg, the ear thickness was suppressed by 63.7% compared to the positive control, the amount of IgE in the serum was suppressed by 60.0%, and the expression level of TSLP in the ear tissue was 58.9. % Was suppressed, and it was confirmed that the amount of IL-31 in the ear tissue was suppressed by 52.2%. Therefore, it could be determined that the material was converted to a material having superior efficacy than the original product by the bioconversion process (see FIG. 6). In addition, it was confirmed that the administration of the bioconverted bellflower fermentation according to the present invention can control the chronic inflammatory reaction by inhibiting the production of TSLP, and can effectively alleviate the itching caused by atopic dermatitis to alleviate atopic dermatitis. I did.

Results 4. Confirmation of increase in the expression level of galectin-9 in serum of atopic dermatitis-induced mice

By measuring the amount of galectin-9 in the serum of atopic dermatitis-induced mice, the effect of controlling the expression of galectin-9 of the bioconverted bellflower fermentation was confirmed. As a result of measuring the amount of galectin-9 in the serum after mouse sacrifice by ELISA, the expression of galectin-9 increased 3.3 times compared to normal mice due to the induction of atopic dermatitis. It was confirmed that the expression of lectin-9 was hardly increased further. On the other hand, it was confirmed that the expression of galectin-9 was significantly increased in the mice to which the bioconverted bellflower fermentation was administered, about 5 times compared to the normal mice and about 2 times compared to the positive control group (see FIG. 7). Galectin-9 can induce activation of Treg cells by increasing the expression of TGF-β1, and it is known as a protein that plays an important role in inhibiting inflammatory responses through Treg cells, so galectin-9 is a biotransformed bellflower fermentation. When water is administered, it has been confirmed that atopic dermatitis can be effectively suppressed through activation of Treg cells.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (14)

A pharmaceutical composition for the treatment of atopic dermatitis, comprising as an active ingredient the polysaccharide obtained after removing the insoluble residue from the fermented product treated with fibrinolytic enzyme after cultured bellflower in liquid form and inoculated with basidiomycete hyphae.
The method of claim 1,
The culture medium is to make bellflower liquid medium after treating the bellflower powder with a hydrolase in a liquid state by powdering bellflower.
The method of claim 2,
The hydrolase is at least one of amylase-based hydrolase and cellulase-based hydrolase, pharmaceutical composition.
The method of claim 1,
The fibrinolytic enzyme is a cellulase alone or a combination of one or more selected from the group consisting of cellulase, hemicellulase, pectinase and glucanase.
The method of claim 1,
The basidiomycete mycelium consists of a mycelium of basidiomycetes selected from the group consisting of shiitake mushrooms, phylum mushrooms, chaga mushrooms, Maitake mushrooms, tree ear mushrooms, snowflake mushrooms, and skirt mushrooms.
The method of claim 1,
The pharmaceutical composition decreases the expression level of IL-4, IL-5, IL-13, TSLP (thymic stromal lymphopoietin), or IL-31 at the site of atopic dermatitis, or To decrease the amount of IgE, or increase the amount of galectin-9 (galectin-9), a pharmaceutical composition.
(a) culturing bellflower in a liquid phase;
(b) performing a biotransformation fermentation process of culture-fermenting by inoculating basidiomycete hyphae in the liquid bellflower medium cultured in step (a);
(c) performing a bioconversion enzyme treatment process of treating fibrin degrading enzyme from the fermented product produced by the bioconversion fermentation process of step (b); And
(d) removing the insoluble residue from the fermented product produced by the bioconversion enzyme treatment process of step (c) and obtaining a polysaccharide; comprising, a method for producing a pharmaceutical composition for the treatment of atopic dermatitis.
The method of claim 7,
In the step (a), the culture medium of bellflower is made into a liquid bellflower medium after the bellflower powder is treated with a hydrolase in a liquid state by powdering bellflower.
The method of claim 8,
The hydrolase is at least one of an amylase-based hydrolase and a cellulase-based hydrolase.
The method of claim 7,
The fibrinolytic enzyme of step (C) is a cellulase alone or a combination of one or more selected from the group consisting of cellulase, hemicellulase, pectinase, and glucanase.
The method of claim 7,
The basidiomycete hyphae of the step (b) is composed of basidiomycete of basidiomycete selected from the group consisting of shiitake mushrooms, phylum mushrooms, chaga mushrooms, maitake mushrooms, wood ear mushrooms, snowflake mushrooms and skirt mushrooms.
The method of claim 7,
The polysaccharide of step (d) decreases the expression level of IL-4, IL-5, IL-13, TSLP (thymic stromal lymphopoietin), or IL-31 in the lesion site of atopic dermatitis. Or, reducing the amount of IgE in the serum, or increasing the amount of galectin-9.
A food composition for improving atopic dermatitis, comprising as an active ingredient a polysaccharide obtained after removing the insoluble residue from the fermented product treated with fibrinolytic enzyme after culture medium of bellflower in liquid form and inoculation of basidiomycete hyphae.
A cosmetic composition for improving atopic dermatitis, comprising as an active ingredient a polysaccharide obtained after removing the insoluble residue from the fermented product treated with fibrinolytic enzyme after culture medium of bellflower in liquid form and inoculation of basidiomycete hyphae.

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