KR20240019003A - Composition for preventing or treating allergic diseases containing sword bean pod - Google Patents

Abstract

The present invention relates to a composition having anti-inflammatory and anti-allergic effects containing peach pod extract as an active ingredient. Specifically, the peach pod extract of the present invention inhibits nitric oxide (NO), iNOS, COX-2, and IL-6. Decreased gene expression, increased expression of anti-inflammatory cytokine IFN-γ gene, decreased expression of inflammatory cytokine proteins (p-IκBα, p-p65), decreased expression of inflammatory mediators iNOS and COX-2 proteins, decreased histamine and IgE expression, It has the effect of reducing the production of inflammatory cytokines (IL-4, IL-5, IL-6) and increasing the production of anti-inflammatory cytokine IFN-γ, making it a pharmaceutical, health functional food and cosmetic composition for preventing and improving allergic diseases. It can be useful.

Description

Composition for preventing or treating allergic diseases containing sword bean pod}

The present invention relates to a composition for preventing or treating allergic diseases containing peach pod extract as an active ingredient.

Allergy is a type of hypersensitivity reaction caused by immune imbalance. The cause of an allergic reaction is caused by the body's immune system reacting to external substances that act as allergens, known as allergens. Allergens are presented on the cell surface by antigen-presenting cells, and the presented antigens are recognized by B lymphocytes, converted into plasma cells, and secrete antibodies (IgE) that specifically recognize the antigen.

Allergic disease is caused by an excessive immune response caused by memory cells due to repeated contact with allergens. IgE that binds to an antigen binds to the receptor (FcεRI) on tissue mast cells, causing degranulation of the mast cells. induces. Degranulation of mast cells secretes histamine, heparin, leukotrienes, prostaglandins, and other anaphylatic agents. These secreted immune-activating factors activate the immune system and cause inflammation and allergic reactions.

The normal human immune system consists of a type 1 T helper cell (T helper cell 1, Th1) response that regulates cellular immunity and a type 2 helper T cell (T helper cell 2, Th2) response that regulates humoral immunity. However, it is balanced. In allergy patients, this balance is disrupted as the type 2 helper cell (T helper cell 2, Th2) response becomes excessive, resulting in various adverse reactions such as an excessive increase in blood IgE (immunoglobulin E) and excessive secretion of histamine.

Hypersensitivity reactions can be broadly divided into four types. Type 1 hypersensitivity reaction, commonly referred to as an allergic reaction, is a reaction that occurs to a specific antigen and is induced by binding of IgE antibodies specific to that antigen to mast cells or basophils. When a large number of IgE antibodies specific for an allergen are produced, these antibodies bind to the high-affinity IgE receptor (high-affinity receptor for IgE, FcεRI) on the surface of mast cells or basophils, and the allergen binds to these cells. is activated, and various bioactive substances are secreted, leading to changes such as vasodilation, increased vascular permeability, smooth muscle contraction, and inflammatory response.

These reactions can occur locally or systemically depending on the type and amount of mediators produced by these cells. In particular, allergens such as fine dust and pollen cause oxidative damage, inflammation, and inflammation in the mucous membranes of the eyes, nose, and bronchi. It can cause allergies, and the particle size of fine dust is so small that it penetrates directly into the human body without being filtered through the nasal mucosa when breathing. Prolonged exposure can cause various respiratory and allergic diseases such as asthma, lung disease, and cardiovascular disease. It can be.

Specific allergic diseases include atopic dermatitis, rhinitis, conjunctivitis, and asthma, and mast cells are very important in various allergic diseases. Mast cells, unlike other cells, contain large and thick heterochromatic granules, and are exposed to antigens when IgE is bound to the high-affinity IgE receptor (high-affinity receptor for IgE, FcεRI), the surface receptor of mast cells. When stimulated, it participates in various immune and inflammatory reactions.

Therefore, mast cells are central to various physiological, immunological, and pathological processes that occur during allergic reactions, such as wound healing, angiogenesis, host response to parasites and neoplasms, acute and chronic inflammatory reactions, and IgE-mediated immediate allergic reactions. plays a role. Mediators secreted when mast cells are activated in allergic reactions include histamine and 5-HT (serotonin), which are biogenic amines that cause vasodilation and pain. In addition, interleukin-1 (IL-1), Interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage migration inhibitory factor (MIF), It secretes cytokines such as tumor necrosis factor (TNF) to induce inflammation and migration and proliferation of leukocytes.

Allergic disease is a common chronic inflammatory disease observed in 10-20% of the entire population, and is increasing day by day as environmental pollution increases due to industrial development and residential environments change. In addition, in Korea, inflammatory allergic diseases such as asthma, allergic rhinitis, and atopic dermatitis are gradually increasing due to rapid economic development, population concentration, environmental pollution, and changes in westernized eating habits, especially in children and the elderly who are sensitive to the environment due to weak immunity. It is rapidly increasing. In Korea, the prevalence of allergic rhinitis disease is on the rise from 11.9% in 2009 to 18.1% in 2020, and it has been confirmed that approximately 6.9 million patients have received treatment, and it has been confirmed that there are measures to prevent and treat fine dust-induced allergic diseases. It can be seen that interest has increased.

These diseases are caused by excessive stimulation of the body's immune system by various allergy-causing factors, causing inflammation-causing substances in cells such as immune cells such as macrophages, epithelial cells, skin cells, and mast cells, so suppress their excessive induction. This can alleviate the symptoms of inflammatory allergic disease.

The method to prevent and treat allergic diseases is to fundamentally prevent allergens (fine dust, etc.) from entering the body, but it is impossible to actually block them. The second method is to use drug therapy. Existing treatments can be broadly divided into steroidal anti-inflammatory drugs or non-steroidal anti-inflammatory drugs and antihistamines or anti-leukotriene drugs. Most of the former have a strong immunosuppressive effect, so they can have a temporary sedative effect in a short period of time, but there are many problems with long-term use, ranging from mild side effects such as nausea to serious side effects such as growth inhibition and osteoporosis. The latter may provide temporary relief, but antihistamines, which are commonly used allergy treatment drugs, only temporarily relieve symptoms and cannot be expected to have a fundamental allergy treatment effect. Side effects such as headache, fatigue, drowsiness, and dry mouth are reported. It is becoming. In addition, non-steroidal anti-inflammatory drugs (NSAIDs), which are used to treat acute or chronic inflammatory diseases such as the above, not only inhibit the COX-2 enzyme, but also inhibit the COX-1 enzyme, thereby preventing various side effects such as gastrointestinal disorders. It is known to represent (Masferrer et al., P. Natl. Acad. Sci. USA. 91:3228-3232, 1994). Therefore, there is a need to discover functional materials derived from natural products and develop health functional foods that can replace allergy drugs and are effective in preventing and relieving allergies in advance, as well as being safe to apply.

Canavalia gladiata (Canavalia gladiata) is a climbing annual plant belonging to the dicotyledonous Rosaceae legume family. Its main origin is distributed in tropical regions of Southeast Asia, Africa, and India. Because the shape of the bean pod resembles a small bean, it is called a small bean. It is also called a small bean.

The leaves of the soybean have a long petiole and are composed of three small leaves. They are egg-shaped, long oval, have a pointed end, are about 10 cm long, and have wavy edges. The flowers bloom between July and August and are light red-purple or white, and long flower stems grow from the leaf axils in summer and hang in racemes. The pods have curved or hook-shaped ends, are about 20 to 30 cm long and 5 cm wide, and contain about 10 to 14 beans. The bean is larger than regular beans, about 3cm in diameter, has a long bean on one side, and is red or white.

Since ancient times, eating soybeans has been used as tea as it is highly effective in relieving inflammation such as hemorrhoids, sinusitis, otitis media, gastritis, and colitis. It has been used to treat the back bronchial tubes, and bean pods are known to be helpful for chronic diarrhea, menstrual cessation, and food loss, and have been used medicinally.

Efficacy in preventing and improving allergic diseases, especially allergic rhinitis, containing soybean or soybean extract or fermented product as an active ingredient (published patent 10-2020-0139422), prevention of inflammation or allergic disease containing fermented soybean extract as an active ingredient and therapeutic compositions (Patent No. 10-1585159), but no anti-inflammatory, anti-allergic effects, or therapeutic agents for allergic diseases of peach pods have been confirmed to date. Accordingly, the present inventors completed the present invention by confirming the efficacy of preventing or treating allergic diseases using the peach pod alcohol extract.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating allergic diseases containing peach pod extract as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving allergic diseases containing peach pod extract as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for preventing or improving allergic diseases containing peach pod extract as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating allergic diseases containing peach pod extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving allergic diseases containing peach pod extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving allergic diseases containing peach pod extract as an active ingredient.

It was confirmed that the composition containing the peach pod extract according to the present invention as an active ingredient is superior to the peach pod extract in suppressing inflammatory substances and cytokines and alleviating hypersensitive immunity, showing excellent effects in preventing or treating allergic diseases. It can be usefully used as pharmaceutical, health functional food, and cosmetic compositions.

Figure 1 is an image showing dodu pods and dodu bean seeds.
Figure 2 is a diagram showing the inhibitory effect of nitric oxide (NO) production of peach pod (SBP) and peach bean (SB) extracts.
Figure 3a is a diagram analyzing the gene expression of the inflammatory mediator iNOS in extracts of soybean pods (SBP) and soybeans (SB).
Figure 3b is a diagram analyzing the gene expression of the inflammation mediator COX-2 in extracts of soybean pods (SBP) and soybeans (SB).
Figure 3c is a diagram analyzing gene expression of the cytokine IL-6 in extracts of soybean pods (SBP) and soybeans (SB).
Figure 3d is a diagram analyzing gene expression of the cytokine IFN-γ in extracts of soybean pods (SBP) and soybeans (SB).
Figure 4 is a diagram analyzing the expression of inflammatory substances (iNOS, COX-2) and NF-κB signaling proteins in extracts of soybean pods (SBP) and soybeans (SB).
Figure 5 is a diagram showing the establishment of a hypersensitivity animal model to evaluate the hypersensitivity immunity alleviation efficacy of soybean pod (SBP) and soybean bean (SB) extracts.
Figure 6a is a diagram analyzing the inhibition of histamine, an allergy mediator, in animals with hypersensitive immunity fed with extracts of peach pods (SBP) and peach bean (SB).
Figure 6b is a diagram analyzing the inhibition of allergy mediator IgE in hyperimmune-induced animals fed with peach pod (SBP) and peach bean (SB) extracts.
Figure 7a is a diagram analyzing the regulation of production of the cytokine IL-4 in hyperimmune-induced animals fed with soybean pod (SBP) and soybean bean (SB) extracts.
Figure 7b is a diagram analyzing the regulation of production of the cytokine IL-5 in hyperimmune-induced animals fed with peach pod (SBP) and peach bean (SB) extracts.
Figure 7c is a diagram analyzing the regulation of production of the cytokine IFN-γ in hyperimmune-induced animals fed with peach pod (SBP) and peach bean (SB) extracts.
Figure 8 is a graph comparing and analyzing the indicator component (pinitol) of extracts of peach pod (SBP) and peach bean (SB) ((a) peach bean (b) peach pod).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating allergic diseases containing peach pod extract as an active ingredient.

The term “allergic disease” used in the present invention refers to a disease caused by allergy. Allergy refers to an antigen-antibody reaction that occurs when a certain type of substance (antigen or allergen) enters the body, antibodies are created against it, and then when an antigen of the same substance enters the body again.

“Allergy” as defined in the present invention includes hypersensitivity, allergic rhinitis, asthma, anaphylactic shock, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, eczema, insect allergy, food allergy, hay fever, and plant allergy. Includes allergies or drug allergies.

In the present invention, allergic reaction refers to type I of four types of allergic reactions: type I, type II, type III, and type IV. In the type I reaction, IgE binds to the surface of mast cells or basophils, and when a specific antigen binds to this IgE antibody, granule escape (degranulation phenomenon) occurs in the mast cells or basophils, and histamine and serotonin are released. , refers to a phenomenon in which chemicals such as bradykinin are released, resulting in increased vascular permeability and stimulation of mucus secretion. Symptoms included include anaphylaxis, bronchial asthma, allergic rhinitis, and hives.

The term “prevention” used in the present invention refers to all actions that suppress or delay the onset of allergic disease by administering the pharmaceutical composition according to the present invention.

As used herein, the term “improvement” means any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.

The dodu pod extract can be extracted by a method known in the art, and the method is not particularly limited.

Preferably, the dodu pod extract may be an extract obtained by extracting the dodu pods with water and/or an organic solvent, and the organic solvent may be a polar organic solvent, a non-polar organic solvent, or a mixed solvent thereof. The polar organic solvent may be a lower alcohol having 1 to 5 carbon atoms, ethyl acetate, or acetone, and the non-polar organic solvent may be ether, chloroform, benzene, hexane, or dichloromethane. For example, the lower alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, or isopropanol.

The dodu pod extract can be used from immature dodu pods, but is not limited thereto.

The immature dodu pods are harvested before the bean seeds swell in the pods, and can be used as a food ingredient.

The dodu pod extract may be 20 to 40% alcohol extract, preferably 25 to 35% alcohol extract, and more preferably 28 to 32% alcohol extract.

The dodu pod extract may include a fraction obtained by fractionating the primary extract extracted using the above-described extraction solvent using an extraction solvent of different polarity. For example, the dodu pod extract may be a fraction extracted with an alcohol having 1 to 5 carbon atoms and then fractionated again with a solvent of different polarity such as ether, benzene, or hexane. Two or more types of solvents can be used during the fractionation, and extracts for each solvent can be prepared by using them sequentially or by mixing them depending on the polarity of the solvents.

In the present invention, the prepared extract or the fraction obtained by performing the fractionation process can be filtered, concentrated, or dried to remove the solvent, and the filtration, concentration, and drying can all be performed. Specifically, the filtration can be performed using filter paper or a vacuum filter, the concentration can be performed under reduced pressure using a vacuum concentrator, etc., and the drying can be performed using a freeze-drying method.

In addition, the prepared extract or the fraction obtained by performing the fractionation process may be subjected to silica gel column chromatography, thin layer chromatography, or high performance liquid chromatography. Additional purified fractions can be obtained by purification using various chromatographies.

According to a preferred experimental example of the present invention, the composition of the present invention is a pharmaceutical composition comprising a pharmaceutically effective amount of the peach pod extract of the present invention and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to achieve the efficacy or activity of the above-described dodu pod extract.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work.

For administration, the composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above. Pharmaceutically acceptable carriers can be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients, and if necessary, antioxidants and buffer solutions. Other common additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed by Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

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

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, and water, in the pharmaceutical composition of the present invention. It can be prepared by mixing sucrose, lactose, gelatin, etc.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It can be. The general dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The composition of the present invention suppresses the amount of nitric oxide (NO) produced.

The nitric oxide (NO) is produced by the action of the inducible nitric oxide synthase (iNOS) enzyme in macrophages, and the produced NO is transformed into phagocytic microbial (pathogen) components and Fe. -Exhibits anti-microbial activity by inhibiting the action of enzymes containing S. Additionally, NO is secreted from activated macrophages and neutrophils to kill extracellular pathogens.

The composition of the present invention reduces gene expression of iNOS, COX-2, and IL-6, and reduces protein expression of iNOS, COX-2, and IL-6.

The inducible nitric oxide synthase (iNOS) oxidizes arginine (L-arginine) to produce citrulline (L-citrulline) and NO, and iNOS is expressed in almost all cells by stimulation such as cytokines. .

Cyclooxygenase-2 (COX-2) is an enzyme that converts arachidonic acid into prostaglandin E2 (PGE2). It is expressed due to inflammation or immune stimulation and produces various cytokines. It is induced in response to growth factors, etc. COX-2 is an enzyme that is expressed temporarily and rapidly within cells by mitogens or cytokines during inflammation or other immune responses.

Interleukin-6 (IL-6) is an inflammatory cytokine produced by many different types of cells. In vivo, stimulated monocytes, fibroblasts, and epithelial cells represent the main source of IL-6. Other cells such as macrophages, T and B lymphocytes, granulocytes, keratinocytes, mast cells, osteoblasts, chondroblasts, glial cells, and smooth muscle cells also produce IL-6 after stimulation.

The composition of the present invention increases anti-inflammatory cytokine IFN-γ gene expression and increases the production amount of cytokine IFN-γ.

The interferon-γ (IFN-γ) is also called type II interferon, and is also called immune interferon because it is produced by CD4 T cells or CD8 T cells and regulates immune responses. IFN-γ can act on T cells, B cells, neutrophil NK cells, and vascular endothelial cells to activate them, and acts as a macrophage activating factor to activate Class 1 and 2 MHC (Class I and II MHC). ) also increases the expression of .

The composition of the present invention reduces the expression levels of p-IκBα and p-p65.

The IκBα is one of a family of cellular proteins that function to suppress the NF-κB transcription factor. It suppresses NF-κB by masking the nuclear localization signal (NLS) of the NF-κB protein and sequestering it in an inactive state in the cytoplasm. do. Additionally, IκBα blocks the ability of the NF-κB transcription factor to bind DNA, which is required for proper function of NF-κB.

The p65 forms the most common heterodimer in the NF-κB signaling pathway and is present in most cell types. p65 is characterized by a Rel homology domain located at the N terminus, which is associated with a transactivation domain at the C terminus. As part of the NF-κB signaling pathway, p65 is typically involved in the body's inflammatory response. These pathways include free radicals, ultraviolet irradiation (UV), tumor necrosis factor-α (TNF-α), interleukin 1-beta (IL-1β), pathogen-associated molecular patterns (PAMPs), or bacterial lipopolysaccharide (LPS). ) can be induced by stressful stimuli including.

The composition of the present invention reduces histamine and IgE.

The histamine is a representative hormone in the living body and is made from the amino acid histidine. The histamine is mainly secreted from the terminals of mast cells, basophils, or nerve cells and causes allergic reactions and peripheral blood vessels. It is a substance that induces many physiological actions such as dilatation, secretion of gastric acid, decomposition of glycogen in the liver, synthesis of glucose, and nerve transmission. In addition, the physiological action of histamine is mediated by histamine receptors present on the surface of cells that exhibit the action, and when histamine binds to the histamine receptor, a signal is transmitted to express the physiological action.

When the IgE is cross-linked by an allergen and then binds to the high-affinity IgE receptor (FcεRI) on mast cells and basophils, the inflammatory cells are activated and release mediators of allergic inflammation, thereby initiating an IgE-mediated allergic reaction. is a glycoprotein molecule mainly expressed on the cell membrane of tissue cells and basophils and plays an important role in type I allergic reactions for the activation of these cells. When antigen-specific IgE cross-links with the corresponding multivalent antigen, i.e., allergen, FcεRI aggregates and signal transduction mechanisms begin to operate, activating mast cells. As a result, degranulation of cells occurs, leading to new synthesis and release of new cotriene and prostaglandin, while releasing chemical mediators such as histamine and serotonin, causing a type I allergic reaction.

The composition of the present invention reduces the production of inflammatory cytokines (IL-4, IL-5).

The interleukin-4 (IL-4) is a cytokine that induces differentiation from unactivated helper T cells (Th0 cells) into Th2 cells. IL-4 has many biological functions, such as stimulating the proliferation of activated B cells and T cells or differentiating B cells into plasma cells, and is an important regulator of humoral immunity and adaptive immunity. In addition, IL-4 not only induces class switching from B progenitor cells to B cells capable of producing IgE and IgG4, but also promotes the production of MHC class II. Excessive production of IL-4 is associated with allergies, and IL-4 mediates pro-inflammatory effects important in the induction of asthma, such as IgE isoform rearrangement and vascular cell adhesion molecule 1 (vascular cell adhesion molecule 1). These include expression of adhesion molecule 1 (VCAM-1), migration of eosinophils through epithelial cells, mucus secretion, and cytokine secretion by Th2.

The interleukin-5 (IL-5) is an interleukin produced by type 2 helper T cells (T helper cell 2, Th2) and mast cells. Through binding to the interleukin-5 receptor, IL-5 is activated by B cells. Stimulates growth and increases secretion of immunoglobulins (mainly IgA). Additionally, IL-5 is a key mediator of eosinophil activation, expressed by eosinophils and observed in mast cells of asthmatic airways by immunohistochemistry, and IL-5 expression is regulated by several transcription factors including GATA3. do.

The composition of the present invention has a pinitol content that is 15% to 25% higher than that of soybeans, preferably 18% to 20% higher.

The pinitol (or D-pinitol, pinitol) is a natural substance extracted from soybeans, alfalfa, pine trees, etc. and has an insulin-like action that lowers blood sugar levels (Narayan et al., Curr. Sci. 56: 139-141, 1987; Bates et al. ., Br. J. Pharmacol. 130: 1944-1948, 2000; Davis et al., Diabetes Care 23: 1000-1005, 2000) as well as cardiovascular disease reduction effect (Kim et al., Eur. J. Clin. Nutr 59: 456-458, 2005). In addition, pinitol promotes muscle development and recovery by activating the supply of creatine and other nutrients to muscle cells (Bates et al., Br. J. Pharmacol. 130: 1944-1948, 2000), and helps prevent cancer. It is also reported to be effective in treating wasting diseases in AIDS patients (US Patent No. 5,827,896), and is known to have various functionalities such as immune disease and heart disease treatment effects, anti-inflammatory, and anticancer effects.

In particular, pinitol is effective in preventing and treating inflammatory diseases by inhibiting the start of the NF-kB signaling pathway that causes inflammation in psoriatic mouse skin (Journal of Environmental Pathology, Toxicology and Oncology, Volume 38, Issue 3, 2019, pp . 285-295) is known to exist. Accordingly, the present inventors confirmed the anti-inflammatory effect of the dodu pods by measuring the pinitol content of the dodu pods. Pinitol is an ingredient that has secured specificity and stability among the various components contained in the dodu pods and has reported functionality, and is an indicator of the dodu pods. Appropriate as an ingredient.

D-pinitol contained as an active ingredient in the composition according to the invention has the structure of Formula 1 below.

[Formula 1]

In another aspect, the present invention provides a health functional food composition for preventing or improving allergic diseases containing peach pod extract as an active ingredient.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. In general, when manufacturing a food or beverage, the health functional food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials, but is not limited thereto.

In another aspect, the present invention provides a cosmetic composition for preventing or improving allergic diseases containing peach pod extract as an active ingredient.

In the present invention, the cosmetic composition is a formulation selected from the group consisting of solution, suspension, emulsion, paste, gel, cream, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray. It may have, but is not limited to this.

The cosmetic composition of the present invention may contain ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and flavoring agents, and carriers. .

Additionally, the composition of the present invention may be used by mixing an organic sunscreen. The organic sunscreen includes glyceryl faba, drometrisol trisiloxane, drometrizole, digalloyl trioleate, disodium phenyldibenzimidazoletetrasulfonate, diethylhexylbutamidotriazone, and diethylamino. Hydroxybenzoylhexylbenzoate, DA-methoxycinnamate, mixture of Lawson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthylanthranilate, benzophenone. -3 (oxybenzone), benzophenone-4, benzophenone-8 (deoxyphebenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenolmethoxyphenyltriazine, cynoxate, ethyldihydroxypropylfaba, Octocrylene, ethylhexyldimethylfava, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltriazone, isoamyl-p-methoxycinnamate, polysilicon-15 (dimethicodiethylbenzalmalonate) , terephthalylidene dicamphorsulfonic acid and its salts, TEA-salicylate, and aminobenzoic acid (faba) can be used.

According to specific examples and experimental examples of the present invention, the present invention prepares a 30% alcohol extract of dodu pods and dodu beans (see Example 1), and compares and analyzes the content of pinitol as an indicator component of the dodu pods and dodu bean extracts. As a result, it was confirmed that the dodu pods contained a higher content of pinitol (see Example 2: Table 1 and Figure 8).

To confirm the effect of the extract of the peach pod on improving inflammation, RAW 264.7 cells were treated with LPS, cultured, and then an in vitro experiment was performed (see Experimental Example 1). As a result of analyzing the efficacy of nitric oxide (NO) inhibition, it was confirmed that the SBP extract treatment group had a much better NO production inhibition effect compared to the SBP extract treatment group (FIG. 2). As a result of analyzing the expression levels of inflammatory mediators nitric oxide synthesis-inducing factor (iNOS), cyclooxygenase-2 (COX-2), and cytokines IL-6 and IFN-γ genes, the SBP extract treatment group It was confirmed that the effect of lowering iNOS, COX-2, and IL-6 gene expression was significant compared to this extract of soybean bean (SB) (Figures 3a, 3b, and 3c), and the anti-inflammatory cytokine IFN-γ was found in peach pod (SBP). ) It was confirmed that the extract treatment group significantly increased compared to the soybean (SB) extract (Figure 3d). A hypersensitive immune animal model was established by injecting a mixed solution of ovalbumin (OVA) and Alum (aluminium hydroxide) into the mouse abdominal cavity (Figure 5), and the normal group, negative control group, positive control group, and 100 mg/kg extract of soybean were treated. Seven groups were created with a group, a group treated with 200 mg/kg extract of soybeans, a group treated with 100 mg/kg extract of dodu pods, and a group treated with 200 mg/kg extract of dodu pods, and the efficacy of suppressing the production of allergy mediators was analyzed (Experimental Example 2 and Table 2). As a result, it was confirmed that histamine and IgE levels were significantly decreased in the soybean pod (SBP) extract treatment group compared to the soybean bean (SB) extract treatment group (Figures 6a and 6b). Lastly, a significant reduction effect of IL-4 and IL-5 was confirmed in the SBP treatment group (Figures 7a and 7b), and the anti-inflammatory cytokine IFN-γ, which was reduced due to hypersensitivity, was decreased in the SBP treatment group. It was confirmed that there was a significant increase in the dodu pod extract treatment group, and in particular, a greater increase effect was confirmed in the dodu pod extract treatment group (Figure 7c).

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples are only for illustrating the present invention, and the present invention is not limited by the following examples and experimental examples.

<Example 1> Preparation of peach pod and peach bean alcohol extract

In the present invention, dodu pods and dodu beans were prepared with alcohol extract, and the dodu pods were collected from immature dodu pods (Figure 1). 30% alcohol was added to dried soybean pods (SBP) and soybeans (SB) powder and stirred at 80°C for 8 hours to perform primary extraction. After the first extraction, the extract was recovered, 30% alcohol was added to the residue, and the second extraction was performed at 80°C for 4 hours. The extract was filtered and concentrated under reduced pressure, and maltodextrin was mixed in an amount equal to the solid content of the concentrate and spray dried. Dodu pod extract and dodu bean extract were prepared by dissolving the extract powder of dodu pods and dodu beans in PBS to a final treatment concentration of 100, 200 μg/mL or 100, 200 mg/kg.

<Example 2> Comparative analysis of pinitol (indicator component) content of dodu pod and dodu bean extract

The present inventors measured the pinitol content in dodu beans and dodu pods, and as a result, it was confirmed that the pinitol content in dodu pods was about 19% higher than that in dodu beans. This means that the solid content (%) of the soybeans and the dodu pods are similar, and the pinitol content compared to 100% solid content is 34.87 mg/g for the soybeans and 41.45 mg/g for the dodu pods, indicating that the dodu pods contain a higher content of pinitol. This was confirmed (Table 1). In addition, the pinitol content of dodu beans or dodu pods contains a higher content compared to the standard material. In particular, the peak of the dodu pod appears higher, indicating that the dodu pod contains a higher content of pinitol than the dodu bean and the standard material. It was confirmed that (FIG. 8).

Sample Pinitol content
(mg pinitol/g sample) Solid content (%) Pinitol content compared to 100% solids
(mg/g) Dodu beans 11.10 31.83 34.87 Dodu pods 13.48 32.53 41.45

<Experimental Example 1> Inflammation improvement effect of Dodu pod extract

<1-1-1> Cell culture and nitric oxide (NO) concentration measurement

To confirm the inflammation-improving effect of the peach pod extract, an in vitro experiment was performed on LPS-induced RAW 264.7 cells. RAW264.7 cells were distributed in a 96-well plate at a concentration of 1x10 ⁶ cells/well, stabilized for 2 hours, then treated with peach pod or peach bean extract at various concentrations (100, 200 μg/mL), and 1 hour later, LPS (Lipopolysaccharides, 1μg/mL) and cultured for 24 hours. After completion of cultivation, the cell culture supernatant was recovered and the amount of NO production was measured according to the Griess reagent system method. Add 50 μL of sulfanilamide reagent to 50 μL of the recovered cell culture supernatant, react in the dark for 10 minutes, and add N-(1-Naphthyl)-ethylenediamine (NED). 50 μL of solution was added and the absorbance (540 nm) was measured after dark reaction for 10 minutes. The amount of NO was calculated by creating a standard curve using sodium nitrite and presented as a quantitative value.

<1-1-2> Nitric oxide (NO) inhibition efficacy analysis

LPS untreated group, inflammation-inducing group (LPS treated), peach pod (SBP) extract 100 μg/mL treated group, peach pod (SBP) extract 200 μg/mL treated group, peach bean (SB) extract 100 μg/mL treated group, peach bean (SB) A group treated with 200 μg/mL extract was prepared.

In the peach pod (SBP) extract and peach bean (SB) extract treatment groups, the amount of nitric oxide (NO) production was reduced in a concentration-dependent manner (Figure 2). In particular, in the peach pod (SBP) extract treatment group, peach bean ( SB) Compared to the extract treatment group, the effect of suppressing NO production was significantly increased. In particular, the group treated with 200 μg/mL of peach pod extract inhibited NO production by about 58% compared to the inflammation-inducing group (+LPS), and SB) The extract treatment group was suppressed by about 30%, confirming that the SBP extract had a better effect of suppressing NO production (Figure 2).

<1-2> Evaluation of inflammatory mediators and inflammation-related cytokine gene expression regulation

<1-2-1> Cell culture and measurement of inflammatory mediator and cytokine gene expression levels

To confirm the regulation of inflammatory mediators and inflammation-related cytokine gene expression by the peach pod extract, an in vitro experiment was performed in RAW 264.7 cells induced by LPS. RAW264.7 cells were distributed in a 12-well plate at a concentration of 5x10 ⁵ cells/well, cultured for 2 hours, treated with peach pod or peach bean extract at various concentrations (100, 200 μg/mL), and 1 hour later treated with LPS, resulting in 24 cells. cultured for some time. Afterwards, total RNA is extracted from the cultured cells using the RNeasy mini kit, and cDNA is synthesized from the extracted RNA using a reverse transcription system kit. The synthesized cDNA used SYBR green mix to express the inflammatory mediators nitric oxide synthesis-inducing factor (iNOS), cyclooxygenase-2 (COX-2), and cytokines IL-6, IL-1β, and IFN-γ genes. The amount was measured by real-time PCR. All experimental results were corrected using GAPDH, an internal control.

<1-2-2> Gene expression analysis of iNOS, COX-2, and cytokines IL-6 and IFN-γ

As a result of the experiment, both peach pod and peach bean extract decreased the gene expression of iNOS and COX-2. In particular, the group treated with 200 μg/mL of soybean pod (SBP) extract decreased iNOS and COX-2 gene expression by 36% and 22%, respectively, compared to the pro-inflammatory group (+LPS), which was compared to the 200 μg/mL of soybean pod (SB) extract. It was confirmed that the iNOS and COX-2 gene expression was significantly lower than that of the treatment group, which decreased by 19.6% and 16.3%, respectively. SBP extract suppresses the inflammatory response by inhibiting the activities of iNOS and COX-2. It was found that there was an excellent effect (Figures 3a and 3b).

IL-6, an inflammatory cytokine, was reduced by 20.5% in the peach pod treatment group and by 18% in the peach bean treatment group compared to the inflammation-inducing group (+LPS), showing a lower decrease in IL-6 gene expression in the peach pod (Figure 3c). .

It is known that the amount of anti-inflammatory cytokine IFN-γ decreases when an inflammatory response is induced, and its expression increases when the inflammatory response is alleviated. Compared to the inflammation-inducing group (+LPS), the gene expression level of the soybean (SB) treated group increased by about 10.4%, but the soybean pod (SBP) treated group increased by about 94%, showing significantly higher gene expression than the soybean. It was confirmed that there was an increase (Figure 3d).

<1-3> Evaluation of inflammation mediators and NFκB signaling protein expression

<1-3-1> Cell culture and measurement of inflammatory mediators and NFκB signaling protein expression

To confirm the expression levels of inflammatory mediators and inflammation-related cytokine proteins in the extract of SBP, an in vitro experiment was performed in RAW 264.7 cells induced by LPS. RAW264.7 cells were distributed in a 6-well plate at a concentration of 1x10 ⁶ cells per well, and after 2 hours, samples were added at each concentration, cultured for 1 hour, treated with LPS, and cultured for 24 hours. Afterwards, the cells were washed with PBS, recovered with a cell scraper, and washed with RIPA buffer (RIPA buffer: 0.5% NP-40, 0.5% TritonX-100, 0.1% sodium deoxycholate, 50 mM Tris-HCl, It was dissolved with 150 mM NaCl, 1 mM EDTA), left on ice for 40 minutes, and then centrifuged (12,000 g, 20 min) to quantify the protein using bicinchoninic acid (BCA). The quantified proteins were separated using a 4-20% SDS-PAGE gel, transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% skim milk for 1 hour. The membrane was washed three times with TBST, and the primary antibody was incubated overnight at 4°C. Afterwards, the membrane from which the primary antibody was removed was washed three times with TBST, reacted with the secondary antibody for 1 hour at room temperature, and protein expression was evaluated using enhanced chemiluminescence (ECL) reagent and Chemi-doc equipment. did. When an inflammatory response is initiated, IκBα is phosphorylated and activates NF-κB to produce inflammatory mediators.

<1-3-2> Expression analysis of inflammatory mediators iNOS and COX-2 protein and NFκB signaling protein

It was confirmed that the expression of iNOS and COX-2 proteins, which are inflammatory mediators, was significantly reduced in the peach pod extract (Figure 4). Compared to the inflammation-inducing group (+LPS), the p-IκBα and p-p65 expression levels were decreased in the peach pod (SBP) extract treatment group, and the p-IκBα and p-p65 expression levels were decreased in the peach bean (SB) treatment group. There was no change in the amount, confirming the effect of suppressing inflammation in the dodu pod extract treatment group (Figure 4).

<Experimental Example 2> Hypersensitivity immunity alleviation effect of peach pod and peach bean extract

<2-1-1> Preparation of hyperimmune induction mouse model

BALB/c mice (6 weeks old) were acclimatized for 1 week under constant temperature and humidity (22 ± 2°C, 55 ± 1%) and light/dark cycle (12 hours) conditions. During the breeding period, water and food were provided ad libitum and experiments were performed in compliance with the laboratory animal performance regulations. To establish an animal model of hypersensitive immunity, a solution of 50 ㎍ of Ovalbumin (OVA) and 2 mg of Alum (aluminium hydroxide) mixed in a 1:1 ratio was placed in the mouse abdominal cavity at 200 ㎕ at the 1st and 3rd weeks. Injections were administered sequentially. Four days after the second intraperitoneal injection, 5% ovalbumin (OVA) intranasal challenges were performed for 30 minutes each for 3 days to establish an ovalbumin (OVA)-induced hypersensitivity animal model (Figure 5). The experimental animals were divided into 7 groups and the experiment was conducted. The normal group was orally administered phosphate-buffered saline (PBS) without inducing hypersensitivity. The negative control group (NC) was orally administered 200 mg/kg of maltodextrin, an excipient, while inducing hypersensitivity immunity. The group treated with soybean and peach pod extracts was orally administered at a concentration of 100 or 200 mg/kg while inducing hypersensitive immunity, and the positive control group was orally administered the anti-inflammatory agent Dexamethasone at a concentration of 0.5 mg/kg while inducing hypersensitive immunity. administered (Table 2).

group Treatment (mg/kg) Normal group Phosphate-buffered saline (PBS) Ovalbumin (OVA)/Alum Negative control group (NC) Maltodextrin (200) Positive control group (PC) Dexamethasone (0.5) SB100 Dodu Beans (100) SB200 Dodu Beans (200) SBP100 Dodu pods (100) SBP200 Dodu pods (200)

Blood collected after 4 weeks of animal testing was centrifuged (3000g, 15min) to separate plasma. The separated plasma was measured for the production of allergy mediators histamine and IgE using a plate coated with capture antibody in the following manner.

For histamine, 100 μL of plasma was placed on a plate, 50 μL of histamine tracer and antibody were added each, reacted at room temperature for 1 hour, and washed three times with wash buffer. Afterwards, 200 μL of the conjugate was added and reacted at room temperature for 30 minutes, washed once more, 200 μL of substrate was added, and after 30 minutes of reaction, the reaction was terminated with a stop solution and the absorbance (450 nm) was measured. To determine the amount of IgE produced in the blood, 100 μL of plasma was dispensed onto a plate, reacted at room temperature for 1 hour, and washed four times with washing buffer. Afterwards, 100 μL of conjugate was added and reacted in the dark for 1 hour. 100 μL of substrate was added and reaction was performed for 10 minutes. Then, a reaction stop solution was added and the absorbance (450 nm) was measured.

<2-1-2> Analysis of efficacy in suppressing production of allergy mediators

The histamine and IgE levels of the group treated with 200 mg/kg extract of SBP were significantly reduced to 167.0 ng/mL and 58.3 ng/mL, respectively, compared to the negative control group (NC) treated with maltodextrin (Figure 6a and Figure 6b). On the other hand, the group treated with 200 mg/kg extract of soybean bean (SB) showed no significant decrease in histamine compared to the negative control group (NC) at 195.9ng/mL, and IgE significantly decreased to 79.4ng/mL, but It was confirmed that the amount of change was smaller than that of the group (Figures 6a and 6b).

<2-1-3> Efficacy in regulating cytokine secretion

50 μL of separated plasma was added to a plate coated with capture antibodies, 50 μL of antibody cocktail was added, and reacted at room temperature for 1 hour. After completion of the reaction, the reaction was washed with a wash buffer, the substrate was added, and the reaction was dark at room temperature for 10 minutes. The reaction was terminated by adding 100 μL of a reaction stop solution, and the cytokine control efficacy was evaluated by measuring the absorbance (450 nm). .

<2-1-4> Cytokine (IL-4, IL-5, IFN-γ) secretion analysis

As a result of observing the production of inflammatory cytokines (IL-4, IL-5) in the blood of mice inducing hypersensitivity, the amount of cytokine production decreased in a concentration-dependent manner in the groups treated with extracts of soybean bean (SB) and soybean pods (SBP). Significant reduction efficacy was confirmed in the peach pod (SBP) treatment group compared to the (SB) treatment group (Figures 7a and 7b). In the case of the anti-inflammatory cytokine IFN-γ, it was confirmed that the amount of cytokine production decreased due to hypersensitivity and then increased significantly due to soybeans and dodu pods (Figure 7c).

<Experimental Example 3> Human application test

(1) Human application test design

A single-center, randomized, double-blind, placebo-controlled human application trial was conducted for 6 weeks on patients with confirmed allergic nasal symptoms. The protocol for human application testing was approved by the Institutional Review Board of Dankook University Hospital.

(2) Selection of subjects for human application testing

Semi-healthy adult men and women aged 19 to 65 were recruited, and were finally selected after a visit to confirm medical history, physical examination, and blood and urine tests to confirm disqualification reasons. Those whose WBC is within the normal range (4~ ¹⁰ People who agreed not to receive drug therapy, non-drug therapy, or other treatments for rhinitis treatment were selected as subjects for human application testing.

(3) Administration method

The human application test subjects who went through the above selection process were randomly divided into 2 groups, a placebo group (capsules of the same formulation as the test group), and a test group (polypod pod extract capsules), and the test was conducted for 6 weeks. The schedule for evaluation consisted of three visits at week 0 (including a visit for testing to select subjects for human application testing), week 3, and week 6. During the 6-week evaluation period, participants in the human application test took 3 tablets every day for a set period of time. Test drugs were provided by human application researchers every three weeks, and rTNSS symptoms were evaluated in the morning and evening during the test drug intake period and were instructed to record them in a diary.

(4) Outcome measurement

Changes in concentration of cytokines (INF-γ, TNF-α, IL-4, IL-5), change in white blood cell count, change in concentration of immunoglobulin IgE, change in concentration of ECP, Histamine, Prostaglandin, 3 and 6 weeks after baseline. We plan to check the change in leukotriene concentration.

Claims (13)

A pharmaceutical composition for preventing or treating allergic diseases, characterized in that it contains peach pod extract as an active ingredient. The method of claim 1, wherein the allergic disease includes hypersensitivity, allergic rhinitis, asthma, anaphylactic shock, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, eczema, insect allergy, food allergy, hay fever, and plant allergy. A pharmaceutical composition for preventing or treating allergic diseases, characterized in that it prevents or improves allergies or drug allergies. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the extract is obtained from immature peach pods. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the extract is 20 to 40% alcohol extract. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the composition suppresses NO production.
The method of claim 1, wherein the composition reduces gene expression of iNOS, COX-2, and IL-6, and reduces the expression of proteins of iNOS, COX-2, and IL-6. Pharmaceutical composition for therapeutic use. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the composition increases gene expression of the anti-inflammatory cytokine IFN-γ and increases production of the cytokine IFN-γ. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the composition reduces the expression levels of p-IκBα and p-p65. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the composition reduces histamine and IgE. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the composition reduces the production of inflammatory cytokines (IL-4, IL-5). The pharmaceutical composition for preventing or treating allergic diseases according to claim 1, wherein the composition has a pinitol content that is 15% to 25% higher than that of soybeans. A health functional food composition for preventing or improving allergic diseases, characterized in that it contains peach pod extract as an active ingredient. A cosmetic composition for preventing or improving allergic diseases, characterized in that it contains peach pod extract as an active ingredient.