KR20220081768A - Composition comprising trifuhalol A or its physiologically acceptable salt, and use thereof - Google Patents

Abstract

Provided are a composition comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient, and uses thereof.

Description

Composition comprising trifuhalol A or its physiologically acceptable salt, and use thereof

A composition comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient, and uses thereof.

Unlike the immune response that tries to protect the invading part when a foreign antigen invades the body, hypersensitivity refers to an abnormal immune response to something that is not harmful to our body, and is a type of immune disorder that destroys tissues. Immune diseases are caused by dysfunction of the immune system, and include autoimmune diseases, immunodeficiency disorders, and allergic diseases.

Allergic diseases are increasing every year, and in the 2000s, it increased to more than 5%, and it is estimated to exceed 10% if you include potential patients. Allergy is a pathological process in the living body that appears as a result of an antibody reaction with an antigen called allergen that causes hypersensitivity reaction, and the most common allergic reaction is immediate type hypersensitivity that causes bronchial asthma, allergic rhinitis, hay fever, and atopic dermatitis. There is a type 1 reaction, which is a reaction, and a type 4 reaction, such as a chronic inflammatory response.

Allergic reactions such as anaphylaxis include binding of antigens to Fc receptors such as mast cells, anti-IgE antibodies, lectins, etc., stimulation of anaphylotoxins, etc., stimulation of the synthetic cortex such as calcium ionophore, compound 48/80, codeine, etc. It is caused by drugs such as hormones. When blood vessels are dilated due to an abnormal immune response caused by a foreign antigen, blood flows in well, but instead, blood pressure is lowered. As it is secreted in large amounts at the same time, the blood vessels of the whole body, not just a part, expand, and then the blood pressure suddenly drops and shock can occur. In addition, as histamine is secreted throughout the body, the airways also become enlarged, blocking the only pathway for oxygen-carbon dioxide exchange, which can result in suffocation. The airways include the tongue, airways and bronchi. Asthma is a chronic inflammatory disease of the airways characterized by paroxysmal dyspnea, persistent cough, wheezing, bronchial hyperresponsiveness, and reversible airflow limitation. It is a common allergic respiratory disease in modern society. The pathological characteristics of asthma include constriction of airway smooth muscle, airway atresia due to airway hypersensitivity, hypersecretion of airway mucus, mucosal thickening due to edema and cell infiltration, accumulation of abnormally thick and sticky mucus in the airway lumen, chronic There are airway inflammation, immunoglobulin E antibody formation, and the like, and it is known that airway resistance is increased due to the above pathological characteristics, causing repeated and intermittent coughing, dyspnea, chest tightness, and wheezing.

Allergic rhinitis is a disease in which symptoms such as paroxysmal and repetitive sneezing, clear runny nose, stuffy nose, and itchy nose appear due to hypersensitivity of the inner part of the nose to allergens. Recently, with the increase of environmental pollution and pollution, allergic rhinitis is increasing worldwide. This disease is so common that 5-20% of the total population suffers from it. Drug therapy for the treatment of allergic rhinitis includes antihistamine pills, nasal sprays, leukotriene receptor antagonists, steroid nasal sprays, and nasal mucosal constrictors.

Atopic dermatitis is a chronic, recurrent inflammatory skin disease accompanied by itching, dry and flaky skin, inflammation, increased skin permeability, and sensitivity to common and innocuous factors. disease that is susceptible to

Lymphocytes in atopic dermatitis lesions have an increased CD4:CD8 ratio of about 7:1 and have strong reactivity to interleukin-4 (IL-4), so that the immune response by Th2 helper T cells is dominant. do. Histologically, hyperkeratosis, dyskeratosis, acanthosis, intercellular edema, dilatation of small blood vessels, and perivascular cell infiltration such as eosinophils, lymphocytes, and histiocytic cells appear in the upper dermis. Recently, in addition to immunological abnormalities, damage to the skin barrier function is considered as one of the important factors. The damage to the barrier function is due to a decrease in ceramide in the stratum corneum lipids, and the moisture retention and skin barrier function of the skin are lowered, which can increase the skin permeability of allergens or irritants and cause or aggravate the inflammatory reaction of the skin.

In addition, CCL26 is a member of the eotaxin subfamily consisting of CCL11/eotaxin-1, CCL24/eotaxin-2, and CCL26/eotaxin-3, and is well known for its major chemotactic role on eosinophils and basophils. . Eotaxin is upregulated in skin lesions of atopic patients and plays an important role in the pathogenesis of atopic dermatitis.

The major factors of atopic dermatitis known to date include genetic factors, immunological factors, and environmental factors. For example, interleukin-4, interleukin-5, interleukin produced when Th2 helper T cells are activated by a factor caused by a filaggrin gene mutation that causes defects in the skin barrier due to a deficiency in filaggrin gene expression. -13 stimulates B cells to promote the production of Immunoglobulin E (IgE) and release histamine from mast cells to induce hypersensitivity reactions. Immunological factors caused by Th1/Th2 imbalance, stimulating skin or antigen It is known that atopic dermatitis develops due to complex interactions such as factors caused by the environment exposed to bacteria and bacteria.

When mast cells are activated by an overactive immune response, pruritogens such as histamine are secreted, which causes the skin barrier to collapse. Therefore, if a substance that can minimize side effects and suppress itch mediators is discovered, it will be effective in controlling various allergic diseases including atopic dermatitis.

A treatment for hypersensitivity allergic disease has not yet been established. According to the severity of atopic dermatitis, there are moisturizers, topical steroids, topical immunomodulators, antihistamines, and antibiotics. Since most of the currently used hypersensitivity allergy treatments are not fundamental treatments, the cure rate is low and the recurrence rate is high, so there is a risk for long-term use for chronic recurrent allergic diseases that require long-term treatment. Therefore, there is a need to develop a therapeutic agent for hypersensitivity allergy disease that can replace it, can reduce the use of existing therapeutic agents, and have no side effects even after long-term use.

One aspect provides a pharmaceutical composition for use in preventing or treating allergic diseases and diseases associated with vasodilation, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient.

Another aspect provides a food composition for use in improving allergic diseases and diseases associated with vasodilation, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient.

Another aspect provides a cosmetic composition for use in maintaining or improving skin moisture content comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient.

Another aspect provides a method for preventing or treating allergic diseases and diseases associated with vasodilation in a subject, comprising administering the pharmaceutical composition to the subject.

Another aspect provides a method for making-up an individual, comprising administering the cosmetic composition to the individual.

One aspect provides a pharmaceutical composition for use in preventing or treating allergic diseases and diseases associated with vasodilation, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient.

The allergic disease may be a hypersensitivity allergic disease. The allergic disease may be selected from the group consisting of atopic dermatitis, asthma, rhinitis, anaphylactic shock, and itch.

The disease associated with vasodilation may be a disease including symptoms caused by vasodilation. The disease associated with vasodilation may be one selected from the group consisting of hot flashes, nasal congestion, and hypotension.

The tripuharol A or a physiologically acceptable salt thereof constricts dilated blood vessels to reduce the outflow of disease-causing substances from blood vessels, to reduce water loss from the skin, and to increase skin water content , and reducing the skin or epidermal thickness at the dermatitis lesion site.

The tripuharol A or a physiologically acceptable salt thereof may be in the form of an extract or a fraction thereof, containing the same.

In the present specification, the extract has the meaning commonly used as a crude extract in the art, but in a broad sense also includes a fraction obtained by additionally fractionating the extract. Accordingly, the extract may be a crude extract, a fraction, or a combination thereof. The crude extract may be one obtained by contacting a plant with fermented seaweed and an extraction solvent. The fraction may be obtained by separating a material containing a specific component with respect to the crude extract. The separation may be by filtration, immersion, centrifugation, chromatography, or a combination thereof. The chromatography is prepared for separation according to various conditions, ie, size, charge, hydrophobicity or affinity, and is ion exchange chromatography, affinity chromatography, size exclusion chromatography, HPLC, high-performance flow chromatography, column chromatography, reversed phase column chromatography or a combination thereof. In the extract, the low-polarity solvent used for extraction may be removed after extraction.

The extraction may include incubating the plant in a solvent for a predetermined time. The extraction may be performed without stirring or stirring, or may include heating. The incubation may be carried out without stirring or stirring at room temperature to reflux temperature. The incubation temperature may be appropriately selected depending on the selected solvent. For example, it may be room temperature to reflux temperature, 30 °C to reflux temperature, 40 °C to reflux temperature. The heating may include heating to 50°C, 60°C, 70°C, 80°C, or reflux temperature. The heating may be 50°C to reflux temperature, 60°C to reflux temperature, 70°C to reflux temperature, 80°C to reflux temperature, or heating to reflux temperature. The extraction time may vary depending on the selected temperature, for example, from 1 hour to 2 months, for example, from 1 hour to 1 month, from 1 hour to 15 days, from 1 hour to 10 days, from 1 hour to 5 days, from 1 hour to 3 days. , 1 hour to 2 days, 1 hour to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 days, 5 hours to 1 day, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days, or 10 hours to 2 days. The extraction may be reflux extraction of the plant in a solvent. The solvent may have a volume of 1 times, 2 times, 5 times, 10 times, or 15 times or more with respect to the weight of the plant. The solvent may have a volume of 1 to 15 times, 2 to 15 times, 5 to 15 times, 10 to 15 times, or about 15 times the weight of the plant. The plant may be dried in the shade.

As the extraction method, conventional methods in the art, such as filtration, hot water extraction, immersion extraction, immersion extraction, microwave extraction, reflux cooling extraction, pressure extraction, subcritical extraction, supercritical extraction, and ultrasonic extraction, may be used. For example, it may be an immersion extraction method. Immersion extraction may be immersion at room temperature or warm immersion, and may be extracted 1 to 5 times. The seaweed plant may be in contact with 0.1 to 10 times or 1 to 6 times the extraction solvent. The cold extraction temperature may be in the range of 20°C to 40°C. The hot extraction or heat extraction temperature may be 40 °C to 100 °C. The cold extraction time may be 24 to 120 hours, and the hot or heated extraction temperature may be 0.5 to 48 hours.

The extraction may also include removing the solvent from the obtained extract by a known method such as concentration under reduced pressure. The extraction may also include preparing a dry extract by drying the obtained extract, such as freeze-drying. The reduced pressure concentration may be using a vacuum reduced pressure concentrator or a vacuum rotary evaporator. In addition, drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying.

The extract may be obtained by extracting the seaweed extract with a low-polarity solvent. The low polar solvent is acetone, methyl acetate, ethyl acetate, n-butyl acetate, t-butyl methyl ether, dimethyl sulfoxide, diethyl ether, heptane, pentane, methyl ethyl ketone, methyl isobutyl ketone, cyclohexane, petroleum ether , dichloromethane, dichloroethane, 1,2-dichloroethene, dichloromethane, hexane, or a combination thereof, or an aqueous solution thereof. The aqueous solution may be an aqueous solution in which the content of the solvent mixed with water is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

The fraction may be a fraction of hexane, ethyl acetate, dichloroethane, or water.

The physiologically acceptable salt may be a pharmaceutically acceptable salt.

The tripuharol A or a physiologically acceptable salt thereof may be isolated as a single component, rather than including a complex component, such as an extract of fermented seaweed.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier may be any carrier known in the art. The carrier may be, for example, a diluent, lubricant, thickener, disintegrant, buffer, preservative, flavoring agent, or binder.

The pharmaceutical composition may be in an oral or parenteral dosage form. The oral dosage form may be a tablet, patient, powder, granule, capsule, or troche. The parenteral dosage form may be a liquid formulation.

Another aspect provides a food composition for use in improving allergic diseases and diseases associated with vasodilation, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient.

In the food composition, if there is no mention of "triphuharol A or a physiologically acceptable salt thereof", "allergic disease" and "disease associated with vasodilation", it is the same as described for "pharmaceutical composition" . The physiologically acceptable salt may be a food acceptable salt.

The food composition may include a food pharmaceutically acceptable carrier. The carrier may be any carrier known in the art. The carrier may be, for example, a diluent, lubricant, thickener, disintegrant, buffer, preservative, flavoring agent, or binder.

Another aspect provides a cosmetic composition for use in maintaining or improving skin moisture content comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient.

In the food composition, if there is no mention of "triphuharol A or a physiologically acceptable salt thereof", "allergic disease" and "disease associated with vasodilation", it is the same as described for "pharmaceutical composition" . The physiologically acceptable salt may be a cosmetically acceptable salt.

The cosmetic composition may include a cosmetically acceptable carrier. The carrier may be any carrier known in the art. The carrier may be, for example, a diluent, lubricant, thickener, disintegrant, buffer, preservative, flavoring agent, or binder.

Another aspect provides a method for preventing or treating allergic diseases and diseases associated with vasodilation in a subject, comprising administering the pharmaceutical composition to the subject.

In the method, the subject may be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat.

In the method, the dosage may be an amount effective for preventing or treating allergic diseases and diseases associated with vasodilation. The dosage may vary depending on the selected subject, the subject's age, weight, sex, dosage form, health status, and disease severity.

The administration may be oral administration or parenteral administration. The administration may be, for example, intravenous, intraperitoneal, intradermal, subcutaneous, epithelial or intramuscular administration.

The allergic disease may be selected from the group consisting of atopic dermatitis, asthma, rhinitis, anaphylactic shock, and itch. The disease associated with vasodilation may be one selected from the group consisting of hot flashes, nasal congestion, and hypotension.

Another aspect provides a method for making-up an individual, comprising administering the cosmetic composition to the individual. The method may be for maintaining or improving skin moisture content.

According to the pharmaceutical composition according to an aspect, it can be used to prevent or treat allergic diseases and diseases associated with vasodilation in a subject.

According to the food composition according to one aspect, it can be used to improve allergic diseases and diseases associated with vasodilation in a subject.

According to the cosmetic composition according to an aspect, it can be used to maintain or improve skin moisture content in an individual.

According to the method for preventing or treating allergic diseases and diseases associated with vasodilation in an individual according to an aspect, it is possible to effectively prevent or treat allergic diseases and diseases associated with vasodilation in an individual.

According to the method of putting on makeup on an object according to an aspect, it is possible to efficiently put on makeup on the object.

1 is a graph showing the result of measuring the degree of degranulation after treatment with seaweed in RBL-2H3 cells.
Figure 2 is a graph of the result of measuring the degree of CCL26 after treatment with seaweed in HaCaT cells.
3 is a graph showing the results of measuring the degree of degranulation after tripuharol A treatment in RBL-2H3 cells.
4 is a graph showing the result of measuring the degree of CCL26 after tripuharol A treatment in HaCaT cells.
5 is a graph showing the hypersensitivity reaction inhibitory effect after induction of anaphylactic shock with compound 48/80 after treatment with seaweed in ICR mice.
6 is a graph showing the hypersensitivity reaction inhibitory effect after induction of anaphylactic shock with compound 48/80 after tripuharol A treatment in ICR mice.
7 is a graph showing the inhibitory effect of itch after inducing itch with compound 48/80 after treatment with fern seaweed in ICR mice.
8 is a graph showing the inhibitory effect of itch after induction of itch with compound 48/80 after treatment with tripuharol A in ICR mice.
9 is a photograph showing the difference in blood vessels by treating the fertilized egg with an extract and its fractions from fertilized egg.
10 is a graph showing the reduction rate of microvessels by treating the fertilized egg with an extract and its fractions from fertilized egg.
11 is a graph showing the diameter of blood vessels by treating a fertilized egg with an extract and a fraction thereof.
12 is a graph showing the reduction rate of microvessels by treating fertilized eggs with minoxidil to dilate blood vessels, and then treating the fertilized egg with an extract and its fractions.
13 is a graph showing the diameter of blood vessels by treating the fertilized egg with minoxidil to dilate the blood vessels, and then treating the fertilized egg with an extract and its fractions.
14 is a photograph showing the phenotype of mice and the like over time after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
15 is a graph showing the inhibitory effect of itch after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
16 is a graph showing the degree of visual improvement of dermatitis symptoms after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
17 is a graph showing the results of measuring the amount of transdermal water loss (TEWL) over time after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
18 is a graph showing the results of measuring the moisture content (hydration) of the skin over time after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
19 is a graph showing the results of measuring the amount of IL-4 in plasma after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
20 is a graph showing the results of measuring the amount of IgE in plasma after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
FIG. 21 is a photograph in which the skin tissue was stained with hematoxylin and eosin after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
22 is a graph showing the results of measuring the epidermal thickness after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
23 is a photograph showing toluidine blue staining of skin tissue after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.
24 is a graph showing the result of measuring the number of mast cells after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

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

Example 1. Preparation of Acetone Extract

The seaweed used in the present invention was directly collected and used in December 2015 from the coast of Gangneung, Gangwon-do, Republic of Korea. After collection, sand and salt were washed several times with tap water to remove them, rinsed with purified water, and then dried in a dark place at room temperature for 5 days. After grinding the dried seaweed, it was added to 70% acetone at a ratio of 35mL of 70% acetone to 1 g of seaweed and was heated at 30℃ using an ultrasonic device (Flexonic-500, Mirae Ultrasound Technology, Korea) for 5.75 hours. The extraction was conducted by irradiating ultrasonic waves. Ultrasonic irradiation conditions were 35 kHz and 56.58 W/cm ² . The extract thus obtained was concentrated under reduced pressure and then lyophilized to be used as a sample. (Yield: 9.9%)

Example 2. Preparation of fractions from fermented wakame seaweed

After suspending the 70% acetone extract of Example 1 in the same amount of water, n-hexane, dichloroethane (DCE), and ethyl acetate (ethyl acetate, EA) were performed in the order of fractionation (consecutive solute fractionation) to obtain four fractions. got it That is, the extract suspended in the same amount of purified water + n-hexane as a fractionation solvent was added, shaken vigorously several dozen times, and then submerged to obtain an n-hexane layer. This process was performed three times in total. The remaining DCE and EA fractions were obtained in the same manner. The remaining fraction was concentrated to obtain a water fraction. The yield of each fraction was 10.9%, 23.9%, 11.7%, and 53.4%, respectively, based on the acetone extract.

Example 3. Isolation of tripuharol A

The EA fraction of fermented seaweed of Example 2 was used for compound isolation. Sephadex LH-20 Column while flowing the solvent in a stepwise gradient of water/methanol (1/1 -> 1/3 -> 0/1) and methanol/acetone (5/1 -> 3/1 -> 1/1) A total of 6 sub-fractions were obtained by chromatography. Among them, subfractions 5 and 6 were collected and concentrated under reduced pressure/lyophilized, and it was confirmed that the main component was tripuharol A through HPLC/MS, ¹ H NMR, and ¹³ C NMR.

H NMR (600 MHz, CD ₃ OD): 5.89 (d, 2H, J = 2.6 Hz, Ph-H), 5.90 (s, 2H, Ph-H), 5.92 (s, 2H, Ph-H). ¹³ C NMR (150 MHz, CD ₃ OD): 95.54 (2C, CH), 95.54 (2C, CH), 98.11 (2C, CH), 125.13 (1C, C), 125.78 (1C, C), 147.99 (1C) , C), 152.48 (2C, C), 152.62 (2C, C), 152.71 (2C, C), 158.77 (1C, C), 158.78 (1C, C), 160.75 (1C, C). HPLC-DAD- QMS m/z : 413.1 (M+Na ⁺ ).

Example 4. Confirmation of suppression of immune cell degranulation of seaweed and tripuharol A

In order to confirm whether the fermented seaweed inhibits the degranulation of immune cells to release inflammatory substances, beta-hexosaminidase activity was measured.

Immune cells, rat basophilic leukemia cells (rat basophilic leukemia cells), RBL-2H3 cells were purchased from Korea Cell Line Bank (Korea Cell Line Bank, Seoul, Korea) was used. After suspending RBL-2H3 cells in DMEM (Cat. No. sh30284.01, HYCLONE, USA) containing 10% FBS, aliquots at a density of 5x10 ⁵ cells/well in a 24-well plate at 37°C, 5% CO It was cultured under the conditions of ₂ . After 16 hours of incubation, an anti-DNP-IgE antibody (Cat. No. D8406, Sigma, USA) was added to the wells at a concentration of 100 ng/ml, followed by incubation at 37° C. and 5% CO ₂ conditions for 4 hours. Cells were then washed twice with siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, 5.6 mM glucose, 1 mM CaCl ₂ , and 0.1% BSA). and 50 µM of ketotifen, 10 µg/mL or 30 µg/mL or 100 µg/mL of 70% acetone obtained in Example 1 or EA fraction, or 10 µM or 30 µM or 100 µM of Each 200 μl of ciraganian buffer containing tripuharol A was added and cultured at 37° C., 5% CO ₂ for 1 hour. PIPES stands for piperazine-N,N'-bis(2-ethanesulfonic acid). Thereafter, DNP-BSA as an antigen was added at a concentration of 100 ng/ml, reacted for 1 hour, and left in an ice bath for 10 minutes to terminate the reaction. DNP stands for dinitrophenyl. Then, each reaction solution was obtained and centrifuged at 2,000 rpm in the supernatant to be beta-hexosaminidase substrate 4-nitrophenyl N-acetyl-β-D-glucosaminide (4-Nitrophenyl N-acetyl-β -D-glucosaminide) was added at 1 mM, reacted at 37° C. for 1 hour, and 0.1M NaCO ₃ /NaHCO ₃ solution was added to stop the reaction. The absorbance of each reaction solution was measured with a microplate spectrophotometer (BioTek, USA) at a wavelength of 405 nm, and the beta-hexosaminidase activity of each treated group was calculated as a percentage of the activity of the untreated group. Table 1 and FIG. 1 .

sample Beta-hexosaminidase activity (%) untreated group 100.00 IgE + DNP-BSA 297.13 IgE + DNP-BSA + Ketotifen 50 uM 183.11 IgE + DNP-BSA + 10 µM 177.61 IgE + DNP-BSA + 30 μM 162.69 100 uM of IgE + DNP-BSA + Seaweed extract 160.12 10 uM of IgE + DNP-BSA + EA fraction 202.06 30 uM of IgE + DNP-BSA + EA fraction 177.93 100 uM of IgE + DNP-BSA + EA fraction 166.46

1 is a graph showing the results of measuring the degree of degranulation after treatment with seaweed in RBL-2H3 cells.

As a result, as shown in Table 1 and Fig. 1, it was confirmed that the extract and the EA fraction significantly inhibited degranulation in RBL-2H3 cells. This means that seaweed can be helpful in alleviating symptoms of atopic dermatitis, such as itching or a rapid allergic reaction caused by immune cell degranulation.

In addition, the same activity was measured and confirmed for tripuharol A isolated from seaweed.

sample Beta-hexosaminidase activity (%) untreated group 100.00 IgE + DNP-BSA 333.46 IgE + DNP-BSA + Ketotifen 50 uM 255.51 IgE + DNP-BSA + tripuharol A 10 uM 245.59 IgE + DNP-BSA + Tripuharol A 30 uM 229.39 IgE + DNP-BSA + Tripuharol A 100 uM 171.09

3 is a graph showing the results of measuring the degree of degranulation after tripuharol A treatment in RBL-2H3 cells.

As a result, as shown in Table 2 and FIG. 3, it was confirmed that tripuharol A significantly inhibited degranulation in RBL-2H3 cells. This means that tripuharol A can be helpful in alleviating symptoms of atopic dermatitis, such as itching or a rapid allergic reaction caused by immune cell degranulation.

Example 5. Confirmation of suppression of CCL26 expression of seaweed and tripuharol A

CCL26 plays a major chemotactic role for eosinophils. In addition, the expression of CCL26 is up-regulated in the lesions of hypersensitivity allergic disease patients, and plays an important role in the pathogenesis of hypersensitivity allergic disease. It was confirmed whether or not seaweed inhibits the expression of CCL26.

Specifically, human HaCaT keratinocytes were purchased from Accegen biotechnology (Cat. No. ABC-TC536S, New Jersey, USA) and used. HaCaT cells were suspended in DMEM containing 10% FBS and then aliquoted at a density of 5x10 ⁵ cells/well in a 6-well plate and cultured at 37° C. and 5% CO ₂ conditions. After 16 hours of incubation, the sample was pretreated in DMEM containing 1% charcoal-dextran-treated (CDT) FBS at a concentration of 10ug/ml, 30ug/ml, or 100ug/ml for 1 hour. , IL-4 (Cat. No. SRP4137, Sigma, USA) was added to the wells at a concentration of 20 ng/ml, followed by incubation at 37° C. and 5% CO ₂ conditions for 24 hours. The samples are the 70% acetone extract of Example 1, the EA fraction of Example 2, and tripuharol A of Example 3. Cells were mounted on a QIAsymphony or QIAQube (Qiagen) instrument using RNeasy mini kit (Qiagen) to extract RNA. The extracted RNA was checked for integrity using an Agilent 2100 BioAnalyzer (Agilent Technologies, SantaClara, CA, USA). cDNA was synthesized from 1 ug RNA as cDNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). 0.02 μl Ex taq polymerase (TAKARA, Otsu, Shiga, Japan), 2 μl Ex taq polymerase buffer, 1.6 μl dNTP (10 mM), 2 μl forward primer (20 μM), 2 μl reverse primer (20 μM), 11.38 μl of nuclease-free water and 1 μl of synthesized first-strand cDNA were well mixed and PCR was performed in a total volume of 20 μl. The expression of CCL26 in each treatment group was calculated as a ratio to the activity of the untreated group, and is shown in Table 3 and FIG. 2 .

sample CCL26 mRNA expression untreated group One IL-4 28.65 IL-4 + 10 uM fermented seaweed extract 18.65 IL-4 + Seaweed extract 30 uM 21.35 IL-4 + Seaweed extract 100 uM 19.66 IL-4 + seaweed EA fraction 10 uM 22.43 IL-4 + Seaweed EA fraction 30 uM 21.77 IL-4 + Seaweed EA fraction 100 uM 17.65

Figure 2 is a graph of the result of measuring the degree of CCL26 after treatment with seaweed in HaCaT cells.

As a result, as shown in Table 3 and FIG. 2 , it was confirmed that the seaweed remarkably inhibited CCL26 in HaCaT cells, which means that it could be helpful in alleviating symptoms of itch and atopic dermatitis.

In addition, it was confirmed whether tripuharol A isolated from fermented seaweed inhibits the expression of CCL26.

sample CCL26 mRNA expression untreated group One IL-4 35.72 IL-4 + Tripuharol A 10 uM 23.66 IL-4 + Tripuharol A 30 uM 20.87 IL-4 + Tripuharol A 100 uM 15.64

4 is a graph showing the result of measuring the degree of CCL26 after tripuharol A treatment in HaCaT cells.

As a result, as shown in Table 4 and Figure 4, it was confirmed that tripuharol A significantly inhibited the expression of CCL26 in HaCaT cells. This means that tripuharol A may help relieve itching and atopic symptoms.

Example 6. Confirmation of anaphylactic shock inhibitory effect in compound 48/80 induced hypersensitivity ICR mouse of fermented seaweed and tripuharol A

In order to confirm the effects of extracts, fractions, and compounds on anaphylaxis hypersensitivity reaction using compound 48/80, the experiment was conducted as follows.

8-week-old ICR mice (Orient Bio, Korea) were separated into 5 groups of 9 mice each. After oral administration, 70% acetone extract, EA fraction, and tripuharol A were dissolved in PBS at 200 mg/kg and 1 hour after oral administration, compound 48/80 (sigma-aldrich, USA) was dissolved in PBS at 10 mg/kg. By intraperitoneal administration, an anaphylactic reaction was induced, and the mouse mortality was measured in units of 5 minutes for a total of 1 hour, and the results were shown.

sample Survival (%) untreated group 100 Compound 48/80 10mg/kg 33.3 Seaweed extract 200mg/kg 77.7 Seaweed EA fraction 200mg/kg 66.6

5 is a graph showing the hypersensitivity reaction inhibitory effect after induction of anaphylactic shock with compound 48/80 after treatment with seaweed in ICR mice.

As a result, as shown in Table 5 and Figure 5, the survival rate was increased in the group treated with the acetone extract and EA fraction of the seaweed seaweed compared to the group induced by anaphylaxis with the compound 48/80. As a result, it was confirmed that the extracts and fractions of the seaweed extract have a protective effect against anaphylactic shock. In addition, isolated from the seaweed

The survival rate for anaphylactic shock induced by compound 48/80 by oral administration of tripuharol A 200mg/kg was confirmed. In FIG. 5 , ACAE indicates an extract of fermented seaweed, and ACEAF indicates an EA fraction of the extract of fermented seaweed.

sample Survival (%) untreated group 100 Compound 48/80 10mg/kg 33.3 Tripuharol A 200mg/kg 66.6

6 is a graph showing the hypersensitivity reaction inhibitory effect after induction of anaphylactic shock with compound 48/80 after tripuharol A treatment in ICR mice.

As a result, as shown in Table 6 and FIG. 6 , the survival rate was increased in the tripuharol A-administered group compared to the group induced by anaphylaxis with compound 48/80. As a result, it was confirmed that tripuharol A had a synergistic effect on anaphylactic shock.

Example 7. Confirmation of itch inhibitory effect in compound 48/80 induced itch ICR mice of seaweed and tripuharol A

The experiment was carried out as follows to confirm the effect of the extract and the compound of the compound 48/80 on histamine-mediated itchiness.

Five-week-old ICR mice (Orient Bio, Korea) were separated into three groups in five groups. A positive control group (terfenadine; sigma-aldrich, 4mg/kg), PBS, and a seaweed extract (20mg/kg) were orally administered, and after one hour, the compound 48/80 was dissolved in PBS at 50ug/kg and then the untreated group Except for terfenadine, PBS, and the extract group of fermented seaweed extract group, it was subcutaneously administered to the back of the neck. Scratching was observed for 1 hour after itch induction, and the results were shown by measuring the mouse scratching 3 to 4 times using its hind paws.

sample Number of scratching untreated group 18 PBS + Compound 48/80 treatment group 81.66 Terfenadine + Compound 48/80 treatment group 28.66 Seaweed extract 20mg/kg + Compound 48/80 treatment group 39.33

7 is a graph showing the inhibitory effect of itching after inducing itching with compound 48/80 after treatment with the extract of fermented seaweed in ICR mice.

As a result, as shown in Table 7 and FIG. 7, it was confirmed that the itchiness was improved by the pruritus extract group as the scratching behavior was reduced compared to the mouse induced with the compound 48/80.

In addition, the effect of improving the itchiness induced by compound 48/80 was confirmed by oral administration of tripuharol A (20 mg/kg) isolated from seaweed.

sample scratching untreated group 18 PBS + Compound 48/80 treatment group 81.66 Terfenadine + Terfenadine treatment group 28.66 Tripuharol A 20mg/kg + Compound 48/80 treatment group 51.33

8 is a graph showing the inhibitory effect of itch after induction of itch with compound 48/80 after treatment with tripuharol A in ICR mice.

As a result, as shown in Table 8 and FIG. 8, it was confirmed that the tripuharol A administration group showed a decrease in scratching compared to the compound 48/80 induction group, so that tripuharol A could help improve itching.

Example 8. Confirmation of fertilized egg serous vasoconstrictor effect of fertilized oviduct seaweed and tripuharol A

In order to evaluate the effect of fermented seaweed extract and its fractions on vasoconstriction and microvessels, macroscopic changes in blood vessels in the chorionic allantoic membrane of fertilized eggs were measured on the 10th day of hatching. After culturing the fertilized eggs at 37.5±0.5°C and 55±5% for 10 days, air chambers were observed to select only those with well-developed ileus. Using the selected eggs, the air chamber was opened, 0.9% sodium chloride solution was put in, and the inner membrane and the intestinal membrane were separated at 37°C for 30 minutes. The serous seaweed extract and its fractions were suspended in 0.9% sodium chloride solution at concentrations of 1% and 2% in the separated enteric membrane, 300 μl of the sample was applied, and reacted for 30 seconds, and stimulation of the urinary membrane with 0.9% sodium chloride solution washed so as not to The degree of vasoconstriction was evaluated at 30 sec, 60 sec, 120 sec, and 300 sec after washing.

The number of microvessels was measured using the HKBASIC program (KOPTIC, korea) by taking pictures of the serous membrane before and after sample processing, and the rate of microvascular change was the number of microvessels in the serous membrane before sample processing and the number of microvessels in the serous membrane after sample processing. was compared as a percentage. (Equation 1: 100 x number of microvessels after sample processing / number of microvessels before sample processing).

The blood vessel diameter was measured using the HKBASIC program (KOPTIC, korea) to measure the diameter of the largest blood vessel in the serous membrane, and the diameter of the blood vessel before and after sample treatment was measured and expressed as a percentage based on the diameter before treatment. . (Equation 2: 100 x vessel diameter after sample treatment / vessel diameter before sample treatment). Tables 9 and 10 show the changes in microvessels and diameters of each treatment group after 300 seconds as a percentage of those before treatment. The microvascular change rate and the blood vessel diameter change rate were calculated according to Equations 1 and 2 above.

sample Microvascular change rate (%) untreated group 100 1% seaweed extract 59.6 Seaweed extract 2% 32.0 Seaweed EA fraction 1% 22.2 Seaweed EA fraction 2% 48.4

sample Blood vessel diameter change rate (%) untreated group 100 1% seaweed extract 69.61 Seaweed extract 2% 75.06 Seaweed EA fraction 1% 84.41 Seaweed EA fraction 2% 96.82

9 is a photograph showing the difference in blood vessels by treating the fertilized egg with the extract and fractions of fertilized egg.

10 is a graph showing the reduction rate of microvessels by treating the fertilized egg with an extract and fractions from fertilized egg.

11 is a graph showing the diameter of blood vessels by treating the fertilized egg with an extract and fractions of fertilized egg.

As a result, as shown in Tables 9 and 10 and FIGS. 9, 10 and 11 , the extract and its fractions from porphyria reduced the number of microvessels and reduced the diameter of large blood vessels. This means that other allergic secondary reactions accompanying vasodilation by histamine secretion in hypersensitivity reactions can be suppressed. In addition, It indicates that the extract and fractions thereof can prevent, delay, ameliorate or treat diseases associated with vasodilation. The disease associated with vasodilation may be telangiectasia, erythema and/or flushing, nasal congestion, and hypotension. The disease associated with vasodilation may or may not be associated with inflammation-induced vasodilation.

9 is a photograph showing the difference in blood vessels by treating the fertilized egg with an extract and its fractions from fertilized egg.

In addition, in a model in which blood vessels were dilated with minoxidil (Sigma-aldrich, USA), the degree of effect of the extract and its fractions on the blood vessels dilated by minoxidil was evaluated.

Specifically, the effect on blood vessels was measured by processing the sample 3 minutes after treatment with minoxidil. The number of microvessels was measured using the HKBASIC program (KOPTIC, korea) by taking pictures of the serous membrane before and after sample processing. It is expressed as a percentage by comparison. The microvascular change rate was calculated according to Equation 1 below.

Equation 1: 100 x number of microvessels after sample processing / number of microvessels before sample processing

The blood vessel diameter was measured using the HKBASIC program (KOPTIC, korea) to measure the diameter of the largest blood vessel in the serous membrane, and the diameter of the blood vessel before and after sample treatment was measured and expressed as a percentage based on the diameter before treatment. .

Equation 2: 100 x vessel diameter after sample processing / vessel diameter before sample processing

Tables 11 and 12 show the changes in microvessels and diameters of each treatment group after 300 seconds of sample treatment as a percentage of those before treatment. The microvascular change rate and the blood vessel diameter change rate were calculated according to Equations 1 and 2 above.

sample Microvascular change rate (%) Minoxidil 1% + distilled water treatment group 130 1% minoxidil + 1% seaweed extract 72.41 1% minoxidil + 2% seaweed extract 18.18 1% minoxidil + 1% seaweed EA fraction 62 Minoxidil 1% + Seaweed EA fraction 2% 84.4

sample Blood vessel diameter change rate (%) Minoxidil 1% + distilled water treatment group 98.60 1% minoxidil + 1% seaweed extract 80.34 1% minoxidil + 2% seaweed extract 85.65 1% minoxidil + 1% seaweed EA fraction 91.65 Minoxidil 1% + Seaweed EA fraction 2% 87.64

Figure 12 is a graph showing the reduction rate of microvessels by treating fertilized chicken eggs with minoxidil to dilate blood vessels, and then treating the extract and its fractions from fertilized fertilized chicken. After dilating the blood vessels with minoxidil for 3 minutes, the sample was processed and measured after 300 seconds.

13 is a graph showing the diameter of blood vessels by treating fertilized chicken eggs with minoxidil to dilate blood vessels, and then processing the extract and its fractions from fertilized fertilized chicken.

As a result, as shown in Tables 11 and 12 and FIGS. 12 and 13 , the extract and its fractions from porcini seaweed reduced the number of microvessels increased by minoxidil and reduced the diameter of large blood vessels. This means that in hypersensitivity reaction, it is possible to suppress other secondary allergic reactions accompanied by vasodilation by histamine secretion.

In addition, it means that hot flashes, nasal congestion, hypotension, etc., which are diseases caused by the expansion of other blood vessels not related to inflammation-induced vasodilation, such as hypersensitivity allergy, can be improved.

Example 9. Confirmation of hypersensitivity allergy disease improvement effect of tripuharol A in NC/Nga mice with house dust mite-induced atopic dermatitis

The following experiment was performed to confirm the improvement of hypersensitivity and allergic diseases of tripuharol A, a compound isolated from seaweed in atopic dermatitis NC/Nga mice.

9-1. House dust mite-induced atopic dermatitis NC/Nga mouse model production and drug administration

6-week-old (female, 23-27 g) NC/Nga mice (Orient Bio, Korea) were separated into three groups: untreated group, house dust mite + vehicle treated group, and house dust mite + tripuharol A treated group (20 mg/kg). In the house dust mite + vehicle treatment group, house dust mites were applied to the hair-removed mice and then the vehicle was treated. it will be spread The vehicle is a solution of 2% carboxyl cellulose (CMC) and distilled water in a ratio of 1:3. The house dust mite is an AD Biostir (Cat. No. AD001, Central Experimental Animal, Seoul, Korea). The tripuharol A was dissolved in a solution of 2% carboxyl cellulose (CMC) and distilled water in a ratio of 1:3 to 20mg/kg/10ml. The untreated group was not treated with anything such as mice with hair removed.

Specifically, after group separation, after acclimatization for 7 days, 100 mg of house dust mite ointment was applied to the back of the mouse. When applying the second house dust mite ointment 4 days after the first application of the ointment, 4% SDS aqueous solution was applied and then 100 mg of house dust mite ointment was applied twice a week for a total of 6 times. The 4% SDS aqueous solution is to increase the skin permeability of house dust mites.

Tripuharol A 20mg/kg/10ml solution was orally administered to mice from the 1st day after the last house dust mite application to the 23rd day once a day in an amount of 20 mg/kg mice.

14 is a photograph showing the phenotype of mice and the like over time after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis. In FIG. 14 , non, HDM+vehichle, and HDM+TriA represent an untreated group, a house dust mite + vehicle treated group, and a house dust mite + tripuharol A treated group, respectively. In FIG. 14 , the horizontal axis represents the number of days elapsed from the day the house dust mite was applied to the hair-removed mouse and the like. In FIG. 14 , day 0 represents just before application of house dust mites.

As a result, as shown in FIG. 14 , compared to the house dust mite + vehicle treatment group, the amount of keratin on the skin of the mice was reduced in the house dust mite + tripuharol A treatment group, and eczema or appearance was eliminated.

9-2. Measurement of itch improvement effect due to hypersensitivity allergic disease

In order to confirm the improvement effect on itching caused by hypersensitivity allergic disease, in the house dust mite + vehicle treatment group described in 9-1, and the house dust mite + tripuharol A treatment group, after tripuharol A application, 1 hour on the last day of the experiment Scratching behavior was observed during the period, and the results were shown by measuring once when the mouse scratched 3 to 4 times using its hind paws.

number of scratching untreated group 16 house dust mite + vehicle 121 House dust mite + triphuharol A 61

As a result, as shown in Tables 13 and 15, the house dust mite + tripuharol A treatment group showed a decrease in scratching compared to the house dust mite + vehicle treatment group, so it was confirmed that tripuharol A improved itching.

15 is a graph showing the inhibitory effect of itch after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis. In FIG. 15 , non, vehichle, and TriA indicate an untreated group, a house dust mite + vehicle treated group, and a house dust mite + Trituharol A treated group, respectively.

9-3. Measurement of gross improvement effect on dermatitis symptoms among symptoms of hypersensitivity allergic disease

Since atopic dermatitis exhibits symptoms such as erythema, bleeding, scarring, and edema, the house dust mite induction group 23 days after application of the house dust mite in the house dust mite + vehicle treatment group described in 9-1, and the house dust mite + tripuharol A treatment group The score was converted based on the severity of the symptoms below. Erythema, bleeding, scarring, edema, erosion, etc. of mice were converted into dermatitis scores and compared. The dermatitis score was calculated according to Equation 5 below.

The dermatitis score was expressed as an average of erythema/bleeding 1-3 points, scarring 1-3 points, edema 1-3 points, and abrasion/scorching 1-3 points, plus the scores for each item.

Dermatitis score untreated group 0.33 house dust mite + vehicle 8 House dust mite + triphuharol A 0.33

16 is a graph showing the degree of visual improvement of dermatitis symptoms after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis. In FIG. 16 , non, vehichle, and TriA represent an untreated group, a house dust mite + vehicle treated group, and a house dust mite + Trituharol A treated group, respectively.

As a result, as shown in Tables 14 and 16, the house dust mite + tripuharol A treatment group significantly relieved symptoms such as erythema, bleeding, scarring, and edema compared to the house dust mite + vehicle treatment group.

9-4. Measurement of transdermal water loss and skin water content

For the mouse skin in the house dust mite + vehicle treatment group described in 9-1, and the house dust mite + tripuharol A treatment group, transEpidermal Water Loss (TEWL, TransEpidermal Water Loss) was measured using AquaFlux (Biox, London, UK). was measured, and skin hydration was measured using Skin-O-Mat (COSOMEP, Berlin, Germany). The percutaneous moisture loss and skin moisture content were measured before (day 0), 7 days, 14 days, and 21 days after induction of atopic dermatitis, a hypersensitivity allergic disease, by the house dust mite cream for the first time. The amount of percutaneous moisture loss by group is shown in Tables 15 and 17, and the skin moisture content is shown in Tables 16 and 18.

TEWL (g/m ² /h) untreated group 42.99 house dust mite + vehicle 83.48 House dust mite + triphuharol A 63.16

Moisture content (%) untreated group 32.8 house dust mite + vehicle 20.1 House dust mite + triphuharol A 27.6

17 is a graph showing the results of measuring the amount of transdermal water loss (TEWL) over time after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

18 is a graph showing the results of measuring the moisture content of the skin over time after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

As a result, as shown in Tables 15 and 17, tripuharol A lowered the amount of transdermal water loss by 24.3% compared to the control vehicle in NC/Nga mice induced by atopic dermatitis, a hypersensitivity allergic disease, by house dust mites. Confirmed. In addition, as shown in Tables 16 and 18, tripuharol A increased the water content by 35% or more compared to the control vehicle in NC/Nga mice induced by atopic dermatitis, a hypersensitivity allergic disease, by house dust mites. . Therefore, it was found that tripuharol A could significantly increase the moisture content of the skin by strengthening the skin barrier function compared to the vehicle in the NC/Nga mouse model of atopic dermatitis, a hypersensitivity allergic disease. This means that it is possible to prevent or improve hypersensitivity allergic diseases by strengthening the skin barrier and preventing the penetration of allergens.

9-5. Measurement of IgE and IL-4 levels in plasma

Marker of hypersensitivity allergic disease in mouse plasma 23 days after the experiment was over, that is, 23 days after application of house dust mite to mice treated with house dust mite + vehicle, and hair-removed mice in the house dust mite + tripuharol A treatment group as described in 9-1 As a result, the amounts of interleukin-4 (IL-4) and immunoglobulin E (IgE) were measured.

Specifically, after anesthetizing mice in each group, blood was collected from the abdominal aorta using a syringe containing 30 ul of 0.18 M EDTA. For plasma separated by centrifugation at 4°C and 8,000 rpm for 15 minutes using a centrifuge, IL-4 ELISA quantification kit (Bethyl Laboratories, USA) and IgE ELISA quantification kit (Bethyl Laboratories, USA) were used to determine IL- 4 and the amount of IgE were measured, the IL-4 measurement results are shown in Tables 17 and 19, and the total IgE measurement results are shown in Tables 18 and 20.

IL-4 (pg/mL) untreated group 13.15 house dust mite + vehicle 71.59 House dust mite + triphuharol A 38.64

IgE (ng/mL) untreated group 51.65 house dust mite + vehicle 242.31 House dust mite + triphuharol A 126.44

19 is a graph showing the results of measuring the amount of IL-4 in plasma after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

20 is a graph showing the results of measuring the amount of IgE in plasma after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

As a result, as shown in Table 17 and Figure 19, tripuharol A significantly reduced the amount of IL-4 in plasma compared to the control vehicle in NC/NGA mice induced by atopic dermatitis, a hypersensitivity allergic disease, by house dust mites. and as shown in Table 18 and FIG. 20, it was confirmed that tripuharol A also reduced the amount of total IgE.

9-6. Confirm histological changes

In order to confirm the epidermal thickness and histological changes in mice, the mice whose drug administration has been completed, i.e., house dust mites + vehicle treatment group described in 9-1, and house dust mites + triphuharol A treatment group, have hair removed from mice, etc. After 23 days of application of the tick, the mice were sacrificed, the back skin tissue was excised, fixed with 10% formalin, and then stained with hematoxylin and eosin. The stained tissue was photographed using an inverted microscope (TE-2000U, Nikon, Japan) and shown in FIG. 21, and the thickness of the epidermal layer was measured using a program (Leica application suite) and shown in Tables 19 and 22.

Epidermal thickness (μm) untreated group 9.32 house dust mite + vehicle 30.33 House dust mite + triphuharol A 15.29

FIG. 21 is a photograph in which the skin tissue was stained with hematoxylin and eosin after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

22 is a graph showing the results of measuring the epidermal thickness after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

As a result, as shown in Table 19 and FIGS. 21 and 22 , tripuharol A reduced the thickness of the skin and epidermal layer compared to the control vehicle in NC/Nga mice induced by atopic dermatitis, a hypersensitivity allergic disease, by house dust mites. It was confirmed to decrease. This means that it is possible to prevent or improve hypersensitivity allergic diseases by preventing the invasion of external allergens by tightening the gap between epidermal cells.

9-7. Confirmation of the number of infiltrated mast cells in the lesion site

In order to determine the degree of mast cell infiltration in the mouse lesion site, mice whose drug administration has been completed, that is, mice with hair removed from the house dust mites + vehicle treatment group described in 9-1, and house dust mites + tripuharol A treatment group After 23 days of application of house dust mites on the back, the mice were sacrificed, the back skin tissue was extracted, fixed with 10% formalin, and then stained with toluidine blue. The stained tissue was photographed using an inverted microscope (TE-2000U, Nikon) and shown in FIG. 23, and the number of mast cells was measured using the Image J program and shown in Tables 20 and 24.

number of mast cells untreated group 13 house dust mite + vehicle 44.3 House dust mite + triphuharol A 22.3

23 is a photograph of toluidine blue staining of skin tissue after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

24 is a graph showing the results of measuring the number of mast cells after tripuharol A treatment in NC/Nga mice with house dust mite-induced atopic dermatitis.

As a result, as shown in Table 20, 23, and 24, tripuharol A was infiltrated at the lesion site compared to the control vehicle in NC/Nga mice, in which atopic dermatitis, a hypersensitivity allergic disease, was induced by house dust mites. It was confirmed that the number of mast cells was significantly reduced. This means that it is possible to prevent or improve the symptoms of hypersensitivity allergic disease caused by mast cells.

Claims (20)

A pharmaceutical composition for use in preventing or treating allergic diseases and diseases associated with vasodilation, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient. The composition of claim 1, wherein the allergic disease is selected from the group consisting of atopic dermatitis, asthma, rhinitis, anaphylactic shock, and itch. The composition of claim 1, wherein the disease associated with vasodilation is selected from the group consisting of hot flashes, nasal congestion, and hypotension. The method according to claim 1, wherein tripuharol A or a physiologically acceptable salt thereof constricts dilated blood vessels to reduce the outflow of disease-causing substances from blood vessels, reduce water loss from skin, and skin moisture content The composition is one or more of increasing the skin or epidermal thickness at the dermatitis lesion site. The composition according to claim 1, wherein the tripuharol A or a physiologically acceptable salt thereof is in the form of an extract or a fraction thereof comprising the same. The method according to claim 5, wherein the extract is a composition obtained by extracting the seaweed extract with a low-polarity solvent. The method according to claim 6, wherein the low-polarity solvent is acetone, methyl acetate, ethyl acetate, n-butyl acetate, t-butyl methyl ether, dimethyl sulfoxide, diethyl ether, heptane, pentane, methyl ethyl ketone, methyl isobutyl ketone, Cyclohexane, petroleum ether, dichloromethane, dichloroethane, 1,2-dichloroethene, dichloromethane, hexane, or a combination thereof, or an aqueous solution thereof. The composition of claim 5, wherein the fraction is a hexane, ethyl acetate, dichloroethane, or water fraction. A food composition for use in improving allergic diseases and diseases associated with vasodilation, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient. The composition of claim 9, wherein the allergic disease is selected from the group consisting of atopic dermatitis, asthma, rhinitis, anaphylactic shock, and itch. The composition of claim 9, wherein the disease associated with vasodilation is selected from the group consisting of hot flashes, nasal congestion, and hypotension. The method according to claim 9, wherein tripuharol A or a physiologically acceptable salt thereof constricts dilated blood vessels to reduce the outflow of disease-causing substances from blood vessels, reduce water loss from skin, and skin water content The composition is one or more of increasing the skin or epidermal thickness at the dermatitis lesion site. The composition according to claim 9, wherein the tripuharol A or a physiologically acceptable salt thereof is in the form of an extract or a fraction thereof comprising the same. The composition according to claim 13, wherein the extract of the seaweed extract is obtained by extracting the plant with a low polarity solvent. The method according to claim 14, wherein the low-polarity solvent is acetone, methyl acetate, ethyl acetate, n-butyl acetate, t-butyl methyl ether, dimethyl sulfoxide, diethyl ether, heptane, pentane, methyl ethyl ketone, methyl isobutyl ketone, Cyclohexane, petroleum ether, dichloromethane, dichloroethane, 1,2-dichloroethene, dichloromethane, hexane, or a combination thereof, or an aqueous solution thereof. The composition of claim 13, wherein the fraction is a hexane, ethyl acetate, dichloroethane, or water fraction. A cosmetic composition for use in maintaining or improving skin moisture content, comprising tripuharol A or a physiologically acceptable salt thereof as an active ingredient. A method for preventing or treating allergic diseases and diseases associated with vasodilation in a subject, comprising administering the pharmaceutical composition of claim 1 to the subject. The method according to claim 18, wherein the allergic disease is selected from the group consisting of atopic dermatitis, asthma, rhinitis, anaphylactic shock, and pruritus, and the disease associated with vasodilation is selected from the group consisting of hot flashes, nasal congestion, and hypotension. how to be. A method of making-up a subject, comprising administering the cosmetic composition of claim 17 to the subject.

Images