Composition for preventing or treating atopic dermatitis comprising synergistic complex extract

Abstract

The present invention relates to a composition for preventing or treating / alleviating symptoms of atopic dermatitis, the composition comprising a naturally derived extract which can be safely used without toxicity and exhibits synergy when co-administered. More specifically, one aspect of the present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis, the composition containing a complex extract of a Daphne genkwa extract and a Euphorbia kansui radix extract.

Description

Composition for preventing or treating atopic dermatitis comprising a synergistic complex extract

[0001] The present invention relates to a composition for preventing or treating / improving symptoms of atopic dermatitis, comprising an extract derived from a natural product, which can be safely used without toxicity and exhibits a synergistic effect when administered in combination.

[0002] Atopic dermatitis is a chronic allergic inflammatory skin disease. It is characterized by severe itching that interferes with daily life, hypersensitivity to external stimuli or allergens, rashes, dry skin, redness, blisters, lichenification, and characteristic eczema. It can also cause bleeding, cracks, and secondary infections. When the condition worsens, acute lesions such as redness, swelling, blisters, exudation, and peeling appear. It is known that patients with severe atopic dermatitis experience exacerbations for more than 30% of the year. It is commonly called "taeyol" in infants, and 70-80% of newborns in Korea experience "taeyol." While most of these cases improve with growth, the condition can persist through childhood, adolescence, and adulthood, with recurring periods of improvement and exacerbation, sometimes leading to a chronic course. The global prevalence of atopic dermatitis in children is approximately 10-30%, and in those over 18 years of age, the prevalence is approximately 1%.

[0003] As can be seen from the etymology of "atopy," which means "unknown" or "strange," the exact cause of atopic dermatitis is unknown, but it is understood to be influenced by genetic predisposition and environmental factors. Genetically, a family history has been confirmed in 70-80% of atopic patients, and according to reports, if both parents have atopic dermatitis, the child has a 75% chance of developing it. Recently, the importance of environmental factors, such as the increase in allergens due to indoor and outdoor pollution and the increase in instant food consumption due to the trend toward nuclear families, has been emphasized.

[0004] Treatment of atopic dermatitis primarily relies on topical steroid ointments, and oral medications such as antihistamines, steroids, and antibiotics. While steroids are highly effective due to their anti-inflammatory and immunosuppressive properties, long-term use can lead to side effects such as skin weakening and atrophy, systemic hormonal symptoms, and addiction. Antihistamines and antibiotics also have side effects such as photosensitivity and bacterial resistance, limiting their use. Therefore, alternatives are needed.

[0005] Daphne genkwa is a plant native to southern Korea and coastal areas of southern China and Japan. In oriental medicine and folk remedies, it has been used as a diuretic, antitussive, expectorant, and edema treatment. The flowers, called wonhwa, are harvested when still in their buds and dried in the sun before use, and the roots are sliced ​​and used whole. Daphne genkwa extract is known to be effective for neurodegenerative diseases and atopic dermatitis (Korean Patent No. 10-1364234), and is also known to have antiviral properties against the influenza virus. In addition, genkwdaphnine N or yuanhuacine, which are daphne diterpenoids, have been identified as the active ingredients in Daphne genkwdaphnine extract.

[0006] Euphorbia kansui is distributed throughout Korea, Japan, northeastern China, Sakhalin, and the southern Kuril Islands. In traditional Oriental medicine, Euphorbia kansui is used for its laxative effect (stimulating the intestines to promote peristalsis and induce diarrhea), cardiotonic effect, treatment of epilepsy, esophageal cancer, pleurisy, diabetes, beriberi, boils caused by kidney qi gathering in the lower part, and urinary incontinence. Substances such as euphol (-euphorbol, -euphor, eupadienol), tirucalol, -euphorbol, 20-deoxyingenol, ingenol derivatives, 13-oxyingenol derivatives, and cansuinin A and B have been isolated and are known to exist. Korean Patent Publication No. 10-2009-0109769 proposes anticancer and anti-inflammatory agents containing compounds derived from euphorbia extract as active ingredients.

[0007] Korean Patent Publication No. 10-2020-0109026 reported that a compound extract of azuki bean and persimmon exhibited a remarkable synergistic effect in enhancing immune function compared to either ingredient alone. Atopic dermatitis is an allergic disease characterized by excessive immune enhancement, and this patent indicates that the compound extract may actually be detrimental to atopic dermatitis.

[0008] The present invention aims to solve the problems of the prior art and to provide a composition useful for the prevention and treatment / symptom improvement of atopic dermatitis through a synergistic effect as a complex extract of natural materials that is safe with low side effects.

[0009] Even if not mentioned above, the technical challenges addressed by the present invention will be readily apparent to those skilled in the art. Furthermore, when describing the invention, detailed descriptions of known technologies related to the invention will be omitted if they are deemed to unnecessarily obscure the essence of the invention.

[0010] One aspect of the present invention for achieving the aforementioned purpose relates to a pharmaceutical composition for preventing or treating atopic dermatitis containing a composite extract of a red bean extract and a persimmon extract.

[0011] In the present invention, the "extract" of azuki bean and persimmon can be extracted from at least one of callus or callus culture derived from the stem, root, leaf or flower of azuki bean and persimmon, respectively, or from one or more tissues thereof. The callus can be induced, for example, by inoculating each segment of petals, leaves, stems or roots, etc., on MS basal medium supplemented with BA 1.0 to 3.0 mg / L, preferably 2.0 mg / L, and NAA 0.1 to 0.3 mg / L, preferably 0.2 mg / L, in a dark culture room at 20 to 30°C, preferably 25°C. The culture can be a culture cultured for 4 weeks or more in the same medium used for callus induction.

[0012] An extract of azuki bean blossom or persimmon includes all forms in which the useful components of azuki bean blossom or persimmon are concentrated. A composite extract can be prepared by preparing azuki bean blossom extract and persimmon extract separately and then mixing them, or by mixing azuki bean blossom and persimmon and extracting them together. In the simplest case, the extract can be a dried product or dried powder from which moisture has been removed, or a juice extracted without a separate solvent. Alternatively, it can be a solvent extract in which the useful components are extracted using a separate solvent. The preparation of a solvent extract can be carried out using a known extraction method used for extracting natural products. For example, extraction can be carried out using water, a C1-C3 alcohol, or a mixture thereof as a solvent, but other organic solvents such as acetone or ethyl acetate can also be used. Extraction can be carried out by simply immersing in a solvent at room temperature, but to increase the extraction efficiency in a short period of time, extraction can be carried out using ultrasound, heating, or stirring, as in the following examples. In addition, it is more desirable to crush or grind the azuki bean or persimmon sample into small pieces before extraction so that the extraction can be more efficient.

[0013] The azuki bean or persimmon used for extraction may be a raw material as collected, dried, frozen, or further fermented to further increase the content of useful substances or facilitate absorption. The amount of solvent used during the extraction can be a volume ratio of 2 to 50 times the weight of the azuki bean or persimmon or a mixture thereof. As the extraction efficiency increases as the amount of solvent used increases, there is no problem with using more than 50 times the solvent, but considering the efficiency and economy of the process, it is preferable that the amount of solvent be 50 times or less. In addition, in order to increase the extraction efficiency while using the same amount of solvent, it is better to increase the number of extractions.

[0014] If the above extract is a juice or solvent extract, the liquid extract may be used as is, or it may be concentrated / dried to increase the content of the active ingredient, or it may be fractionated using an additional solvent.

[0015] Among the above complex extracts, the weight ratio of the azuki bean extract and the persimmon extract is preferably 1:3 to 3:1. Even when the ratio is outside the above range, the anti-atopic effect is exhibited, but the synergistic effect is reduced.

[0016] The composition of the present invention exhibited a synergistic inhibitory effect on the release of histamine, which triggers allergic reactions in mast cells, key cells that cause inflammatory responses in allergic diseases, and on the expression of CCL17 / TARC, which is abnormally increased by atopic factors in keratinocytes. This suggests that the composition may exhibit a more significant effect in the prevention or treatment of atopic dermatitis than either azuki bean extract or persimmon extract, respectively.

[0017] The dosage of the pharmaceutical composition of the present invention can be appropriately selected by those skilled in the art to obtain the maximum effect with the minimum amount without side effects, taking into consideration all of the sex, age, weight, severity of pathological condition of the subject to be administered, drug form, administration route, frequency of administration, administration period, etc. The composition of the present invention can be prepared by a method known in the pharmaceutical field for use as a therapeutic agent, and can be used on its own or mixed with a pharmaceutically acceptable carrier, forming agent, diluent, etc. The composition of the present invention can be formulated as a preparation for oral or parenteral administration and used as an agent for the treatment and prevention of atopic dermatitis. Specifically, the composition of the present invention can be formulated as an injection, granule, tablet, pill, capsule, gel, syrup, suspension, emulsion, drop, ointment, or liquid. In addition, the composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and when administered in combination with other therapeutic agents, they can be administered sequentially or simultaneously. Administration may be given once a day or divided into several doses. The general dosage may be 0.001 mg / kg·day to 10 g / kg·day, specifically 0.1 mg / kg·day to 1 g / kg·day, and more specifically 1 mg / kg·day to 200 g / kg·day, but the scope of the present invention is not limited thereto. The composition of the present invention can be usefully used not only for humans but also for livestock and companion animals.

[0018] Another aspect of the present invention relates to a health functional food composition for preventing or improving symptoms of atopic dermatitis, comprising a composite extract of an azuki bean extract and a persimmon extract. The composite extract may be added to the functional food composition of the present invention, preferably in an amount of 0.01 to 100 wt%, and in addition, various additives used in the manufacture of functional foods, such as thickeners, stabilizers, preservatives, colorants, flavoring agents, etc., may be further included. The functional food composition of the present invention may be provided as a health functional food in the form of a tablet, capsule, pill, or liquid, or may be formulated as a food or beverage.

[0019] In explaining the functional food of the present invention, specific descriptions of the azuki bean extract, the persimmon extract, and the efficacy against atopic dermatitis may be referred to the description of the pharmaceutical composition.

[0020] In particular, when the composition of the present invention is used to prevent or improve symptoms of atopic dermatitis in livestock or companion animals, it can be used as a feed additive composition.

[0021] Another aspect of the present invention relates to a cosmetic composition for preventing or improving symptoms of atopic dermatitis, comprising a complex extract of a red bean extract and a persimmon extract. The composition of the present invention does not exhibit cytotoxicity, and thus can be used continuously for prevention even in the absence of atopic dermatitis without concern for side effects. The cosmetic composition of the present invention may contain the complex extract in a ratio of 0.1 to 100 wt%. The composition may be used together with other additives widely used in the cosmetic field, and may be included in formulations such as skin, skin lotion, skin softener, skin toner, astringent, lotion, nutritional lotion, milk lotion, moisture lotion, nutritional cream, massage cream, moisture cream, hand cream, essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body cleanser, body lotion, shampoo, milky lotion, foundation, pressed powder, loose powder, eye shadow, etc.

[0022] As described above, the composition of the present invention is derived from a natural product and does not exhibit cytotoxicity, so it can be used safely without side effects. In addition, it has a remarkable effect by synergistically suppressing the release of histamine and the expression of CCL17 / TRAC, which are inducing factors of atopic dermatitis, and therefore can be usefully used in pharmaceutical compositions, health functional food compositions, feed additive compositions, and cosmetic compositions for preventing and treating / improving symptoms of atopic dermatitis.

[0023] Figure 1 is a graph showing that the composition of the present invention exhibits a synergistic inhibitory effect on histamine release.

[0024] Figure 2 is a graph showing the histamine release inhibition effect according to the composition of the present invention.

[0025] Figure 3 is a graph showing that the composition of the present invention exhibits a synergistic inhibitory effect on the expression of CCL17 / TRAC.

[0026] The present invention will be described in more detail below with reference to the attached examples. However, these drawings and examples are merely examples intended to easily illustrate the content and scope of the technical concept of the present invention and are not intended to limit or alter the technical scope of the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible within the scope of the technical concept of the present invention based on these examples.

[0027] [Example]

[0028] Example 1: Preparation of extract

[0029] 1) Preparation of azuki bean extract

[0030] The entire azuki bean tree, including the flowers, stems, and roots, was thoroughly washed with tap water and distilled water, and then dried in a 42℃ dryer for 72 hours. The dried azuki bean tree was cut into pieces using a mixer, and then ground into 20-50 mesh sizes using a mortar and pestle to use as an extraction sample. The obtained powder was soaked in 30 times (w / v) volume of alcohol for one day and then extracted using ultrasonic waves. The extract was passed through a first 0.45 μm membrane filter and a second 0.22 μm membrane filter to remove solid components, and then concentrated to 1 / 10 using an evaporator in a 50℃ water bath. The concentrate was recovered and dried using a freeze dryer, and the obtained powder (yield ~5%) was used in the following examples.

[0031] 2) Preparation of sensitive extract

[0032] The entire plant, including flowers, leaves, stems, and roots, was thoroughly washed with tap water and distilled water and then dried in a dryer at 42°C for 72 hours. An extract was prepared using the same process as for the preparation of the azuki bean extract in 1) using the dried plant. The powder obtained by drying using a freeze dryer (yield ~3.7%) was used in the examples below.

[0033] Example 2: Cytotoxicity evaluation

[0034] To determine whether the extracts of the red bean flower tree and / or persimmon exhibit cytotoxicity, experiments were conducted as follows. Human mast cells (HMC-1, human mast cells) or human keratinocyte cells (HaCat, human keratinocyte cell line) were seeded at 6.0 × 10 in a 96-well plate. 4 After dispensing at a concentration of 10 cells / well, the cells were cultured for 24 hours at 37°C and 5% CO2. Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was used as the medium. The cultured cells were treated with azuki bean and / or persimmon extracts at a concentration of 10 or 500 μg / ml for 24 hours, and the cell viability was measured using the MTS assay method.

[0035] As a result, it was confirmed that all of the azuki bean extract, the persimmon extract, and the combined extract (azuki bean extract: persimmon extract 10:1, 1:1, and 1:10 w / w) did not significantly affect the proliferation of HMC-1 cells and HaCat cells in the above concentration range, and thus did not exhibit cytotoxicity.

[0036] Example 3: Evaluation of histamine release inhibition effect

[0037] Mast cells are key cells responsible for acute and chronic inflammatory diseases, including atopic dermatitis. Therefore, the histamine release-inhibiting effect of the extract prepared in Example 1 was evaluated using mast cells.

[0038] Specifically, HMC-1 cells were seeded in 24-well plates at 10 5Cells were seeded at a concentration of 10 cells / well and cultured for 24 hours at 37°C and 5% CO2. The cultured cells were pretreated with 20 or 50 μg / mL of azuki bean extract (DG), persimmon extract (EK), or azuki bean and persimmon combined extract (DG+EK, 1:1 w / w) for 1 hour, and then stimulated with 30 ng / mL of phorbl-12-myrsitate-13 acetate (PMA), an allergic reaction-inducing substance, and 5 μM A23187, and cultured for 12 hours. As a control group, only the same amount of DMSO, the solvent of the extract, was treated instead of the extract. The normal group refers to the experimental group that was not stimulated with PMA or A23187 or treated with the extract.

[0039] The supernatant of the culture medium was taken, and the amount of histamine production was measured using an ELISA kit (Biolegend, USA) according to the manufacturer's manual, and the results are shown in Fig. 1. According to Fig. 1, both the azuki bean extract and the persimmon extract showed an inhibitory effect on histamine release, but the persimmon extract had a rather minimal effect. When the azuki bean extract and the persimmon extract were mixed in a 1:1 weight ratio, the same dose showed a significant inhibitory effect compared to not only the persimmon extract but also the persimmon extract, confirming that the combined extract showed a synergistic effect.

[0040] In order to confirm the synergistic effect according to the composition of the complex extract, the histamine production inhibitory effect was evaluated using the same method as above, except that the complex extract with a mixed weight ratio of azuki bean extract and persimmon extract of 10:1, 3:1, 1:1, 1:3, or 1:10 was treated at a concentration of 50 μg / ㎖.

[0041] Example 4: Evaluation of the inhibitory effect of CCL17 / TRAC expression

[0042] TARC (thymus and activation-regulated chemokine) is a protein that induces cell migration and infiltration into inflammatory sites. When keratinocytes are stimulated with IFN-γ (interferon-γ) or TNF-α (tumor necrosis factor-α), CCL17 / TARC expression abnormally increases. Therefore, the anti-atopic efficacy of the extract was tested by suppressing CCL17 / TRAC expression.

[0043] HaCaT cells were seeded in 24-well plates at 10 5 Cells were seeded at a concentration of 10 cells / well and cultured for 24 hours at 37°C and 5% CO2. The cultured cells were pretreated with 50 μg / mL of azuki bean extract (DG), persimmon extract (EK), or azuki bean and persimmon combined extract (DG+EK, 1:1 w / w), and then cultured for 24 hours after stimulation with 10 ng / mL of TNF-α and 10 ng / mL of IFN-γ. The culture solution was centrifuged, and the supernatant was collected, and the content of TARC in the culture solution was measured using a human CCL17 / TARC kit (R&D systems) according to the manufacturer's manual.

[0044] In Fig. 3, the normal group represents the experimental group that was not treated with TNF-α / IFN-γ and the extract, the control group represents the experimental group that was treated only with TNF-α / IFN-γ and not the extract, and DG, EK, and DG+EK represent the experimental groups that were treated with TNF-α / IFN-γ after treatment with a mixture of the azuki bean extract, persimmon extract, and complex extract, respectively. As can be seen in Fig. 3, both the azuki bean extract and the persimmon extract inhibited the expression of CCL17 / TARC, and when treated with the same amount of the complex extract, a clear synergistic effect was shown for the inhibition of the expression of CCL17 / TARC. This indicates that the complex extract, together with the histamine inhibitory effect of Example 3, is a composition that has a synergistic effect on the treatment of atopic dermatitis.

Claims

1. A pharmaceutical composition for preventing or treating atopic dermatitis containing a complex extract of azuki bean extract and persimmon extract.

2. In claim 1, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the above azuki bean extract and persimmon extract are each extracted from at least one of a stem, root, leaf, flower, or a callus or a culture of a callus derived from one or more of these tissues.

3. In claim 1, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the above azuki bean extract and persimmon extract are each in the form of a dry powder or juice, or are extracted using water or C1 to C3 alcohol or a mixture thereof as a solvent.

4. In any one of claims 1 to 3, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the weight ratio of the azuki bean extract and the persimmon extract among the above complex extracts is 1:3 to 3:

1.

5. In any one of claims 1 to 3, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the above complex extract exhibits a synergistic inhibitory effect on histamine release and CCL17 / TRAC expression compared to each of the azuki bean extract and the persimmon extract.

6. A health functional food composition for preventing or improving symptoms of atopic dermatitis containing a complex extract of azuki bean extract and persimmon extract.

7. In claim 6, A health functional food composition for preventing or improving symptoms of atopic dermatitis, characterized in that the weight ratio of the azuki bean extract and the persimmon extract among the above extracts is 1:3 to 3:

1.

8. A feed additive composition for preventing or improving symptoms of atopic dermatitis in livestock or companion animals, containing a composite extract of azuki bean extract and persimmon extract.

9. A cosmetic composition for preventing or improving symptoms of atopic dermatitis containing a complex extract of azuki bean extract and persimmon extract.

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