Composition for preventing, ameliorating or treating pemphigus comprising Eclipta prostrata extract as effective component

Abstract

The present invention relates to a composition for the prevention, improvement, or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient. The *Hyeoncho* extract of the present invention has an excellent effect of inhibiting the dissociation of keratinocytes induced by AK23 (Anti-Desmoglein 3) and has an effect of inhibiting the activity of MLK3 (mixed lineage kinase 3), so it can be usefully used for the prevention, improvement, or treatment of pemphigus.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient. Background Technology

[0002] Pemphigus is a chronic blistering disease characterized by the formation of blisters on the skin and mucous membranes. Histologically, it is distinguished by acantholysis, a phenomenon in which the junctions between epithelial cells are broken. Most patients possess autoantibodies against antigens present on the surface of epithelial cells in their serum, and given that these autoantibodies are directly involved in the development of pemphigus, it can be considered a representative autoimmune disease caused by autoantibodies.

[0003] Based on clinical and histological findings, pemphigus can be classified into pemphigus vulgaris, proliferative pemphigus, pemphigus foliaceus, and pemphigus erythematosus. Among these, pemphigus vulgaris is the most common type, with an equal incidence rate between men and women, and it is most prevalent in middle-aged individuals. In cases of pemphigus vulgaris, erosion of the oral mucosa occurs in almost all patients, and oral lesions appear as the first symptom in most cases. It frequently occurs on the buccal mucosa, palate, gums, and tongue; rather than appearing as vesicular lesions, it usually manifests as ruptured erosions or cloudy spots resulting from the shedding of epithelial tissue. If the affected area is extensive, even minor irritation causes severe pain, leading to significant difficulty in eating; if it persists for a long time, it may induce hypoproteinemia, weight loss, or anemia. Depending on the patient, there are cases where only a small number of localized oral lesions develop, followed by a persistent disease course that fluctuates between exacerbation and remission over several years; in other cases, oral lesions may remain as intractable residual lesions even after extensive skin lesions have disappeared through treatment. Generally, blisters begin to appear on the skin four months after the onset of oral lesions, and the condition frequently occurs on the face, trunk, groin, and axillae. Most cases of pemphigus vulgaris start with a few lesions in localized areas such as the mouth, but after several months, widespread lesions develop throughout the body, and if left untreated, it can be fatal. Therefore, it is essential to prevent further progression by treating the condition with sufficient oral steroids in the early stages when the lesions are localized.

[0004] Treatment for pemphigus involves administering systemic steroids for a certain period, and may include immunosuppressants or plasmapheresis; it is also advisable to use antibiotics, vitamins, and antihistamines as adjuvants. However, while systemic steroids are the primary treatment for pemphigus, long-term use can lead to side effects. Immunosuppressants are used in combination with steroids but may cause side effects such as reduced blood cell count, infection, hemorrhagic cystitis, infertility, digestive disorders, hepatitis, and hair loss. Therefore, there is a need to develop natural materials that can overcome the shortcomings of existing treatments and provide excellent improvement in pemphigus without side effects.

[0005] As prior art related to the improvement of pemphigus, Korean Published Patent No. 2011-0094327 discloses a 'pemphigus treatment containing an anti-FAS ligand antibody' and Korean Published Patent No. 2022-0148804 discloses a 'method for treating pemphigus disorder' using a human neonatal Fc receptor antagonist; however, there is no disclosure regarding the 'composition for the prevention, improvement, or treatment of pemphigus containing *Hyeoncho* extract as an active ingredient' of the present invention. The problem to be solved

[0006] The present invention was derived from the above requirements and provides a composition for the prevention, improvement, or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient, and the present invention was completed by confirming the effect of the *Hyeoncho* extract in inhibiting the dissociation of keratinocytes induced by AK23 (Anti-Desmoglein 3) and the effect of inhibiting the activity of MLK3 (mixed lineage kinase 3). means of solving the problem

[0007] In order to solve the above problem, the present invention relates to a nasturtium plant ( Eclipta prostrata Provides a health functional food composition for the prevention or improvement of pemphigus containing an extract as an active ingredient.

[0008] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient.

[0009] In addition, the present invention provides a quasi-drug composition for the prevention or improvement of pemphigus containing a *Hyeoncho* extract as an active ingredient.

[0010] In addition, the present invention provides a herbal medicine composition for the prevention or treatment of pemphigus containing a *Hallyeocho* extract as an active ingredient.

[0011] In addition, the present invention provides a veterinary composition for the prevention or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient.

[0012] In addition, the present invention provides a feed additive for the prevention or improvement of pemphigus containing a nasturtium extract as an active ingredient. Effects of the invention

[0013] The present invention relates to a composition for the prevention, improvement, or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient. The *Hyeoncho* extract of the present invention has an excellent effect of inhibiting the dissociation of keratinocytes induced by AK23 (Anti-Desmoglein 3) and has an effect of inhibiting the activity of MLK3 (mixed lineage kinase 3), so it can be usefully used for the prevention, improvement, or treatment of pemphigus. Brief explanation of the drawing

[0014] Figure 1 shows the results of confirming the cytotoxicity of the *Hallyeocho* extract in human skin cells, HaCaT. Figure 2 shows the results of confirming the inhibitory effect of keratinocyte dissociation by treatment with Erythronium japonicum extract. Keratinocyte dissociation was induced by treatment with AK23, and IgG was used as a negative control for AK23. Specific details for implementing the invention

[0015] To achieve the purpose of the present invention, the present invention (Euryale ferox) Eclipta prostrataProvides a health functional food composition for the prevention or improvement of pemphigus containing an extract as an active ingredient.

[0016] The extraction solvent of the above-mentioned *Hallyeocho* extract is preferably water, a C1-C4 lower alcohol, or a mixture thereof, more preferably ethanol, and even more preferably 70% (v / v) ethanol, but is not limited thereto.

[0017] In one embodiment of the present invention, the nasturtium extract has the effect of inhibiting the activity of MLK3.

[0018] The above composition is preferably prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

[0019] When the health functional food composition of the present invention is used as a food additive, the active ingredient may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to its purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw material. However, when consumed for a long period for the purpose of health and hygiene or for health control, the amount may be less than the above range, and since there are no issues regarding safety, the active ingredient may be used in an amount greater than the above range.

[0020] There are no specific restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned active ingredients may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0021] When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotensin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the above natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.

[0022] The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

[0023] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient.

[0024] The composition of the present invention is preferably prepared in any one of the formulations selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto.

[0025] The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient, and may be in various oral or parenteral formulations. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds.

[0026] In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, emulsions, syrups, and aerosols; in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa dough, laurin dough, and glycerogelatin may be used as bases for suppositories. When administering parenterally, it is preferable to select a method of topical application or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura mater, or cerebral blood vessel injection.

[0027] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0028] The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses.

[0029] It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, taking into account all of the aforementioned factors, and this can be easily determined by a person skilled in the art.

[0030] In addition, the present invention provides a quasi-drug composition for the prevention or improvement of pemphigus containing a *Hyeoncho* extract as an active ingredient.

[0031] The term "quasi-drug" as used in the present invention refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, among which the effect is milder than that of pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are defined as articles excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans or animals, and products that have a mild effect on the human body or do not act directly on it. The quasi-drug composition of the present invention is used for the purpose of treating skin wounds and is not particularly limited in its formulation; for example, it may be a transdermal formulation such as a lotion, ointment, gel, cream, patch, or spray.

[0032] In addition, for each formulation, the quasi-drug composition may arbitrarily select and combine other ingredients according to the formulation or purpose of use of other quasi-drugs. The amount of the active ingredient can be appropriately determined according to the purpose of use (inhibition or alleviation). For example, it may include conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0033] In addition, the present invention provides a herbal medicine composition for the prevention or treatment of pemphigus containing a *Hallyeocho* extract as an active ingredient.

[0034] The herbal medicine composition of the present invention means that it is manufactured according to a Korean medical prescription, but is not limited thereto.

[0035] In addition, the present invention provides a veterinary composition for the prevention or treatment of pemphigus containing a *Hyeoncho* extract as an active ingredient.

[0036] The veterinary composition of the present invention may further include suitable excipients and diluents according to conventional methods. Examples of excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0037] The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0038] The effective amount of the veterinary composition according to the present invention can be appropriately selected according to the individual animal. It may be determined based on the severity of the disease or condition, the sensitivity to the active ingredient of the present invention according to the individual's age, weight, health status or gender, the route of administration, the duration of administration, factors including other compositions combined with or used simultaneously with the composition, and other factors well known in the physiological or veterinary field.

[0039] In addition, the present invention provides a feed additive for the prevention or improvement of pemphigus containing a nasturtium extract as an active ingredient.

[0040] The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act. In the present invention, the term "feed" may refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest. The types of said feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0042] The present invention will be described in more detail below using manufacturing examples and embodiments. These manufacturing examples and embodiments are intended solely to explain the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0044] Preparation Example 1. Preparation of Nasturtium extract

[0045] Dried Erythronium was purchased domestically from Omniherb. Ten times the amount of 70% (v / v) ethanol was added to 1 part by weight of dried Erythronium, and extraction was performed using a reflux extractor (MS-DM609) for 3 hours. After extraction, the solution was filtered using a 5㎛ vacuum filter and concentrated using a rotary vacuum concentrator (EC-1020). The concentrate was dried (freeze-dryer: Ilshin Bio LP20), homogenized, and used as a sample for the experiment.

[0047] Example 1. Confirmation of cytotoxicity of *Hallyeocho* extract

[0048] Human skin cells, HaCaT cells (AddexBio, CA, USA), were cultured in DMEM / high glucose containing 10% (v / v) fetal bovine serum and 1% (v / v) antibiotic-antifungal solution in an incubator maintained at 37°C and 5% CO2.

[0049] To confirm cytotoxicity, HaCaT cells were seeded into a 24-well plate at a density of 500,000 cells per well and stabilized for 24 hours. After confirming that a monolayer of cells had formed in each well, the cells were treated with the *Hallyeocho* extract of Preparation Example 1 and cultured for 24 hours. Subsequently, cytotoxicity was confirmed by treating with the CCK-8 reagent (Dojindo, USA) and measuring cell viability.

[0050] As a result, as disclosed in Figure 1, it was confirmed that the extract of *Hallyeocho* had no toxicity to HaCaT cells at a concentration of 20 μg / mL.

[0052] Example 2. Confirmation of the inhibitory effect of *Hyeoncho* extract on keratinocyte dissociation

[0053] HaCaT cells were seeded into a 24-well plate at a concentration of 500,000 cells per well and stabilized for 24 hours. After confirming that a monolayer had formed in each well, the cells were treated with a *Hallyeocho* extract at a concentration confirmed to be non-cytotoxic in Example 1. Subsequently, AK23 antibody (MBL, Japan) was added to each well at a concentration of 5 µg / mL and cultured for 24 hours. Human IgG antibody was used as a negative control for AK23. Afterward, each well was washed with PBS, and 150 µl of Hanks buffered saline (HBSS, Gibco) containing dispase II reagent (2.4 U / mL, Sigma) was added and reacted at 37°C for 20 minutes. Then, 200 µl of HBSS was added, and the cells were exposed to mechanical stress by pipetting 15 times with a 1 mL pipette to shear the cell monolayer. After that, the cells were stained with MTT (1 mg / mL, Sigma) for 20 minutes, and then images were captured using a microscope.

[0054] As a result, as disclosed in FIG. 2, the extract of *Hallyeocho* of the present invention had an excellent effect in inhibiting the dissociation of keratinocytes induced by AK23 treatment.

[0056] Example 3. Evaluation of MLK3 enzyme inhibitory activity of Eclipta prostrata extract

[0057] To confirm the inhibitory effect on kinase activity of MLK3, recombinant human MLK3 protein was mixed with 40 μg / mL of the *Hyeoncho* extract from Preparation Example 1 in a reaction solution (8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.33 mg / mL myelin basic protein, 5 mM DTT), and then 10 mM magnesium acetate and gamma- 33A P-ATP mixture was added and reacted at room temperature for 40 minutes. Afterward, the reaction was terminated by the addition of 0.5% phosphoric acid, 10 µl of the total mixture was spotted onto a P30 filter mat, washed four times with 0.425% phosphoric acid for 4 minutes each, and then washed once with methanol. The activity of MLK3 was then measured using scintillation counting and evaluated as a % value relative to the control group.

[0058] As a result, as disclosed in Table 1 below, the MLK3 activity of the *Hallyeocho* extract was about 26% compared to the control group, showing an inhibitory effect of about 74%.

[0059] Control group(-) Nasturtium extract MLK3 activity (% of control group) 100 26

Claims

Claim 1 Nasturtium Eclipta prostrata A health functional food composition for the prevention or improvement of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, containing an ethanol reflux extract as an active ingredient. Claim 2 delete Claim 3 delete Claim 4 A health functional food composition for the prevention or improvement of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, characterized in that, in claim 1, the composition is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. Claim 5 Nasturtium Eclipta prostrata A pharmaceutical composition for the prevention or treatment of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, containing an ethanol reflux extract as an active ingredient. Claim 6 A pharmaceutical composition for the prevention or treatment of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, characterized in that, in addition to the active ingredient, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. Claim 7 A pharmaceutical composition for the prevention or treatment of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, characterized in that, in claim 5, the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. Claim 8 Nasturtium Eclipta prostrata A quasi-drug composition for the prevention or improvement of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, containing an ethanol reflux extract as an active ingredient. Claim 9 Nasturtium Eclipta prostrata A herbal medicine composition for the prevention or treatment of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, containing an ethanol reflux extract as an active ingredient. Claim 10 Nasturtium Eclipta prostrata A veterinary composition for the prevention or treatment of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, containing an ethanol reflux extract as an active ingredient. Claim 11 Nasturtium Eclipta prostrata A feed additive for the prevention or improvement of pemphigus induced by the activation of MLK3 (mixed lineage kinase 3) and the dissociation of keratinocytes, containing an ethanol reflux extract as an active ingredient.

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