A composition for improving or treating pemphigus comprising extracts of Torilidis Fructus

Abstract

The present invention relates to a composition for improving or treating pemphigus containing a Torilidis Fructus extract, and more specifically, it is expected that the Torilidis Fructus extract can be usefully utilized in the treatment of pemphigus.

Description

Technology Field

[0001] The present invention relates to a composition for improving or treating pemphigus containing a Saposhnikovia divaricata extract.

[0002] The present invention relates to a health functional food for improving or preventing pemphigus containing a *Sophora* extract. Background Technology

[0003] Pemphigus refers to a group of autoimmune blistering diseases of the skin and mucous membranes associated with autoantibodies against the cell surface of keratinocytes, resulting in a loss of cell-to-cell adhesion of keratinocytes. Pemphigus can be divided into four major types: pemphigus vulgaris, pemphigus foliaceus, pemphigus paraneoplastic, and IgA pemphigus. Among these, the classic types of pemphigus are pemphigus vulgaris (PV) and pemphigus foliaceus (PF). In both PF and PV, pathogenic IgG antibodies primarily target desmogleins (Dsg), which are transmembrane glycoproteins of desmosomes; the target antigen for PV is Dsg 3, while the target antigen for PF is Dsg 1 (Bolognia JL, et al., 2018).

[0004] In pemphigus, the disruption of cell-cell adhesion is currently thought to be mediated by direct inhibition by autoantibodies and subsequent signaling via antibody binding. The role of the p38 mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of pemphigus is well established (Lu HT et al., 1999). As a member of the MAPK family, p38 plays a crucial role in cellular responses to various infections and environmental stimuli, as well as osmotic and oxidative stress (Mao X et al., 2011). In vitro studies using PV serum IgG and mAbs, as well as in vivo studies using the skin of pemphigus patients, have activated p38, making it a promising target for pharmacological treatment. However, p38 possesses four different isoforms (α, β, γ, δ) and is involved in various cellular processes. Therefore, targeting p38 with p38 inhibitors can induce off-target effects and systemic toxicity (Mao X et al., 2014).

[0005] Current treatment strategies for pemphigus vary depending on the ability to induce disease control (DC) and sustained clinical remission (CR). Corticosteroids rapidly affect PV symptoms, but high daily doses (e.g., 1–1.5 mg / kg / day of oral prednisone) must be administered to achieve an effect. If DC is not reached after 3–4 weeks, the prednisone dose must be increased, and in patients with very active disease, an intravenous (IV) bolus of corticosteroids (e.g., methylprednisolone) may be preferable at the start of treatment; however, there remains a need for safer, faster-acting drugs that can control the disease early and maintain clinical remission in pemphigus patients using or without corticosteroids at minimum doses (e.g., 20 mg / day of prednisone or less of 10 mg / day).

[0006] The inventors completed the present invention by confirming that the Saposhnikovia divaricata extract used in this study exhibits antipemphigus activity that dramatically inhibits fragmentation by AK23. Prior art literature

[0007] Bolognia JL, Schaffer JV, Cerroni L, Callen JP, Cowen EW, et al., editors. Dermatology. 4th Ed. China: Elsevier, 494-508 (2018)Mao X, Sano Y, Park JM, Payne AS. p38 MAPK activation is downstream of the loss of intercellular adhesion in pemphigus vulgaris. J Biol Chem. 14;286(2), 1283-91 (2011)Mao X, Li H, Sano Y, Gaestel M, Mo Park J, Payne AS. MAPKAP kinase 2 (MK2)-dependent and -independent models of blister formation in pemphigus vulgaris. J Invest Dermatol., 134(1), 68-76 (2014)Payne AS, Stanley JR. Vesiculobullous disorders. In: Kang S, Amagai M, Bruckner AL, Enk AH, Margolis DJ, McMichael AJ, et al., editors. Fitzpatrick's dermatology. 9th Ed. New York, McGraw-Hill, 909-925 (2019) The problem to be solved

[0008] The objective of the present invention is to provide a composition for improving or treating pemphigus containing a Saposhnikovia divaricata extract.

[0009] The objective of the present invention is to provide a health functional food for improving or preventing pemphigus containing a *Sophora* extract. means of solving the problem

[0010] The present invention relates to a pharmaceutical composition for the prevention or treatment of pemphigus, characterized by comprising a natural composition.

[0011] The present invention relates to a health functional food for preventing or improving pemphigus, characterized by comprising a natural composition.

[0012] The above pemphigus may be a pharmaceutical composition for the prevention or treatment of pemphigus selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, pemphigus paraneoplastic, IgA pemphigus, and pemphigoid.

[0013] The above composition for the prevention or treatment of pemphigus may be a black cohosh extract.

[0014] Saposhnikovia divaricata (Torilis japonica Decandolle) warms the kidneys and boosts Yang energy, making it useful for treating conditions such as kidney deficiency impotence in men and infertility in women. It is effective for uterine coldness in women, severe itching or leukorrhea caused by Trichomonas vaginitis, and pruritus. Additionally, Saposhnikovia divaricata has therapeutic effects on skin diseases and is used externally for eczema, allergic dermatitis, and oozing. It stimulates male hormones to increase the weight of the uterus and ovaries, inhibits skin bacteria, and acts as an anthelmintic. To harvest, the entire plant is cut down when the fruit ripens and turns yellow, the fruit is picked, and dried in the sun. Pharmacological studies have identified anti-trichomoniasis, sex hormone, and antifungal effects, but its mechanism of action or efficacy regarding pemphigus are unknown.

[0015] The extract selected from Torilidis Fructus of the present invention is obtained by separating active ingredients contained in the extraction material by contacting a solvent and the extraction material under specific conditions, and the extract may contain active ingredients within the raw material through an extraction process for Torilidis Fructus. The extract may be extracted using a conventional solvent under conditions of conventional temperature and pressure, according to a conventional method known in the art for extracting extracts from herbal medicines. For example, the Torilidis Fructus extract may be extracted using water, alcohol, ethyl acetate, acetone, hexane, dichloromethane, or a mixture thereof, and preferably water, alcohol, or an aqueous alcohol solution may be used. Furthermore, the extraction method may utilize various methods such as hot water extraction, cold maceration extraction, reflux extraction, and ultrasonic extraction, and preferably, extraction may be performed using water, (C1-C4) alcohol, or an aqueous solution of (C1-C4) alcohol, but is not limited thereto.

[0016] In the composition for preventing or treating pemphigus according to the present invention, the water, (C1-C4) alcohol, or (C1-C4) alcohol aqueous extract concentrate may be any one selected from the hexane fraction, methylene chloride fraction, ethyl acetate fraction, and n-butanol fraction after additionally adding water. Additionally, the fraction of the present invention may be purified using a column purification method, wherein the column may be, for example, an aromatic type synthetic adsorbent column.

[0017] The extract composition selected from the above Torilidis Fructus may be a pharmaceutical composition for the prevention or treatment of pemphigus, formulated for systemic or topical administration.

[0018] The above composition may be a pharmaceutical composition for the prevention or treatment of pemphigus administered orally in the form of a tablet, capsule, liquid, suspension, or powder.

[0019] In addition, the above composition may be a pharmaceutical composition for the prevention or treatment of pemphigus, characterized in that it is in the form of a gel, hydrogel, ointment, cream, foam, spray, lotion, liquid, or skin patch.

[0020] In addition, the above composition may be an injectable pharmaceutical composition for the prevention or treatment of pemphigus administered by injection.

[0021] The above composition provides a pharmaceutical composition for the prevention or treatment of pemphigus comprising a Torilidis Fructus extract and a pharmaceutically acceptable excipient. The Torilidis Fructus extract may be added at a concentration of 0.001% to 20% by weight of the total weight of the composition.

[0022] The pharmaceutical composition according to the present invention can be formulated into a suitable form with a commonly used pharmaceutically acceptable carrier. “Pharmaceutically acceptable” means a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness. Additionally, the composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, each according to conventional methods.

[0023] Carriers, excipients, and diluents that may be included in the above composition may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl parahydroxybenzoate, propyl parahydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it is prepared using commonly used fillers, stabilizers, binders, disintegrants, surfactants, diluents, or excipients. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms are prepared by mixing at least one excipient, for example, starch, microcrystalline cellulose, sucrose or lactose, low-substituted hydroxypropyl cellulose, hypromellose, etc., with a composition containing the Saposhnikovia divaricata extract of the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, for example, humectants, sweeteners, flavorings, and preservatives, may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, 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 paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.To formulate a parenteral administration formulation, a composition containing Saposhnikovia divaricata extract may be mixed with water to form a solution or suspension with sterile or preservatives, stabilizers, hydrating agents or emulsifying promoters, salts or buffers for osmotic pressure regulation, and other therapeutically useful substances, and this may be prepared in an ampoule or vial unit administration form.

[0024] The above pharmaceutical composition may be administered to mammals, such as rats, livestock, and humans, via various routes. Any mode of administration is expected, for example, orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, intracerebrovascular injection, or topical application. The dosage will vary depending on the age, sex, and body weight of the subject to treatment, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the time of administration, the route of administration, the absorption, distribution, and excretion rates of the drug, the types of other drugs used, and the judgment of the prescriber. The determination of the dosage based on these factors is within the level of a person skilled in the art, and generally, the dosage ranges from 0.01 mg / kg / day to approximately 500 mg / kg / day. A preferred dosage is 0.1 mg / kg / day to 200 mg / kg / day, and a more preferred dosage is 1 mg / kg / day to 200 mg / kg / day. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0025] Furthermore, the above composition may be a pharmaceutical composition as well as a health functional food composition. The above health functional food refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the above functionality may refer to consumption for the purpose of obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The above health functional food may include conventional food additives, and unless otherwise stipulated, the suitability of the above food additives may be determined in accordance with the specifications and standards for the relevant items, in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0026] The items listed in the above Food Additives Codex may include, for example, chemically synthesized compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline additives for noodles, preservative preparations, and tar dye preparations. The above health functional food may be used in various ways in foods and beverages for the improvement of pemphigus, for example, in various foods, beverages, chewing gum, tea, vitamin complexes, health functional supplements, and food additives. The above health functional food may contain 1.0 to 10.0 weight% of the above composition relative to the total weight for the purpose of improving pemphigus. If the above composition is less than 1.0 weight%, the effect of improving pemphigus may not be sufficiently realized, and if it exceeds 10.0 weight%, the inherent quality of the product may not be realized or cost efficiency may be reduced. In addition, the above-mentioned health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of improving pemphigus. Specifically, the above-mentioned health functional food in tablet form may be manufactured by granulating a mixture of the *Sophora triphylla* extract or its fractions, excipients, binders, disintegrants, and other additives by a conventional method, and then adding a lubricant or the like and compression molding, or by directly compression molding the mixture. In addition, the above-mentioned health functional food in tablet form may contain a binder or the like as needed, and may be coated with a suitable coating agent as needed. Among the above-mentioned health functional food in capsule form, hard capsules may be manufactured by filling a conventional hard capsule with a mixture of *Sophora triphylla* extract and additives such as excipients, or the granules thereof, or coated granules thereof, and soft capsules may be manufactured by filling a capsule base such as gelatin with a mixture of *Sophora triphylla* extract or its fractions and additives such as excipients. The above soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.The above-mentioned pill-shaped health functional food may be prepared by molding a mixture of Saposhnikovia divaricata extract or its fractions, excipients, binders, disintegrants, etc., using an appropriate method. If necessary, the pill may be coated with sucrose or other suitable coating agents, or coated with starch, talc, or other suitable substances. The above-mentioned granular-shaped health functional food may be prepared into granules by forming a mixture of Saposhnikovia divaricata extract or its fractions, excipients, binders, disintegrants, etc., into granules using an appropriate method. If necessary, it may contain flavoring agents, stimulants, etc. Furthermore, the definitions of terms regarding the excipients, binders, disintegrants, lubricants, stimulants, flavoring agents, etc., as described in literature known in the art, may include those with identical or similar functions. Although embodiments of the present invention are described in detail, it is obvious that the present invention is not limited by the following examples. Effects of the invention

[0027] The present invention relates to a composition for improving or treating pemphigus containing an extract of Torilidis Fructus, and more specifically, it has been confirmed that the said Torilidis Fructus extract is effective in treating pemphigus.

[0028] Through this, it is expected that the Torilidis Fructus extract composition of the present invention can be usefully utilized in the treatment of pemphigus. Brief explanation of the drawing

[0029] Figure 1 is an experimental result showing that the Saposhnikovia divaricata extract used in the present invention is non-toxic to HaCaT cell survival. Figure 2 shows the results confirming the anti-pemphigus activity of Saposhnikovia divaricata extract in HaCaT cells. Specific details for implementing the invention

[0030] Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein is provided to be thorough and complete and to sufficiently convey the concept of the present invention to those skilled in the art.

[0031] <Example 1. Preparation of Torilidis Fructus Extract>

[0032] The plant extract (CA01-099) used in this study was obtained from the Korean Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea).

[0033] The voucher specimen (PBC-194) is stored at the Herbarium of the Korea Research Institute of Biotechnology and Bioengineering. Saposhnikovia divaricata (53 g) was air-dried and powdered, then 1 L of 95.0% ethyl alcohol (GR grade) was added, and extraction was performed at room temperature for 30 cycles (40 KHz, 1500 W, 15 min ultrasonic treatment - 120 min incubation / cycle). Saposhnikovia divaricata extract (4.0 g) was obtained by filtering using an ultrasonic extractor (SDN-900H, SD-ULTRASONIC CO., LTD) (Qualitative Filter No. 100, HYUNDAI MICRO CO., LTD) followed by vacuum drying.

[0034] <Experimental Example 1. Confirmation of Non-Toxic Concentration of Saposhnikovia Extract>

[0035] 1-1. HaCaT Cell Culture

[0036] Immortalized human skin keratinocytes, HaCaT cells (AddexBio, CA, USA), were cultured in DMEM / high glucose with 10% fetal bovine serum and 1% antibiotic-antifungal solution at 37°C and 5% CO2. All materials for cell culture were purchased from HyClone (UT, USA).

[0037] 1-2. Cell Viability Analysis

[0038] HaCaT cells (4x10 3Cells (cells / well) were seeded into a 96-well plate and cultured for 24 hours, then treated with different concentrations of Saposhnikovia divaricata extract for 3 days. Cell viability was evaluated triplicate using the Cell Counting Kit-8 (Dojindo Molecular Technologies, ML, USA) according to the manufacturer's protocol. Absorbance was measured at 450 nm using a HIDEX Sense microplate reader (Hidex, Finland).

[0039] 1-3. Experimental Results

[0040] As shown in Figure 1, it was observed that the extract of *Sasaengja* up to a treatment concentration of 10 μg / ml was not toxic to HaCaT cells, which are immortalized human skin keratinocytes.

[0041] <Experimental Example 2. Confirmation of Antipemphigus Activity of Saposhnikovia Extract>

[0042] 2-1. Dispase-based Dissociation Analysis

[0043] Ref.(Heupel WM, et al ., Pemphigus vulgaris IgG cause loss of desmoglein-mediated adhesion and keratinocyte dissociation independent of epidermal growth factor receptor. Am J Pathol. The analysis was performed by referring to the method of (2009) 174(2):475-85.). Briefly, HaCaT cells (5x10 5Cells (in a well) were seeded into a 24-well plate and incubated for 24 hours, followed by treatment with 5 μg / ml AK23 along with the extract of the present invention at the indicated non-toxic concentration for 24 hours. Then, the cells were washed with pre-warmed PBS and incubated at 37°C for 20 minutes with 150 μl of Hanks buffered saline (HBSS, Gibco) containing Dispase II solution (2.4 U / ml; Sigma). After adding 200 μl of HBSS, the cell monolayer was exposed to mechanical stress by pipetting 15 times with a 1 ml pipette for monolayer shearing. Subsequently, the cells were stained with MTT (5 mg / ml; Sigma) for 20 minutes. The number of generated fragments was counted, and images were captured under a microscope. Dissociation analysis was performed three times, and statistical differences were analyzed using Student's t-test. A p<0.05 value was considered significant.

[0044] 2-2. Experimental Results

[0045] Dispase-based dissociation assays have been reported using a representative in vitro pemphigus model with a monolayer of cells such as HaCaT cells. Evidently, treatment with AK23 on a monolayer of HaCaT cells increased the number of fragments exhibiting a loss of intercellular adhesion, a key characteristic of autoimmune blistering skin diseases such as pemphigus. Since the possibility that chemical toxicity itself may cause a loss of cell adhesion cannot be ruled out, the non-toxic concentration (up to 10 μg / ml) of the *Sophora triphylla* extract of the present invention was applied to HaCaT cells for dispase-based dissociation assays.

[0046] As shown in Figure 2, AK23 alone strongly induced fragmentation of the HaCaT cell monolayer, but the Saposhnikovia divaricata extract used in this study exhibited antipemphigus activity that dramatically inhibited fragmentation caused by AK23.

[0047] Through the above experimental results, it was confirmed that the Saposhnikovia divaricata extract is effective in inhibiting pemphigus without toxicity in human skin keratinocytes.

[0048] <Formulation Example 1. Pharmaceutical Formulation>

[0049] Formulation Example 1-1. Preparation of Tablets

[0050] Example 1 of the present invention: 200 mg of *Sophora triphylla* extract was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silica. A 10% gelatin solution was added to this mixture, and the mixture was ground and passed through a 14-mesh sieve. This was dried, and the mixture obtained by adding 160 g of potato starch, 50 g of active ingredient, and 5 g of magnesium stearate was made into tablets.

[0051] Formulation Example 1-2. Preparation of Injectable Solution

[0052] Example 1 of the present invention: 100 mg of Saposhnikovia divaricata extract, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20°C for 30 minutes.

[0053] <Preparation Example 2. Topical preparation>

[0054] Preparation Example 2-1. Preparation of an ointment

[0055] Example 1 of the present invention was prepared by uniformly mixing and stirring 2.0 wt% of Saposhnikovia divaricata extract, 8.0 wt% of diethyl sebacate, 5.0 wt% of brazil, 6.0 wt% of polyoxyethylene oleyl ether phosphate, 0.1 wt% of sodium benzoate, and the remainder of Vaseline with high-speed stirring.

[0056] Formulation Example 2-2. Preparation of cream

[0057] Example 1 of the present invention was prepared by uniformly mixing and stirring 0.5 wt% of Saposhnikovia divaricata extract, 15.0 wt% of stearic acid, 1.0 wt% of cetanol, 0.7 wt% of potassium hydroxide, 5.0 wt% of glycerin, 3.0 wt% of propylene glycol, 0.05 wt% of a preservative, and the remaining amount of purified water with high-speed stirring.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of pemphigus vulgaris characterized by containing an extract of Torilidis Fructus as an active ingredient. Claim 2 A pharmaceutical composition for the prevention or treatment of pemphigus vulgaris according to claim 1, characterized in that the Torilidis Fructus extract comprises water, (C1-C4) alcohol, or an aqueous (C1-C4) alcohol extract as an active ingredient. Claim 3 delete Claim 4 A health functional food for the prevention or improvement of pemphigus vulgaris, characterized by containing Torilidis Fructus extract as an active ingredient. Claim 5 A health functional food for the prevention or improvement of pemphigus vulgaris, characterized in that, in claim 4, the above-mentioned Torilidis Fructus extract comprises water, (C1-C4) alcohol, or an aqueous (C1-C4) alcohol extract as an active ingredient. Claim 6 delete

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