KR20210021846A - Composition for preventing or treating Sjogren's syndrome comprising Adenophrae Radix - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Sjogren′s syndrome containing an Adenophora stricta Miq. extract as an active component. The extract has effects of increasing the expression of aquaporin 5 (AQP5), reducing the production of anti-Ro/SSA and anti-La/SSB, which are autoantibodies, and reducing the secretion of inflammatory cytokines TNF-α, IFN-γ or IL-6, thereby being able to be usefully used for preventing or treating Sjogren′s syndrome.

Description

Composition for preventing or treating Sjogren's syndrome comprising Adenophrae Radix as an active ingredient

The present invention relates to a composition for the prevention or treatment of Sjogren's syndrome comprising a sasamic extract as an active ingredient.

Sasam (Adenophrae Radix) refers to the root of the stalk belonging to the Campanulaceae family. Sasam is a perennial herb that grows in Korea, Japan, China, etc., with a height of 50-100 cm. The hairs are dense throughout the ground, the roots are enlarged, and the flowers bloom in a purple bell shape in July-September.

Sjogren's syndrome is a chronic and systemic autoimmune disease caused by the infiltration of lymphocytes and autoimmune antibodies into exocrine glands such as salivary and lacrimal glands. Dry eye and xerostomia appear as a result of inflammation and destruction in the salivary and lacrimal glands. Dry mouth can cause dysphagia, difficulty chewing, discomfort in conversation, changes in taste, increased tooth decay, and serious physical, functional, social and psychological effects. Sjogren's syndrome is the most common autoimmune disease with a prevalence of 0.5% to 1.0%, but the exact cause and pathological mechanism are not fully elucidated.

Treatment of Sjogren's syndrome mainly relieves dryness by using artificial tears, topical lubricants (artificial saliva and vaginal lubricants) and stimulants such as pilocarpine, cevimeline, prednisone, and immunosuppressants (azathioprine, cyclosporine). However, such treatment may cause side effects such as sweating, frequent urination, redness, sinusitis, and nausea when administered for a long period of time, and symptom relief is limited in that it is temporary. Therefore, there is a need to develop drugs for fundamental treatment without side effects.

Korean Patent Publication No. 10-2017-0045762

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of Sjogren's syndrome comprising a sasamin extract as an active ingredient.

Another object of the present invention is to provide a health functional food for the prevention or improvement of Sjogren's syndrome comprising a sasamin extract as an active ingredient.

Another object of the present invention is to provide a method for increasing the expression of AQP5 (Aquaporin 5), comprising administering to a subject a ginseng extract.

Another object of the present invention is to provide a method for reducing the production of anti-Ro/SSA and anti-La/SSB in serum, comprising administering a sasamin extract to a subject.

Another object of the present invention is to provide a method for reducing the secretion of inflammatory cytokines TNF-α, IFN-γ or IL-6, comprising the step of administering a sasamin extract to an individual.

Another object of the present invention is to provide a method of increasing the expression of AQP5 (Aquaporin 5) comprising the step of treating a sample with an extract of Adenophrae Radix in vitro.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of Sjogren's syndrome comprising a sasamin extract as an active ingredient.

In one embodiment of the present invention, the extract may be extracted with water, an organic solvent, or a mixture thereof, and the organic solvent may be methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, It may be one or more selected from the group consisting of ethyl acetate, methylene chloride, hexane, and cyclohexane.

In one embodiment of the present invention, the extract may be one to increase the expression of AQP5 (Aquaporin 5).

In an embodiment of the present invention, the extract may be to reduce the production of anti-Ro/SSA and anti-La/SSB.

In one embodiment of the present invention, the extract may be one to reduce the secretion of inflammatory cytokines TNF-α, IFN-γ or IL-6.

In one embodiment of the present invention, the extract may be administered in an amount of 0.1 to 1000 mg/kg, preferably 1 to 1000 mg/kg, more preferably 10 to 1000 mg/kg May be, more preferably 100 to 1000 mg/kg, but is not limited thereto.

In addition, the present invention provides a health functional food for the prevention or improvement of Sjogren's syndrome, comprising the sasamin extract as an active ingredient.

In addition, the present invention provides a method for increasing the expression of AQP5 (Aquaporin 5), comprising administering to a subject a sasamin extract.

In addition, the present invention provides a method for reducing the production of anti-Ro/SSA and anti-La/SSB in serum, comprising administering to a subject an extract of Sasamin.

In addition, the present invention provides a method for reducing the secretion of inflammatory cytokines TNF-α, IFN-γ or IL-6, comprising the step of administering a sasamin extract to an individual.

In addition, it provides a method for increasing the expression of AQP5 (Aquaporin 5) comprising the step of treating a sample with an extract of Adenophrae Radix in vitro.

The extract according to the present invention increases the expression of AQP5 (Aquaporin 5), reduces the production of autoantibodies anti-Ro/SSA and anti-La/SSB, and inflammatory cytokines TNF-α, IFN-γ or IL Since it has the effect of reducing the secretion of -6, it can be usefully used in the prevention or treatment of Sjogren's syndrome.

1 is a result of measuring the cell viability after treating NS-SV-AC cells with sasamic extract and 2 μM of 5-Aza for 48 hours (data are expressed as mean±SD (n=3); # #: p <0.01, significant compared to normal; and *: p <0.05, **: p <0.01, ***: p <0.001, significant compared to control ).
Figure 2 is a result of measuring the fluid secretion rate after treatment of NS-SV-AC cells with sasamic extract and 2 μM of 5-Aza (data are expressed as mean±SE (n=3); #: p <0.05, significant compared to the normal group; and *: p <0.05, significant compared to the control).
Figure 3A is a result of measuring the expression level of AQP5 protein through Western blot after treating NS-SV-AC cells with four ginseng extract and 2 μM of 5-Aza, and 3B is a result of standardized quantification of the result of Western blot. Results (data are expressed as mean±SE (n=3); #: p <0.05, significant compared to normal; And *: p <0.05, significant compared to control One case).
FIG. 4 is a result of measuring the expression pattern of AQP5 protein through immunofluorescence analysis after treatment of NS-SV-AC cells with sasam extract and 2 μM of 5-Aza, and the left line indicates positive AQP5 with red dots. (dot), and the right line shows DAPI (blue) and AQP5 together (A, B: normal; C, D: control treated with only 5-Aza; E, F: Group treated with 5-Aza and 100 μg/ml of Ginseng extract; Magnification: × 200; and Bar = 100 μm).
5 is a result of measuring the amount of salivary secretion after treatment with sasam extract in NOD/SCID mice (N: normal group; C: control group; 100, 500, 1000: 100, 500, 1000 mg/kg of ginseng extract Treated group; #: p <0.05, significant compared to normal group; and *: p <0.05, **: p <0.01, significant compared to control).
6A is a result of measuring the expression levels of AQP 5 protein and M3R protein through Western blot after treatment with a sasam extract in NOD/SCID mice, and 6B is a result of standardized quantification of the expression level of AQP 5 protein, 6C is the result of quantification of the expression level of M3R protein by standardization (N: normal group; C: control group; 100, 500, 1000: group treated with 100, 500, 1000 mg/kg of sassam extract; data is mean± Denoted as SD (n=3); #: p <0.05, significant compared to the normal group; and *: p <0.05, significant compared to the control).
Figure 7 is a result of measuring the amount of autoantibodies anti-Ro/SSA (A) and anti-La/SSB (B) in mouse serum through ELISA after treatment with a ginseng extract in NOD/SCID mice (N : Normal group; C: control group; and 100, 500, 1000: group treated with 100, 500, 1000 mg/kg of sasamin extract).
Figure 8 is a result of measuring the amount of TNF-α (A), IFN-γ (B) and IL-6 (C) in mouse serum through ELISA after treatment with a ginseng extract in NOD/SCID mice (N : Normal group; C: Control group; 100, 500, 1000: Group treated with 100, 500, 1000 mg/kg of Sasamin extract; Data expressed as mean±SD (n=6); ###: p < 0.001, significant compared to normal group; and *: p <0.05, **: p <0.01, significant compared to control).
Figure 9 is a result of observing the histological changes of salivary gland tissue after treatment with the ginseng extract in NOD/SCID mice (A: normal group; B: control group; C: group treated with ginseng extract 100 mg/kg; D: group treated with sasam extract 500 mg/kg; E: group treated with sasam extract 1000 mg/kg; arrows: inflammatory lesions; magnification: × 200; and Bar = 100 μm).

The extract according to the present invention may be extracted and separated from nature using a method known in the art for extraction and separation, and the "extract" as defined in the present invention is used as an appropriate solvent. ) Extracted from, for example, a crude extract of Adenophorae Radix, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

As a suitable solvent for extracting the extract from the sasam, any organic solvent may be used as long as it is a pharmaceutically acceptable organic solvent, and water or an organic solvent may be used, but is not limited thereto, for example, purified water, methanol ( alcohols having 1 to 4 carbon atoms, including methanol), ethanol, propanol, isopropanol, butanol, etc., acetone, ether, benzene, chloroform ( Various solvents such as chloroform), ethyl acetate, methylene chloride, hexane, and cyclohexane may be used alone or in combination. Preferably, water can be used.

As the extraction method, any one of methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation method, ultrasonic extraction method, elution method, and compression method may be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method. There is no limitation on the preparation method of the sasamin extract of the present invention, and any known method may be used.

For example, the sasamin extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying of the primary extract extracted by the hot water extraction or solvent extraction method described above. Further, the primary extract was further purified by using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get

Therefore, in the present invention, the sasamin extract is a concept including all extracts, fractions, and purified products obtained in each step of extraction, fractionation or purification, and their dilutions, concentrates, or dried products.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.

In addition, the pharmaceutical composition of the present invention is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. I can. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.

The term "individual" refers to all animals including humans, such as rats, mice, and domestic animals. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.

The food composition of the present invention may include all foods in a conventional sense, and may be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the term "functional" It refers to ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients for structure and function or for physiological effects.

The term "health functional food" of the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, and mixing specific ingredients contained in food ingredients or using specific ingredients as a raw material for the purpose of supplementing health. It refers to foods designed and processed to sufficiently exert biological control functions such as biological defense, biological rhythm control, disease prevention and recovery, etc., by ingredients, and the health food composition is used to prevent diseases and recover diseases. Can perform functions related to etc.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition of the present invention can be prepared by mixing suitable other auxiliary ingredients that may be included in food and known additives according to the choice of a person skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the extract according to the present invention and a fraction thereof as a main component, such as juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formulas, infant foods, etc.), processed meat products, fish meat products, tofu, rice cakes, noodles (e.g., ramen, noodles, etc.), health supplement foods. , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, sweets (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, as in ordinary beverages. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin. , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Experimental method

1.1. Preparation of Adenophorae Radix Extract

Sasam was purchased and used from Bonchomaru (Seoul, Korea). 50 g of dried sasamic sample was added with 1 L of water and boiled at 100° C. for 2 hours, the suspension was filtered, and then distilled under reduced pressure. The filtrate was lyophilized to obtain 1.5 g of powder. The dried extract was dissolved in distilled water (Millipore, Billerica, MA, USA) and stirred at room temperature for 2 minutes to be used.

1.2. Cell culture

Human salivary gland acinar cells (“NS-SV-AC cells”) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). NS-SV-AC cell line DMEM (Dulbecco's minimal essential medium, Gibco-BRL, Grand Island, NY, added with 10% FBS (fetal bovine serum, Gibco-BRL), 100 U/mL penicillin and 100 μg/mL streptomycin) USA) at 37° C. and 5% CO ₂ incubator.

1.3. Cell viability and cell proliferation assay

Cell proliferation was determined by the MTT assay. NS-SV-AC cells were starved for 24 hours, then several concentrations of sasamic extract (1, 5, 10, 50 or 100 μg/mL) and 2 μM of 5-aza-2'-deoxycytidine (hereinafter, "5 -Aza", Sigma-Aldrich Corp. St. Louis, MO, USA) were treated simultaneously. After 48 hours, the medium was removed and treated with MTT. Thereafter, spectrophotometric analysis was performed at 570 nm using a microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA).

1.4. Animal experiment

20-week-old NOD/SCID (non-obese diabetic mice) female mice were used as the Sjogren's syndrome mouse model, and 20-week-old normal Balb/c female mice were used as the normal model (Saron Bio Inc., Seoul, Korea). , Korea). Animals were grown in a sterile environment with a 12-hour light-dark cycle at the Center for Laboratory Animal Care and Use at Kyunghee University. The protocol for the use of mice in the present invention was approved by the Institutional Animal Care & Use Committee (IACUC), Kyunghee University (KHUASP(SE)-16-113).

Mice were randomly classified into 5 groups: normal group (N, Balb/c, n=6); Control (C, NOD/SCID, n=6) and three ginseng extract treatment groups (100, 500 or 1000 mg/kg, o.p., n=6/each group). Four ginseng extract was treated 5 days a week for 4 weeks. Animals were weighed weekly to control the amount of gavage and to check their general health.

1.5. Fluid secretion measurement

The net fluid secretion rate in control cells and cells treated with 5-Aza (2 μM) for 48 hours was measured in a modified form of the conventional method. Briefly, NS-SV-AC cells were cultured in six-well Transwell-Col culture chambers (Coster, Cambridge, MA, USA). The apical fluid was replaced with 0.4 ml of hyperosmotic medium (400 mOsm), and the medium in the basal chamber was replaced with isosmotic medium (300 mOsm). After 4 hours, the liquid at the apex was collected and the volume was measured with a graduated pipette.

In an in vivo study, each female mouse was anesthetized by intraperitoneal injection of urethane (1.3 g/kg). Saliva was collected at the bottom of the mouth with a capillary paper plug for 10 minutes, and the weight of the paper plug was measured before and after collection of the salivary glands. The amount of saliva was normalized to milliliters (mL) of saliva per gram of body weight per 10 minutes.

1.6. ELISA analysis

IgG detection was performed according to the manufacturer's instructions using a mouse anti-Ro/SSA and anti-La/SSB ELISA kit (Signosis, Inc., Santa Clara, CA, USA). All serum samples were diluted 1:50.

The amounts of TNF-α, IFN-γ and IL-6 in NOD/SCID mouse serum were performed according to the manufacturer's instructions using mouse TNF-α, IFN-γ and IL-6 ELISA kit (R&D).

1.7. Western blot analysis

For total protein extraction, the salivary glands of NS-SV-AC cells and NOD/SCID mice were lysed by treatment with lysis buffer at 4° C. for 1 hour. The insoluble material was centrifuged at 13,000 x g for 20 minutes at 4° C. using a Smart R17 Refrigerated Micro Centrifuge (Hanil BioMed, Inc. Seoul. Korea) and discarded. Protein concentration in cell lysates was determined using Protein Assay. Equal amounts of protein were separated by 10% SDS-PAGE at 120 V for 2 hours. The separated protein was transferred to the membrane and blocked with 5% skim milk powder (in TBST, Tris-buffered saline containing 0.5% Tween-20 (10 mM of Tris-Cl, pH 7.4)) at room temperature for 1 hour. Thereafter, the membrane was rabbit anti-AQP 5 (1:1000) (Santa Cruz Biotechnology, Inc., Texas, USA), anti-M3R (1:500) (Abcam, Cambridge, England) and anti-β-tubulin (1 :1000) (Santa Cruz Biotechnology, Inc).

1.8. Immunofluorescence staining

NS-SV-AC cells were dispensed on a slide glass, and cultured for 48 hours with or without treatment with 5-Aza and sasam extract. Cells were fixed with 2% paraformaldehyde and treated with anti-AQP 5 antibody (Santa Cruz Biotechnology, Inc., Texas, USA) for 4 hours at room temperature. The salivary gland tissue was fixed with 4% paraformaldehyde and then embedded. Thereafter, Cyanine dyes (Cy3)-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Invitrogen, Carlsbad, CA, USA) was used as the secondary antibody. Immunostained cells were treated with a mounting solution containing DAPI (4'6-diamidino-2-phenylindole), and visualized using an Olympus Immunofluorescence microscope (Carl Zeiss, Oberkochen, Germen).

1.9. Histological analysis of salivary glands

Salivary glands were separated at the time of euthanasia of each group of mice, fixed with 4% paraformaldehyde, embedded with O.C.T compound, and cut to a thickness of 5 μm. Each section was stained with hematoxylin dye.

1.10. Statistical analysis

Statistical analysis was performed using GraphPrism 4.0.3 software (GraphPad Software, Inc., San Diego, CA, USA). Data were expressed as mean±standard deviation (S.D.) or mean±standard error (S.E.), and were analyzed using SPSS software version 12.0 (SPSS, Chicago, IL, USA).

Example 2. Effect of increasing cell proliferation by sasam extract in NS-SV-AC cells

The present inventors performed an experiment through an MTT assay measuring the metabolic activity of living cells in order to confirm whether there is an effect on cell proliferation in NS-SV-AC cells by the sasam extract. NS-SV-AC cells were cultured in starvation medium for 24 hours before the experiment to stop their growth, and then, sasamin extract and 2 μM of 5-Aza were treated for 48 hours. The normal control (normal) was treated with PBS, the control (control) was treated with only 5-Aza.

As a result, it was confirmed that the cell viability was significantly increased in the group treated with 5-Aza and sasam extract compared to the control group treated with only 5-Aza (FIG. 1). Specifically, when the concentrations of Sasamin extract were treated with 1, 5, 10, 50, and 100 μg/mL, the cell viability was 57.4 ± 10.8%, 48.1 ± 8.9%, 54.6 ± 10.7%, 62.4 ± 6.8%, and 79.7 ± 6.3, respectively. As a %, it was increased compared to the group treated with 5-Aza alone, 36.8 ± 4.3%.

From the above results, it was confirmed that the sasamin extract has an effect of improving the cell proliferation of acinar cells and protecting acinar cells from cytotoxicity induced by 5-Aza.

Example 3. Effect of increasing the fluid secretion rate by sasamic extract in NS-SV-AC cells

The present inventors conducted an experiment to confirm whether the sasamin extract has an effect of increasing the fluid secretion rate in NS-SV-AC cells. As a result, it was confirmed that in the cells treated with only 5-Aza, the total fluid secretion rate was 1.1 ± 0.3 μl/cm 2 .h, which was significantly reduced compared to the control (1.6 ± 0.2 μl/cm 2 .h) (FIG. 2 ). However, in the case of simultaneous treatment of 100 μg/ml of Sasamin extract with 5-Aza, the total fluid secretion rate was 1.7 ± 0.2 μl/cm 2 .h, which was significantly higher than that of the group treated with 5-Aza alone. It was confirmed that it was increased (FIG. 2).

From the above results, it was confirmed that the sasamin extract has an effect of increasing the fluid secretion rate in acinar.

Example 4. Effect of increasing the expression level of AQP 5 protein by sasam extract in NS-SV-AC cells

The present inventors performed western blot and immunofluorescence analysis in order to confirm whether the sassam extract changes the expression of the membrane channel protein AQP5 (Aquaporin 5) protein.

As a result, it was confirmed that in the cells treated with only 5-Aza, the expression level of the AQP5 protein was 51.7 ± 20.2%, which was significantly reduced compared to the control (100%) (FIG. 3). However, in the case of simultaneous treatment of 100 μg/ml of Sasamin extract with 5-Aza, the expression level of AQP5 protein was 87.2%, and it was confirmed that the expression level of AQP5 protein was significantly increased compared to the group treated with only 5-Aza ( Fig. 3).

In addition, in immunofluorescence analysis in the same manner as the Western blot result, it was confirmed that the expression of the AQP5 protein was increased compared to the group treated with only 5-Aza when 100 μg/ml of Sasamin extract was simultaneously treated with 5-Aza (FIG. 4 ).

From the above results, it was confirmed that the sasamin extract has an effect of increasing the expression level of AQP 5 protein in NS-SV-AC cells.

Example 5. Effect of increasing saliva secretion by sasamin extract in NOD/SCID mice

The NOD/SCID mouse is an animal model in which pancreatic B cells are destroyed by an autoimmune mechanism, and is used as an animal model of Sjogren's syndrome. The present inventors conducted an experiment to confirm whether there is an effect of increasing salivation by sasam extract even in vivo through NOD/SCID mice. As a result, it was confirmed that in the control group (C), which is a NOD/SCID mouse, which is a Sjogren syndrome mouse model, salivation was 5.8 ± 1.3 μl/10 min/g, which was significantly reduced compared to the normal mouse group (N) ( Fig. 5). However, in the group treated with 100, 500, and 1000 mg/kg of Sasamin extract in NOD/SCID mice, the amount of salivation was 12.2 ± 2.7, 14.2 ± 2.2, and 12 ± 3.2 μl/10 min/g, respectively, and the control group It was confirmed that saliva secretion was significantly increased compared to (C).

From the above results, it was confirmed that the Sasamin extract has an effect of increasing the amount of salivation in an animal model of Sjogren syndrome, and thus has a therapeutic effect on Sjogren syndrome.

Example 6. Effect of increasing the expression levels of AQP 5 protein and M3R protein by sasamic extract in NOD/SCID mice

Salivary secrection is associated with a membrane channel protein aquaporin-related mechanism, and AQP5 is a major water migration channel protein present in acinar cells, and anti-muscarinic receptor type III (M3R). Is a protein that is highly expressed in pancreatic B cells and is known as a regulator of blood glucose homeostasis. Accordingly, the present inventors performed western blot to confirm whether the AQP 5 protein and M3R protein expression levels were affected by the sasam extract in NOD/SCID mice.

As a result, it was confirmed that the expression level of AQP 5 protein was 81.7% in the control group (C) of NOD/SCID mice, which was significantly reduced compared to the normal mouse group (N, 100%) (FIGS. 6A and 6B). By the way, it was confirmed that the expression level of AQP 5 protein was 93% in the group treated with the ginseng extract in the amount of 1000 mg/kg in NOD/SCID mice, and that the expression level of the AQP 5 protein was increased compared to the control group (C) (Fig. 6B).

In addition, in the control group (C) of NOD/SCID mice, it was confirmed that the expression level of M3R protein was 81.7%, which was significantly reduced compared to the normal mouse group (N, 100%) (FIGS. 6A and 6C ). However, in the group treated with 500 and 1000 mg/kg of Sasamin extract in NOD/SCID mice, the expression level of M3R protein was 95% and 95.1%, respectively, indicating that the expression level of M3R protein was increased compared to the control group (C). Confirmed (Fig. 6C).

From the above results, it was confirmed that the sasamin extract has a therapeutic effect on Sjogren syndrome by increasing the expression levels of the AQP 5 protein and M3R protein in the Sjogren syndrome animal model.

Example 7. Effect of reducing the production amount of autoantibody by sasamin extract in NOD/SCID mice

Anti-Ro/SSA and anti-La/SSB are known as autoimmune biomarkers associated with damage to salivary and lacrimal glands by severe lymphocyte infiltration. About 70% of Sjogren syndrome patients have anti-Ro/SSA and about 60% have anti-La/SSB. Accordingly, the present inventors performed ELISA to determine whether the sasamin extract has an effect of reducing the production of anti-Ro/SSA and anti-La/SSB, which are autoantibodies, in the serum of NOD/SCID mice, which is an animal model of Sjogren's syndrome. I did.

As a result, it was confirmed that the production of anti-Ro/SSA and anti-La/SSB was significantly increased in the control group (C) as NOD/SCID mice compared to the normal mouse group (FIG. 7). However, in the group treated with 100, 500, and 1000 mg/kg of Sasamin extract in NOD/SCID mice, the production of anti-Ro/SSA and anti-La/SSB was significantly reduced compared to the control group (C). Confirmed (Fig. 7).

From the above results, it was confirmed that the sasamin extract has a therapeutic effect on Sjogren syndrome by remarkably reducing the production of autoantibodies anti-Ro/SSA and anti-La/SSB in the Sjogren syndrome animal model.

Example 8. Inhibitory Effect of Inflammatory Cytokines by Four Ginseng Extract in NOD/SCID Mice

The balance of Th1 cells and Th2 cells plays an important role in autoimmune diseases. IFN-γ, an inflammatory cytokine secreted by Th1 cells, is known to be overexpressed in Sjogren syndrome patients. In addition, TNF-α and IL-6 activate B cells and induce an inflammatory response, and in particular, IL-6 has been reported to be associated with overactivity of B cells. Accordingly, the present inventors have conducted ELISA to determine whether sasam extract has an effect of inhibiting the amount of inflammatory cytokines TNF-α, IFN-γ and IL-6 in the serum of NOD/SCID mice, which is an animal model of Sjogren's syndrome. Was performed.

As a result, in the control group (C), which is NOD/SCID mice, the amount of TNF-α was 116.6 ±19.0 pg/ml, which was significantly increased compared to the normal mouse group (N), and the ginseng extract was 100, 500 and 1000 mg/ml. In the group treated with kg, it was confirmed that the effect was significantly reduced compared to the control group as 61.4 ± 27.6, 59.3 ± 22.9, and 58.2 ± 17.4 pg/ml, respectively (FIG. 8A).

In addition, in the case of IFN-γ, 90.7 ± 10.9 pg/ml in the control group (C), which was significantly increased compared to the normal mouse group (N), and 31.4 ± in the group treated with 100 and 1000 mg/kg sasamin extract, respectively. It was confirmed that there is a remarkably reduced effect as 20.4 and 40.8 ± 21 pg/ml (FIG. 8B).

In addition, in the case of IL-6, in the control group (C), it was 203.7±24.6 pg/ml, which was significantly increased compared to the normal mouse group (N), and in the group treated with 100 mg/kg of Sasamin extract, 97.42±27 pg/ml It was confirmed that there is a remarkably reduced effect as ml (Fig. 8C).

From the above results, it was confirmed that the sasamin extract has a therapeutic effect on Sjogren syndrome by remarkably reducing the amount of inflammatory cytokines TNF-α, IFN-γ, and IL-6 in the antibody of the Sjogren syndrome animal model.

Example 9. Histopathological changes in salivary gland tissues by salivary gland extract in NOD/SCID mice

The present inventors performed an experiment to confirm whether the salivary gland extract gives histological changes to the salivary gland tissue. As a result, in the control group (C), which is NOD/SCID mice, lymphocyte infiltration and inflammatory lesions were found compared to the normal mouse group (N) (FIGS. 9A and 9B ). On the other hand, it was confirmed that cell damage due to infiltration and inflammation was remarkably improved in the group treated with the sasamin extract compared to the control group (FIGS. 9C, 9D and 9E).

From the above results, it was confirmed that the salivary gland extract has a therapeutic effect on Sjogren syndrome through the effect of recovering cell damage caused by infiltration and inflammation in the salivary gland tissue of the Sjogren syndrome animal model.

Claims (12)

A pharmaceutical composition for the prevention or treatment of Sjogren's syndrome, comprising an extract of Adenophrae Radix as an active ingredient. The method of claim 1,
The composition, characterized in that the extract is extracted with water, an organic solvent, or a mixture thereof. The method of claim 2,
The organic solvent is a composition characterized in that at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane. The method of claim 1,
The composition, characterized in that the extract increases the expression of AQP5 (Aquaporin 5). The method of claim 1,
The composition, characterized in that the extract reduces the production of anti-Ro/SSA and anti-La/SSB. The method of claim 1,
The extract is a composition, characterized in that to reduce the secretion of inflammatory cytokines TNF-α, IFN-γ or IL-6. The method of claim 1,
The composition, characterized in that the extract is administered in an amount of 0.1 to 1000 mg / kg. Health functional food for the prevention or improvement of Sjogren's syndrome, which contains the extract of Adenophrae Radix as an active ingredient. A method for increasing the expression of AQP5 (Aquaporin 5), comprising administering an extract of Adenophrae Radix to a subject. A method for reducing the production of anti-Ro/SSA and anti-La/SSB in serum, comprising administering to a subject an extract of Adenophrae Radix. A method for reducing the secretion of inflammatory cytokines TNF-α, IFN-γ or IL-6, comprising administering an extract of Adenophrae Radix to an individual. A method for increasing the expression of AQP5 (Aquaporin 5) comprising the step of treating a sample with an extract of Adenophrae Radix in vitro.

Images