Pharmaceutical composition for preventing or treating obesity comprising Dictyopteris divaricata extract

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of obesity comprising a smooth-stemmed nettle extract as an active ingredient. It has been confirmed that the smooth-stemmed nettle extract has an inhibitory effect on the accumulation of fat and triglycerides in 3T3-L1 preadipocytes and inhibits the expression of lipogenesis transcription factors and fatty acid transport proteins in 3T3-L1 preadipocytes, and thus can be utilized as a therapeutic agent for obesity or in health functional food compositions.

Description

Technology Field

[0001] The present invention provides a pharmaceutical composition for the prevention or treatment of obesity comprising a smooth-bone nettle extract as an active ingredient. Background Technology

[0002] The World Health Organization (WHO) declared obesity a "disease that needs to be treated" and subsequently defined it as a "new infectious disease of the 21st century." Obesity is determined by the Body Mass Index (BMI), calculated by dividing body weight (kg) by the square of height (m), and a BMI of 25 or higher is considered obese. As of 2014, it is estimated that 39% of the world's population aged 18 and older is obese or overweight. In particular, the obesity rate among children and adolescents has increased rapidly; between 1999 and 2008, childhood and adolescent obesity in the United States more than tripled from 5% to 16.9%.

[0003] According to the '2017 Obesity White Paper' published by the National Health Insurance Service, the obesity rate among the 13.95 million individuals who underwent general health checkups and life-stage health examinations in Korea last year was 33.55%. Among them, men accounted for 41.29% and women 23.74%, showing a gender gap. For men, only 29.99% were classified as normal weight, while a large portion were overweight (25.64%), obese (35.74%), severely obese (5.31%), and morbidly obese (0.24%), indicating a significant population that is either obese or at high risk of becoming obese. In particular, 46.26% of men in their 30s had a BMI of 25 or higher. Meanwhile, for women, the normal weight rate was relatively high at 50.03%, while overweight accounted for 18.33%, obese 19.54%, severely obese 3.59%, and morbidly obese 0.61%. The rates of severe and morbid obesity were consistently higher for both men and women as income decreased.

[0004] The national and social costs associated with obesity are also substantial and are increasing over time. According to the 'Research Report on Improvement Measures for Insurer Obesity Management Programs to Enhance Healthy Life Expectancy' recently published by the Health Insurance Policy Research Institute under the National Health Insurance Service, the socio-economic costs of obesity nearly doubled from 4.7654 trillion won in 2006 to 9.1506 trillion won in 2015. A detailed breakdown of the 9.1506 trillion won shows that medical expenses accounted for 58.8% (5.3812 trillion won), premature death costs 17.9% (1.6371 trillion won), productivity loss 14.9% (1.3654 trillion won), caregiving costs 5.3% (486.4 billion won), and transportation costs 3.1% (280.4 billion won).

[0005] While dietary improvements and regular exercise are required for the prevention and treatment of obesity, major efforts to control it are focused on anti-obesity drugs and health supplements due to the characteristics of modern society. With global interest in obesity intensifying, there is an urgent need to develop treatments that can more effectively diagnose, prevent, or treat the condition. Prior art literature

[0006] 1. Republic of Korea Registered Patent No. 10-2177035 (Published on Feb. 22, 2017) The problem to be solved

[0007] The objective of the present invention is the smooth-bone nettle ( Dictyopteris divaricata The invention provides a pharmaceutical composition for the prevention or treatment of obesity containing an extract as an active ingredient.

[0008] Another objective of the present invention is to provide a health functional food composition for reducing body fat that includes a smooth-bone nettle extract as an active ingredient.

[0009] Another objective of the present invention is to provide a method for preparing a smooth-stemmed nettle extract, comprising: a first step of drying and grinding the smooth-stemmed nettle to produce it in powder form; a second step of adding water, a C1 to C4 alcohol, or an aqueous solution thereof to the powder-form smooth-stemmed nettle and performing ultrasonic extraction to produce a substrate solution; a third step of filtering the substrate solution to obtain a filtrate; and a fourth step of concentrating the filtrate under reduced pressure and drying it. means of solving the problem

[0010] To achieve the above objective, the present invention relates to a smooth-bone nettle ( Dictyopteris divaricata Provides a pharmaceutical composition for the prevention or treatment of obesity comprising an extract as an active ingredient.

[0011] In addition, the present invention provides a body fat reduction health functional food composition comprising a smooth-bone nettle extract as an active ingredient.

[0012] In addition, the present invention provides a method for preparing a smooth-stemmed nettle extract, characterized by comprising: a first step of drying and grinding the smooth-stemmed nettle to produce it in powder form; a second step of adding water, a C1 to C4 alcohol, or an aqueous solution thereof to the powder-form smooth-stemmed nettle and performing ultrasonic extraction to produce a substrate solution; a third step of filtering the substrate solution to obtain a filtrate; and a fourth step of concentrating the filtrate under reduced pressure and drying it. Effects of the invention

[0013] The present invention relates to a pharmaceutical composition for the prevention or treatment of obesity comprising a smooth-stemmed nettle extract as an active ingredient. It has been confirmed that the smooth-stemmed nettle extract has an inhibitory effect on the accumulation of fat and triglycerides in 3T3-L1 preadipocytes and inhibits the expression of lipogenesis transcription factors and fatty acid transport proteins in 3T3-L1 preadipocytes, and thus can be utilized as a therapeutic agent for obesity or in health functional food compositions. Brief explanation of the drawing

[0014] Figure 1 shows the cytotoxicity results of the extract of *Solidago virulencephala*. Figure 2 shows the inhibitory effect of the extract on intracellular fat accumulation and the results of electron microscopy of intracellular fat. Figure 3 shows the triglyceride content in fat cells of the smooth bone extract. Figure 4 shows the results of the lipogenesis transcription factor and fatty acid transport protein expression inhibitory effects of the extract of *Solidago virosa*. Specific details for implementing the invention

[0015] The present invention will be described in more detail below.

[0017] Smooth-bone net-like ( Dictyopteris divaricata Provides a pharmaceutical composition for the prevention or treatment of obesity comprising an extract as an active ingredient.

[0018] The above-mentioned smooth-bone nettle extract can be extracted with any one solvent selected from the group consisting of water, C1-C4 alcohols, and mixtures thereof.

[0019] The above-mentioned smooth-bone nettle extract can inhibit the accumulation of fat and triglycerides in 3T3-L1 preadipocytes.

[0020] The above-mentioned smooth-bone nettle extract can inhibit the expression of lipogenesis transcription factors and fatty acid transport proteins in 3T3-L1 preadipocytes.

[0021] The above-mentioned lipogenesis transcription factor is one or more selected from the group consisting of PPARγ, C / EBPα, and SREBP-1, and the above-mentioned fatty acid transport protein may be FABP4, but is not limited thereto.

[0022] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, leavening agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions. Specifically, the carrier, excipient, and diluent may be 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, and mineral oil. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations 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 suspensions. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as the base for suppositories.According to one embodiment of the present invention, the pharmaceutical composition may be administered to a subject in a conventional manner via intravenous, intra-arterial, intraperitoneal, intramuscular, intra-arterial, intraperitoneal, intrasternal, transdermal, nasal, inhalation, topical, rectal, oral, ocular, or intradermal routes. The dosage of the active ingredient according to the present invention may vary depending on the subject's condition and body weight, the type and severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, more preferably 0.1 mg / kg to 100 mg / kg. Administration may be performed once a day or divided into several doses, and the scope of the present invention is not limited by this.

[0024] In addition, the present invention provides a body fat reduction health functional food composition comprising a smooth-bone nettle extract as an active ingredient.

[0025] The above-mentioned health functional food may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable beverages. These ingredients may be used independently or in combination. Furthermore, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, chewing gum, ice cream, soup, beverage, tea, functional water, drinkables, alcohol, and vitamin complexes. In addition, the above-mentioned health functional food may additionally include food additives, and unless otherwise stipulated, suitability as a "food additive" is determined according to the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Examples of items listed in the above-mentioned "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, kohlrabi dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkali agents added to noodles, preservative preparations, and tar dye preparations. At this time, the content of the active ingredient added to the food during the manufacturing process of the health functional food may be appropriately increased or decreased as needed, and preferably, it may be added in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

[0027] In addition, the present invention provides a method for preparing a smooth-stemmed nettle extract, characterized by comprising: a first step of drying and grinding the smooth-stemmed nettle to produce it in powder form; a second step of adding water, a C1 to C4 alcohol, or an aqueous solution thereof to the powder-form smooth-stemmed nettle and performing ultrasonic extraction to produce a substrate solution; a third step of filtering the substrate solution to obtain a filtrate; and a fourth step of concentrating the filtrate under reduced pressure and drying it.

[0028] The above second step may additionally include a step of extracting by repeating it 6 to 10 times.

[0030] Hereinafter, to aid in understanding the present invention, examples and the like will be described in detail. However, the following examples and the like are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0032] <Example 1> Sample Acquisition and Extract Preparation

[0033] *Smooth-skinned nettle* was collected in Gangneung, Gangwon-do, washed with water, and frozen at -80°C for 1 day. The frozen samples were dried using a freeze-dryer for 4 days. The dried samples were ground using a grinder to produce a powder. 70% ethanol was added to the dried powder, and the sample was extracted 8 times using an ultrasonic extractor for 1 hour at room temperature. Each extraction was filtered using filter paper, and the filtered extract was concentrated using a vacuum concentrator. To ensure complete drying, the concentrated sample was frozen at -80°C and then freeze-dried for 4 to 5 days. The dried extract was converted into a powder and frozen at -80°C until use in the experiment.

[0035] <Example 2> Observation of cytotoxicity in 3T3-L1 preadipocytes

[0036] To confirm the cytotoxicity of *Solidago virosa* extract on 3T3-L1 preadipocytes, 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% bovine serum and 1% antibiotics. Subsequently, the extract was treated at different concentrations (10, 50, and 100 μg / mL) and cultured for 48 hours. After adding MTT working solution and culturing for an additional 4 hours, the MTT working solution was removed. Cells were lysed with DMSO, and cytotoxicity was confirmed by measuring absorbance at 540 nm using a microplate reader (Multiskan™ GO, Thermo Scientific™, Waltham, MA, USA).

[0037] As a result, according to Figure 1, the cytotoxicity results of the extract of *Solidago virulencephala* were shown. All extract-treated groups exhibited a cell viability of over 80% compared to the untreated group, confirming that no toxicity was observed against 3T3-L1 preadipocytes. Subsequently, efficacy analysis was conducted at concentrations of 10, 50, and 100 μg / mL.

[0039] <Example 3> Observation of the inhibitory effects on fat accumulation and triglycerides in 3T3-L1 preadipocytes

[0040] To confirm the efficacy of *Solidago virosa* extract in inhibiting differentiation (inhibition of fat accumulation) in 3T3-L1 preadipocytes, Oil Red O staining was performed. 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics. After 2 days of culture, the medium was changed to the same medium and cultured for an additional 2 days. Subsequently, the medium was changed to differentiation medium (DMEM containing 10% fetal bovine serum, 0.5 mM IBMX, 0.25 μM dexamethasone, and 10 μg / mL insulin) and the extract was applied at different concentrations (10, 50, and 100 μg / mL). After 2 days, the differentiation medium was changed to adipocyte growth medium (DMEM supplemented with 10% FBS and 5 μg / mL insulin) and changed at 2-day intervals. After adipocyte differentiation was complete, the cells were washed twice with 1X PBS (Phosphate Buffer Saline) and fixed in 10% formalin at room temperature. The fixed cells were washed with 60% isopropanol, completely dried, and then stained with Oil Red O solution for 1 hour at room temperature. After washing three times with distilled water, the stained cells were observed and photographed using an electron microscope (DMI6000, Leica, Wetzlar, Germany). Subsequently, the cells were dissolved in 100% isopropanol, and the absorbance was measured at 500 nm using a microplate reader (Multiskan™ GO, Thermo Scientific™, Waltham, MA, USA).

[0041] In addition, an ELISA kit (PicoSens™ Triglyceride Assay Kit, Biomax, Korea) was used to measure triglyceride content. After adipocyte differentiation was complete, the cells were washed twice with 1X PBS (Phosphate Buffer Saline), lysed using cell lysis buffer, and the triglyceride content in the adipocytes was measured using the method provided by the kit manufacturer.

[0042] As a result, according to Figure 2, the inhibitory effect of the extract on intracellular fat accumulation and the electron microscope image of intracellular fat can be seen, and as the content of the extract on

[0043] In addition, according to Figure 3, which shows the triglyceride content in adipocytes of the smooth-bone nettle extract, it was confirmed that as the content of the smooth-bone nettle extract increased, the triglyceride content decreased compared to the untreated group.

[0045] <Example 4> Analysis of the Efficacy of Inhibiting Adipogenesis Transcription Factor and Fatty Acid Transport Protein Expression in 3T3-L1 Preadipocytes

[0046] Western blot analysis was performed to confirm the inhibitory effect of *Solidago virosa* extract on the expression of transcription factors (PPARγ, C / EBPα, SREBP-1) and fatty acid transport protein (FABP4) associated with 3T3-L1 preadipocyte differentiation (adipogenesis). Following the completion of adipocyte differentiation as in Example 3, the cells were washed twice with 1X PBS (Phosphate Buffer Saline), lysed in cell lysis buffer (20 mM Tris, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% NP-40, 10 mg / mL aprotinin, 10 mg / mL leupeptin, and 1 mM PMSF) for 60 minutes, and then centrifuged at 12,000 rpm at 4°C for 10 minutes. Protein concentration was confirmed using a BCATM protein analysis kit. Cell lysates were electrophoresed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and proteins were transferred to a nitrocellulose membrane. Then, the membrane was blocked with 5% non-fat dry milk-TBST (25 mM Tris-HCl, 137 mM NaCl, 2.65 mM KCl, 0.05% Tween 20, pH 7.4) for 2 hours. Subsequently, the membrane was hybridized with target primary antibodies (1:1000 dilution) overnight at 4°C. The hybridized membrane was washed with TBST and then hybridized with secondary antibodies (1:3000 dilution) for 2 hours at room temperature. The membrane after the antibody reaction was completed was treated with a chemiluminescent detection reagent (ECL) to induce luminescence, and visualized using a Davinch-Chemi Imager™ (CAS400SM, Core Bio, Seoul, Korea).

[0047] As a result, according to Figure 4, the results showed that the extract of *Solidago virosa* inhibited the expression of lipogenesis transcription factors and fatty acid transport proteins. It was confirmed that the expression of lipogenesis transcription factors PPARγ, C / EBPα, SREBP-1 and fatty acid transport protein FABP4 was reduced compared to the untreated group.

[0049] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0050] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 Smooth-bone net-like ( Dictyopteris divaricata A pharmaceutical composition for the prevention or treatment of obesity comprising an aqueous ethanol extract as an active ingredient. Claim 2 delete Claim 3 A pharmaceutical composition for preventing or treating obesity according to claim 1, wherein the ethanol aqueous extract of *Solidago virosa* inhibits the accumulation of fat and triglycerides in 3T3-L1 preadipocytes. Claim 4 A pharmaceutical composition for the prevention or treatment of obesity according to claim 1, wherein the ethanol aqueous extract of *Solidago virosa* inhibits the expression of lipogenesis transcription factors and fatty acid transport proteins in 3T3-L1 preadipocytes. Claim 5 A pharmaceutical composition for preventing or treating obesity according to claim 4, wherein the lipogenesis transcription factor is one or more selected from the group consisting of PPARγ, C / EBPα, and SREBP-1, and the fatty acid transport protein is FABP4. Claim 6 A body fat reduction health functional food composition comprising an ethanol aqueous extract of *Solidago virosa* as an active ingredient. Claim 7 A method for preparing an ethanol aqueous extract of *Solidago virosa*, characterized by comprising: a first step of drying and grinding *Solidago virosa* to produce a powder form; a second step of adding an aqueous ethanol solution to the powdered *Solidago virosa* and ultrasonically extracting it to produce a substrate solution; a third step of filtering the substrate solution to obtain a filtrate; and a fourth step of concentrating the filtrate under reduced pressure and drying it. Claim 8 A method for preparing an ethanol aqueous extract of *Smooth-bone-netweed* according to claim 7, further comprising the step of repeating the second step 6 to 10 times for extraction.

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