KR102534210B1 - Pharmaceutical Composition comprising Nidus vespae extract as an active ingredient for Preventing or Treating obesity - Google Patents

Abstract

The present invention relates to a composition for the prevention, treatment, or improvement of obesity containing Nobongbang . In addition, it was confirmed that the expression of a transcription factor or adiponectin involved in fat cell formation and differentiation was suppressed, and adipogenesis was suppressed and fatty liver was significantly improved in mice on a high fat diet. Therefore, Nobongbang of the present invention can be usefully used as a pharmaceutical composition and health functional food for preventing, treating, or improving obesity.

Description

Pharmaceutical composition for preventing or treating obesity comprising Nobongbang extract as an active ingredient

The present invention relates to a composition for the prevention, improvement or treatment of obesity containing an extract of Nobongbang ( Nidus vespae ) as an active ingredient.

Obesity generally refers to a state in which adipose tissue is excessive in the body, and is a phenomenon in which energy consumed from food is not balanced with energy consumed through physical activities and the like, and surplus energy is accumulated as body fat. Obesity has a wide variety of causes, such as genetic factors, irregular eating habits, lack of exercise, environmental influences from westernized diets, depression, boredom, psychological effects caused by excessive stress, and pathological factors caused by changes in thyroid or adrenal cortical hormones. known to be caused by In recent years, there have been many changes in eating habits in accordance with economic growth and lifestyle changes, and busy modern people tend to be overweight and obese due to high-calorie diets such as fast food and low exercise.

In addition, since obesity is caused by hypertrophy of adipocytes, inhibition of adipocyte differentiation may be helpful in treating obesity. Previously, anti-obesity studies related to adipocyte differentiation and inhibition of lipid accumulation have been reported, and in this case, 3T3-L1 mouse fibroblasts are a well-characterized model for preadipocyte differentiation and are generally used for in vitro studies. .

The seriousness of obesity is recognized more because of various complications that can be caused by obesity than the risk itself. When body fat is abnormally high due to energy imbalance over a long time, diabetes, hyperlipidemia, heart disease, stroke, and arteriosclerosis , various metabolic diseases such as fatty liver and adult diseases are induced. In addition, obesity can cause not only physical diseases but also mental diseases such as social isolation or alienation, lack of confidence, and depression. is being recognized

Obesity can be treated through diet, regular exercise, lifestyle improvement such as behavioral therapy, and drugs such as appetite suppressants and fat absorption inhibitors. Since obesity is a chronic disease, long-term use is required when attempting drug treatment. Currently, products approved for long-term use for more than 3 months in Korea are sibutramine, an appetite suppressant, and orlistat, a lipolytic enzyme inhibitor. ) is there. However, since most of these obesity treatment drugs are psychoactive drugs that control appetite by acting on the central nervous system, they are accompanied by side effects such as headache and vomiting, and there are problems such as concerns over abuse. Therefore, studies are being actively conducted to develop materials having high stability and excellent anti-obesity effects that can solve the side effects of the commercially available anti-obesity drugs.

On the other hand, it is not known whether Nobongbang is effective in treating obesity.

Patent Publication No. 10-2020-0056367

Therefore, the inventors of the present invention have tried to develop a composition for preventing or treating obesity with high stability and excellent anti-obesity effect that can solve the problems of conventional anti-obesity agents, and as a result, it was confirmed that the Nobongbang extract of the present invention has an excellent anti-obesity effect. The invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity, or lipid-related metabolic diseases comprising an extract of Nobongbang ( Nidus vespae ) as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving obesity, or lipid-related metabolic diseases, comprising Nobongbang extract as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention Nobongbang ( Nidus vespae ) Provides a pharmaceutical composition for preventing or treating obesity, or lipid-related metabolic diseases comprising an extract as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving obesity, or lipid-related metabolic diseases, comprising Nobongbang extract as an active ingredient.

In one embodiment of the present invention, the food composition may be a health functional food composition, but is not limited thereto.

In another embodiment of the present invention, the extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate , methylene chloride, hexane, cyclohexane, petroleum ether, dichloromethane, subcritical fluid, and supercritical fluid by one or more solvents selected from the group consisting of It may be, but is not limited thereto.

In another embodiment of the present invention, the metabolic disease may be one or more selected from the group consisting of diabetes, hyperlipidemia, fatty liver, arteriosclerosis, hypertension, cardiovascular disease, and metabolic syndrome in which the above diseases occur simultaneously. , but is not limited thereto.

In another embodiment of the present invention, the nobongbang extract may inhibit the expression of a transcription factor or adiponectin involved in adipocyte formation and differentiation, but is not limited thereto.

In another embodiment of the present invention, the transcription factor may be peroxisome proliferator-activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C/EBPa), but is not limited thereto.

In another embodiment of the present invention, the Nobongbang extract may inhibit differentiation of adipocytes, but is not limited thereto.

In another embodiment of the present invention, the Nobongbang extract may reduce intracellular lipid accumulation, but is not limited thereto.

In addition, the present invention provides a composition for inhibiting adipocyte differentiation in vitro comprising Nobongbang extract as an active ingredient.

In addition, the present invention provides a composition for inducing differentiation into beige adipocytes or brown adipocytes in vitro, comprising Nobongbang extract as an active ingredient.

In addition, the present invention provides a method for preventing, improving and/or treating obesity or lipid-related metabolic diseases, comprising administering to a subject a composition containing Nobongbang extract as an active ingredient.

In addition, the present invention provides a use of a composition comprising Nobongbang extract as an active ingredient for preventing, improving and/or treating obesity or lipid-related metabolic diseases.

In addition, the present invention provides a use for producing a drug used for obesity or lipid-related metabolic diseases of Nobongbang extract.

The present inventors found that Nidus vespae extract effectively inhibits preadipocyte differentiation without cytotoxicity, reduces lipid accumulation in adipocytes, and inhibits the expression of transcription factors or adiponectin involved in adipocyte formation and differentiation. It was confirmed, and it was confirmed that the high-fat diet suppressed adipogenesis in mice and significantly improved fatty liver. Therefore, Nobongbang of the present invention can be usefully used as a pharmaceutical composition and health functional food for preventing, treating, or improving obesity.

Figure 1a shows the result of measuring the body weight of mice after completing the animal experiment (left), and representative images (right) of the abdominal organs of the mouse group.
Figure 1b records the feed intake every 3 days during the experiment.
Figure 1c shows adipose tissue weights of eWAT, iWAT fat (left), and representative images of eWAT and iWAT (right). **p < 0.01 and *p < 0.05.
Figure 1d shows HDL, LDL, TG and CHO contents in plasma. **p < 0.01 and *p < 0.05.
Figure 2a is an image of iWAT (top) and liver (bottom) tissues observed at 20x magnification after staining with H&E. **p < 0.01 and *p < 0.05.
Figure 2b shows the results of measuring the size of fat cells using Image J software. **p < 0.01 and *p < 0.05.
Figure 2c is the result of measuring the mRNA expression level of UCP-1 in iWAT. **p < 0.01 and *p < 0.05.
Figure 2d is the result of measuring the mRNA expression level of PGC1α in iWAT. **p < 0.01 and *p < 0.05.
Figure 2e is the result of measuring the mRNA expression level of PRDM16 in iWAT. **p < 0.01 and *p < 0.05.
3a is an image of differentiated adipocytes at 40x magnification. NC and CON were treated with the same amount of DMSO as NV.
Figure 3b shows the result of measuring the content of Oil Red O solution by 450 nm absorbance. NC and CON were treated with the same amount of DMSO as NV.
Figure 3c shows the results of measuring the intracellular triglyceride content by absorbance at 570 nm. NC and CON were treated with the same amount of DMSO as NV.
Figure 3d shows the result of measuring the viability of 3T3-L1 preadipocytes by MTT assay at 595 nm absorbance. NC and CON were treated with the same amount of DMSO as NV.
Figure 4a shows the results of measuring the protein expression levels of p-IRβ, IRβ, p-AKT, AKT, and β-actin by Western blotting.
Figure 4b shows the p-IRβ / IRβ ratio
Figure 4c shows the p-AKT/AKT ratio.
Figure 4d shows the results of measuring the mRNA expression levels of PPARγ, C/EBPa and adiponectin.
Figure 4e shows the results of measuring the protein expression levels of PARγ, C/EBPa and adiponectin.

The present invention provides a composition for preventing, improving or treating obesity or lipid-related metabolic diseases, comprising Nobongbang extract as an active ingredient.

In addition, the present invention provides a composition for inhibiting adipocyte differentiation in vitro comprising Nobongbang extract as an active ingredient.

In addition, the present invention provides a composition for inducing differentiation into beige adipocytes or brown adipocytes in vitro, comprising Nobongbang extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

Nobongbang ( Nidus vespae ) of the present invention is a beehive of the wasp family (Vespidae) and is known as a medicine with geopung poison (祛風攻毒) and insecticidal pain (殺蟲止痛), and is known to contain propolis . However, the use of Nobongbang for treating obesity has not been known. Usually disk-shaped ~ irregular and flat mass shape, but sometimes close to federal (蓮房) shape, the diameter is about 3 ~ 20 cm. The outer surface is gray to grayish brown, and hexagonal holes with a diameter of 3 to 8 mm are regularly drilled on one side, and some have black tips. The weight is light, and the quality is elastic, so it does not break even when rubbed.

The nobongbang of the present invention may be a nobongbang built by a wasp, particularly a black wasp, but is not limited thereto.

The nobongbang of the present invention may be used as it is without damaging its original form, or may be used after performing a pretreatment process in consideration of the process speed and process (manufacturing) efficiency intended by those skilled in the art, and the pretreatment process may be, for example, conventional It may go through steps such as sorting, washing with water, cutting, powdering, and drying.

The extract of Nobongbang of the present invention may be obtained from the whole or part of Nobongbang body, and the Nobongbang of the present invention may be directly collected or commercially purchased and used.

Nobongbang extract of the present invention can be extracted by a known natural product extraction method. For example, it may be extracted with one or more solvents selected from the group consisting of water, organic solvents having 1 to 6 carbon atoms, and subcritical or supercritical fluids. The organic solvent having 1 to 6 carbon atoms is alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride chloride), hexane, dichloromethane, cyclohexane, and petroleum ether, but may be one or more selected from the group consisting of, but is not limited thereto. As the extraction method, a conventional extraction method such as cold acupuncture, warm acupuncture, and heating using the solvent may be used.

The nobongbang extract of the present invention may inhibit the expression of a transcription factor or adiponectin involved in adipocyte formation and differentiation, but is not limited thereto.

The transcription factor of the present invention may be peroxisome proliferator-activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C/EBPa), but is not limited thereto.

The Nobongbang extract of the present invention may inhibit differentiation of adipocytes, but is not limited thereto.

The Nobongbang extract of the present invention may reduce intracellular lipid accumulation, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that Nobongbang extract inhibits adipogenesis in HFD-induced obese mice and reduces plasma lipid levels (see Example 1).

In another embodiment of the present invention, it was confirmed that Nobongbang extract induces brown adipogenesis and differentiation in HFD-induced obese mice and exhibits an effect of improving fatty liver (see Example 2).

In another embodiment of the present invention, it was confirmed that Nobongbang extract inhibited the differentiation of 3T3-L1 preadipocytes without cytotoxicity (see Example 3).

In another embodiment of the present invention, the expression of p-IRβ and p-AKT is suppressed in 3T3-L1 preadipocytes treated with Nobongbang extract, and the mRNA expression of PPARγ, C/EBPa, and adiponectin is also inhibited in a dose-dependent manner. It was confirmed (see Example 4).

The term "obesity" of the present invention refers to a state in which fat cells are proliferated and differentiated in the body due to metabolic disorders, resulting in excessive accumulation of fat, and metabolism accompanied by hypertension, diabetes, and dyslipidemia. It can lead to related complications including syndromes. When the amount of energy absorbed increases relative to the amount consumed, the number and volume of fat cells increase, and as a result, the mass of adipose tissue increases.

The term "metabolic disease" of the present invention may be a lipid-related metabolic disease, for example, from the group consisting of diabetes, hyperlipidemia, fatty liver, arteriosclerosis, hypertension, cardiovascular disease, and metabolic syndrome in which the above diseases occur simultaneously. It may be one or more selected, but is not limited thereto.

As the term of the present invention, "white adipocytes" are cells that have the function of accumulating a large amount of fat energy in the body as neutral fat. It can also be the cause, and generally, “adipocytes” means “white adipocytes”.

The term of the present invention, "beige adipocyte" may be derived from white adipose tissue, and is a cell having a beige color due to the presence of iron in mitochondria. Compared to white adipocytes, brown adipocytes are adipocytes that can generate heat by consuming energy. Beige adipocytes have the function of converting fat into energy and produce UCP-1 in response to cold temperatures or certain hormones. produce

The term of the present invention, "differentiation" refers to a phenomenon in which structures and functions are specialized from each other while cells divide and proliferate, that is, cells, tissues, etc. of organisms change in form or function to perform a given task. says that In general, it is a phenomenon in which a relatively simple system is separated into two or more qualitatively different subsystems. For example, in ontogeny, there is a qualitative difference between initially homogeneous parts of a living system, such as a distinction between initially homogeneous egg parts such as head or body, or a cell such as muscle cell or nerve cell. Differentiation occurs, or the state of being divided into qualitatively distinguishable subregions or subsystems as a result is called differentiation.

The composition of the present invention is characterized in that it increases the activity of brown adipocytes or increases the expression of one or more selected from the group consisting of UCP-1, PGC1α and PRDM16.

As used herein, the term "brown adipocyte" is a cell having a function of converting fat into energy, similar to beige adipocytes, and has a tan to reddish brown color when viewed with the naked eye. As the number of brown adipocytes increases, body fat decreases, and as adults age, brown adipocytes decrease. Brown adipocytes are generated from stem cells that differentiate into muscle cells, while beige adipocytes are differentiated in that they are generated within the white adipocyte layer.

As used herein, the term "differentiation inducing composition" refers to a composition capable of inducing a process in which cells at an early stage have characteristics as each tissue, and for the purpose of the present invention, white adipocytes are classified as beige adipocytes or brown adipocytes. It refers to a composition capable of inducing differentiation into adipocytes.

The content of the Nobongbang extract in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition. It may be, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods. , tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta agents, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external agents are creams, gels, patches, sprays, ointments, and warning agents. , lotion, liniment, pasta, or cataplasma may have formulations such as the like.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl excipients such as cellulose, 1928, 2208, 2906, 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, A lubricant such as sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, 1828, 2906, 2910, etc. may be used as the suspending agent according to the present invention. Surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried 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 suspending agents.

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 effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. It may be a mammal of, but is not limited thereto.

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of a desired disease, and “treatment” means that the desired disease and its resulting metabolic abnormality are improved or improved by administration of the pharmaceutical composition according to the present invention. All actions that are advantageously altered are meant, and "improvement" means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administration of the composition according to the present invention.

In addition, the present invention provides a food composition containing Nobongbang extract as an active ingredient.

In addition, Nobongbang extract may be added to food for the purpose of preventing or improving obesity or lipid-related metabolic diseases.

In the present invention, food includes functional food and health functional food.

When the Nobongbang extract of the present invention is used as a food additive, the Nobongbang extract may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the Nobongbang extract of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in a conventional sense.

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, A carbonation agent used in carbonated beverages and the like may be contained. In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Experimental Example 1. Preparation of Nobongbang extract (NV)

Nobongbang ( Nidus vespae ) was collected from a tree branch at a height of about 10 m in Daegu, Korea in September 2018. The collected bee produced by Nobongbang was identified as V. v. nigrithorax. There were about 1,200 adults in the nest, and the adults and larvae were removed from the nobongbang and the nest was stored in the shade at 25℃ for 7 days. Nobongbang was pulverized into pellets less than 1 cm in length. The pellet (836 g) was extracted with 15 L of a solvent mixture consisting of dichloromethane and MeOH (1:1, v/v) at room temperature. The extract was filtered and concentrated under reduced pressure. The resulting crude extract (10.03 g) was suspended in 1 L of MeOH and an equal volume of hexane was added. The sample was partitioned between the hexane layer (3.0 g) and the MeOH layer using a separatory funnel. The MeOH layer (6.27g) obtained by concentrating the MeOH layer under reduced pressure was dissolved in 1L of distilled water (DW). The DW suspension was extracted twice with 1 L of ethyl acetate (EA). The solvent was removed from the EA layer (1.66 g) under reduced pressure. The remaining DW suspension was extracted twice with 1 L of butanol (BuOH). After evaporating the solvent under reduced pressure, the resulting extract was freeze-dried to obtain 8.3 g of powder.

Experimental Example 2. Reagents

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and neonatal calf serum (NBCS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Insulin, Indomethacin, Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), 3-(4,5)-dimethylthiazo-2-yl-2,5-diphenyl Tetrazolium bromide (MTT) and Oil Red O solutions were purchased from Sigma-Aldrich (St Louis, MO, USA). 3T3-L1 preadipocytes were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). A high-fat diet with 60% fat (HFD, D12492) is available from Research Diet Inc. (New Brunswick, NJ). For Western blotting, the following antibodies were used: PPARγ, adiponectin (Abcam, Cambridge, MA, USA), C/EBPα, p-IRβ, IRβ, (Cell Signaling Technology, Beverly, USA), p-AKT, AKT, and β-actin (Santa Cruz Biotechnology, CA, USA).

Experimental Example 3. Administration of animals and Nobongbang extract (NV)

6-week-old male C57BL/6 mice were purchased from Hyochang Science (Daegu, Korea). Mice were housed in cages at 25-30 °C for acclimatization for 1 week in a 12-hour light/dark cycle. After 1 week of acclimatization, mice were randomly divided into 4 groups (n=7); (1) normal diet (ND); (2) HFD; (3) HFD supplemented with NV 100 mg/kg/day (NV 100) and (4) HFD supplemented with NV 200 mg/kg/day (NV 200). NV was orally administered daily in the NV treatment group, whereas mice in the control group were fed the same amount of sterile water. Food and water were provided ad libitum for 8.5 weeks. At the end of the experimental process, organs and blood were collected and used for downstream processing. Animal experiments were approved by the Animal Research Committee of Kyungpook National University, Daegu, Republic of Korea (approval number: KNU 2019-0152).

Experimental Example 4. Tissue Heatoxylin and Eosin (H&E) staining

Inguinal white adipocyte tissue and liver were embedded in paraffin, sectioned (5 μm thick), stained with H&E according to standard protocols, and examined under a microscope.

Experimental Example 5. Blood biochemical analysis

Plasma was prepared from whole blood by centrifugation. GOT (glutamic oxaloacetic transaminase), GPT (alanine aminotransferase), ALP (alkaline phosphatase), T-BIL (total bilirubin), BUN (Blood Urea Nitrogen), CREA (creatinine), HDL (high-density lipoprotein), LDL (low-density lipoprotein) in plasma Lipoprotein), TG (triglyceride) and CHO (cholesterol) levels were measured using an Olympus AU 400 (Olympus Optical, Tokyo, Japan) analyzer according to the manufacturer's procedure.

Experimental Example 6. Adipocyte Differentiation

Mouse 3T3-L1 preadipocytes were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% BCS and 1% penicillin-streptomycin in a humidified 5% CO ₂ incubator at 37 °C. Prior to experiments, 3T3-L1 preadipocytes were seeded and grown to confluence for 2 days. On day 2 after confluence, to initiate adipocyte differentiation, medium was supplemented with adipogenic cocktail (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 μM dexamethasone (DEX), 167 nM insulin, 100 DMEM containing μM indomethacin and 10% FBS). The stimulated cells were then maintained in medium supplemented with 1.72 nM insulin and 10% FBS for an additional 6 days by replacing half of the medium every 2 days. To investigate the effect of NV, 3T3-L1 cells were treated with NV at concentrations of 100 or 200 μg/ml every 2 days during the course of differentiation (8 days).

Experimental Example 7. Oil Red O staining and triglyceride (TG) analysis

3T3-L1 preadipocytes were induced to differentiate in the presence or absence of NV (100 or 200 μg/mL). 3T3-L1 preadipocytes and differentiated adipocytes were washed with phosphate buffered saline (PBS), fixed with 4% formaldehyde for 1 hour, and then placed in a filtered 0.3% Oil Red O solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes at room temperature. Subsequently, the stained cells were washed three times with distilled water and photographed using a microscope at 400X magnification. Cells were stained with Oil Red O dissolved in isopropyl alcohol, and lipid accumulation at 450 nm was quantified.

Triglyceride content was measured with a colorimetric/fluorescent assay kit (Biovision, Milpitas, CA, USA). 3T3-L1 preadipocytes were seeded in 6-well plates with differentiation medium and NV (100 and 200 μg/mL) for up to 8 days. After differentiation, cells were lysed in 5% NP-40 lysis buffer and triglycerides were converted to fatty acids and glycerol using lipase enzyme. The absorbance of the released glycerol was measured at 570 nm.

Experimental Example 8. Cell viability analysis

3T3-L1 preadipocytes were seeded in 96-well plates at a density of 5×10 ⁴ cells per well. After culturing for 24 hours, cells were treated with NV at concentrations of 100 or 200 μg/mL for 48 hours. MTT (3-(4,5)-dimethylthiazo-2-yl-2,5-diphenyltetrazolium bromide) solution was added to each well and incubated for 3 hours. The reaction product was dissolved in isopropyl alcohol (Duksan Pure Chemicals, Korea) for 1 hour. The absorbance of the reaction was measured at 595 nm.

Experimental Example 9. Western blot analysis

After differentiation of 3T3-L1 preadipocytes in the absence or presence of NV (100 and 200 μg/mL), tissues or cells were lysed with a radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase inhibitors and protease inhibitors. Total protein content (30 μg) was separated on a 10% SDS-polyacrylamide gel by electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked with 5% skim milk in PBS for 1 hour, incubated overnight at 4° C. with relevant primary antibodies, and then washed 5 times with TBS-T buffer. The membrane was then incubated with HRP-conjugated secondary antibody for 1 hour and again washed 5 times with TBS-T buffer (10 mM tris, 150 mM NaCl and 0.05% Tween 20). Protein bands were visualized using ECL and detected on a Fusion Solo detector (Vilber Lourmat, Marne La Vallee, France). The total amount of protein was assessed by evaluating protein bands using Image-J software and normalizing to β-actin.

Experimental Example 10. Real-time reverse transcription polymerase chain reaction (RT-PCR)

Total RNA from adipose tissue and 3T3-L1 adipocytes were isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) and RNAiso Plus reagent (TaKaRa Bio, Japan) according to the manufacturer's instructions, respectively. 250 ng of total RNA was used for complementary DNA (cDNA) synthesis using the Prime-Script RT reagent kit (TaKaRa Bio). RT-PCR was performed with the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using SYBR Green (TOYOBO, Japan).

After differentiating 3T3-L1 cells with SME (100 or 200 μg/mL), total RNA was extracted from adipocytes using Trizol reagent. The cDNA library was synthesized with the PrimeScript ^TM RT Reagent Kit (TaKaRa Bio). The mRNA expression level was quantified by analysis of cDNA implemented by the iCycler iQTM Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using SYBR Green (TOYOBO, Japan). The sequences of the PCR primers used in the experiment are as shown in Table 1.

Experimental Example 11. Statistical Analysis

Statistical data analysis was performed using SPSS 23 software (SPSS, Chicago, IL, USA). Statistical Analysis Data are expressed as mean±SEM and SD and analyzed using one-way ANOVA. A p-value of less than 0.05 was considered statistically significant.

Example 1. Confirmation of fat expansion inhibition and plasma lipid level reduction effect of Nobongbang extract (NV) in HFD-induced obese mice

As shown in Figure 1a, the anti-obesity effect of NV was investigated in obese mice induced by HFD. There was no significant difference in total body weight between the NV 100 or 200 group and the HFD group despite a slight decrease in body weight by NV administration. Nevertheless, it was confirmed that the NV 200 group showed reduced visceral and subcutaneous adipose tissue compared to the HFD group on the photograph.

Also, as shown in Fig. 1b, the NV 200 group showed a higher feed intake than the HFD group. In other words, despite the higher feed intake, the weight gain of the NV 200 group was similar to that of the normal diet group, and visceral and subcutaneous fat were significantly reduced, and Nobongbang extract had a significant effect on obesity, especially in suppressing lipogenesis. indicates

In addition, based on the above results, it was hypothesized that NV administration could affect HFD-induced metabolic abnormalities such as fat accumulation and blood lipid levels.

As shown in Fig. 1c, the NV 200 group showed significantly reduced weight in inguinal (iWAT), epididymal (eWAT) and retroperitoneal (rWAT) white adipose tissue. In particular, NV administration significantly reduced the weight of iWAT compared to HFD at all doses ranging from 100 to 200 (mg/kg/day).

In addition, the effect of NV on plasma lipid profiling was analyzed.

As shown in Figure 1d, the LDL, TG, and CHO contents of the NV group were significantly lower, and the HDL contents increased in a dose-dependent manner compared to the HFD group.

In addition, as shown in Table 2, GOT and GPT are biochemical markers of liver dysfunction, and the levels of these markers were measured at lower levels in a dose-dependent manner in the NV group than in the HFD group. Meanwhile, nephrotoxicity markers such as ALP, T-BIL, BUN, and CREA were not significantly different between NV 100 or NV 200 and the HFD group.

Taken together, these results suggest that NV exhibits anti-obesity effects and helps control abnormal fat gain and lipid content, especially in HFD-induced obese mice.

Example 2. Confirmation of brown fat induction and fatty liver improvement effects of Nobongbang extract (NV) in HFD-induced obese mice

Next, we investigated whether NV administration affects fatty liver, a common metabolic complication associated with adipocyte hypertrophy and obesity.

As shown in Fig. 2a, both adipocyte size and hepatic lipid deposition in adipose tissue decreased in the NV 100 and 200 groups compared to the HFD group.

In addition, as shown in Fig. 2b, the NV group had a similar pattern in adipocyte size distribution to that of the ND group in a dose-dependent manner.

On the other hand, the size of adipocytes reflects the type of adipocytes and a decrease in size is associated with browning of adipocytes. Beige adipocytes of white adipose tissue are characterized by the presence of multilayered lipid droplets similar to brown adipocytes. Given the histological characteristics of the NV group, we hypothesized that NV could induce fat browning by overexpression of a thermogenic gene.

As shown in Figures 2c to 2e, it was confirmed that the NV group showed a dose-dependent increase in the expression of UCP-1, PGC1α and PRDM16. These results imply that administration of NV affects lipid accumulation in metabolic organs and fat browning in white adipose tissue.

Example 3. Confirmation of the effect of Nobongbang extract on the survival rate of 3T3-L1 preadipocytes

Considering the results showing the anti-obesity effect of NV in the adipose tissue of obese mice induced by HFD, it was confirmed whether NV treatment could regulate adipocyte differentiation in vitro. 3T3-L1 preadipocytes were treated in differentiation medium with or without NV at 100 or 200 μg/mL for 8 days.

As shown in Figures 3a to 3c, NV significantly inhibited adipocyte differentiation and intracellular triglyceride accumulation in a dose-dependent manner.

In order to confirm whether the anti-adipocyte differentiation effect of NV is due to apoptosis or proliferation inhibition, the cytotoxicity of NV to preadipocytes was confirmed.

As shown in Figure 3d, NV treatment at concentrations of 100 and 200 μg/ml for 48 hours did not affect the viability of preadipocytes.

The above results indicate that NV can exert a strong anti-differentiation effect on 3T3-L1 adipocytes without cytotoxicity on preadipocytes.

Example 4. Confirmation of adipogenesis-related gene expression during 3T3-L1 cell differentiation

Finally, the mechanism of the anti-adipocyte differentiation effect of NV was investigated. Insulin is important in mediating adipogenesis by triggering the activation of the insulin receptor (IR) and AKT. As a result, AKT is upstream of key adipogenesis-regulating genes such as PPARγ, C/EBPα, and adiponectin.

As shown in Figures 4a to 4c, NV treatment significantly inhibited the expression of p-IRβ and p-AKT compared to the control group in a dose-dependent manner.

In addition, as shown in Figure 4d, NV decreased the mRNA expression of PPARγ, C/EBPa and adiponection. Protein expression of these proteins was reduced in NV-treated adipocytes, consistent with mRNA expression.

These results indicate that NV inhibits adipogenesis in 3T3-L1 cells through downregulation of the insulin signaling pathway.

In summary of the above examples, the present inventors found that Nidus vespae extract effectively inhibits differentiation of preadipocytes without cytotoxicity and reduces lipid accumulation in adipocytes, as well as transcription factors involved in formation and differentiation of adipocytes. Or it was confirmed that the expression of adiponectin was inhibited, and it was confirmed that adipogenesis was inhibited and fatty liver was significantly improved in high-fat diet mice. and health functional foods and materials for their development.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

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Claims (13)

Nobongbang ( Nidus vespae ) A pharmaceutical composition for preventing or treating obesity comprising an extract as an active ingredient.
According to claim 1,
The extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane (hexane), cyclohexane (cyclohexane), petroleum ether (petroleum ether), dichloromethane (dichloromethane), subcritical fluid, characterized in that the extract by one or more solvents selected from the group consisting of, and supercritical fluid, pharmaceutical composition.
delete According to claim 1,
The pharmaceutical composition, characterized in that the nobongbang extract inhibits the expression of a transcription factor or adiponectin involved in the formation and differentiation of adipocytes.
According to claim 4,
Characterized in that the transcription factor is peroxisome proliferator-activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C/EBPa), the pharmaceutical composition.
According to claim 1,
The pharmaceutical composition, characterized in that the nobongbang extract inhibits the differentiation of adipocytes.
According to claim 1,
The nobongbang extract is characterized in that to reduce intracellular lipid accumulation, a pharmaceutical composition.
A food composition for preventing or improving obesity comprising Nobongbang extract as an active ingredient.
According to claim 8,
The composition is a food composition, characterized in that the health functional food composition.
According to claim 8,
The extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane (Hexane), cyclohexane, petroleum ether (petroleum ether), dichloromethane (dichloromethane), subcritical fluid, characterized in that the extract by one or more solvents selected from the group consisting of, and supercritical fluid, food composition .
delete delete delete

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