KR20190106587A - Pharmaceutical composition comprising the extract of Phlomis umbrosa Turczaninow for preventing or treating of obesity - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating obesity, containing a Phlomis umbrosa Turczaninow extract as an active component. The Phlomis umbrosa Turczaninow extract according to the present invention is confirmed to have effects of having no or little side effects by using natural product, suppressing the increase of body weight and body fat by high fat diet, suppressing the production of fatty liver by high fat diet, reducing fasting blood sugar and total cholesterol, and also browning of adipose tissues by increasing UCP1 of subcutaneous fat tissues. Therefore, the Phlomis umbrosa Turczaninow extract is expected to be used in various fields such as medicinal, quasi-drug, functional foods and the like for preventing and treating obesity and metabolic diseases such as non-alcoholic fatty liver, type 2 diabetes and the like due to obesity.

Description

Pharmaceutical composition comprising the extract of Phlomis umbrosa Turczaninow for preventing or treating of obesity

The present invention relates to a composition for the prevention, improvement or treatment of obesity, including a fast-acting extract as an active ingredient.

Obesity refers to a phenomenon in which excess calories are accumulated in the form of fat in the body by ingesting excessive calories compared to calories consumed. According to the World Health Organization (WHO), some 400 million people over 15 years old are classified as obese and over 1.6 billion are overweight (announced in September 2006). Is not just a disease, but a serious social problem. Obesity is known to be caused by genetic effects, environmental effects of westernized diet, psychological effects of stress, etc., but the exact cause or mechanism is not clear.

There are two main goals of treatment for obesity: the first is to burn excess fat to lose weight, and the second is to improve metabolic imbalance. Abdominal obesity is closely related to symptoms such as insulin resistance, type 2 diabetes, hypertension, and lipid metabolism abnormalities and acts as a powerful risk factor for early arteriosclerosis, ischemic heart disease and cerebrovascular disease (Kopleman PG, Nature, 404, 2000; Manson et al., New England J. Med., 333, 1995; Mustet al., JAMA, 282, 1999). Therefore, the current treatment of obesity is aimed at improving metabolic abnormalities by removing factors that cause cardiovascular disease early as well as weight loss.

On the other hand, studies to suppress obesity through the regulation of dietary intake and energy consumption has been actively made. The hypothalamus, motor nervous system, autonomic nervous system and peripheral nervous system are all involved in organs that control food intake. Especially in the pathogenesis of obesity, especially the hypothalamus plays an important role. Neuropeptide Y, POMC / CART, Melano Cortin receptors, norepinephrine and serotonin are representative factors secreted by the hypothalamus. Current strategies for developing obesity treatments include reducing meals, suppressing caloric intake, promoting exothermic reactions, regulating energy metabolism, and regulating signaling through the nervous system (Park, Mi-Jung, Korean J Pediatr 48 (2), 2005).

Representative drugs for reducing weight due to obesity are sibutramine (sibutramine), which suppresses appetite, and orlistat, which suppresses the absorption of fat contained in food by inhibiting lipase in the body. However, in the case of sibutramine, side effects such as blood pressure rise, insomnia, dry mouth, and dizziness are relatively common. In addition, sibutramine has disadvantages that cannot be used in patients with cardiovascular disease or uncontrolled hypertension. In addition, in the case of olistat, side effects such as diarrhea, fatty stool, and lost money are common, and the effect of the drug is not obvious when fat intake is less than that of Westerners such as Koreans. Therefore, the necessity of steady research on safer and anti-obesity drugs has been raised.

)’라고도 불린다. 한속단은 몸에 열을 내리고 부기를 가라앉히는 효능이 있는 것으로 알려져 있으나, 한속단에 체중 및 체지방 증가 억제 효과와 혈당 증가 억제 효과가 있음에 대해서는 전혀 개시된바 없다. Meanwhile, Phlomis umbrosa Turczaninow) is a medicinal herb that dried the roots of the genus belonging to the labiatae.

Also called. Speed is known to have the effect of lowering heat and swelling in the body, but it has never been disclosed that the effect of inhibiting weight and body fat increase and blood sugar increase in the speed limit.

The technical problem to be achieved by the present invention is to provide a composition having no side effects and safe and effective in the prevention or treatment of obesity, and a pharmaceutical or functional food containing the same.

Accordingly, it is an object of the present invention to provide a composition for the prevention, treatment or improvement of obesity, and a method for producing the same, including a fast-acting extract as an active ingredient.

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

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity, including a fast-acting extract as an active ingredient.

In another aspect, the present invention provides a food composition for improving obesity, including a fast-acting extract as an active ingredient.

In one embodiment of the present invention, the composition may be to reduce the weight and fat mass.

In another embodiment of the present invention, the composition may be to reduce the dietary efficiency.

In another embodiment of the present invention, the composition may be to suppress the increase in blood sugar.

In another embodiment of the present invention, the blood sugar may be fasting blood sugar.

As another embodiment of the present invention, the fast-acting extract is water, C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and these It may be extracted with one or more solvents selected from the group consisting of a mixed solvent, preferably C ₁ to C ₄ lower alcohol, more preferably 70% alcohol may be extracted as a solvent.

In addition, the present invention comprises the steps of (a) ultrasonic extraction after adding the extraction solvent in one speed stage; And (b) concentrating the extract extracted from the above under reduced pressure and lyophilizing.

In addition, the present invention provides a method for preventing or treating obesity, comprising administering a rapid extract to a subject.

The present invention also provides the use of a sessile extract for producing a medicament for preventing or treating obesity.

Fast-acting extract according to the present invention using natural products have no or little side effects, suppress the increase in body weight and body fat by high fat diet, dietary obesity-induced mouse model, inhibit fatty liver production by high fat diet, fasting blood sugar And it was confirmed that there is an effect to reduce the plasma total cholesterol, and also the browning effect of the white adipose tissue by increasing the UCP1 of subcutaneous fat tissue. Therefore, Hansokdan extract is expected to be used in various fields such as medicinal, quasi-drug, and functional foods for the prevention and treatment of metabolic diseases such as non-alcoholic fatty liver and type 2 diabetes due to obesity and obesity.

1 is a view confirming through the ORO staining that the formation of adipocytes is inhibited according to the fast-acting extract treatment to 3T3-L1 adipocyte.
2 is a diagram confirming through RT-qPCR that the expression level of brown fat-specific markers increased in accordance with the Hansokdan extract treatment to 3T3-L1 adipocyte.
Figure 3 is a schematic diagram showing an experimental design using a dietary obesity-induced mouse model in order to evaluate the anti-obesity effect of the Han Sokdan extract.
Figure 4 is a graph showing the body weight change according to the 12-week breeding period of the normal diet group (ND), high-fat diet group (HFD), and fast-acting extract feeding group (PUE) mice.
FIG. 5 shows the body weight gain for 12 weeks in normal diet group (ND), high fat diet group (HFD), and fast-acting extract feeding group (PUE) mice, in terms of weight gain per day (g / day). The graph shown.
6 is a graph showing the average energy intake (kcal / day) after 12 weeks of dietary supplementation of the normal diet group (ND), high-fat diet group (HFD), and fast-acting extract feeding group (PUE) mice.
7 is a food efficiency ratio (FER) showing the weight gain with respect to energy intake after 12 weeks of dietary supplementation of the normal diet group (ND), high-fat diet group (HFD), and fast-acting extract feeding group (PUE) mice ) Is a graph.
FIG. 8 shows the normal diet group (ND), the high-fat diet group (HFD), and the fast-acting extract feeding group (PUE) mice after 12 weeks of dietary administration, and the liver and muscle were extracted and weighed. The graph shows the weight per 100g body weight.
9 is fed to the normal diet group (ND), high-fat diet group (HFD), and fast-acting extract feeding group (PUE) mice for 12 weeks, and then extracted white adipose tissue for each site and weighed by weight per 100g body weight It is a graph.
FIG. 10 is a graph showing the fasting blood glucose levels at 12 weeks after dietary feeding for 12 weeks of normal diet group (ND), high fat diet group (HFD), and fast-acting extract feeding group (PUE) mice.
FIG. 11 is a graph showing plasma total cholesterol levels measured at week 12 after 12 weeks of dietary feeding for normal diet group (ND), high fat diet group (HFD), and fast-acting extract feeding group (PUE) mice.
FIG. 12 is a diagram illustrating morphological changes of liver tissues at 12 weeks after diet for 12 weeks of normal diet group (ND), high fat diet group (HFD), and fast-acting extract feeding group (PUE) mice.
FIG. 13 is a diagram illustrating morphological changes of subcutaneous fat tissue at 12 weeks after diet for 12 weeks of normal diet group (ND), high fat diet group (HFD), and fast-acting extract feeding group (PUE) mice.

The present inventors confirmed that the 3D3-L1 adipocytes inhibited the formation of fat globules and increased the expression of brown fat-specific genes by treatment with the fast-acting extract, and the fast-acting extract was extracted from the mouse model induced by obesity by the high-fat diet. As a result of mixing and feeding, to confirm the excellent anti-obesity effect of the Han Sokdan extract to complete the present invention.

)’라고도 불린다. 한속단은 몸에 열을 내리고 부기를 가라앉히는 용도로 이용되어 온 약재로서 이에 항비만 효능에 대해서는 알려진 바가 없다. In the present invention the 'speed limit ( Phlomis umbrosa Turczaninow) is a medicinal herb that dried the roots of the genus belonging to the labiatae.

Also called. Hansokdan is a medicine that has been used to reduce heat and reduce swelling in the body, so there is no known anti-obesity effect.

Accordingly, the present inventors confirm the inhibitory effect and the hypoglycemic effect of increasing the body weight and body fat of the fast-acting extract, and provide a pharmaceutical composition for preventing or treating obesity, including the fast-acting extract as an active ingredient.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

In addition, the pharmaceutical composition for the prevention or treatment of obesity, including the fast-acting extract according to the present invention, external preparations and sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like according to conventional methods, respectively It may be used in the form of a solution, preferably having a formulation of a cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma. Carriers, excipients and diluents that may be included in the composition comprising the fast-acting extract include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes such as parenteral, oral, and all modes of administration can be expected, for example, oral, It may be administered by rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the oral dosage form may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 100 mg / kg may be administered once to several times a day. The dosage may also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

In addition, the present invention provides a food composition for improving obesity, including the Han Sokdan extract as an active ingredient. In addition, fast-acting extract may be added to food for the purpose of reducing body fat or improving obesity. When the fast-acting extract of the present invention is used as a food additive, the fast-acting extract can be added as it is or used together with other food or food ingredients, and can be suitably used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the fast-acting extract of the present invention in the preparation of food or beverage is added in an amount of up to 15% by weight, preferably up to 10% by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or for health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all foods in a conventional sense.

The health beverage composition according to the present invention may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in the general beverage. Natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of said natural carbohydrate is generally about 0.01-0.20g, preferably about 0.04-0.10g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually chosen in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a composition for reducing the weight or body fat, including a fast-acting extract as an active ingredient.

The fast-acting extract of the present invention can be extracted according to conventional methods known in the art for extracting extracts from natural products, that is, using conventional solvents under the conditions of conventional temperature and pressure. For example, the fast-acting extract in the present invention is selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof. Extraction can be performed using one or more solvents selected, preferably alcohols having 1 to 4 carbon atoms, more preferably 70% alcohol. In addition, the method of extracting the extract from the speed limit may be extracted through various methods such as hot water extraction, cold needle extraction, reflux extraction, and ultrasonic extraction, but is not limited thereto, and preferably, an ultrasonic extraction method may be used. .

The prepared extract can then be filtered or concentrated or dried to remove the solvent, can be carried out both filtration, concentration and drying, the filtration, concentration or drying process can be carried out several times. For example, the filtration may be performed using a filter paper or a vacuum filter, the concentration may be a vacuum concentrator, the drying may be performed by a spray drying method, a freeze drying method, but is not limited thereto.

In addition, the extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of butanol, n-hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. , But not limited to this.

In addition, the present invention comprises the steps of (a) ultrasonic extraction after adding the extraction solvent in one speed stage; And (b) concentrating the extract extracted from the above under reduced pressure and then freeze-drying.

In one embodiment of the present invention, it was confirmed that adipocyte formation of 3T3-L1 adipocyte was inhibited by the Hansokdan extract treatment (see Example 1-1), and it was confirmed that brown fat-specific gene expression was increased (execution). See Example 2-2).

In one embodiment of the present invention, in order to evaluate the anti-obesity effect of the fast-acting extract extracted by the above method, experiments were conducted using 3T3-L1 adipocyte and dietary obesity-induced mouse model. Specifically, it was classified into normal diet group, high fat diet group, and fast-acting extract supplemented group and raised for 12 weeks, and the weight change was measured. (Refer to Example 3-2). After 12 weeks of breeding, liver, muscle, and various white adipose tissues were separated from each group of mice and weighed. It was confirmed that the effect of inhibiting weight loss of muscle was excellent (see Example 3-3), and the effect of inhibiting body fat increase was also excellent, indicating that the anti-obesity effect was excellent (see Example 3-4).

In another embodiment of the present invention, as a result of comparing the fasting glucose extract supplementation on the fasting blood glucose, it was confirmed that the fasting glucose increase due to the high-fat diet is suppressed by the fasting extract supplement (see Example 4) .

In addition, in another embodiment of the present invention, as a result of comparing the effect of the fasting extract extract on the plasma total cholesterol, it was confirmed that the plasma total cholesterol increase caused by the high fat diet is suppressed by the fasting extract supplement (Example 5).

Therefore, through the results of the embodiment of the present invention, it was found that the fast extract extract has the effect of suppressing the weight and body fat mass, liver tissue weight increase due to high fat diet, and improve fasting blood glucose.

The obesity, which is a disease to be prevented or treated according to the present invention, refers to a condition in which fat cells proliferate and differentiate due to metabolic disorders and thus fat is accumulated excessively, and hypertension, diabetes mellitus, dyslipidemia, etc. Related complications, including metabolic syndrome, may be caused. When energy absorption increases relative to consumption, the mass and volume of fat cells increase, resulting in an increase in the mass of fat tissue.

On the other hand, in another embodiment of the present invention, by observing the morphological changes of liver tissue and adipose tissue by supplementation of the fast-acting extract, the increase in fat accumulation of liver tissue by high-fat diet is reduced by supplementation of the fast-acting extract , It was confirmed that the increase in the size of fat cells by the high-fat diet was restored to the normal diet group level by supplementation of the fast-acting extract (see Example 6).

Therefore, through the results of the embodiment of the present invention, it was found that the fast-acting extract has an effect of inhibiting the increase of total cholesterol in the blood and the increase of the size of fat cells due to the high-fat diet. In addition to the prevention or treatment of obesity can be used for the prevention or treatment of metabolic diseases including fatty liver, type 2 diabetes, cardiovascular atherosclerosis, hypertension, etc. The metabolic disease may be induced by obesity, The fatty liver may be non-alcoholic fatty liver, but is not limited thereto.

In particular, it was confirmed through the specific embodiment of the present invention that the accumulation of fat globules around the portal vein of the liver tissue by the high fat diet was increased, and the number and size of the fat globules accumulated in the liver tissue by supplementation of the fast-acting extract were increased. As confirmed that the reduced, the fast-acting extract of the present invention may be effective in preventing or treating fatty liver, and in the present invention, fatty liver is a cause of excessive intake of fat or increase in endogenous intrahepatic fat synthesis or decrease in its discharge. As long as the fat is excessively accumulated in the liver tissue, it is not limited, but preferably means non-alcoholic fatty liver due to excessive intake of fat.

As used herein, the term "prevention" means any action that inhibits obesity or delays the onset by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "treatment" means any action that improves or advantageously changes the condition for obesity by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “improvement” means any action that reduces the parameters associated with obesity, eg the extent of symptoms, by administration of a composition according to the invention.

The term "body fat" used in the present invention refers to adipose tissue constituting the body and is widely distributed in subcutaneous tissue, mammary gland, and kidneys, and functions as a storage fat in addition to being used for energy, and protects the intestines. Excessive accumulation of stored fat is called obesity. In obesity, the amount of body fat is more important than weight in terms of prevention of complications. Accumulation of visceral fat in the abdominal cavity is more associated with glucose, lipid metabolism, hypertension, and coronary artery disease than subcutaneous fat. In the present invention, body fat includes both subcutaneous fat and visceral fat.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1. Preparation of Hansokdan Extract

2 kg of the speed limit was added to 140 liters of 70% alcohol and heated to 40 ° C. to 60 ° C., and the active ingredient of the raw material was ultrasonically extracted at least twice for 5 hours under 25 Kw at 7 to 800 KHz. The extract obtained by the above method was filtered through a 60 mesh screen to remove the residue, and then filtered through a finer filter (CP 0.1 micron) to completely remove the residue. The residue was removed from the extract was constantly supplied to a condenser in a vacuum (vacuum degree: 760mmHg), the water vapor evaporated therein was collected in a cooling capacitor (30 ~ 48 ℃) to obtain a primary concentrate. The obtained concentrate was adjusted to 70% concentration to precipitate residues, contaminants, and abnormal substances, and then a supernatant was obtained to carry out secondary concentration. The secondary concentrate was obtained by adding 70 liters of 95% alcohol to 25 liters of the primary concentrate, allowing the mixture to stand for 12 hours after cooling and precipitation for 12 hours, and then obtaining a supernatant. Then, the fast extract obtained by freeze-drying the secondary concentrate was used in the following experiment. The freeze drying was performed under the conditions of freezing temperature -45 ℃, shelf temperature 30 ℃, vacuum degree 5mTorr, trap temperature -75 ℃.

Example  2. Speed limit  3T3-L1 by extract of adipocyte  Inhibition of Adipocyte Formation and Expression of Brown Fat Specific Gene Expression

2-1. Inhibitory Effects of 3T3-L1 Adipocytes on Adipocyte Formation

In order to examine the inhibitory effect of adipocyte formation by the fast-acting extract, the fast-acting extract was treated with MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μg / ml insulin) as a differentiation drug in 3T3-L1. After differentiation, ORO (Oil-Red-O) staining was performed after 8 days.

As a result, as shown in Figure 1, it was confirmed that the intracellular fat globule formation was inhibited compared to the control (CON) in the fast-acting extract treatment group (PUE). Through the above results, it was found that the fast-acting extract inhibits fat cell formation.

2-2. Expression of Brown Fat Specific Genes in 3T3-L1 Adipocytes

RT-qPCR confirmed the difference in the expression level of brown fat-specific genes when the Sokcho extract was added to the adipocytes. More specifically, 3T3-L1 cultured by the method of Example 2-1 to 100 μg / mL of the Han Sok-dan extract, and treated the sample and cultured for 8 days after the differentiation of the cells TRIzol (Invitrogen, Grand Island, NY) ) And RNA was extracted according to the manufacturer's protocol. RNA concentration was quantified using nonodrop (260/280 nm), and cDNA was synthesized using QuantiTect® reverse transcription kit (Qiagen, Hilden, Germany). Template cDNA was diluted in RNAse free water to use 50 ng / μL, SYBR Premix Ex Taq II (TAKARA BIO INC., Japan) was used for gene expression analysis. Primers capable of analyzing the expression of each gene were purchased from Genotech Co., Ltd. (Daejeon, Korea). The composition of the reaction solution is 10 μL of SYBR Green, 2 μL of template, and 10 μM of primer, respectively, and RNAse free water is added to a final volume of 25 μL, and then dehydrated at 94 ° C for 15 seconds in a denaturation step. 30 seconds at 55 ~ 58 ° C in the annealing step and 30 seconds at 72 ° C in the extension step, these three steps were repeated amplified 35-40 times. At this time, by analyzing the threshold cycle (Ct) appearing by monitoring the fluorescence signal of each cycle, mRNA expression between each experimental group was quantitatively analyzed by CFX96 Real time system (Bio-rad, USA). MRNA levels of all genes were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the base sequences of PCR primers for each gene are shown in Table 1 below.

Primer Forward (5'-3 ') SEQ ID NO: Reverse (5'-3 ') SEQ ID NO: Cpt1α ATC TGG ATG GCT ATG GTC AAG GTC One GTG CTG TCA TGC GTT GGA AGT C 2 Gapp AAG GTC ATC CCA GAG CTG AA 3 CTG CTT CAC CAC CTT CTT GA 4 Pgc-1α AGC CGT GAC CAC TGA CAA CGA G 5 GCT GCA TGG TTC TGA GTG CTA AG 6 Prdm16 CAG CAC GGT GAA GCC ATT C 7 GCG TGC ATC CGC TTG TG 8 Ucp1 ACT GCC ACA CCT CCA GTC ATT 9 CTT TGC CTC ACT CAG GAT TGG 10

As a result, as shown in Figure 2, PGC-1α and PRDM16 mRNA expression significantly increased according to the treatment of the fast-acting extract, UCP1 mRNA expression was also significantly increased compared to the control. In addition, the expression of CPT1a mRNA, a fat oxidation-related gene, was also significantly increased by the fast-acting extract treatment. Through the above results, it was confirmed that the brown fat-specific gene expression increase effect of the adipocytes by Hansokdan extract treatment.

Example 3. Analysis of weight change and long-term weight change by Hansokdan extract

3-1. Experimental animal models

In order to confirm the effect of the fasting extract extract on the weight gain inhibition using a dietary obesity-induced mouse model, the experiment was designed and proceed as shown in the schematic diagram shown in FIG. Specifically, four-week-old male C57BL / 6J mice (n = 30) were purchased from Jackson, USA, and adapted to breed in Lab chow for one week, normal diet group (ND, n = 10) and high fat diet group (HFD, n = 10), one-speed extracts were grouped into groups (PUE, n = 10) and bred for 12 weeks.

3-2. Weight change analysis

In order to examine the effects of the fasting extract on the weight gain in dietary obesity-induced mouse model, as shown in Example 3-1, the normal diet group (ND), high-fat diet group (HFD), and fast-acting The body weight was measured at weekly intervals for 12 weeks in the breeding group (PUE).

As a result, as shown in Figure 4, in the case of high-fat diet group (HFD) compared to the normal diet group (ND), the weight increased rapidly, whereas in the fast-acting extract pay group (PUE) salary 6 weeks from high-fat diet group A significant decrease in body weight was observed over (HFD).

In addition, as shown in FIG. 5, when the body weight gain is converted into the weight gain amount (g / day) for 12 weeks, the weight gain of the fast-acting extract supplemented group (PUE) is higher than that of the high-fat diet group (HFD). It was confirmed that was significantly suppressed.

On the other hand, as shown in Figure 6, the average energy intake (energy intake) for 12 weeks was increased in the high-fat diet group (HFD) compared to the normal diet group (ND), while the high-fat diet group (HFD) and fast-acting extract group (PUE) There was no difference in liver energy intake.

According to the above, as shown in Figure 7, the food efficiency ratio (FER) indicating the weight gain with respect to energy intake was also observed to be significantly lower in the fast-acting extract group (PUE) than the high-fat diet group (HFD) In the same energy intake environment, sokcho extract supplement can be seen to have a significant effect on reducing weight.

3-3. Long-term weight analysis

In addition to the weight change analysis of Example 3-2, to investigate the effect of the fast-acting extract intake on organ weight in a dietary obesity-induced mouse model. To this end, mice in each group bred for 12 weeks by the method of Example 3-1 were fasted for 12 hours, anesthetized through inhalation with isoflurane (Baxter, USA), sacrificed and organs were removed. Liver and muscle tissue of each mouse was rinsed several times with phosphate buffered saline (PBS), drained, weighed, and expressed as weight per 100 g of body weight.

As a result, as shown in Figure 8, the weight of the high-fat diet group (HFD) significantly increased compared to the normal diet group (ND) in the liver, while the fast-acting extract feeding group (PUE) than the high-fat diet group (HFD) Liver weight was significantly reduced. In the case of muscle, it was significantly decreased in the high-fat diet group (HFD) compared to the normal diet group (ND), and the muscle weight in the fast-acting extract fed group (PUE) significantly increased compared to the high-fat diet group (HFD). Could. Through the above results, it was confirmed that the fast-paced extract inhibits liver weight increase and also inhibits muscle weight loss.

3-4. Gravimetric Analysis of Adipose Tissue

In order to analyze the change in weight of adipose tissue by ingestion of fast-acting extract, white adipose tissue, that is, perirenal WAT and subcutaneous white fat, were obtained from each mouse in the same manner as in Example 3-3. (Subcutaneous WAT), mesenteric white fat (Mesentric WAT) and interscapular white fat (Interscapular WAT) tissue was extracted and weighed and compared to the weight per 100g body weight.

As a result, as shown in Figure 9, in the high-fat diet group (HFD), the weight of the periphery white fat, subcutaneous white fat, mesenteric white fat and scapula white fat significantly increased compared to the normal diet group (ND), while In the PUE, the peripheral WAT, the subcutaneous WAT, the mesenteric WAT and the interscapular WAT were higher than those of the HFD. It was confirmed that the weight was all significantly reduced. Through the above results, it was found that the Han Sokdan extract has a body fat reduction effect by significantly reducing the amount of fat for each site.

Example 4 Analysis of the Effect of Hansokdan Extract on Blood Glucose Change

As a result of Example 2 confirmed that the fast-acting extract intake has the effect of reducing weight, in addition to this it was to find out whether it affects the blood sugar change. To this end, after 12 weeks of breeding by the method of Example 3-1, mice were fasted for 12 hours and blood was collected from the tail to measure fasting blood glucose.

As a result, as shown in FIG. 10, fasting blood glucose levels were increased in the high-fat diet group (HFD) compared to the normal diet group (ND), while fasting in the fast-acting extract feeding group (PUE) was higher than the high-fat diet group (HFD). It was confirmed that the blood glucose level was significantly reduced. Through the above results, it was found that supplementation of Hansokdan extract has an excellent hypoglycemic effect.

Example 5 Analysis of the Effect of Hansokdan Extract on Plasma Cholesterol Change

In order to determine whether the fast-acting extract intake also affects plasma lipid levels, after 12 weeks of breeding by the method of Example 3-1, mice were fasted for 12 hours and plasma was obtained by centrifuging whole blood collected from the abdominal vein. Plasma total cholesterol was measured using a total cholesterol measurement solution (Asan Pharmaceutical kit). Plasma cholesterol is present in two forms, cholesteryl ester (CE) and free cholesterol, so that the cholesterol esterase is used to convert CE to fatty acids and free cholesterol in order to quantify all of them. Thus the glass transition cholesterol using cholesterol oxidase (cholesterol oxidase), was converted to the △ 4-cholestenon and H ₂ O _2, a peroxidase (peroxidase) to H ₂ O ₂ wherein the phenol (phenol), 4-amino- After mixing with antiptrine, the color was red, and the absorbance at 500 nm was measured and compared with the standard cholesterol solution (300 mg / dL).

As a result, as shown in Figure 11, the high-fat diet group (HFD) increased the plasma total cholesterol level compared to the normal diet group (ND), while the fast-acting extract fed group (PUE) than the high-fat diet group (HFD) It was confirmed that the plasma total cholesterol level was significantly reduced. Through the above results, it was found that the fast-acting extract has a plasma total cholesterol lowering effect.

Example 6 Analysis of the Effect of Hansokdan Extract on Morphological Changes of Liver and Adipose Tissue

To observe the morphological changes of the tissues by the fast extract, liver and adipose tissues were fixed in 10% formaldehyde solution for 24 hours, then exchanged twice with the same solution, dehydrated with double ethanol and embedded in paraffin. . Thereafter, 5 μm-thick tissue sections treated with poly-L-lysine were prepared, stained with H & E (hematoxylin-eosin) according to a known method, and observed at 200 magnification with an optical microscope. 13 is shown.

As shown in FIG. 12, as a result of observing the liver tissue, the high fat diet group (HFD) group showed the largest accumulation of lipid droplets around the portal vein of the liver tissue, The experimental group (PUE) ingesting Hansokdan extract showed a decrease in the size and number of fat globules compared to the high-fat diet group (HFD).

In addition, as shown in Figure 13, subcutaneous fat tissue (Subcutaneous WAT) as a result of observation, it was observed that the fat cell size of the high-fat diet group (HFD) compared to the normal diet group (ND), intake of the fast-acting extract The experimental group (PUE) was found to have a smaller fat cell size than the high-fat diet group (HFD). Furthermore. Fat bulge size of high fat diet group (HFD) was increased in the scapula brown adipose tissue (HFD) group than the finishing diet group (ND) group, and the fast-acting extract feeding group (PUE) was similar to the normal diet group (ND) group. Small fat globules accumulation was observed.

On the other hand, the expression of UCP1 in brown fat and subcutaneous fat of the fasting extract extract group (PUE) was observed by immunohistochemical staining. It was confirmed that the HFD than the group, and through this it was observed the browning phenomenon of subcutaneous fat by supplementation with Hansokdan extract.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Pharmaceutical composition comprising the extract of Phlomis          umbrosa Turczaninow for preventing or treating of obesity <130> MP17-301 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cpt1-alpha_Primer_F <400> 1 atctggatgg ctatggtcaa ggtc 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Cpt1-alpha_Primer_R <400> 2 gtgctgtcat gcgttggaag tc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh_Primer_F <400> 3 aaggtcatcc cagagctgaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh_Primer_R <400> 4 ctgcttcacc accttcttga 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Pgc-1alpha_Primer_F <400> 5 agccgtgacc actgacaacg ag 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Pgc-1alpha_Primer_R <400> 6 gctgcatggt tctgagtgct aag 23 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Prdm16_Primer_F <400> 7 cagcacggtg aagccattc 19 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Prdm16_Primer_R <400> 8 gcgtgcatcc gcttgtg 17 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ucp1_primer_F <400> 9 actgccacac ctccagtcat t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ucp1_primer_R <400> 10 ctttgcctca ctcaggattg g 21

Claims (17)

A pharmaceutical composition for the prevention or treatment of obesity, including a fast-acting extract as an active ingredient. The method of claim 1,
The fast-acting extract is one selected from the group consisting of water, C ₁ to C ₄ lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof A pharmaceutical composition for preventing or treating obesity, characterized in that extracted with the above solvent. The method of claim 1,
The composition is characterized in that to reduce the body weight and body fat, obesity prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is characterized in that reducing the dietary efficiency, obesity prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is characterized in that to suppress the increase in blood sugar, obesity prevention or treatment pharmaceutical composition. The method of claim 5,
The blood sugar is fasting blood sugar, characterized in that for preventing or treating obesity. The method of claim 1,
The composition is characterized in that to suppress the increase in total plasma cholesterol, obesity preventive or therapeutic pharmaceutical composition. The method of claim 1,
The composition is characterized in that to promote the browning of subcutaneous white adipose tissue, obesity prevention or treatment pharmaceutical composition. Containing fasting extract as an active ingredient, food composition for improving obesity. The method of claim 9,
The fast-acting extract is one selected from the group consisting of water, C ₁ to C ₄ lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof Food composition for improving obesity, characterized in that extracted with the above solvent. The method of claim 9,
The composition is characterized in that reducing the weight and body fat, obesity improvement food composition. The method of claim 9,
The composition is characterized in that reducing the dietary efficiency, food composition for improving obesity. The method of claim 9,
The composition is characterized in that to suppress the increase in blood sugar, food composition for improving obesity. The method of claim 13,
The blood sugar is fasting blood sugar, characterized in that for improving obesity food composition. The method of claim 9,
The composition is characterized in that to suppress the increase in total cholesterol, food composition for improving obesity. The method of claim 9,
The composition is characterized in that to promote the browning of subcutaneous white adipose tissue, food composition for improving obesity. (a) ultrasonic extraction after adding the extraction solvent at one speed stage; And
(b) a step of lyophilizing and then extracting the extract extracted from the above under reduced pressure, a method for producing a rapid extract.

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