KR102696677B1 - A composition for improving, preventing and treating of obesity metabolic disease comprising Rosa multiflora root extract - Google Patents

Abstract

The present invention relates to a composition for improving, preventing or treating obesity and metabolic diseases, and more specifically, by including an extract of the root of the thorn tree as an effective ingredient, the composition can improve, prevent or treat diseases that may be caused by obesity and metabolic diseases, and thus can be utilized as a food composition, further a health functional food or a pharmaceutical composition.

Description

{A composition for improving, preventing and treating of obesity metabolic disease comprising Rosa multiflora root extract}

The present invention relates to a composition for improving, preventing or treating metabolic diseases, comprising an extract of the root of the thistle tree as an effective ingredient.

In Korea and around the world, the average life expectancy of humans is increasing and the quality of life is improving rapidly due to economic development and medical technology development, so an active response strategy for the era of well-being that matches this is urgently needed. In particular, in terms of protecting national health and reducing government and individual medical expenses due to the accelerated aging and occurrence of adult diseases, the development of effective and safe medicines and health functional foods is necessary.

Cardiovascular diseases such as heart attack, hypertension, arteriosclerosis, and stroke account for 30% of all mortality worldwide. In particular, obesity, diabetes, hypertension, arteriosclerosis, heart disease, and stroke appear to be different diseases, but they are understood as a syndrome with a common cause: insulin resistance due to increased body fat.

Obesity is one of the most common nutritional disorders worldwide. According to WHO statistics, approximately 250 million people are currently classified as obese, and it is predicted that approximately 300 million people will suffer from obesity in 20 years.

The above obesity refers to a state in which body fat is excessively accumulated due to an imbalance in calorie metabolism caused by excessive energy intake or decreased energy consumption, and recently, the incidence of obesity has been rapidly increasing due to modern people's Western eating patterns and lack of exercise. In addition, in terms of numerical concepts, obesity is defined as a BMI (body mass index) of 25 or higher, and the BMI measurement method is an obesity measurement method that estimates the amount of body fat by dividing the weight (kg) by the square of the height (㎡).

Furthermore, obesity is known to occur when normal adipocytes undergo abnormal development in which preadipocytes differentiate into adipocytes. Excessive differentiation into adipocytes and accumulation of fat within adipocytes increase the number and size of adipocytes, which in turn worsens obesity symptoms. When preadipocytes reach confluence, the cell cycle is arrested, cell proliferation ceases, and differentiation into adipocytes (adipogenisis) is induced by hormones such as 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone (DEX), and insulin, and obesity is caused by hypertrophy and hyperplasia of adipocytes within the tissue. The initial differentiation process of preadipocytes begins with the induction of sterol regulatory element binding proteins-1c (SREBP-1c) expression by increasing the concentration of cAMP by DEX, followed by the mutual expression of peroxisome proliferator activated receptor-γ (PPAR-γ) and CCAAT enhancer binding protein-α (CEBP-α) by IBMX to promote differentiation. Their expression induces differentiation into adipocytes together with the expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1), which convert saturated fatty acids into monounsaturated fatty acids when unsaturated fatty acids are synthesized into saturated fatty acids.

Among the obesity treatment drugs, appetite suppressants include Hutermin, Phentermine, and Lorcaserin, and their side effects include anxiety, dizziness, and insomnia, and lipolytic enzyme inhibitors include Orlistat, Lorcaserin, and Mazindol, and their side effects include oily stool, abdominal pain, and fecal incontinence. As the side effects of the above commercialized drugs are being reported, there is a demand for natural obesity treatment drugs that are more effective and have fewer side effects.

Republic of Korea Publication Patent No. 10-2021-0066370 Republic of Korea Patent No. 2172316

The purpose of the present invention is to provide a food composition for improving or preventing metabolic diseases, which contains an extract of the root of the thistle tree as an effective ingredient.

In addition, another object of the present invention is to provide a food composition for weight loss containing a thorn tree root extract as an effective ingredient.

In addition, another object of the present invention is to provide a food composition for improving liver damage containing a thorn tree root extract as an effective ingredient.

In addition, another object of the present invention is to provide an appetite suppressing food composition containing a thistle root extract as an effective ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases, which contains an extract of the root of the thistle tree as an effective ingredient.

The food composition of the present invention, which can improve or prevent metabolic diseases to achieve the above-mentioned purpose, may contain an extract of the root of the thistle tree as an effective ingredient.

The above metabolic disease may be any one selected from the group consisting of obesity, metabolic syndrome, insulin deficiency, insulin-resistance related disorders, diabetes including type 2 diabetes, glucose intolerance, dyslipidemia, atherosclerosis, non-alcoholic fatty liver disease, hyperglycemia, fatty liver, dyslipidemia, immune system dysfunction associated with overweight and obesity, high cholesterol, increased triglycerides, inflammatory immune diseases, and atherogenic dyslipidemia.

The above-mentioned extract of the root of the thistle may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The lower alcohol having 1 to 4 carbon atoms may be methanol, ethanol, butanol or propanol.

The above-mentioned extract of the root of the thistle may be extracted with an ethanol aqueous solution of 20 to 80%.

The extraction temperature of the above-mentioned thistle root extract may be 40 to 80°C, and the extraction time may be 3 to 10 hours.

In addition, the weight loss food composition of the present invention for achieving the other purposes mentioned above may contain a thorn tree root extract as an effective ingredient.

In addition, the food composition for improving liver damage of the present invention to achieve another purpose mentioned above may contain an extract of the root of the thistle tree as an effective ingredient.

The above liver damage may be any one selected from the group consisting of acute alcoholic liver damage, chronic alcoholic liver damage, non-alcoholic lipid accumulation, non-alcoholic inflammation, and non-alcoholic liver damage.

In addition, the appetite suppressing food composition of the present invention to achieve the above-mentioned another purpose may contain a thorn tree root extract as an effective ingredient.

In addition, the pharmaceutical composition of the present invention for preventing or treating metabolic diseases to achieve another purpose described above may contain an extract of the root of the thistle tree as an active ingredient.

In addition, the method for treating a metabolic disease of the present invention to achieve another purpose mentioned above can administer a pharmaceutical composition containing a thistle root extract as an active ingredient to a patient with a metabolic disease.

The composition for improving, preventing or treating metabolic diseases, which comprises the extract of the root of the present invention as an effective ingredient, inhibits lipid accumulation, reduces triglyceride content, increases adrenergic receptor beta-2 content and increases cAMP content, and thus can improve, prevent or treat diseases that may be caused by obesity, and can also be utilized as a food composition, a health functional food or a pharmaceutical composition.

In addition, the extract of the root of the thistle of the present invention can be used as an effective ingredient in a food composition for weight loss, a food composition for improving or preventing dyslipidemia, a food composition for improving or preventing hypercholesterolemia, a food composition for improving liver damage, a food composition for improving or preventing diabetes, a food composition for suppressing appetite, or a pharmaceutical composition for preventing or treating metabolic diseases.

Figure 1 is a graph measuring intracellular lipid accumulation when treated with extracts prepared according to Examples 1 and 2 of the present invention.
Figure 2a is a graph measuring the intracellular triglyceride content when treated with extracts prepared according to Example 1, Example 2, and Comparative Examples 1 to 4 of the present invention.
Figure 2b is a graph measuring the intracellular triglyceride content when treated with extracts prepared according to Examples 1 to 6 of the present invention.
Figure 3 is a graph showing the energy consumption pathway of the ADRB2-cAMP pathway.
FIG. 4 is a graph measuring the intracellular adrenergic receptor beta-2 content when treated with extracts prepared according to Examples 1 and 2 of the present invention.
Figure 5 is a graph measuring the intracellular cAMP content when treated with extracts prepared according to Examples 1 and 2 of the present invention.
FIG. 6a is a Western blot showing the expression of PGC-1α protein within cells when treated with extracts prepared according to Examples 1 and 2 of the present invention, and FIG. 6b is a graph quantitatively expressing the PGC-1α protein expressed in FIG. 6a.
FIG. 7a is a Western blot showing the expression of CPT-1 protein within cells when treated with extracts prepared according to Examples 1 and 2 of the present invention, and FIG. 7b is a graph quantitatively expressing the CPT-1 protein expressed in FIG. 7a.
FIG. 8a is a Western blot showing the expression of UCP1 protein within cells when treated with extracts prepared according to Examples 1 and 2 of the present invention, and FIG. 8b is a graph quantitatively expressing the UCP1 protein expressed in FIG. 8a.
Figure 9 is a graph measuring intracellular lipid accumulation when treated with an extract prepared according to Example 2 of the present invention.
Figure 10 is a graph measuring the intracellular PGC 1α content when treated with an extract prepared according to Example 2 of the present invention.
Figure 11 is a graph measuring the intracellular AMPK content when treated with an extract prepared according to Example 2 of the present invention.
Figure 12 is a graph measuring the intracellular PPARα content when treated with an extract prepared according to Example 2 of the present invention.
Figure 13 is a graph measuring the intracellular COL1A1 content when treated with an extract prepared according to Example 2 of the present invention.
Figure 14 is a graph measuring the intracellular ACTA2 content when treated with an extract prepared according to Example 2 of the present invention.
Figure 15a is a graph showing the body weight gain per feed intake of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; Figure 15b is a graph showing the body weight over time of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; Figure 15c is a graph showing the body weight gain of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group.
FIG. 16a is a graph showing neutral lipid (TG) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; FIG. 16b is a graph showing total cholesterol (t-cholesterol) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; FIG. 16c is a graph showing low-density lipoprotein cholesterol (LDL-cholesterol) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; and FIG. 16d is a graph showing high-density lipoprotein cholesterol (HDL-cholesterol) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group.
Figure 17 is a graph showing (a) AST, (b) ALT, (c) GTT, (d) ALP, and (e) LDH in the blood of normal group 1, control group 1, positive control group 1, example 1, and example 2.
Figure 18 is a graph showing (a) glucose, (b) insulin, (c) leptin, and (d) adiponectin in the blood of normal group 1, control group 1, positive control group 1, example 1, and example 2.
Figure 19 is a graph showing the body weight over time of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200) group, and example 2 (150) group.
Figure 20a is a photograph taken using dual energy X-ray absorptiometry (DEXA) of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200) group, and example 2 (150) group; Figure 20b is a graph showing the body fat mass of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200) group, and example 2 (150) group.
Figure 21a is a photograph showing the peritesticular adipose tissue (eWAT) of normal group 2, control group 2, positive control group 2, positive control group 3, Example 2 (200), and Example 2 (150); Figure 21b is a graph showing the measurement of fat cell size of normal group 2, control group 2, positive control group 2, positive control group 3, Example 2 (200), and Example 2 (150).
Figure 22a is a photograph showing staining of accumulated fat in liver tissues of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200), and example 2 (150); Figure 22b is a graph showing measurements of the area of accumulated fat in liver tissues of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200), and example 2 (150).

The present invention relates to a composition for improving, preventing or treating metabolic diseases, comprising an extract of the root of the thistle tree as an effective ingredient.

The above metabolic disease may be any one selected from the group consisting of obesity, metabolic syndrome, insulin deficiency, insulin-resistance related disorders, diabetes including type 2 diabetes, glucose intolerance, dyslipidemia, atherosclerosis, non-alcoholic fatty liver disease, hyperglycemia, fatty liver, dyslipidemia, immune system dysfunction associated with overweight and obesity, high cholesterol, increased triglycerides, inflammatory immune diseases, and atherogenic dyslipidemia.

In addition, the present invention contains the extract of the root of the thorn tree as an effective ingredient, and thus can be used as a composition for weight loss since it suppresses an increase in body weight; Since it reduces the contents of neutral lipid (TG), total cholesterol (t-cholesterol) and low-density lipoprotein cholesterol (LDL-cholesterol) in the blood and increases the content of high-density lipoprotein cholesterol (HDL-cholesterol), it can be used as a composition for improving blood flow, a composition for preventing, treating or improving dyslipidemia, or a composition for preventing, treating or improving hypercholesterolemia; Since it has excellent AST, ALT, GTT, ALP and LDH activities in the blood, it can be used as a composition for improving any one of liver damage selected from acute alcoholic liver damage, chronic alcoholic liver damage, non-alcoholic lipid accumulation, non-alcoholic inflammation and non-alcoholic liver damage; Since it reduces blood blood sugar and increases insulin, it can be used as a composition for preventing, treating or improving diabetes; Since it reduces leptin, it can also be used as a food composition for appetite suppression.

The above composition for prevention, treatment or improvement may be a pharmaceutical composition or a food composition.

Hereinafter, the present invention will be described in detail.

The composition of the present invention contains a thorn bush root extract as an active ingredient.

The root of the Rosa multiflora tree above is good for treating postpartum pain, edema, blood stasis, and arthritis, and the Rosa multiflora mushroom that grows on the root is not only the best medicine for treating convulsions and epilepsy in children, but also has an excellent effect in suppressing the occurrence of various types of cancer.

In the present invention, the root of the thistle tree is used. If other parts of the thistle tree, such as flowers, stems, or fruits, are used, the anti-obesity effect may be reduced.

The above-mentioned thorny root is mixed with an extraction solvent in a weight ratio of 1:5 to 25, preferably 1:8 to 15, and extracted at 40 to 80°C, preferably 55 to 65°C, for 3 to 10 hours, preferably 5 to 7 hours, and then concentrated under reduced pressure to prepare an extract. When the weight ratio of the above-mentioned thorny root and the extraction solvent is out of the above range, the effective ingredient of the thorny root may be extracted in a small amount in the extract.

If the extraction temperature and extraction time are below the lower limit, the effective ingredient of the root of the thistle may be extracted in small amounts, and the effect of improving, preventing, or treating obesity may be reduced.

The extraction solvent for extracting each of the above extracts is water, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The lower alcohol may include 20 to 80% methanol, ethanol, butanol, or propanol.

Although the above extraction solvent is not particularly limited, an extract extracted with a 20 to 80% ethanol aqueous solution is preferably effective in improving, preventing, or treating obesity.

The term ‘extract’ used in the present invention includes extracts obtained by extracting components contained in the root of the thistle using the solvent, fractions separated therefrom, concentrates obtained by further concentrating these extracts or fractions, and purified products obtained by purifying or separating them, as well as dried products obtained by drying the extracts, fractions, concentrates or purified products, or powders obtained by pulverizing them.

In order to produce the above purified product, various purification methods may be additionally implemented, such as passing the product through an ultrafiltration membrane having a molecular weight cut-off value, or separation by various chromatographies (designed for separation by size, charge, hydrophobicity or affinity).

The present invention relates to a food composition for preventing or improving obesity, comprising an extract of the root of the thistle tree as an effective ingredient.

The above ‘food composition’ contains, in addition to the extract of the root of the thistle tree as an effective ingredient, food raw materials that can be used as food as described in the standards and specifications for food commonly used in food manufacturing (‘Food Code’) and food additives described in the Food Additive Code.

Although not particularly limited, examples thereof include proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. The carbohydrates may include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, lactose, etc.; oligosaccharides or polysaccharides such as dextrin, corn syrup, cyclodextrin, etc.; sugar alcohols such as xylitol, sorbitol, erythritol, etc. The flavoring agent may include natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.).

When manufacturing a food composition using the above-mentioned thistle root extract as an effective ingredient, the thistle root extract does not need to be particularly limited as long as it has an amount that can prevent or treat obesity, but may be included in amounts of, for example, 0.1 to 99 wt%, 0.5 to 95 wt%, 1 to 90 wt%, 2 to 80 wt%, 3 to 70 wt%, 4 to 60 wt%, and 5 to 50 wt%.

In the above food composition, the active ingredient, the extract of the root of the thorn tree, may vary depending on the condition, weight, presence or absence of disease, degree, and duration of the ingestion, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, still more preferably 20 to 800 mg, and most preferably 50 to 500 mg. The number of administrations need not be particularly limited, but may be adjusted by a person skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range.

The above food composition need not be particularly limited, but may be, for example, in the form of powder, granules, tablets, capsules, pills, extracts, jelly formulations, tea bags, or beverage formulations.

In addition, the extract of the root of the thistle may be added to general foods to provide functionality for preventing or improving obesity, and the foods to which it can be added are not particularly limited, but for example, it may be added to confectionery, bread or rice cakes, processed cocoa products or chocolates, processed meat or eggs, processed fish products, tofu or jelly products, noodles, tea, coffee, beverages, special-purpose foods, soy sauce, seasoned foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, dried foods, and other foods listed in the standards and ingredient specifications for livestock products (‘Livestock Products Codex’) pursuant to Article 7 of the Food Sanitation Act. In addition, it may be added to processed milk products, processed meat products, packaged meat products, and processed egg products listed in the processing standards and ingredient specifications for livestock products pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, a food composition containing the extract of the root of the thistle tree as an active ingredient can be used alone as a “health functional food for preventing or improving obesity.”

The above ‘health functional food’ refers to a food (Article 3, Paragraph 1 of the Health Functional Food Act) manufactured (including processed) in accordance with legal standards using raw materials or ingredients that have functionality useful to the human body. The terminology and scope of the above ‘health functional food’ may differ depending on the country, but it can correspond to ‘dietary supplement’ in the United States, ‘food supplement’ in Europe, ‘health functional food’ or ‘food for special health use (FoSHU)’ in Japan, and ‘health food’ in China.

The above food composition or health functional food may additionally contain food additives, and suitability as a food additive shall be determined in accordance with the general provisions and general test methods of the ‘Food Additives Code’ for the relevant item, unless otherwise provided for.

In addition, in the above health functional food, the raw materials notified as ‘functional raw materials’ used for “improving blood neutral fat” or individually recognized raw materials, such as Univex bamboo leaf extract, mackerel refined oil, DHA concentrated oil, indigestible maltodextrin, vegetable oil diglyceride, refined squid oil, and globin hydrolysate, are used in combination; or the raw materials notified as ‘functional raw materials’ used for “improving blood cholesterol” or individually recognized raw materials, such as aloe extract, aloe complex extract, bamboo leaf extract, spirunina, sugar cane wax alcohol, linseed, pu-erh tea extract, red yeast rice, barley beta-glucan extract, Changnyeong onion extract, green tea extract, and red grape fermented concentrate are used in combination; Raw materials notified as ‘functional raw materials’ used for ‘body fat reduction’ or individually recognized raw materials, including Lactobacillus gasseri BNR17, L-carnitine tartrate, Garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green mate extract, green coffee bean extract, perilla leaf extract, green tea extract, soybean germ extract, etc. complex, Gol-oe leaf alcohol extract powder, lactoferrin (milk refined protein), lemon balm extract mixed powder, mate hot water extract, seaweed, etc. complex extract (xanthigen), fermented vinegar pomegranate complex, pu-erh tea extract, seomoktae (mouse-eye bean) peptide complex, vegetable oil diglyceride, wild mango seed extract, oil containing medium chain fatty acid (MCFA), Health functional food ingredients related to body fat reduction, such as coleus forskohlii extract, chitosan, chitooligosaccharides, green apple extract polyphenol, fingerroot extract powder, and hibiscus complex extract, may be used in combination; or raw materials notified as “functional raw materials” used for “improving liver health or alcoholic liver damage” or individually recognized raw materials, such as broccoli sprite powder, shiitake mushroom mycelia extract, milk thistle extract, bokbunja extract powder, fermented turmeric, platycodon root extract, lactic acid bacteria-fermented garlic extract, walnut fruit extract, and lactic acid bacteria-fermented kelp extract, may be used in combination with health functional food ingredients related to liver health or alcoholic liver damage improvement.

The present invention also provides a method for treating obesity by administering the composition to a human or an animal other than a human.

The present invention also provides a novel use of a thorny root extract for the manufacture of a medicament for the prevention or treatment of obesity, or a medicament for animals.

The above ‘pharmaceutical composition’ or ‘medicine’ may further contain, in addition to the extract of the root of the thistle tree as an active ingredient, an appropriate carrier, excipient and diluent commonly used in the manufacture of pharmaceutical compositions, etc.

The above ‘carrier’ is a compound that facilitates the addition of a compound into a cell or tissue. The above ‘diluent’ is a compound that not only stabilizes the biologically active form of the target compound, but is also diluted in water to dissolve the compound.

The carrier, excipient and diluent need not be specifically limited, but examples thereof include lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

The dosage of the above pharmaceutical composition, medicament, veterinary pharmaceutical composition or veterinary medicament may vary depending on the age, sex and weight of the patient or animal to be treated, and will depend on, among other things, the condition of the subject to be treated, the specific category or type of the disease to be treated, the route of administration and the properties of the therapeutic agent used.

The above pharmaceutical composition, medicament, veterinary pharmaceutical composition or veterinary medicament is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age and weight, sex and condition of the patient or animal to be treated, the severity of the disease to be treated, etc., but is generally preferably administered at 0.1 to 1,000 mg/kg per day, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg. The unit dosage form preparation formulated in this manner can be administered several times at regular intervals as necessary.

The above pharmaceutical composition, medicament, veterinary pharmaceutical composition or veterinary medicament may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The above pharmaceutical composition, medicine, veterinary pharmaceutical composition or veterinary medicine can be formulated and used as oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups and aerosols, respectively, according to conventional methods. When formulating, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents and surfactants can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations can be prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be included.

The above method for treating obesity is to administer the composition to a human or an animal other than a human, particularly a mammal, for example, to administer the composition to an obese subject to be treated.

The dosage, administration method, and number of administrations for the above treatment may refer to the dosage, administration method, and number of administrations of the pharmaceutical composition, medicament, veterinary pharmaceutical composition, or veterinary medicament.

Hereinafter, preferred examples are presented to help understand the present invention, but the following examples are only illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and technical idea of the present invention, and it is natural that such changes and modifications fall within the scope of the appended patent claims.

Example 1. 50% ethanol aqueous solution

A mixture of the root of the thorn bush and a 50% ethanol aqueous solution in a weight ratio of 1:10 was extracted at 60°C for 5 hours to obtain a root extract of the thorn bush bush (yield: 7.30%).

Example 2. 70% ethanol aqueous solution

A mixture of the root of the thorn bush and a 70% ethanol aqueous solution in a weight ratio of 1:10 was extracted at 60°C for 5 hours to obtain a root extract of the thorn bush bush (yield: 7.11%).

Example 3. 3 hours extraction_50% ethanol aqueous solution

A thorn root extract was obtained by mixing the thorn root and a 50% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 3 hours.

Example 4. 3 hours extraction_70% ethanol aqueous solution

A thorn root extract was obtained by mixing the thorn root and 70% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 3 hours.

Example 5. 7 hours extraction_50% ethanol aqueous solution

A thorn root extract was obtained by mixing the thorn root and a 50% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 7 hours.

Example 6. 7 hours extraction_70% ethanol aqueous solution

A thorn root extract was obtained by mixing the thorn root and 70% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 7 hours.

Comparative Example 1. Thorn Stem_50% Ethanol Solution

A thorn stem extract was obtained by mixing the thorn stem and a 50% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 5 hours.

Comparative Example 2. Thorn Stem_70% Ethanol Solution

A thorn stem extract was obtained by mixing the thorn stem and a 70% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 5 hours.

Comparative Example 3. Thistle fruit_50% ethanol aqueous solution

A thorny tree fruit extract was obtained by mixing thorny tree fruit and 50% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 5 hours.

Comparative Example 4. Thistle fruit_70% ethanol aqueous solution

A thorny tree fruit extract was obtained by mixing thorny tree fruit and 70% ethanol aqueous solution in a weight ratio of 1:10 and extracting at 60°C for 5 hours.

<Experimental Example Ⅰ> In vitro ( In vitro )

Cell culture

3T3-L1 preadipocytes were purchased from the American type culture collection (ATCC, Manassas, USA) and cultured in Dulbecco's modified eagle's medium (DMEM, Welgene, Daegu, Korea) supplemented with 10% bovine calf serum (Welgene, Daegu, Korea) and 1% penicillin-streptomycin (Welgene, Daegu, Korea) at 37°C under 5% _CO2 conditions.

*, p < 0.05; **, p < 0.01; ***, p < 0.001

Test Example 1. Confirmation of Lipid Accumulation

To differentiate 3T3-L1 preadipocytes into adipocytes, ^5X105 cells/well were seeded into a 6-well plate and cultured until the cells were completely confluent. The medium was replaced and cultured for 2 more days. Differentiation was initiated by culturing for 2 days in DMEM medium containing MDI solution (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 uM dexamethasone, 10 ug/mL insulin) and 10% FBS, and then the medium was replaced with DMEM medium containing 10 ug/ml insulin and 10% FBS, and differentiation was progressed for 2 days. After that, differentiation was completed into adipocytes that form lipid droplets through intracellular fat accumulation by culturing for 4 days in DMEM medium containing only 10% FBS. For measuring lipid accumulation, the extracts of Example 1 and Example 2 were treated simultaneously at concentrations of 0.05, 0.1, and 0.2 mg/mL, respectively, with MDI solution and at each medium exchange, and cultured until differentiation induction was completed. The cells after differentiation induction were washed twice with PBS, treated with 10% formalin, fixed at 4°C for 1 hour, washed, and treated with 60% isopropanol solution to stain the adipocytes. The stained cells were washed with PBS, oil red O was eluted using 100% isopropanol, and the absorbance was measured at 520 nm to confirm lipid accumulation by comparing it with the control group.

Figure 1 is a graph measuring intracellular lipid accumulation when treated with extracts prepared according to Examples 1 and 2 of the present invention.

As shown in Fig. 1, the effect of the extracts of Examples 1 and 2 on lipid accumulation in the process of inducing adipocyte differentiation was confirmed, and it was confirmed that lipid accumulation was reduced in a concentration-dependent manner compared to adipocytes when treated with the extracts of Examples 1 and 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 101.1%, 72.3%, and 35.6% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 95.9%, 68.7%, and 37.1% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

Test Example 2. Measurement of Triglyceride Content

Intracellular triglyceride contents were measured using the EZ-Triglyceride Quantification Assay Kit. To differentiate 3T3-L1 preadipocytes into adipocytes, 5 × 10 ⁵ cells/well were seeded in a 6-well plate, and the cells were cultured until completely confluent. The medium was replaced and cultured for 2 more days. Differentiation was initiated by culturing for 2 days in DMEM medium containing MDI solution (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 uM dexamethasone, 10 ug/mL insulin) and 10% FBS, and then the medium was replaced with DMEM medium containing 10 ug/ml insulin and 10% FBS, and differentiation was performed for 2 days. After that, differentiation was completed into adipocytes that form lipid droplets through intracellular fat accumulation by culturing for 4 days in DMEM medium containing only 10% FBS. For TG measurement, the extracts of Examples 1 and 2 were treated simultaneously at concentrations of 0.05, 0.1, and 0.2 mg/mL, respectively, with MDI solution and at each medium exchange, and cultured until differentiation induction was completed. The cells with completed differentiation induction were washed twice with PBS, homogenized using NP40, heated at 100 ℃ to dissolve intracellular triglycerol, and centrifuged at 13,000 rpm for 2 minutes to remove insoluble substances. For the lysate and standard, 50 uL and 2 uL of lipase were added to a 96-well plate and reacted at room temperature for 20 minutes. Then, 46 uL of triglyceride assay buffer, 2 uL of triglyceride enzyme mix, and 2 uL of triglyceride probe were added respectively, reacted at room temperature for 30 minutes, and the absorbance was measured at 570 nm to compare with the control group, thereby confirming the TG contents.

Figure 2a is a graph measuring the intracellular triglyceride content when treated with extracts prepared according to Example 1, Example 2, and Comparative Examples 1 to 4 of the present invention.

As shown in Fig. 2a, the effects of the extracts of Example 1, Example 2, and Comparative Examples 1 to 4 on the triglyceride content in the process of inducing adipocyte differentiation were confirmed. As a result, it was confirmed that the triglyceride content was significantly reduced in a concentration-dependent manner compared to adipocytes when treated with the extracts of Example 1 and Example 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 74.0%, 52.2%, and 25.9% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 67.8%, 48.9%, and 26.4% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

On the other hand, it was confirmed that the extracts of Comparative Examples 1 to 4 did not decrease the triglyceride content compared to the extracts of Examples 1 and 2, and in particular, it was confirmed that the triglyceride content of Comparative Examples 3 and 4 rather increased.

Figure 2b is a graph measuring the intracellular triglyceride content when treated with extracts prepared according to Examples 1 to 6 of the present invention.

As shown in Fig. 2b, it was confirmed that the triglyceride content was significantly reduced in a concentration-dependent manner compared to adipocytes when treated with the extracts of Examples 1, 2, 5, and 6 of the present invention.

In addition, it was confirmed that the extracts of Examples 3 and 4 did not reduce the triglyceride content compared to the extracts of Examples 1, 2, 5, and 6.

Through the experiments of the above Test Examples 1 and 2, it was confirmed that the extracts of Examples 1 and 2 reduced lipid accumulation and triglyceride content, so the energy consumption pathway of the ADRB2-cAMP pathway was confirmed as shown in Fig. 3.

Test Example 3. Measurement of adrenergic receptor beta-2 contents

Intracellular Adrenergic receptor beta 2 levels were analyzed using a mouse beta-2 adrenergic receptor ELISA kit. Differentiated 3T3-L1 adipocytes were treated with the extracts of Examples 1 and 2 at concentrations of 0.05, 0.1, and 0.2 mg/mL, respectively, and cultured for 24 hours. The medium was recovered, centrifuged at 3,000 rpm for 10 minutes, and used in the experiment. 100 uL of culture medium was dispensed into a 96-well plate, incubated for 90 minutes at 37°C, and 100 uL of biotin-detection antibody working solution was added. After incubation for 60 minutes at 37°C, the cells were washed three times with wash buffer, 100 uL of SABC working solution was added, and incubated for 30 minutes at 37°C. The cells were washed five times with wash buffer, 90 uL of TMB substrate was added, and incubated for 30 minutes at 37°C. To terminate the reaction, 50 uL of stop solution was added, and the absorbance at 450 nm was measured and compared with the control group to confirm the adrenergic receptor beta 2 contents.

FIG. 4 is a graph measuring the intracellular adrenergic receptor beta-2 content when treated with extracts prepared according to Examples 1 and 2 of the present invention.

As shown in Fig. 4, the effect of the extracts of Examples 1 and 2 on intracellular ADRB2 was confirmed, and it was confirmed that the intracellular ADRB2 content increased compared to adipocytes when treated with the extracts of Examples 1 and 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 124.7%, 142.1%, and 130.5% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 135.4%, 115.5%, and 134.1% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

Test Example 4. Measurement of cAMP content

The measurement of intracellular cyclic AMP contents was analyzed using a cAMP assay kit (Competitive ELISA). Differentiated 3T3-L1 adipocytes were treated with the extracts of Examples 1 and 2 at concentrations of 0.05, 0.1, and 0.2 mg/mL, respectively, and cultured for 24 hours. The medium was recovered and centrifuged at 3,000 rpm for 10 minutes and used in the experiment. 50 uL of neutralizing buffer was mixed with 100 uL of medium, and 5 uL acetylating reaction mix (Acetylating Reagent A: Acetylating Reagent B = 1:2) was mixed and reacted at room temperature for 10 minutes. After 10 minutes, 845 uL 1X assay buffer was mixed to prepare the sample to be used in the experiment. Add 50 uL sample, standard, and 10 uL reconstituted cAMP antibody to a Protein G coated 96-well plate and react with stirring at room temperature for 1 hour. Do not discard the reaction solution, but add 10 uL cAMP-HRP to each well and react with stirring at room temperature for 1 hour. After 1 hour, wash 5 times with 200 uL 1X assay buffer and add 100 uL HRP developer and react with stirring at room temperature for 1 hour. To terminate the reaction, add 100 uL 1 M HCl and measure the absorbance at 450 nm.

Figure 5 is a graph measuring the intracellular cAMP content when treated with extracts prepared according to Examples 1 and 2 of the present invention.

As shown in Fig. 5, the effect of the extracts of Examples 1 and 2 on intracellular cAMP was confirmed, and it was confirmed that the intracellular cAMP content increased compared to adipocytes when treated with the extracts of Examples 1 and 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 128.5%, 107.3%, and 107.1% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 131.2%, 140.9%, and 143.2% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

Test Example 5. Measurement of protein expression

Differentiated 3T3-L1 adipocytes were treated with the extracts of Examples 1 and 2 at concentrations of 0.05, 0.1, and 0.2 mg/mL, respectively, and cultured for 24 hours. After culture, the cells were scraped with a cell scraper, centrifuged at 5,000 rpm for 10 minutes, and lysed using RIPA buffer. The lysed cells were mixed with a sample buffer containing sodium dodecyl sulfate (SDS) and β-mercapto-ethanol in a 3:1 ratio with the same amount of protein quantified by the Bradford method, and then heated at 100 °C for 10 minutes. The protein sample was electrophoresed by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (0.45 μm, PVDF transfer membrane, Thermo, Rockford, IL, USA). Membranes were blocked in tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk for 2 hours. Then, they were reacted overnight in buffer containing primary antibodies against PGC-1α (1:1000), CPT-1 (1:1000), UCP-1 (1:1000), and β-actin (1:5000), and washed three times for 5 minutes each with TBS-T (TBS containing 0.1% Tween 20). Then, they were reacted for 1 hour in buffer containing anti-rabbit IgG, HRP-linked antibody (1:1000), and then exposed to X-ray film using the enhanced chemiluminescence method. The intensity of the detected bands was measured using imageJ (National Institutes of Health, Bethesda, MD, USA) software, and the quantitative content was calculated.

5-1. Measurement of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) protein expression

FIG. 6a is a Western blot showing the expression of PGC-1α protein within cells when treated with extracts prepared according to Examples 1 and 2 of the present invention, and FIG. 6b is a graph quantitatively expressing the expressed PGC-1α protein.

As shown in FIGS. 6A and 6B, the effect of the extracts of Examples 1 and 2 on the expression of PGC-1α protein within cells was confirmed. As a result, it was confirmed that the expression of PGC-1α protein within cells increased compared to adipocytes when treated with the extracts of Examples 1 and 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 116.4%, 131.1%, and 149.6% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 125.8%, 136.7%, and 144.6% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

5-2. Measurement of Carnitine palmitoyltransferase 1 (CPT-1) protein expression

Figure 7a is a Western blot showing the expression of CPT-1 protein in cells when treated with extracts prepared according to Examples 1 and 2 of the present invention, and Figure 7b is a graph quantitatively expressing the expressed CPT-1 protein. The control group of Figure 7b is adipocytes.

As shown in FIGS. 7a and 7b, the effect of the extracts of Examples 1 and 2 on the expression of intracellular CPT-1 protein was confirmed, and it was confirmed that the expression of intracellular CPT-1 protein increased compared to adipocytes when treated with the extracts of Examples 1 and 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 114.1%, 123.6%, and 131.4% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 134.3%, 138.1%, and 139.7% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

5-3. Measurement of Uncoupling Protein-1 (UCP1) protein expression

FIG. 8a is a Western blot showing the expression of UCP1 protein within cells when treated with extracts prepared according to Examples 1 and 2 of the present invention, and FIG. 8b is a graph quantitatively expressing the expressed UCP1 protein.

As shown in FIGS. 8a and 8b, the effect of the extracts of Examples 1 and 2 on the expression of UCP1 protein within cells was confirmed, and it was confirmed that the expression of UCP1 protein within cells increased compared to adipocytes when treated with the extracts of Examples 1 and 2.

Specifically, it was confirmed that the extract of Example 1 exhibited 148.3%, 137.2%, and 131.9% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively; and it was confirmed that the extract of Example 2 exhibited 140.3%, 128.2%, and 124.8% when treated at 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, respectively.

<Experimental Example Ⅱ> In vitro ( In vitro )

Cell culture

HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% heat-inactivated Fetal Bovine Serum (Gibco, Life Technologies, Grand Island, NY, USA) and 1% penicillin-streptomycin (P/S, Gibco) at 37°C and 5% _CO2 .

Test Example 6. Confirmation of Lipid Accumulation

HepG2 cells were cultured overnight in a 12-well plate at ^5x105 cells/well, and then replaced with DMEM Media containing FFA-free bovine serum albumin. 250 uM Palmitic acid and the extract of Example 2 were simultaneously treated at concentrations of 0, 12.5, 25, 50, and 100 ug/ml, and the cells were cultured for 24 hours. After completion of the culture, the cells were washed at least twice with DPBS and fixed by treating with 4% PFA (paraformaldehyde) solution for 30 minutes. After completion of the fixation, the cells were washed at least twice and reacted with 60% isopropanol for 5 minutes, and then the lipids accumulated within the cells were stained using Oil Red O staining solution. The stained cells were washed at least twice, and ORO stained in the cells was eluted using 100% isopropanol. The cells were then transferred to a 96-well plate and the absorbance at 510 nm was measured to confirm lipid accumulation.

Figure 9 is a graph measuring intracellular lipid accumulation when treated with an extract prepared according to Example 2 of the present invention.

As shown in Fig. 9, after inducing intracellular lipid accumulation in HepG2 cells by treating them with 250 uM palmitic acid, efficacy was evaluated by treating them with 12.5 ug/ml, 25 ug/ml, 50 ug/ml, and 100 ug/ml of the extract of Example 2. As a result of confirming intracellular lipid through Oil-Red-O Staining, it was confirmed that lipid accumulation was reduced in a concentration-dependent manner.

Gene expression measurement

HepG2 cells were cultured overnight in a 6-well plate at ^1x106 cells/well, and then treated with palmitic acid and the extract of Example 2 simultaneously, and cultured for 24 hours. After culture, the cells were washed with DPBS, lysed using the lysis buffer included in the RNeasy mini kit (Qiagen, Hilden, Germany), and intracellular RNA was isolated using an RNeasy spin column. The concentration of the isolated RNA was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was synthesized using a TOPscript™ RT DryMIX kit (Enzynomics, Daejeon, Korea) by reacting at 50 °C for 60 minutes and then at 95 °C for 5 minutes, and then qPCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). mRNA was detected using the Biorad CFX96 real-time PCR detection system, and gene expression was analyzed using the △△CT calculation method.

Test Example 7. Measurement of PGC 1α (peroxisome proliferator-activated receptor γ coactivator 1-α) content

Figure 10 is a graph measuring the intracellular PGC 1α content when treated with an extract prepared according to Example 2 of the present invention.

As shown in Figure 10, PGC 1α is a thermogenic indicator, and when its expression increases, fat within the tissue is consumed as heat, thereby reducing body fat. When the extract of Example 2 was treated at 10 ug/ml and 50 ug/ml, it was confirmed that PGC1α gene expression increased by 0.95 times and 1.41 times, respectively, compared to the vehicle control.

Test Example 8. Measurement of AMPK (AMP-activated kinase) content

Figure 11 is a graph measuring the intracellular AMPK content when treated with an extract prepared according to Example 2 of the present invention.

As shown in Figure 11, when AMPK is activated, the activity of enzymes involved in lipogenesis is inhibited. When the extract of Example 2 was treated at 10 ug/ml and 50 ug/ml, it was confirmed that AMPK gene expression increased by 1.31-fold and 1.49-fold, respectively, compared to the control group (vehicle control).

Test Example 9. Measurement of PPARα (peroxisome proliferator-activated receptors) content

Figure 12 is a graph measuring the intracellular PPARα content when treated with an extract prepared according to Example 2 of the present invention.

As shown in Fig. 12, PPARα is a gene that promotes fat catabolism and promotes fatty acid oxidation through UCP in adipose tissue and muscle. When HepG2 cells were treated with 10 ug/ml and 50 ug/ml of the extract of Example 2, it was confirmed that the expression of the PPARα gene increased by 1.23 times and 1.51 times, respectively, compared to the control group (vehicle control).

Test Example 10. Measurement of COL1A1 (collagen type I alpha 1 chain) content

Figure 13 is a graph measuring the intracellular COL1A1 content when treated with an extract prepared according to Example 2 of the present invention.

As shown in Figure 13, COL1A1 is a liver fibrosis-related gene. When analyzing the gene expression of HepG2 cells treated with Palmitic acid and then treated with 100 ug/ml of the extract of Example 2, it was confirmed that the gene expression increased to 1.3 when treated with Palmitic acid (PA) and decreased to 0.5 after treatment with the extract of Example 2.

Test Example 11. Measurement of ACTA2 (actin alpha 2) content

Figure 14 is a graph measuring the intracellular ACTA2 content when treated with an extract prepared according to Example 2 of the present invention.

As shown in Figure 14, the ACTA2 (α-SMA) gene is known to be highly expressed in the liver injury model and to be hardly expressed under normal conditions. It was confirmed that the gene expression, which increased to 1.3 when treated with palmitic acid (PA), decreased to 0.4 after treatment with the extract of Example 2.

<Experimental Example III> In vivo ( In vivo )

Animal testing

SD male 4-week-old (130-150 g) rats were used in the experiment after acclimation for 1 week under the conditions of temperature 22±1℃, humidity 55±3%, and 12-hour light/dark cycle (8:00 am/8:00 pm). For 4 weeks, normal group 1 (normal) was fed a formulated feed (AIN-76A), and control group 1 was fed a high-fat (60% fat) feed to induce obese individuals and used for the test. The remaining groups were fed a high-fat diet and orally administered the control substance and the extracts of Examples 1 and 2. After the experiment, blood was collected from the experimental animals and analyzed using an automated chemistry analyzer (Thermo, Thermo).

-Rats in 5 groups-

Normal group 1: Normal diet + CMC (sodium carboxymethyl cellulose) administration

Control group 1: High-fat diet + CMC

Positive control group 1: High-fat diet + Garcinia cambogia 200 mg/kg

Example 1: High-fat diet + 100 mg/kg of the extract of Example 1

Example 2: High-fat diet + 100 mg/kg of the extract of Example 2

The extracts of Examples 1 and 2 and Garcinia cambogia were diluted in CMC and used.

Test Example 12. Measurement of growth rate

Figure 15a is a graph showing the body weight gain per feed intake of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; Figure 15b is a graph showing the body weight over time of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; Figure 15c is a graph showing the body weight gain of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group.

As shown in Fig. 15a, it was confirmed that the weight gain according to feed intake was high in the order of normal group 1 > example 2 group > example 1 group > control group 1 = positive control group 1.

In addition, as shown in Figures 15b and 15c, it was confirmed that body weight increased in all groups, and the amount of weight gain increased in the order of positive control group 1 > control group 1 > example 1 group > example 2 group > normal group 1.

Test Example 13. Blood Lipid Analysis

FIG. 16a is a graph showing neutral lipid (TG) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; FIG. 16b is a graph showing total cholesterol (t-cholesterol) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; FIG. 16c is a graph showing low-density lipoprotein cholesterol (LDL-cholesterol) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group; and FIG. 16d is a graph showing high-density lipoprotein cholesterol (HDL-cholesterol) in the blood of normal group 1, control group 1, positive control group 1, example 1 group, and example 2 group.

As shown in FIGS. 16a to 16d, it was confirmed that the groups that took the extracts of Examples 1 and 2 had decreased levels of neutral lipid (TG), total cholesterol (t-cholesterol), and low-density lipoprotein cholesterol (LDL-cholesterol) in their blood, and increased levels of high-density lipoprotein cholesterol (HDL-cholesterol), compared to the control group.

Through this, it was confirmed that the content of neutral fat (TG), total cholesterol (t-cholesterol), and low-density lipoprotein cholesterol (LDL-cholesterol) increased by a high-fat diet was reduced, and the content of high-density lipoprotein cholesterol (HDL-cholesterol) decreased by a high-fat diet was increased.

The extracts of Examples 1 and 2 reduce the contents of neutral fat (TG), total cholesterol (t-cholesterol) and low-density lipoprotein cholesterol (LDL-cholesterol) in the blood, and increase the content of high-density lipoprotein cholesterol (HDL-cholesterol), and therefore can be used as a health functional food composition for improving blood flow; a composition for preventing, treating or improving dyslipidemia; or a composition for preventing, treating or improving hypercholesterolemia. The composition for preventing, treating or improving may be a pharmaceutical composition or a food composition.

Test Example 14. Liver Function Analysis

Figure 17 is a graph showing (a) AST, (b) ALT, (c) GTT, (d) ALP, and (e) LDH in the blood of normal group 1, control group 1, positive control group 1, example 1, and example 2.

As shown in Figure 17, the group that took the extracts of Examples 1 and 2 showed AST, ALT, GTT, ALP, and LDH similar to those of normal group 1, confirming that no hepatotoxicity occurred.

The extracts of Examples 1 and 2 have excellent AST, ALT, GTT, ALP and LDH activities in blood, and therefore can be used as a food or pharmaceutical composition for improving any one of liver damage selected from acute alcoholic liver damage, chronic alcoholic liver damage, non-alcoholic lipid accumulation, non-alcoholic inflammation and non-alcoholic liver damage.

Test Example 15. Blood Sugar and Hormone Analysis

Figure 18 is a graph showing (a) glucose, (b) insulin, (c) leptin, and (d) adiponectin in the blood of normal group 1, control group 1, positive control group 1, example 1, and example 2.

As shown in Fig. 18, the groups that took the extracts of Examples 1 and 2 showed decreased blood sugar, increased insulin, and decreased leptin compared to Control Group 1. It was confirmed that adiponectin was similar in all groups.

The extracts of Examples 1 and 2 can be used as compositions for preventing, treating or improving diabetes because they reduce blood sugar levels and increase insulin; and can be used as food compositions for suppressing appetite because they reduce leptin. The compositions for preventing, treating or improving can be pharmaceutical compositions or food compositions.

<Experimental Example IV> In vivo ( In vivo )

Animal testing

C57BL6J male 6-week-old mice were obtained from Coretech and used in the experiment after acclimation for 1 week under the conditions of temperature 22±1℃, humidity 55±3%, and 12-hour light/dark cycle (am/pm 8 PM). For 8 weeks, normal group 2 (normal) was fed a general feed (Orient), and control group 2 was fed a high-fat feed (fat 60%, Research diet. D16042106) to induce obese individuals and used for the test. The remaining groups were fed a high-fat diet and orally administered the control substance and the extract of Example 2.

*, p < 0.05; **, p < 0.01; ***, p < 0.001

-6 group mouse-

Normal group 2: Normal diet + distilled water

Control group 2: High-fat diet + distilled water

Positive control group 2: High-fat diet + Garcinia cambogia 400 mg/kg

Positive control group 3: High-fat diet + Cissus antarctica 65 mg/kg

Example 2 (200): High-fat diet + 200 mg/kg of the extract of Example 2

Example 2 (150): High-fat diet + 150 mg/kg of the extract of Example 2

Test Example 16. Measurement of growth rate

Figure 19 is a graph showing the body weight over time of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200) group, and example 2 (150) group.

As shown in Figure 19, the positive control group 2, positive control group 3, Example 2 (200) group, and Example 2 (150) group had similar average body weights, but it was confirmed that the control group 2 had an increase in body weight compared to the other groups.

Test Example 17. Body fat measurement using DEXA

On the day of autopsy of each group of mice above, body fat was measured using dual energy X-ray absorptiometry (DEXA) before organ removal. The radiographs of body fat were displayed in three modes according to low-density fat (blue), medium-density fat (yellow), and high-density fat (red).

Figure 20a is a photograph taken using dual energy X-ray absorptiometry (DEXA) of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200) group, and example 2 (150) group; Figure 20b is a graph showing the body fat mass of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200) group, and example 2 (150) group.

As shown in Figures 20a and 20b, it was confirmed that a large amount of fat was accumulated in the body cavity and subcutaneous tissue of the control group 2, positive control group 2, positive control group 3, Example 2 (200) group, and Example 2 (150) group compared to the normal group 2 (red).

However, it was confirmed that the body fat of group Example 2 (200) was significantly reduced compared to other groups except for group Normal 2.

Test Example 18. Changes in the size of fat cells

Peri-testicular adipose tissue (eWAT) was extracted at autopsy. The eWAT was fixed with 10% formalin solution, embedded in paraffin, and sectioned. After staining with hematoxylin and eosin, the stained tissue was observed and photographed using a light microscope (Olympus, Japan). Afterwards, the adipocyte size was measured using ImageJ software (National Institutes of Health, Bethesda, MD).

Figure 21a is a photograph showing the peritesticular adipose tissue (eWAT) of normal group 2, control group 2, positive control group 2, positive control group 3, Example 2 (200), and Example 2 (150); Figure 21b is a graph showing the measurement of fat cell size of normal group 2, control group 2, positive control group 2, positive control group 3, Example 2 (200), and Example 2 (150).

As shown in Figures 21a and 21b, the size of fat cells in Control Group 2 increased by about twice compared to Normal Group 2 due to fat accumulation due to a high-fat diet, and it was confirmed that the size of fat cells in Example 2 (200), Positive Control Group 2, and Positive Control Group 3 significantly decreased.

Test Example 19. Fat accumulation in liver tissue

Liver tissues extracted at autopsy were fixed with 10% formalin, embedded in OCT compound, frozen, and then cryosectioned. After that, lipid components accumulated in cells were sufficiently stained with pre-prepared Oil Red O solution, and observed and photographed using an optical microscope (Olympus, Japan). The area (%) of lipid vesicles stained with Oil Red O was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Figure 22a is a photograph showing staining of accumulated fat in liver tissues of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200), and example 2 (150); Figure 22b is a graph showing measurements of the area of accumulated fat in liver tissues of normal group 2, control group 2, positive control group 2, positive control group 3, example 2 (200), and example 2 (150).

As shown in Figures 22a and 22b, in the control group 2, fat was accumulated in the liver tissue due to the high-fat diet, and numerous stained fat vesicles were observed compared to the normal group 2, and it was confirmed that the area of fat accumulated in the liver tissue was significantly reduced in the Example 2 (200) group, the Example 2 (150) group, the positive control group 2, and the positive control group 3.

Below, a formulation example of a composition containing the powder of the present invention is described, but the present invention is not intended to be limited thereto but is merely intended to be described specifically.

Preparation Example 1. Preparation of a mountain product

500 mg of extract powder obtained in Example 1

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled into a sealed bag to produce a powdered preparation.

Preparation Example 2. Preparation of tablets

300 mg of extract powder obtained in Example 1

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by pressing them according to the usual method for manufacturing tablets.

Preparation Example 3. Preparation of capsules

200 mg of extract powder obtained in Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above ingredients are mixed according to the conventional capsule manufacturing method and filled into a gelatin capsule to manufacture a capsule.

Preparation Example 4. Preparation of injection

600 mg of extract powder obtained in Example 1

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

It is manufactured with the above ingredient content per ampoule according to the manufacturing method of a conventional injection.

Preparation Example 5. Preparation of liquid preparation

4 g of extract powder obtained in Example 1

10 g of isoflavone

Mannitol 5 g

Appropriate amount of purified water

According to the usual method for manufacturing a liquid, each ingredient is dissolved in purified water, an appropriate amount of lemon flavor is added, the above ingredients are mixed, purified water is added, and the total amount is adjusted to 100 g by adding purified water, and then the liquid is filled into a brown bottle and sterilized.

Preparation Example 6. Preparation of granules

1,000 mg of extract powder obtained in Example 1

Vitamin mixture appropriate amount

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate monobasic 15 mg

55 mg of dibasic calcium phosphate

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a preferred example of mixing ingredients relatively suitable for granules, but the mixing ratio may be arbitrarily modified, and granules may be manufactured by mixing the above ingredients according to a conventional method for manufacturing granules, and then used to manufacture a health functional food composition according to a conventional method.

Preparation Example 7. Manufacturing of functional beverages

1,000 mg of extract powder obtained in Example 1

1,000 mg of citric acid

100 g of oligosaccharide

2 g of plum concentrate

1 g of taurine

Add purified water to make a total of 900 mL

The above ingredients are mixed according to a conventional health beverage manufacturing method, stirred and heated at 85°C for about 1 hour, the resulting solution is filtered, placed in a sterilized 2 L container, sealed and sterilized, then refrigerated and used to manufacture the functional beverage composition of the present invention.

The above composition ratio is a preferred example of mixing ingredients suitable for relatively preferred beverages, but the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demand country, and intended use.

Claims (12)

A food composition for improving or preventing metabolic diseases, characterized by containing an extract of the root of the thistle tree as an effective ingredient.
The above metabolic disease is obesity,
A food composition for improving or preventing metabolic diseases, characterized in that the above-mentioned thistle root extract is an extract of thistle root in an ethanol aqueous solution of 20 to 80%. delete delete delete delete A food composition characterized in that in claim 1, the extraction temperature of the root extract of the thornbush is 40 to 80°C, and the extraction time is 3 to 10 hours. A food composition for improving or preventing metabolic disease, characterized in that in claim 1, the obesity is improved or prevented by inducing weight loss. delete delete A food composition for improving or preventing metabolic disease, characterized in that in claim 1, the obesity is improved or prevented by inducing appetite suppression. A pharmaceutical composition for the prevention or treatment of metabolic diseases, characterized by containing an extract of the root of the thistle tree as an active ingredient.
The above metabolic disease is obesity,
A pharmaceutical composition for preventing or treating metabolic diseases, characterized in that the above-mentioned thistle root extract is an extract of thistle root in an ethanol aqueous solution of 20 to 80%. delete