KR20230139404A - Pharmaceutical composition for preventing or treating metabolic diseases comprising a mixture extract of Erigeron canadensis L, gardenia fruit and Gentian - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic diseases, and more specifically, by containing mixed extracts of turmeric, gardenia, and gentian root, it can exhibit the effect of inhibiting fat accumulation or foam cell formation, and ultimately prevents metabolic diseases. It can be useful for prevention, improvement or treatment.

Description

Pharmaceutical composition for preventing or treating metabolic diseases comprising a mixture extract of Erigeron canadensis L, gardenia fruit and Gentian}

The present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic diseases containing mixed extracts of turmeric, gardenia, and gentian root.

Recently, due to changes in rich and diverse diets and lifestyles, nutritional intake can easily become unbalanced, and mechanized modern life has led to lack of exercise. Accordingly, metabolic diseases are also increasing. Metabolic disease is a general term for diseases caused by imbalance in metabolism of carbohydrates, lipids, etc. in the body, and may include obesity, diabetes, insulin resistance, fatty liver, hyperlipidemia, arteriosclerosis, or complications thereof. The incidence of metabolic diseases has recently been rapidly increasing in Korea.

Obesity refers to a condition in which energy intake and consumption are unbalanced due to lifestyle or genetic causes and excess energy is accumulated as fat, resulting in an abnormal increase in body fat and metabolic abnormalities. Obesity is common among modern people due to the improvement in living standards due to economic development, and is a major cause of increasing the risk of high blood pressure, hyperlipidemia, arteriosclerosis, heart disease, and diabetes.

Diabetes is a disease in which insulin secretion is insufficient or normal function is not achieved. It is characterized by hyperglycemia, which increases the concentration of glucose in the blood. Hyperglycemia causes various symptoms and signs and causes glucose to be excreted in the urine. Additionally, over a long period of time, it can cause vascular disorders and dysfunction of nerves, kidneys, and retina, which can even lead to loss of life.

Arteriosclerosis refers to a disease in which oil increases in the blood, thickens the blood vessel walls, narrows the inside of the blood vessels, and causes blood clots to form, narrowing the lumen of the blood vessels and reducing the elasticity of the arteries. Depending on the area of occurrence, it can lead to stroke, angina, myocardial infarction, or peripheral blood vessels. Divided into diseases, etc. Vascular abnormalities caused by arteriosclerosis can be a significant cause of death.

Fatty liver disease is a pathological condition in which fat exceeds 5% of the total liver weight, and liver diseases including it are known to be the second most serious cause of death after cancer in adults in their 40s and 50s in developed countries. The causes of fatty liver include excessive drinking, obesity, diabetes, and hyperlipidemia, and fatty liver is known to have a very high risk of worsening into hepatitis, cirrhosis, and liver cancer.

To date, drugs to treat metabolic diseases have been developed, but no satisfactory treatment method or drug has been developed. Accordingly, there is a need to develop new natural materials that can prevent or treat metabolic diseases without side effects.

Korean Patent No. 2299407

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases.

The purpose of the present invention is to provide a food composition for preventing or improving metabolic diseases.

The purpose of the present invention is to provide a method for preventing or treating metabolic diseases.

1. A pharmaceutical composition for the prevention or treatment of metabolic diseases containing mixed extracts of Mandarinwort, Gardenia and Gentian herb.

2. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to 1 above, wherein the mixed extract is extracted by mixing 0.5 to 1.5 parts by weight of Mandarinwort, 0.5 to 1.5 parts by weight of Gardenia, and 0.5 to 1.5 parts by weight of Gentian herb.

3. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to 1 above, wherein the mixed extract is extracted by mixing Mungcholy, Gardenia, and Gentian herb at a weight ratio of 1:1:1.

4. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to 1 above, wherein the mixed extract is a mixture of Manchuria, Gardenia, and Gentian herb and extracted with water or alcohol having 1 to 4 carbon atoms.

5. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to 1 above, wherein the mixed extract is a mixture of Manchuria, Gardenia, and Gentian herb and extracted with water and alcohol having 1 to 4 carbon atoms.

6. The pharmaceutical composition for the prevention or treatment of metabolic diseases according to 1 above, wherein the mixed extract is a mixture of Chinese pepper, gardenia, and gentian root at a weight ratio of 1:1:1 and extracted with water.

7. The pharmaceutical composition for preventing or treating metabolic diseases according to 1 above, wherein the metabolic disease is at least one selected from the group consisting of obesity, diabetes, fatty liver, dyslipidemia, and arteriosclerosis.

8. The pharmaceutical composition for preventing or treating metabolic diseases according to item 7 above, wherein the metabolic disease is at least one selected from the group consisting of fatty liver disease and arteriosclerosis.

9. The pharmaceutical composition for preventing or treating metabolic diseases according to 1 above, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

10. A food composition for preventing or improving metabolic diseases containing mixed extracts of Manchuria, Gardenia and Gentian herb.

The composition containing the mixed extract of Manchuria, Gardenia, and Gentian herb of the present invention can exhibit the effect of inhibiting fat accumulation or inhibiting foam cells, and can ultimately be useful in preventing, improving, or treating metabolic diseases such as fatty liver or arteriosclerosis. there is.

Figure 1 shows the results of GC-MS analysis of a mixed extract of Mandarinwort, Gardenia, and Gentian chinensis according to an example.
Figure 2 shows the results of GC-MS analysis of an extract of Manchurian root alone according to an example.
Figure 3 shows the results of GC-MS analysis of a single gardenia extract according to an example.
Figure 4 shows the results of GC-MS analysis of a single extract of Gentian herb according to an example.
Figure 5 shows the results of confirming the cytotoxicity and fat accumulation inhibition efficacy of a mixed extract of Mandarinwort, gardenia, and Gentian herb according to an example.
Figure 6 shows the results of confirming the cytotoxicity and fat accumulation inhibitory effect of the extract of Mandarinwort alone according to an example.
Figure 7 shows the results of confirming the cytotoxicity and fat accumulation inhibition efficacy of the gardenia extract alone according to an example.
Figure 8 shows the results of confirming the cytotoxicity and fat accumulation inhibition efficacy of Gentian herb extract alone according to an example.
Figure 9 shows the foam cell inhibitory effect of a mixed extract of Chinese herbaceous plant, gardenia, and gentian root according to an example.
Figure 10 shows the results of confirming the effectiveness of the mixed extract of Mandarinwort, gardenia, and Gentian chinensis in inhibiting the expression level of the PPAR-γ gene according to an example.
Figure 11 shows the results of confirming the effectiveness of the mixed extract of turmeric, gardenia, and gentian root in inhibiting the expression level of the CD36 gene according to an example.

The present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases containing a mixed extract of Chinese herb, Gardenia, and Gentian herb.

The term “extract” refers to a substance separated by dissolving the substance to be extracted in a solvent, and specifically, an extract obtained by extraction treatment of the extraction subject, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, and a freeze-dried product of the extract. , it may include the extract itself, such as a crude product or purified product of the extract, or a mixture thereof, and extracts of all formulations that can be formed using the extract.

The mixed extract may be extracted after mixing turmeric, gardenia, and gentian root. For example, the mixed extract is 1 to 10: 1 to 10: 1 to 10, 1 to 5: 1 to 5: 1 to 5, 1 to 4: 1 to 4: 1 to 4, 1 to 3. : 1 to 3: 1 to 3, 1 to 2: 1 to 2: 1 to 2, or may be mixed and extracted at a weight ratio of 1:1:1. According to one embodiment, the mixed extract may be extracted by mixing turmeric, gardenia, and gentian root in a weight ratio of 1:1:1.

The mixed extract may be an extract obtained by mixing 0.5 to 10 parts by weight of ginseng, 0.5 to 10 parts by weight of gardenia, and 0.5 to 10 parts by weight of gentian root, for example, 1 to 10 parts by weight of ginseng, 1 to 10 parts by weight of gardenia, and 1 to 1 part by weight of gentian root. to 10 parts by weight; 1 to 5 parts by weight of Chinese pepper, 1 to 5 parts by weight of gardenia, and 1 to 5 parts by weight of gentian root; 1 to 2 parts by weight of Chinese pepper, 1 to 2 parts by weight of gardenia, and 1 to 2 parts by weight of gentian root; Alternatively, it may be extracted by mixing 0.5 to 1.5 parts by weight of Chinese pepper, 0.5 to 1.5 parts by weight of gardenia, and 0.5 to 1.5 parts by weight of gentian root.

Aster is a plant belonging to the Asterales order, Asteraceae family, and Erigeron genus. It is classified as an animal or plant that can be used as a main ingredient in the Food Code. It can also be expressed as Erigeron canadensis L. or Conyza canadensis . there is. Gardenia can mean the gardenia tree or its fruit, Gardenia jasminoides or It can also be expressed as 梔子. Gentian plant is a perennial herb belonging to the Gentian family, Gentiana scabra for. It can also be expressed as scabra, dragon root, choyongdam, and gentian.

Garlic, gardenia, and gentian root can be purchased independently commercially, or collected or cultivated in nature.

The plant itself, leaves, stems, flowers, roots, fruits, or a combination of these can be used independently for Manchuria, gardenia, and gentian root, and the extraction site is not limited.

The type of extraction solvent used to extract Mandarinwort, gardenia, and gentian root is not particularly limited, and any solvent known in the art can be used. Extraction solvents include water, alcohols having 1 to 4 carbon atoms, or mixed solvents thereof, and these may be used alone or in combination of one or more types.

The mixed extract may be extracted with one or more extraction solvents selected from water, alcohol with 1 to 4 carbon atoms, or mixtures thereof. For example, the extraction solvent may be 100% (v/v) water, distilled water, or 10 to 100% (v/v) ethanol, but is not limited thereto.

The mixed extract may be a mixture of seaweed, gardenia, and gentian root in a 1:1:1 weight ratio and extracted with water.

According to one embodiment, the mixed extract may be obtained by extracting ginseng, gardenia, and gentian root with distilled water.

The extraction method is not particularly limited and can be extracted according to methods commonly used in the art. For example, the extraction method may be using reduced pressure high temperature extraction, boiling water extraction, reflux extraction, hot water extraction, cold needle extraction, room temperature extraction, ultrasonic extraction, or steam extraction, or a combination thereof, but is not limited thereto.

The extraction method may be performed by adding 1 to 20 times, 5 to 20 times, 10 to 20 times, 5 to 15 times, or 12 to 14 times the extraction solvent relative to the weight of the mixture of Chinese herb, gardenia, and gentian root. The mixed extract may be extracted by adding 1 to 20 times, 5 to 20 times, 10 to 20 times, 5 to 15 times, or 12 to 14 times the extraction solvent based on the total weight of the mesquite, gardenia, and gentian root. there is.

Reduced pressure concentration of the extract may be done using a vacuum concentrator or vacuum rotary evaporator, and drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying.

According to one embodiment, it was confirmed that the mixed extract of Manchuria, Gardenia, and Gentian herb exhibited an effect of inhibiting fat accumulation in hepatocytes and inhibiting foam cell formation. The term “foam cells,” also called lipid-filled macrophages, is a type of cell that contains cholesterol. Foam cells can form plaques that can lead to atherosclerosis and cause heart attacks and strokes. Foam cells are fatty M2 macrophages containing low-density lipoprotein (LDL).

The mixed extract of Manchuria, Gardenia, and Gentian herb of the present invention can prevent, improve, or treat fatty liver by inhibiting fat accumulation in hepatocytes. The mixed extract of Manchuria, Gardenia, and Gentian herb of the present invention can prevent, improve, or treat atherosclerosis or arteriosclerosis by inhibiting foam cells.

According to one embodiment, it was confirmed that the mixed extract of Manchuria, Gardenia, and Gentian chinensis exhibits an effect of inhibiting the expression of CD36 and PPAR-γ, which are known as representative markers of metabolic diseases.

In addition, according to one embodiment, it was confirmed that the mixed extract of Manchuria, Gardenia, and Gentian herb showed a significantly superior fat accumulation inhibitory effect compared to the individual extracts of Manchuria, Gardenia, and Gentian herb.

In light of these results, it was confirmed that the mixed extract of Manchuria, gardenia, and Gentian herb can be used for the treatment of metabolic diseases.

The term “metabolic disease” is a general term for diseases caused by metabolic disorders in the body, and may include obesity, diabetes, dyslipidemia, fatty liver, etc.

Metabolic diseases may include, but are not limited to, obesity, diabetes, fatty liver, hypertension, arteriosclerosis, dyslipidemia, hyperlipidemia, liver disease, cardiomyopathy, myocardial infarction, cerebral infarction, hyperinsulinemia, hyperglycemia, or insulin resistance disease.

Fatty liver may be non-alcoholic or alcoholic fatty liver, but is not limited thereto.

The term “obesity” may refer to a state of excessive accumulation of fat in the body. The standard for obesity is that body fat is more than 25% of body weight, or more than 30-35% for women. A common measurement method is body mass index (BMI), which is expressed as weight (kg)/height (m) ² . Body Mass Index) is widely used. For Westerners, a body mass index exceeding 30 kg/m ² is defined as obesity, and a body mass index of 25 to 30 kg/m ² is defined as overweight. For Asians, a body mass index exceeding 28 kg/m ² , which is two degrees lower, is defined as obesity, and a body mass index exceeding 23 to 28 kg/m ² is defined as obesity. can be said to be overweight. In addition, obesity can be defined based on waist to hip ratio (WHR) or abdominal fat, and this application includes all cases of obesity defined by conventional standards, including the above-mentioned standards. do.

The term “dyslipidemia” may refer to a state in which lipid components such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids in the blood are increased or HDL cholesterol is decreased. For example, the dyslipidemia may be any one or more selected from the group consisting of hyperlipidemia, hyperlipidemia, hyperlipidemia, hypertriglyceridemia, and hypolipidemia.

The term “arteriosclerosis” refers to a disease in which blood vessels become narrowed or blocked, causing blood flow to the periphery. The arteriosclerosis may include, but is not limited to, coronary artery sclerosis and atherosclerosis.

The pharmaceutical composition of the present invention may improve liver damage caused by metabolic diseases. Additionally, the pharmaceutical composition may improve cardiovascular damage caused by metabolic disease.

The term “prevention” refers to any action that inhibits or delays the progression of metabolic disease by administering the mixed extract or composition of the present invention.

The term “treatment” refers to any action in which symptoms of a metabolic disease are improved or beneficially changed by administration of the mixed extract or composition of the present invention.

The pharmaceutical composition of the present invention contains mixed extracts of turmeric, gardenia, and gentian root as active ingredients and may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be those commonly used in formulation, such as saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and antioxidant. However, it is not limited to this.

The pharmaceutical composition of the present invention can be formulated into an injectable formulation, pills, capsules, granules, or tablets, but is not limited thereto.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) according to the desired method, and can be appropriately selected by a person skilled in the art.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times.

The effective amount of the pharmaceutical composition of the present name may vary depending on the patient's age, gender, condition, weight, absorption, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitant drug, and is generally 0.001 per kg of body weight. From 150 mg to 150 mg, preferably from 0.01 to 100 mg, can be administered daily or every other day, or divided into 1 to 3 times per day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a method for preventing or treating metabolic diseases, comprising administering a mixed extract of Chinese herbaceous plant, gardenia, and gentian root to an individual.

Depositphotos, gardenia, gentian root, mixed extracts, and metabolic diseases have been described above, so detailed explanations will be omitted.

The term “administration” means introducing a pharmaceutical composition or mixed extract into a subject by any suitable method.

The term “individual” refers to all animals, including rats, mice, and livestock, including humans, that have or may develop metabolic diseases. The animals may be not only humans but also mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require prevention or treatment of similar symptoms, but are not limited thereto.

In addition, the present invention provides a food composition for preventing or improving metabolic diseases comprising a mixed extract of Chinese herb, gardenia, and gentian root.

Depositphotos, gardenia, gentian root, mixed extracts, and metabolic diseases have been described above, so detailed explanations will be omitted.

The food composition of the present invention can be consumed on a daily basis, and unlike general drugs, it is made from natural products and has the advantage of having no side effects that may occur during long-term use of the drug, so it is useful for the purpose of preventing or improving metabolic diseases. can be used

The term “improvement” refers to any action that reduces the degree of symptoms of a parameter, such as a metabolic disease, that is related to the condition being treated by ingestion of the food composition.

Food compositions can be manufactured without limitation in various forms as long as they are recognized as food. For example, foods include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and health products. It may be functional food or health food, and includes all foods in the conventional sense.

Food compositions can be manufactured by methods commonly used in the art.

The food composition may further include raw materials and ingredients commonly added in the art in addition to the mixed extracts of Manchuria, Gardenia, and Gentian herb. For example, the food composition may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. . In addition, the food composition includes minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu); and amino acids such as lysine, tryptophan, cysteine, and valine.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. However, the examples below are provided only for illustrative purposes to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

1. Extract preparation

(1) Manufacture of mixed extracts of turmeric, gardenia and gentian root

50 g each of Manchuria, Gardenia, and Gentian herb were added to 2000 ml of distilled water, boiled for 3 hours, and then filtered. The filtrate was concentrated under reduced pressure to 100 ml using a rotary evaporator and then freeze-dried to obtain 37.5 g of mixed extract (3Hb) (yield 25%).

(2) Manufacture of ginseng extract

It was prepared in the same manner as in 1(1) above, except that 150 g of Manchuria, Gardenia and Gentian chinensis alone was added to 2000 ml of distilled water to obtain a Manchurian extract (yield 21.8%).

(3) Gardenia extract production

It was prepared in the same manner as in 1(1) above, except that 150 g of only gardenia among the Chinese pepper, gardenia, and gentian root were added to 2000 ml of distilled water to obtain a gardenia extract (yield 18%).

(4) Manufacture of Gentian herb extract

It was prepared in the same manner as in 1(1) above, except that 150 g of only Gentian herb among Chinese herb, gardenia and Gentian herb was added to 2000 ml of distilled water to obtain a Gentian herb extract (yield 26.4%).

2. Confirmation of the effect of inhibiting fat accumulation in liver cells

(1) Experimental materials and methods

Human hepatocellular carcinoma cell line (HepG2) was purchased from the Korea Cell Line Bank. HepG2 cells were cultured in an environment of 5% CO ₂ and 37°C using MEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (Penicilin-steptomycin).

Palmitic acid (PA) was used as a fatty acid to accumulate lipids. 20mM of PA was mixed with NaOH 0.01M and maintained at 70°C for 30 minutes to create a suspension. Then, it was mixed with 10% fatty acid free BSA solution in a 1:3 ratio and conjugated at 37°C. The PA produced in this way was used to make 500 μM PA using MDM media.

In order to confirm the cytotoxicity of the analysis sample to hepatocyte cell lines, it was analyzed using WST-1. Cells were distributed in 96 wells and cultured for 24 hours, and treated with Manchuria extract alone, Gardenia alone extract, Gentian herb extract alone, and mixed extract of Manchuria, Gardenia and Gentian herb (3Hb) at a concentration of less than 10 mg/mL. Afterwards, 20ul of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) was treated on the culture medium incubated for 24 hours and incubated for 2 hours. After further culturing, the absorbance was measured at 450 nm using a microplate reader. The measured absorbance was quantified relative to the untreated group, converted to percentage, and graphed. The analyzed results were recorded as IC ₅₀ , the cytotoxicity level and the concentration at which 50% of cells were killed.

Next, to analyze the inhibitory effect of PA-induced lipid accumulation, it was measured using an HT-1S microscope. Specifically, HepG2 cells (1X 10 ⁵ cells/well) were seeded in Tomo Dish. Afterwards, 500 μM PA and a concentration of 500 μM, which were confirmed to be non-toxic, were treated with 500 μM of Manchurian extract alone, gardenia alone, Gentian chinensis alone, and mixed extract of Manchuria, Gardenia, and Gentian chinensis (3Hb) for 24 hours, respectively. Lipid accumulation was acquired in live cells using a Tomocube HT-1S microscope. Based on the three-dimensional refractive index distribution of cells, lipid accumulation was analyzed using Tomostudio software. Specifically, PA-treated liver cells were image-processed (tomostudio software) from density difference to cell inclusions (fat) in red, then AI-based quantification was performed using a 3D tomolipid analysis program, followed by anova test and post-hoc. A Turkey test was conducted. In Figures 5B, 6B, 7B, and 8B, red color represents fat.

(2) Experiment results

The cytotoxic IC ₅₀ of the mixed extract of Manchuria, Gardenia, and Gentian chinensis was 1801 ug/mL, and the non-toxic concentration was defined as 500 ug/mL (see Figure 5A). The group treated with mixed extracts of Manchuria, Gardenia, and Gentian herb showed significant lipid accumulation inhibition efficacy. Specifically, the group treated with mixed extracts of Manchuria, gardenia, and Gentian herb (3Hb) had significantly reduced lipid accumulation compared to the group in which only lipid accumulation was induced by PA and not treated with the mixed extract, and lipid accumulation was not induced by PA. The degree of fat accumulation was lower than that of the group (see Figure 5B).

Meanwhile, the group treated with Mango root extract alone (treated at 500 ug/mL, which was confirmed to be non-toxic) did not show a significant lipid accumulation inhibition effect compared to the group in which only lipid accumulation was induced with PA (see Figure 6). In addition, the group treated with gardenia extract alone (500 ug/mL treatment, which was confirmed to be non-toxic) did not show a significant lipid accumulation inhibition effect compared to the group in which only lipid accumulation was induced with PA (see Figure 7). In addition, the group treated with Gentian herb extract alone (500 ug/mL treatment, which was confirmed to be non-toxic) did not show a significant lipid accumulation inhibition effect compared to the group in which only lipid accumulation was induced with PA (see Figure 8).

In Figures 5 to 8, the volume ratio was calculated as lipid volume/cell volume (um ³ ). Data are expressed as mean percentages relative to the untreated group ± standard deviation. *: P < 0.05 compared to untreated group.

3. Confirmation of the effect of inhibiting foam cell formation

(1) Experimental materials and methods

A mouse-derived immune cell line (Murine monocyte) was purchased from the Korea Cell Line Bank. HepG2 cells were cultured in an environment of 5% CO ₂ and 37°C using MEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (Penicilin-streptomycin).

To confirm the cytotoxicity of the analysis sample, Raw 264.7 cells were distributed in a 96-well plate at a concentration of 1x10 ⁴ cells/well and cultured for 24 hours to form a monolayer. To enable use in the test, the medium was removed from the cultured cells, and the mixed extract (3Hb) of Manchuria, Gardenia, and Gentian chinensis was treated at a concentration within 300 μg/mL. After culturing in a cell culture environment (5% CO ₂ , 37 ± 1)°C for 24 hours, WST-1 was treated and cultured for an additional 2 hours, and the absorbance was measured at 450 nm with a microplate reader. The measured absorbance was quantified relative to the untreated group, converted to percentage, and graphed.

Next, to confirm the efficacy of inhibiting foam cell formation induced by OX-LDL (Oxidized low-density lipoprotein), Raw 264.7 cells (1X 10 ⁵ cells/well) were seeded. Afterwards, the mixture was treated with 50 ng/mL OX-LDL and mixed extracts of Manchuria, Gardenia, and Gentian herb (3Hb) for 24 hours. Lipid accumulation was acquired in live cells using a Tomocube HT-1S microscope. Based on the three-dimensional refractive index distribution of cells, lipid accumulation was analyzed using Tomostudio software. Specifically, ox-LDL-treated liver cells were image-processed (tomostudio software) from the density difference to cell inclusions (fat) in red, then AI-based quantification was performed using a 3D tomolipid analysis program, followed by anova testing and post-hoc verification. hoc) Turkey test was conducted. In Figure 9B, red color represents fat.

(2) Experiment results

30ug/mL, which is within the non-toxic concentration range, of the mixed extract (3Hb) of Manchuria, Gardenia, and Gentian chinensis was treated and cultured for 24h. As a result, lipid accumulation occurred due to the treatment with OX-LDL, but lipid accumulation was inhibited in the group treated with the mixed extract of Chinese herbaceous plant, gardenia and gentian root (3Hb) (see Figure 9).

In Figure 9, the volume ratio was calculated as lipid volume/cell volume (um ³ ). Data are expressed as mean percentages relative to the untreated group ± standard deviation. *: P < 0.05 compared to untreated group.

4. Confirmation of the effect of inhibiting the expression of metabolic disease-related markers

(1) Experimental method

To verify the efficacy of 3Hb on lipid accumulation, the mRNA expression levels of PPAR-γ and CD36, which are known markers related to metabolic diseases and foam cell formation, were analyzed. Raw 264.7 cells (1X 10 ⁵ cells/well) were seeded. Afterwards, the mixture was treated with 50 ng/mL OX-LDL and 100 μg/mL of a mixed extract of Manchuria, Gardenia, and Gentian herbacea (3Hb) for 24 hours. RNA was extracted using trizol, and total RNA was extracted using chloroform and iso-propanol. cDNA was synthesized using total RNA. The PCR reaction consisted of initial denaturation at 95°C for 2 minutes, followed by amplification 30 times: denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute. Afterwards, electrophoresis was performed on a 1% agarose gel and the expression level was confirmed through UV irradiation.

(2) Experiment results

As a result of confirming the gene expression of PPAR-γ and CD36, genes related to lipid accumulation, in the immune cell line in which fat accumulation was induced by OX-LDL, the expression of PPAR-γ and CD36 was found in the immune cell line in which fat accumulation was induced by OX-LDL compared to the control group. The expression levels of each gene were significantly increased. Meanwhile, the expression levels of both genes were significantly reduced in the group treated simultaneously with OX-LDL and the mixed extract of Manchuria, Gardenia, and Gentian herb (3Hb) (see Figures 10 and 11). In Figures 10 and 11, the mRNA expression of PPAR-γ and CD36 was measured by reverse transcription PCR. Data are expressed as mean percentages relative to the untreated group ± standard deviation. *: P < 0.05 compared to untreated group. Primers used for reverse transcription PCR are shown in Table 1 below.

gene Primer sequence (5'->3') PPAR-γ Forward CAA TCC GAA TTT TTC AAG GGT GCC A (SEQ ID NO: 1) Reverse GAG CAC CTT GGC GAA CAG CTG AGA G (SEQ ID NO: 2) CD36 Forward GAG CCA TCT TTG AGC CTT CA (SEQ ID NO: 3) Reverse TCA GAT CCG AAC ACA GCG TA (SEQ ID NO: 4) GAPDH Forward GAC AAG CTT CCC GTT CTC AG (SEQ ID NO: 5) Reverse GAG TCA ACG GAT TTG GTC GT (SEQ ID NO: 6)

<110> Semyung University Division of Industry-Academy Cooperation Group <120> Pharmaceutical composition for preventing or treating metabolism diseases comprising a mixture extract of Erigeron canadensis L, gardenia fruit and Gentian <130> SP22A045 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma forward primer <400> 1 caatccgaat ttttcaaggg tgcca 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma reverse primer <400> 2 gagcaccttg gcgaacagct gagag 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD36 forward primer <400> 3 gagccatctt tgagccttca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD36 reverse primer <400> 4 tcagatccga acacagcgta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 gacaagcttc ccgttctcag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 gagtcaacgg atttggtcgt 20

Claims (10)

A pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a mixed extract of Chinese herbaceous plant, gardenia and gentian root.
The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the mixed extract is extracted by mixing 0.5 to 1.5 parts by weight of Mandarinwort, 0.5 to 1.5 parts by weight of Gardenia, and 0.5 to 1.5 parts by weight of Gentian herb.
The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein the mixed extract is obtained by mixing and extracting Chinese pepper, gardenia, and gentian root at a weight ratio of 1:1:1.
The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein the mixed extract is a mixture of Chinese herb, Gardenia, and Gentian herb and extracted with water or alcohol having 1 to 4 carbon atoms.
The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein the mixed extract is a mixture of Mandarinwort, Gardenia, and Gentian herb and extracted with water and alcohol having 1 to 4 carbon atoms.
The pharmaceutical composition for the prevention or treatment of metabolic diseases according to claim 1, wherein the mixed extract is a mixture of sagebrush, gardenia, and gentian root at a weight ratio of 1:1:1 and extracted with water.
The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the metabolic disease is at least one selected from the group consisting of obesity, diabetes, fatty liver, dyslipidemia, and arteriosclerosis.
The pharmaceutical composition for preventing or treating metabolic diseases according to claim 7, wherein the metabolic disease is at least one selected from the group consisting of fatty liver disease and arteriosclerosis.
The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
A food composition for preventing or improving metabolic diseases comprising a mixed extract of Manchuria, Gardenia and Gentian herb.