KR102025572B1 - Composition for preventing, ameliorating or treating metabolic diseases comprising mixture of Diospyros lotus leaf and grape fruit stem extract as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of metabolic diseases, which contains a mixture of the cedar leaf and grape cluster stem extract as an active ingredient, the mixture of the cedar leaf and grape cluster stem extract of the present invention Inhibition of differentiation and excellent effect of improving obesity, hyperlipidemia, arteriosclerosis and cardiovascular disease, hyperinsulinemia and fatty liver in a high fat diet animal model, and thus, contains a mixture of the extracts of the horseradish leaf and the grape cluster stem as an active ingredient The composition may be usefully used as a material for treating a metabolic disease or a nutraceutical composition for preventing or improving metabolic disease.

Description

Composition for preventing, ameliorating or treating metabolic diseases comprising mixture of Diospyros lotus leaf and grape fruit stem extract as effective component }

The present invention relates to a composition for the prevention, amelioration or treatment of metabolic diseases, which contains a mixture of the gingko biloba leaf and grape cluster stem extract as an active ingredient.

Recently, the development of metabolic diseases (Metabolic Disease, Metabolic Syndrome), including various diseases such as obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes or liver disease is increasing rapidly according to economic development and changes in eating habits. Although these diseases occur individually, they are usually closely related to each other and often accompanied by various symptoms.

Obesity is widely known to cause chronic diseases such as fatty liver, high blood pressure, diabetes, and cardiovascular disease. As of 2007, 1.7 billion people, or 25% of the world's population, are overweight (BMI> 25). One out of two children is obese, and the number of childhood obesity is increasing rapidly, which is a serious social problem. The obesity drugs sold at home and abroad are orallyat, which is the main ingredient of orlistat, which has been approved by the US FDA. However, xenical, which inhibits lipase action, has side effects such as fatty stool, gas production, and decreased absorption of fat-soluble vitamins. There is this.

Hyperlipidemia is a condition in which more fatty substances are present in the blood and accumulated on the walls of blood vessels, causing inflammation, resulting in cardiovascular disease. As lipid components such as cholesterol in the blood increase, blood flow is not smooth, and lipid components adhere to arterial walls, causing chronic inflammatory reactions and atherosclerosis, and the resulting blood clots cause cardiovascular or cerebrovascular diseases. This can cause myocardial infarction, stroke or cerebral infarction.

The arteriosclerosis is a fatty substance (plaque) containing cholesterol, phospholipids, calcium, etc. in the vascular lining to reduce the elasticity of the arteries and harden, the blood supply is inhibited or the pressure is increased due to the narrowed arteries rupture, peeling, etc. It tells you what happens.

Currently, drugs for treating hyperlipidemia or arteriosclerosis are 'statin'-based drugs with inhibitory activity against HMG-CoA reductase, which plays an important role in the synthesis of cholesterol in the liver. It is known to have side effects such as muscle toxicity.

Hyperinsulinemia is a condition associated with obesity or diabetes, such as high levels of insulin in the blood, which promotes sympathetic activity or promotes the absorption of sodium in the kidneys, and has fewer side effects and does not cause weight gain. There is an urgent need for the development of therapeutic agents that can lower the number of teeth.

The liver is an organ that plays a central role in nutrient metabolism.Any abnormalities in liver function cause problems in the nutrient metabolism of the living body, making glucose into glycogen, converting proteins into albumin, or breaking down unnecessary substances into the bile. There is an abnormality in the function of the liver.

Among them, fatty liver is a disease that causes abnormal liver accumulation, such as triglycerides in liver cells, causing liver dysfunction. It is known that the initial condition is a simple fatty liver in which only fat deposition occurs on hepatocytes, and then the condition progresses to fatty hepatitis including liver fibrosis, cirrhosis and the like.

Fatty liver is classified into alcoholic fatty liver and non-alcoholic fatty liver depending on the cause. Although alcoholic fatty liver disease progresses from early simple fatty liver to fatty hepatitis and cirrhosis, nonalcoholic fatty liver disease stays in simple fatty liver and the condition does not progress, but recently, even in non-alcoholic fatty liver disease, simple fatty liver It is reported that the condition may progress due to fatty hepatitis or cirrhosis.

Most patients with nonalcoholic fatty liver disease are accompanied by insulin resistance, obesity, diabetes and hyperlipidemia. In particular, 69-100% of nonalcoholic fatty liver patients are obese, 20-40% of obese patients are accompanied by nonalcoholic fatty liver, and in particular, the prevalence of liver disease in male obese patients is higher than that of female obese patients. To date, there are two major therapeutic agents used in these patients. The first is to treat and improve fatty liver by correcting risk factors such as obesity, insulin resistance, or hyperlipidemia, and the second is to repair damaged liver cells. Drugs that are responsible for the function of hepatoprotective, antioxidants or nutritional support, etc. are applicable. However, until now, there is no effective drug treatment for nonalcoholic fatty liver disease, but only basic treatments such as weight loss through diet therapy and exercise therapy are recommended. In particular, since non-alcoholic steatohepatitis is more likely to develop cirrhosis or hepatocellular carcinoma, more active drug treatment is essential, and oxidative stress and insulin resistance, which are considered to be important for the development and progression of non-alcoholic steatohepatitis, are particularly important. Although the treatment aimed at this has been attempted, there is a need for the development of a high-efficiency drug for non-alcoholic fatty liver disease since there is no sufficient scientific evidence for the treatment.

On the other hand, Diospyros lotus ) is a deciduous plant that grows naturally in Korea, China, and Asia. In traditional medicine, it has been used to soothe, pain, convergence and constipation. Fatty acid, sugars, flavonoids and non-volatile substances have been reported as biochemical components of horsetail trees, and recently, blood anticoagulation, brain cell protective action, antioxidant and anticancer effects have been reported.

The physiological activity effect of the fern is mainly targeted to the fruits and seeds of the fern, and the evaluation of the physiological activity on the leaves of the fern is inadequate. In addition, the fruit of the cedar is not only small compared to persimmon, but also has a lot of seeds, and can be collected in late autumn, which is not easy to use industrially, and there is a limit to procure material in large quantities. Therefore, if the bioactive activity of the gingko biloba is identified and utilized, it will be very easy to achieve industrialization by overcoming the limitations on the utilization of the gingko biloba fruit and seeds.

Meanwhile, the grape (Vitis vinifera ) is a deciduous tree belonging to the family of grapes and is one of the most grown fruits with about 30% of the world's fruit production. Grapes contain a variety of organic acids such as glucose, fructose, sucrose, citric acid, tartaric acid, acetic acid and malic acid, and vitamins A, B and C. The skin of grapes contains peonidine, delphinidine, malvidin and petunidine. Although anthocyanin-based components are well known, few studies have been conducted on the effects of grape stem stem extract on the treatment of metabolic diseases.

On the other hand, Korean Patent Publication No. 2015-0027676 discloses a composition for the prevention or treatment of metabolic syndrome-related diseases, including grape and Schisandra chinensis extract, but the mixture of the cedar leaf and grape cluster stem extract of the present invention as an active ingredient There is no disclosure of a composition for preventing, ameliorating or treating metabolic diseases.

The present invention has been made by the above-mentioned demands, and provides a composition for the prevention, improvement or treatment of metabolic diseases, which contains a mixture of gingko leaf and grape stem extract as an active ingredient, the composition is compared to a single extract The present invention was completed by inhibiting the differentiation of adipocytes and confirming that the high fat diet animal model has an effect of improving obesity, hyperlipidemia, arteriosclerosis and cardiovascular disease, hyperinsulinemia and fatty liver.

In order to solve the above problems, the present invention provides a health functional food composition for the prevention or improvement of metabolic diseases containing a mixture of the gingko leaf and grape cluster stem extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases, which contains a mixture of horseradish leaves and grape cluster stem extract as an active ingredient.

In another aspect, the present invention provides a feed additive for the prevention or improvement of metabolic diseases in animals other than humans containing a mixture of horseradish leaves and grape cluster stem extract as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of metabolic diseases, which contains a mixture of the cedar leaf and grape cluster stem extract as an active ingredient, the mixture of the cedar leaf and grape cluster stem extract of the present invention Inhibits differentiation, reduces weight gain compared to extracts alone in high fat diet animal models, reduces weight of liver and adipose tissues, and lowers total cholesterol content by reducing blood low density lipoprotein cholesterol In addition, the effect of reducing the content of triglycerides in blood was excellent. In addition, the effect of reducing blood leptin, GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyruvate transaminase) content was excellent, fat content of liver tissue and collagen fiber decreased, fat cell size of adipocytes around epididymis Was reduced, the MDA (malondialdehyde) in the liver tissue, GSH (glutathione) content is increased, the activity of SOD (superoxide dismutase) and GPx (glutathione peroxidase) was excellent.

Figure 1 shows the differentiation inhibitory effect (A) and triglyceride content (B) of the adipocytes by the extract treatment of the present invention. Adipocyte differentiation was induced by treatment with MDI (0.5 mM IBMX, 1 μM dexamethasone, and 1 μg / ml insulin), Control was negative control, DLE was treated with horseradish leaf extract, and MGFE was treated with grape stem extract. , DLE + MGFE is a group treated with a mixture of horsetail leaves and grape stem extract. *, ** means that the value of DLE + MGFE administration significantly reduced compared to DLE, MGFE, * is p <0.05, ** is p <0.01.
Figure 2 shows the total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol content in the blood of the high fat diet animal model ingesting the extract of the present invention. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group. *, **, *** means that the value of DLE + MGFE is significantly reduced compared to DLE, MGFE, * is p <0.05, ** is p <0.01, *** is p <0.001 .
Figure 3 shows the arteriosclerosis index (A) and cardiovascular risk factors (B) of a high fat diet animal model ingesting the extract of the present invention. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group. * Indicates a significant decrease in DLE + MGFE administration compared to DLE and MGFE, and * is p <0.05.
Figure 4 shows the blood insulin concentration (A) and leptin concentration (B) of a high fat diet animal model ingesting the extract of the present invention. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group. * Indicates a significant decrease in DLE + MGFE administration compared to DLE and MGFE, and * is p <0.05.
Figure 5 shows the blood GOT (A) and GPT (B) content of the high fat diet animal model ingesting the extract of the present invention. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group. * Indicates a significant decrease in DLE + MGFE administration compared to DLE and MGFE, and * is p <0.05.
Figure 6 shows the histological changes of the liver in a high fat diet animal model ingesting the extract of the present invention under a microscope. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group.
Figure 7 confirms the degree of collagen fiber production in the liver of a high fat diet animal model ingesting the extract of the present invention, A is the result observed at 100 magnification, B is the result observed at 200 magnification. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group.
Figure 8 confirms the MDA (A) and GSH concentration (B), SOD (C) and GPx activity (D) in the liver tissue of the high fat diet animal model ingested the extract of the present invention. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group. *, ** means that the value of DLE + MGFE is significantly reduced or increased compared to DLE and MGFE, * is p <0.05 and ** is p <0.01.
9 is a microscope confirming the histological changes of the adipose tissue around the epididymis in a high fat diet animal model ingesting the extract of the present invention. Control is a normal diet group, HFD is a high fat diet group, DLE is a high fat diet and horseradish leaf extract group, MGFE is a high fat diet and grape cluster stem extract group, and DLE + MGFE is a high fat diet and GCE is a high fat diet and garcinia cambogia extract administration group.

The present invention relates to a nutraceutical composition for the prevention or improvement of metabolic diseases, which contains a mixture of horseradish leaves and grape cluster stem extract as an active ingredient.

The metabolic disease refers to one or more diseases selected from the group consisting of hyperlipidemia, obesity, arteriosclerosis, cardiovascular disease, hyperinsulinemia and nonalcoholic fatty liver disease, wherein the nonalcoholic fatty liver disease is simple fatty liver, nutrient fatty liver, hunger fatty liver, Obese fatty liver, diabetic fatty liver, fatty hepatitis, liver fibrosis or cirrhosis, but is not limited thereto.

In one embodiment of the present invention, the mixture is a mixture of the horseradish leaves: grapevine stem extract in a 0.5 to 2: 0.5 to 2 weight ratio, more preferably the horseshoe leaves: grapevine stem extract 1: Although weight ratio of 1 is mixed, it is not limited to this.

In one embodiment of the present invention, the extract of the horseradish leaves and grape vine stem is preferably extracted using water, a lower alcohol of C ₁ to C ₄ or a mixture thereof as a solvent, more preferably ethanol solvent Extraction using, but is not limited to this.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately used depending on the purpose of use (prevention or improvement). In general, in the preparation of food or beverages, the nutraceutical composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the raw material. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of dietary supplement. Examples of foods to which the health functional food composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, drinks, tea Drinks, alcoholic beverages, vitamin complexes, and the like, and include all health foods in the conventional sense.

In addition, the nutraceutical composition of the present invention may be prepared as a food, in particular a functional food. Functional foods of the present invention include ingredients that are commonly added in food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates can be monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g. dextrins, cyclodextrins, etc.) or sugar alcohols (e.g. , Xylitol, sorbitol, erythritol and the like). The flavourant may be a natural flavourant (eg, taumartin, stevia extract, etc.) and a synthetic flavourant (eg, saccharin, aspartame, etc.).

Various nutritional supplements, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid The carbonation agent etc. which are used for a drink can be contained further. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic diseases, which contains a mixture of horseradish leaves and grape cluster stem extract as an active ingredient.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, which is commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals Oils and the like, but are not limited thereto. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

Suitable dosages of the pharmaceutical compositions according to the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the inhibition and treatment of metabolic diseases.

The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time, etc., and is not limited to a specific range of concentration.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a dose container. In this case, the formulation may be prepared in any one formulation selected from injections, creams, sprays, ointments, warnings, lotions, linings, pasta and cataplasma, and may further include a dispersing or stabilizing agent.

In addition, the present invention relates to a feed additive for the prevention or improvement of metabolic diseases in animals other than humans, which contains a mixture of horseradish leaves and grape cluster stem extract as an active ingredient.

The feed additive may be in the form of a high concentrate, powder or granules containing a mixture of 20-90% by weight of horseradish leaves and grape cluster stem extract.

The feed additive of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins and alpha. It may further include one or more selected from natural antioxidants such as tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.

Animal feed additives and mixtures containing the mixture of the gingko biloba leaf and grape stem extract of the present invention are amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, digestion and absorption as auxiliary components It can be used with materials such as enhancers, growth promoters, disease prevention agents and the like.

The feed additive may be administered to the animal alone in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered as a top dressing or directly mixed into animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients. Typically, single or divided daily dosages can be used as is well known in the art. When the feed additives are administered separately from the animal feed, dosage forms of the extracts, as is well known in the art, can be prepared in immediate release or sustained release formulations in combination with non-toxic pharmaceutically acceptable edible carriers. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the extract may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like.

In addition, the animal feed comprising a mixture of horseradish leaves and grape cluster stem extract as a feed additive may be any protein-containing organic cereal meal commonly used to meet the dietary needs of animals. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures. The feed additive may be used by adding to the animal feed by dipping, spraying or mixing. The veterinary composition or feed additive of the present invention can be applied to the diet of a companion animal.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples. These preparations and examples are only intended to explain the present invention more specifically, it is apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Materials and methods

1. Preparation of Extract

Cypress leaves were collected from Singi Village, Suhang-ri, Bugwi-myeon, Jinan-gun, Jeollabuk-do, and then commissioned by Professor Kim Hong-jun of the Department of Herbal Medicine, College of Oriental Medicine, Woosuk University. The identified specimens were stored in the Atokyu & A Research Institute's specimen storage room.

The prepared cedar leaves were chopped and dried at 40 ° C. for 16 hours. Dried dried cedar leaves (100 g) were added to 50% (v / v) ethanol (2,000 mL), extracted for 3 days, filtered using a 0.45 μm filter, and the filtrate was lyophilized and recovered. It was used for the experiment while storing at 20 ℃.

Grape stem stems of Muscat Bailey A variety were purchased from Baeksan-ri Hope Farm, Sintaein-eup, Jeongeup-si, Jeonbuk, Korea, and then chopped and dried at 40 ° C for 16 hours. The dried grape cluster stem (100 g) was ground and added to 80% (v / v) ethanol (2,000 ml), extracted for 3 days, filtered using a 0.45 μm filter, and the filtrate was lyophilized and recovered. It was used for the experiment while storing at 20 ℃.

2. Cell Culture and Differentiation

3T3-L1 cell lines derived from mouse embryos were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA). Culture of 3T3-L1 during the progenitor cell phase was performed with 10% (v / v) FBS, 0.15% (w / v) sodium bicarbonate and 1% (v / v) penicillin-streptomycin. The DMEM medium was used to incubate at 37 ℃ in 5% CO ₂ incubator, in order to induce adipocyte differentiation in the culture medium of the progenitor progenitor cells (MDI, 0.5 mM IBMX, 1 μM dexamethasone And 1 μg / ml insulin) were prepared and incubated for 3 days, followed by incubation for 4 days in a medium to which 1 μg / ml insulin was added.

The manufactured cedar leaf extract (100 μg / ml), grape stem extract (100 μg / ml) and mixtures thereof (50 μg / ml of cedar leaf extract and 50 μg / ml grape stem extract) are The mixtures were treated with MDI medium and insulin containing medium.

3. Oil Red  O dyeing

Differentiated 3T3-L1 adipocytes were stained with PBS for Oil Red O staining and fixed by injecting 10% (v / v) neutral formalin solution (pH 7.2) for 60 minutes at room temperature. After removing the formalin solution and washing with sterile distilled water, 60% (v / v) isopropyl alcohol (isopropyl alcohol) was added and left for 5 minutes. After that, isopropyl alcohol was completely removed, and Oil Red O solution was injected and stained for 20 minutes. After washing four times with distilled water to remove the extracellular residual Oil Red O was observed under a microscope (× 100, Olympus, Tokyo, Japan). Thereafter, 0.5 ml of 100% isopropyl alcohol was added per well to elute the intracellular dye. The eluted dye solution was measured for absorbance at 540nm using a microplate reader (Molecular devices, USA).

4. Triglyceride Analysis in 3T3-L1 Cells

Triglyceride content analysis was determined using a commercially available assay kit (Cayman Chemical., USA) according to the manufacturer's protocol.

5. Experimental animals  Breeding and Diet

The experimental animals were purchased from the Central Experimental Animal Co., Ltd., 4 weeks old C57BL / 6 male mice (18-20 g), and were adapted to the nursery environment for one week. The temperature of the kennel was maintained at 18 ~ 24 ℃ and the relative humidity was 50 ~ 60%, and the contrast was adjusted by 12 hour cycle (09: 00 ~ 21: 00).

Mice were fed the normal diet (control group), high fat diet (control group, HFD), high fat diet and horseradish leaf extract group (DLE 200mg / kg / day), high fat diet and grape stem extract group (MGFE 200mg / kg / day), a high fat diet, a mixture of cedar leaf and grape stem extract (DLE 100mg / kg / day + MGFE 100mg / kg / day), a high fat diet and garcinia cambogia extract administration group (GCE 200mg / kg / day) 6 groups of 8 animals were randomly divided for 16 weeks. Each extract was administered orally once a day at a final concentration of 200 mg / kg. D12450B (10% kcal fat) for general feed and D12492 (60% kcal fat) for high-fat feed were purchased from Central Experimental Animal. High-fat diets were fed refrigerated at 4 ° C., while drinking water and feed were free. Feed intake and body weight were measured at weekly intervals. This study was conducted in accordance with the regulations of the Animal Experiment Ethics Committee of Jeonju University.

6. Blood Collection and Storage

Blood was fasted for 12 hours at the end of the last 16 weeks of the experiment, and blood was collected from the heart after anesthesia. The collected blood was separated by centrifugation at 3,000 rpm for 15 minutes at 4 ° C, and the separated serum was used while being stored at -80 ° C until analysis.

7. Biochemical Analysis of Blood

The levels of triglyceride, total cholesterol, HDL (High Density Lipoprotein) cholesterol, and GOT-GPT in serum were analyzed using a measurement kit (Asan) .Low Density Lipoprotein (LDL) cholesterol content was measured by Friedewald et al. (1972, Clin Chem). Was calculated as in the following formula (1).

[Equation 1]

LDL Cholesterol = (Total Cholesterol-HDL Cholesterol) -Total Triglyceride / 5

In addition, the arteriosclerosis index and cardiovascular risk factors were calculated by the following equations (2) and (3).

[Equation 2]

Atherosclerosis index = (total cholesterol-HDL cholesterol) / HDL cholesterol

[Equation 3]

Cardiovascular Risk Factors = Total Cholesterol / HDL Cholesterol

8. Insulin and Blood Leptin  Concentration measurement

Serum insulin concentrations and leptin concentrations were measured using the ultra sensitive mouse insulin ELISA kit (Crysral Chem Inc., Elk Grove Village, IL, USA) and the leptin Quantikine ELISA kit, R & D systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol.

9. Weighing liver tissue, fat accumulation, collagen observation and biochemical analysis

Fasting for 12 hours in the last 16 weeks at the end of the experiment, extracting liver tissue, measuring the total weight with a precision electronic balance, and then freezing part of the liver tissue rapidly (-40 ° C.) for further experiments. Some were washed with brine to remove water, and fixed with 4% (v / v) paraformaldehyde (paraformaldehyde, pH 7.4) was produced as a paraffin block through a series of processes.

The paraffin block was cut to a thickness of 5 μm and subjected to deparaffin and water hydration. Fat accumulation of liver tissue was stained with H & E and observed under a microscope (× 200, Olympus, Tokyo, Japan). Stained with a trichrome staining kit (Abcam, Inc., Cambridge, UK) to confirm collagen fiber production was observed under a microscope (× 100, × 200).

Lipid peroxidation in liver tissue was measured according to the manufacturer's protocol using the MDA ELISA kit (Cell Biolabs., USA), and liver GSH (glutathione) levels were measured using the GSH assay kit (United States Biological., USA). Measured according to the manufacturer's protocol, liver superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity was measured using a commercially available assay kit (Cayman Chemical., USA) according to the manufacturer's protocol.

10. Observation of weight and fat size around the epididymis

At the end of the experiment, the adipose tissue around the epididymis was extracted and weighed with a precision electronic balance. After washing with saline, the water was removed, and then fixed with 4% (v / v) paraformaldehyde (pH 7.4). The paraffin block was produced through the process. The paraffin block tissue was cut to a thickness of 5 μm, and the cut tissue sections were treated with deparaffin (deparaffin) and hydrated, and stained with H & E to confirm the size of fat under a microscope (× 100, Olympus, Tokyo, Japan). .

Example  1. Inhibitory Effect of Adipocyte Differentiation by Mixtures of Extracts of Grape Leaf and Grape Stem

Adipocyte differentiation inhibitory effect according to the treatment of the mixture of the horseradish leaf and grape cluster stem extract of the present invention was confirmed using 3T3-L1, a mouse fat precursor cell. As a result, as shown in FIG. 1, it was confirmed that lipid production was inhibited and the content of triglycerides was reduced when adipocyte differentiation was performed by the treatment of the leaves of the horseradish, the extract of the grape vine or the mixture thereof. Inhibition of differentiation of adipocytes was excellent.

Example  2. Weight Gain and Tissue Weight Measurement by the Mixture of Cucurbita Leaves and Grape Stem Extracts in a High Fat Dietary Animal Model

It was confirmed that the effect of the mixture of horseradish leaves and grape cluster stem extract of the present invention on the weight gain and tissue by the high fat diet. As a result, as shown in Table 1, it was confirmed that the body weight of the mouse was increased by the high fat diet, and the increase in body weight was decreased by the administration of the horseradish leaf extract, the grape cluster stem extract, or a mixture thereof. In particular, the effect of reducing the weight gain was the best when the mixture of the extract of the horseradish leaves and the grape cluster stems.

The weights of liver tissue and adipocytes around the epididymis were also decreased by the administration of the extract, and the most effective effect was the administration of the mixture of the extract of horseradish leaves and grape vine stems.

group Weight (g) Weight (g) Liver weight (g) Epididymal fat mass (g) Before the experiment After the experiment Control 21.58 ± 1.58 ^a 28.32 ± 2.78 ^c 6.74 ± 2.46 ^c 0.97 ± 0.14 ^c 1.20 ± 0.20 ^c HFD 20.65 ± 0.52 ^a 48.78 ± 1.36 ^a 28.13 ± 1.67 ^a 2.12 ± 0.31 ^a 2.39 ± 0.34 ^a HFD + DLE 21.76 ± 2.04 ^a 46.22 ± 1.69 ^b 24.46 ± 1.09 ^b 1.56 ± 0.26 ^b 1.87 ± 0.17 ^b HFD + MGFE 21.96 ± 1.49 ^a 45.94 ± 0.96 ^b 23.98 ± 0.92 ^b 1.62 ± 0.23 ^b 1.89 ± 0.21 ^b HFD + DLE + MGFE 21.86 ± 0.65 ^a 44.16 ± 1.78 ^c 21.80 ± 1.18 ^c 1.44 ± 0.23 ^b 1.85 ± 0.21 ^b HFD + GCE 21.72 ± 1.14 ^a 45.87 ± 1.40 ^b 24.15 ± 1.58 ^b 1.51 ± 0.27 ^b 1.99 ± 0.14 ^b

The letters a to c in the column mean that there is a significant difference from each other, p <0.05.

Example 3. High Fat  Changes of Blood Cholesterol and Triglyceride by Dietary Mixture of Cucurbita Leaves and Grape Stem Extracts in Dietary Animal Models

The effect of the mixture of horseradish leaf and grape stem extract of the present invention on the content of blood cholesterol and triglycerides in a high fat diet animal model was confirmed. As a result, the content of total cholesterol and triglycerides increased by high fat diet was decreased by the administration of horseradish leaf extract, grape stem extract or mixtures thereof, and the reduction effect by the administration of the mixture was the best (FIG. 2A, B).

In addition, the content of HDL cholesterol in the total cholesterol did not change significantly, but the content of LDL cholesterol was reduced by the administration of horseradish leaf extract, grape vine stem extract, or mixtures thereof. 2C, D).

The atherosclerosis index and cardiovascular risk factors calculated by Eqs. 2 and 3, using the measured total cholesterol, triglyceride, and HDL cholesterol levels, were also reduced by the administration of cedar leaf extract, grape stem extract, or mixtures thereof. , Showed a significant reduction effect upon administration of the mixture (FIG. 3).

Example  4. Insulin Serum Levels by Administration of Mixture of Leafy Grape Leaf and Grape Stem Extract in High Fat Dietary Animal Model Leptin  Change in content

The effect of the mixture of horseradish leaf and grape stem extract of the present invention on the blood insulin and leptin content of the high fat diet animal model was confirmed.

Blood insulin content was increased by high fat diet and showed a tendency to decrease by the administration of horseradish leaf extract, grape vine stem extract or mixtures thereof (FIG. 4A).

In addition, the content of leptin in blood was also increased by high-fat diet, and it showed a tendency to decrease by the administration of cedar leaf extract, grape stem extract or a mixture thereof. 4B).

Example 5 High Fat  Changes in Indicators of Improving Blood Liver Function by Mixtures of Cucurbita Leaves and Grape Stem Extracts in Dietary Animal Models

Blood GOT and GPT contents were analyzed to determine the effect of the mixture of the horseradish leaves and grape cluster stem extracts of the present invention on the improvement of liver function in a high fat diet animal model.

As a result, GOT and GPT increased by high-fat diet were decreased by the administration of cedar leaf extract, grape vine stem extract, or mixtures thereof. It was confirmed that the most effective in improving the liver function (Fig. 5).

Example 6 High Fat  Pathological Analysis of Liver Tissue by Mixture of Cucurbita Leaves and Grape Stem Extracts in Dietary Animal Models

The effect of high fat diet and extract administration on liver tissue was analyzed pathologically. As a result of H & E staining of the extracted liver tissue at 200-fold magnification, it was confirmed that the increased fat content in the liver tissue was reduced by administration of horseradish leaf extract, grape vine stem extract or a mixture thereof by high fat diet. When administered, the effect of reducing fat content was the best, showing a form similar to that of the general diet group (FIG. 6).

In addition, as a result of confirming the expression of collagen fibers affecting the liver fibrosis, the expression of collagen fibers increased by high-fat diet decreased when administration of horseradish leaf extract, grape vine stem extract or a mixture thereof, and collagen fibers when the mixture was administered Expression reduction effect was the best (Fig. 7).

Example 7 High Fat  Analysis of Biochemical Changes in Liver Tissue by Mixtures of Cucurbita Leaves and Grape Stem Extracts in Dietary Animal Models

MDA concentration was measured to determine the peroxidation of liver. MDA concentrations increased by high-fat diet decreased with the administration of cedar leaf extract, grape vine stem extract, or mixtures thereof, and showed the best effect of reducing the oxidative damage of liver tissue. 8A).

In addition, the GSH concentration decreased by the high fat diet was increased when administration of the horseradish leaf extract, grape cluster stem extract or a mixture thereof, showing the best increase effect when the mixture administration (Fig. 8B).

In addition, analysis of liver antioxidant enzymes SOD and GPx activity showed that the enzyme activity decreased by high-fat diet was increased by the administration of horseradish leaf extract, grape vine stem extract or a mixture thereof. (FIG. 8C, D).

Example 8 High Fat  Analysis of Changes in Adipose Tissues around Epididymis by Mixtures of Cucumber Leaf and Grape Stem Extract in Dietary Animal Model

The effect of high fat diet and extract administration on adipose tissue around epididymis was confirmed. As a result, the size of the adipocytes in the adipose tissue enlarged by the high fat diet was reduced when the cedar leaf extract, grape vine stem extract or a mixture thereof was administered, and the reduction effect was the best when the mixture was administered (FIG. 9).

Claims (7)

For the prevention or improvement of hyperlipidemia, obesity, arteriosclerosis, or nonalcoholic fatty liver disease, which contains a mixture of the gingko biloba leaf and grape stalk ethanol extract in a 1: 1 weight ratio. Health food composition. delete The hyperlipidemia of claim 1, wherein the nonalcoholic fatty liver disease is any one selected from the group consisting of simple fatty liver, nutrient fatty liver, hunger fatty liver, obese fatty liver, diabetic fatty liver, fatty hepatitis, liver fibrosis and liver cirrhosis. Health functional food composition for the prevention or improvement of obesity, arteriosclerosis or non-alcoholic fatty liver disease. delete delete Cypress Leaf: For the prevention or treatment of hyperlipidemia, obesity, arteriosclerosis or nonalcoholic fatty liver disease, which contains a mixture of cypress leaves and grape stem ethanol extracts mixed with 1: 1 weight ratio of vine stem ethanol extract Pharmaceutical composition. Cholesterol leaf: Hyperlipidemia, obesity, arteriosclerosis, or nonalcoholic fatty liver disease in animals other than humans, which contains a mixture of cypress leaves and grape stem ethanol extracts in a 1: 1 weight ratio Feed additives for the prevention or improvement of

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