KR20200070615A - A hyperlipemia improvement health functional food composition containing hot water extract of Ulmus macrocarpa Hance as an active ingredient and its preparing method - Google Patents

Abstract

The present invention relates to a hyperlipidemia alleviation-health functional food composition containing a hot water extract of Ulmus macrocarpa Hance as an active ingredient and a manufacturing method thereof and, specifically to an Ulmus macrocarpa Hance health functional food composition manufactured by enzymatically treating an Ulmus macrocarpa Hance extract extracted with hot water and freeze-drying the extract to form a powder. Therefore, there is no cytotoxicity by using a natural hot water extract. The present invention can regulate the ability to inhibit fat accumulation and the expression of genes related to triglyceride synthesis and degradation in hepatocytes, can be involved in regulating the concentration of total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in the blood, and can regulate the expression of proteins related to the synthesis and degradation of total cholesterol and triglyceride in liver tissues.

Description

A hyperlipemia improvement health functional food composition containing hot water extract of Ulmus macrocarpa Hance as an active ingredient and its preparing method

The present invention relates to a health functional food composition and a method for improving hyperlipidemia comprising a Yolk elm hot water extract as an active ingredient, and in detail, it is prepared by enzymatically treating, freeze-drying, and pulverizing the hot water extracted Yolk elm extract. In addition, the present invention relates to a hyperlipidemia-improving health functional food composition and a method of manufacturing it, comprising as an active ingredient an elm tree hydrothermal extract having functions such as suppressing fat accumulation, regulating fat synthesis and degradation-related gene expression, and controlling cholesterol concentration in liver tissue. .

The Ulm macrocarpa Hance grows at the foot of the mountain and develops cork on small branches and fine hairs when young. The leaves are misaligned, and the egg is upside-down, wide or long oval, and the tip is suddenly pointed. It is 3.5-15.5cm long and 2-9cm long. Both sides of the leaves are rough, serrated on the edges, and petioles are 3-6mm long and hairy.

The flowers bloom in May, gathered in the inflorescence, and the fruit is elongated in an egg upside-down shape and is 2~3cm long and ripens in late May. It can be used for feng shui, vehicle materials, furniture materials, flowers can be used as wheat plants, and young leaves, bark and fruit can be consumed. It has the effect of insecticidal, 消積 (droplet), 蟲積腹痛 (allergic abdominal pain), 腸風痔漏 (jangpungchiru), 小兒疳瀉 (children's appreciation-malnutrition, lower anemia of infants caused by parasites (hari)) , 冷痢 (cool), 疥癬 (improvement), 惡瘡 (sound) is known to treat the function.

Hyperlipidemia is a condition in which more cholesterol and fat-based substances are present in the blood and accumulate on the walls of blood vessels, causing inflammation and consequently cardiovascular diseases. If it persists, there is a problem that there is a high possibility of deterioration due to cardiovascular diseases such as arteriosclerosis and stroke resulting in myocardial infarction angina.

Although there are many types of lipids, cholesterol is often increased more than normal, and this condition is called hypercholesterolemia, which is not only an important cause of atherosclerosis or ischemic heart disease, but also a cause of other diseases. Cholesterol must be properly in our body to make the cells that make up our body and make some hormones, but too much cholesterol sticks to blood vessels to block blood vessels and can be the culprit of fatal diseases such as myocardial infarction or cerebral infarction due to blood clots. have.

'Republic of Korea Patent Publication No. 10-2014-0145666' discloses a composition for the prevention or treatment of obesity comprising a natural complex of brown algae, lactose and low molecular alginic acid as active ingredients, but synthesis of total cholesterol and triglyceride in liver tissue , There is no disclosure about the regulation of the expression and synthesis of proteins related to decomposition, so there is a problem that it does not clearly show the effect as a practical health functional food for prevention or treatment of obesity.

(Patent Document 1) KR 10-2014-01456766 A (Patent Document 2) KR 10-1492196 B1

In order to solve the above problems, an object of the present invention is to provide a health functional food composition for improving hyperlipidemia, which includes an elm hydrothermal extract as an active ingredient.

In addition, an object of the present invention is to provide a method for preparing a health functional food composition for improving hyperlipidemia, which includes a Yoshino elm hot water extract as an active ingredient.

In addition, an object of the present invention is to provide a health functional food for improving hyperlipidemia, which includes a hot elm tree extract as an active ingredient.

In order to solve the above object, the present invention,

Provided is a health functional food composition for improving hyperlipidemia, which includes the elm hydrothermal extract as an active ingredient.

The elm hot water extract is extracted by mixing the dried elm and purified water in a weight ratio of 1: 5 to 1: 10.

In addition, the hot water extract of elm is extracted at 75 to 98 ℃.

The present invention to solve the above other object,

In the elm extract,

Drying the elm tree at room temperature to chop;

Injecting the shredded elm and purified water into the extractor:

Heating and extracting the shredded elm in the extractor for 6 hours to generate an extract;

Filtering the extract solution to inactivate the enzyme for 2 hours and then inactivating it for 10 minutes to generate an enzyme-treated solution;

Filtering the enzyme-treated liquid and concentrating it in a vacuum reduced pressure condition to produce a concentrated liquid;

Sterilizing the concentrate for 30 minutes and then freeze-drying; And

Characterized in that it comprises the step of pulverizing the lyophilized concentrate,

Provides a method for manufacturing a health functional food composition for improving hyperlipidemia, which includes Yoshino elm hot water extract as an active ingredient.

The extracted liquid is extracted by mixing the chopped elm and purified water in a weight ratio of 1: 5 to 1: 10.

In addition, the extract is extracted at 75 to 98°C.

The enzyme is one or more selected from the group consisting of starch degrading enzyme and pectin degrading enzyme.

The present invention to solve the above another object,

Provides a health functional food for improving hyperlipidemia, which includes Yoshino elm hot water extract as an active ingredient.

The present invention provides a health functional food composition and a method for improving hyperlipidemia using a hot elm tree extract as an active ingredient, and has no cytotoxicity using natural hot water extract, and inhibits fat accumulation in hepatocytes and synthesizes and decomposes triglycerides. It can regulate the expression of genes, can be involved in the regulation of total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, and can control the expression of total cholesterol and triglyceride synthesis and degradation-related proteins in liver tissue. It works.

Figure 1 is a graph showing the cell viability according to the concentration of the elm hydrothermal extract according to an embodiment and a comparative example.
2 is a photomicrograph showing the degree of fat accumulation inhibition in HepG2 hepatocytes according to one embodiment and a comparative example.
Figure 3a is a graph showing a comparison of the expression level of Acetyl-CoA Carboxylase1 (ACC1), a gene related to triglyceride synthesis in HepG2 hepatocytes according to an embodiment and a comparative example.
Figure 3b is a graph showing the comparison of the expression level of SREBP1c, a gene related to triglyceride synthesis in HepG2 hepatocytes according to one embodiment and a comparative example.
Figure 3c is a graph showing a comparison of the expression level of the cholesterol biosynthesis related gene HMG-reducatase (HMGCR) in HepG2 hepatocytes according to one embodiment and a comparative example.
Figure 3d is a graph showing the comparison of the expression level of AMPK, a gene related to triglyceride synthesis in HepG2 hepatocytes according to one embodiment and a comparative example.
4 is a graph comparing the concentrations of AST and ALT, which are hepatotoxicity indicators in blood, of rats according to one embodiment and a comparative example.
Figure 5 is a graph showing the comparison of the content of total cholesterol, neutral lipids, LDL-cholesterol, and HDL-cholesterol in blood serum of rats according to one embodiment and a comparative example.
6 is a Western blot band showing the expression level of triglyceride synthesis and degradation-related proteins in liver tissue of rats according to one embodiment and a comparative example.
7 is a graph showing a comparison of expression levels of triglyceride synthesis and degradation-related proteins in liver tissue of rats according to one embodiment and a comparative example.

Hereinafter, the present invention will be described in more detail.

In the present specification, when a part is to “include” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.

In the present invention, the term "active ingredient" means to include the main ingredient as a substance or group of substances expected to directly or indirectly express the efficacy and effect of the health functional food.

In the present invention, the term "improvement" refers to all actions in which symptoms of hyperlipidemia are improved through taking the above-mentioned health functional food composition.

In the present invention, the term "health functional food" refers to a food product manufactured and processed using ingredients or ingredients having useful functionality for the human body according to Act 6727 on the Health Functional Food, and is referred to as "functional" Refers to ingesting for the purpose of obtaining useful effects for health purposes such as adjusting nutrients or physiological effects on the structure and function of the human body. Various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and neutralizers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids It may contain thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like, and may contain flesh for the preparation of natural fruit juice, fruit juice beverage, and vegetable beverage. These ingredients can be used independently or in combination.

According to an aspect of the present invention, there is provided a health functional food composition for improving hyperlipidemia, comprising a Yoshino elm hot water extract as an active ingredient.

Hyperlipidemia is a disease in which many cholesterol and fat components are present in the blood and accumulate on the walls of blood vessels, causing inflammation and consequently cardiovascular disease. This abnormal blood lipid state is also defined as dyslipidemia. Strictly, hyperlipidemia refers to a condition in which cholesterol and triglycerides are increased in the blood, and dyslipidemia includes a condition in which high-density lipoprotein cholesterol (HDL cholesterol) is reduced as well as an increase in lipids such as cholesterol and triglycerides.

The fats in the blood can be divided into four types: total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides.

Low-density lipoprotein cholesterol (LDL cholesterol) is a bad cholesterol that induces arteriosclerosis, which causes cardiovascular and cerebrovascular diseases by accumulating on the walls of blood vessels, and high-density lipoprotein cholesterol (HDL cholesterol) serves to transport cholesterol accumulated in blood vessel walls to the liver. Cholesterol contained in high-density lipoprotein is known as good cholesterol because it has the effect of preventing atherosclerosis. Total cholesterol is a name that combines low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, and triglycerides are produced to store excess energy consumed as food, and are normally stored in fat cells and then released when needed. The fat used.

The elm hot water extract is extracted by mixing the dried elm powder and purified water in a weight ratio of 1: 5 to 1: 10. When the weight ratio of dried elm powder and purified water is less than 1: 5, extraction is not easy, and when it exceeds 1: 10, the yield may be lowered.

In addition, the hot water extract of elm is extracted at 75 to 98 ℃. If the hot water extraction temperature is less than 75°C, the yield is lowered, and if it is higher than 98°C, the efficiency may be lowered.

Catechin, one of the active ingredients of elm, is water-soluble and shows a better extraction effect from hot water extraction than ethanol extraction, and it is a raw material used for food. It is 1: 5 or 1: 10. As the price is less competitive, processing by hot water extraction can take advantage of efficiency and economics.

According to another aspect of the present invention, in the elm extract, drying the elm at room temperature to chop and powder; Injecting the shredded elm and purified water in an extractor: heating and extracting the shredded elm in the extractor for 6 hours to generate an extract; Filtering the extract solution to inactivate the enzyme for 2 hours and then inactivating it for 10 minutes to generate an enzyme-treated solution; Filtering the enzyme-treated liquid and concentrating it in a vacuum reduced pressure condition to produce a concentrated liquid; Sterilizing the concentrate for 30 minutes and then freeze-drying; And characterized in that it comprises the step of pulverizing the lyophilized concentrate, provides a method for producing a hyperlipidemia improved health functional food composition comprising a Yoshino elm hot water extract as an active ingredient.

The extract is mixed with finely divided elm and purified water in a weight ratio of 1: 5 to 1: 10 to extract. In addition, the extract is extracted at 75 to 98°C.

The enzyme is one or more selected from the group consisting of starch degrading enzyme and pectin degrading enzyme. The starch degrading enzyme is typically an amylase, and may preferably be at least one selected from the group consisting of α-amylase, β-amylase, β-gluconase, isoamylase, glucoamylase, and oligosaccharide-producing amylase. Pectinase is one of the enzymes that hydrolyze glycosidic bonds and is distributed in microorganisms such as bacteria, fungi and yeast, higher plants, and reptiles. Preferably, it may be one or more selected from the group consisting of pectinase, polygalacturonase, and pectin esterase, which are enzymes involved in the degradation of pectin.

In the case of lyophilization after general hot water extraction, the drying process is carried out in the most concentrated state in consideration of the manufacturing cost and workability, and this is mainly done near 25brix to 30brix. In the case of Yom Elm, it is difficult to concentrate the concentration of the concentrate more than 5 brix because it is a mucilage (combination of starch and pectin), and it is difficult to concentrate the concentration of the concentrate over 5 brix. It is sold in a simple powder or extract form, rather than concentrated and processed form. For this reason, by decomposing and concentrating the viscous material using an enzyme, it is possible to concentrate the concentration of the concentrate more than 20 brix, so that the drying cost can be lowered more than three times, and the efficiency of concentration and drying efficiency can be increased.

According to another aspect of the present invention, to provide a health functional food for improving hyperlipidemia, comprising a Yolk elm hot water extract as an active ingredient.

Health functional food is a food prepared by using ingredients or ingredients (functional ingredients below) that are useful for the human body or nutrients that are prone to deficiency in everyday meals. It refers to products made with these functional ingredients by recognizing the functional raw materials by evaluating the scientific basis such as animal tests and human application tests by the Ministry of Food and Drug Safety.

It can be said to be a product that focuses on the bio-regulatory functions that help maintain and promote health. It is a product made through a certain procedure according to the regulations on health functional food, and has the phrase'health functional food' or a certification mark. In this regard, the names such as'health food','natural food', and'natural food' can be considered different from'health functional food'.

Hereinafter, examples will be described in detail to specifically describe the present specification. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not interpreted to be limited to the embodiments described below. The embodiments of the present specification are provided to more fully describe the present specification to those skilled in the art.

<Example>

Example 1. Preparation of Yolk Elm Extract

After drying naturally, the Cortex Ulmus macrocarpa Hance was finely chopped, and a certain amount of the powdered Elm was measured using an electronic scale (CAS). After that, the purified water was added to the extractor according to the mixing ratio (10 times the weight of the raw material), and then the extraction temperature was set to 95°C and heated/extracted for 6 hours while circulating the purified water. The extract was filtered, transferred to an enzyme reactor, and then enzyme (0.4% Viscozyme L, Novozymes, Denmark) was added according to the mixing ratio, followed by enzymatic reaction for 2 hours and inactivation at 100°C for 10 minutes. The enzyme-treated liquid was filtered using a 0.1 μm mesh filter, concentrated under vacuum reduced pressure, and sterilized at 90° C. or higher for 30 minutes. After the sterilization liquid was powdered with a freeze dryer, the dried block was powdered using a grinder. It was angry.

Example 2. Cytotoxicity check of elm extract

In order to measure the cytotoxicity of the elm extract prepared according to the embodiment, an MTT assay system was used. The cells used HepG2 (human hepatocellular carcinoma cell line, American Type Culture Collection, Manassas, VA.USA) hepatocytes, and the cells were 10% fetal bovine serum (FBS), 100 units/ml in DMEM (Dulbecco's modified Eagle medium). Penicillin and streptomycin (GIBCO BRL, Eggenstein, Germany) were added and cultured at 37° C. and 5% CO ₂ conditions. First, HepG2 hepatocytes were planted in a 96-well plate (SPL life science inc., Pocheon, Korea) at a concentration of 1×10 ⁵ cells/well, and cultured for 1 day. 25, 50, 100, 200 μg/ml). This was incubated for 2 days at 37°C, and 20 µl of the MTT reaction solution was added to each well, followed by reaction for 4 hours. After 4 hours, the supernatant was removed and 150 μl of dimethyl sulfoxide (DMSO) was added to each well, and absorbance was measured at 570 nm using an enzyme immunoassay reader.

Example 3. Confirmation of fat accumulation inhibitory ability of elm extract

Oil red O staining was used to measure the ability to inhibit fat accumulation in hepatocytes of the elm extract prepared according to the Examples. More specifically, HepG2 hepatocytes were cultured in a CO ₂ incubator at 37° C. and 5% so as to be 80% saturated in a 35 mm culture dish, and then treated with DMEM medium without FBS in saturated cells for 1 day. It was further cultured. Subsequently, it was exchanged with a medium containing oleic acid (OA) (0.5% FBS, DMEM containing 600 μM OA), and the concentration of the elm extract prepared according to the above example (25, 50, 100 μg/ml) After treatment with a medium containing OA, the cells were cultured for 1 day to achieve fat accumulation. The cells were washed with a phosphate buffered saline (PBS), then treated with 10% formalin for 20 minutes. After the formalin was completely removed, rinsed with 60% isopropanol, oil red O solution was added and stained at room temperature for 10 minutes. In this case, the oil red O solution was prepared by dissolving 0.35 g oil red O powder (SIGMA) in 100 ml of isopropanol, diluting it in distilled water at a ratio of 6:4, and then filtering. After staining, the oil red O solution was completely removed, and then washed 4 times with distilled water to check the staining state of the cells under a microscope.

Example 4. Triglyceride synthesis and degradation of genes related to elm extract

Measuring the expression control of AMPK genes involved in triglyceride synthesis genes Acetyl-CoA Carboxylase1 (ACC1), SREBP1c, HMG-reducatase (HMGCR) and triglyceride in hepatocytes of the elm extract prepared according to the Examples For this, it was measured using real time PCR analysis. After treating OA and elm extract HepG2 hepatocytes in the same manner as in Example 3, total RNA was separated using a TRIzol (Invitrogen, USA) solution. Total RNA amount was measured by absorbance at 260 nm and 280 nm using a UV spectrophotometer. Total RNA of 1 μg was reverse transcribed into cDNA using SuperScript II (Invitrogen) reverse transcriptase and oligo dT(15) (Promega, USA). The primers of each enzyme are shown in Table 1 below.

ACC1 forward primer 5'-GGATCCGGCGCCTTACTT-3' reverse primer 5'-CTCCGATCCACCTCATAGTTGAC-3' SREBP1c forward primer 5'-GCTGTCCACAAAAGCAAATCTCT-3' reverse primer 5'-GTCAGTGTGTCCTCCACCTCAGT-3' HMGCR forward primer 5'-TTCTGACAATAACACGATGCATAGC-3' reverse primer 5'-AAGGCCAGCAATACCCAAAA-3' AMPK forward primer 5'-TCTCAGGAGGAGAGCTATTTGATTATATC-3' reverse primer 5'-TGGAACAGACGCCGACTTTC-3' GAPDH forward primer 5'-CTGCCCCCTCTGCTGATG-3' reverse primer 5'-AGGAGGCARTGCTGATGATCTT-3'

For real-time PCR conditions, 5 ng cDNA was amplified in StepOnePlus real-time PCR system (Applied Biosystems) with 0.33 μM forward and reverse primers of each gene using SYBR Green PCR Master Mix (Applied Biosystems). The analysis was performed using StepOne Software v2.3 (Applied Biosystems). The amount of each gene expression was corrected by the amount of GAPDH, a housekeeping gene.

Example 5. Animal experiment

5-1. Experiment group design

5 weeks old 120-150g male rats (Central Laboratory Animals) were purchased and purified for 7 days to conduct animal experiments. The rats were kept in a constant environment of 24 ± 1℃ constant temperature, 50% humidity, and 12 hours photoperiod, and the elm extract prepared according to the example was administered, and a high cholesterol diet was induced to induce hyperlipidemia (#D12336, Research Diets) solid feed was orally administered. The experimental group was designed as shown in Table 2 below, and the feed and drinking water were freely consumed for 6 weeks during the experiment period.

Classification Test substance Treatment concentration
(mg/kg) Experiment
Number of animals (n) Dietary Test group U25* Elm
Hot water extract 25 10 Hyperlipidemia-inducing diet U50 50 10 U100 100 10

5-2. Blood analysis

In order to confirm the effect of the elm extract prepared according to the example on the changes in the components of the blood of the rat, after fasting for 12 hours after the experiment, anesthesia with diethyl ether, and then opened to collect blood from the abdominal vein Thereafter, plasma was separated. The isolated plasma was analyzed using a blood biochemical analyzer such as aminotransferase (AST), alanine aminotransferase (ALT), triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-cholesterol) and low density lipoprotein cholesterol (low density). Blood levels of lipoprotein cholesterol (LDL-cholesterol) were measured, respectively.

5-3. Expression and activity of triglyceride synthesis and degradation-related proteins in liver tissue

To confirm the effect of the elm extract prepared according to the examples on the expression and activity of triglyceride synthesis and degradation-related proteins in the liver tissue of rats, it was analyzed using a Western blot method. After the end of the experiment, the liver tissue was removed by autopsy, and finely ground to remove protein elution buffer (50 mM TrisHCl, pH 7.5, 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 80 μg/ml leupeptin, 3 mM NaF and 1 mM DTT) was added and treated at 4° C. for 30 minutes. After electrophoresis of 10 μg of liver tissue eluate on a 10% TrisHCl gel, the protein was electrically transferred to a nitrocellulose membrane. Then, 10% skim milk is pretreated on a nitrocellulose membrane and primary antibodies against the target protein (anti-p-AMPK, anti-AMPK, anti-p-ACC, anti-ACC, anti-SREBP1, anti- After treatment with HMGCR, anti-beta-Actin), and secondary antibody treatment, the target protein was detected using a chemiluminescent ECL kit (Amersham Pharmacia Biotech). The western blot band was quantified using an image analyzer (ThermoFisher Scientific).

Comparative Example 1. Confirmation of cytotoxicity of elm extract

The experiment was conducted in the same manner as in Example 2, and at this time, in the step of incubating for 2 days after treating with the medium containing the elm extract by concentration, distilled water of the same amount as the dose of the elm extract was put in the medium. Cultured for 2 days (normal control). Subsequently, through the same procedure as in Example 3, toxicity was confirmed in HepG2 hepatocytes.

Comparative Example 2. Confirmation of the ability to inhibit fat accumulation of elm extract

The experiment was carried out in the same manner as in Example 3, wherein the elm extract was treated with a medium containing OA for each concentration and then incubated for 1 day to become fat accumulation, and the same amount as the dose of the elm extract After distilled water was added and treated with a medium containing OA, the cells were cultured for 1 day to accumulate fat (positive control). In addition, a normal control group in which the medium containing OA was not exchanged, that is, was not fat-accumulated in hepatocytes, was prepared, and then the same procedure as in Example 3 was performed to confirm the ability to inhibit fat accumulation in HepG2 hepatocytes.

Comparative Example 3. Triglyceride synthesis and degradation of genes related to the elm extract

The experiment was conducted in the same manner as in Example 4, wherein the positive control treated only with hepatocyte OA and the normal control not treated with liver cells were used as comparative examples. Acetyl-CoA Carboxylase1 (ACC1), SREBP1c, HMG-reducatase (HMGCR) and expression regulation of AMPK genes involved in triglyceride degradation were compared and measured.

Comparative Example 4. Animal experiment

4-1. Control design

5 weeks old 120-150g male rats (Central Laboratory Animals) were purchased and purified for 7 days to conduct animal experiments. Rats were kept in a constant environment of 24±1℃ constant temperature, 50% humidity, and 12 hours photoperiod, and each control substance was administered, and a high cholesterol diet (#D12336, Research Diets) was used as a solid feed to induce hyperlipidemia. Was added. The normal control group was used as a basic comparative example by introducing a basic diet rather than a hyperlipidemia-inducing diet. The control group was designed as shown in Table 3 below, and the feed and drinking water were freely consumed for 6 weeks during the experiment period.

Classification Test substance Treatment concentration
(mg/kg) Experiment
Number of animals (n) Dietary Control Normal (N) - - 10 Basic diet Control (C) - - 10 Hyperlipidemia-inducing diet positivity
Control ATO Atorvastatin 1.023 10 Hyperlipidemia-inducing diet O3 Omega 3 170 10 Hyperlipidemia-inducing diet

4-2. Blood analysis

AST in the blood of the controls according to Comparative Example 4-1 (Normal, N), positive control (Control, C), positive control (Atorvastatin, ATO), and another positive control (Omega 3, O3)) Blood levels of (aminotransferase), ALT (alanine aminotransferase), triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-cholesterol) and low density lipoprotein cholesterol (LDL-cholesterol) In order to measure each, blood components were analyzed in the same manner as in Example 5-2.

4-3. Expression and activity of triglyceride synthesis and degradation-related proteins in liver tissue

The controls according to Comparative Example 4-1 (Normal, N), positive control (Control, C), positive control (Atorvastatin, ATO), and another positive control (O3) were neutral in the liver tissue of the rat. Western blot method was performed in the same manner as in Example 5-3 to confirm the effect on the expression and activity of the protein related to fat synthesis and degradation.

<Evaluation and Results>

Results 1. Preparation of Elm Extract

According to Example 1, 250 μg, 500 μg, 1 mg, and 2 mg of the powdered elm were weighed and dissolved in 10 ml of distilled water, respectively, and then filtered through a 0.2 μm filter to 25 μg. /ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml, respectively.

Results 2. Cytotoxicity evaluation of elm extract

In order to confirm the cytotoxicity by concentration of elm extract according to Examples and Comparative Examples, the MTT Assay System was used, and when the elm extracts were treated, the degree of change in cell proliferation was calculated as a percentage compared to the comparative example. Fig. 1 shows the result.

As a result, it was shown that even if the concentration of the elm extract was increased to 25, 50, 100, or 200 μg/ml, the cell viability was not affected even when compared with the comparative example in which no treatment was performed. Therefore, within the concentration, it was confirmed that the elm extract did not show any toxicity to the cells and did not affect the cell viability. This suggests that the elm hot water extract is harmless to the human body and can be used as a health functional food without an upper limit of its concentration.

Result 3. Analysis of fat accumulation inhibitory ability of elm extract

Oil Red O staining was used to measure and compare the ability to inhibit fat accumulation in hepatocytes of the elm extract prepared according to Examples and Comparative Examples, and the results are shown in FIG. 2. The staining state of the cells was confirmed under a microscope, and as the fat accumulation increased, the degree of oil red O staining increased.

As a result, fat accumulation did not occur in the hepatocytes (normal control) of the comparative example without any treatment, and significant fat accumulation was observed in the comparative example (positive control) with no treatment after fat accumulation. In addition, it was confirmed that fat accumulation was suppressed in a concentration-dependent manner in cells treated with the elm extract according to the embodiment.

Results 4. Expression of genes related to triglyceride synthesis and degradation of elm extract

According to Examples and Comparative Examples, the expression regulation of AMPK genes involved in the triglyceride synthesis related genes Acetyl-CoA Carboxylase1 (ACC1), SREBP1c, HMG-reducatase (HMGCR) and triglyceride was compared and measured. 3 to 6 are shown.

ACC1 is an enzyme involved in the synthesis of malonyl-CoA from acetyl-CoA and is known as a rate-regulating enzyme in the fatty acid synthesis process. When the expression of the ACC1 gene decreases, fatty acid synthesis decreases. SREBP-1c is a transcription factor that plays an important role in the synthesis of triglycerides in liver tissue. When the expression of the SREBP-1c gene is suppressed, fatty acid synthesis is reduced and consequently, the accumulation of triglycerides in liver tissue is suppressed.

In liver cells, HMG-reducatase is involved in the cholesterol biosynthesis process, and when the expression of the HMGCR gene decreases, the concentration of cholesterol in the blood decreases. When the expression of AMPK in the liver cells increases, SREBP, a transcription factor that plays an important role in triglyceride synthesis It has the effect of inhibiting triglyceride synthesis by reducing -1 expression. It is also known to inhibit the expression of acetyl-CoA carboxylase (ACC), a rate-regulating enzyme in the process of synthesizing the target gene and fatty acid of AMPK.

3A to 3C, the expression of each of the ACC1, SREBP1c, and HMGCR genes showed a significant increase in the positive control group of the comparative example compared to the normal control group of the comparative example, and elm extract 25, 50, and 100 μg/ml When the concentration of OA was treated with OA, it was confirmed that the concentration was decreased in a concentration-dependent manner compared to the positive control.

As shown in Fig. 3d, the expression of the AMPK gene showed a significant decrease in the positive control of Comparative Example 2 compared to the normal control of Comparative Example, and the concentrations of the elm extract 25, 50, and 100 μg/ml were treated with OA. When it was confirmed that the concentration-dependent increase compared to the control group.

It was confirmed that the elm extract of the present invention showed an effect of improving hyperlipidemia by suppressing the expression of ACC1, SREBP1c, and HMGCR genes and increasing the expression of the AMPK gene.

Results 5. Rat blood analysis

The concentrations of AST and ALT, which are hepatotoxicity indicators in the blood of rats tested according to Examples and Comparative Examples, were analyzed and the concentrations thereof are shown in FIG. 4.

As a result, the group administered the elm extract with a high cholesterol diet showed a significant decrease in the concentrations of AST and ALT used as indicators of hepatic liver toxicity compared to the high cholesterol diet group, and the positive control group ATO group. The O3 group also showed a significant reduction effect. The effect of improving the fatty liver of the elm extract of the Example was confirmed by significantly reducing the concentration of AST and ALT in the blood.

The content of total cholesterol, neutral lipid, LDL-cholesterol, and HDL-cholesterol in serum in the blood of rats tested according to Examples and Comparative Examples was analyzed and illustrated in FIG. 5.

As a result, the group administered the elm extract with the high cholesterol diet decreased the total cholesterol, neutral lipid and LDL-cholesterol content in the serum and showed an increase in the HDL-cholesterol content compared to the high cholesterol diet group. According to these results, it was confirmed that the hot elm extract could not only inhibit the progression of fatty liver, but also prevent it.

Results 6. Expression and activity of triglyceride synthesis and degradation-related proteins in experimental and control groups in rat liver tissue

The expression levels of triglyceride synthesis and degradation-related proteins in liver tissue of rats tested according to Examples and Comparative Examples are shown in FIGS. 6 to 7.

It is known that activation of AMPK in the liver inhibits the synthesis of fatty acids and cholesterol and promotes the oxidation of fatty acids. This AMPK activation inhibits acetyl-CoA carboxylase (ACC) activity, a rate-regulating enzyme, during the fatty acid synthesis process through phosphorylation, and reduces the expression of SREBP-1, a transcription factor that plays an important role in triglyceride synthesis, in the liver. Inhibits triglyceride synthesis. HMGCR is a rate-limiting enzyme in the cholesterol biosynthesis process, and it can be seen that when HMGCR activity decreases in the liver, cholesterol concentration decreases.

As shown in Figures 6 to 7, the elm extract group was significantly increased AMPK activity compared to the CON group, it was confirmed that the ACC activity is inhibited. In addition, it was confirmed that the expression of SREBP-1 and HMGCR was significantly decreased compared to the CON group due to the increase in AMPK expression. Therefore, it was confirmed that the elm extract inhibits the synthesis of fatty acids, triglycerides and cholesterol through the AMPK mechanism.

From the above experimental results, the elm extract of the present invention reduced the total cholesterol, triglyceride and LDL-cholesterol contents, increased HDL-cholesterol content, and increased the synthesis of triglycerides, fatty acids and cholesterol compared to when fed only a high cholesterol diet. By controlling the mechanism of decomposition, it was confirmed that it is effective in preventing and treating fatty liver and hyperlipidemia.

Claims (8)

A health functional food composition for improving hyperlipidemia, which includes Yoshino elm hot water extract as an active ingredient. According to claim 1,
The elm heat water extract is characterized in that the dried elm and purified water are mixed in a weight ratio of 1: 5 to 1: 10 to extract, health functional food composition for improving hyperlipidemia. According to claim 1,
The elm hot water extract is characterized in that the extraction at 75 to 98 ℃, hyperlipidemia improvement health functional food composition. In the elm extract,
Drying the elm tree at room temperature to chop;
Injecting the shredded elm and purified water into the extractor:
Heating and extracting the shredded elm in the extractor for 6 hours to generate an extract;
Filtering the extract solution to inactivate the enzyme for 2 hours and then inactivating it for 10 minutes to generate an enzyme-treated solution;
Filtering the enzyme-treated liquid and concentrating it in a vacuum reduced pressure condition to produce a concentrated liquid;
Sterilizing the concentrate for 30 minutes and then freeze-drying; And
Characterized in that it comprises the step of pulverizing the lyophilized concentrate,
A method for manufacturing a health functional food composition for improving hyperlipidemia, which includes Yoshino elm hot water extract as an active ingredient. The method of claim 4,
The extract is a method of manufacturing a health functional food composition for improving hyperlipidemia, characterized in that the shredded elm and purified water are mixed and extracted in a weight ratio of 1: 5 to 1: 10. The method of claim 4,
The extract is characterized in that the extraction at 75 to 98 ℃, hyperlipidemia improvement method for health functional food composition. The method of claim 4,
The enzyme is characterized in that at least one selected from the group consisting of starch degrading enzyme and pectin degrading enzyme, a method for producing a health functional food composition for improving hyperlipidemia. A health functional food for improving hyperlipidemia, which includes Yoshino elm hot water extract as an active ingredient.

Images