KR20220115192A - Food composition for preventing or improving obesity comprising of extracts from Lizard's tail and Green tea - Google Patents

Abstract

A composite composition containing a Saururus chinensis extract and a green tea extract as active ingredients and preventing, treating, or improving obesity, and a method for manufacturing the same are disclosed in the present invention. The method for manufacturing the composite composition comprises the steps of: (a) grinding a dried natural raw material and obtaining a dry raw material powder; (b) adding a hydrophilic organic solvent to the dried raw material powder of the step (a), extracting an extract with stirring, concentrating, freezing, and drying the extract; and (c) manufacturing a mixed composition by blending the frozen and dried extract of the natural raw material obtained in the step (b) at a predetermined ratio, wherein a pretreatment for a high pressure treatment is additionally performed in a high pressure emulsifier in the step (a). The composite composition manufactured according to the manufacturing method of the present invention is a useful invention with a special advantage of making it possible to develop a food material which is excellent and more effective in preventing, treating, or improving obesity than a single material by complexly using the Saururus chinensis extract effective in suppressing a fat accumulation and the green tea extract having an effect of suppressing a rise in blood sugar by inhibiting an absorption of a carbohydrate.

Description

Food composition for preventing or improving obesity comprising of extracts from Lizard's tail and Green tea

The present invention relates to a food composition for improving obesity prevention and a method for manufacturing the same, and more particularly, it can prevent or improve obesity symptoms caused by excessive accumulation of body fat through suppression of postprandial blood glucose rise by inhibiting body fat reduction and carbohydrate absorption. A food composition containing a mixture prepared to contain the same amount (w / w) of the ginseng extract and the green tea extract as a food composition with an excellent effect is provided as a functional mixture composition for weight control in obesity prevention and individuality.

Obesity refers to a state in which adipose tissue is excessively accumulated due to an increase in the number and quantity of adipocytes in the body. On the other hand, carbohydrates are essential nutrients used as an energy source in the body. In order to maintain a healthy body function, it is ideal to consume carbohydrates to account for 55 to 70% of the total daily intake of nutrients. According to the National Health and Nutrition Examination Survey, the minimum daily intake of carbohydrates is about 100 ~ 130g, but the average carbohydrate intake of Korean adults is 298.1g, which is more than twice the minimum intake. When carbohydrates are consumed excessively, the blood sugar level increases and insulin is secreted. As a result, the excess carbohydrates are converted into fat and accumulated in the body, leading to obesity.

The World Health Organization (WHO) has declared the “War on Obesity” and is promoting the seriousness of obesity around the world, and has been selected as one of the top 10 health risk factors in the world. Even in Korea, the number of obese people is on the rise due to reduced activity and westernized eating habits. Obesity accumulates a lot of fat and sugar in the blood, making it vulnerable to diabetes, high blood pressure, dyslipidemia, and cardiovascular disease.

The present inventors measured 50 kinds of natural products in vitro toxicity test, fat accumulation inhibitory effect, and α-glucosidase activity inhibitory effect as a new food material with safety and anti-obesity efficacy. Three hundred seconds as a material and green tea as a material for inhibiting carbohydrate absorption in the body were finally selected (Fig. 1, Table 1).

Efficacy evaluation of adipocyte differentiation and inhibition of α-glucosidase activity Material fat cells
differentiation(%) α-glucosidase
inhibition(%) Material fat cells
differentiation(%) α-glucosidase
inhibition(%) thorny agapi fruit 113.4 - upper leaf 99.3 - Ecklonia 87.6 99.1 loofah cucumber 100.7 19.6 mistletoe 117.0 - fresh herb 105.7 - mustard greens 97.3 4.7 amaranth 95.7 - gyeomyeongja 112.0 18.2 Eoseongcho 94.8 52.6 goji berry 124,3 26.8 bitter melon 35.5 - Guarana 100.2 - Lotus root 96.1 - guava leaves 41,9 99.8 lotus leaf 98.8 48.5 me 95.4 - Audi 110.5 - green tea 66.4 99.9 sumac 93,6 - snow horse riding 91.6 17.1 Citron 106.9 - ginseng 125.9 - wormwood 93.7 45.5 Dalgae 95.2 23.5 grapefruit 117.9 33.5 wrap around 96.1 - peony 90.6 93.7 round horse 100,5 22,0 cloves 55.9 97.7 round 98.8 - rhubarb 94.1 - rose hip 100.7 - dermis 90.4 - Maekmun-dong 93.6 - yam 98.7 11.0 mint 120.7 77.3 Chi distance 105.8 39.7 Bokbunja 35.5 99.7 Tobokryeong 90.9 99.0 vitamin tree 103.8 - green tea 103.9 - corn milk 96.3 34.6 Glyphs 71.2 98.1 three hundred seconds 54.0 - hojanggeun 26.3 96.7 sangbaekpi 98.9 - Mr. Hong 106.6 -

(-: no effect)

Saururus chinesis (Lour.) Bail) is a perennial herbaceous plant that is distributed in China, Japan, and Korea and is a perennial herb belonging to the cotyledonous plant Pepperaceae, sometimes called 'Cheonseongcho'. It is an excellent medicinal herb for various diseases such as antioxidant, anti-aging, detoxification, anticancer, diuretic, and blood circulation, and has been used as a folk medicine for various medicinal effects since ancient times. As the main ingredient contained in ginseng plant, it contains essential oil of methyl n-nonyl ketone and various flavonoids. As for the research on ginseng, there have already been mainly studies related to antibacterial, skin whitening and anti-aging.

Green tea ( Camellia sinensis ) is an evergreen broad-leaved shrub that is the most widely consumed beverage worldwide. Green tea is largely divided into Ujeon, Sejak, Jungjak, and Daejak depending on the harvesting time and season, and it is known that the later the harvesting period, the more catechin components are contained (Yang et al., 2012). Catechin, a major component of green tea, is a kind of polyphenol (-)-epigalloe-catechin 3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatchin 3-O-gallate (ECG) , (-)-epicatechin (EC), etc., and its action has already been reported to reduce blood pressure, antithrombotic, anticancer, antiobesity, antidiabetic, anti-inflammatory, and cardiovascular diseases.

Research on green tea with various effects has been actively carried out, and green tea extract has already been widely used as a material for body fat reduction health functional food with the effect of improving obesity and weight loss. In the present invention, green tea extract inhibits α-glucosidase activity to delay digestion and absorption of ingested carbohydrates, thereby reducing absorption in the body and preparing green tea extract to act as a mechanism to prevent obesity.

There are various extraction methods for extracting natural products using polar solvents such as water, methanol, ethanol, etc. However, the present inventors attempted to increase the extraction of the active ingredients contained in the natural product by additionally applying a high-pressure extraction method in addition to the solvent extraction or physical extraction in order to efficiently extract the active ingredient of the natural product. The high-pressure extraction method is a method of increasing active ingredients by splitting natural product particles by passing through a narrow nanocell (diamond) based on high pressure.

The present invention searches for anti-obesity materials that do not have side effects on the human body after selecting natural resource materials to reduce the accumulation of triglycerides, which is the cause of obesity, and inhibit the absorption of carbohydrates in the body. It provides an anti-obesity functional food composition having an effect of preventing and improving obesity that can be used, for example.

Korean Patent Publication No. 10-2019-0103665 discloses the anti-obesity effect of a solvent extract of ginseng plant, and Korean Patent Registration No. 10-161447 discloses 10:6:10 : A functional composition for improving and preventing obesity of a 4 (w/w) complex extract is disclosed.

In addition, Korean Patent Registration No. 10-1461098 discloses the anti-obesity effect of a 2:1:1 (w/w) mixed composition of alcohol extracts of ginseng chives, goji berries and eoseongcho. On the other hand, Korean Patent Publication No. 10-2007-19395 discloses the anti-obesity effect of a complex powder of green tea powder, Spirulira and corn starch.

1. Patent Publication No. 10-2019-0103665 2. Patent registration 10-1621447 3. Patent registration 10-1461098 4. Patent Publication 10-2007-19395

Therefore, it is an object of the present invention to provide a natural product complex extract consisting of an optimal blending ratio of ginseng and green tea.

Another object of the present invention is to provide a food composition for preventing and improving obesity, which contains a natural product complex extract for preventing obesity and weight control as an active ingredient by inhibiting the body absorption of carbohydrates and materials excellent for reducing body fat in the body to suppress postprandial blood sugar rise there is

Another object of the present invention is to find the most efficient method of extracting the active ingredient within the alcohol concentration range (30 to 70%) of the concentration of 30 to 70%, extract each extract, and extract each extract based on the corresponding solid content. An object of the present invention is to provide a method for preparing a natural product complex composition that maximizes the effect of reducing body fat by mixing the extract.

The above object of the present invention is to provide a method for preparing a mixed composition for preventing and improving obesity containing a trifolieae extract and green tea extract as active ingredients, comprising the steps of: (a) pulverizing each dried natural raw material as the food material to obtain a dry raw material powder; (b) adding a hydrophilic organic solvent to the dry raw material powder obtained in the grinding step (a), extracting with stirring, and then concentration and freeze-drying; (c) in the method for producing a triticale and green tea extract comprising the step of preparing a mixed composition by mixing the freeze-dried extract of the natural product obtained in the freeze-drying step (b) in a certain ratio;

In step (a), a hydrophilic organic solvent is added to each dry raw material powder, and a pretreatment step of crushing natural product particles for 1 to 3 hours at a high pressure of 10,000 to 30,000 psi in a high pressure emulsifier is further added to improve the extraction efficiency and safety of active ingredients. characterized by augmentation.

According to the present invention, in the step (b), the hydrophilic organic solvent is water or alcohol or a mixture thereof, and the hot water extraction method is extracted 1 to 3 times for 60 minutes to 240 minutes at a temperature of 40 to 60 ° C. The amount of purified water used should be 10 to 30 times the weight of the raw material (w/w), which is efficient in consideration of the safety of the process and the stability of the active ingredient contained in the raw material, and it was preferable to simplify the extraction process. In addition, when using the organic solvent, it is preferable to use ethanol having a relatively high polarity, and it is preferable to mainly use 30 to 70% (w/w) of ethanol by mixing with purified water.

In the above, the pressure of 10,000 psi or less is not suitable for splitting the dry and pulverized natural material raw material, and when it exceeds 30,000 psi, the pulverized material is agglomerated.

As described above, according to the production method of the present invention, the extracts of ginseng extract and green tea extract, which are each involved in the suppression of postprandial blood glucose rise due to the mechanism of inhibiting the differentiation of fat cells or the accumulation of fat, and inhibiting the absorption of carbohydrates into the body, were specially selected and optimal. By using the composite composition mixed in the mixing ratio, there is an effect of confirming the anti-obesity effect and the lipid improvement effect, and furthermore, there is an excellent effect of providing a food composition for preventing or improving obesity.

1 is a view showing the results of the basic experiment for selecting the raw material of the present invention,
Figure 2 is a diagram of the manufacturing process of three hundred seconds and green tea extract showing the most preferred embodiment of the present invention,
3 is a graph of the cell viability of Sambaekcho extract (TM-1) and green tea extract (TM-2) prepared according to an embodiment of the present invention;
4 is an α-Glucosidase inhibitory activity (AGI) graph of green tea extract (TM-2) prepared according to an embodiment of the present invention;
Figure 5 is a graph of adipocyte differentiation (fat accumulation) confirmed by treating 3T3-L1 cells with the extract (TM-1) prepared according to an embodiment of the present invention;
6 is a graph showing the content of triglycerides confirmed by treating 3T3-L1 cells induced to differentiate with the extract (TM-1) prepared according to an embodiment of the present invention;
7 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. A graph showing the change in body weight measured by treatment with a high fat diet obese model rat and breeding for 8 weeks, and a graph showing the final body weight measured before sacrifice,
8 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. A graph showing the weight gain and dietary efficiency measured after 8 weeks of breeding for obese model mice treated with a high-fat diet,
Figure 9 is according to an embodiment of the present invention green tea extract (TM-2), a 1:1 (w / w) mixture of green tea extract and green tea extract (TM-3) treated with high-fat diet obese model mice 8 Oral glucose tolerance measurement graph and area graph under the blood glucose response curve measured after weekly breeding;
10 is a treatment with a high-fat diet obese model rat with a 1:1 (w/w) mixture (TM-3) of green tea extract (TM-2), Sambaekcho extract and green tea extract prepared according to an embodiment of the present invention; The glycated hemoglobin content measured after breeding for 8 weeks,
11 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. Graph showing the weight of organs (liver, total white adipose tissue) after 8 weeks of treatment with high-fat diet obese model rats,
12 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. A graph showing the size and cell number of adipocytes in epididymal adipose tissue after 8 weeks of treatment with high-fat diet obese model mice,
13 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. Changes in tissue morphology of epididymal adipose tissue after 8 weeks of treatment in high-fat diet obese model rats,
14 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. After 8 weeks of treatment in high-fat diet obese model rats, serum glucose, triglyceride, total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol), high-density lipoprotein cholesterol (HDL-cholesterol) ) a graph showing the measurement result,
15 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. A graph showing the results of measuring the content of insulin and leptin in serum after 8 weeks of treatment with high-fat diet obese model mice,
16 is a 1:1 (w/w) mixture of Sambaekcho extract (TM-1), green tea extract (TM-2), Sambaekcho extract and green tea extract (TM-3) prepared according to an embodiment of the present invention. A graph showing the mRNA expression level of fat metabolism-regulating protein after 8 weeks of treatment in high-fat diet obese model mice,
17 is a view showing a chromatogram of the results of HPLC analysis to measure the active ingredient content of the composition prepared according to an embodiment of the present invention.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to the accompanying drawings. Such modifications fall within the scope of the present invention.

Example 1. Preparation of natural extracts and mixtures thereof

The manufacturing process of the mixed extract of Sambaekcho and green tea prepared as an example in the present invention is shown in FIG. 2 .

1-1. Preparation of extract of ginseng

The three hundred seconds selected in the present invention were purchased from a dried plant through a three hundred second production farm (Bohyeonsan Clean Herb Farming Association Corporation). Purchased three hundred seconds is pulverized using a blender to produce three hundred seconds raw materials, put into an extractor, 30% alcohol 30 times the weight of three hundred seconds dried raw materials (w/w) is added, and the filtrate obtained by extraction at 60° C. for 4 hours. Concentrated and lyophilized through a lyophilizer to prepare an extract powder of 300 sec.

1-2. Preparation of green tea extract

For the green tea selected in the present invention, green tea was purchased through a green tea producer (agricultural company Boseong Green Tea Sundawon). Purchased green tea is pulverized using a blender to produce green tea raw materials, then put into an extractor, 30% of alcohol 20 times the weight of green tea raw materials (w/w) is added, and the filtrate obtained by extraction at 60° C. for 4 hours. Concentrated and freeze-dried green tea extract powder through a freeze dryer was prepared.

1-3. Preparation of mixed composition

1-1 above. and 1-2., the extract and green tea extract prepared in 1 to 9:9 to 1 (w/w) were mixed with respect to the solid content. The present inventors have applied this ratio in the following experiments because 1:1 (w/w) mixture was the most effective for achieving the mechanism and anti-obesity effect of the present invention as a result of a number of experiments.

1-4. Preparation of high-pressure emulsified extracts of ginseng and green tea (comparative example)

In the same manner as in Example 1-2., three hundred seconds and green tea raw materials were mixed with 30% alcohol, respectively, and a pretreatment of splitting particles at 10,000 to 30,000 psi in a high pressure emulsifier for 1 to 3 hours was additionally performed, and then the natural product extraction process Concentrated the filtrate obtained through the lyophilizer to prepare an extract powder of each extract lyophilized. A complex mixture was obtained in which the extraction powder of each extract obtained according to this Example 1-4. was formulated in a 1:1 (w/w) mixing ratio, and this was used as a test material.

Example 2. Measurement of α-Glucosidase Inhibition Effect to Check Blood Sugar Control Effect

The α-glucosidase inhibitory activity was measured according to the method of Watanabe et al. After putting 20 μL of liquid sample and 20 μL of 100 mM phosphate buffer (pH 6.8), dissolve 0.2 unit/mL α-glucosidase, an enzyme, in 10 mM phosphate buffer (pH 6.8) and add 20 μL, and 2.5 dissolved in 100 mM phosphate buffer (pH 6.8) as a substrate 20 μL of mM p-nitrophenyl-D-glucopyranoside (p-NPG) was added, and after incubation at 37° C. for 15 minutes, 80 μL of 0.2 M sodium carbonate solution was mixed to terminate the reaction, and absorbance was measured at 405 nm. α-Glucosidase inhibitory activity (AGI) was calculated using the following equation.

Depending on the type of green tea (powder/leaf) or harvesting period (thickness/magnificence), the effect of inhibiting α-glucosidase activity was confirmed. was selected (FIG. 3). In addition, the α-glucosidase inhibitory activity was checked for each concentration, and the IC50 (concentration of the material that reduces the activity of α-glucosidase by 50%) was confirmed to be 20.0 ug/mL.

Example 3. To check the effect of preventing, treating and improving obesity in vitro cell experiment

3-1. cell culture

3T3-L1 cells, which are preadipocytes derived from mice, were purchased from the Korea Cell Line Bank and used. 3T3-L1 cells were cultured in DMEM medium (Welgene) with 10% bovine calf serum (BCF), 100 units/mL penicillin, and 100 ug/mL streptomycin added to a cell culture medium (complete DMEM culture medium) in a humidified CO ₂ incubator at 37°C. (5% CO ₂ /95% air). When the cells were about 80% full of the culture dish, the cell monolayer was washed with phosphate buffer saline (PBS, pH 7.4), and then the cells were removed and subcultured by adding trypsin-2.65 mM EDTA, and the medium was changed every 2 days. .

3-2. Cell viability measurement

3T3-L1 cells were seeded in a 24-well plate so as to become 3 x 10 ⁴ cells/well, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, each test substance was exchanged with a cell culture medium containing various concentrations (0, 10, 25, 50, 100, 200 ug/mL), and the cells were cultured for 72 hours. After culturing the cells for 72 hours, MTT assay (Denizot F and Lang R. J Immunological Method 89: 271-277, 1986) was performed to measure the number of viable cells. The MTT assay method is based on the principle that mitochondrial dehydrogenase reduces MTT (Amresco) to make formazan, a blue substance. In this test, formazan was dissolved in isopropanol and absorbance was measured at a wavelength of 570 nm (FIG. 4).

3-3. Cell differentiation induction and test substance treatment

3T3-L1 cells were seeded in a 24 well plate at a concentration of 1 x 10 ⁵ cells/w. After the cells reached a state of confluence, the cells were cultured to induce differentiation into adipocytes by sequentially exchanging the cell culture medium with three types of differentiation inducing medium (DM). That is, the cell culture medium was exchanged with the differentiation inducing culture medium containing DMI (1 uM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 ug/mL insulin) added to DMEM medium containing 10% FBS for 2 days. differentiation was stimulated. After 2 days, the DMEM medium containing 10% FBS was replaced with a new differentiation-inducing culture medium containing 5 ug/mL insulin to stimulate differentiation for another 2 days. After stimulating differentiation for a total of 4 days, cells were maintained in DMEM medium containing 10% FBS for about 4 to 8 days to induce differentiation into adipocytes, during which the cell culture medium was exchanged every 2 days. To investigate the effect of the test substance on adipocyte differentiation, the test substance was added to the differentiation inducing culture medium at different concentrations (6.25, 12.5, 25, 50, 100, 200 ug/mL) and treated with the cells.

3-4. Measurement of adipocyte differentiation (fat accumulation) (Oil red-O staining and quantification)

After inducing differentiation of 3T3-L1 cells and treating the test substance, the cells were rinsed with DPBS (Welgene) and then 4% paraformaldehyde (PFA, Biosesang) was added to fix the cells at room temperature for 1 hour. After fixing the cells, they were treated with oil Red O (Sigma-Aldrich Co.) solution and stained at room temperature for 1 to 2 hours. After observing the degree of staining of adipocytes, the cells were rinsed with distilled water and the adipocytes were observed through a microscope. In order to quantitatively analyze the degree of adipocyte differentiation, the absorbance was measured at a wavelength of 490 nm by dissolving in isopropanol after staining with oil red O (FIG. 5).

3-5. Measurement of triglyceride content in adipocytes

3T3-L1 cells were dispensed into a 6-well plate so as to become 3 x 10 ⁵ cells/well, and the test substance was treated while inducing differentiation. Then, the cell culture medium was removed and the cells were rinsed with DPBS * Welgene. 300 μL of distilled water was added to the cells, and the cells were collected in a 1.5 mL Eppen tube using a cell scraper, and then homogenized with a homogenizer. To the homogenized cell solution, 900 μL of Folchchloroform : methanol = 2:1 (v/v) solvent was added, mixed well, and reacted at 37° C. for 30 minutes. After 30 minutes, centrifuge (5,000 rpm) for 10 minutes, transfer the lower layer containing lipids to a new 1.5 mL tube, and centrifuge for 2 hours using a high-efficiency centrifugal concentrator (GeneVac) to evaporate and remove the chloroform layer. After drying, a pellet was obtained. 20 μL of chloroform was added to this pellet and the lipid was dissolved again, and used as a test solution. For the test solution, triglyceride assay kit (Asan Pharmaceutical) was used to measure triglycerides according to the method suggested by the manufacturer (FIG. 6).

3-6. Statistical processing

All analysis values were expressed as mean±standard error of the mean (SEM). In order to compare the difference between the control group and the test substance administration group, analysis of variance (ANOVA) was used and the significance was verified using the Duncan' multiple range test in SAS software version 9.4 (SAS 　 institute) (ρ < 0.05).

Example 4. To check the effect of preventing, treating and improving obesity in vivo animal testing

4-1. Experimental animal breeding environment

Experimental animals were 5-week-old without specific pathogens, and male C57BL/6 mice were purchased from Dooyeol Bio Co., Ltd., and after 1 week of quarantine and adaptation procedures, healthy experimental animals without weight loss were selected and used for the experiment. Experimental animals are bred in a breeding environment set at 23±3℃, 50±10% relative humidity, 10 ∼ 15 times/hour of ventilation, 12 hours of illumination (08:00 ∼ 20:00), and 150∼300Lux of illumination. During the entire period of the test, the experimental animals were allowed to freely ingest solid feed and drinking water for experimental animals.

4-2. Test substance administration

According to the egg mass method, (G1) control diet group, (G2) high-fat diet control group, (G3) high-fat diet + trifoliea extract: 1:1 mixture of green tea extract ( TM-3), (G4) high-fat diet + positive control (Garcinia cambogia extract, HCA) administration group, and 10 experimental animals were used in each test group. During the pre-test period, the control group (10% of kcal from fat, Research Diets Inc) and the high-fat diet group (60% of kcal from fat, Research Diets Inc) were supplied, respectively, and negative water was allowed to be consumed ad libitum. The test substance was dissolved in negative water and orally administered at a fixed time for 8 weeks.

4-3. Weight and dietary intake measurement

During the test period, the body weight of the experimental animals was measured at a fixed time every week. The dietary intake of each experimental animal was measured at 2-day intervals, and the daily food intake and daily energy intake were calculated by calculating the amount consumed during the test period. Food Efficiency Ratio (FER) was calculated as follows by dividing the weight gain during the test period by the amount of food consumed during the same period.

The change in body weight of the animals measured during the test period showed a steady increase in all groups as shown in FIG. 7 to indicate a normal weight change. Compared to the control group, the high-fat diet increased body weight more in the control group, and the high-fat diet increased the body weight. Compared with the control group, all groups treated with the test substance (TM-1, TM-2, TM-3, HCA) showed a decrease in body weight increase compared to the high-fat diet control group. The test substance TM-3 administration group showed significant weight loss from the 1st week, and in particular, the test substance TM-3 administration group showed the smallest weight gain ( FIG. 8 ).

4-4. Oral glucose tolerance test (OGTT)

To check the blood glucose level, oral glucose tolerance was measured for the control group, the high-fat control group, and the test substances TM-2 and TM-3 administered groups. 30 minutes after the test substance was administered, 2 g/kg BW glucose was orally administered. After fasting for 12 hours 3 days before the end of the test, after oral administration of glucose, blood was taken from the tail of the experimental animal 30, 60, and 120 minutes, and blood glucose was measured using a blood glucose meter (ACCU-CHEK, Roche Diagnostics Korea). measured.

The blood glucose level before glucose administration (0 min) and 30 min blood glucose level was significantly higher in the high-fat control group compared to the control group, and the test substance TM-3 administration group showed a low blood glucose level, but there was a significant difference. there was no The 60-minute and 120-minute blood sugar levels were significantly lower in the test substance TM-3 administration group compared to the high-fat diet control group. In the area under the curves (AUC), the AUC was significantly increased in the high-fat diet control group compared to the control diet group, and the AUC was significantly increased by administration of the test substance TM-3. decreased (Fig. 9).

4-5. laboratory animal blood collection

Before sacrifice, the experimental animals were anesthetized using an anesthetic prepared by diluting tribromoethanol with tertiary amylalcohol, and then orbital blood was collected. The blood was placed in a plasma sepatate tube (Becton Dickinson) or a serum separate tube (Becton Dickinson) and left at room temperature for 30 minutes, then centrifuged at 5,000 rpm for 10 minutes to separate plasma and serum, and stored at -70°C until analysis.

4-6. Glycated hemoglobin (HbA1C) measurement

The glycated hemoglobin content, which is an indicator of long-term blood glucose level, was measured for the control diet group, the high-fat diet control group, and the test substances TM-2 and TM-3 administration groups using the plasma collected at the end of the test. HbA1C in plasma was measured using a measurement kit purchased from Mybiosource and according to the method suggested by the manufacturer. Compared with the control diet group (G1), the glycated hemoglobin content of the high-fat diet control group was significantly increased, and the glycated hemoglobin content of the test substance TM-3 administered group was significantly decreased compared to the high-fat diet control group (FIG. 10).

4-7. Liver and adipose tissue analysis

After blood was collected from the experimental animals, liver and adipose tissue (eg, epididymal fat, retroperitoneal fat, visceral fat, groin fat, and scapular fat) were extracted and the weight of each was measured. For each histological analysis, a portion of each tissue was cut and fixed in 4% paraformaldehyde (Sigma-Aldrich Co.), and the remaining tissue was stored at -70°C for further analysis in the future.

As shown in FIG. 11, the weight of the liver was significantly increased in the control group with the test method compared to the control group, which

Significantly increased, which significantly decreased liver weight in the test substances TM-2 and TM-3 administration groups (FIG. 11).

Body fat is divided into white adipose tissue and brown adipose tissue according to its shape and action. White adipose tissue mainly stores excess energy in the body as fat, brown adipose tissue functions to produce heat, and the weight of white fat increases. as the weight increases. The weight of the white adipose tissue, epididymal fat, retroperitoneal fat, visceral fat, and groin fat, all significantly increased in the high-fat diet control group compared to the control diet group, and the total weight of white fat increased in the high-fat diet control group was the test substance. It was significantly decreased in the TM-3 administration group.

In addition, the high-fat diet increased the adipocyte size of epididymal adipose tissue and decreased by all test substances, and in particular, the increased adipocyte size decreased in the test substance TM-3 administration group. In addition, the number of total adipocytes compared to the same area was significantly increased in all test substance-administered groups, and in particular, the TM-3 administration group significantly increased (Fig. 12).

4-8. Analysis of glucose and lipid components in blood (serum)

The serum glucose, triglyceride, total cholesterol, LDL-cholesterol, and HDL-cholesterol levels were measured using a blood biochemistry analyzer (KoneLab 20 XT, Thermo Fisher Scientific). Blood glucose was significantly increased in the high-fat diet control group compared to the control diet group, and decreased significantly in the test substances TM-2 and TM-3 administration groups. The blood triglyceride content showed a tendency to increase in the high-fat diet control group compared to the control diet group, but did not show a significant difference. decreased. Blood total cholesterol content and HDL-cholesterol content were significantly increased in the high-fat diet control group compared to the control diet group, and significantly decreased in the test substances TM-2 and TM-3 administration groups (FIG. 14).

4-9. Analysis of hormones related to fat metabolism in blood (serum)

Leptin is a hormone produced and secreted by adipose tissue. It is an important substance that regulates energy intake and storage, insulin sensitivity, and metabolic rate. Long-term high-fat intake leads to a chronic rise in blood sugar and induces insulin resistance, and when body fat accumulates excessively, hyperinsulinemia, in which the insulin concentration in the blood is high, continues. The leptin content in serum was measured using a measurement kit purchased from R&D Systems, and the insulin content was measured using a measurement kit purchased from Millipore Corporation according to the method suggested by each manufacturer.

As a result of measuring the blood leptin and insulin content of animals in this study, the high-fat diet significantly increased in the control group compared to the control diet group, and the leptin content in the blood was significantly decreased in the test substances TM-2 and TM-3 administered groups. . The blood insulin content showed a tendency to decrease in the test substance administration group, but no significant difference was found ( FIG. 15 ).

4-10. Analysis of mRNA expression of genes related to fat synthesis and energy metabolism in adipose tissue

The effects of test substances (TM-1, TM-3) on lipogenesis, degradation, and energy metabolism-related mRNA expression in epididymal adipose tissue were analyzed. Total RNA was isolated from epididymal adipose tissue collected at the end of the test using TRIzol REAGENT (Thermo Fisher Scientific), quantified using a micro-volume spectrophotometer (BioSpec-nano, SHIMADZU), and RNA was used for the experiment. After cDNA was obtained from total RNA, real-time PCR was performed using Rotor-Gene 300 PC (Corbett Research) and Roter-Gene SYBR Green Kit (QIAGEN).

The expression of lipogenesis-related genes SREBP-1, PPAR-γ, C/EBPa, FAS, aP2, and ACC1 mRNA was significantly increased in the high-fat diet control group compared to the control diet group, and test substances TM-1, TM- It decreased in the 3 administration group. On the other hand, the expression of CPT-1 mRNA, a gene related to lipolysis, decreased in the high-fat diet control group and increased in the test substances TM-1 and TM-3 administration group.

Example 5. Active ingredient content analysis

Sauchinone, an active ingredient in ginseng plant, and EGCG, an active ingredient in green tea, were analyzed using HPLC-DAD, and the analysis conditions are as follows. Weigh each sample accurately in an appropriate amount, pour it into a volumetric flask, and fill with 70% methanol up to the marked line on the flask. After sonication for 10 minutes, it was filtered using a syringe filter (0.45μ) and then injected into the HPLC device. The standard was prepared at 5 or more concentrations and analyzed by HPLC, and the peak calculated by analyzing each concentration with the HPLC device The content was calculated by substituting the area of into the functional formula obtained by connecting linearly.

HPLC analysis conditions for Sauchinone Item Condition injection volume 10 μL detector wavelength 240 nm column temperature 35 ℃ mobile phase A: 0.1% phosphoric acid solution
B: acetonitrile flow rate 1.0 mL/min

Mobile phase conditions for sauchinone hours (minutes) A solution (%) Solution B (%) 0.0 50 50 40.0 50 50

Conditions for HPLC analysis of EGCG Item Condition injection volume 10 μL detector wavelength 275 nm column temperature 35 ℃ mobile phase A: 0.1% phosphoric acid solution
B: Acetonitrile containing 0.1% phosphoric acid flow rate 1.0 mL/min

Mobile phase conditions of EGCG hours (minutes) Mobile phase (%) A solution B solution 0.0 90 10 5.0 90 10 10.0 87 13 20.0 85 15 25.0 70 30 30.0 70 30 31.0 90 10 35.0 90 10 alcohol extraction Alcohol extraction after high pressure emulsification Sauchinone (mg/g) 6.8 8.2 (temporary) EGCG (mg/g) 136.8 163.3 (temporary)

Claims (5)

(a) pulverizing the dried natural product raw material to obtain a dry raw material powder;
(b) adding a hydrophilic organic solvent to the dry raw material powder obtained in the pulverization step (a), stirring and extracting, filtering and concentrating the extract;
(c) freeze-drying the concentrate of the natural product obtained in the concentration step (b) and secondary grinding;
(d) in the second grinding step (c) in the method for producing a mixed composition of the extract comprising the step of mixing the dry powder of the natural product to obtain a composite composition; In the step (a), a hydrophilic organic solvent is added to each dry powder and a pretreatment step of crushing natural product particles for 1 to 3 hours at a high pressure of 10,000 to 30,000 psi in a high pressure emulsifier is further added. . According to claim 1, wherein 30% ethanol (v/v) is added to the natural raw material dried in the pulverization step (b) 10 to 30 times the weight of the raw material (w/w), and stirred at a temperature of 25 to 60 ° C. Method, characterized in that the extraction for 60 minutes to 240 minutes. The method according to claim 1, wherein 30% ethanol (v/v) is added to the natural raw material dried in step (a) by 10 to 30 times the weight of the raw material (w/w), followed by high-pressure extraction. A complex extract prepared by the method of claim 1 or 2, wherein the extract of Sambaekcho extract and green tea extract are mixed in a 1:1 (w/w) ratio. A food composition for preventing and improving obesity containing the complex extract of claim 4.

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