KR20120108071A - Composition containing soybean by product for anti-obesity - Google Patents

Abstract

PURPOSE: A composition containing soybean-processed by-products is provided to reduce accumulation of body fat and to prevent and treat obesity without side effect. CONSTITUTION: A composition for preventing or treating obesity contains 0.001-30 wt% of soybean-processed by-product as an active ingredient. The by-products are soybean soaking water, soybean curd whey, or a mixture thereof. The soybean soaking water is prepared by dipping soybeans at 10-30 Deg. C. for 3-24 hours. The active ingredient suppresses the accumulation of cholesterol in body, the accumulation of neutral fat, the differentiation of preadipocytes to adipocytes, the generation of fat, the activation of acetyl-CoA carboxylase(ACC), and the activation of AMPK(AMP-activated protein kinase). The composition contains one or more pharmaceutically acceptable carriers or excipient.

Description

Composition for preventing or treating obesity using soybean by-products as an active ingredient {Composition containing soybean by product for anti-obesity}

The present invention relates to a composition for preventing or treating obesity using the soybean processing by-product as an active ingredient, and more particularly to a composition for preventing or treating obesity containing pure soybean, soybean immersion water or a mixture thereof as an active ingredient. It is about.

In modern society, the ingestion of vegetable foods is gradually reduced due to the westernization of diet due to the improvement of living standard, and the change in dietary patterns, while the intake of animal foods containing saturated fatty acid or cholesterol is increasing. It is a problem (Kim et al., 1993).

Obesity is a metabolic disorder caused by excessive accumulation of body fat due to an imbalance in energy intake and consumption (Song et al., 2009), which is associated with a variety of causes, including genetic, environmental and social structural changes. As a syndrome (Grundy, 1998; Albu et al., 1997), excessive energy intake causes metabolic activity in the body to be consumed, resulting in accumulation of triglycerides in the adipose tissue. Affects physiological and biochemical functions (Huh, 1990). In addition, the exact mechanism of obesity is not known, the higher the obesity is known to increase the risk of cardiovascular disorders. Obesity has attracted much attention worldwide because obesity can cause diseases such as cardiovascular disease, diabetes, respiratory disease, and osteoarthritis, as well as its own problems (Antipatis and Gil, 2001).

Obesity is a combination of excessive energy consumption, lack of exercise, as well as endocrine factors, drug causes, genetic factors, etc. As a way to improve obesity, diet, exercise, behavioral therapy or drug treatment is used. In drug therapy, drugs such as reductil, which exhibits an appetite suppressing effect, and Xenical, which suppresses digestion and absorption of fats, are used in clinical practice depending on the mechanism of action of obesity (Kim, 2004). However, as these drugs are known to cause side effects such as oily stools, abdominal bloating, dizziness, dry mouth, constipation and increased blood pressure (Reddy and Chow, 1998), the development of safer and more potent natural functional substances is required. There is a need for a lot of research on natural bioactive substances.

In Korea, many natural substances are marketed as health foods that are effective in preventing obesity, but most of them are not scientifically verified, and they are often social problems because they cause health food abuse.

Meanwhile, soybean pure water and soybean immersion water are by-products generated during the tofu production process. Antioxidant Activity (Kim and Lee, 2007), Functional Properties (Kim et al., 2006), and Changes of Soybean Property by Soak Stock (Jang et al., 1995) However, studies on the physiological activity of soybean by-products and soaked water are very poor.

Therefore, there is an urgent need for an effort to objectively verify natural foods that can be eaten as foods and to develop natural materials having a therapeutic and preventive effect on obesity.

Therefore, the present inventors are continuing to study the development of natural substances having a therapeutic and preventive effect of obesity, soybean by-products and soybean immersion water is a natural high-fat diet for improving lipid metabolism and anti-obesity of the mouse by high-fat diet Confirming the physiological activity efficacy, the present invention was completed.

An object of the present invention is to provide a natural food extract that is harmless to the human body and can reduce the accumulation of fat in the body by a high fat diet.

It is also an object of the present invention to provide a soybean processing by-product having a prophylactic and therapeutic effect of obesity.

In another aspect, the present invention is to provide a composition for the prevention or treatment of obesity, the soybean by-products as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing or treating obesity, and more particularly, a composition for preventing or treating obesity, using soybean by-product as an active ingredient.

In another aspect, the present invention provides a dietary supplement comprising the composition.

Hereinafter, the present invention will be described in detail.

The composition according to the present invention is characterized in that it contains soybean processing by-products, and more particularly, to provide a soybean processing by-product and a composition for preventing or treating obesity containing the same for the purpose of preventing and treating obesity due to a high fat diet. It features.

The soybean processing by-product of the present invention is characterized in that the soybean soaking water (soybean soaking water), soybean curd whey or a mixture thereof.

As used herein, the term "prevention" refers to inhibiting the occurrence of a disease or condition in an animal that has never been diagnosed as having a disease or condition but is prone to such disease or condition. As used herein, the term “treatment” refers to (a) inhibiting the development of a disease or condition; (b) alleviation of the disease or condition; And (c) elimination of the disease or condition.

The present invention provides a composition for preventing or treating obesity, containing soybean processing by-products as an active ingredient.

The soybean processing by-products are soybean soaking water, soybean curd whey or a mixture thereof, and more preferably soybean soaking water.

In the present invention, the soybean curd whey is more specifically, the supernatant obtained by centrifuging the liquid pure water generated during tofu production at 10,000 to 14,000 rpm, using a filter paper to remove the solids, Concentrate of the supernatant, or dried product prepared by freeze-drying the concentrate.

In the present invention, the soybean soaking water is obtained by immersing soybean in water at room temperature, more specifically, soybean soaked in water at a temperature of 10 to 30 ℃ without hydrothermal heating for 3 to 24 hours Filtrate obtained after immersion, concentrate of the filtrate, or dried product prepared by freeze drying the concentrate.

The composition of the present invention may be prepared by containing the active ingredient, soybean soaking water, soybean curd whey or a mixture thereof in an amount of 0.001 to 30% by weight of the total composition.

The active ingredient is fat degeneration and fat accumulation inhibition of liver and epididymal tissue; Inhibition of accumulation of cholesterol in the body; Inhibition of the accumulation of triglycerides, differentiation of fat cells into adipocytes, proliferation of adipocytes; Inhibition of lipogenesis; Inhibition of the activity of acetyl-CoA carboxylase (ACC); And increasing the activity of AMP-induced activated protein kinase (AMPK), characterized in that it controls the underlying cause for obesity through at least one action selected from.

More specifically, the active ingredient is characterized by having an anti-obesity effect by inhibiting the activity of peroxisome proliferator activated receptor (PPARγ).

The compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / 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 multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of a powder, granules, tablets, capsules, and may further include a dispersant or stabilizer.

The composition of the present invention can be developed as a dietary supplement. The dietary supplement is one or more selected from a drink, caramel, chocolate, diet bar formulation, and confectionary, and includes ingredients commonly added in the manufacture of food, for example, proteins, carbohydrates, fats, nutrients, Seasonings and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when prepared with a drink, in addition to staniocalcin 2 of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, and the like may be further included. Given the easy access to food, the food of the present invention is very useful for the treatment or prevention of obesity, and for improving cognitive function.

The composition according to the present invention is harmless to the human body using extracts of natural foods, has an excellent effect of reducing the accumulation of fat in the body, and also prevents obesity caused by high fat intake by inhibiting the promotion of fatty acid oxidation. It can be effectively used without side effects in the treatment, there is an advantage that can greatly contribute to the prevention of various diseases such as metabolic and cardiovascular, cerebrovascular disease, cancer caused by obesity.

1 is a result confirming the histological changes of hepatocytes for the soybean by-product intake diet group according to the present invention,
(A: normal diet group, B: high fat diet group, C: soybean pure water diet group, D: soybean immersion diet group)
Figure 2 is a result confirming the histological changes of fat cells for the soybean by-product intake diet group according to the present invention,
(A: normal diet group, B: high fat diet group, C: soybean pure water diet group, D: soybean immersion diet group)
Figure 3 is a result confirming the phosphorylation of acetyl-CoA carboxylase (acetyl-CoA carboxylase) for soybean by-product intake diet group according to the present invention,
(ND: normal diet, HFD: high fat diet, H-SCW: soybean pure diet, H-SSW: soybean immersion diet)
Figure 4 is a result confirming the distribution of mouse abdominal adipose tissue for soybean by-product intake diet group according to the present invention,
(A: normal diet group, B: high fat diet group, C: soybean pure water diet group, D: soybean immersion diet group)
5 is a cell survival rate of the cells according to the soybean processing by-product treatment concentration of the present invention,
Figure 6 confirms the fat content in fat cells according to the soybean processing by-product treatment concentration of the present invention,
Figure 7 is a result of confirming the state before and after differentiation (A) and differentiation of fat cells through a microscope,
8 is a result of confirming the differentiation capacity of the cells according to the soybean processing by-product treatment concentration of the present invention,
9 is a result confirming the expression of the peroxysome growth factor activator receptor gamma (PPARγ) according to the soybean processing by-product treatment concentration of the present invention,
10 is a result of confirming the expression of fat metabolism related genes according to the soybean processing by-product treatment concentration of the present invention by RT-PCR,
11 is a result of quantifying the expression of fat metabolism related genes according to the soybean processing by-product treatment concentration of the present invention using an ImageQuant program,
(A: APOE, B: adipsin, C: Fabp4, D: Scd1)
12 shows the results of confirming the expression of adipokine related genes according to the soybean by-product treatment concentration of the present invention.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

[Manufacturing Example]

(1) soybean curd whey

The pure water sample used in the experiment was centrifuged at 12,000 rpm of the liquid pure water generated in the preparation of tofu, and then the solids were removed using a filter paper (whatman No. 1) to obtain a supernatant, and the obtained supernatant was concentrated, Lyophilization was used to dry samples. On the other hand, the liquid sample was taken as the pure water of the liquid generated when manufacturing tofu as a sample.

(2) soybean soaking water

Soybeans were immersed in water at 15 ° C. for 8 hours without hot water, filtered, concentrated, and lyophilized.

[ Experimental Example  One]

Three-week-old male mice (C57BL / 6) used in this experiment were purchased from Samtaco Co., Ltd., and were bred under constant conditions (temperature 21.4 ± 0.05 ° C, humidity 61 ± 1%, light and shade for 12 hours) in the breeding room. The diet was incubated for one week with sufficient intake of water (distilled water) and feed (Dyets feed (AIN-93G Purified Rodent Diet) purchased from central experimental animals).

After the treatment, the animals were fed a high-fat diet (40% Beef Tallow + AIN-76A Diet) according to each experimental group. The diet and drinking water were taken freely, and all experimental diets were refrigerated during the breeding period and used in the experiments.

The experimental group consisted of four groups: normal diet (fat: 11.7%), ND, high fat diet (fat: 40%), HFD, and 10% soybean pure diet (high fat). fat: 40%) + 10% soybean pure, H-SCW] prepared in the above preparation, 10% soybean immersion diet group [high fat (fat: 40%) + after 10% soybean immersion prepared in the above preparation , H-SSW] divided the feed of different compositions as shown in Table 1 for about 7 weeks. In addition, the weight was measured at a certain time every day to observe the weight change during the experiment, and the amount of change in feed and drinking water was checked.

In this experiment, all animal experiments were performed according to the National Institutes of Health (Principle of Laboratory Animal Care) and Andong National University experimental animal care and use guidelines.

On the last day of the experiment, the animals were fasted for about 12 hours, anesthetized with ether, and blood was collected from the carotid artery. Blood was collected in serum tubes and centrifuged at 1600 x g for 15 minutes to obtain serum, and frozen at -80 ° C until analysis. After blood collection, the experimental animals were opened and the epididymal adipose tissue and liver tissue were removed, washed with physiological saline, and then wiped dry with filter paper. Electronic tissues were weighed and stored frozen at -80 ° C until analyzed.

(1) Adipose tissue mass and histological changes in liver and epididymal adipose tissue

To determine the effect of soybean sprouts and soybean immersion on obesity in mice caused by high fat diet, liver and epididymal adipose tissue were weighed and compared.

In order to measure the weight of the adipose tissue, liver tissue and epididymal adipose tissue were immediately extracted after laparotomy, washed with phosphate buffer solution, and then water was removed with a filter paper, and the weight thereof was shown in Table 2 below.

As can be seen in Table 2, the weight of liver tissue in each experimental group was 83.2% higher in the high fat diet group than the normal diet group. On the other hand, the soybean pure water diet and soybean immersion diet group decreased 24.8% and 68.3%, respectively, compared to the high fat diet group. The weight of epididymal fat was 210% higher in the high fat diet group than in the normal diet group, and the soybean pure diet group and the soybean immersion diet group were reduced to 24.3% and 76.9%, respectively.

In addition, in order to confirm the fat cell size of the adipose tissue, the gravimetric fat tissue was fixed with 4% formaldehyde solution. After fixing, washing with water and dehydration, and then treated with a paraffin (paraffin) solution to make a paraffin block (section) to a thickness of 4 to 6 ㎛. The sectioned tissue was double-stained with hematoxylin and eosin, and then observed under an optical microscope (Olympus BX50, Japan), and photographed using Olympus DP-71 to observe the adipose tissue of each experimental group. (Heo et al., 2009; Choietal., 2006).

As a result, in the high fat diet group, excess fat and cholesterol were not normally discharged in vitro, leading to the accumulation of fat in the liver, resulting in hypertrophy.The weight of the epididymal adipose tissue representing body fat accumulation increased due to the high fat diet. The diet of pure water and immersion decreased fat accumulation. In the high fat diet group, excess fat and cholesterol were not normally excreted in vitro, leading to fat accumulation in the liver, resulting in enlargement of liver and epididymal adipose tissue, while soybean pure water and soybean immersion water accumulated liver fat and epididymal fat due to high fat diet. It is also the result of confirming that it is effective in reducing the

In particular, soybean immersion significantly inhibited the accumulation of fat in the liver and epididymal adipose tissue due to high fat diets, and it was found that immersion water is more effective than pure water to prevent abdominal obesity and metabolic diseases caused by high fat diet. Could.

(2) Pathological changes in liver and epididymal adipose tissue

The liver is a digestive system that plays a role in synthesizing and storing glucose as glycogen. Glycogen produced by assimilation is used to control blood sugar, but when it is excessively converted, it is stored in the form of fat. Cause change (Friedman et al., 1991). Also, since obesity is characterized by an increase in adipose tissue weight and an increase in blood fat content, the investigation of fat in the epididymis is an important indicator for examining weight changes and obesity. All increase (Fujioka, 2002).

In this regard, the effects of pure water and immersion on the histological changes of liver and epididymal fat in mice caused by high-fat diets can be seen in FIG. 1. As shown in FIG. 1, the liver nucleus of the normal diet group is generally round and centered. Cooper cells between the hepatocytes were found to be evenly spread throughout. On the other hand, high fat diet-induced obesity resulted in severe fat degeneration and phobia around the nucleus, which caused the nucleus in the cytoplasm to be pushed to the periphery and significantly reduced Cooper cells observed in the normal group.

In contrast, the soybean pure diet group had hepatocellular hypertrophy due to intracellular fat accumulation compared to the normal diet group, but the fat phobia of the hepatocyte volume such as the high-fat diet group was not obvious. Only the fat vesicles of were identified, and the majority of hepatocytes and Cooper cells were similar to normal hepatocytes, indicating no serious abnormalities of liver tissue such as high fat diet group.

In addition, as a result of examining the effect of soybean pure water and soybean immersion on morphological changes of the epididymal fat, which is white fat, as shown in FIG. 2, the high fat diet group has a very large fat cell size due to the accumulation of fat. It was confirmed that the larger.

On the other hand, the soybean pure and soybean submerged diet groups had smaller fat cells than the high-fat diet group. Especially, the soybean submerged diet group had more fat cells than normal diet group, but the cell size was normal. Almost similar to the fat cells were confirmed.

In the recent study, Moon et al. (2003) found that natural ingredients composed of herbal medicines are effective in anti-obesity by reducing epididymal adipose tissue volume and adipocyte size, and Yuan et al. (2009). As reported to have an effect of reducing the accumulation of epididymal fat, soybean pure water and soybean immersion of the embodiment was found to be effective in suppressing the fat degeneration of liver and epididymal tissue due to high-fat diet, further anti-obesity effect It is also the result of confirming that there is.

(3) biochemical analysis of blood components

Obesity is often accompanied by various dyslipidemia due to abnormal flow of fat metabolism and glucose metabolism, and previous studies have reported results of elevated triglycerides, cholesterol and blood glucose levels in obese diet-induced animals. (Lee et al., 2006).

In addition, changes in the activity of GOT and GPT enzymes in plasma are an indicator of liver tissue damage as hepatic cell degeneration and necrosis progress, resulting in the release of aminotransaminase into the blood.

In this experiment, the collected blood was used for analysis of plasma centrifuged at 3,000 rpm for 10 minutes. Isolated plasma contains amino acid transferases (GOT, GPT), total cholesterol, high density lipoproteins, low density lipoproteins, total lipids and triglycerides. The content was analyzed in a blood analyzer (Hitachi 7600).

As a result, as can be seen in Table 3, the GOT activity in serum was 27.8% in the high-fat diet group, 57.1% in the soybean meal group, and 20.6% in the soybean immersion diet group when compared to the normal diet group and each experimental group. GPT activity was increased in both high fat and soybean meal groups compared to normal diet group, but the soybean immersion diet group showed similar values to normal diet group.

Serum total cholesterol levels were 93.6% in the high fat diet group, 71.2% in the soybean pure water group, and 13.2% in the soybean immersion diet group, compared to the normal diet group. Soybean immersion diet group showed 247.9% and soybean immersion diet group and 82.8% of soybean immersion diet group. It was confirmed that it works effectively to reduce the increase rate of fat. In addition, HDL-cholesterol levels were higher in the high fat diet group than in the normal diet group, and LDL-cholesterol levels were the lowest in the soybean immersion diet group compared to the high fat diet group and the soybean pure diet group.

Soybean immersion in the tofu by-products of the above examples was based on a number of studies (Park et al., 2005; Wat et al., 2009; Davignon and Cohn, 1996) that reducing blood lipid content reduces the risk of coronary heart disease. It is also confirmed that it is effective in reducing the cardiovascular risk caused by obesity compared to soybean pure water.

(4) phosphorylation ratio of obesity target protein

In this experiment, we investigated the expression of p-AMPK and p-ACC in liver tissues to determine the activity of AMPK and ACC, the target proteins of obesity.

In liver tissues, phospho-AMPK and phospho-ACC were identified by western blot. Liver tissue was homogenized using lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.25% sucrose, 0.4 mg / ml digonin and 1.5 mM PMSF), and protein quantification was performed using Bio-Rad assay reagent (Bio- Rad, USA). 40 μg of protein extracted from liver was electrophoresed and treated with primary antibodies (p-AMPK-alpha, p-Acetyl-CoA) (Cell signaling Technology, Beverly, USA) and secondary antibodies (anti-rabbit IgG-HRP). ) (Santa cruz biotechnology, Santa Cruz, USA) to determine the phosphorylation ratio of AMPK and ACC.

As can be seen in Figure 3, the result of examining the phosphorylation ratio of the experimental group relative to the expression of p-ACC of the normal diet group, the p-ACC of the high-fat diet group was 23.0%, whereas the soybean pure water diet group 83.6%, soybean saliva The index diet group was 164.2%.

 From the above results, it was confirmed that soybean pure water and soybean immersion water acted more effectively on the phosphorylation of ACC than high fat diet. Especially, soybean immersion water has a high phosphorylation ratio of ACC. It was confirmed that the anti-obesity effect was very high by inhibiting the promotion of oxidation.

(5) distribution of adipose tissue

To determine the abdominal fat volume of 12-week-old male C57BL / 6 mice, using in vivo micro-CT system (In-vivo Micro-CT, Skyscan 1076, SKYSCAN NV, Belgium) Pictures were taken up to (L2 to L5). Ketamine (Ketamine, Huons, 1.5 ml / kg) and xylazine (Xylazine, Bayer, 0.5 ml / kg) were used for anesthesia of the mouse. To obtain abdominal fat imaging, the voxel size of the in vivo microtomography system was set to 35, the filter was 1.0 mm aluminum, the exposure time was 790 ms, the voltage was 100 kV, the current was 100, and the rotation angle was 1.2 ° (deg). It was.

Gray values corresponding to the tissues in the abdomen of male C57BL / 6 mice are calculated using the Mimics 13.0 (materialize NV, Belgium) program from the images obtained using the in vivo microtomography system. It was. Based on the gray values of the tissues measured using the threshold method, each region is identified by the spine (lumbar vertebral), the lean tissue, the adipose tissue, and the skin in the image. The abdominal adipose tissue extracted was reconstituted into a three-dimensional structure and the volume was measured therefrom. The body weight, total abdominal fat volume, abdominal subcutaneous fat volume and abdominal visceral fat volume of each subject measured are shown in Table 4 below.

As shown in Table 4 and FIG. 4, the distribution of abdominal adipose tissue in the mouse was confirmed through an in vivo microtomography system. It was confirmed to occupy. However, the 10% soybean meal group was found to have more abdominal fat distribution than the high-fat diet group, and the 10% soybean immersion diet group decreased by about 1.7 to 2.7 times as compared to the soybean pure and high-fat diet groups.

In addition, when comparing the body weight and abdominal fat of each experimental group, the body weight was highest in the high fat diet group (40.5 ± 0.5 g), but the volume of visceral fat and subcutaneous fat was the total volume of abdominal fat in the soybean pure diet group. The highest value was 4205.7 ± 511.2 수치. On the other hand, the soybean submerged diet group showed a significant decrease in the volume of abdominal fat compared to the high-fat diet and the soybean meal diet. Especially, the subcutaneous fat volume was 502.0 ± 48.5 ㎣ and 431.8 ± in the normal diet group. The figure was similar to 129.7 kPa.

From the above results, it was confirmed that the soybean immersion diet group was more effective in suppressing the accumulation of abdominal fat due to the high fat diet than the soybean pure diet group.

[ Experimental Example  2]

(One) Soybean Processing  By concentration of by-product treatment Cell-like  Cell survival rate

The 3T3-L1 pre-adipocyte cells used in the experiment were purchased from the American Type Culture Collection (ATCC) and used in Dulbecco's modified Eagle's medium (DMEM, Gibco Inc., USA) medium with 10% fetal bovine serum. 1% antibiotics-antimycotic was added and used in the experiment while subcultured every 2 to 3 days in a 37 ℃ cell incubator containing 5% CO ₂ .

Concentration dependent cell viability by soybean by-product treatment was measured by MTT assay. Incubate the cultured 3T3-L1 cells in a 96 well plate at 5 × 10 ³ cells / well, remove the supernatant after incubation for 24 hours, and extract soybean by-products with 10 ppm, 25 ppm, 100 ppm concentration. Treated with.

The soybean by-product was treated by concentration and incubated for 24 hours, 10 μl of MTT reagent (5 mg / ml) was added per well, and further cultured for 3 hours. Then, the supernatant was removed, and 150 μl of DMSO was dispensed into each well, stirred for 30 minutes, and the absorbance was measured at 570 nm using a microplate reader (Infinite200, Tecan Trading AG, Switzerland). This absorbance represents the amount of MTT reduced by cells and is proportional to the number of cells that survived each well.

Cell viability (%) = (absorbance of sample treated / absorbance of control) × 100

As a result, as can be seen in Figure 5, soybean immersion water showed a cell viability of 95% or more when treated with a high concentration of 100 ppm concentration, 95.21% at 10 ppm, 92.12% at 25 ppm when processing soybean pure water. Cell viability (P <0.05) of 88.65% was shown at ppm, indicating that cell viability was partially reduced when high soybean by-products were treated.

(2) Effect on the effect of inhibiting fat differentiation

In order to analyze the effect on the differentiation inhibitory effect of 3T3-L1 adipocyte cells (cultured cells), soybean immersion water and soybean pure water were treated with differentiation-inducing substances at concentrations of 10 ppm, 25 ppm and 100 ppm, respectively. . After the differentiation induction process, the cells were observed under a microscope by Oil Red O staining and fat content was quantified. Cells not treated with differentiation-inducing substances and cells treated with differentiation-inducing substances were used as controls.

As a result, as can be seen in Figure 6, compared with the cells treated with differentiation-inducing substance, the group treated with soybean pure water extract showed 94.42% at 10 ppm, 87.09% at 25 ppm, and 79.14% at 100 ppm, increasing the treatment concentration. As a result, it was confirmed that fat content in adipocytes was significantly decreased.

In addition, even when observed under the microscope after Oil Red O staining, it can be seen that as the concentration of soybean pure water and soybean immersion water increased, the dyed fat content decreased.

From the above results, it was confirmed that the soybean pure water showed an effect of inhibiting adipocyte differentiation as the concentration increased, and soybean immersion water showed 85.54% of adipocyte differentiation inhibitory effect even at a low concentration of 10 ppm. This is also the result of confirming that soybean processing by-products have an effect of inhibiting the differentiation of fat cells into fat cells to form fat cells.

[ Experimental Example  3]

(1) Differentiation-induced final transcription factor PPAR Effect on the expression of γ

To investigate the effects on the expression of genes involved in differentiation induction of 3T3-L1 adipocyte cells, differentiation-induced control, 10 ppm, 25 ppm and 100 ppm soybean sprouts and soybean immersion water, respectively, The induced experimental groups were compared.

Cells are known to induce the expression of the final transcription factor, PPARγ, by appropriate differentiation stimulation and expression of adipocyte induction regulators. The peroxisome proliferator activated receptor (PPARγ) belongs to the nuclear hormone receptor superfamily, and PPAR family, PPARα, PPARγ, and PPARζ are known. These PPAR transcription factors form heterodimers with Retinoid X receptor α to recognize the peroxisome proliferator response element (PPRE) of target genes and to regulate the expression of adipose cell differentiation-related genes involved in fat formation. Therefore, in order to confirm this, the expression of PPARγ, which is the final transcription factor of adipocyte formation according to differentiation induction and soybean byproduct treatment, was confirmed.

First, total RNA was extracted using TRIzol (Gibco Inc., USA), and then purified using Total RNA Clean up kit (Ambion, USA). Single stranded cDNA was prepared using the SuperScript ™ kit (Invitrogen, USA) as a template for purified RNA. In order to specifically amplify genes related to adipocyte differentiation, primers described in Table 5 were prepared, and the expression level of each gene was compared with that of the GAPDH gene. Gene expression was analyzed using ImageQuant 5.2 (GE Healthcare, USA) program after electrophoresis.

 As can be seen in Figure 9 as a result of confirming the PPARγ mRNA expression pattern for the treatment group treated with soybean sprout and soybean immersion water, compared to the control group induced the differentiation of adipocytes, the treatment group treated with soybean sprout and soybean immersion water mRNA expression was reduced.

Soybean immersion treatment showed less than 50% reduction in all treatment concentrations, but some concentration-independent phenomena were observed. In addition, in the case of soybean sprouts, the concentration of PPARγ decreased in concentration-dependent manner as the treatment concentration was increased, which was in agreement with the cell observation results through Oil Red O staining.

From the above results, it was confirmed that the inhibition of adipocyte differentiation of soybean sprouts and soybean immersion water was achieved by suppressing the expression of PPARγ.

(2) Effect of Soybean Processing By-Products on Local Metabolism

As the process of inducing differentiation of adipocytes progresses, expression of adipose metabolism-related genes is increased by the regulation of PPARγ, a transcriptional regulator. Increased expression of PPARγ is known to increase the absorption of sugar in the body and cause differentiation of adipocytes. In addition, apolipoprotein E (APOE), fatty acid binding protein 4 (Fabp 4), adipsin and stearoyl-coenzyme A desaturase 1 (Scd 1), which are involved in fat metabolism, are regulated by transcriptional regulators such as PPARγ. In this experiment, the expression level of these genes was analyzed to confirm the inhibition of adipocyte differentiation of soybean sprouts and soybean immersion water.

As a result, as can be seen in Figures 10 and 11 it was confirmed that the expression of APOE is reduced to less than 30 to 40% during soybean pure water and soybean immersion treatment compared to the control. Expression of Scd1 gene was increased compared to undifferentiated at induction of differentiation, but after treatment with soybean pure water and soybean immersion water, 50 to 85% of gene expression was confirmed according to the treatment concentration, respectively.

On the other hand, Fabp 4 mRNA expression did not change significantly when the soybean sprouts and soybean immersion treatment compared to the control group. Adipsin is a serine protease that promotes complement peptides by stimulating triglyceride acylation, which is mainly found in cultured fat cells, and is known to induce expression in relation to energy storage signals during differentiation induction. . When soybean sprouts and soybean immersions were treated, adipsin expression was suppressed as a whole in adipose-derived cells. In particular, when 10 ppm of soybeans were treated, the expression level was less than 50% compared to the adipocyte differentiation control group. Decreased to. In soybean immersion treatment, adipsin expression was reduced to 45% and less than 28% at 100 ppm concentration, respectively.

The above result of suppressing mRNA expression of some genes of fat metabolism-related genes is also a result of confirming that soybean pure water and soybean immersion regulate fat process by controlling fat metabolism process.

(3) Effect of Soybean Processing By-Products on Adipocaine

Adipose cells are known to be able to induce various adult diseases such as obesity and diabetes through secretion of various hormones and cytokines as well as the synthesis and storage functions of triglycerides. As adipokines secreted from such adipocytes, resistin (RESTin, RETN), adiponectin (ADIPOQ) and the like are known.

Resistin (RESTin) is present in the cytoplasm of fat and is only expressed in adipose tissue, and is known to be reduced in expression by thialidinedione (TZD), a synthetic ligand of PPARγ. Therefore, the expression level of these adipokines was analyzed.

As a result, as can be seen in Figure 12, the resistin (resistin, RETN), which is expressed when adipocyte differentiation is induced, did not show a significant difference in expression compared to the adipocyte differentiation control group when soybean by-products were treated.

Adiponectin (ADIPOQ), on the other hand, was found to rapidly reduce mRNA expression at all treatment concentrations when soybean immersion and soybean pure were treated.

From the above results, it was confirmed that soybean immersion water and soybean pure water were involved in regulating energy metabolism related to adipogenesis of adipocytes by reducing the amount of adiponectin (ADIPOQ) in adipokines. It was confirmed that the differentiation of adipocytes and the formation of adipocytes were inhibited when the cells were differentiated into fat cells by treatment.

In addition, the expression of adipsin, Fabp4, and adiponectin (ADIPOQ), which regulate fat metabolism and energy metabolism, was inhibited during adipocyte differentiation. Consistent with Oil-Red O staining results, the treatment of soybean by-products was found to be involved in some of the regulation at the liposynthesis stage.

In addition, in view of the result of the decrease in the expression of PPARγ by soybean processing by-products, the inhibition of adipocyte differentiation induced by soybean processing by-products appears mainly through the inhibition of differentiation-inducing transcription factors and at the same time, it inhibits adipogenesis of adipocytes. I could confirm it.

In conclusion, from the above results, the inhibition of adipocyte differentiation and adipogenesis by soybean processing by-products is regulated through the expression of genes related to adipose differentiation and fat metabolism regulation. It could be confirmed that it can be used.

Claims (9)

As an active ingredient, a composition for preventing or treating obesity containing soybean processing by-products. The method of claim 1,
The soy processing by-products are soybean soaking water, soybean curd whey or a mixture thereof, characterized in that the composition for preventing or treating obesity. The method of claim 2,
The soybean immersion water is a composition for preventing or treating obesity, characterized in that obtained by immersing soybean in water at a temperature of 10 to 30 ℃ for 3 to 24 hours. 3. The method according to claim 1 or 2,
The active ingredient is a composition for preventing or treating obesity, characterized in that 0.001 to 30% by weight of the total composition. The method of claim 4, wherein
The active ingredient is fat degeneration and fat accumulation inhibition of liver and epididymal tissue; Inhibition of accumulation of cholesterol in the body; Inhibition of the accumulation of triglycerides, differentiation of fat cells into adipocytes, proliferation of adipocytes; Inhibition of lipogenesis; Inhibition of the activity of acetyl-CoA carboxylase (ACC); And increasing the activity of AMP-induced activated protein kinase (AMPK). The composition for preventing or treating obesity, characterized in that it is carried out by at least one action selected from. 6. The method of claim 5,
The active ingredient is a composition for preventing or treating obesity, characterized in that it inhibits the activity of peroxysome growth factor activating receptor gamma (PPARγ, peroxisome proliferator activated receptor). 3. The method according to claim 1 or 2,
The composition is a composition for preventing or treating obesity, characterized in that it comprises one or more pharmaceutically acceptable carriers or excipients. A dietary supplement for the prevention or treatment of obesity, comprising the composition of claim 1. The method of claim 8,
The dietary supplement is a dietary supplement for the prevention or treatment of obesity, characterized in that at least one selected from drink, caramel, chocolate, diet bar formulation, and confectionary.

Images