KR102657151B1 - A composition for anti-obesity containing Rosa rugosa extract - Google Patents

Abstract

The present invention relates to an anti-obesity composition containing a glycolysis extract, which inhibits fat accumulation, reduces triglyceride content, and reduces the expression of PPARγ and C/EBPα proteins that regulate adipogenesis by containing glycolysis stem extract as an active ingredient. , a composition with excellent anti-obesity efficacy by reducing the expression of SREBP-1c and SCD-1 proteins that regulate fatty acid synthesis (lipogenesis) and increasing the expression of AMPK phosphorylated protein that inhibits adipogenesis and fatty acid synthesis (lipogenesis). It was intended to be provided.

Description

Anti-obesity composition containing glycolic acid extract as an active ingredient {A composition for anti-obesity containing Rosa rugosa extract}

The present invention relates to an anti-obesity composition containing a glycolic flower extract as an active ingredient, and more specifically, to confirm the anti-obesity functionality of a glycolytic flower stem extract, establish optimal extraction conditions, and provide a composition containing a glycolytic flower extract with excellent anti-obesity effect. It relates to an anti-obesity composition.

Obesity refers to a condition in which an excessive amount of body fat accumulates in the body, and is recognized as one of the important diseases that modern humanity must overcome. Obesity is a phenomenon in which excess energy is accumulated in the form of body fat due to low energy consumption compared to the nutrients consumed chronically. The World Health Organization (WHO) defines obesity as an abnormal or excessive accumulation of fat that poses a health risk. A body mass index (BMI) of 25 or more was defined as overweight, and a body mass index (BMI) of 30 or more was defined as obesity.

Meanwhile, global obesity has nearly tripled since 1975, and in 2016, more than 1.9 billion adults aged 18 or older were overweight, of which more than 650 million were obese. In addition, as of 2020, 39 million children under the age of 5 are overweight or obese, and the number of obesity patients is rapidly increasing, and the situation is worsening due to the recent COVID-19 situation.

Obesity is caused by a very complex genetic or phenomenological relationship between various neuroendocrine substances and various elements related to energy metabolism, including irregular eating habits, excessive food intake, lack of exercise, endocrine diseases, genetic factors, and mental health. The causes are very diverse due to factors and drugs. If obesity occurs and continues, it causes various complications such as high blood pressure, diabetes, hyperlipidemia, fatty liver, obstructive sleep apnea, and degenerative arthritis, and also increases the risk of various cancers such as colon cancer, pancreatic cancer, prostate cancer, and breast cancer.

Accordingly, various treatments for obesity are being studied, but the available treatments for obesity are very limited due to various side effects. As of 2020, obesity treatments available in Korea include drugs approved for short-term use such as phentermine, diethylpropion, phendimetrazine, and mazindol, orlistat, naltrexone/bupropion sustained-release tablets, phentermine/topiramate capsules, and liraglutide. There are medications approved for long-term use, such as: The obesity treatment market has increased from USD 1.2 billion in 2016 to USD 1.75 billion in 2020, and various obesity treatments are being researched at home and abroad (Ministry of Trade, Industry and Energy 「Bio-industry Technology Development Project」 information provision report, 2021.07., Korea Pharmaceutical Bio Association).

Accordingly, in the present invention, we attempted to prepare a composition that can effectively improve obesity using the natural product Rosa rugosa THUNB. By including glycolysis stem extract, it inhibits fat accumulation, reduces triglyceride content, and improves PPARγ and C/ We sought to develop a composition that exhibits excellent anti-obesity effects by reducing EBPα protein expression, reducing SREBP-1c and SCD-1 protein expression, and increasing AMPK phosphorylated protein expression.

Republic of Korea Patent No. 10-1407150 (registered on June 5, 2014) describes a composition for the treatment and prevention of diseases related to exercise and physical stress containing glycolic flower extract as an active ingredient. Republic of Korea Patent Publication No. 10-2021-0122167 (published on October 8, 2021) discloses a composition for preventing, improving or treating female menopausal syndrome containing glycolic acid extract as an active ingredient.

The present invention provides an anti-obesity composition that inhibits fat accumulation, reduces triglyceride content, and regulates the expression of proteins related to adipogenesis and fatty acid synthesis in 3T3-L1 adipocytes by containing glycolysis extract as an active ingredient. We want to develop and provide it.

The present invention provides an anti-obesity composition containing an extract of Rosa rugosa THUNB. as an active ingredient.

In the anti-obesity composition of the present invention, the extract may be characterized by extracting glycolytic stems.

In the anti-obesity composition of the present invention, the extract includes the steps of (a) washing and pulverizing the glycolysis stems; After step (a), extraction is performed for 5 to 7 hours by adding 9 to 11 times the amount of water or ethanol aqueous solution compared to the pulverized glycolysis stems; (b); After step (b), (c) cooling at room temperature and then centrifuging; After step (c), filtering the supernatant and then concentrating it under reduced pressure (d); After step (d), freeze-drying step (e); It may be characterized in that it is manufactured including.

In the anti-obesity composition of the present invention, the composition may be characterized as being a food composition.

The present invention relates to an anti-obesity composition containing a glycolysis extract. By containing glycolysis stem extract as an active ingredient, it inhibits fat accumulation, reduces triglyceride content, reduces PPARγ and C/EBPα protein expression, SREBP-1c and SCD-1. It can provide a composition with excellent anti-obesity efficacy by reducing protein expression and increasing AMPK phosphorylated protein expression.

Figure 1 is a diagram showing the results of confirming the effect on the cell viability of 3T3-L1 preadipocytes by glycolysis extraction site and extraction conditions.
Figure 2 is a diagram showing the results of confirming the effect on lipid accumulation by glycolysis extraction site and extraction conditions.
Figure 3 is a diagram showing the results of confirming the effect on triglyceride contents by glycolysis extraction site and extraction conditions.
Figure 4 is a diagram showing the results of confirming the effect of glycolysis stem extract on PPARγ protein expression.
Figure 5 is a diagram showing the results of confirming the effect of glycolysis stem extract on C/EBPα protein expression.
Figure 6 is a diagram showing the results of confirming the effect of glycolysis stem extract on SREBP-1c protein expression.
Figure 7 is a diagram showing the results of confirming the effect of glycolysis stem extract on SCD-1 protein expression.
Figure 8 is a diagram showing the results of confirming the effect of glycolysis stem extract on AMPK phosphorylated protein expression.

The present invention provides an anti-obesity composition containing an extract of Rosa rugosa THUNB. as an active ingredient.

Rosa rugosa THUNB. is a deciduous broad-leaved shrub belonging to the Rosaceae family, and is also called maegoe. The young shoots of this flower are eaten as a vegetable and the roots are known to be good for diabetes, toothache, and arthritis. The flowers are used for pain relief and hemostasis, and are also used as a raw material for perfume. It doesn't matter what type of soil it is, but it grows well in fertile sandy loam with moderate humidity, and it mainly grows on sandy soil along the coast. In addition, it can overwinter in the open field, can be cultivated anywhere in the country, and is resistant to drought and salt damage.

Meanwhile, in the anti-obesity composition of the present invention, the extract may be extracted from any one or more parts selected from the stem, root, or fruit of the glycolic flower, but it is preferable to extract the stem of the glycolic flower.

In the anti-obesity composition of the present invention, the extract includes (a) washing and pulverizing the glycolysis stems; After step (a), extracting for 5 to 7 hours by adding 9 to 11 times the weight of water or ethanol aqueous solution compared to the weight of the pulverized glycosylated stems; After step (b), (c) cooling at room temperature and then centrifuging; After step (c), filtering the supernatant and then concentrating it under reduced pressure (d); After step (d), freeze-drying step (e); It is recommended to manufacture it including. At this time, more preferably, the extraction in step (b) is performed for 6 hours by adding 10 times the weight of water or ethanol aqueous solution compared to the weight of the glycolysis stem.

Meanwhile, in the anti-obesity composition of the present invention, the composition may be characterized as a food composition.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes all modifications of the technical idea equivalent thereto.

[Example 1: Preparation of glycolic acid extract]

In this example, glycolic flower stems and roots were extracted according to each condition to prepare glycolic flower stem extract and glycolic flower root extract, respectively.

First, to prepare each extract, the glycolytic stems and roots were washed and pulverized, then 10 times the weight of the extraction solvent was added and extracted for 6 hours according to the conditions in Table 1 below. Then, after cooling at room temperature, centrifugation was performed at 3,000 , Tokyo, Japan). Then, each glycolysis extract was prepared by drying with a freeze dryer (FD 8508, Ilshin, Korea).

Extraction conditions for producing glycolic flower stem extract and glycolic flower root extract T1 T3 T5 menstruum 100% water 70% water / 30% ethanol 50% water / 50% ethanol temperature 100℃ 60℃ 60℃ hour 6hrs 6hrs 6hrs

[Experimental Example 1: Confirmation of cell viability of glycolic flower stem and glycolic flower root extract]

In this experiment, we sought to determine the effect of the extracts prepared in Example 1 on the cell viability of 3T3-L1 preadipocytes by the glycolysis extraction site and extraction conditions.

3T3-L1 preadipocytes were purchased from the American type culture collection (ATCC, Manassas, USA) and cultured in dulbecco's supplemented with 10% bovine calf serum (Welgene, Daegu, Korea) and 1% penicillin-streptomycin (Welgene, Daegu, Korea). Cultured in modified eagle's medium (DMEM, Welgene, Daegu, Korea) at 37°C and 5% CO ₂ conditions. The toxicity of glycolytic stem extract and glycolic root extract on 3T3-L1 cells was assessed using the 3-[4,5-dimethylthiazole-2-yl]-2,5-di-phenyl-tetrazolium bromide (MTT) reduction method. did.

First, undifferentiated 3T3-L1 cells were distributed in a 96 well plate at 100 uL at 1×10 ⁵ cells/mL and cultured for 24 hours. Then, the material was diluted in medium without BCS and injected into the cells. After treatment, the cells were cultured for 24 hours. The culture medium containing the material was removed, MTT (0.5 mg/mL) was dissolved in the culture medium, 100 uL was dispensed, and the culture medium was removed after culturing at 37°C for 4 hours. Then, 100 μL of DMSO was added to the formazan formed in each well and dissolved using a shaker. After 30 minutes, the formazan was measured using a UV/vis spectrophotometer (Opiwen 2120UV plus, Mecasys Co. Ltd., Korea) was used to measure the absorbance at 570 nm, and cytotoxicity was confirmed based on the absorbance value of the control group.

Figure 1 is a graph showing the effect of glycolysis stem extract and glycolysis root extract on the cell viability of 3T3-L1 preadipocytes by concentration. As a result of the experiment, as shown in Figure 1, it was confirmed that glycolysis stem and glycolysis root extracts were not toxic to 3T3-L1 preadipocytes up to a concentration of 0.2 mg/mL.

[Experimental Example 2: Confirmation of the effect on fat accumulation of extracts by glycolysis extraction site and extraction conditions]

In this experiment, we attempted to determine the effect on lipid accumulation of the extracts prepared from the glycolysis extraction site and extraction conditions prepared in Example 1.

To differentiate 3T3-L1 preadipocytes into adipocytes, 5 × 10 ⁵ cells/well were dispensed into a 6 well plate and cultured so that the cells were completely dense. After culturing for 2 more days, the cells were cultured in MDI solution. Differentiation was initiated by culturing for 2 days in DMEM medium containing (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 uM dexamethasone, 10 μg/mL insulin) and 10% FBS, followed by 10 μg/ml insulin. Differentiation was carried out for 2 days by replacing the medium with DMEM containing (insulin) and 10% FBS. After that, differentiation into adipocytes forming lipid droplets was completed by intracellular fat accumulation by culturing them in DMEM medium containing only 10% FBS for 4 days. To measure lipid accumulation, glycolytic stem extract and glycolytic root extract were simultaneously treated at concentrations of 0.05, 0.075, and 0.1 mg/mL every time the MDI solution and medium were changed, and cultured until differentiation induction was completed. Cells after completion of differentiation induction were washed twice with PBS, treated with 10% formalin, fixed at 4°C for 1 hour, washed, and treated with 60% isopropanol solution to stain adipocytes. Stained cells were washed with PBS, oil red O was eluted using 100% isopropanol, and absorbance was measured at 520 nm to confirm lipid accumulation compared to the control group.

Figure 2 is a graph showing the effect of glycolysis stem extract and glycolysis root extract on lipid accumulation by concentration. As a result of the experiment, as shown in Figure 2, it was confirmed that the glycolytic stem extract was superior to the glycolytic root extract in suppressing fat accumulation in 3T3-L1 adipocytes.

[Experimental Example 3: Confirmation of the effect on the triglyceride content of the extract by glycolysis extraction site and extraction conditions]

In this experiment, we attempted to determine the effect on triglyceride contents of the extracts prepared by the glycolysis extraction site and extraction conditions prepared in Example 1.

Triglyceride contents were measured using the EZ-Triglyceride Quantification Assay Kit. To differentiate 3T3-L1 preadipocytes into adipocytes, 5 × 10 ⁵ cells/well were dispensed into a 6 well plate and cultured so that the cells were completely dense. After culturing for 2 more days, the cells were cultured in MDI solution. Differentiation was initiated by culturing for 2 days in DMEM medium containing (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 uM dexamethasone, 10 ug/mL insulin) and 10% FBS, followed by 10 ug/ml insulin. Differentiation was carried out for 2 days by replacing the medium with DMEM containing (insulin) and 10% FBS. After that, differentiation into adipocytes forming lipid droplets was completed by intracellular fat accumulation by culturing them in DMEM medium containing only 10% FBS for 4 days. Lipid accumulation was measured by simultaneously treating glycolytic stem extract and glycolytic root extract at concentrations of 0.05, 0.075, and 0.1 mg/mL every time the MDI solution and medium were changed, and culturing the cells until differentiation induction was complete. Cells after completion of differentiation induction were washed twice with PBS, homogenized using NP40, heated at 100°C to dissolve triglycerol present in the cells, and centrifuged at 13,000 rpm for 2 minutes to remove insoluble substances. For lysate and standard, add 50 uL of lipase and 2 uL of lipase to a 96 well plate, react for 20 minutes at room temperature, and then add 46 uL of triglyceride assay buffer, 2 uL of triglyceride enzyme mix, and 2 uL of triglyceride probe. After each addition, they were reacted at room temperature for 30 minutes, and the absorbance was measured at 570 nm to check the triglyceride content (TG contents) compared to the control group.

Figure 3 is a graph showing the effect of glycolysis stem extract and glycolysis root extract on triglyceride contents by concentration. As a result of the experiment, as shown in Figure 3, it was confirmed that the glycolytic stem extract was superior to the glycolytic root extract in suppressing triglyceride content in 3T3-L1 adipocytes.

[Experimental Example 4: Confirmation of the effect of each extraction condition on glycolysis stem extract on intracellular protein expression]

In this experiment, in order to determine the effect of the glycolysis stem extract prepared in Example 1 on intracellular protein expression according to the extraction conditions, the glycolysis stem extract was tested for adipogenesis and fatty acid synthesis (lipogenesis) in 3T3-L1 adipocytes. An experiment was conducted to confirm the effect on the expression of the regulating protein.

3T3-L1 adipocytes that had completed differentiation were treated with glycolysis stem extract at concentrations of 0.05 and 0.1 mg/mL and cultured for 24 hours. Cells after completion of culture were scraped with a cell scraper, centrifuged at 5,000 rpm for 10 minutes, and lysed using RIPA buffer. For the lysed cells, the same amount of protein quantified by the Bradford method was mixed 3:1 in a sample buffer containing sodium dodecyl sulfate (SDS) and β-mercapto-ethanol, and then heated at 100°C for 10 minutes. Protein samples were electrophoresed using SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (0.45 μm, PVDF transfer membrane, Thermo, Rockford, IL, USA). The membrane was blocked in tris-buffered saline (TBS) containing 0.1% tween 20 and 5% skim milk for 2 hours. Then PPARγ (1:1000), C/EBPα (1:1000), SREBP-1c (1:1000), SCD-1 (1:1000), pAMPK (1:1000), AMPK (1:1000) ( Cell signaling technology, Danvers, MA, USA) reacted overnight in buffer containing primary antibody and washed three times for 5 minutes each with TBS-T (TBS containing 0.1% tween 20). Then, it was reacted for 1 hour in a buffer containing horseradish peroxidase-conjugated secondary antibody (1:1000) (Cell signaling technology, Danvers, MA, USA) and then spotted on x-ray film using the enhanced chemiluminescence method. Sensitized. The intensity of the detected band was analyzed using imageJ (National institutes of health, Bethesda, MD, USA) software.

First, as a result of confirming the effect of the glycolysis stem extract on the expression of PPARγ and C/EBPα proteins that regulate adipogensis, as shown in Figures 4 and 5, the glycolysis stem extract prepared in Example 1 was all concentration-dependent. This reduced PPARγ and C/EBPα protein expression.

Meanwhile, as a result of confirming the effect of glycolysis stem extract on the expression of SREBP-1c and SCD-1 proteins that regulate fatty acid synthesis (lipogenesis), as shown in Figures 6 and 7, all glycolysis stem extracts prepared in Example 1 were It reduced SREBP-1c and SCD-1 protein expression in a concentration-dependent manner.

In addition, as a result of confirming the effect of the glycolysis stem extract on the expression of AMPK phosphorylated protein that inhibits adipogenesis and fatty acid synthesis (lipogenesis), as shown in Figure 8, all glycolysis stem extracts prepared in Example 1 showed AMPK phosphorylation. Increased protein expression.

In this way, the glycolysis stem extract of the present invention inhibits fat accumulation, reduces triglyceride content, reduces PPARγ and C/EBPα protein expression, reduces SREBP-1c and SCD-1 protein expression, and increases AMPK phosphorylated protein expression, thereby preventing obesity. It was confirmed that it has an excellent effect on prevention and improvement. In addition, it was found that a composition with excellent anti-obesity effect could be prepared by containing the glycolytic stem extract of the present invention as an active ingredient.

Claims (4)

Contains Rosa rugosa THUNB. stem extract as an active ingredient and has the effect of inhibiting fat accumulation in adipocytes.
The extract is,
Step (a) of washing and pulverizing the glycolysis stem;
After step (a), extracting at 60°C for 6 hours by adding 9 to 11 times more 50% (v/v) ethanol aqueous solution than the pulverized glycosylated stem;
After step (b), (c) cooling at room temperature and then centrifuging;
After step (c), filtering the supernatant and then concentrating it under reduced pressure (d);
After step (d), freeze-drying step (e); An anti-obesity composition comprising:
delete Step (a) of washing and pulverizing the stems of Rosa rugosa THUNB.;
After step (a), extracting at 60°C for 6 hours by adding 9 to 11 times the amount of 50% (v/v) ethanol aqueous solution compared to the pulverized glycosylated stem;
After step (b), (c) cooling at room temperature and then centrifuging;
After step (c), filtering the supernatant and then concentrating it under reduced pressure (d);
After step (d), freeze-drying step (e); Including the process of producing glycolysis ( Rosa rugosa THUNB.) stem extract,
A method for producing an anti-obesity composition, wherein the glycolytic stem extract has an effect of inhibiting fat accumulation in adipocytes.
According to paragraph 1,
The composition is,
A composition for anti-obesity, characterized in that it is a food composition.