KR20150053321A - Anti obesity Composition comprising anthocyanin originated from black bean peels as an active ingredient - Google Patents
Anti obesity Composition comprising anthocyanin originated from black bean peels as an active ingredient Download PDFInfo
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Abstract
The present invention relates to a natural food-derived anti-obesity functional food material which improves the side effects of obesity and obesity treatment drugs, which are common causes of various chronic diseases, and exhibits pharmacological activity for the treatment of obesity. The extract of the black bean husks according to the present invention contains cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G), cyanidin-3-galactoside (cy3gal), petunidin-3-glucoside (pt3glc), pelargonidine -3-glucoside (pg3glc) and peonidin-3-glucoside (pn3glc), which have excellent effects on improving lipid metabolism and preventing and treating obesity.
Description
The present invention relates to an anti-obesity functional food material derived from a natural food having excellent pharmacological activity in consideration of adverse effects of drugs for prevention of obesity and treatment of obesity related to lipid metabolism, which are common causes of various chronic diseases.
Obesity is a condition in which excess fat tissue is accumulated in the body. The main cause of obesity is the increase in body weight due to lack of consumption energy, especially exercise, after consuming high calorie or high fat foods (Bray GA, Popkin BM): Dietary fat affects obesity. Am J Clin Nutr 68: 1157-1173 (1998), Bray GA, Popkin BM. Am J Clin Nutr 70: 572-573 (1999)).
In Korea, the obesity population is steadily increasing, and the global increase in childhood obesity is three times as high as 10 years, which is expected to lead to an increase in the number of obese adults. In addition, obesity causes severe adult diseases such as diabetes, arteriosclerosis, cardiovascular disease, hypertension and hyperlipidemia due to weight gain, and the incidence of various cancers such as colorectal cancer, rectal cancer, prostate cancer, breast cancer and ovarian cancer is increased. (Gastroenterol Clin. North Am 20: 1-19 (1991), Wada K, Ikeda Y.: "Epidemiology and natural history of gallstone disease. J Med Sys 22: 19-25 (1998)).
Studies on anti-obesity functional foods have been carried out all over the world, and orlistat, which is currently approved for weight loss, is accompanied by many side effects such as fatness, abdominal bloating, insomnia, headache rash and the effect on visceral fat loss is unknown . In addition, the existing obesity inhibitors (pethamin, pendimetrazine, diethipropion, marginal) are focused on weight loss, affecting not only the body weight but also the amount of muscle and bone, and the side effects are serious. Therefore, there is a growing interest in the study of the mechanism of action of natural food functional dietary materials, because of the side effects of obesity treatment drugs.
Especially in Korea, researches are focused on traditional foods. Among them, various physiologically active ingredients contained in soybeans, which are the main ingredients of fermented foods, are combined with various metabolites to function as health functional ingredients, thus exhibiting preventive and therapeutic effects on various adult diseases such as cancer, cardiovascular disease, osteoporosis and kidney disease Cancer Lett 114: 183-184 (2003). Effects of capsaicin on chemically induced two-stage mouse skin carcinogenesis. Cancer Lett 114: 183-184 1997), Rhee SH, Kong KR, Jung KO, Park KY. Decreasing effect of Kochujang on body weight and lipid levels of adipose tissues and serum in rats fed a high-fat diet. J Food Sci Nutr 32: 882-886 2003).
The black soybeans contained anthocyanin pigment in the skin (Bae EA, Moon GS: A study of the antioxidative activities of Koeran soybeans. J Korean Soc Food Sci Nutr 26: 203-208 (1997) The antioxidative effect of soybean was higher than that of yellow soybean, but it was higher than that of yellow soybeans. (RDAJ Crop Sci 39: 35-38 (1997)). The antioxidant effect of soybean was highly related to anthocyanin content, which is a major pigment of soybean husk (Son JH, Choung MG, Choi HJ, Jang UB, Last GB, Byun MW, Choi C.: Physiological effect of Korean black soybean pigment. Korean J Food Sci Technol 33: 764-768 (2001)). In particular, the black part of the black soybean skin showed antioxidative properties. The darker the black color, the better the antioxidant effect. The test group containing methanol extract of black bean chungkukjang showed higher antioxidant ability than the test group containing methanol extract of soybean chungkukjang (Pratt DE, Birac PM.: Sources of antioxidant activity of soybeans and soy products. J Food Sci 44: 1720-1727 (1979))
The extracts of alcoholic beverages containing anthocyanin and / or anthocyanidin for the prevention and treatment of diabetes are disclosed in Korean Laid-Open Publication No. 10-2012-0090089. In this document, as the target plant, black bean pellets, asiaberry, bilberry, black currant and the like are contained, and 50% v / v of ethanol / water is used as an extracting solvent of the fruit, and the extract is used for immune diseases and metabolic syndrome It is aimed at diabetes.
In addition, the anti-diabetes or obesity treatment effect of various phytochemicals obtained from the hot-water extract obtained by steaming the various bean sprouts including black soybean grains is disclosed in U.S. Publication No. 2004-0131749. U.S. Patent Nos. 8,198,249 and 8,329,743 disclose a composition for inhibiting lipogenesis including a stilbene derivative or anthocyanin together with a benzophenone derivative of a soybean plant or a composition for inhibiting insulin secretion comprising purified C3G (cyanidin-3-glucoside) And a method for promotion.
However, none of these documents have suggested or taught the lipid metabolism or anti-obesity effects of the pigment compounds C3G (cyanidin-3-glucoside) and D3G (delphinidin-3-glucoside)
In addition, Korean Patent Laid-Open Publication No. 10-2009-0124274 discloses an anti-obesity active drink containing anthocyanin extract derived from black soybean kernel or genistein as an active ingredient. However, it is different from the present invention and uses a different compound It is only doing.
Therefore, the inventors of the present invention found that anthocyanin compounds derived from alcohol extracts of black bean husks, especially cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G), cyanidin-3-galactoside (cy3gal), petunidin- ), pelargonidin-3-glucoside (pg3glc), and peonidin-3-glucoside (pn3glc) compounds and their related gene and protein expression effects of visceral fat loss, fat accumulation and lipolysis And the present invention has been completed.
3-glucoside (C3G), cyanidin-3-galactoside (cy3gal), petunidin-3-glucoside (pT3GLc), pelargonidin- 3-glucoside (pG3glc) And peonidin-3-glucoside (pn3glc) compounds have not been tested for their improved lipid metabolism and anti-obesity efficacy.
It is an object of the present invention to provide a spirit extract containing an anthocyanin pigment compound derived from a pea shell.
Another object of the present invention is to provide an anthocyanin pigment which comprises cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G), cyanidin-3-galactoside (cy3gal), petunidin- -glucoside (pg3glc) and peonidin-3-glucoside (pn3glc) as an active ingredient.
It is still another object of the present invention to provide a pharmaceutical composition for preventing and treating obesity containing the above-mentioned compound as an active ingredient.
The above object of the present invention can be achieved by a method for producing a black soybean skin extract, Confirming the peak by HPLC chromatogram of the alcohol extract; Progenitor adipocytes and lipolytic cells to confirm lipolysis ability.
As described above, the present invention has an effect of providing a novel alcoholic extract derived from a black bean skin. Further, the present invention has an effect of providing a novel alcoholic extract derived from a black bean skin, and furthermore, cyanidin-3-glucoside (C3G), delphinidin- , petunidin-3-glucoside (pt3glc), pelargonidin-3-glucoside (pg3glc) and peonidin-3-glucoside (pn3glc) as effective ingredients and an anti-obesity food composition .
FIG. 1 is an HPLC chromatogram of the alcohol extract of the black bean husk according to the present invention. FIG.
2 is a schematic diagram of a 3T3-L1 precursor adipocyte culture protocol according to the present invention.
3 is a schematic diagram of an experimental animal dietary schedule according to the present invention.
Figure 4 is a graph showing the effect of cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G), cyanidin-3-galactoside (cy3gal), petunidin-3-glucoside (pt3glc), pelargonidin- (A) MTT assay and (B) Oil red-O staining of 3T3-L1 precursor adipocytes with a composition containing a mixture of peonidin-3-glucoside (pn3glc)
FIG. 5 shows results of western blotting of 3T3-L1 cells (Day 8) according to the present invention.
FIG. 6 is a graph showing the weight of mesentery fat of a mouse induced by a 45% high fat diet according to the present invention and a photograph showing mesentery fat.
FIG. 7 is an H & E staining photograph of epididymal fat of a mouse according to the present invention. FIG.
8 is a graph (A) showing the Troglyceride level measured in the liver of the mouse according to the present invention and a graph (B) showing the total cholesterol content in the blood.
FIG. 9 is a graph (A) showing the measurement of the lipid peroxide level in the mouse liver according to the present invention and a graph (B) showing the ALT in the blood.
FIG. 10 shows the results of measurement of the expression of genes (PPARγ, CEBP / α and ACC) involved in lipogenesis in the mouse mesentery fat induced by the 45% high fat diet according to the present invention.
FIG. 11 shows the results of measurement of the expression of proteins (HSL and AMPK) involved in lipid degradation in mouse mesentery fat induced by the 45% high fat diet according to the present invention.
According to the present invention, the black bean skin is preferable for the improvement of obesity. However, Celia 3 was the most preferred for the anti - obesity effect. On the other hand, the ratio of the alcohol extraction solvent was preferably 50 to 100%. However, the highest yield of anthocyanin components was detected in 80% of the alcohol solution, which proved to be the most desirable. In carrying out the present invention, one or more black bean cultivars may be selected and used so that the C3G component and the D3G component are contained at a ratio of about 3: 1 (w / w) in the group consisting of the black beans described in the following [Table 1].
Obtaining an 80% alcohol extract of the bark of Black
However, the scope of the present invention is not limited to the following examples and experimental examples.
Example 1. Preparation and composition analysis of black bean skin extract
1 kg of black bean (Celadon No. 3, origin: Gyeongsangnam-do, Milyang) was extracted with 15 times solvent (80% ethanol, 0.1% formic acid) in the dark room for 24 hours. The extract (15.24 L) obtained above was filtered through a filter paper, and the filtrate was concentrated using a vacuum rotary condenser at 35 ° C or lower to completely remove ethanol. It was freeze-dried and homogenized by a pulverizer at a temperature of 30 캜 or higher. The crude extract extracted from the above was subjected to HPLC for the analysis of its components, and the resultant chromatogram is shown in FIG. The major components of the extract of the present invention were 3G (cyanidin-3-glucoside) and D3G (delphinidin-3-glucoside) and the content ratio (w / w) was about 3: 1.
Experimental Example 1. Cultivation of pre-adipocytes and treatment of extracts
3T3-L1 progenitor adipocytes were maintained in DMEM medium containing 10% BCS, and after 2 days in confluence, differentiation was induced in media containing 10% FBS, 10 ug / mL Insulin, 0.5 mM isobutyl-methylxanthin and 1 μM Dexamethasone (Fig. 1). The fresh medium was replaced every 2 days from the date of induction of differentiation, and the 80% ethanol extract prepared in Example 1 was treated with 100 and 300 ug / mL, respectively.
Experimental Example 2. MTT assay And Oil red -O staining
One) MTT assay
MTT assays were performed on precursor adipocytes and differentiated cells (
2) Oil red -O staining
The fully differentiated 3T3-L1 cells (Day 8) were fixed in formaldehyde solution, stained with Oil Red-O solution for 20 min, and dissolved in Isopropylalcohol, and the absorbance was measured at 520 nm. As a result of the experiment, it was confirmed that the extract of black bean bark of the present invention had an effect on lipid accumulation of adipocytes because the decrease of lipid accumulation amount was larger than the decrease of cell number. However, hydroxycitrate (HCA), a target of Garcinia cambogia known as an anti-obesity material, did not affect cell viability and lipid content (Fig. 4B).
Experimental Example 3. Western Blotting
Western blotting was performed on fully differentiated 3T3-L1 cells (Day 8). As a result, it was confirmed that AMPK related lipid degradation was significantly increased in the extract of black bean bark of the present invention at a concentration of 300 μg (FIG. 5A). In addition, it was confirmed that PPARγ and ACC involved in lipogenesis were significantly decreased in the extract of black bean bark extract having a concentration of 300 μg (FIG. 5B).
Experimental Example 4. Breeding conditions of experimental animals
The animals used in the present invention were 4 week old male C57BL / 6N mice of 12-15 g, which were adapted for 1 week after the purchase and then sacrificed at a high fat group (HF, n = 31) were induced with diet-induced obesity (DIO) at 45% high fat diet for 10 weeks. After the induction of DIO, the high-fat diet group was divided into HF-control (HF n = 10), HF + 1 g / kg black soybean extract (BB n = 9) and HF + 30 mg / kg Orlistat The group was orally administered for 12 weeks (Fig. 3). During the experimental period, the incubation temperature was maintained at 20 ~ 22 ℃, the light cycle was maintained at 12 hour intervals, and the humidity was maintained at 55 ± 1%.
After sacrifice by anesthetizing with ethyl ether, blood was collected from the heart, and liver and adipose tissues (epididymal fat, mesentery fat, subcutaneous fat) were extracted, weighed and placed in liquid nitrogen, and stored at -80 ° C until analysis . Western blotting, RT-PCR, and hematoxylin and eosin stain (H & E stain) were performed by conventional methods for the analysis of proteins, enzymes, and genes involved in lipid synthesis and degradation.
Experimental Example 5. Experimental animal groups Dietary efficiency
The body weight gain and the fat tissue weight of the experimental animals according to the present invention are shown in Table 2 below.
In obesity - induced rats, high - fat diet group had the highest weight gain and lowest group O group. There was no significant difference between the high fat diet group and the black soybean skin intake group. However, the final weight and weight gain of the black soybean skin extract group (1 g / kg) of the present invention was lower than that of the high fat dietary group. In addition, the dietary intake was the lowest in the group fed with the extract of black bean husk extract of the present invention. Dietary efficiency was the lowest in group O, and the intake of the extract of black bean husk extract of the present invention tended to be lower than that of high fat dietary group, but it was not significant.
Experimental Example 6. Characterization of adipose tissue in organ or tissue of experimental animals
1) Weight of mesenteric fat tissue
As shown in Fig. 6, the ingestion of black soybean skin suppressed the increase of mesenteric fat. That is, there was a significant decrease compared to the highest fat diet group, and there was a significant difference in all four groups.
2) H & E of the epididymis Staining
The paraffin section of the extracted epididymal fat was 5 ㎛ and the fat tissue size was compared by H & E staining. As a result, it was observed that the extract of the present invention was reduced from the fat content of the high fat diet group and the O group ).
3) Measurement of blood and liver lipid levels
Triglyceride (TG) levels in the liver were the highest in the high fat diet group and lowest in the black crab shell diet extract group of the present invention (p <0.0001) (Fig. 8A). The total cholesterol (TC) content in the blood was also highest in the high fat dietary group, while the ingestion of the black soybean husk extract of the present invention was higher than that of the O group but lower than that of the high fat dietary group and the control group (FIG. However, there was no significant difference between total cholesterol content and total cholesterol content in the liver according to the treated material.
4) Peroxide content and Degree of damage
As a result of the measurement of MDA, a lipid peroxide in the liver, the extract of black bean skin extract according to the present invention was significantly lower than that of the high fat dietary group (FIG. 9A). We measured alanine aminotransferase (ALT), which is more sensitive to liver damage than aspartate aminotransferase (AST), in liver damage (hepatocyte necrosis and tissue destruction). Experimental results showed that the group of black bean peel extract of the present invention had the lowest serum ALT level. Thus, it was confirmed that there was no hepatotoxicity (Fig. 9B). Especially, the ALT normal value range of mouse was 17-77 U / I, so it was confirmed that the hepatotoxicity of all treatment groups was not shown.
5) Lipid synthesis lipogenesis ) Related gene expression measurement
The degree of gene expression of lipid synthesis related factors (PPARγ, CEBP / α, ACC) in mesenteric fat was measured by the qPCR method. Experimental results showed that the expression of PPARγ was not different between all treatment groups, but was lowest in O group. The extract of black bean skin extract of the present invention significantly inhibited the expression of C / EBPa and ACC gene in the high fat dietary group, and particularly inhibited the oritate level of the obesity drug (FIG. 10).
6) Lipolysis ( lipolysis ) Related protein expression
Western blot analysis was used to identify protein expression (p-HSL, AMPK) of factors involved in lipid degradation in mesenteric fat. As a result, p-HSL was higher than that of the high fat diet group, but there was no significant difference between the groups. In addition, the expression of AMPK was the highest in the group fed with the extract of the black bean husk extract of the present invention (Fig. 11).
The present invention relates to a method for producing cyanidin-3-glucoside (C3G), cyanidin-3-glucoside (D3G), cyanidin-3-galactoside (cy3gal), petunidin-3-glucoside (pt3glc), pelargonidin- ) And peonidin-3-glucoside (pn3glc), and thus it is a very useful invention in the biomedical industry using natural materials.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170054686A (en) | 2015-11-10 | 2017-05-18 | 순천향대학교 산학협력단 | Pharmaceutical composition for treatment of obesity comprising delphinidin or pharmaceutically acceptable salts thereof as an effective component |
| KR20180042920A (en) | 2016-10-19 | 2018-04-27 | 학교법인혜화학원 | Extracts from red bean shell having anti obesity and Compositions comprising the same as an active ingredient |
| CN110548039A (en) * | 2019-09-23 | 2019-12-10 | 长春理工大学 | Weight-losing medicine for food-borne obesity and preparation method thereof |
| EP3586857A1 (en) * | 2018-06-29 | 2020-01-01 | Korea Institute of Science and Technology | Seed of new soybean cultivar scel-1, plant body of the seed and part of the plant body, and extract obtained from the seed |
-
2013
- 2013-11-07 KR KR1020130135066A patent/KR20150053321A/en active Application Filing
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170054686A (en) | 2015-11-10 | 2017-05-18 | 순천향대학교 산학협력단 | Pharmaceutical composition for treatment of obesity comprising delphinidin or pharmaceutically acceptable salts thereof as an effective component |
| KR20180042920A (en) | 2016-10-19 | 2018-04-27 | 학교법인혜화학원 | Extracts from red bean shell having anti obesity and Compositions comprising the same as an active ingredient |
| EP3586857A1 (en) * | 2018-06-29 | 2020-01-01 | Korea Institute of Science and Technology | Seed of new soybean cultivar scel-1, plant body of the seed and part of the plant body, and extract obtained from the seed |
| US10716822B2 (en) | 2018-06-29 | 2020-07-21 | Korea Institute Of Science And Technology | Seed of new soybean cultivar SCEL-1, plant body of the seed and part of the plant body, and extract obtained from the seed |
| CN113163729A (en) * | 2018-06-29 | 2021-07-23 | 韩国科学技术研究院 | Novel seeds of soybean cultivar SCEL-1, plants of said seeds and parts of said plants, and extracts from said seeds |
| CN113163729B (en) * | 2018-06-29 | 2023-07-28 | 韩国科学技术研究院 | Seed, plant body and part of plant body of soybean cultivar SCEL-1, and extract extracted from the seed |
| CN110548039A (en) * | 2019-09-23 | 2019-12-10 | 长春理工大学 | Weight-losing medicine for food-borne obesity and preparation method thereof |
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