KR20180013716A - Composition for treating obesity comprising fermented steam-dried ginseng berry extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing and treating obesity, which contains oleic acid contained in a fermented and steam-dried ginseng berry extract as an effective component, wherein the fermented and steam-dried ginseng berry extract is obtained by fermenting a steam-dried ginseng berry extract using Lactobacillus plantarum.

Description

[0001] The present invention relates to a composition for treating obesity comprising an extract of Fusobacterium ginseng as an active ingredient,

The present invention relates to a composition for treating obesity, and more particularly, to a composition for treating obesity containing an extract of Fusarium spp.

Eating habits that are westernized and failure to control weight in adults lead to a rapid increase in obesity rates and result in many negative consequences for our health and quality of life, and social costs are increasing as obesity-related metabolic diseases increase. to be. Obesity refers to the excess accumulation of fat in the body caused by genetic cause or lifestyle habits and causes diseases such as adult diseases and chronic degenerative diseases. In Korea, the socio-economic cost of obesity The total cost of health care costs was estimated to be 1.97 trillion won, including direct costs of 1.77 trillion won and indirect costs of 715.2 billion won as of 2005. In particular, considering the increase in medical expenses and the steady increase in obese population, the socio- It is urgent to develop an active medical treatment method for obesity estimated to be 300 billion won. According to a report by GBI Research, the anti-obesity market is expected to grow at an annual rate of 20.7% from US $ 750 million in 2012 to US $ 2.6 billion in 2019. However, In particular, the increase of obese patients and the bundle of related diseases such as type 2 diabetes accelerate the social burden, and it is urgent to develop a new, more safe and effective treatment for obesity. Korean Patent Publication No. 0877604 discloses a composition for preventing and treating obesity containing processed ginseng extract.

However, the above-mentioned prior art has a problem that it does not show sufficient anti-obesity activity to inhibit accumulation of adipocytes.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a therapeutic agent for obesity which is safe and efficacious by applying a special processing to ginseng fruit. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a method of producing a fermented ginseng fruit low-alcohol extract, which comprises fermenting the ginseng fruit of Lactobacillus plantarum , removing the solid content from the fermentation broth, and then extracting the dried fermented ginseng fruit extract with C1 to C4 lower alcohol, A composition for prevention and treatment of obesity is provided.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating obesity, which comprises oleic acid as an active ingredient.

According to another aspect of the present invention, there is provided a fermented guinea ginseng fruit lower alcohol extract obtained by fermenting ginseng fruit of Lactobacillus plantarum , removing the solid content from the fermentation broth, and then extracting the dried fermented ginseng fruit extract with C1 to C4 lower alcohol A health functional food for improving obesity is provided.

According to one aspect of the present invention, there is provided a method of producing a fermented ginseng fruit low-alcohol extract, which comprises fermenting the ginseng fruit of Lactobacillus plantarum , removing the solid content from the fermentation broth, and then extracting the dried fermented ginseng fruit extract with C1 to C4 lower alcohol, A health functional food for weight control is provided.

According to one aspect of the present invention, there is provided a health functional food for improving obesity containing oleic acid as an active ingredient.

According to another aspect of the present invention, there is provided a health functional food for weight control comprising oleic acid as an active ingredient.

According to one embodiment of the present invention as described above, the ginseng fruit is safer and more effective than the conventional anti-obesity agent through a special processing process, and thus has the effect of replacing the conventional chemical synthesis agent with many side effects. Of course, the scope of the present invention is not limited by these effects.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a process for producing an ethanol extract of ginseng fruit of fermented ginseng according to an embodiment of the present invention. FIG.
FIG. 2 is a photograph of a Lactobacillus plantarum strain isolated from a cherry kernel used in fermentation of ginseng fruit according to an embodiment of the present invention.
FIG. 3 is a nucleotide sequence comparison chart of Lactobacillus plantarum strains isolated from cherry peel husks used in fermentation of ginseng fruit according to an embodiment of the present invention.
FIG. 4 is a chromatogram showing the results of GG / MS chromatography on non-fermented ginseng fruit extract (hereinafter abbreviated as 'SGB') prepared according to an embodiment of the present invention. The numbers of the respective peaks represent the compounds shown in Table 1 below.
FIG. 5 is a chromatogram showing the result of GC / MS chromatography of the fermented ginseng fruit extract (hereinafter abbreviated as 'FSGB') prepared according to an embodiment of the present invention. The numbers of the respective peaks represent the compounds shown in Table 1 below.
FIG. 6 is a chromatogram showing the results of GG / MS chromatography on an ethanol extract (hereinafter abbreviated as 'SEE') of a non-fermented ginseng fruit extract prepared according to an embodiment of the present invention. The numbers of the respective peaks represent the compounds shown in Table 2 below.
FIG. 7 is a chromatogram showing the result of GC / MS chromatography of ethanol extract (hereinafter referred to as "FEE") of the fermented ginseng fruit extract prepared according to one embodiment of the present invention. The numbers of the respective peaks represent the compounds shown in Table 2 below.
FIG. 8 is a graph showing cell viability (A) and cytotoxicity (B) for 3T3-L1 as a lipid precursor cell using FEE prepared according to an embodiment of the present invention.
FIG. 9 is a graph showing changes in the expression of PPARγ (A), FABP4 (B) and leptin (C) according to treatment with oleic acid or FEE during treatment with MDI as a differentiation inducer and 3T3-L1 as a lipid precursor cell.
FIG. 10 is a graph showing the expression level of Acrp30 gene according to treatment of FEE concentration, inducing the differentiation of 3T3-L1 cell line by treating MDI as an inducer of lipid differentiation according to an embodiment of the present invention.
FIG. 11 is a photograph showing immunoreactivity of 3T3-L1, a lipogenic precursor cell, to PPARγ expression level after treatment with MDI and FEE as inducers of differentiation through fluorescence staining.
Figure 12 is a graph of food intake over time for various experimental mice:
Ctrl: Control,
HFD: high fat diet
+ PTM: positive control group (PTM high fat diet group)
+ FEE: High fat diet with FEE administration.
Figure 13 is a micrograph of a section of adipose tissue extracted from the scapular region after administration to various experimental mice according to one embodiment of the present invention;
Ctrl: Control
HFD: It's a highland system.
+ PTM: an appetite suppressant
+ SEE: non-fermented ginseng fruit ethanol extracts
+ FEE: Ginseng fruit ethanol extract from fermentation supplements.
FIG. 14 is a graph comparing and measuring fat area in the experiment of FIG.
FIG. 15 is a graph showing the expression levels of leptin gene measured in adipose tissue of high-fat diet-fed group administered with SEE and FEE.
16 is a graph showing changes in body weight following administration of various drugs to high fat diets;
Ctrl: Negative control,
HFD: high fat diet group,
HFD-AD: positive control group (AD-treated high fat diet group, an appetite suppressant)
HFD-FSGB Low: Low concentration (100 mg / kg) FSGB administration High-fat diet group,
HFD-FSGB High: High dose (200 mg / kg) FSGB administration High-fat diet group,
HFD-FEE: FEE (100 mg / kg) high fat diet group.
17 is a graph showing in vitro test results comparing oleoyl acid and FEE inhibitory activity against alpha glucosidease activity.

Definition of Terms:

As used in this document, " Panax ginseng CA Meyer" is a herb that has been used in Korea, China, and Japan for over 2,000 years. It is used for the purpose of preventing diseases and prolonging the life span. The effects and effects of ginseng which have been known so far include the action on the central nervous system, the anticarcinogenic action, the anticancer activity action, the immune function control action, the anti-diabetic action, the hepatic function enhancement effect, the improvement of the cardiovascular disorder, Control effect, improvement of menopausal disorder, effect on osteoporosis, antistress action, antipyretic action, antioxidant activity, aging inhibitory effect, etc. are known.

As used in this document, "ginseng berry" is a red fruit that is only opened once in four years after the ginseng was cultivated. It is known as an antioxidant containing higher nutrients than ginseng roots. Four-year-old ginseng Has been recognized only as a ginseng seed since it is a rare fruit that is only opened for a week in the middle of July. However, it has been found out that it has superior efficacy to ginseng roots through clinical experiments and research results in the medical fields such as the USA, Japan and Korea.

As used herein, "oleic acid" is an omega-9 fatty acid that is widely found in animals and plants. It is an unsaturated fatty acid having one double bond between carbon atoms and is effective for wound healing, immunity and inflammation diseases. Has been reported to be effective.

As used herein, "PPARγ" (Peroxisome proliferator-activated receptor gamma) is the most expressed in adipose tissue and is known to regulate the transcriptional cascade involved in lipid differentiation, as well as uptake of fatty acids Or trapping genes that are known to be involved in the pathogenesis of cancer.

As used herein, "FABP4 (Fatty acid binding protein 4)" is known as a binding protein that binds to fatty acids during the differentiation of adipocytes and acts mainly on fat accumulation and fatty acid oxidation in cells. Can be controlled.

As used herein, "Acrp30 (adipocyte complement-related protein of 30kD)" is a protein hormone that regulates many metabolic processes, including glucose regulation and fatty acid oxidation, and is rapidly secreted into the blood during differentiation into adipocytes. In particular, Acrp30 is known as a protein specifically synthesized in adipocytes and can be used as an adipocyte differentiation factor.

As used herein, "GLUT4 (glucose transporter 4)" is a protein required for the differentiation and maturation of all adipocytes and plays an important role in glucose metabolism. Thus, regulation of GLUT4 in progenitor adipocytes such as 3T3-L1 can be used as a marker for mast cell differentiation.

As used herein, "leptin" is a hormone which is a hormone derived from adipocytes. It is a hormone that regulates energy balance by inhibiting hunger and is a hormone that is synthesized during the differentiation of adipocytes with GLUT4. It triggers the accumulation of triglyceride, so it is a factor that can check the level of leptin expression and confirm the differentiation into adipocytes.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a method for producing a fermented ginseng fruit low-alcohol extract, which comprises fermenting the ginseng fruit with Lactobacillus plantarum , removing strains and solids from the fermentation broth, and then extracting the dried fermented ginseng fruit extract with C1- A composition for preventing and treating obesity, which is contained as an active ingredient, is provided.

In the composition for preventing and treating obesity, the fermented ginseng fruit extract of the present invention is characterized by: i) Ii) hot water extraction of the ginseng fruits; Iii) fermenting L. plantarum in the ginseng fruit extract; And iv) removing the solid content from the ginseng fruit fermentation broth and drying it.

In the composition for preventing and treating obesity, the lower alcohol may be ethanol.

In the composition for prevention and treatment of obesity, the lower alcohol extract of the fermented ginseng fruit comprises at least 25%, 26%, 27%, 28%, 29%, or preferably 30% Or more. The lower alcohol extract of the fermented ginseng fruit may contain 25 to 41%, 27 to 39%, 29 to 37%, or 31 to 35% of oleic acid as an index material.

In the composition for preventing and treating obesity, the ginseng is selected from the group consisting of Panax ginseng , Panax quinquefolia , Panax notoginseng , Panax japonica , Panax trifolia , Panax pseudoginseng , Panax vietnamensis , Panax elegatior , Panax wangianus , or Panax bipinratifidus .

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating obesity containing oleic acid as an active ingredient.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient or diluent may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, water, tylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. A diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant or the like which is generally used in the formulation can be used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like can be mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories.

Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition for preventing and treating obesity according to the present invention may vary depending on the age, sex and body weight of the patient, but it is generally 0.01 to 50 mg / kg, preferably 0.1 to 10 mg / kg, Kg can be administered once or several times per day. Also, the dosage of the pharmaceutical composition may be increased or decreased according to the route of administration, degree of disease, sex, weight, age, and the like.

According to another aspect of the present invention, there is provided a fermented guinea ginseng fruit lower alcohol extract obtained by fermenting ginseng fruit of Lactobacillus plantarum , removing the solid content from the fermentation broth, and then extracting the dried fermented ginseng fruit extract with C1 to C4 lower alcohol A health functional food for improving obesity is provided.

In the health functional food for obesity improvement, the fermented ginseng fruit extract of the present invention is characterized in that: (i) the ginseng fruit is bred; Ii) hot water extraction of the ginseng fruits; Iii) fermenting Lactobacillus plantarum in the ginseng fruit extract; And iv) removing the solid content from the ginseng fruit fermentation broth and drying it.

In the health functional food for obesity improvement, the lower alcohol may be ethanol.

In the health functional food for obesity improvement, the lower alcohol extract of the fermented ginseng fruit comprises at least 25%, 26%, 27%, 28%, 29%, or preferably 30% Or more. The lower alcohol extract of the fermented ginseng fruit may contain 25 to 41%, 27 to 39%, 29 to 37%, or 31 to 35% of oleic acid as an index material.

According to one aspect of the present invention, there is provided a method of producing a fermented ginseng fruit low-alcohol extract, which comprises fermenting the ginseng fruit of Lactobacillus plantarum , removing the solid content from the fermentation broth, and then extracting the dried fermented ginseng fruit extract with C1 to C4 lower alcohol, A health functional food for weight control is provided.

According to one aspect of the present invention, there is provided a health functional food for improving obesity containing oleic acid as an active ingredient.

According to another aspect of the present invention, there is provided a health functional food for weight control comprising oleic acid as an active ingredient.

The inventors of the present invention found from the preliminary study that the ginseng fruit contained a large amount of ginsenoside compared with the roots and that the extract had an efficacy for the improvement of the antidiabetic activity, the promotion of the secretion of neurotransmitters, the anti-dementia and the liver function improvement However, it has been found through the animal experiments that weight gain is slowed when the ginseng fruit weight is administered in an obesity rat (db / db) model. As a result, the fermented steam- The lower alcohol extract of the ginseng fruit is very useful for suppressing obesity and controlling the body weight to an appropriate level. It is confirmed that oleic acid, which is contained in a large amount of the lower alcohol extract of the fermented ginseng fruit, is an effective ingredient and an index component Thereby completing the present invention.

 Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Preparation of ginseng fruit extract (SGB)

According to one embodiment of the present invention, the content of ginsenoside composition was observed through processing of ginseng fruit. Specifically, 1 kg of immature fruit of ginseng cultivated in the Ginseng Special Division located in Yeoncheon, Gyeonggi Province was re - collected and washed with purified water to remove foreign matter. Then, the above-mentioned fruits were placed in a steamer, placed in a steamer, and heated for 1 hour while maintaining an average temperature of 100 ° C for 2 hours. The cooked ginseng fruit was placed in a dryer and dried for 24 hours while heating to maintain the temperature at 50 ° C. The first dried ginseng fruit was again put into a booster, heated for 2 hours while maintaining an average temperature of 100 ° C, and then put in a drier and dried for 24 hours while heating to maintain the temperature at 50 ° C . After the ginseng fruit was dried four to nine times, the composition and composition of the ginsenosides were compared and analyzed. As a result, it was observed that the ginsenosides Rf, Rh1, Rg3, M1 and Rh2 increased as the number of times of addition was increased. In the case of Re, Rc and Rd, the content was highest in the four times of application, . Table 1 shows the results of comparing the content of each ginsenoside composition according to the number of times of administration.

Content (% W / W) Four times Five times 6th exhibition 7 times 8 times 9 times Ginsenoside Re 7.46 3.64 2.82 1.64 1.07 0.58 Ginsenoside Rf 7.25 8.25 7.74 9.25 9.25 9.01 Ginsenoside Rh1 3.48 3.84 3.32 4.04 3.87 3.83 Ginsenoside Rg2 2.33 2.26 1.74 1.22 0.96 1.78 Ginsenoside Rb1 2.43 2.03 1.57 1.49 1.13 0.97 Ginsenoside Rc 4.62 3.54 2.85 2.41 1.82 0.94 Ginsenoside Rd 11.68 10.04 9.04 8.78 7.95 7.06 Ginsenoside Rg3 24.92 27.87 28.82 29.7 28.79 29.12 Ginsenoside M1 8.26 8.83 9.62 9.02 9.98 10.07 Ginsenoside Rh2 8.52 9.75 10.99 10.73 11.19 12.18

Thus, the inventors of the present invention found that the ginseng fruit having the highest contents of Re, Rg2, Rb1, Rc and Rd was mixed with the 7th seed ginseng fruit having the highest total ginsenoside content and then pulverized. No. 2 filter paper to remove the precipitate and prepare the ginseng fruit extract (SGB).

Example 2: Ginseng fruit extract (FSGB) obtained by fermentation

Lactobacillus plantarum , a biocompatible strain identified by itself from the cherry kernel, was inoculated into the ginseng fruit extract (SGB) prepared in Example 1 of the present invention at 1 × 10 ⁷ CFU / mL and fermented at 30 ° C. for 72 hours. The L. plantarum strain was identified as 16s RNA gene analysis disclosure L. plantarum strain and 99% strain with a same-sex (Figs. 2 and 3). After the fermentation process, the mixture was centrifuged at 3,500 rpm for 1 minute. 2 strains were removed by filtration using filter paper, and further sterilization was carried out at 121 ° C for 15 minutes, and then the supernatant was cultured to confirm the presence of the strain. The filtration and sterilization process was repeated if it was confirmed that there was a residual strain. The obtained fermented product was freeze-dried to obtain a fermented ginseng fruit extract, which was named 'FSGB' for convenience.

As a result, as shown in FIG. 1, the yield of the ginseng fruit extract (FSGB) obtained from the ginseng fruit of 100 g obtained through the above-mentioned propagation and fermentation process was 28.01 g on average, Which is about 28% of the weight of the first ginseng fruit.

Example 3: Ginseng fruit ethanol extract (FEE) obtained by fermentation

100 g of FSGB was immersed in 100% of ethanol and stirred for 3 hours at room temperature for 6 hours. The residue was removed and ethanol was evaporated to obtain a fermented ginseng fruit ethanol extract (47.08 g). For convenience, FEE '. The final yield of the FEE was 47.1%, which was very high. The solution was stored at -20 ° C until dissolved in 100% ethanol as a storage solution. The lyophilized SGB prepared in Example 1, which had not been fermented as a control, was extracted with 100% ethanol by the same method to obtain a non-fermented ginseng fruit ethanol extract at a dry yield of 20.2%, which was referred to as 'SEE' Respectively.

Experimental Example 1: Component analysis

The present inventors analyzed the main components contained in the various ginseng fruit extracts (SGB, FSGB, FEE and SEE) prepared in Examples 1 to 3 by gas chromatography (GC / MS chromatography). Specifically, GC-MS (Agilent Technologies 5975C) equipped with a CTC CombiPAL autosampler system (Palo Alto, CA, USA) was used for component analysis and 1 μl of sample was transferred to HP-5 column (Agilent Technologies, 250 μm × 0.25 μm × 30 m). The injector temperature was 250 ° C and the GC oven was measured at 310 ° C at 3 ° C intervals per minute after stopping at 50 ° C for 2 minutes. Data were collected and analyzed after 5 minutes in consideration of the solvent peak, and main components of SGB, FSGB, FEE and SEE were analyzed using Wiley7N library data (see Tables 2 and 3 and FIGS. 4 to 7).

As a result, as shown in Table 2 and FIG. 4, the main component present in the SGB was octadecyl dimethylallyl ether in an amount of about 13.77%, and the next largest component was cycloheptasiloxane and tetradecamehyl- in an amount of about 7.03% , And no 9-octadecenoic acid was detected at all. On the other hand, in the FSGB fermented with Lactobacillus plantarum , the content of oleic acid, which was not present in the SGB at all, was detected as 14.13% as shown in Table 2 and FIG. 5, and octadecanoic acid, butyl ester and the like were detected. This is judged by the composition change due to bioconversion according to the fermentation process.

As a result of SEE and FEE analysis of the SGB and FSGB extracted with ethanol, SEE was found to contain hexadecanoic acid (10.88%), oleic acid (18.67%), , And 9,12-Octadecadienoic acid (Z, Z, 19.71%). Interestingly, the content of oleic acid, which was not detected in SGB, increased by 18.67%. In addition, as shown in Table 2 and FIG. 7, in the case of FEE, it was confirmed that the content of oleic acid was as high as 33.17%, indicating that the content of oleic acid was more than twice that of FSGB. However, the content of hexadecanoic acid did not change much, and many components which were not detected in FSGB were also confirmed. Therefore, it can be seen that FEE is not simply a concentrated substance of FSGB.

Comparison of SGB and FSGB contents by GC / MS chromatography peak Ginseng fruit extract (SGB) Fermented ginseng fruit extract (FSGB) Retention time
(min) compound area
(%) quality
(%) Retention time
(min) compound area
(%) quality
(%) One 17.722 Cycloheptasiloxane, tetradecamethyl- 7.03 93 17.838 Cycloheptasiloxane, tetradecamethyl- 1.50 93 2 23.061 Hexadecanoic acid 4.44 99 23.061 Hexadecanoic acid 12.79 99 3 24.651 9,12-Octadecadienoic acid (Z, Z) - 2.79 95 4 24.680 9-Octadecenoic acid (oleic acid) 14.13 90 5 24.960 Hexadecanoic acid, 1,1-dimethylethyl ester 2.37 90 6 25.065 9-Octadecenamide, (Z) - 1.81 86 7 26.607 Octadecanoic acid, butyl ester 10.85 93 8 33.922 Stigmast-5-en-3-ol 6.70 72 9 33.353 Octadecyl dimethylsilyl ether 13.77 87

Comparison of SEE and FEE content by GC / MS chromatography peak SGB Ethanol Extract (SEE) FSGB Ethanol Extract (FEE) stay
time
(min) compound area
(%) quality
(%) Retention time (min) compound area
(%) quality
(%) One 22.608 Hexadecanoic acid 10.88 99 22.724 Hexadecanoic acid 12.75 99 2 24.661 9-Octadecenoic acid (oleic acid) 18.67 99 24.391 9-Octadecenoic acid (oleic acid) 33.17 99 3 24.979 9,17-Octadecadienal 2.09 97 24.989 9-17-Octadecadienal 3.55 93 4 27.205 Benzene, (3-nitropropyl) 3.41 46 5 27.504 (2R, 5E) -caryophyll-5-en-12-al 2.33 43 6 28.978 9,12-Octadecadienoic acid (Z, Z) 19.71 90 7 29.836 2,6,10,14,18,22-Tetra-cosahexaene 4.52 98 8 30.231 otochilone 2.90 43 9 31.185 2- (Acetoxymethyl) -3- (methoxycarbonyl) biphenylene 2.25 55 10 31.908 4,14-Bis (hydroxymethyl) - [2,2] metacyclophane 0.94 46 11 32.023 D-α-Tocopherol 1.44 95 12 33.845 γ-Sitosterol 3.93 99 33.854 γ-Sitosterol 4.44 97

Experimental Example 2: Cytotoxicity test

Cell viability (3T3-L1) and cytotoxicity assay (J774A) were performed to determine the drug toxicity of the FEE prepared in Example 3, respectively.

Specifically, 3T3-L1, a lipogenic precursor cell, was purchased from ATCC (American Type Culture Collection, USA) and subcultured with DMEM (containing 10% Bovine Serum, 100 units / ml penicillin, 100 μg / ml streptomycin) The cell viability was analyzed by treatment with FEE concentration. The cell survival rate was determined by measuring the color material of formazan as a final substance as a method for analyzing the enzymatic activity of intracellular mitochondria. For the analysis, CCK-8 (CCK-8, Dojindo, Japan) was used. After the completion of the reaction, CCK solution: cell culture solution was added at 1:10 to each plate well. Absorbance was measured at 450 nm wavelength by photometry and compared.

As a result, also, in the 3T3-L1 100 ㎍ / mL concentration in the cell it is confirmed to be activated to be somewhat lower, but the concentration in the experiment as cell concentrations do not meet the IC ₅₀ Toxicity was not observed as shown in 8A. Cell viability and cytotoxicity were assayed using the LDH release assay (CytoTox 96 Kit, Promega, USA), which measures the amount of lactate hypoglycemia (LDH) released into the culture medium by cell membrane injury. As shown in FIG. 8B, similar to the results observed in the cell activity, it showed slight cytotoxicity at the high concentration of FEE (100 ㎍ / mL) but it was found to be very low toxicity compared to the positive control group of hydrogen peroxide.

Experimental Example 3: Analysis of adipocytes (3T3L-1)

In accordance with one embodiment of the present invention, the differentiation of 3T3-L1 cell line was induced and the expression level of the gene was monitored by FEE treatment. Specifically, 3T3-L1 undifferentiated adipocyte cell line was distributed on a plate for adipocyte differentiation, and when cells became 100%, MDI (methyllisobutylxanthine, dexamethasone, insulin, Adipogenesis Kit, # 10006908; Cayman, Ann Arbor, USA) were added to 10% FBS DMEM culture medium, cultured for 3 days, and insulin alone was added to fresh medium (2 days interval for 7 days) to induce differentiation. 3T3-L1 cells are undifferentiated adipocytes differentiated into adipocytes by mehyllysobutylxanthine, dexamethasone, and insulin. In the differentiation of adipocytes, factors such as PPARγ and C / EBPα, which induce transcription by transferring signals in the nucleus, The accumulation in adipocytes is accompanied by the expression of various adipocyte specific genes such as GLUT4 (glucose transporter4), adiponectin, FABP4 (fatty acid binding protein 4), and leptin.

Thus, the present inventors administered 50 μg / mL of FEE or 10 μM of oleic acid or 50 μM of oleic acid, which is the component most abundant in FEE, to 3T3-L1 cells that are lipid precursor cells treated with MDI (methylisobutylxanthine, dexamethasone, insulin) The expression level of the adipocyte differentiation-related gene was analyzed by RT-PCR and immunocytochemistry after treatment with μM (corresponding to 14.123 μg / mL).

As a result, as shown in Fig. 9, the FEE of the present invention significantly inhibited the expression of PPARγ, FABP4 and leptin genes, which are representative fat-related genes. On the other hand, analysis of the inhibitory activity against Acrp30 expression showed that FEE inhibited Acrp30 expression in a concentration-dependent manner, especially at a concentration of 100 μg / mL (FIG. 10). In addition, as shown in FIG. 9, oleic acid, which is a major component of the FEE, also decreased PPARγ, FABP4 and leptin expression levels in a concentration-dependent manner. However, the reduction level was not superior to that of FEE at 50 μg / mL. In any event, the above results demonstrate that oleic acid can also be used for the prevention and treatment of obesity.

Experimental Example  4: Immunocytochemistry ( Immunocytochemistry ) analysis

In the 3T3-L1 cell line differentiated according to an embodiment of the present invention, immunocytochemical analysis was performed to confirm the expression level of PPARγ, which is an adipocyte differentiation-related factor, through fluorescent staining. Specifically, the 3T3-L1 cell line was inoculated and cultured on a 4-well chamber slide, and the cells were treated with insulin and FEE (50, 100 / / mL) for 72 hours during the induction period of mast cell differentiation after initial MDI treatment for 2 days , Fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 for 30 minutes. After incubation at 4 ° C overnight, the cells were reacted with secondary antibody (anti-rabbit IgG-Alexa Fluor 488, 1: 1000; Cell signaling, Beverly, USA) with a primary antibody (1: 500; Cell signaling Beverly, USA) ) Was reacted at room temperature for 2 hours. The nuclei of the cells were cross-stained using Hoechst 33342 and the stained cells were observed under a microscope (AX-70, Olympus, Tokyo, Japan).

As a result, the expression level of PPARγ in the cytoplasm of the experimental group treated with MDI was found to be abruptly increased, while the expression of PPARγ was suppressed in the FEE treatment group. Therefore, FEE seems to inhibit the differentiation of adipocytes and accumulation of fat bodies by controlling both mRNA transcription and protein expression of PPARy (Fig. 11).

Experimental Example 5: Evaluation of animal efficacy

According to one embodiment of the present invention, the anti-obesity activity effect of FEE administration was observed in high fat diabetic mice. Specifically, 5 week old C57BL / 6J male mice were sacrificed for one week, and their blood glucose levels were measured and fed a high fat diet (60 kcal%) for 8 weeks. In the experimental group, PTM (Phentermine, 0.625 mg / kg) (AD) and low dose FSGB (HFD-FSGB Low, 100 mg / SEF and HFD-FEE, 100 mg / kg), and then administered orally at the same time intervals each day. Dietary intake, body weight, and blood sugar changes were measured periodically during the experiment period. The blood glucose was measured at the interval of 3 days after the purifying process and immediately before feeding the high fat diet. After 8 weeks of obesity induction, obesity was observed by weighing. After ingesting each test substance, fat was extracted from the area around the anterior / interscapular to detect the difference in degree of fat formation. The star weights were measured and compared. The animal test was approved by the GWNU-2015-40 (Approval no .: GWNU-2015-40) of Wonju National University, and the experimental procedure was conducted by the IACUC Standard Operation Guideline (Food and Drug Administration, Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters, 2011) .

As a result of comparing the feed intakes of the first group, there was no significant change in the intake of silver in the negative control group as shown in FIG. 12, but in the case of the high-fat diet group, the feed intake increased with time. On the other hand, in the case of the PTM administration method and the FEE administration method according to the present invention, the food intake was decreased compared with the control, and the food intake was found to be lower with the lapse of time. This shows that the FEE of the present invention can inhibit appetite in obese patients.

On the other hand, microscopic photographs were taken of the scapular fat tissue sections obtained from each experimental group and fat area was calculated based on the results. As shown in FIGS. 13 and 14, fat area increased in the high fat diet group, Of the FEE-treated group showed a reduction in the fat area compared to the positive control PTM and a decrease in the fat area to almost the control level. On the other hand, the SEE decreased the fat area compared to the high fat diet group but the fat reduction was less than the PTM.

In addition, the expression level of leptin gene in adipose tissue of each experimental group was quantified by RT-PCR. As shown in FIG. 15, the FEE of the present invention significantly decreased the expression of leptin gene. On the other hand, in the case of PTM and SEE, the expression of leptin gene was decreased in comparison with that of high fat diet, but its efficiency did not reach FEE.

Finally, as shown in FIG. 16, HFD showed a relatively rapid increase in body weight, whereas the control group (ctrl) and other experimental groups showed a large difference in body weight between normal C57BL / 6 mice . On the other hand, the experimental group treated with AD, SGB and FSGB showed that the weight gain rate was slower than that of the HFD group, whereas the FEE group showed almost no weight gain after 36 days It was found that the effect was remarkable. Furthermore, the FEE-treated group showed a better weight control effect than the FSGB-administered group (HFD-FSGB High, 200 mg / kg) despite the relatively low dose of 100 mg / kg. This is to prove that the ethanol extract of the fermented ginseng fruit of the present invention is very useful for the treatment and improvement of obesity by inhibiting weight gain even when the dietary habit of excessive energy supply is maintained.

Experimental Example 6: Mechanism analysis of FEE and oleic acid

As a result of the above experiment, the present inventors confirmed that the high fat diet of oleic acid, which is one of the main components of the FEE of the present invention, is effective in increasing the body weight of an animal and inhibiting obesity.

The blood level of insulin produced and secreted by the pancreas is recognized as one of the major causes of obesity. Blood insulin levels are regulated by glucose, which is absorbed into the blood after ingestion of food. Therefore, the absolute concentration of glucose produced in the small intestine can be regulated to control diabetes as well as obesity. Therefore, the higher the inhibitory activity of the enzyme activity in the sugar chain, the easier it is to develop the desired anti-obesity / anti-diabetic material. Therefore, in order to investigate whether FEE and oleic acid have the ability to inhibit the activity of the sugar chain enzymes, the present inventors investigated the inhibitory activity of FEE and oleic acid on alpha glococidal activity. Specifically, the inhibitory activity of alpha glucosidease activity was determined by mixing 0.15 unit / mL of 67 mM potassium phosphate buffer, α-glucosidase and oleic acid (100 μg / mL) or FEE (100 μg / mL) After incubation with 10 mM p-Nitrophenyl α-glucoside solution, substrate activity was measured and absorbance was measured at 405 nm for 5 minutes at intervals of 5 minutes.

As shown in FIG. 17, the oleic acid standard reagent effectively inhibited the activity of the enzyme, and FEE also completely inhibited the activity of the enzyme after 30 minutes of measurement, and the sugar inhibitory activity of the FEE was increased by oleic acid . Thus, these results support the results of FIG. 9, indicating that oleic acid is also effective in controlling blood sugar and inhibiting obesity by inhibiting sugar disaccharide activity.

On the other hand, when considering the results of other experiments, it is considered that oleic acid treated with the same amount of oleic acid has higher alpha-glucosidase inhibitory activity than FEE, but in the case of inhibiting the expression of lipogenic- FEE was more effective than other oleic acids in the FEE.

Therefore, the fermented ginseng fruit lower alcohol extract according to one embodiment of the present invention exhibits a very strong anti-obesity effect in an in vitro test and an animal test, and thus it is possible to develop it as a health food for obesity treatment and obesity improvement Very high.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (3)

A pharmaceutical composition for preventing and treating obesity, which comprises oleic acid as an active ingredient. A health functional food for improving obesity containing oleic acid as an active ingredient. Oleic acid as an active ingredient, a health functional food for weight control.

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