KR102197241B1 - Composition for antioxidant and lipid differentiation inhibitory activity by ampk phosphorylation from fraction of ceramium kondoi - Google Patents

Abstract

The present invention relates to a composition for antioxidant and lipid differentiation inhibitory activity using ethanol extracts and fractions of Ceramium kondoi. By ingesting the Ceramium kondoi fractions, it is possible to obtain an effect of alleviating obesity through antioxidant and lipid differentiation inhibitory activity.

Description

Composition for inhibiting fat differentiation through antioxidant and AMPK phosphorylation of silkworm fraction {COMPOSITION FOR ANTIOXIDANT AND LIPID DIFFERENTIATION INHIBITORY ACTIVITY BY AMPK PHOSPHORYLATION FROM FRACTION OF CERAMIUM KONDOI}

The present invention relates to silkworm, and more specifically, to a composition for antioxidant and lipodifferentiation inhibitory activity using a silkworm ethanol extract or fraction.

Obesity is caused by an imbalance of excessive nutritional intake and energy consumption, and causes the normal body shape to be transformed by increasing the size of fat cells and the number of fat cells. When obesity occurs, it causes muscle and skeletal disorders and acquired body transformation, and has a fatal effect on health as well as beauty. Studies on anti-obesity use 3T3-L1 adipocytes notified by the Ministry of Food and Drug Safety to confirm the level of expression of transcription factors related to fat measurement in adipocyte differentiation stage. Therefore, research is being conducted in the aspect of preventive medicine using components that inhibit the differentiation of adipocytes from predipocytes, and major factors in adipocyte accumulation are peroxisome proliferator-activated receptors (PPARs), adipocyte protein 2 (aP2), AMP-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK), AMP-activated protein kinase α (AMPKα), acetyl-CoA carboxylase (ACC) and CCAAT-enhancer-binding proteins (C/EBPs) ), etc.

As studies on the relationship between oxidation and adipocyte differentiation have recently been published, various studies are being conducted to reduce adipocytes based on antioxidant components. Oxidation causes physiological or pathological problems as active oxygen (O ₂ , H ₂ O _2, ·OH, etc.) is generated in the process of energy metabolism using oxygen necessary for human life. The human body removes free radicals by using antioxidants that have enzymatic defense mechanisms such as superoxide dismutase and catalase, but there is a limit, so studies are being made to develop various materials with excellent antioxidant effects. The past tert -butylhydroxyanisol (BHA), tert- butylhydroquinone synthetic antioxidants are antioxidants developed that are excellent in the effect is limited to use as a matter of economic safety being able to replace them, such as (BHQ) and tert -butylhydroxytoluene (BHT) Is required. Therefore, studies on natural materials using plants (herbs) for the purpose of disease treatment or prevention have been conducted in the Eastern region, including Korea. In particular, polyphenol compounds contained in plants have anti-inflammatory, anti-tumor, and antibacterial activities as well as antioxidant activity. And various physiological activities such as anti-diabetes have been reported.

Ceramium kondoi is an annual herb. It grows commonly in fields or roadsides, and 1,600 species are distributed worldwide, and 11 species of silkworm are distributed in Korea. It is called silk grass, meaning that it grows by covering it like silk on the ground, and other names are called by various names such as spotted grass, bedbug, naemocho, nowcho, sundocho, now, yeonji, and jangpancho. The taste of silkworm is spicy and bitter, and has effects such as detoxification, blue fever, eczema, burns, lack of breast milk, and blood circulation, and is mainly used as a folk remedy to treat cuts using medicinal or stem milk. There are reports of anticancer, antibacterial, anti-inflammatory, hemostasis, antioxidant and fatty acid composition of silkworm, and flavonoids, tannins, and terpenoids are reported as component studies.

The present invention is a study to find the efficacy of the use of silkworm, and a number of studies related to antioxidants of silkworm have been confirmed, but since antiobesity-related studies are insufficient, a mechanism for inhibiting differentiation of adipocytes was attempted.

The matters described as the above-described background art are only for enhancing understanding of the background of the present invention, and should not be taken as acknowledging that they correspond to the prior art already known to those of ordinary skill in the art.

Patent Document 1: KR 10-1793822 B1 (2017.10.30) Patent Document 2: KR 10-2011-0120813 A (2011.11.04)

The present invention is intended to help improve obesity by inhibiting the differentiation of adipocytes by ingestion of silkworm.

The main object of the present invention is to provide a food composition showing antiobesity efficacy by containing a silkworm ( Ceramium kondoi ) extract or fraction.

Another object of the present invention is to provide a composition containing a silkworm ( Ceramium kondoi ) extract or fraction to show weight loss effect.

Another object of the present invention is to provide a composition that contains an extract or fraction of Ceramium kondoi and exhibits lipodifferentiation inhibitory activity through AMPK phosphorylation.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

In order to achieve the above object, the present invention provides a food composition for anti-obesity or a composition for weight loss comprising an extract or fraction of silkworm ( Ceramium kondoi ) as an active ingredient.

In the present invention, the extract or fraction of the silkworm ( Ceramium kondoi ) is characterized in that it inhibits the differentiation of adipocytes.

In the present invention, the term "extract" refers to a preparation obtained by squeezing a herbal medicine into an appropriate leachate and evaporating the leachate. The extract obtained by extraction treatment, the diluted or concentrated solution of the extract, the dried product obtained by drying the extract, and the preparation thereof Or it may be a purified product.

In the present invention, the silk grass is pulverized, extracted with ethanol, filtered with filter paper, concentrated under reduced pressure, and freeze-dried.

The dried extract powder is mixed with distilled water and fractionated with an equal amount of n- hexane, and concentrated under reduced pressure and freeze-dried. In the same way, fractionation was performed with chloroform, ethyl acetate, and n- butanol, and concentrated under reduced pressure and freeze-dried. Finally, the remaining water layer is concentrated under reduced pressure.

In the present invention, the antioxidant of silkworm was higher in DPPH radical scavenging activity (3.4 ± 0.1ug/mL RC ₅₀ ) and reducing power (13.2 ± 0.3 RPC) than the extract in ethyl acetate layer, which is total phenol (278.15±1.14 mg/g). ) And flavonoids (188.22±2.31 mg/g) were found to have a correlation ( p <0.001). In addition, it was confirmed that the ethyl acetate layer, which had a high antioxidant effect, was not toxic when treated with less than 10 ug/mL in Lx2 and 3T3-L1 cells. It was confirmed that the adipogenic differentiation and triglyceride production of 3T3-L1 cells were also statistically significantly decreased compared to the extract ( p <0.05, p <0.001), and p-AMPK protein was increased in the adipocyte differentiation mechanism to inhibit adipocyte differentiation. I confirmed that.

In the present invention, the term "obesity" refers to a state with excess body fat, and refers to a state in which body fat is 25% (male), 30% (female) or more of body weight.

In the present invention, the term "anti-obesity" means to treat, prevent or ameliorate an obese state.

In addition, the extract or fraction of the present invention may be used for the prevention, improvement or treatment of obesity, abdominal obesity or visceral fat accumulation in each part of the body, and preferably may be used for the prevention, improvement or treatment of visceral fat accumulation. have. The abdominal obesity or the accumulation of visceral fat includes, but is not limited to, those caused by a long time sitting in a chair, lack of exercise, alcohol (alcohol) intake, stress, and the like.

According to an aspect of the present invention, the present invention provides a food, health functional food or pharmaceutical composition comprising the extract or fraction of the silkworm.

In the present invention, the blending ratio of the extract or fraction of the present invention can be appropriately selected according to the kind and the kind, amount, form, etc. of the other ingredients to be blended. Usually, the silkworm of the present invention for the total amount of medicine or food Fractions may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight.

The extract or fraction prepared by the present invention may be used by mixing a certain amount when preparing a general food. In particular, general foods may be mixed with confectionery, cookies, breads, noodles, rice cakes, biscuits, flour dough itself, and the like, but is not limited thereto, and a suitable blending form, amount, etc. can be selected when manufacturing known foods.

When the composition of the present invention is prepared as a food or health functional food composition, it includes, as an active ingredient, the silkworm extract or fraction according to the present invention, as well as ingredients commonly added during food production, for example, protein, carbohydrate , Fats, nutrients, seasonings and flavoring agents. Examples of the above-described carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc., in addition to the silkworm extract or fraction according to the present invention, are additionally added. Can be included.

When the composition of the present invention is prepared as a pharmaceutical composition, it may be prepared in a pharmaceutical formulation according to a conventional method. In the preparation of the formulation, it is preferable to mix or dilute the active ingredient with a carrier or encapsulate it in a carrier in the form of a container. When the carrier is used as a diluent, it may be a solid, semi-solid or liquid substance that acts as a carrier, excipient, or medium for the active ingredient. Accordingly, the formulation may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injections, sterile powders, and the like. The compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

Here, the pharmaceutical composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, intraperitoneal, muscle, topical application, patch, and iontophoresis, among which topical application and oral administration Is preferred. The actual dosage of the active ingredient of the present invention should be determined in light of various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, and the dosage should be determined by any method. It does not limit the scope of.

By ingesting the silkworm fraction according to the present invention, it is possible to obtain an effect of improving obesity by inhibiting the differentiation of antioxidant and adipocytes. The present invention can be used in various ways as a cosmetic, food, health functional food or pharmaceutical composition using the silkworm fraction.

1 is a graph analyzing the reducing power of ethanol extracts and fractions of Ceramium kondoi according to the present invention. (Results are expressed as mean ± SD of three independent experiments. AE: Ceramium kondoi ethanol extract, H: n -hexane fraction , C: chloroform fraction, E: ethyl acetate fraction, B: butanol fraction, W: water fraction.AT: α-tocopherol, AC: ascorbic acid)
2 is a graph analyzing the differentiation capacity of adipocytes of the ethanol extract and fractions of silkworm ( Ceramium kondoi ) according to the present invention using the 3T3-L1 cell line.
(A) Fatty Oil-red O staining in 3T3-L1 cells (B) Oil-red O staining degree (C) Triglyceride content in 3T3-L1 cells
(AE: ceramium kondoi ethanol extract, H: n -hexane fraction, C: chloroform fraction, E: ethyl acetate fraction, B: butanol fraction, W: water fraction.MDI: adipogenic inducer)
3 is a graph analyzing the ability to inhibit the production of adipogenic differentiation by regulating the AMPK signal of the silkworm ( Ceramium kondoi ) ethyl acetate fraction according to the present invention. (A) Western blot analysis showing adipogenesis-related protein activity in 3T3-L1 cells (B) Western blot analysis showing adipogenesis-related protein activity in 3T3-L1 cells treated with compound C

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

1. Experimental materials

Silk grass was purchased at Nonaegol Farm (Hwachon-myeon, Hongcheon-gun, Gangwon-do) and the above-ground part was used as a sample for this study. The dried silk grass was pulverized, and 686.9 g was extracted in 1 L of 80% ethanol for 48 hours. The extract was filtered through filter paper (Advantech, circles 300 mm, Japan), then concentrated under reduced pressure at 45°C and freeze-dried. The dry extract powder (118.3 g) was mixed with 2 L of distilled water for a third period, fractionated with the same amount of n- hexane, concentrated under reduced pressure, and freeze-dried to obtain 8.4 g of a fraction. In the same manner, chloroform was fractionated to obtain a fraction of 43.8 g, ethyl acetate was fractionated to obtain a fraction of 23.5 g, and n- butanol was fractionated to obtain a fraction of 20.1 g. Finally, the remaining water layer was concentrated under reduced pressure to obtain 10.5 g. In the experiment, it was concentrated under reduced pressure and freeze-dried and used in the experiment.

2. DPPH radical scavenging activity

1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA) The radical scavenging activity of the extracts and fractions of silkworm in ethanol (99.9%, pure grade, Duksan Pharmaceutical Co. Ltd., KyongKido) , Korea) and 100 uL of a 60 mM DPPH solution were dispensed into a 96 well plate and reacted at room temperature for 30 minutes, and then antioxidant was measured at 540 nm using a Wallac Victor2 plate reader. DPPH radical scavenging ability was calculated as a 50% inhibitory concentration (RC ₅₀ ) value of the measured concentration.

3. Reduction power experiment

The method for measuring reducing power is a 0.2M sodium phosphate buffer (pH 6.6, Sigma-Aldrich, St. Louis, MO, USA) 100 uL and 1% potassium in 100 uL concentrations of 0.01, 0.1 and 1 mg/mL of silkworm extract and fractions. 100 uL of ferricyanide (Sigma-Aldrich, St. Louis, MO, USA) was added and reacted in a heat block at 50°C for 20 minutes, followed by 100 uL of 10% trichloroacetic acid (Sigma-Aldrich, St. Louis, MO, USA). Added. Thereafter, 20 uL of 0.1% ferric chloride was added, reacted at 37°C for 20 minutes, and then centrifuged at 13,000 rpm for 10 minutes. Then, 100 uL of the supernatant was measured for reducing power at 700 nm of the Wallac Victor2 plate reader. As a control, α-tocopherol (Sigma-Aldrich, St. Louis, MO, USA) and ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) were used.

4. Total phenol content

For the total phenol content, 50 μL of 2 N Folin-Ciocalteu phenol reagent (Sigma-Aldrich Co., St. Louis, MO, USA) was added to 100 μL of 1 mg/mL concentration of the extract and fraction, followed by stirring for 3 minutes. Thereafter, 300 μL of 20% Na ₂ CO ₃ (Sigma-Aldrich, St. Louis, MO, USA) was added and reacted in a heat block at 37° C. for 30 minutes. 1 mL of distilled water was added to the final sample, and the absorbance was measured at 765 nm of the Wallac Victor2 plate reader. Quantitative analysis of total polyphenol was calculated using gallic acid (Sigma-Aldrich, St. Louis, MO, USA) as a standard material.

5. Total flavonoid content

For the total flavonoid content, 900 uL of 80% ethanol was added to 100 μL of 1 mg/mL concentration of the silkworm extract and fractions, followed by stirring for 30 seconds. Take 500 uL of this solution and 10% aluminum nitrate (Sigma-Aldrich, St. Louis, MO, USA) 100 uL, 1 M potassium acetate (Sigma-Aldrich, St. Louis, MO, USA) 100 uL, 80% ethanol 4.3 Each mL was mixed and reacted for 40 minutes in a 37℃ heat block. The change in absorbance was measured at 415 nm using a Wallac Victor2 plate reader, and the total flavonoid content was calculated from a standard curve prepared using quercetin (Sigma-Aldrich, St. Louis, MO, USA) for quantification of total flavonoids. .

6. Cell culture

3T3-L1 (Korea Cell Line Bank, Seoul, Korea) cell line 10% bovine calf serum (Gibco-BRL, Grand Island, NY, USA), DMEM with 1% P/S (Gibco-BRL, Grand Island, NY, USA) 2×10 ⁵ cells/mL cells were dispensed into 100 mm culture dishes (SPL, Pocheon, Korea) and cultured in a 37° C. CO ₂ incubator. Lx2 (ATCC, manassas, VA, USA) cell line 3×10 ⁵ cells/mL in DMEM medium supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA) and 1% P/S After dispensing with, it was cultured in a 37° C. CO ₂ incubator.

7. Cyto-X analysis

Cyto-X (LPS solution, Daejeon, Pocheon, Korea) reagent was used to test cell toxicity using 3T3-L1 and Lx2 cell lines. The cells were cultured in a 96 well plate (SPL, Pocheon, Korea) for 12 hours at 1×10 ⁴ cells, and then the extracts and fractions of silkworm were treated at 1, 10 and 100 μg/mL. After 24 hours of incubation, Cyto-X reagent was added to each well at a concentration of 1/10, and then wrapped with foil and treated in an incubator for 4 hours. It was measured and quantified at 450 nm absorbance using a Wallac Victor2 plate reader (Perkin Elmer Corp., Nerwalk, CT).

8. 3T3-L1 adipocyte differentiation

Dispense the 3T3-L1 cell line into a 6-well plate (SPL, Pocheon, Korea) at 4×10 ⁵ cells/mL cell count into a 12 well plate (SPL, Pocheon, Korea) and then 10% bovine until confluent. Incubate in DMEM medium supplemented with calf serum, 1% P/S, and 37°C CO ₂ incubator. Adipogenic inducer (5 μg/mL insulin, 1 μM dexamethasone, 0.5 mM IBMX (MDI)) in DMEM medium supplemented with 10% fetal bovine serum and 1% P/S when cells are confluent (Day 0, start of differentiation) It was exchanged for a medium containing and cultured for 3 weeks. The medium was repeatedly replaced after 4 days of culture, and differentiation into adipocytes was induced. On Day 0, silkworm extract and fractions were treated with 10 μg/mL, cultured until Day 10, and stained with Oil-red-O. The fat mass was confirmed through a microscope, and the stained cells were stained with isopropanol, and the fat mass was confirmed at 520 nm using a Wallac Victor2 plate reader.

9. Measurement of triglyceride content in cells

The triglyceride content in cells was measured using the Triglyceride Quantification Colorimetric Kit (BioVision, Milpitas, CA, USA). On Day 0, silkworm extract and fractions were treated with 10 μg/mL and cultured for 3 weeks to induce differentiation. After the differentiation induction experiment, the cells were homogenized with 5% NP-40, heated slowly at 80-100° C., and cooled at room temperature. The supernatant and TG standard obtained by centrifugation for 2 minutes were dispensed into a 96 well plate, and then the TG assay buffer was dispensed at a predetermined volume. 2 μL of lipase was dispensed into each well, mixed, and left at room temperature for 20 minutes, and 50 μL of TG reaction mix was dispensed into each well. After 60 minutes, the optical density was measured at 570 nm using a Wallac Victor2 plate reader.

10. Total protein expression analysis

The 3T3-L1 cell line was cultured in 60 mm cell culture dishes (3 pieces) at 5×10 ⁵ cells for 12 hours, and then 10 μg/mL silkworm extract and fractions were treated for 12 hours to collect cells. After quantification using Bio-Rad protein assay (Bio-Rad, Richmond, CA), 20 μg of protein was confirmed by SDS-PAGE. Gel-transferred proteins were blocked in 5% skim milk diluted in PBS-T for 1 hour, and then the primary antibodies were treated in a refrigerator for 12 hours at a concentration of 1:1000, respectively. After washing with PBS-T three times, the secondary antibody was treated at room temperature at a concentration of 1:5000, and then washed with PBS-T three times. It was measured by X-ray film color development using Amersham ECL Western blotting detection reagent (GE Healthcare, Milwaukee, Wisconsin, USA). In the experiment, ACC, phosphor-AMPK, AMPKα1/2, C/EBPα and PPARγ (Santa Cruz, CA) antibodies were used.

11. Statistical analysis method

All physiological activity experiments were quantified through 3 replicate experiments, and statistical analysis was performed with a one-way ANOVA with a confidence interval p <0.05. The statistical program was used with Graph Pad Prism 5 software (Graph Pad Software, Inc., La Jolla, CA, USA).

[Experimental Example 1] Total polyphenol and flavonoid content

As shown in Table 1, the total polyphenol content of the silkworm extract and fractions was observed highest in the chloroform layer at 295.12±0.58 mg/g, the ethyl acetate layer 278.15±1.14 mg/g, and the nucleic acid layer 251.14±2.14 mg/g , Extract 243.21±0.51 mg/g, butanol layer 221.54±1.33 mg/g, and water layer 15.25±0.14 mg/g in that order.

The polyphenol content of ethanol extract among the antioxidant activities of the extract according to the extract of silkworm extract was reported very similarly as 261.27 mg/g (Kwon et al. , 2016), because 80% ethanol extract was used in this experiment . It is believed that a difference of around 20 mg/g occurred. As such, the difference in antioxidants depending on the extraction solvent is already known in extraction experiments using natural products, and is the reason for the different extraction content of substances according to the solvent (Kim et al. , 2010).

Polyphenols are contained in many plants, such as flowers, stems, fruits, and seeds, and have various functions such as antioxidant and anti-inflammatory effects. Particularly, as a result of quantifying quercetin, a kind of flavonoid as a standard, the highest observed in the chloroform layer was 201.51±1.43 mg/g, similar to the total phenol content, and the ethyl acetate layer was 188.22±2.31 mg/g, and the nucleic acid layer 77.65± The correlation was 1.08 mg/g, extract 75.02±1.08 mg/g, butanol layer 30.87±0.22 mg/g, water layer 6.20±0.27 mg/g in order (Table 1, p <0.001).

Values represent the mean ± SD of three independent expreiments.

TP: total polyphenol content, F: flavonoid content

AE: ceramium kondoi ethanol extract, H: n -hexane fraction, C: chloroform fraction, E: ethyl acetate fraction, B: butanol fraction, W: water fraction

Phenolic substances are the secondary metabolites of plants and are a generic term for aromatic compounds having a hydroxyl group. Phenolic substances have various structures and molecular weights such as phenol, phenylpropanose, benzoic acid derivatives, flavoids, tannins, and lignans (An et al. , 2006). Phenolic compounds are ester-linked with cell wall polysaccharides and lignin, or exist in the form of polymers, and various antioxidant properties are known. In particular, flavonoids are secondary plant phenolic substances having antioxidant and chelating properties, and react as free radical inhibitory mediators of intracellular proteins, nucleic acids and lipids of reactive oxygen species (ROS). Flavonoids in food are mainly present as 3-O-glycosides and polymers. The importance of antioxidants such as flavonoids contained in plants in the diet and prevention of various diseases that are problematic through oxidation are increasing in recent years. In general, the glycosidic unit is glucose, but there are forms of glucorhamnose, galactose, arabinose, and rhamnose.Because these β-linkage substances are not degraded by the enzymes of the pancreas, it is estimated that they are degraded by intestinal microbes and absorbed in the intestine (Heim et al. , 2002). The antioxidant properties of flavonoids depend on hydroxyl group, O-methylation, 2-3 double bonds, 4-oxo function, carbohydrate properties and degree of polymerization. We tried to measure the antioxidant effect of extracts and fractions of silkworm.

[Experimental Example 2] Antioxidant Activity of Silkworm

DPPH radical scavenging activity is stabilized by hydroxyl groups among antioxidant properties. DPPH is not a naturally occurring radical and is highly reactive and relatively stable causing oxidative damage in biological systems. DPPH radical scavenging ability is a method mainly used for the analysis of natural products or antioxidants because of its simple method and low cost.

As shown in Table 2, the DPPH radical RC ₅₀ value of the silkworm extract and fractions was 11.2 ± 0.3ug/mL of silkworm nucleic acid layer, 10.3 ± 0.3ug/mL of chloroform layer, 6.5 ± 0.1ug/mL of water layer, 4.6 ± of butanol layer. It was observed in the order of 0.2ug/mL, extract 5.3±0.3ug/mL, ethyl acetate layer 3.4±0.1ug/mL. The ethyl acetate layer was found to have an RC ₅₀ value similar to that of α-Tocopherol. DPPH radical scavenging activity among the extraction solvents of silkworm was found to be highest in ethanol extracts among ethanol, methanol and water extracts.

Values represent the mean ± SD of three independent experiments. RC ₅₀ : reaction concentration of 50% value. TP: total phenols content, F: flavonoids content, AE: ceramium kondoi ethanol extract, H: n -hexane fraction, C: chloroform fraction, E: ethyl acetate fraction, B: butanol fraction, W: water fraction.

As shown in Figure 1, the reducing power is an antioxidant method that measures the degree to which iron ions are reduced to Fe ²⁺ from Fe ³⁺ by the antioxidant component in the ^sample.Ethyl acetate layer 13.2 ± 0.3 RPC at a concentration of 1 mg/mL of the sample. (reducing power control value), 12.1 ± 0.4 RPC of the butanol layer, 11.4 ± 0.1 RPC of the extract, and 10.4 ± 0.3 RPC of the water layer were observed. In contrast, it was confirmed that the nucleic acid and chloroform layers had very low RPC values.

The reducing power is a measure of antioxidant activity that is closely related to the antioxidant activity, and the antioxidant activity is expressed by the free radical scavenging ability by the reducing power. Antioxidant reducing agents provide reducing power by providing hydrogen to stop the free radical chain reaction, and the reducing power found in plant extracts has a close relationship with antioxidant activity. The antioxidant activity is thought to contain a large number of antioxidants through the fact that the ethyl acetate layer exhibits high activity in both DPPH radical scavenging and reducing power.

[Experimental Example 3] Cytotoxicity and treatment concentration setting of silkworm

Cell viability was measured using 3T3-L1 (mouse adipocyte cell line) and Lx2 (human hepatic stellate cell line) cells to confirm the cytotoxicity of the extracts and fractions.

The Lx2 cell line is one of the hepatocytes that regulate the toxicity of food or drugs introduced into the body as human hepatic stellate cells. Cytotoxicity of silkworm was not confirmed in all extracts and fractions when treated at a concentration of 1 ug/mL in the Lx2 cell line. Under the same conditions, 3T3-L1 cells did not show toxicity at less than 10 ug/mL, and 3T3-L1 differentiation experiments had to induce the differentiation of mast cells for a long time, so a toxicity test was conducted 4 days (96 hours) before adipocyte differentiation experiment. When proceeding, it was found that no cell toxicity was observed at a concentration of 10 ug/mL (data not shown). Based on the above results, the anti-obesity efficacy was confirmed by setting the concentration of the extracts and fractions up to 5 to 10 ug/mL as the experimental conditions, and did not significantly affect the inhibition of the proliferation of the extract and fractions.

[Experimental Example 4] Effect on Lipid droplet (local) generation

Fatty is produced by accumulating triglyceride and cholesterol ester on the double membrane of the endoplasmic reticulum and forming a layer, and is produced when the fat is separated from the endoplasmic reticulum. Stimulation of adipogenesis is known as MAPK, APMKα and ACC, and adipocytes generated by stimulation are regulated by important adipogenic transcription factors such as C/EBPα and PPARγ in the differentiation process by C/EBPß and C/EBPδ. Is being reported. Fatty formation induces obesity, is involved in the development of metabolic diseases such as inflammation, arteriosclerosis, and type 2 diabetes, and causes diseases such as cancer (Jo et al. , 2015). Therefore, using the 3T3-L1 cell line was observed using Oil red O staining to determine how the extracts and fractions of silkworm affect the differentiation ability of adipocytes.

As shown in Fig. 2, the untreated control group can confirm the formation of large adipocytes in the cytoplasm when inducing differentiation. However, it was found that lipid formation was remarkably suppressed in the nucleic acid, chloroform and ethyl acetate layer. Oil red O staining was eluted with isopropanol and the staining degree was measured. As a result, there was a statistically significant difference in the whole extract ( p <0.05), and there were statistically significant differences in the nucleic acid, chloroform, ethyl acetate, butanol and water layers. ( p <0.001). The layers showing the strong adipocyte inhibitory effect were the nucleic acid, chloroform and ethyl acetate layers, and the butanol and water layers showed the adipocyte differentiation inhibitory effect after the extract.

As studies on the components present in silkworm, studies on tannins, phenols, flavonoids, and terpenoids have been reported (Chung & Kim, 1985; Tanaka et al. , 1990; Lee et al. , 1991; Fang et al. , 1993; Tanaka & Matsunaga, 1999; An et al. , 2007). Terpenoids are natural single substances contained in medicinal plants and are known to regulate PPARγ in 3T3-L1 and HepG2 cells (Takahashi et al. , 2002), and flavonoids and tannins components also inhibit C/EBPα and PPARγ in 3T3-L1 cells. It is known to inhibit the formation of fat by regulating (Hsu & Yen, 2007). It has already been reported that natural single substances in various medicinal plants inhibit the differentiation of fat and control various diseases (Yadav et al. , 2019).

[Experimental Example 5] Inhibition of triglyceride production of silkworm fraction

Triglyceride, a triglyceride, is used as an important energy source for cells along with glucose, but excess triglyceride due to excessive food intake is stored in fat cells, causing obesity and various diseases (Goldberg, 2012). Therefore, to measure the content of triglyceride in adipocytes, the content of triglyceride in fat eluted with isopropanol was measured. In the silkworm extract, nucleic acid, and butanol layers, as shown in the oil red O staining results, when the content of triglyceride was compared to the untreated control, statistically significant differences were also observed in the whole extract, nucleic acid and butanol layers ( p <0.05). The extract, nucleic acid, and butanol layers showed stronger inhibition ( p <0.01), and the chloroform and ethyl acetate layers showed the strongest inhibition ( p <0.001). On the other hand, the content of triglyceride in the ethyl acetate layer showed no statistical difference when compared to the undifferentiated group. Silkworm contains isoquercitrin as a single substance, and isoquercitrin has a mechanism of reducing the triglyceride level of 3T3-L1 at a concentration of 30 uM (Hassan et al. , 2014; Zhang et al. , 2018). The decrease in the triglyceride level in the fraction used in this experiment appears to be dependent on the isoquercitrin contained in the fraction. Looking at the above results, the inhibitory effect of triglyceride production, which is related to the inhibition of fat formation of the silkworm ethyl acetate layer, was the fractional layer with the highest antioxidant effect, and it is thought that antioxidants effectively inhibit the differentiation of adipocytes and regulate the accumulation of triglycerides. do.

[Experimental Example 6] Inhibition of adipogenic differentiation by regulation of AMPK signal of silkworm fraction

In the process of inhibiting adipogenesis by the silkworm ethylacetate layer, we looked at the changes in the expression of ACC acting in the lower stages of AMPK and p-AMPK, and if we inhibited the activity of AMPK, we checked what changes were observed in the changes caused by the silkworm ethylacetate layer. I did.

As shown in FIG. 3, the change in the expression of AMPK and ACC confirmed the increase in phosphorylation of AMPK and ACC when differentiation was induced, and it was confirmed that phosphorylation of AMPK was increased by a layer of silkworm ethyl acetate. This could be expected to be involved in the activation of AMPK in the inhibition of adipogenesis by the silkworm ethylacetate layer.

의 발현이 다시 증가되는 것을 알 수 있었다. In addition, the expression of AMPK was equally increased after the concentration-dependent treatment of the ethyl acetate layer, which is believed to be due to the effect of various phenolic compounds present in the ethyl acetate layer. On the other hand, when the AMPK inhibitor compound C was treated and then treated with a layer of silkworm ethyl acetate, the expression of pAMPK was suppressed again, and PPARγ, C/EBPα and C/EBP, which were inhibited during treatment with the silkworm ethylacetate layer.

It was found that the expression of was increased again.

Resveratrol (3,5,4'-trihydroxystilbene), a natural component, is a polyphenol component present in grapes and other plants. It increases the expression of p-AMPKα in 3T3-L1 cells, and PPARγ, C / EBPα and SREBP-1c It was confirmed that adipose formation was suppressed by reducing the mRNA and protein expression of (Chen et al. , 2011). In addition, pronuciferine and nuciferine extracted from lotus leaves have been confirmed to have a regulatory effect on fat production by controlling phosphorylation of AMPK in the form of aporphine alkaloids (Novo et al. , 2015). AMPKα siRNA pretreatment was found to increase adipogenesis and expression of PPARγ, C/EBPα and SREBP-1c proteins in 3T3-L1 cells, and various studies show that by increasing phosphorylation of AMPK, it reduces adipogenesis and differentiation of adipocytes. (Wu et al. , 2013; Wang et al. , 2014; Tan et al. , 2017).

AMPK is a complex that detects the concentration of ATP, ADP, and AMP to confirm the cellular energy state, and is activated by metabolic stress that inhibits mitochondrial ATP production or promotes ATP consumption (Hardie et al. , 2012). AMPK prevents fatty acid production in adipocytes, regulates the accumulation of triglycerides, and regulates ACC to promote fatty acid oxidation, thereby regulating the expression of genes related to adipogenesis. In addition, it is a target that regulates PPARγ and C/EBPα through AMPK and has a major influence on fat differentiation (Choi et al. , 2011).

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art to which the present invention pertains, these specific technologies are only preferred embodiments, and the scope of the present invention is not limited thereto. It is obvious. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (3)

The dried silk grass was pulverized and 686.9 g was extracted in 1 L of 80% ethanol for 48 hours,
The extract was filtered through filter paper (Advantech, circles 300 mm, Japan), concentrated under reduced pressure at 45°C, and freeze-dried,
The dry extract powder (118.3 g) was mixed with 3 liter of distilled water, fractionated with an equal amount of n- hexane, concentrated under reduced pressure, and freeze-dried to prepare a fraction of 8.4 g, and the chloroform fraction was fractionated in the same manner to obtain 43.8 g And fractionating ethyl acetate to prepare a fraction of 23.5 g, fractionating n- butanol to prepare a fraction of 20.1 g, and concentrating the remaining water layer under reduced pressure to prepare 10.5 g,
Anti-obesity food composition comprising an extract or fraction of silkworm ( Ceranium kondoi ) as an active ingredient.
The method of claim 1,
Anti-obesity food composition, characterized in that the extract or fraction of the silkworm ( Ceramium kondoi ) inhibits the differentiation of adipocytes.
The dried silk grass was pulverized and 686.9 g was extracted in 1 L of 80% ethanol for 48 hours,
The extract was filtered through filter paper (Advantech, circles 300 mm, Japan), concentrated under reduced pressure at 45°C, and freeze-dried,
The dry extract powder (118.3 g) was mixed with 3 liter of distilled water, fractionated with an equal amount of n- hexane, concentrated under reduced pressure, and freeze-dried to prepare a fraction of 8.4 g, and chloroform was fractionated in the same manner to obtain a fraction of 43.8 g. And fractionating ethyl acetate to prepare a fraction of 23.5 g, fractionating n- butanol to prepare a fraction of 20.1 g, and concentrating the remaining water layer under reduced pressure to prepare 10.5 g,
A composition for weight loss comprising an extract or fraction of silkworm ( Ceramium kondoi ) as an active ingredient.

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