KR20230163048A - Composition for Anti-obesity or Anti-diabetes Using an Extract of Ulva pertusa - Google Patents

Abstract

The present invention shows that the extract of Sulfuria chinensis inhibits the expression of transcription factors related to adipogenesis in 3T3-L1 adipocytes, which are mouse preadipocytes, and in an experimental evaluation using obese animal models due to a high-fat diet, reduced body weight and liver. Disclosed is an anti-obesity or anti-diabetic composition using an extract of Sulfuria chinensis, which has the activity of improving function, reducing blood sugar levels, improving oral glucose tolerance and insulin resistance, reducing the weight of organs related to obesity, and inhibiting the production of fat droplets in liver tissue. do.

Description

Anti-obesity or anti-diabetic composition using Ulva pertusa extract {Composition for Anti-obesity or Anti-diabetes Using an Extract of Ulva pertusa}

The present invention relates to an anti-obesity or anti-diabetic composition using an extract of Siberian seaweed.

In modern society, the obese population is increasing due to excessive nutritional intake and a decrease in physical activity due to a convenient living environment due to rapid automation and an increase in processed foods and eating out. The World Health Organization (WHO) classifies obesity as a disease rather than a phenomenon or symptom, and has reported that by 2015, the number of obese people worldwide will increase to 1.5 billion, making it a serious health problem (Ann Epidemiol. 2005. 15:87-97; J Life Sci. 2013. 23:69-78). The cause of obesity is not clearly known, but it is not caused by a single condition but by various causes such as inappropriate eating and lifestyle habits, genetic factors, and reduced physical activity (Korean J. Food Science Nutrition 22(1) 110-12.2009;Korean J.Food Science Nutrition 23(5):363-367.1990).

Obesity is an imbalance between energy intake and consumption, resulting in excessive accumulation of body fat and an increase in the number and size of fat cells (Cell.104:531-543 2001). Energy in the body is stored in fat cells in the form of triglyceride. When the stored energy source is depleted, the stored fat is broken down into free fatty acids and glycerol and used as an energy source, but excessive intake of energy promotes the differentiation of fat cells and increases the amount of stored fat in the body, which is a direct cause of obesity. (Metabolism. 30: 425-427 1981; Biochem J. 1;435(3):723-32 2011). Obesity not only changes body shape due to fat accumulation in the internal organs and abdomen, but also acts as a risk factor that increases the incidence of various diseases (Korean J. Pediatr.48:126-137. 2005) When excessive visceral fat accumulates, the body's sugar level increases. Problems with metabolism occur, and symptoms such as abnormal hormone secretion and cytokine secretion occur. Due to obesity, the increase in triglycerides and LDL-cholesterol and the decrease in HDL-cholesterol lead to abnormal fat metabolism in the body, decrease the insulin receptors present in the tissues, and also reduce insulin sensitivity, which inhibits the transport of glucose into cells. This can cause high blood sugar and diabetes. In addition, obesity is known to be closely related to the occurrence of metabolic diseases such as hyperlipidemia, cardiovascular disease, cancer, respiratory disorders, stroke, and osteoarthritis (Med. Int. 22 385-388 1994;. Ann. Intern. Med . 103:1024-1029 1985).

Diabetes is divided into type 1 diabetes and type 2 diabetes depending on its cause. Type 1 diabetes occurs when pancreatic cells are destroyed due to genetic defects or infections and insulin is not secreted. Type 2 diabetes occurs when insulin is secreted normally or in excess due to obesity, but its action is not effective. This is called insulin resistance and indicates relative insulin deficiency. Hyperinsulinemia, a common characteristic of obesity, is caused by insulin resistance caused by excessive accumulation of adipose tissue, especially abdominal obesity. This is a result of a failure of the insulin signaling transduction system due to a failure of the post-transporter insulin receptor substrate-1 (IRS-1) in fat cells, which are insulin target cells, resulting in hyperglycemia due to a failure of glucose transporter 4 (GLUT4), and to compensate for this. Hyperinsulinemia has been reported to occur.

The present invention discloses the anti-obesity or anti-diabetic effects of the L. oleraceae extract.

The purpose of the present invention is to provide an anti-obesity or anti-diabetic composition using an extract of Siberian seaweed.

Other or specific purposes of the present invention will be presented below.

The present invention, as confirmed in the Examples and Experimental Examples below, shows that in an in vitro test using 3T3-L1 mouse pre-adipocytes, the extract of Siberia chinensis inhibits differentiation into adipocytes and inhibits the expression of transcription factors related to adipogenesis. In addition, in a high-fat diet animal model test, it was confirmed that weight loss, improvement in liver function, reduction in blood sugar levels, improvement in oral glucose tolerance and insulin resistance, reduction in the weight of organs related to obesity, and inhibition of fat droplet production in liver tissue were completed. It has been done.

Considering the above-described experimental results, the present invention can be viewed in one aspect as an anti-obesity or anti-diabetic composition containing a Corolla extract as an active ingredient, and in another aspect, a hyperlipidemia composition containing the Corolla extract as an active ingredient. It can be understood as a composition for improving (hypercholesterolemia and/or hypertriglyceridemia) or a composition for improving liver function (i.e. for improving fatty liver, especially for improving non-alcoholic fatty liver disease).

In this specification, “seaweed” refers to seaweed whose scientific name is Ulva pertusa .

In this specification, the term “Seaweed seaweed extract” refers to the seaweed seaweed as the extraction target, and as an extraction solvent, water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, and chloroform. , extract obtained by leaching using ethyl acetate, butylacetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, carbon dioxide, It refers to an extract obtained using a supercritical extraction solvent such as pentane, or a fraction obtained by fractionating the extract. The extraction method is cold immersion, reflux, heating, ultrasonic radiation, and supercritical, considering the polarity, degree of extraction, and degree of preservation of the active substance. Any method, such as extraction, can be applied. In the case of a fractionated extract, the extract is suspended in a specific solvent and then mixed with a solvent of different polarity to form a fraction obtained by adsorbing the crude extract onto a column filled with silica gel and then mixed with a hydrophobic solvent, a hydrophilic solvent, or a mixture of these. It is meant to include fractions obtained using the mobile phase. In addition, the meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, and spray drying. Preferably, it refers to an extract obtained using water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof as an extraction solvent, and more preferably, an extract obtained using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms as an extraction solvent. do.

In addition, in this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

Also, in this specification, “anti-obesity” means reducing body fat, suppressing body fat accumulation, and/or reducing body weight. Therefore, it includes the prevention of obesity and the treatment of obesity, and further includes reducing body weight/body fat for beauty or health purposes (aka diet purposes) even though body weight is not classified as obesity or overweight.

Also, in this specification, “anti-diabetic” means alleviating, preventing, or treating the symptoms of diabetes.

In addition, in this specification, the term "obesity" refers to a state in which adipose tissue is abnormally increased, whether it is obesity due to genetic factors or obesity due to environmental factors, and according to the classification of body mass index (BMI), the term "obesity" refers to a state in which fat tissue is abnormally increased. This includes obesity (if BMI is 30.0 or more), obese (if BMI is 25 to 30), and overweight (if BMI is 23 to 25).

In addition, the term "diabetes" in this specification includes insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes) as described above, and furthermore, it occurs due to damage to the pancreas due to other diseases, etc. This means that diabetes, such as hyperthyroidism, hyperadrenocorticism, diabetes caused by growth hormone hyperactivity or catecholamine hyperactivity, and gestational diabetes.

Also in this specification,

The composition for anti-obesity of the present invention may contain the active ingredient in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as the active ingredient can exhibit anti-obesity activity, anti-diabetic activity, etc. Silver will be determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, “effective amount” means that when the composition of the present invention is administered to mammals, preferably humans, to which it is applied during the administration period recommended by medical experts, etc., the intended functional and pharmacological effects, such as anti-obesity effect and anti-diabetic effect, are achieved. It refers to the amount of active ingredient contained in the composition of the present invention that can be expressed. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In addition to the active ingredients, the composition of the present invention is already safe in the art for enhancing and reinforcing anti-obesity and anti-diabetic effects, or for improving the convenience of taking or ingesting through the addition of similar activities such as blood pressure regulating activity. It may further include any compounds or natural extracts that have been validated and known to have the corresponding activity.

These compounds or extracts include compounds, extracts, and pharmaceuticals listed in compendial documents such as the Pharmacopoeia of each country (in Korea, the "Korean Pharmacopoeia") and the Code of Health Functional Foods of each country (in Korea, it is the "Standards and Specifications for Health Functional Foods" notified by the Ministry of Food and Drug Safety). The laws of each country that govern the manufacture and sale of compounds, extracts, and health functional foods that have received product approval in accordance with the laws of each country (in Korea, this is the “Pharmaceutical Affairs Act”) (in Korea, this is the “Health Functional Foods Act”) 」) includes compounds or extracts whose functionality has been recognized.

For example, according to the Korean 「Health Functional Foods Act」, Garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green tea extract, chitosan, and Lactobacillus gasseri are recognized as functional for 'body fat reduction'. BNR17 ( Lactobacillus gasseri BNR17), L-carnitine tartrate, green mate extract, green coffee bean extract, perilla leaf extract, soybean germ extract, etc. complex, ginseng leaf alcohol extract powder, lactoferrin (milk purified protein), lemon balm extract mixed powder , mate thermal water extract, seaweed and other complex extracts (xanthigen), fermented vinegar pomegranate complex, pu-erh tea extract, soybean peptide complex, vegetable oil diglyceride, wild mango seed extract, medium chain fatty acid (MCFA)-containing oil, Complex extracts such as coleus forskohlli extract, chitooligosaccharide, finger root extract powder, hibiscus, etc., GABA-containing powder derived from L-glutamic acid recognized for its functionality in 'blood pressure regulation', katsuobushi oligopeptide, Natto bacteria culture powder, Seomoktae ( Peptide complex, salmon peptide, olive leaf extract, sardine peptide, casein hydrolyzate, coenzyme Q10, grape seed enzymatically decomposed extract powder, Haitai oligopeptide, etc., DHA concentrated oil and globin singer recognized for their functionality in ‘improving blood neutral fat’ Decomposition products, indigestible maltodextrin, bamboo leaf extract, vegetable oil diglyceride, refined sardine fish oil, refined squid oil, etc., L-arabinose, nopal extract, cinnamon extract powder, guava leaf extract, Indigestible maltodextrin, freeze-dried silkworm powder, hemp spirit extract, banaba leaf extract, leaf extract, etc., fermentation-generated amino acid complex recognized for its functionality as 'fatigue improvement', Hovenia vulgaris fruit extract, rhodiola extract, etc., as 'anti-stress' These compounds or extracts include L-theanine, asiaganda extract, milk protein hydrolyzate, and ginseng leaf extract, which are recognized for their functionality.

One or more of these compounds or extracts may be included in the composition of the present invention along with the active ingredient.

The composition of the present invention can be regarded as a food composition in a specific aspect.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated drinks, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, It can be manufactured into foods such as snacks and noodles, and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to the Korean "Act on Health Functional Foods", or confectionery, beans, tea, etc. according to each food type according to the food code of the Korean "Food Sanitation Act" (Ministry of Food and Drug Safety Notification "Food Standards and Specifications"). These may be beverages, special purpose foods, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the Food Additives Code in accordance with each country's laws governing the manufacture and distribution of food (in Korea, it is the "Food Sanitation Act"), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification "Food Additive Standards and Specifications"), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified as sweeteners and flavoring agents in terms of function. , preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents are used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and as synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, etc. may be used.

Preservatives include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, and pectin. etc. may be used, and as acidulants, acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms. As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the laws of each country.

The composition of the present invention may be considered a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art. Here, “pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what is acceptable for the subject of application (prescription).

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, and wafers using a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, Cellulose such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable. Yu, etc. can be mentioned. In the case of formulation, if necessary, diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included in the formulation.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of eye drops, injections, transdermal administration, nasal inhalation, or suppositories along with a suitable carrier according to methods known in the art. When formulating as eye drops, suitable carriers include sterile water, saline solution, and isotonic solutions such as 5% dextrose. If necessary, benzalkonium chloride, methylparaben, ethylparaben, etc. can be added for preservative purposes. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or sterile for injection. Isotonic solutions such as water or 5% dextrose can be used. When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the base is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

As described above, according to the present invention, it is possible to provide an anti-obesity or anti-diabetic composition using an extract of Corolla. The anti-obesity or anti-diabetic composition of the present invention can be commercialized as functional foods, drugs, etc.

Figure 1 shows the results of confirming the cell viability after treating 3T3-L1, a mouse preadipocyte cell, with the extract of L. oleracea at different concentrations (31.25, 62.5, 125, 250, 500 ㎍/㎖).
Figure 2 shows the results of confirming the expression level of adipogenic transcription factors after treating 3T3-L1, a mouse preadipocyte cell, with the extract of Siberian snail at different concentrations (250, 500 μg/ml).
Figure 3 shows a normal group (NC) fed a normal diet to 5-week-old C57BL/6 male mice, an obese group (HFD) fed a high-fat diet, and a high-fat diet administered to 5-week-old C57BL/6 male mice with drinking water. This is the result of confirming the change in body weight of the treatment group (HFD+UA).
Figure 4 shows a normal group (NC) fed a normal diet to 5-week-old C57BL/6 male mice, an obese group (HFD) fed a high-fat diet, and a high-fat diet administered to 5-week-old C57BL/6 male mice with drinking water. This is the result of confirming the feed and water intake of the treatment group (HFD+UA).
Figure 5 shows a normal group (NC) fed a normal diet to 5-week-old C57BL/6 male mice, an obese group (HFD) fed a high-fat diet, and a high-fat diet administered to 5-week-old C57BL/6 male mice with drinking water. This is the result of measuring the concentrations of Triglyceride (TG) and Total Cholesterol (TC) in the serum of the treatment group (HFD + UA).
Figure 6 shows a normal group (NC) fed a normal diet to 5-week-old C57BL/6 male mice, an obese group (HFD) fed a high-fat diet, and a high-fat diet administered to 5-week-old C57BL/6 male mice with drinking water. These are the results of measuring the oral glucose tolerance and insulin resistance of the treatment group (HFD+UA).
Figure 7 shows a normal group (NC) fed a normal diet to 5-week-old C57BL/6 male mice, an obese group (HFD) fed a high-fat diet, and a high-fat diet administered to 5-week-old C57BL/6 male mice with drinking water. This is the result of histological observation of the liver tissue of the treatment group (HFD+UA).

Hereinafter, the present invention will be described with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

<Example> Anti-obesity activity and anti-diabetic activity of Siberian sage extract

1. Sample preparation and experimental method

1.1 Sample preparation - Preparation of seaweed extract

After collecting the raw materials of seaweed (outpost), it was washed at least three times with running water and dried with cold air at 40°C. The dry powder was added to 80% ethanol and extracted for 24 hours. Afterwards, it was filtered and concentrated under reduced pressure. The concentrate was freeze-dried to prepare the final extract.

1.2 Test method

1.2.1 In vitro test

(1) Cell viability measurement method

3T3-L1 cells, a preadipocyte cell line, were cultured in DMEM medium supplemented with 10% bovine serum and 1% penicillin/streptomycin and in an incubator maintained at 37°C and 5% CO ₂ . The cultured 3T3-L1 cells were adjusted to 1×10 ⁵ cells/mL, dispensed at 100 μL each into 96 wells, and cultured for 24 hours at 37°C and 5% CO ₂ conditions. The extract of Siberian snail was prepared at a concentration of 31.25 to 500 μg/mL, dispensed at 200 μL each, and cultured for 3 days at 37°C and 5% CO ₂ conditions. Afterwards, MTT solution (Ambresco, USA) with a concentration of 5 mg/mL was prepared, dispensed at 20 μL each, and incubated at 37°C in 5% CO ₂ for 2 hours. The medium was removed, 200 μL of DMSO (dimethyl sulfoxide) was dispensed, mixed, and the absorbance was measured at 560 nm.

(2) Inhibition of adipocyte differentiation and RNA extraction

Cultured 3T3-L1 cells were inoculated into a 6 well plate (3×10 ⁵ cells/well), and when the cells were 100% confluent, 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma, USA), 1 μM Cultured in 10% FBS-DMEM (MDI medium) containing dexamethasone (Sigma, USA) and 10 μg/mL insulin (Sigma, USA). To confirm the effect of each sample on inhibiting adipocyte differentiation, samples were treated with MDI medium at different concentrations, and after 48 hours, they were replaced with MDI medium containing 10 μg/mL insulin. On the 4th day of differentiation, the medium was replaced with DMEM containing only 10% FBS, and the experiment was conducted for a total of 8 days from the start of differentiation induction. Afterwards, 1 ml of QIAzol lysis reagent (Qiagen science, USA) was dispensed into each well to lyse the cells. 200 μl of chloroform was dispensed into the lysate, mixed for 15 seconds, centrifuged at 12,000g for 15 minutes, and the supernatant was added with 500 μl of isopropanol. It was transferred to the containing tube, mixed, and centrifuged again at 12,000 g for 10 minutes. After removing the supernatant, 1 ml of 75% ethanol prepared by mixing 100% ethanol and 0.1% diethyl pyrocarbonate water (DEPC) at a ratio of 75:25 was dispensed into each tube, centrifuged at 12,000g for 5 minutes, and the supernatant was After removal, it was dried at room temperature. After dissolving nuclease free water in 40 μl portions, 995 μl of 0.1% DEPC water was added to 5 μl of RNA, and the amount of total RNA was quantified by measuring absorbance at 260 nm.

(3) Real-time polymerase chain reaction (qRT-PCR)

1 μg of RNA was qRT-PCR amplified using the One Step SYBR PrimeScript RT-PCR Kit (Takara, Japan), and the sequences of the primers used are shown in Table 1. qRT-PCR was analyzed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and the conditions were 42°C for 5 minutes, 95°C for 10 seconds, followed by 95°C for 3 seconds and 60°C. This was repeated 40 times for 30 seconds at ℃.

Primer sequences used to amplify adipogenesis-related RNA target order sequence number SREBP1c-Forward GCG CTA CCG GTC TTC TAT CA One SREBP1c-Reverse TGT GTG CAC TTC GTA GGG TC 2 FAS-Forward CTA CCA GGC CAT CCG TAG TG 3 FAS-Reverse ACA ATA TCC ACT CCC TGA ATC 4 GAPDH-Forward CCC CTC TGG AAA GCT GTG G 5 GAPDH-Reverse ACA TTG GGG GTA GGA ACA CG 6

1.2.2 Animal model testing

(1) Creation of a model to prevent obesity caused by a high-fat diet

Five-week-old C57BL/6 male mice were purchased and acclimatized for one week while maintaining constant temperature (22 ± 1°C) and constant humidity (45 ± 10%) and adjusting light and dark in a 12-hour cycle. The experimental groups were a normal group (NC) administered a normal diet (ND) and distilled water (DW), a high-fat diet control group (HFD), and a high-fat diet administered with drinking water prepared with a 1% concentration of Corolla extract. (HFD+UA) was divided into two feeding groups. All groups were divided into 4 groups of 6 animals each and the experiment was conducted for 12 weeks.

(2) Weight change and analysis of diet and water intake

Body weight was measured once a week during the experimental period, and diet and drinking water were analyzed by measuring the amount fed and remaining amount every 2 to 3 days and calculating the amount consumed per day by one animal.

(3) Serum test for liver function test

Blood was left at room temperature for more than 30 minutes to coagulate, and then centrifuged (3,000 rpm, 30 min) to obtain glutamic oxaloacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), gamma-glutamyl transferase (GGT), and alkaline phosphatase (ALP) from the serum. ), total cholesterol, triglyceride, and glucose concentrations were measured.

(4) Oral glucose tolerance test (OGTT)

When performing the oral glucose tolerance test, all animals were fasted for more than 12 hours. Glucose solution was administered orally at 2 g per body weight (kg), and then blood was collected from the tail vein of the experimental animals at 0, 30, 60, 90, and 120 minutes, respectively, and changes in blood sugar were measured using a blood glucose meter.

(5) Insulin tolerance test (ITT)

In the insulin tolerance test (ITT), mice were fasted for 6 hours and injected intraperitoneally with 0.75 unit/kg of insulin. Blood glucose was measured before injection (time = 0) and at 30, 60, 90, and 120 minutes after injection. To analyze insulin sensitivity, the area under the ITT curve was calculated.

(6) Organ weight measurement and histopathological analysis (H&E staining)

To measure organ weight, at the end of all experiments, the experimental animals were fasted for 12 hours, anesthetized using an anesthetic, dissected, and the liver, epididymal fat, perirenal fat, and mesenteric fat were extracted. The extracted organs were washed with saline solution, moisture was removed with filter paper, and their weight was measured and analyzed. Liver tissue was fixed in 10% formalin and embedded in paraffin blocks. Afterwards, serial sections were produced by sectioning at a thickness of 3 μm. Each section was stained with hematoxylin and eosin and analyzed under a light microscope.

2. Experimental results

2.1 In vitro evaluation results

(1) Confirmation of cytotoxicity and determination of appropriate concentration of seaweed

By confirming the cytotoxicity of 3T3-L1 cells, the concentration range of UA extract to be used in the experiment was selected. As a result of checking the cell viability by using the extract at concentrations of 31.25, 62.5, 125, 250, and 500 μg/mL on cells grown to 80-90%, the results showed that when treated with the Green Leaf extract, no cytotoxicity was observed at all concentrations below 500 μg/mL. did not (Figure 1).

(2) Measurement of expression level of transcription factors related to adipogenesis

To determine whether the expression of adipogenesis-related transcription factors (SREBP1c and FAS genes) is suppressed by UA, 3T3-L1 preadipocytes were treated with UA extract at various concentrations (250, 500 μg/mL). The cells were cultured for one day and analyzed. As a result, it was confirmed that the expression levels of both SREBP1c and FAS genes were suppressed depending on the concentration of the SREBP1c and FAS extracts (Figure 2).

2. Animal model evaluation results

(1) Analysis of weight change in experimental animals

The effect of preventing obesity was analyzed by administering a 1% concentration of Corianderwort extract in drinking water along with a high-fat diet. In the experimental group administered negative UA, the rate of weight gain was analyzed to be lower than that in the obese group (HFD), and when converted to weight gain and analyzed, the weight gain rate was measured to be significantly lower in the UA-treated group ( Figure 3)

(2) Analysis of feed and water intake of experimental animals

Since it is difficult to clearly determine the obesity prevention effect of the extract if there is a decrease in feed intake or water intake due to extract administration, dietary intake and water intake were analyzed. In the case of dietary intake, the intake was measured every day, and in the case of drinking water, the intake was measured and analyzed at two-day intervals. As a result, there was no difference in dietary and drinking water intake between the obese group (HFD) and the group administered with Corolla extract (HFD+UA). It was judged to be a weight loss effect caused by the extract (Table 2, Figure 4).

Values of food and water intake of experimental animals division Normal group (NC) Obese group (HFD) Group administered with Corianderwort extract (HFD+UA) feed intake 2.51±0.33 2.25±0.13 2.22±0.11 drinking water 4.89±0.63 5.18±0.52 4.95±0.44

(3) Effect of improving liver function through serology test

As a result of serum biochemical analysis, GOT and GPT items related to liver function tended to increase in the obese group (HFD) compared to the normal group (NC). +UA), GOT and GPT levels were significantly decreased. In addition, significant differences in total cholesterol (CHO), triglyceride (TG), and blood sugar (GLU) were confirmed in the group administered the extract of the seaweed (HFD+UA) compared to the obese group (HFD). Blood sugar levels were found to decrease in the extract administered group (Table 3, Figure 5).

division Normal group (NC) Obese group (HFD) Group administered with Corianderwort extract (HFD+UA) GOT 211.40±15.96 U/L 312.83±15.66 U/L 217.17±11.10 U/L GPT 55.40±8.11 U/L 138.50±15.37 U/L 89.83±8.76 U/L GGT 0.78±0.64 U/L 0.51±0.53 U/L 0.73±0.43 U/L ALP 68.88±6.34 U/L 73.82±11.20 U/L 68.80±5.06 U/L CHO 113.25±6.33 mg/dL 193.67±11.32 mg/dL 156.33±9.67 mg/dL TG 34.06±5.12 mg/dL 50.93±5.97 mg/dL 39.00±5.05 mg/dL GLU 204.80±5.88 mg/dL 281.17±9.33 mg/dL 223.67±6.92 mg/dL

(4) Effect of improving oral glucose tolerance

After fasting the experimental animals for more than 12 hours, glucose solution was orally administered at 2 g per body weight (kg), and then blood was collected from the tail vein of the experimental animals at 0, 30, 60, 90, and 120 minutes, respectively, using a blood glucose meter. As a result of measuring the change in blood sugar, the highest blood sugar level was measured in the obese group (HFD) in the blood sugar analysis before administering the glucose solution. After 30 minutes, the blood sugar level was measured to be higher than 300 mg/dL in the obese group (HFD) compared to the group administered the Corolla extract (HFD+UA). As a result of analyzing blood sugar 120 minutes after glucose administration, the group administered the Corolla extract was 0. The blood sugar level was measured similarly to the blood sugar level measured at 0 minutes, but in the case of the obese group (HFD), the blood sugar level did not recover to the 0 minute blood sugar level (FIG. 6).

(5) Effect of improving insulin resistance

For the insulin resistance test, blood sugar was measured by intraperitoneally injecting insulin at a dose of 0.75 unit/kg, and blood was collected from the tail vein of the experimental animal at 0, 30, 60, 90, and 120 minutes as in the oral glucose tolerance test to measure blood sugar. was measured. In the case of the obese group (HFD), blood sugar levels measured 30 minutes after insulin injection showed higher blood sugar levels than normal, confirming insulin resistance. However, in the case of the group treated with the UA extract, blood sugar levels measured 30 minutes later showed blood sugar levels similar to normal, confirming a decrease in insulin resistance. As a result of calculating and analyzing the area under the curve (AUC) using the graph of the insulin resistance test, a significant difference occurred between the normal group and the obese group, and a significant difference was also confirmed between the obese group and the group administered with the AUC extract (Figure 6 ).

(6) Anti-obesity effect through organ weight measurement

The weight of the liver, epididymal fat, mesenteric fat, and perirenal fat was measured and found to be significantly higher in the obese group (HFD) when comparing the normal group (NC) and the obese group (HFD). In the obese group and the experimental group administered the L. aquila extract, the tissue weight of the extract-administered group was measured lower than that of the obese group in white adipose tissue and liver tissue. This showed the same results as the weight analysis results (Table 4).

Results of organ weight measurement for each condition of experimental animals division Normal group (NC) Obese group (HFD) Group administered with Corianderwort extract (HFD+UA) epididymal fat 0.59±0.13 3.01±0.42 2.32±0.17 mesenteric fat 0.59±0.06 2.00±0.29 1.40±0.45 Perirenal fat 0.24±0.08 1.83±0.26 1.48±0.19 liver 1.12±0.07 1.80±0.18 1.44±0.27

(7) Anti-obesity effect through histopathological analysis

As a result of analyzing the liver tissue by performing H&E staining, many fat droplets were observed in the liver tissue of the obese group (HFD), showing the appearance of a fatty liver. In the case of the group administered with the Corianderwort extract (HFD+UA), fewer fat droplets were observed, and fat droplets were observed in the liver tissue. The size also showed a significant difference (Figure 7).

Claims (9)

An anti-obesity food composition containing Ulva pertusa extract as an active ingredient.
According to paragraph 1,
A composition wherein the extract is an extract of water, ethanol, or a mixed solvent thereof.
A pharmaceutical composition for anti-obesity comprising Ulva pertusa extract as an active ingredient.
According to paragraph 3,
A composition wherein the extract is an extract of water, ethanol, or a mixed solvent thereof.
An anti-diabetic food composition containing Ulva pertusa extract as an active ingredient.
According to paragraph 1,
A composition wherein the extract is an extract of water, ethanol, or a mixed solvent thereof.
An anti-diabetic pharmaceutical composition comprising Ulva pertusa extract as an active ingredient.
According to paragraph 3,
A composition wherein the extract is an extract of water, ethanol, or a mixed solvent thereof.
A composition for improving fatty liver comprising Ulva pertusa extract as an active ingredient.