Visceral fat accumulation inhibitor and AKT activation / ACC activity regulator

Abstract

To provide visceral fat accumulation inhibitors, AKT activation / ACC activity regulators, and the like, as agents useful for inhibiting visceral fat accumulation.SOLUTION: A visceral fat accumulation inhibitor is intended to inhibit visceral fat accumulation through oral consumption. It contains extract from Citrus sphaerocarpa peel as an active ingredient, while also containing lecithin. The lecithin contains the extract from Citrus sphaerocarpa peel. Based on 100 pts.mass of the extract from Citrus sphaerocarpa peel, the lecithin is contained by 120 pts.mass-500 pts.mass.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a visceral fat accumulation inhibitor, an AKT activator, an ACC activity regulator, and the like. [Background technology]

[0002] In recent years, the number of obese people has been increasing worldwide, and is said to account for approximately 10% of the world's population. Obesity is a serious health problem and a risk factor for many diseases, including type 2 diabetes, coronary heart disease, hypertension, and cancer. When caloric intake increases excessively, fat cells store energy in the form of triacylglycerol, which results in the promotion of adipogenesis, an increase in the amount of adipose tissue, and obesity. Avoiding and improving obesity is necessary to prevent lifestyle-related diseases, and in recent years, research has been conducted into regulating fat production.

[0003] Here, since preadipocyte 3T3-L1 cells are cells that efficiently differentiate into adipocytes when stimulated with drugs, they have been used in various studies on the mechanisms of adipocyte differentiation and adipogenesis. Furthermore, studies using preadipocyte 3T3-L1 cells have reported that drugs and natural ingredients promote or inhibit adipogenesis.

[0004] The present applicant has been conducting research into the physiological functions of citrus peels. In recent years, in a study using preadipocyte 3T3-L1 cells, it has been reported that an extract from kabosu peel inhibits lipid droplet accumulation in adipocytes (see Patent Document 1). However, Patent Document 1 does not report on in vivo results, nor does it report on the mechanism of action.

[0005] Citrus fruits are known to contain polymethoxyflavones (PMFs), such as nobiletin, as functional components. PMFs are said to be involved in the differentiation and generation of adipocytes. Specifically, they inhibit the activity of intracellular phosphodiesterase E (PDE), thereby excessively increasing intracellular cyclic adenosine monophosphate (cAMP). When cAMP binds to protein kinase A (PKA), PKA is activated, which activates AMP-activated protein kinase (AMPK), which regulates the downstream acetyl-CoA carboxylase (ACC), promoting the lipolysis system and suppressing adipogenesis and accumulation. Thus, activation of the PKA / AMPK signaling pathway is thought to be one way to suppress adipogenesis and accumulation.

[0006] Obesity can be divided into visceral fat obesity and subcutaneous fat obesity. Visceral fat obesity is more likely to cause various lifestyle-related diseases than subcutaneous fat obesity, and from the perspective of preventing lifestyle-related diseases, it is considered particularly important to suppress the accumulation of visceral fat. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2021-183565 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide an agent that is effective in inhibiting the accumulation of visceral fat. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have found that oral ingestion of an extract from kabosu peel inhibits the accumulation of visceral fat in particular. Furthermore, when the mechanism of action was examined, it was found that in liver tissue, activation of the PI3K / AKT signaling pathway due to AKT activation contributes to the inhibition, and further, in white adipose tissue (WAT), activation of ACC present downstream of the AMPK signal is regulated by phosphorylation. Although activation of the PKA / AMPK signaling pathway by polymethoxyflavones (PMFs) contained in citrus fruits has been reported, this time, activation of the PI3K / AKT signaling pathway by citrus fruits has been newly discovered. That is, the present invention has the following configuration.

[0010] The visceral fat accumulation inhibitor of the present invention is an agent for inhibiting visceral fat accumulation by oral ingestion, and is characterized by containing an extract of kabosu peel as an active ingredient.

[0011] The visceral fat accumulation inhibitor is characterized in that it contains lecithin and the extract of kabosu peel is encapsulated in the lecithin, and contains 120 to 500 parts by mass of the lecithin per 100 parts by mass of the extract of kabosu peel.

[0012] The visceral fat is characterized as being fat accumulated in the abdominal cavity. Fat accumulated in the abdominal cavity is a concept that includes fatty liver.

[0013] The AKT activator of the present invention is characterized by containing an extract of kabosu peel as an active ingredient, and has the effect of activating AKT particularly in liver tissue.

[0014] The ACC activity regulator of the present invention is characterized by containing an extract of kabosu peel as an active ingredient. In a specific embodiment, the regulator is an agent that has the effect of activating ACC in white adipose tissue (ACC activator). Effect of the Invention

[0015] The visceral fat accumulation inhibitor, AKT activator, and ACC activity regulator of the present invention contain an extract of kabosu peel as an active ingredient, and as shown in the examples described later, they are particularly effective in inhibiting the accumulation of visceral fat compared to subcutaneous fat, and are effective, for example, in preventing and / or improving visceral fat-type obesity. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the results of an obesity induction test in zebrafish. [Diagram 2] FIG. 1 shows changes in body weight and other parameters in an evaluation test using high-fat diet-induced obese mice. [Diagram 3] FIG. 1 shows micro-CT scan images of mice and their respective fat masses. [Figure 4] FIG. 1 shows analytical images of mouse liver tissue. [Diagram 5] FIG. 1 shows target genes. [Figure 6] FIG. 1 shows changes in the expression of glycolipid metabolism-related genes in liver tissue. [Figure 7] FIG. 1 shows changes in the expression of glycolipid metabolism-related genes in white adipose tissue. [Figure 8] FIG. 1 shows immunofluorescence staining of liver tissue. [Figure 9] FIG. 1 shows immunofluorescence staining of white adipose tissue. [Figure 10] FIG. 1 shows the expression of Ampk protein and its phosphorylation. [Figure 11] FIG. 1 shows the expression of Acc protein and its phosphorylation. [Figure 12] FIG. 1 shows the expression of Pka protein and its phosphorylation. [Figure 13] FIG. 1 shows the expression and phosphorylation of Akt protein. [Figure 14] FIG. 1 shows the expression of FoxO1 protein and its phosphorylation. [Figure 15] FIG. 1 shows immunofluorescence staining of 3T3-L1 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The visceral fat accumulation inhibitor of the present invention contains an extract of kabosu peel (also simply referred to as kabosu extract) as an active ingredient. In the present invention, the "visceral fat accumulation inhibitor" is an agent that inhibits fat (e.g., mesenteric) that accumulates around the internal organs by oral ingestion. It may also have the effect of reducing accumulated visceral fat. Visceral fat is distinguished from subcutaneous fat that accumulates between the skin and muscle.

[0018] The AKT activator of the present invention contains an extract of kabosu peel as an active ingredient. AKT (also called protein kinase B) is a serine / threonine kinase. In the present invention, the "AKT activator" is an agent that has the effect of activating AKT, and particularly activates AKT in internal organs (e.g., liver). AKT is activated by phosphorylation of serine 473 (Ser473) and threonine 308 (Thr308). It is believed that activation of AKT affects proteins and factors present downstream, suppressing the accumulation of visceral fat.

[0019] In general, the signal transduction pathways involved in glycolysis / gluconeogenesis are known to be the insulin signal transduction pathway, the PI3K / AKT signal transduction pathway, the PKA / AMPK signal transduction pathway, and the glucagon signal transduction pathway. This study suggests that kabosu extract activates AKT, particularly in the internal organs, and directly activates the PI3K / AKT signal transduction pathway. It is also possible that kabosu extract acts on other related factors, directly or indirectly activating other transduction pathways.

[0020] The ACC activity regulator of the present invention contains an extract of kabosu peel as an active ingredient. The ACC activity regulator is expressed, for example, by activating AMPK. Activation of AMPK directly activates the PKA / AMPK signal transduction pathway, which regulates the downstream ACC, promotes the lipolysis system, and leads to the inhibition of fat production and accumulation. For example, when ACC is phosphorylated, the lipolysis system is promoted, and when ACC is not phosphorylated (dephosphorylated), the system leans toward the lipogenesis system. The ACC activity regulator promotes the lipolysis system by maintaining the phosphorylated state of ACC and promoting the phosphorylation of ACC.

[0021] The agent of the present invention may have an ACC activity modulating effect in addition to an AKT activation effect, in which case it can be called an agent for modulating both AKT activation and ACC activity.

[0022] Kabosu is a citrus fruit of the genus Rutaceae and its scientific name is Citrus sphaerocarpa. Kabosu is the fruit of a species closely related to yuzu, and is mainly produced in Oita Prefecture in Japan. In the present invention, the peel refers to the endocarp (albedo) and exocarp (flavedo), and refers to the part of the fruit excluding the pulp, sepal membrane, seeds, etc. As the kabosu peel, it is preferable to use the pomace discharged after squeezing the fruit juice from the viewpoint of effective use of resources.

[0023] Methods for extracting kabosu peel include organic solvent extraction, mixed solvent extraction with organic solvent and water, and supercritical extraction. Examples of organic solvents include non-polar solvents such as normal hexane, diethyl ether, cyclohexane, dichloromethane, 1,1,2,2-tetrafluoroethane, 1,1,2-trichloroethene, butane, and propane; and protic polar organic solvents such as methanol, ethanol, propanol, and isopropanol. Among the above organic solvents, normal hexane is preferred because it can efficiently extract the desired components and from the viewpoint of food production. A specific method for extracting kabosu peel will be described later.

[0024] As shown in the Examples below, the kabosu extract of the present invention can also be used as an active ingredient of an agent for promoting or suppressing the expression of genes related to glycolipid metabolism. For example, it can be used as an agent for promoting the expression of the phosphoenolpyruvate carboxykinase 1 (PCK1) gene.

[0025] The various agents of the present invention may be in any dosage form, as long as they can be orally ingested, such as tablets, powders, capsules, granules, liquids, and syrups.

[0026] The amount of the kabosu extract in each agent of the present invention is, for example, 0.001% by mass to 20% by mass, and preferably 0.01% by mass to 10% by mass, based on the total amount of the agent.

[0027] The various agents of the present invention may contain additives other than the kabosu extract, such as excipients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, and the like that are commonly used in foods and pharmaceuticals, as needed.

[0028] The additive preferably contains lecithin, and more preferably contains the lecithin as liposome. The extract of kabosu peel contains a large amount of oil-soluble substances derived from the kabosu peel and exhibits lipophilicity. By forming the kabosu extract into a liposome using lecithin and encapsulating it, the oil-soluble substances contained in the kabosu extract can be efficiently absorbed into the body even when taken orally.

[0029] Here, lecithin refers to a lipid mixture mainly composed of various phospholipids. Examples of phospholipids contained in the lecithin include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylserine (PS). Any lipid mixture mainly composed of these phospholipids can be used as the lecithin. The concentration of phosphatidylcholine (PC) in the lecithin is, for example, 30% by mass or more, and preferably 50% by mass or more. The upper limit of the phosphatidylcholine concentration is, for example, 75% by mass.

[0030] Lecithin can be used without any particular limitation as long as it is obtained from plant seeds or animal raw materials and contains phospholipids as the main component, for example, soybean lecithin, rapeseed lecithin, egg yolk lecithin, sunflower lecithin, etc. In addition, as the lecithin, a paste-like lecithin containing oil, a powdered lecithin obtained by removing the oil from the paste-like lecithin and drying it, a fractionated lecithin obtained by fractionating and purifying the paste-like lecithin and the powdered lecithin, an enzymatically decomposed lecithin (lysolecithin) obtained by decomposing lecithin with an enzyme, etc. can be used.

[0031] In the various agents of the present invention, the content of lecithin relative to the kabosu extract is not particularly limited, but the content of lecithin relative to 100 parts by mass of the kabosu extract is, for example, 10 parts by mass to 1,000 parts by mass, preferably 100 parts by mass to 1,000 parts by mass, and may be 120 parts by mass to 500 parts by mass.

[0032] The method for producing various agents of the present invention includes an extraction step of extracting kabosu peel to obtain an extract. The kabosu peel used as a raw material may contain pulp, juice, etc. that may be mixed in during the processing. The extraction step is carried out, for example, by stirring the kabosu peel in a non-polar solvent and then immersing it at a predetermined temperature. The immersion temperature is not particularly limited, but is preferably 20°C or lower, and more preferably 0°C or lower. The amount of the non-polar solvent is not particularly limited, but is preferably 1 to 10 times, and more preferably 1 to 5 times, the volume of the kabosu peel.

[0033] After soaking for, for example, 10 minutes to 2 hours, the solvent is removed from the treatment liquid at a temperature below room temperature (for example, below 20°C) using a centrifugal membrane concentrator or a vacuum distillation apparatus, etc., to obtain an extract containing oil-soluble substances.

[0034] The obtained kabosu extract can be used as it is as various preparations, or it may be diluted with an appropriate solvent and used, or it may be made into a powder or paste and used as various preparations.

[0035] In addition, it is preferable to perform a pretreatment before the above extraction step in which the kabosu peel is frozen and crushed to obtain a frozen crushed product. For example, the kabosu peel is frozen after squeezing the juice. The freezing temperature is -5°C or lower, and preferably -10°C or lower. The lower limit of the freezing temperature is about -30°C. Freezing can prevent the pomace from deteriorating or spoiling. The frozen product is then crushed using a stirring mill or the like to obtain a frozen crushed product. The crushed size is preferably 5 mm or less. In the case of the pomace, since the pomace itself is in a fibrous state, the extraction step may be performed after freezing without crushing.

[0036] In the present invention, the kabosu extract obtained in the extraction step may be subjected to post-treatment such as a further extraction step or a step of removing impurities.

[0037] The various agents of the present invention can be used in humans (eg, healthy individuals) and non-human animals who regularly consume a high amount of fatty food.

[0038] The various agents of the present invention can be orally ingested, and can be used in combination with any additives, for example, in the form of food and drink, food additives, medicines, supplements, etc. For example, they can be used as food and drink, food additives, supplements, etc. that claim or display the inhibition of visceral fat accumulation or weight loss, etc., for people with visceral fat obesity or those prone to it.

[0039] When the various agents of the present invention are used as foods, the forms include various foods such as breads, cakes, noodles, confectioneries, jellies, frozen foods, ice creams, dairy products, beverages, etc. To prepare foods in various forms, the various agents of the present invention can be used alone or in appropriate combination with other food materials, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc. EXAMPLES

[0040] [1. Preparation of kabosu extract] The kabosu peel was used as the raw material. As the kabosu raw material, the pomace (water content about 80% by mass) of the kabosu peel from which the juice was squeezed was prepared. 60 kg of this pomace was frozen at -10°C or lower. About 1 to 2 times the volume of normal hexane (extraction solvent) was added to this frozen raw material, which was stirred and crushed, and then immersed at 0°C or lower for 15 minutes. The normal hexane solution (layer) was then collected, and normal hexane was removed at a temperature below room temperature using a centrifugal thin-film concentrator and a reduced-pressure distillation apparatus. The obtained kabosu extract (CSP) was a yellow-green, transparent, oil-soluble liquid containing volatile components specific to kabosu.

[0041] [2. Zebrafish obesity induction test] As an in vivo model, the anti-obesity properties were evaluated by the zebrafish obesity test (ZOT) using young zebrafish. The procedure was as follows. 1: Five fish with a body length of approximately 10 mm (30 dpf) were transferred to a 6-well plate, and 5 ml of HO was added per well. 2: 3 mg of boiled egg yolk was added as a high-fat diet, and the mice were reared at 28°C for 24 hours with agitation (150 rpm). 3: After 24 hours, visceral adipose tissue (VAT) was stained with Nile Red, and VAT was observed under a fluorescent microscope and images were taken. 4-1: Test 1: Mice were kept at 28°C without food for 24 hours and then treated with each sample (control group, CSP10 group: CSP was added to wells at 10 μg / mL, CSP20 group: CSP was added to wells at 20 μg / mL). After 24 hours, staining, observation and images were taken as above, and images were taken as above. 4-2: Test 2: Mice were kept at 28°C for 72 hours without feeding and were then treated with each sample (Control group, CSP10 group: 10μg / mL CSP added to wells). After 24 and 72 hours, staining, observation and imaging were performed as described above, and images were taken as described above.

[0042] In young zebrafish, visceral adipose tissue was specifically stained red with Nile Red. In Test Example 1, after ingestion of a high-fat diet and after 24 hours of fasting, and in Test Example 2, after ingestion of a high-fat diet, after 24 hours of fasting, and after 72 hours of fasting, the area and intensity of red staining were depicted using ImageJ software, and the amount of VAT was quantified. The results are shown in Figure 1 together with images.

[0043] As shown in Figure 1, in Test 1, the CSP10 group showed a tendency to reduce visceral fat tissue due to high-fat diet intake, and the CSP20 group significantly reduced visceral fat tissue. In particular, in Test 2, although the kabosu extract was at a lower concentration than in Test 1, the visceral fat tissue was further reduced by treatment for a long period of time. This confirmed the effect of reducing accumulated visceral fat.

[0044] [3. Test on high-fat diet-induced obese mice] (a) Evaluation in feeding trials As an in vivo model, the anti-obesity properties were evaluated using a high-fat diet-induced obese mouse test. Eight-week-old C57BL / 6NCrSlc (B6) male mice were used in the test. The composition of each group (n=5) is as follows:

[0045] Group A: They were fed a normal diet and were given 0.2% by mass of a microemulsion formulation of lecithin (phosphatidylcholine content 70% by mass) dissolved in water and allowed to drink freely for 6 weeks [ND(-)]. Group B: They were fed a normal diet, and lecithin (phosphatidylcholine content 70% by mass) was made into a microemulsion formulation using a high-pressure homoemulsifier, and kabosu extract was encapsulated in it (lecithin 0.2% by mass, kabosu extract 0.1% by mass) which was dissolved in water and given free access to water for 6 weeks [ND+0.1% CSP]. Group C: They were fed a high-fat diet and were given 0.2% by mass of a microemulsion formulation of lecithin (phosphatidylcholine content 70% by mass) dissolved in water and allowed to drink ad libitum for 6 weeks [HFD(-)]. Group D: fed a high-fat diet and allowed to drink ad libitum for 6 weeks. Lecithin (phosphatidylcholine content 70% by mass) was made into a microemulsion formulation using a high-pressure homoemulsifier and encapsulated with kabosu extract (lecithin 0.2% by mass, kabosu extract 0.1% by mass) was dissolved in water (HFD + 0.1% CSP).

[0046] The mice in groups A to D were kept for 6 weeks. After 6 weeks, the body weight, food intake, fasting blood glucose level, plasma TAG, and plasma T-CHO were measured. The results are shown in Figure 2. In each bar graph, groups A to D are shown from the right (same for Figures 3 and 4).

[0047] As shown in Figure 2, weight gain was observed in groups C and D on a high-fat diet (HFD) compared to groups A and B on a normal diet (ND). Furthermore, among groups C and D, group D, which was given a high dose of kabosu extract, lost significantly more weight than group C, which did not receive kabosu extract. This is thought to be because the increase in visceral fat caused by the intake of a high-fat diet was suppressed. No significant differences were observed between groups in terms of food intake, fasting blood glucose level, plasma TAG, or plasma T-CHO.

[0048] Figure 3 shows micro-CT scan images of mice (groups A to D) after six weeks, along with quantitative analysis of visceral fat and subcutaneous fat. As shown in Figures 3(b) and (c), it can be seen that both visceral fat and subcutaneous fat increased due to the ingestion of a high-fat diet (comparison of groups A and C). In contrast, a significant reduction was confirmed, particularly in visceral fat, by ingestion of kabosu extract (group D) (see Figure 3(b)). In other words, it can be said that kabosu extract suppressed the accumulation of visceral fat caused by the ingestion of a high-fat diet compared to subcutaneous fat.

[0049] (b) Analysis of liver tissue by Oil Red O staining The liver tissue was stained with Oil Red O and analyzed according to the following procedure. 1: Liver tissue was fixed in 4% paraformaldehyde solution at 4°C for more than 24 hours. 2: After fixation, the sections were successively immersed in 10%, 15%, and 20% sucrose / PBS. 3: Immersed in tissue mount and frozen in isopentane cooled with liquid nitrogen. 4: Using a cryostat for frozen sections, the tissue was sliced ​​at 0.08 mm at -25°C. 5: The prepared sample was air-dried for one day. 6: Stained with Oil Red O solution and washed with 60% isopropanol. 7: The sections were then stained with hematoxylin / eosin solution and washed with running water. 8: The specimen was air-dried slightly and then mounted with an aqueous mounting medium. 9: The sample was observed under a microscope and images were taken.

[0050] The captured images and the results of quantitative analysis of the stained areas are shown in Figure 4. As shown in Figure 4, a significant reduction in fat accumulation in the liver was observed in Group D, which received a high dose of kabosu extract.

[0051] (c) Analysis of changes in gene expression related to glycolipid metabolism The changes in the expression of genes related to glycolipid metabolism were analyzed by the following procedure: The tissues used for the analysis were liver tissue and epididymal white adipose tissue (eWAT). 1: Total RNA was extracted from liver tissue or white adipose tissue using TRIzol reagent and the RNeasy Lipid Tissue Mini Kit. 2: After measuring the RNA concentration, the RNA was reverse transcribed into cDNA equivalent to 1 ug of RNA using ReverTra Ace qPCR RT Master Mix with random primers. 3: Quantitative PCR was performed using the Step One Plus system with Power Up SYBR Green Master Mix. For each target gene (see Figure 5), forward and reverse primers were used. 4: The target genes were analyzed by comparative quantification using the standard curve method.

[0052] The correspondence between each primer and the sequence number is as follows: Pparg gene: Forward primer (SEQ ID NO: 1), Reverse primer (SEQ ID NO: 2) Fasn gene: Forward primer (SEQ ID NO: 3), Reverse primer (SEQ ID NO: 4) Srebp1 gene: Forward primer (SEQ ID NO: 5), Reverse primer (SEQ ID NO: 6) Cebpa gene: Forward primer (SEQ ID NO: 7), Reverse primer (SEQ ID NO: 8) Cebpb gene: Forward primer (SEQ ID NO: 9), Reverse primer (SEQ ID NO: 10) Ppara gene: Forward primer (SEQ ID NO: 11), Reverse primer (SEQ ID NO: 12) Acox1 gene: Forward primer (SEQ ID NO: 13), Reverse primer (SEQ ID NO: 14) Ppargc1a gene: Forward primer (SEQ ID NO: 15), Reverse primer (SEQ ID NO: 16) Cpt1a gene: Forward primer (SEQ ID NO: 17), Reverse primer (SEQ ID NO: 18) PI3K gene: Forward primer (SEQ ID NO: 19), Reverse primer (SEQ ID NO: 20) Akt gene: Forward primer (SEQ ID NO: 21), Reverse primer (SEQ ID NO: 22) FoxO1 gene: Forward primer (SEQ ID NO: 23), Reverse primer (SEQ ID NO: 24) Pck1 gene: Forward primer (SEQ ID NO: 25), Reverse primer (SEQ ID NO: 26) Prkaca gene: Forward primer (SEQ ID NO: 27), Reverse primer (SEQ ID NO: 28) Prkaa1 gene: Forward primer (SEQ ID NO: 29), Reverse primer (SEQ ID NO: 30) Prkaa2 gene: Forward primer (SEQ ID NO: 31), Reverse primer (SEQ ID NO: 32) Acaca gene: Forward primer (SEQ ID NO: 33), Reverse primer (SEQ ID NO: 34) Pde3b gene: Forward primer (SEQ ID NO: 35), Reverse primer (SEQ ID NO: 36) Gapdh gene: Forward primer (SEQ ID NO: 37), Reverse primer (SEQ ID NO: 38)

[0053] The target genes used were genes involved in lipogenesis, lipolysis, the PI3K / AKT signaling pathway, and the AMPK / ACC signaling pathway. Figure 6 shows the changes in gene expression in liver tissue. For each gene, groups A, B, C, and D are shown from the right (similar to Figure 7). As shown in Figure 6, in high-fat diet-induced obese mice, the intake of kabosu extract (group D) resulted in significant promotion of the expression of the Fasn gene, Ppara gene, Acox1 gene, and Pck1 gene, and an increase in the expression of genes involved in lipolysis was observed. In other words, the agent containing the extract of kabosu peel as an active ingredient is also a promoter of Fasn gene expression, Ppara gene expression, Acox1 gene expression, and Pck1 gene expression in liver tissue. There was no change in the expression of the Akt gene in liver tissue.

[0054] Figure 7 shows the results in white adipose tissue (WAT). As shown in Figure 7, in high-fat diet-induced obese mice, the ingestion of kabosu extract (group D) resulted in a significant promotion of the expression of the Cebpa gene, Cebpb gene, Akt gene, Pck1 gene, Prkaa1 gene, and Prkaa2 gene, and an increase in the expression of genes involved in energy regulation and glucose metabolism was observed. In other words, an agent containing an extract of kabosu peel as an active ingredient is also an agent for promoting the expression of the Cebpa gene, Cebpb gene, Akt gene, Pck1 gene, Prkaa1 gene, and Prkaa2 gene in white adipose tissue. In particular, the expression of the Akt gene was significantly increased in white adipose tissue.

[0055] (d) Analysis of factors and proteins involved in lipid synthesis / degradation by immunofluorescence staining First, the specimens were prepared according to the following procedure. 1: Liver tissue and white adipose tissue were fixed in 4% paraformaldehyde solution at 4°C for 24 hours or more. 2: After fixation, the sections were washed with 1x TBS and immersed in liquid paraffin overnight. 3: The paraffin-embedded samples were sliced ​​to 0.003 mm using a microtome to prepare tissue specimens. 4: The prepared samples were air-dried for at least one day.

[0056] Immunofluorescence staining was performed using the prepared specimens. 1: The tissue specimen was deparaffinized sequentially using xylene, aqueous ethanol, and water. 2: Antigen retrieval: Citrate buffer was added and the mixture was treated in a pressure cooker for 3 minutes. 3: After 10 min, the tissue specimen was immersed in cold running water for 10 min. 4: After antigen retrieval, the plate was washed twice with 0.025% TritonX-100 / 1×TBS for 5 minutes each. 5: Incubated with blocking buffer (10% normal goat serum / 1% BSA / 1×TBS) for 2 hours (RT). 6: After blocking, the sections were incubated with primary polyclonal antibodies in detection buffer (1% BSA / 1×TBS) at 4° C. overnight. 7: After incubation, the plate was washed three times for 5 minutes with 0.025% TritonX-100 / 1×TBS. 8: Next, Alexa Fluor 488-conjugated goat anti-rabbit IgG was used as the secondary antibody and incubated in detection buffer (1% BSA / 1×TBS) for 60 minutes. 9: After incubation, the tissue specimens were washed three times for 5 minutes with 0.025% TritonX-100 / 1×TBS. 10: The tissue specimen was sealed with an anti-fade solution containing DAPI for nuclear counterstaining. 11: The tissue specimen was observed under a fluorescent microscope and images were taken.

[0057] An example of an image of immunofluorescent staining of liver tissue is shown in Figure 8, and an example of an image of immunofluorescent staining of white adipose tissue is shown in Figure 9. In liver tissue and white adipose tissue, the expression and phosphorylation of Ampk, Acc, Pka, Akt, and FoxO1 proteins by treatment with kabosu extract were compared. The results are shown in Figures 10 to 14. In each figure, Group A, Group B, Group C, and Group D are shown from the right.

[0058] In liver tissue, treatment with kabosu extract promoted the phosphorylation of Ampk and the phosphorylation of Akt, i.e., Ampk and Akt were activated by kabosu extract in liver tissue.

[0059] On the other hand, in white adipose tissue, treatment with kabosu extract did not promote the phosphorylation of Ampk or Akt, but promoted the phosphorylation of Acc and FoxO1. In other words, in white adipose tissue, the activity of Acc and FoxO1 was regulated by kabosu extract.

[0060] [4. In vitro evaluation] This was done according to the following procedure. 1: A 13 mm diameter circular cover glass was placed on the bottom of a 24-well plate (TPP), and 3T3-L1 cells were seeded on top of it. The cells were then cultured in an incubator set at 37°C and 5% CO2 using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) until they became confluent (maximum cell density). 2: After confirming that the cells were confluent, the medium was replaced and the cells were cultured for an additional 2 days. 3: A Dulbecco's modified Eagle medium (DMEM) containing 0.25 μM dexamethasone, 500 μM IBMX, and 10 μg / mL insulin was prepared, and the medium was replaced with this to initiate differentiation induction. 4: Two, four, and six days after differentiation induction, the medium was replaced with DMEM containing 5 μg / mL insulin. 5: On the 8th day after differentiation induction, the cells were fixed with 4% paraformaldehyde (PFA) / 1×TBS at room temperature for 10 minutes. 6: 0.1% TritonX-100 / 1×TBS was added, and cell membrane permeabilization was performed at room temperature for 10 minutes. 7:3% bovine serum albumin (BSA) / 1×TBS was added, and blocking was carried out at 37° C. for 1 hour. 8: Antibodies against Acc and pAcc were prepared, and the antibody solutions were added dropwise to the samples and incubated at 4°C overnight. 9: The plate was washed with shaking for 5 minutes three times in 0.05% Tween 20 / 1×TBS (TBST). AlexaFluor 488 goat anti-rabbit IgG solution prepared in 10:1% BSA / 1×TBS was added dropwise to the sample and then incubated at 37° C. for 1 hour. 11: Washing was performed with shaking for 5 minutes three times with 0.05% TBST. 12: A Hoechst 33342 solution diluted 10,000 times with TBS was dropped onto the sample and incubated at room temperature for 5 minutes to stain the cell nuclei. After washing once each with 13:1× TBS and ultrapure water, the cover glass was removed and the sections were mounted on slides with 2.5% DABCO / 90% glycerol / 1× PBS. 14: Each protein was visualized and photographed under a fluorescence microscope (Olympus Corporation, CKX53).

[0061] [Image quantification] This was done according to the following procedure. Ten images of each sample taken at 1:200 magnification were stacked using ImageJ software (Ver. 1.52u), and the sum of the brightness of all images (integrated density) was measured after setting the same threshold. 2: The quantitative values ​​obtained were divided by the number of cells present in each image, and the values ​​were used to quantitatively compare each protein.

[0062] Immunofluorescent staining of 3T3-L1 cells and the quantitative results are shown in Figure 15. In the drug-induced obesity model of 3T3-L1 cells, phosphorylation of Acc was promoted. In other words, the evaluation results of 3T3-L1 cells are considered to show a similar effect in white adipose tissue (WAT).

[0063] From the above test results, the mechanism of action of kabosu extract is inferred as follows. Kabosu extract activates AKT in the PI3K / AKT signaling pathway, especially in liver tissue. Activation of AKT activates, for example, cAMP-responsive element-binding protein (CREB), which in turn increases the expression of carnitine palmitoyltransferase 1 (CPT1), which promotes downstream β-oxidation. In addition, activation of the PKA / AMPK signaling pathway is triggered in white adipose tissue, and the phosphorylation of AMPK regulates the activity of ACC, reducing the production of malonyl-CoA and leading to increased expression of CPT1. Kabosu extract also has the effect of activating AMPK, and it is thought that it may directly act to activate the PKA / AMPK signaling pathway and regulate the activity of ACC, which is downstream of AMPK.

[0064] Furthermore, activation of AKT inactivates FoxO1, which exists downstream of AKT, and controls the expression of genes that affect glycolipid metabolism. In other words, FoxO1 phosphorylated by activated AKT is translocated from the nucleus to the cytoplasm, and is therefore unable to bind to the promoter sequences of related genes in the nucleus, and is therefore unable to promote gene expression.

[0065] It is believed that through this mechanism of action, kabosu extract specifically suppresses the accumulation of visceral fat. [Industrial Applicability]

[0066] The visceral fat accumulation inhibitor of the present invention exhibits an effect of inhibiting the accumulation of visceral fat by containing an extract of kabosu peel, and therefore can be effectively used for the prevention and / or improvement of visceral fat-type obesity, etc. In addition, the extract of kabosu peel is highly safe and is useful, for example, as a food additive or supplement to be taken with meals.

Claims

1. A visceral fat accumulation inhibitor that inhibits the accumulation of visceral fat by oral ingestion, characterized in that it contains an extract of kabosu peel as an active ingredient.

2. 2. The visceral fat accumulation inhibitor according to claim 1, comprising lecithin, and the extract of kabosu peel is encapsulated in the lecithin.

3. 3. The visceral fat accumulation inhibitor according to claim 2, wherein the lecithin is contained in an amount of 120 to 500 parts by mass per 100 parts by mass of the extract of kabosu peel.

4. 3. The visceral fat accumulation inhibitor according to claim 1, wherein the visceral fat is fat accumulated in the abdominal cavity.

5. An AKT activator comprising an extract of kabosu peel as an active ingredient.

6. 1. An ACC activity regulator comprising an extract of kabosu peel as an active ingredient.

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