Fat breakdown promoting components

Abstract

To provide a novel fat cell differentiation inhibitor capable of being safely ingested as a food product: and to provide a lipolysis promoter.SOLUTION: Halocynthiaxanthin included in a fat cell differentiation inhibitor and a lipolysis promoter acts to suppress differentiation to fat cells and suppress obesity. The Halocynthiaxanthin included in the fat cell differentiation inhibitor and the lipolysis promoter suppresses adiposity in the fat cells. In addition, the Halocynthiaxanthin included in the fat cell differentiation inhibitor and the lipolysis promoter suppresses adiposity by a lipolytic action even after differentiation to matured fat cells, and suppresses obesity.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a composition for inhibiting adipocyte differentiation and a composition for promoting lipolysis, which contain halocynthiaxanthin, a type of carotenoid, as an active ingredient.

Background Art

[0002] Fucoxanthin has been reported to have various biological activities. Patent Document 1 describes that fucoxanthin has anti-obesity activity. On the other hand, Hosokawa et al. reported that fucoxanthin induces apoptosis and enhances the anti-proliferative effect of troglitazone, a PPARγ ligand, on colon cancer cells (Non-Patent Document 1). PPARγ is also widely known as a master regulator of adipocyte differentiation (Non-Patent Document 2). Similarly, Patent Document 2 describes that fucoxanthin has anti-tumor activity against liver cancer. Furthermore, Sachindra et al. reported that fucoxanthin and its metabolite, fucoxanthinol, have high antioxidant effects (Non-Patent Document 3).

[0003] As the metabolic pathway of fucoxanthin in humans, it is known that fucoxanthin is sequentially converted to fucoxanthinol and amarouciaxanthin A (Non-Patent Document 4).

[0004] Patent Document 1 describes that fucoxanthinol, a metabolite of fucoxanthin, has an effect of reducing visceral fat. On the other hand, Patent Document 3 describes that fucoxanthinol causes apoptosis in virus-related malignancies and has anti-tumor activity against virus-related malignancies.

[0005] Yim et al. have reported that amarouciaxanthin A, a further metabolite of fucoxanthin, has an inhibitory effect on 3T3-L1 adipocyte differentiation through the downregulation of PPARγ and C / EBPα mRNA expression (Non-Patent Literature 5).

[0006] The anti-obesity and anti-diabetic effects of fucoxanthin are exerted through the induction of uncoupling protein 1 (UCP1) expression in white adipose tissue (WAT) (Non-Patent Literature 6, Non-Patent Literature 4). Furthermore, fucoxanthin improves insulin resistance and blood glucose levels through the downregulation of adipocytokines related to insulin resistance in WAT and the upregulation of glucose transporter 4 (GLUT4) in skeletal muscle (Non-Patent Literature 6).

[0007] Interestingly, studies suggest that the metabolic pathway for fucoxanthin in ascidians differs from that in humans, with fucoxanthin being converted sequentially to fucoxanthinol, then to halocynthiaxanthin, and finally to mytiloxanthin (Non-Patent Literature 7).

[0008] Konishi et al. have reported that halosynthiaxanthin and fucoxanthinol exhibit antiproliferative effects on human leukemia, breast cancer, and colon cancer cells via apoptosis induction (Non-Patent Document 8). Yoshida et al. have reported that halosynthiaxanthin enhances the sensitivity of cancer cells to tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL)-induced apoptosis (Non-Patent Document 9). Furthermore, Patent Document 4 describes that halosynthiaxanthin has an inhibitory effect on tumor cell proliferation.

[0009] However, there are no reports that halosynthiaxanthin has effects such as reducing visceral fat, anti-obesity / anti-diabetic effects, or inhibiting adipocyte differentiation. Furthermore, the role of halosynthiaxanthin in energy metabolism and fat metabolism has not been studied.

[0010] The inventors conducted diligent research and, using the accumulation of lipid droplets and the gene expression of PPARγ and GLUT4 as indicators, discovered that halothiaxanthin suppresses the differentiation of adipocytes, thus completing the present invention. Surprisingly, these effects were stronger than those of fucoxanthin and fucoxanthinol. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Patent No. 5012505 [Patent Document 2] Japanese Patent Application Publication No. 10-158156 [Patent Document 3] Patent No. 4337986 [Patent Document 4] Japanese Patent Application Publication No. 4-120019 [Non-patent literature]

[0012] [Non-Patent Document 1] Biochim Biophys Acta. 2004 Nov 18;1675(1-3):113-9 [Non-Patent Document 2] Physiol Rev. 1998 Jul;78(3):783-809. [Non-Patent Document 3] J Agric Food Chem. 2007 Oct 17;55(21):8516-22. [Non-Patent Document 4] Mar Drugs. 2015 Apr 13;13(4):2196-214. [Non-Patent Document 5] J Agric Food Chem. 2011 Mar 9;59(5):1646-52. [Non-Patent Document 6] J Oleo Sci. 2015;64(2):125-32. [Non-Patent Document 7] Mar Drugs. 2020 Nov 24;18(12):588.

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0013] The problem to be solved by the present invention is to provide a novel composition for inhibiting adipocyte differentiation and a composition for promoting lipolysis that can be safely ingested as a food.

Means for Solving the Problems

[0014] The present invention has the following configuration. [Embodiment 1] A composition for inhibiting adipocyte differentiation, comprising halocynthiaxanthin as an active ingredient. [Embodiment 2] A composition for promoting lipolysis, comprising halocynthiaxanthin. [Advantages of the Invention]

[0015] By the action of halocynthiaxanthin contained in the composition for inhibiting adipocyte differentiation and the composition for promoting lipolysis of the present invention, differentiation into adipocytes is suppressed and obesity is suppressed. Further, halocynthiaxanthin contained in the composition for inhibiting adipocyte differentiation and the composition for promoting lipolysis of the present invention suppresses the accumulation of fat in adipocytes. Furthermore, halocynthiaxanthin contained in the composition for inhibiting adipocyte differentiation and the composition for promoting lipolysis of the present invention suppresses fat accumulation and obesity by its lipolytic action even after differentiating into mature adipocytes. [Brief Description of the Drawings]

[0016] [Figure 1] It is a figure showing the effect of suppressing PPARγ expression by halocynthiaxanthin. In the figure, C, A, 1, 2, and 3 respectively represent control, Retinoic Acid, fucoxanthin, fucoxanthinol, and halocynthiaxanthin. [Figure 2] It is a figure showing the effect of suppressing GLUT4 expression in adipocytes by halocynthiaxanthin. In the figure, C, A, 1, 2, and 3 respectively represent control, Retinoic Acid, fucoxanthin, fucoxanthinol, and halocynthiaxanthin. [Figure 3] It is a figure showing concentration-dependent suppression of fat accumulation in adipocytes by halocynthiaxanthin. [Figure 4] It is a figure showing concentration-dependent increase in the amount of free glycerol by the addition of fucoxanthin. [Figure 5] It is a figure showing concentration-dependent increase in the amount of free glycerol by the addition of fucoxanthinol. [Figure 6]This figure shows the concentration-dependent increase in free glycerol levels upon addition of halosine thiaxanthine. [Figure 7] This figure shows the concentration-dependent increase in the amount of free glycerol upon the addition of chloroquine. [Modes for carrying out the invention]

[0017] The halothiaxanthin used in the present invention may be chemically synthesized or extracted and produced from natural products. Furthermore, the halothiaxanthin used in the present invention may be produced by enzymatically converting commercially available fucoxanthin or fucoxanthinol. In one embodiment, the halothiaxanthin used in the present invention is extracted from sea squirts and purified to a purity of 70% or higher, 80% or higher, 90% or higher, 95% or higher, 98% or higher, or 99% or higher.

[0018] A method for extracting and purifying halosin thiaxanthin from sea squirts is as follows: Generally, sea squirts of the genus Halocynthia, such as Halocynthia japonica and Halocynthia rhinoceros, can be used for extraction. For extraction, the entire sea squirt, parts of the tissue separated from the sea squirt such as the tunic and internal organs, or dried versions thereof can be used. Extraction methods include solvent extraction and supercritical carbon dioxide extraction. For solvent extraction, any organic solvent capable of dissolving halocynthiaxanthin, including aqueous organic solvents, can be used. For example, acetone can be used for extracting carotenoids such as halocynthiaxanthin. To concentrate the carotenoid fraction from the crude extract, liquid-liquid partitioning using organic solvents, or column chromatography using silica gel, modified silica gel, resin, or modified resin as support structures can be used. To purify halocynthiaxanthin from the crudely purified carotenoid fraction, various normal-phase and reverse-phase high-performance liquid chromatography systems and supercritical fluid chromatography can be used. More specifically, for example, the method described in Non-Patent Document 7 can be used. In summary, the following method can be used: An ethanol extract of sea squirt is extracted with acetone at room temperature, partitioned between n-hexane ether (7:3, v / v) and sodium chloride aqueous solution, and the organic layer is concentrated under reduced pressure with sodium sulfate. The concentrated residue is subjected to silica gel column chromatography and eluted with acetone. The acetone-eluted fraction is subjected to preparative high-performance liquid chromatography using an octadecylsilyl column and separated with chloroform:acetonitrile (1:9, v / v) to obtain halosin thiaxanthine.

[0019] The adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention, which contain halosinthiaxanthin as an active ingredient, are not sea squirts themselves. The halosinthiaxanthin content is preferably 0.001% by mass or more, and more preferably 0.01% by mass or more, of the total mass of the adipocyte differentiation inhibitory composition and lipolysis promoting composition. There is no particular upper limit, but 10% by mass or less is preferred. In one embodiment of the present invention, the adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention do not contain an effective amount of fucoxanthin or fucoxanthinol. In one embodiment, the mass of fucoxanthin and fucoxanthinol contained in the adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention is 1 / 10 or less, preferably 1 / 30 or less, and more preferably 1 / 100 or less, of the mass of halosinthiaxanthin. In another embodiment, the number of moles of fucoxanthin and fucoxanthinol contained in the adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention is 1 / 10 or less, preferably 1 / 30 or less, and more preferably 1 / 100 or less, of the number of moles of halothiaxanthin.

[0020] In one embodiment, the adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention, which contain halosinthiaxanthin as an active ingredient, contain other active ingredients having adipocyte differentiation inhibitory activity, fat accumulation inhibitory activity, or lipolysis promoting activity. In another embodiment, the adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention, which contain halosinthiaxanthin as an active ingredient, do not contain other active ingredients having adipocyte differentiation inhibitory activity, fat accumulation inhibitory activity, or lipolysis promoting activity.

[0021] In one embodiment, the adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention, which contain halosinthiaxanthin as an active ingredient, do not contain other components having antiproliferative activity, antitumor activity, antioxidant activity, or apoptosis-inducing activity. [Examples]

[0022] [Example 1] Purification of halosinthiaxanthin 1025.4 mg of ethanol extract from sea squirt (Halocynthia roretzi) was subjected to flash chromatography, fractionated into Fr.1 to Fr.9, and concentrated under reduced pressure using an evaporator. [Table 1]

[0023] Each fraction was separated and developed using thin-layer chromatography (TLC), and it was confirmed that halothiaxanthin was highly present in Fr.5, referring to Patent Document 1. [Table 2]

[0024] Fr.5 (90.67 mg) was subjected to solid-phase extraction using a cartridge column, fractionated into Fr.5-1 to Fr.5-7, and concentrated under reduced pressure using an evaporator. [Table 3]

[0025] Each obtained fraction was developed using TLC, and it was confirmed that fractions Fr.5-2 and Fr.5-3 contained a large amount of highly purified halosynthiaxanthin. Fr.5-2 (11.53 mg) and Fr.5-3 (5.57 mg) were subjected to HPLC to purify halosin thiaxanthin. The purity of the obtained purified product was confirmed to be 95% or higher by UPLC area value. Identification was performed by comparing NMR, UV-Vis, and MS data with (Non-Patent Document 10). [Table 4] [Table 5]

[0026] [Example 2] Evaluation of the suppression of adipocyte differentiation As indicators of adipocyte differentiation, we performed gene expression analysis of cells and quantitative analysis of fat accumulation. (1) Test method 1) Cells used 3T3-L1 cells 2) Test sample Fucoxanthin (Sigma-Aldrich, F6932), fucoxanthinol (Merck, 72723), and halothiaxanthin purified according to the method described above were used as test samples, and the solvent was adjusted to 10 mM using DMSO. Retinoic acid (RA) (Sigma, R2625-50MG) was used as a positive control. Note that the controls in Figures 1-3 are solvent controls and consist only of DMSO. 3)Cell culture Cells were cultured at 37°C, 5% carbon dioxide, and 95% air. The culture medium used was DMEM medium (Invitrogen, 11995-065) (basic medium) supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicilin-streptmycin (Sigma, P4333). 0.05% trypsin-EDTA (Sigma, SLCH9546) was used for cell detachment during subculturing. A 24-well plate was used, with 2-3 × 10⁶ cells per well. 3 cell / cm 2 The seeds were sown and cultured until confluence was reached. To induce differentiation into adipocytes, the culture medium was removed, and the sample (final concentrations of 5, 10, and 20 μM for fat accumulation quantification; final concentration of 20 μM for gene expression analysis) or a positive control (1 μM) was added to differentiation induction medium (basal medium containing Insulin (Sigma, 16634) (10 μg / mL), Dexamethasone (Sigma, D4902) (1 μM), and IBMX (Sigma, 17018) (500 μM)), and the cells were cultured for a further 48 hours. Subsequently, the culture medium was changed every two days using basal medium containing Insulin (10 μg / mL) and the sample.

[0027] (2) Analysis of gene expression in cells 1) RNA purification The cell supernatant was removed two days after differentiation induction, and RNA was purified using the RNeasy Plus mini Kit (Qiagen, 74136). RNA concentration was measured using a Nano DropOne C spectrophotometer (Thermo Fisher Scientific, ND-ONE-W). 2) cDNA synthesis 8 μL of RNA solution adjusted to 45 ng / μL was added to each microcentrifuge tube, 5 μL of 5×Prime Script RT Master Mix was added, and the mixture was heated at 37°C for 15 minutes and 85°C for 5 seconds, then cooled to 4°C. 3) Real-time PCR cDNA was added to a 96-well plate, along with 0.8 μL of forward primers (10 μM) and reverse primers (10 μM) of the target gene, 3.8 μL of ultrapure water, 10 μL of SYBR® Premix Ex Taq II, and 0.4 μL of ROX Reference Dye (50 × conc.). The reaction was carried out at 95°C for 1 minute, followed by 40 cycles of (95°C for 3 seconds, 60°C for 30 seconds). The instrument used was a QuantStudio 5 Real-Time PCR System, Fast96 well (Thermo Fisher Scientific, QS5-96F). Comparative Ct method was used for analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct for the expression of the target gene. [Table 6] 4) Results As shown in Figure 1, halosin thiaxanthin significantly suppressed the expression of PPARγ, a master regulator of adipocyte differentiation. The PPARγ expression inhibitory effect of halosin thiaxanthin was more potent than that of fucoxanthin and fucoxanthinol. Furthermore, as shown in Figure 2, analysis of gene expression in 3T3-L1 cells after halothiaxanthine treatment revealed a decrease in GLUT4 expression. GLUT4 is a glucose transporter expressed after adipocyte differentiation, suggesting a decrease in the amount of mature adipocytes in the cultured cells. This effect was significantly more potent compared to fucoxanthine and fucoxanthinol. These results suggest that halosin thiaxanthin acts before the differentiation of preadipocytes into mature adipocytes, thereby suppressing the differentiation into adipocytes and thus inhibiting obesity.

[0028] (3) Fat accumulation determination 1) Preparation of Oil Red O solution 150 mg of Oil Red O (Sigma, O0625-25G) was dissolved in 50 mL of isopropanol and stirred for 30 minutes. Impurities were removed using a 0.22 μm filter (Millipore, SLGVR33RS) to obtain a 100% Oil Red O solution. The 100% Oil Red O solution was diluted with distilled water to a 60% concentration. 2)Oil Red O staining Six days after differentiation induction as described in (1) above, the cell culture medium was removed, 4% Paraformaldehyde-PBS was added, and the cells were allowed to stand at room temperature for 15 minutes to fix. After washing twice with PBS(-), 60% Isopropanol was added to each well and allowed to stand at room temperature for 1 minute. After removing the supernatant, Oil Red O was added to each well and stained at room temperature for 20 minutes. After washing once with 60% Isopropanol, the cells were washed twice with PBS and observed under a microscope. Next, 100% Isopropanol was added to each well, and the cells were extracted and processed using VersaMax. TM The absorbance at 492 nm was measured using a Tunable Microplate Reader (molecular device, 89429-538). 3) Results As shown in Figure 3, the addition of halosynthiaxanthin resulted in a concentration-dependent suppression of lipid accumulation in 3T3-L1 cells. The lipid accumulation inhibitory effect of halosynthiaxanthin was more pronounced than that of fucoxanthin, and a clear effect was observed even at low concentrations of 5 and 10 μM. These results indicate that halosynthiaxanthin suppresses lipid accumulation in adipocytes.

[0029] [Example 3] Fat breakdown (1) Test method 1) Cells used 3T3-L1 cells 2) Test sample Fucoxanthin, fucoxanthinol, and halothiaxanthin were used as test samples. Each sample was prepared to dilute to 2.5, 5, and 10 mM. Chloroquine diphosphate (Tokyo Chemical Industry, C2301) (25 mM) was used as a positive control. 3)Cell culture Cells were cultured at 37°C, 5% carbon dioxide, and 95% air. The culture medium used was DMEM medium (Sigma, D6429) (basic medium) supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicilin-streptmycin (Wako, 168-23191). Trypsin (gibco, 15090-046) was used for cell detachment during subculturing. A 96-well plate was used, with 1.5 × 10⁶ cells per well. 4 Cells were seeded in 100 μL per well and cultured until confluence was reached. Next, the culture medium was removed from the wells, and basal medium containing insulin (WAKO, 099-06473) (10 μg / mL), dexamethasone (G-Biosciences, API-04) (1 μM), and IBMX (WAKO, 95-03413) (500 μM) was added, and the cells were incubated for another 48 hours. Subsequently, the cells were incubated for 5 to 10 days using basal medium containing 2% FBS, 1% Penicilin-Streptmycin (Wako, 168-23191), and kanamycin solution (Wako, 117-00961) (50 μg / mL), with the medium being changed every 2 to 4 days, to induce differentiation into mature adipocytes.

[0030] (2) Determination of free glycerol Differentiated mature adipocytes were treated with each sample dissolved in DMSO at concentrations of 0, 2.5, 5, and 10 μM, and the positive control at concentrations of 0, 8.3, and 25 μM. After 48 hours of incubation, the supernatant was collected, and a solution for free glycerol quantification was prepared using the Free Glycerol Assay Kit II (abcam, ab155899). Free glycerol was quantified by measuring the absorbance at 450 nm using SpectraMax® M3 (Molecular Devices, S / N MT05846). (3) Results As shown in Figures 4, 5, 6, and 7, the addition of fucoxanthin, fucoxanthinol, halothiaxanthin, and chloroquine resulted in a concentration-dependent increase in free glycerol levels. This result indicates that halothiaxanthin, like fucoxanthin and fucoxanthinol, promotes lipolysis in adipocytes. These results suggest that halothiaxanthin suppresses fat accumulation and inhibits obesity through its lipolytic effect, even after differentiation into mature adipocytes. [Industrial applicability]

[0031] The adipocyte differentiation inhibitory composition and lipolysis promoting composition of the present invention, which contain halosin thiaxanthin as an active ingredient, can be used by adding them to food, beverages, or supplements.

Claims

1. A composition for inhibiting adipocyte differentiation, containing halosine thiaxanthin as an active ingredient.

2. A composition for promoting fat breakdown, containing halosine thiaxanthin as an active ingredient.

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