JP6426991B2 - Body fat reduction promoter - Google Patents

Description

  The present invention relates to a body fat reduction promoter.

  The number of obese people is on the rise worldwide, and obesity has various adverse effects such as an increase in the burden on the heart, an increase in the burden on joints such as knees and hips, and causes of lifestyle-related diseases. In addition, the cause of obesity is neutral fat, excessive accumulation of neutral fat, and the increase / hypertrophy of fat cells make the body larger than the ideal body shape, which also causes a problem in appearance. In order to improve the obesity, it is necessary to break down neutral fat.

  Ingredients having the action of suppressing or degrading accumulation of neutral fat, particularly body fat, such as caffeine have been reported (Non-patent documents 1 to 4), amino acids such as branched amino acids, various plant extracts Supplements and food and drink that have been formulated are sold.

Biochimica et Biophysica Acta, General Subjects, 496 (2), 458-74 (1977) Japanese pharmacology journal, 74, 459-466 (1978) J Appl Physiol 115, 260-267 (2013) Toxicology In Vitro, 27 (6), 1830-1837 (2013)

However, conventional body fat combustion promoters and the like have problems such as their effects being insufficient.
Therefore, the present invention is to provide a body fat reduction promoter having an effect of reducing body fat, particularly both subcutaneous fat and visceral fat.

  Therefore, the present inventor has examined the action of various animal-derived components on subcutaneous fat and visceral fat, and the combination of caffeine and a phospholipid containing ω3 unsaturated fatty acid as a constituent fatty acid is likewise as a constituent fatty acid. Compared with fish oil which is triglyceride containing ω3 unsaturated fatty acid, it promotes the degradation of subcutaneous fat and visceral fat significantly better, suppresses differentiation into subcutaneous fat cells and visceral fat cells, and is useful as a body fat reduction promoter And completed the present invention.

That is, the present invention provides the following [1] to [4].
[1] A body fat reduction promoter containing (a) caffeine and (b) a phospholipid containing ω3 unsaturated fatty acid as a constituent fatty acid.
[2] The body fat reduction promoter according to [1], wherein the body fat is subcutaneous fat and / or visceral fat.
[3] The agent for reducing body fat loss according to [1] or [2], wherein the promotion of body fat loss is promotion of degradation of subcutaneous fat and / or visceral fat, or differentiation suppression of subcutaneous fat cells and / or visceral fat cells.
[4] The body fat reduction promoter according to any one of [1] to [3], wherein the phospholipid containing ω3 unsaturated fatty acid as the constituent fatty acid (b) is derived from chryil oil.

  According to the body fat reduction promoter of the present invention, subcutaneous fat and / or visceral fat are reduced, and differentiation into subcutaneous fat cells and / or visceral fat cells is suppressed, so accumulation of subcutaneous and visceral fat is increased. It is suppressed and obesity is improved.

It is a figure which shows subcutaneous lipolysis promotion effect. It is a figure which shows visceral lipolysis accelerating action. It is a figure which shows the differentiation suppression effect | action to a subcutaneous fat cell. It is a figure which shows the differentiation suppression effect | action to a visceral fat cell.

  The active ingredients of the body fat reduction promoter of the present invention are two components of (a) caffeine and (b) phospholipids containing ω3 unsaturated fatty acid as a constituent fatty acid.

  (A) Caffeine is a chemical name 1, 3, 7-trimethylxanthine, which is a component contained in coffee and the like. It can be extracted from coffee beans, but can also be produced by known synthetic methods. Caffeine has central nervous system excitatory activity, cardiac activity and so on, and is known to have a fat burning effect, but it is known how the action is changed by the combination with (b) phospholipids Not.

(B) Phospholipids containing ω3 unsaturated fatty acid as constituent fatty acid include docosahexaenoic acid which is ω3 highly unsaturated fatty acid, eicosapentaenoic acid, linolenic acid, and phospholipid having α-linolenic acid as constituent fatty acid.
The (b) phospholipid may be pure, or a natural lipid containing the (b) phospholipid may be used. (B) As a natural lipid containing a phospholipid, for example, krill-derived oil, particularly, Antarctic krill-derived oil (krill oil) is preferable. The krill-derived oil preferably contains 35 to 70% by mass of the ω3 polyunsaturated fatty acid-containing phospholipid, and is therefore preferably used. These krill-derived oils can be commercially available products.

  From the viewpoint of the body fat reduction promoting effect, the body fat reduction promoter of the present invention preferably has a content mass ratio (a / b) of (a) component to (b) component of 1/10 to 100/1. 1/50 to 50/1, more preferably 1/20 to 20/1, still more preferably 1/10 to 10/1, and 1/5 to 5/1. It is further preferred that

  The composition containing (a) caffeine and (b) phospholipids has an action to promote subcutaneous lipolysis, an action to promote visceral lipolysis, an action to suppress differentiation into subcutaneous adipocytes, and visceral fat, as shown in the examples described later. Since it has a differentiation inhibitory effect on cells, it is useful as a body fat reduction promoter including subcutaneous fat and visceral fat. Ingestion of the body fat reduction promoter of the present invention promotes the degradation of subcutaneous fat and visceral fat and suppresses the differentiation and maturation into subcutaneous fat cells and visceral fat cells, thereby preventing or improving obesity and reducing obesity. The various lifestyle-related diseases involved are prevented or ameliorated.

  The body fat reduction promoter of the present invention can be used by mixing it with medicines, quasi-drugs, cosmetics and functional foods. Moreover, the administration form is preferably oral administration or transdermal administration.

  When the body fat reduction promoter of the present invention is used as pharmaceuticals, quasi-drugs, cosmetics, functional foods, a mixture of the components (a) and (b) can be used as it is, but it is effective For example, cellulose and derivatives thereof, starch and derivatives thereof, lactose such as natural and synthetic polymers, excipients, stearic acid and salts thereof, lubricants such as natural and synthetic waxes, saccharides, sour taste Various carriers such as agents and perfumes can be blended. In addition, the pharmaceutical, quasi-drug, cosmetic or functional food of the present invention may be in the form of powder, granules, tablets, pills, hard capsules, soft capsules, syrups and the like according to the administration method and administration route. It can be a type.

  In addition, the body fat reduction promoter of the present invention may be blended with other components that contribute to body fat reduction, such as garcinia extract, gymnema, capsaicin, fenugreek seed extract, indigestible dextrin, other dietary fibers, etc. .

  The body fat reduction accelerator of the present invention preferably contains the components (a) and (b) in a total amount of 1 to 100% by mass, more preferably 5 to 80% by mass, and still more preferably 10 to 70% by mass. Moreover, as for the intake of the body fat reduction promoter of this invention, 10-50,000 mg is preferable per day with a total of (a) component and (b) component, and also 100-5,000 mg is preferable.

  The present invention will be further described in detail by way of examples.

Example 1 (lipolysis promoting action)
(1) Preparation of medium The growth medium was prepared by adding all of FBS, L-glutamine, GA-1000 SingleQuats attached to PGM-2 bullet kit to Preadipocyte Basal Medium-2 (500 mL) attached to PGM-2 bullet kit, 10 The final concentration of% FBS, 2 mM L-glutamine, 50 μg / mL Gentamicin, 37 ng / mL Amphotericin-B was prepared.
The differentiation medium was prepared by adding all of insulin, dexamethasone, indomethacin, and isobutyl-methylxanthine included in the PGM-2 bullet kit to 200 mL of growth medium.

(2) Preparation of sample It prepared so that it might become the target sample final concentration, respectively using differentiation media. The caffeine and fish oil-containing medium were sterilized with a 0.45 μm filter and used for the test. Since the krill oil-containing medium did not permeate through the filter, it was aseptically prepared on a clean bench and used for the test without filter sterilization. Krill oil and fish oil were directly added to the medium without solvent, suspended, and tested in the form of an emulsion.
Sample-free differentiation culture medium was used as a negative control. As a positive control, a 10 μM Isoproteenol-containing medium was used (see note below). The 10 μM Isoproteenol-containing medium was prepared by filter-sterilizing the Isoproteenol Solution attached to Adipolysis Assay Kit by adding 1/1000 to the differentiation medium.
(Note) Isoproterenol functions as an agonist of adrenergic beta receptor. When adrenalin binds to the adipocyte beta receptor, protein kinase A is activated, which activates hormone sensitive lipase. This hormone sensitive lipase converts neutral fat into free fatty acid and free glycerol. By such a mechanism, Isoproterenol promotes the decomposition of neutral fat.

(3) Pre-culture of human preadipocytes Human preadipocytes (subcutaneous fat and visceral fat) were allowed to sleep in a T-75 flask using a growth medium and cultured until they reached 80% confluence. The medium was changed every two days, cells were detached using 0.25% Trypsin / EDTA, and the cells were recovered and used for this test. Purchased cell number 2 was used for the test.

(3) Differentiation induction of adipocytes and sample treatment Pre-adipocytes are seeded in 96-well plate at 10,000 cells / 100 μL / well using growth medium in a CO ₂ incubator (5% CO ₂ , 37 ° C.) Incubated for 24 hours. After culture, it was confirmed that the culture was 100% confluent, and was replaced with 200 μL of differentiation medium, and cultured in a CO ₂ incubator for 10 days to induce differentiation into adipocytes. On the 10th day after induction of differentiation, the medium was changed to a sample-containing differentiation medium, cultured in a CO ₂ incubator, and treated with a sample. At the end of culture, cells were observed with a phase-contrast microscope, and the medium was collected and stored at -20 ° C, and the amount of lipolysis was measured on the subsequent day.

(4) Determination of free glycerol (absorptiometry)
Neutral fats (triglycerides) are hydrolyzed to give fatty acids and glycerol. By measuring the concentration of glycerol, it is possible to evaluate lipolysis in vivo. The determination of glycerol was basically carried out by absorption method using Adipolysis Assay Kit (see below). In addition, in order to correct the effect of glycerol contained in the culture medium or the sample or the coloration of the culture medium by the sample, measurement was also performed on the sample-containing culture medium (n = 3), and cell processing was performed using the average value as a reference. It was subtracted from the measured value.
(Note) A method in which glycerol is treated with various enzymes to produce quinoneimine dye that can be detected by measuring absorbance at 540 nm.

(5) Statistical analysis A significant difference test was performed in the comparative test interval (no addition zone vs. sample addition zone). The test was performed by two-sided test using Student's t-test, and the one with P <0.05 (less than 5% null hypothesis) is shown in the figure. Graph data are shown as mean ± standard error. When describing the significance level, * P <0.05, ** P <0.01, *** P <0.001.

(6) Reagent test substance caffeine (extract) (Swan Pharmaceutical Co., Ltd.)
・ Krill oil (SEIKO PHARMACEUTICAL CO., LTD., Containing 41% by mass of ω3 highly unsaturated fatty acid phospholipid)
・ Fish oil (NOF Corporation, San Omega DHA 27)
(7) Cells-Human preadipocytes (subcutaneous fat) (Lonza, Cat. No. PT-5020, Lot. No. 0000279746, 38 years old, Asian, female, BMI = 26)
・ Human preadipocytes (visceral fat) (Lonza, Cat. No. PT-5005, Lot. No. 0000310955, 92 years old, Caucasian, female, BMI = 19)
・ Human preadipocytes (visceral fat) (Lonza, Cat. No. PT-5005, Lot. No. 8 F3482, 46 years old, Caucasian, male, BMI = 26)

(8) Results i) The subcutaneous lipolysis promoting action is shown in FIG.
As shown in FIG. 1, when caffeine and krill oil were used in combination three hours after the treatment, a remarkable subcutaneous lipolysis promoting action was exhibited as compared with the case where caffeine and krill oil were caused to act alone.

ii) Visceral lipolysis promoting action is shown in FIG.
As shown in FIG. 2, when caffeine and krill oil were used in combination at 24 hours and 48 hours after the treatment, a remarkable visceral lipolysis promoting action was exhibited as compared with the case where caffeine and krill oil were caused to act alone.
Isoproterenol is a positive control sample.

Example 2 (Adipocyte differentiation inhibitory action)
(1) Media preparation, sample preparation, pre-culture of human progenitor cells, statistical analysis, and reagents were performed in the same manner as in Example 1.

(2) Differentiation induction of adipocytes and sample treatment Pre-adipocytes are seeded in 96-well plates at 10,000 cells / 100 μL / well using a growth medium in a CO ₂ incubator (5% CO ₂ , 37 ° C.) Incubated for 24 hours. After culture, it was confirmed that the culture was 100% confluent, and was replaced with 200 μL of a sample-containing differentiation medium, and cultured in a CO ₂ incubator to start induction of differentiation into adipocytes. The cells were replaced with fresh sample-containing differentiation medium at 4 and 7 days after induction of differentiation. Various sample analysis performed oil red staining on the 4th, 7th, and 10th days after the differentiation induction start, and the adipocyte differentiation rate was analyzed sequentially. Each n = 5 was tested. Concentration-effect analysis of caffeine and krill oil was conducted by oil red staining on day 10 to analyze the adipocyte differentiation rate. Each n = 3 was tested.

(3) Oil Red Dyeing An oil red solution was prepared by dissolving 150 mg of oil red O in 50 mL of 100% 2-propanol, adding 33 mL of deionized water and filtering it through a filter paper. Oil red staining was performed according to the following protocol, and staining was followed by phase contrast microscopy and quantification.

(4) Results (i) The action of suppressing differentiation into subcutaneous adipocytes is shown in FIG. Than 3, when combined with caffeine and Krill oil showed differentiation inhibiting effect on remarkable subcutaneous fat cells.

(Ii) The action of suppressing differentiation into visceral fat cells is shown in FIG. When used with the caffeine and krill oil from FIG. 4 shows the differentiation inhibiting effect on remarkable visceral fat cells.
LiCl is a positive control.

Claims (3)

A visceral lipolysis promoter comprising (a) caffeine and (b) krill oil . The visceral lipolysis promoter according to claim 1, wherein the content mass ratio (a / b) of (a) caffeine to (b) krill oil is 1/5 to 5/1. The visceral lipolysis promoter according to claim 1 or 2, wherein the visceral lipolysis promoter is a drug for promoting visceral lipolysis, a quasi drug, a cosmetic or a functional food composition.