JP4809611B2 - Fat cell fat accumulation inhibitor - Google Patents

Description

The present invention relates to a precursor adipocyte differentiation inhibitor, and more particularly, to a food, a pharmaceutical or a cosmetic that eliminates and prevents obesity by suppressing the number of mature fat cells and preventing accumulation of systemic and / or local excess fat. .

Conventionally, obesity due to abnormal fat deposition in body adipose tissue and various organs, or hyperlipidemia is considered to be closely related to the development of various lifestyle-related diseases such as hypertension, arteriosclerosis, diabetes, etc. Yes.

Obesity is also a major problem in the field of beauty because it loses the body's balance and significantly impairs its appearance. In subcutaneous adipose tissue, local enlargement of adipocytes causes swelling of the subcutaneous connective tissue, causing unevenness of the skin surface similar to orange peel, ie cellulitis.

Obesity is said to be caused by constitutional factors, dietary factors, mental factors, central factors, metabolic factors, lack of exercise, etc., resulting in calorie intake exceeding calorie consumption and fat accumulation. ing. In obesity, the amount of fat, that is, triglyceride accumulated in individual adipocytes in a living body is increased and the cells are enlarged. In recent years, it has become clear that the number of adipocytes increases even after adulthood, suppressing the differentiation of preadipocytes into mature adipocytes, reducing the number of mature adipocytes, and suppressing fat accumulation in mature adipocytes. It is expected to suppress obesity progression and improve obesity.

There are not many foods or pharmaceuticals or cosmetics that suppress the increase in the number of fat cells and exhibit an anti-obesity effect. For example, those containing preadipocyte differentiation-inhibiting peptides as active ingredients (see Patent Document 1), activated milk There exists what uses Kiyoshi as an active ingredient (refer patent document 2). There is also an attempt to apply ω-3 highly unsaturated fatty acid as an active ingredient to an external preparation for skin (see Patent Document 3). In addition, alterenol acts on β3 adrenergic receptors distributed in adipocytes, causing protein kinase A (PKA) activation and hormone sensitive lipase (HSL) activation, and the degradation of neutral fat accumulated as oil droplets Has been reported (see Non-Patent Document 1). However, there is still no sufficient effect, and a novel preadipocyte differentiation inhibitor has been desired.

The compound represented by the formula (I) of the present invention is not known to have an effect of inhibiting fat accumulation.

The investigator has been continually researching foods, medicines, and cosmetics that suppress the increase in the number of fat cells and show anti-obesity effects, such as Kawaratake, Kanokosou, Uwamasamana, Paschaka, Paiko, Agrayaho, Gajutsu, Camille, Precursor fat containing mushrooms or ingredients extracted from plants consisting of guava leaves, caffa lime, juniper berries, nutmeg, basil, mace, lemongrass, rosemary, bamboo leaves, gymnema sylvestre, green senryu, lemon verbena A cell differentiation inhibitor has been invented (see Patent Document 4). In the present invention, as a result of further research and development, Bisabolol oxide-A-β-glucoside, which is a compound represented by the chemical formula (I), was isolated from chamomile and developed a fat cell fat accumulation inhibitor.
Japanese Unexamined Patent Publication No. 6-293796 JP 2000-37738 A JP-A-11-130656 JP 2004-75640 A Furutani, Y .; Karasawa, T. Protein Nucleic Acid Enzyme 2000, 45, 935-940.

An object of the present invention is to provide an effective and highly safe preadipocyte differentiation inhibitor, an adipocyte fat accumulation inhibitor, an anti-obesity agent, a cellulitis ameliorating agent, and a food containing these, without fear of side effects, To provide medicine and cosmetics.

（ここで、式中Ｒはグルコース残基を示す。） The present inventor isolated a compound represented by the chemical formula (I), which is a novel compound, from chamomile, found a strong adipocyte accumulation inhibitory activity, and completed the present invention.

(Here, R represents a glucose residue.)

That is, the present invention provides: Compound represented by chemical formula (I). 2. A preadipocyte differentiation inhibitor comprising a compound represented by the formula (I) as an active ingredient. 3. A fat cell fat accumulation inhibitor comprising a compound represented by the formula (I) as an active ingredient. 4). An antiobesity agent comprising a compound represented by the chemical formula (I) as an active ingredient. 5 . The agent according to any one of claims 2 to 4 , wherein the compound represented by the chemical formula (I) is derived from a plant. 6 . The compound according to any one of claims 2 to 4 , wherein the compound represented by the chemical formula (I) is derived from chamomile. 7 . A medicament containing one of the agents of claims 2-6. 8 . Cosmetics containing either agent according to claim 2-6. About.

Since the compound represented by the chemical formula (I) of the present invention strongly suppresses fat cell fat accumulation, it can contribute to the suppression and improvement of fat accumulation and obesity, and is caused by local fat cell hypertrophy. It is effective in preventing and treating cellulitis.

The compound represented by the chemical formula (I) of the present invention can be obtained by extraction and purification from chamomile (Matricaria chamomilla L.) or other plants containing the compound represented by the chemical formula (I) of the present invention.

Chamomile (Matricaria chamomilla L.) is a dicotyledon of the Asteraceae family, distributed from Europe to West Asia and cultivated for medicinal purposes. In Europe, it is used as a tea and as a folk medicine for anti-inflammatory, gallant, stomach upset, sweating, mental instability, etc. The production area of chamomile (Matricaria chamomilla L.) used in the present invention is not particularly limited.

The plant containing the compound represented by the formula (I) of the present invention or a part thereof is a dried product obtained by drying itself, a pulverized product thereof, a juice obtained by squeezing and extracting water, water or alcohol, A crude extract using an organic solvent such as ether and acetone, and an extract fraction obtained by stepwise purification by various chromatographies such as partition chromatography and column chromatography can be used. . These may be used alone or in combination of two or more.

For example, an extract obtained by adding 3 L of 99.5% ethanol to 1 kg of a dried product of the compound represented by the chemical formula (I) and immersing it overnight at room temperature is used as it is as a preadipocyte differentiation inhibitor and a food containing them. You may use as a pharmaceutical or cosmetics, and you may use the thing refine | purified combining various chromatography.

The compound represented by the chemical formula (I) of the present invention is a novel compound, and it has been found that the present invention is excellent in fat accumulation inhibitory activity of adipocytes.

The compound represented by the chemical formula (I) of the present invention has excellent fat cell fat accumulation inhibitory activity, fat accumulation inhibitor, anti-obesity agent, cellulitis ameliorating agent, and foods and medicines containing these And can be used as a cosmetic.

Production of the compound represented by the chemical formula (I) of the present invention as a preadipocyte differentiation inhibitor, fat accumulation inhibitor, anti-obesity agent, cellulitis ameliorating agent, and foods, medicines and cosmetics containing them. Can do.

As an application method as a medicine, either oral administration or parenteral administration can be adopted. In administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared by conventional means. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, Examples include albumin, water, and physiological saline. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents and the like can be appropriately added as necessary.

As a food, it is eaten as a health food or a food material useful for treatment and prevention of cellulitis or obesity as it is, or with various nutritional components added or contained in food or drink. For example, after adding the above-mentioned appropriate auxiliaries, it may be used for edible by using conventional means, forming into an edible form, for example, granular, granular, tablet, capsule, paste, etc. It can also be used in various foods such as processed foods such as ham and sausage, processed fish foods such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, fermented milk products, water, fruit juice, milk, You may add and use for drinks, such as a soft drink.

The effective dose is appropriately selected and determined according to the patient's age, weight, symptom, patient grade, administration route, administration schedule, formulation form, intensity of inhibitory activity of the material, etc. The compound represented by the chemical formula (I) of the present invention is preferably about 0.001 to 1000 mg / kg body weight per day, and may be divided into several times a day. In the case of the hot water extract of chamomile (Matricaria chamomilla L.), a weight of about 1-1000 mg / kg body weight per day is preferable.

Further, when used as a cosmetic or a cosmetic material, the compound represented by the chemical formula (I) of the present invention is added to wheat germ oil or olive oil to obtain a fat cell fat accumulation inhibitor-containing composition, which is used as a cosmetic. Can be used as a material. The amount added is not particularly limited, but as an example, it is 0.1% by mass to 60% by mass, preferably 0.5% by mass to 50% by mass with respect to the weight of wheat germ oil or olive oil. It is.

In addition, it can be directly used as a cosmetic ingredient to produce a cosmetic having an effect of inhibiting differentiation of preadipocytes and inhibiting fat accumulation of adipocytes. Although it does not specifically limit as cosmetics, From a functional surface, the emulsion for face or body, a cosmetic liquid, a cream, a lotion, an essence, a pack, a sheet | seat etc. are preferable, for example.

Such a cosmetic can be produced according to a conventional method. The amount added in the cosmetic is not particularly limited, but as an example, an appropriate amount is about 0.01% by mass or more and 20% by mass or less of the total weight of the cosmetic.

Separation of fat accumulation inhibitory active substance <br/> Fat to mouse fetus-derived white adipocytes 3T3-L1 cells using soft extract obtained by concentrating extracts extracted from chamomile (Matricaria chamomila L.) with 30% ethanol aqueous solution Separation was performed using the accumulation inhibitory activity as an index.

24.1 g of soft extract of chamomile (Matricaria chamomila L.) was distributed between ethyl acetate (1.2 L × 3) and water (1.2 L), and the ethyl acetate layer was concentrated under reduced pressure (FIG. 1). 997.2 mg of the obtained ethyl acetate layer was partitioned between 90% ethanol (200 mL) and hexane (200 mL × 3), and the 90% ethanol layer was concentrated under reduced pressure to obtain 847.5 mg as a brown oil. The obtained 90% ethanol layer was subjected to ODS silica gel column chromatography [Cosmosil 75 C ₁₈ OPN (90 g), methanol / water (7/3 → 8/2 → 9/1) → methanol → methanol / chloroform (9/1 )] Was separated into four fractions (Fr. 1-1 to 1-4). Of these, 102.8 mg of brown oily substance was obtained as Fr. 1-2. The obtained Fr. 1-2 was subjected to 6-step analysis using reversed-phase high-performance liquid column chromatography [Develosil ODS HG-5 (φ20 × 250 mm), methanol / water (65/35), 5 mL / min, UV 215 nm]. Separated into minutes (Fr. 2-1 to 2-6). Among them, 25.2 mg of the compound of the formula (I) of the present invention (Fr. 2-6, yield 0.10% based on the weight of the aqueous ethanol extract) was isolated as a brown oil.

A schematic of the fat accumulation inhibitory activity test method mouse embryos derived white adipocytes preadipocytes (preadipocytes) fat accumulation inhibitory activity test method using 3T3-L1 cells shown in FIG. First, 3T3-L1 cells are cultured in a growth medium. Since it reaches confluence on the 4th to 7th days, it is replaced with a differentiation-inducing medium (insulin-containing) supplemented with a sample. Spherical fine lipid droplets appear in the cells and become fully differentiated fat cells on the 11th to 14th days. Therefore, in order to calculate the fat accumulation rate, 0.2% Triton X solution was added and allowed to stand at room temperature for 30 minutes, followed by ultrasonication for 1 minute, and then added to the 96-well plate to which Triglyceride E-Test Wako was previously added. After incubation for 30 minutes, the absorbance at 630 nm was measured. On the other hand, in order to calculate the cell viability, CellCounting Kit-8 was added to the previously fully differentiated adipocytes, and the absorbance at 450 nm was measured after culturing for 4 hours.

These preadipocytes have the property of accumulating fat after differentiation. In this study, insulin induces differentiation of 3T3-L1 preadipocytes, but the sample added at the same time inhibits preadipocyte differentiation or promotes post-differentiation metabolism and suppresses fat accumulation. We examined whether or not. In this study, two parameters, fat accumulation rate and cell viability, were used for evaluation. The fat accumulation rate was calculated by the following formula.

また細胞生存率は次式で算出した。

The cell viability was calculated by the following formula.

Control refers to differentiated cells that have been induced to differentiate 3T3-L1 preadipocytes with insulin. In this test, when a cytotoxic sample is used, the cells may be killed or inhibited, so it may seem that the fat accumulation rate is low, but the cell viability is also low. turn into. Therefore, in order to be able to judge the presence or absence of this cytotoxicity, evaluation by cell viability was introduced in this test, and the state of fat accumulation was observed with a microscope as needed. It was devised so that a biologically active substance having weak cytotoxicity and strongly inhibiting fat accumulation can be assayed efficiently.

Structural Analysis <br/> shape brown oil molecular formula of extracts of Fr. 2-6 obtained in Example 1 C ₂₁ H ₃₆ O ₇
High resolution mass spectrometry measurement: [M + Na] ⁺ 423.2349 (Δ -1.0 mmu) mode is ESI positive
Analyzer QSTAR
Calculated value for [M + Na] ⁺ : completely in agreement with 423.2349, and the molecular formula of the extract of Fr. 2-6 obtained in Example 1 was found to be C ₂₁ H ₃₆ O ₇ .

The ¹ H and ¹³ C NMR spectrum data of the extract of Fr. 2-6 are shown below. By analysis of ¹ H NMR spectrum, ¹³ C NMR spectrum, and HMBC spectrum measured in deuterated methanol, the terpenoid glycoside Bisabolol oxide, in which the extract of Fr. 2-6 is a compound represented by the chemical formula (I) of the present invention -A-β-glucoside was determined. The structure of the terpenoid glycoside Bisabolol oxide-A-β-glucoside is shown in chemical formula (II).

NMR spectrum data of the extract of Fr. 2-6 of Example 1 ¹ H NMR (600 MHz, CD ₃ OD)
δ 1.05 (3 H, s), 1.20 (1 H, m), 1.25 (3 H, s), 1.27 (3 H, s), 1.29 (1 H, m), 1.62 (3 H, br s), 1.70 (1 H, m), 1.80 (1 H, ddt, J = 12.0, 4.2, 1.8 Hz), 1.87 (1 H, m), 1.89 (1 H, m), 1.92 (1 H, m), 1.96 (1 H, m), 1.96 (1 H, m), 1.98 (1 H, m), 2.00 (1 H, m), 3.18 (1 H, dd, J = 9.0, 7.8 Hz), 3.23 (1 H , ddd, J = 9.0, 5.4, 2.4 Hz), 3.27 (1 H, dd, J = 9.0, 8.4 Hz), 3.32 (1 H, t, J = 9.0), 3.43 (1 H, d, J = 7.8 , 3.6 Hz), 3.65 (1 H, dd, J = 12.0, 5.4 Hz), 3.84 (1H, dd, J = 12.0, 2.4 Hz), 4.31 (1 H, d, J = 7.8 Hz), 5.34 (1 H, br dd, J = 4.2 , 1.8 Hz) signal. 4 hydroxyl groups (-O H) was not observed.

¹³ C NMR (150 MHz, CD ₃ OD)
δ 23.6 (q), 23.8 (q), 24.1 (t), 24.5 (t), 25.6 (q), 28.4 (t), 30.1 (q), 31.2 (t), 32.1 (t), 44.1 (d) , 62.8 (t), 71.7 (d), 75.5 (d), 75.9 (s), 76.8 (s), 77.8 (d), 78.2 (d), 83.5 (d), 106.2 (d), 122.0 (d) , 134.9 (s)

Preadipocyte fat accumulation inhibitory activity of the compound of the formula (I) of the present invention obtained in Example 1 According to the method shown in Example 1, the compound of the formula (I) of the present invention obtained in Example 1 The preadipocyte fat accumulation inhibitory activity of the compound was examined. As a result, the compound of the chemical formula (I) of the present invention showed a strong fat accumulation inhibitory activity against mouse embryo-derived white adipocytes (precursor adipocytes) 3T3-L1 (Table 1).

Reference substance (-)-alterenol (+)-tartrate hydrate (see Non-Patent Document 1) Concentration of 12.5 μg / mL had a cell viability of 100% and a fat accumulation rate of 67.2%. On the other hand, the compound represented by the chemical formula (I) of the present invention obtained in Example 1 showed a high activity with a cell survival rate of 100% and a fat accumulation rate of 58% when added at a concentration of 12.5 μg / mL. . It was also confirmed that the safety was high.

Formulation Example 1
[Manufacture of tablets]
(Composition) (Composition: Mass%)
Chamomile extract * 10
Lactose 60
Cornstarch 29
Guar gum 1
* Contains 1% by mass of the compound of the formula (I) of the present invention.

Formulation example 2
[Manufacture of juice]
(Composition) (Composition: Mass%)
Frozen concentrated Wenzhou orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Fragrance 0.2
Dye 0.1
Chamomile extract * 1.0
Wed 82.48
* Contains 1% by mass of the compound of the formula (I) of the present invention.

Formulation Example 3
[Manufacture of face cream]
(Composition) (Composition: Mass%)
Isopropyl isostearate 8.0
Jojoba oil 6.0
Cetanol 8.0
Stearyl alcohol 2.0
Polyoxyethylene lauryl ether 1.5
Propylene glycol 6.0
Sorbitol 1.0
Paraben 0.4
Chamomile extract * 1.0
Vitamin E 0.5
Fragrance 0.1
Purified water 65.5
* Contains 1% by mass of the compound of the formula (I) of the present invention.

Separation of fat accumulation inhibiting active substances (liquid-liquid extraction separation, chromatographic separation) Outline of test method for fat accumulation inhibition using mouse embryo-derived white adipocytes (preadipocytes) 3T3-L1 cells

Claims (8)

The following chemical formula (I)

(Wherein, R represents a glucose residue).   A preadipocyte differentiation inhibitor comprising the compound represented by the chemical formula (I) according to claim 1 as an active ingredient.   An adipocyte fat accumulation inhibitor comprising the compound represented by the formula (I) according to claim 1 as an active ingredient.   The antiobesity agent which uses the compound shown by chemical formula (I) of Claim 1 as an active ingredient. The agent according to any one of claims 2 to 4 , wherein the compound represented by the chemical formula (I) according to claim 1 is derived from a plant. 5. The agent according to any one of claims 2 to 4 , wherein the compound represented by the chemical formula (I) according to claim 1 is derived from chamomile. A medicament containing one of the agents of claims 2-6. Cosmetics containing either agent according to claim 2-6.

Images