JP5800635B2 - Anti-obesity agent - Google Patents

Description

  The present invention relates to an anti-obesity agent with enhanced adipocyte differentiation-inhibiting action and fat accumulation-inhibiting action, which is obtained by subjecting an alcoholic extract of gramineous plant seeds to a specific purification treatment.

  Currently, in Japan and Western countries, an increasing number of patients have a condition called metabolic syndrome in which symptoms such as diabetes, hypertension and arteriosclerosis overlap. It is thought that obesity is deeply involved in the metabolic syndrome. Obesity refers to a state where fat has accumulated excessively due to an increase in the number of fat cells or enlargement of the fat cells themselves due to excessive intake of energy due to overeating or a decrease in energy consumption due to lack of exercise. Various adipocytokines are secreted from hypertrophic fat cells that have accumulated fat excessively, and as a result, insulin resistance, hypertension, hyperlipidemia, and the like are induced. In order to control obesity and metabolic syndrome, it is important to elucidate the mechanism of the differentiation of fat precursor cells into hypertrophic adipocytes and the accumulation of fat, and to suppress the differentiation into fat cells and the accumulation of fat Can also help prevent obesity.

  Conventionally, substances such as berberine have been known as substances useful for the prevention of obesity, but these substances are pharmaceuticals and cannot be continuously taken in effective amounts without a doctor's prescription. .

  Patent Document 1 discloses a PPAR (peroxisome proliferator-responsive receptor) ligand agent containing a grass plant extract as an active ingredient, and the ligand activity of PPARα and PPARγ ligand activity by the ligand agent is observed. Has been issued. The ligand activity of PPARα affects lipid metabolism, and the ligand activity of PPARγ is involved in adipocyte differentiation. However, Patent Document 1 does not show whether the PPAR ligand agent described in the document actually suppresses fat cells from accumulating fat.

  Patent Document 2 discloses an adipocyte differentiation inhibitor containing, as an active ingredient, a seed of a gramineous plant or an extract of aboveground foliage. However, there was no dose-dependent effect on its differentiation-inhibiting action, and in a study in which diabetic mice were fed a high-fat diet, the amount of visceral fat was reduced, but blood lipids were not affected at all. The effect is considered to be weak or limited.

JP 2004-161656 A JP 2005-247695 A

  Accordingly, an object of the present invention is to provide an anti-obesity agent that is easy to be taken on a daily basis, has a high anti-obesity effect, has a correlation between dose and activity, easily realizes the effect, and is highly safe. It is in.

  As a result of intensive studies, the present inventors, as shown in a test example to be described later, include a component that inhibits fat accumulation in the mere extract of the gramineous plant seed as well as a component having an action of suppressing fat accumulation. Therefore, it has been found that fat accumulation increases conversely when the amount of extract applied is increased. Therefore, it is considered that a PPAR ligand agent containing, for example, a grass plant extract described in Patent Document 1 as an active ingredient does not exhibit fat accumulation inhibitory activity even if it exhibits PPAR ligand activity.

  As a result of further investigations, the present inventors have conducted a refinement treatment of gramineous plant seed extract using partition chromatography and concentrated the active ingredient to obtain a highly effective fat accumulation inhibitor. The present invention has been successfully achieved.

  That is, the present invention provides an anti-obesity agent containing, as an active ingredient, a peak component of partition chromatography of an alcoholic extract of a grass plant seed.

  According to the present invention, it is possible to provide an anti-obesity agent that is easy to take on a daily basis, has a high anti-obesity effect, has a correlation between dose and activity, easily realizes the effect, and is highly safe. .

1 is a chromatogram of partition chromatography when the anti-obesity agent of Example 1 was obtained. FIG. 2-1 shows the relationship between the amount of anti-obesity agent added and the amount of intracellular triglyceride when mouse preadipocytes (3T3-L1 cells) were induced to differentiate in the medium supplemented with the anti-obesity agent of Example 1 and cultured. It is the graph which showed. FIG. 2-2 is a graph showing the relationship between the amount of composition added and the amount of intracellular triglyceride when 3T3-L1 cells were induced to differentiate and cultured in a medium to which the composition of Comparative Example 1 or 2 was added. . FIG. 3 shows (a) 3T3-L1 cells after induction of control differentiation, (b) 3T3-L1 cells to which the anti-obesity agent of Example 1 was added during differentiation induction, and (c) during differentiation induction. It is the optical microscope photograph (all 400 times) which each showed 3T3-L1 cell which added the composition of the comparative example 1. FIG.

  Hereinafter, the antiobesity agent of the present invention will be described in detail based on preferred embodiments.

  The gramineous plant used for the anti-obesity agent of the present invention is not particularly limited, and examples thereof include wheat, durum wheat, rye, rye wheat, barley, oats, corn, corn, rice, millet, millet, millet and the like. Wheat plants such as wheat and durum wheat are preferable, and wheat is more preferable. These may be used alone or in combination of two or more. The grass seeds may be any form of grass seeds, for example, grass seeds (preferably seed hulls; mosses) themselves; those obtained by cutting, grinding or pulverizing the grass seeds; It may be a product obtained by drying the grass seeds; a product obtained by drying or crushing or powdering the grass seeds. Preferable examples including a grass seed hull include bran, powder, rice husk, bran and the like, and a seed with a hull.

  The method for extracting with alcohol is not particularly limited, and examples thereof include a method of immersing, stirring or refluxing the grass seeds in various forms described above, a supercritical fluid extraction method, and the like. In the case of a method of immersing, stirring or refluxing in alcohol, the extraction temperature is preferably 2 to 100 ° C., the extraction time is preferably 30 minutes to 72 hours, and the amount of alcohol used is 50 parts per 100 parts by mass of the grass seed. -2000 mass parts is preferable.

  Examples of the alcohol that can be used for extraction include monovalent lower alcohols (preferably having 1 to 4 carbon atoms) such as methanol, ethanol, n-propanol, isopropanol, and n-butanol, and 1,3- Examples include alcohols that are liquid at room temperature (25 ° C.) such as polyhydric alcohols such as butylene glycol, propylene glycol, and glycerin, and ethanol is preferred from the viewpoint of operability and environmental properties. In addition, the alcohol includes water-containing alcohols further containing aqueous components (water, pure water, distilled water, tap water, acidic water, alkaline water, neutral water, etc.). The alcohol content in the hydrous alcohol is usually 70% by volume or higher, preferably 80% by volume or higher, more preferably 90% by volume or higher.

  In the present invention, the partition chromatography may be of any type as long as the active ingredient according to the present invention can be obtained, but the normal phase chromatography method using a non-aqueous solvent as the mobile phase is preferable, the open column method, A method such as a pressure column method or high performance liquid chromatography can be appropriately selected.

  The mobile phase includes monovalent lower alcohols (preferably having 1 to 4 carbon atoms) such as methanol, ethanol, n-propanol, isopropanol, and n-butanol, and 1,3-butylene glycol, propylene glycol, and glycerin. Alcohols that are liquid at room temperature (25 ° C.) such as polyhydric alcohols such as; ethers such as diethyl ether and propyl ether; esters such as butyl acetate and ethyl acetate; ketones such as acetone and ethyl methyl ketone; hexane; Acetonitrile; as well as chloroform. These are used alone or in combination. When combining a plurality, both an isocratic mode in which the mixing ratio is constant and a gradient mode in which the mixing ratio is changed can be employed.

  As the carrier, any carrier can be used as long as it can carry and release the target active ingredient, and generally silica gel, polyacrylamide gel, dextran gel and the like can be mentioned.

  A detection wavelength should just be 170-320 nm, Preferably it is 190-280 nm.

  Among these, it is preferable to fractionate a peak component when silica gel is used as a carrier and a hexane-ethyl acetate mixed solvent is used as a mobile phase. In particular, a silica gel as a carrier and a hexane-ethyl acetate mixed solvent as a mobile phase are hexane-sized. -It is preferable to use the peak component at the detection wavelength of 254 nm in the gradient mode to low, because the effective component according to the present invention can be obtained efficiently.

  The anti-obesity agent of the present invention contains the active ingredient according to the present invention and, if necessary, various pharmaceutically acceptable carriers, excipients, other additives, and other components. The anti-obesity agent of the present invention can be formulated by a conventional method. In this case, the dosage form of the anti-obesity agent of the present invention is an oral preparation such as a tablet, powder, granule, capsule or the like. In addition, as other components, other components having medicinal effects, anti-inflammatory drugs, various vitamins, herbal medicines, and minerals can be appropriately blended.

  The content of the active ingredient in the anti-obesity agent of the present invention is not particularly limited, and can be appropriately changed depending on the dosage form of the anti-obesity agent, the symptom or age sex of the person to be administered or ingested, etc. In general, it is preferable that the dose or intake of the active ingredient of the anti-obesity agent of the present invention is 0.01 to 10 g per person per day.

  The anti-obesity agent of the present invention may be administered or ingested as it is, or the anti-obesity agent of the present invention may be added to a food or drink or animal feed for administration or ingestion. Examples of foods and drinks to be added include confectionery such as breads, noodles, tablets, and candy, and beverages such as soft drinks, juices, and energy drinks, but are not limited thereto. The timing of addition to food and drink is not particularly limited, and it may be added during the production process of the food or drink, may be added to the produced food or drink, and the same applies to animal feed. is there.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited only to these Examples.

[Example 1]
After pulverizing 2 g of wheat seeds with a multi-bead shocker (multiple specimen cell crusher / MB301 (S): manufactured by Yasui Kikai Co., Ltd.), 5 times the amount of ethanol was added, and the conditions were 150 rpm and room temperature for 2 hours. Extracted with shaking. Subsequently, the mixture was centrifuged at 3500 rpm and room temperature for 15 minutes, and the supernatant was dried with a centrifugal concentrator to obtain a wheat ethanol extract.
The wheat ethanol extract was then purified by medium pressure chromatography. Medium pressure chromatography conditions are as follows. The peak component appearing 12 to 14 minutes after the start of elution was collected and the solvent was distilled off to obtain 3 mg of the anti-obesity agent of the present invention. The chromatogram at this time is shown in FIG. The horizontal axis of the chromatogram represents the time (minutes) from the start of elution.

<Medium pressure chromatography conditions>
Column: Silica gel (injection column S, high flash column M, 60 mm, 40 μm, Yamazen Corporation)
Mobile phase: Hexane / ethyl acetate (volume ratio) = 90/10 for 3 minutes, 80/20 for 5 minutes, 60/40 for 10 minutes Detection wavelength: 254 nm

[Comparative Example 1]
After pulverizing 200 mg of wheat seeds with a multi-bead shocker (multiple specimen cell crusher / MB301 (S): manufactured by Yasui Kikai Co., Ltd.), 5 times the amount of ethanol was added and the conditions were 150 rpm and room temperature for 2 hours. Extracted with shaking. Subsequently, the mixture was centrifuged for 15 minutes at 3500 rpm and room temperature, and the supernatant was dried with a centrifugal concentrator. The weight of the obtained concentrate was measured and dissolved in ethanol so as to be 160 mg / mL to obtain 4.8 mg of a comparative composition.

[Comparative Example 2]
After pulverizing 200 mg of wheat seeds with a multi-bead shocker (multi-sample cell crusher / MB301 (S): manufactured by Yasui Kikai Co., Ltd.), 5 times the amount of hexane was added and the conditions were 150 rpm and room temperature for 2 hours. After degreasing by shaking, hexane was removed. Five times the amount of ethanol was added, and the mixture was extracted by shaking for 2 hours at 150 rpm and room temperature. Subsequently, the mixture was centrifuged for 15 minutes at 3500 rpm and room temperature, and the supernatant was dried with a centrifugal concentrator. The weight of the obtained concentrate was measured and dissolved in ethanol so as to be 87 mg / mL to obtain 2.6 mg of a comparative composition.

[Test example]
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), 10 U / mL penicillin, 10 μg / mL streptomycin, glucose 4.5 g / L, DMEM, manufactured by Sigma-Aldrich) The suspension was suspended at a density of 4 × 10 ⁴ cells / mL, and 1 mL each was seeded in a 24-well plate. Subsequently, after culturing at 37 ° C. in the presence of 5% CO _{2 for} 3 days, it was confirmed that the cells had become confluent under a microscope. Then, in order to induce differentiation into adipocytes, the medium was changed to 10 μg / mL insulin ( Human origin), 10% FBS-DMEM (differentiation induction medium) containing 250 nM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine, and 2 days after differentiation induction, the medium was 10% FBS-DMEM containing 10 μg / mL insulin. It was replaced with (maintenance medium) and further cultured for 6 days.

  In the above culture, the anti-obesity agent of Example 1 or the composition of Comparative Example 1 or 2 is appropriately dissolved in ethanol, and the addition amount as a dry solid content is a predetermined amount (see the description of FIGS. 2-1 and 2-2). ) And added to the differentiation-inducing medium and the maintenance medium. As a control, culture was also performed in a system in which neither the anti-obesity agent of Example 1 nor the compositions of Comparative Examples 1 and 2 was added.

In order to evaluate the adipocyte differentiation inhibitory action, each cultured cell 2 days after differentiation induction was observed under an optical microscope. The optical micrograph at that time is shown in FIG. 3 (a) is an optical micrograph of the control, and FIG. 3 (b) is an optical micrograph when 0.04 mg of the anti-obesity agent of Example 1 is added, FIG. 3 (c). These are optical micrographs when 1.6 mg of the composition of Comparative Example 1 is added.
Moreover, in order to evaluate intracellular fat accumulation inhibitory properties, the amount of triglyceride (TG) in the cultured cells after completion of the culture in the maintenance medium was measured according to the following measuring method. The results when the anti-obesity agent of Example 1 is used are shown in FIG. 2-1, and the results when the composition of Comparative Example 1 or 2 is used are shown in FIG. In addition, in FIGS. 2-1 and 2-2, a result is represented by the relative value which made the triglyceride production amount in no addition (control) 100%.

(Measurement method of triglyceride content)
The cultured 3T3-L1 cells were washed twice with 500 μL / well of PBS (−), 300 μL of 2-propanol was added, and the mixture was shaken at 80 rpm for 20 minutes to extract intracellular triglycerides. The amount of extracted triglyceride was quantified using Triglyceride E-Test Wako (manufactured by Wako Pure Chemical Industries).

From FIG. 2-1, it is clear that the anti-obesity agent of the present invention can reduce the amount of triglyceride accumulated in the cells, and the effect becomes greater as the addition amount increases. On the other hand, as is clear from FIG. 2-2, the composition of Comparative Example 1 or 2, that is, the mere extract, does not show the effect of the anti-obesity agent of the present invention, but rather increases as the amount added increases. The amount of triglyceride accumulated in the cells has increased.
In addition, in the micrograph of Fig. 3 (a), which became adipocytes by induction of differentiation, and the micrograph of Fig. 3 (c) to which the composition of Comparative Example 1 was added, accumulation of many lipid droplets was observed in the cells. However, in the micrograph of FIG. 3 (b) with the addition of the anti-obesity agent of the present invention, it is clear that the differentiation of preadipocytes into adipocytes is suppressed and the number of lipid droplets accumulated in the cells is small. .

Claims (2)

Contains as an active ingredient the peak component of the partition chromatography of the alcohol extract of the seeds of the genus Wheat ,
The alcohol of the alcohol extract is ethanol alone or hydrous ethanol having an ethanol content of 90% by volume or more,
An antiobesity agent , wherein the peak component is a peak component when silica gel is used as a carrier and a hexane-ethyl acetate mixed solvent is used as a mobile phase . Anti-obesity agent according to claim 1 Symbol placement and has a fat accumulation inhibitory action.