JP2012001524A - Preadipocyte differentiation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preadipocyte differentiation inhibitor using a compound belonging to safe and effective carotenoids as active principle, and to provide pharmaceutical compositions and cosmetic compositions exhibiting the action to inhibit the differentiation of preadipocytes to adipocytes.SOLUTION: It is found that siphonaxanthin has inhibitory effect on the differentiation of preadipocytes to adipocytes. There are provided preadipocyte differentiation inhibitors, pharmaceutical compositions, and cosmetics composition using siphonaxanthin as active principle.

Description

  The present invention relates to a preadipocyte differentiation inhibitor containing siphonaxanthin as an active ingredient, and a composition containing the agent.

  In recent years, obesity has been rapidly increasing, triggered by excessive intake of calories associated with the enrichment of dietary habits around the world and lack of exercise due to the development of transportation means. Obesity is known to cause adult diseases such as hyperlipidemia, diabetes, and hypertension, especially because of the combination of factors such as hyperlipidemia, hypertension, and hyperinsulinism due to obesity. Even if it is a symptom, there is a high risk of developing seriousness such as ischemic heart disease in the future, which is a social problem as a metabolic syndrome.

  Obesity is simply caused by excessive intake of fat, and more fat than necessary may accumulate in the body, but fat precursor cells that differentiate into fat cells in the body respond to various differentiation stimuli. It may differentiate into cells and cause obesity due to an increase in the number of fat cells. Generally, when the BMI value is 25 or more, it is considered obese.

  Therefore, in recent years, there has been a demand for the discovery of effective functional ingredients that suppress the increase in fat cells that cause obesity, and flavonoids such as catechin and flavangenol have been found as active ingredients that suppress obesity. Various products have been put into practical use.

Furthermore, it was discovered that fucoxanthinol belonging to carotenoids has an effect of suppressing differentiation of preadipocytes into adipocytes (Patent Document 1), and various carotenoids are expected to have the same effect. However, only carotenoids having an allene structure (= C =) such as neoxanthine, fucoxanthin and fucoxanthinol have been reported to have an inhibitory effect. That is, it has been reported that carotenoids having no allene structure have no effect of differentiation from preadipocytes into adipocytes. (Patent Document 2)
It has already been reported that siphonaxanthin, which is abundant in green algae, is a carotenoid having no allene structure and can be used as an antitumor agent (Patent Document 3).

International Patent Publication WO / 2006/126325 JP 2008-280281 A JP 2009-227642 A

  The main object of the present invention is to provide a safe and excellent anti-adipocyte differentiation inhibitor. Another object of the present invention is to provide a pharmaceutical composition, a food composition, a cosmetic composition, and the like that exhibit an action of suppressing differentiation of preadipocytes into adipocytes.

  As a result of intensive studies in view of the above problems, the present inventors have found that siphonaxanthin belonging to carotenoids is superior in fat compared to β-carotene and fucoxanthin, which are other compounds belonging to the same category. It has been found that it has an action of suppressing the differentiation of progenitor cells into adipocytes.

  Furthermore, siphonaxanthin is another compound belonging to the same class, and it has been clarified that the toxicity to cells is lower than that of neoxanthine, which has an action of suppressing the differentiation of preadipocytes into adipocytes. That is, the present invention has been completed based on such knowledge, and includes the embodiments shown below.

Item 1. A preadipocyte differentiation inhibitor comprising siphonaxanthin as an active ingredient.
Item 2. An anti-obesity agent containing siphonaxanthin as an active ingredient.
Item 3. An external preparation containing siphonaxanthin as an active ingredient.
Item 4: An internal preparation containing siphonaxanthin as an active ingredient.
Item 5. An internal preparation containing siphonaxanthin.
Item 6. A cosmetic composition comprising siphonaxanthin as an active ingredient Item 7. A pharmaceutical composition comprising siphonaxanthin as an active ingredient.
Item 8. A food composition comprising siphonaxanthin.

The present invention is described in detail below.
Siphonaxanthin Siphonaxanthin of the present invention has the following formula (1):

に示される化合物である。

It is a compound shown by these.

  The siphonaxanthin of the present invention can be obtained using natural products as raw materials even if it is obtained by chemical synthesis such as listed in the multi-step oxidation reaction process using lutein as a raw material and synthesis of loroxanthin. Even those derived from natural products may be obtained by using a siphonaxanthin precursor derived from a natural product as a raw material and then chemically synthesizing it. From the viewpoint of environment and animal safety, siphonaxanthin derived from natural products is preferred.

  Specific examples of the natural product as the raw material described above include algae such as brown algae, red algae and green algae, preferably green algae. Specific examples of green algae include mills (Codium fragile [Suringer] Hariot), japonica (Ulva japonica), and firefly (Caulerpa lentilifera), and are preferably japonicas that inhabit relatively shallow places. The japonica contains about 30 μg per gram of dry algae, and the mill contains about 200 μg of siphonaxanthin.

  The natural product-derived siphonaxanthin can be obtained by a method of isolation using at least one of the algae as a raw material. As an example of a specific method, the raw material is left as it is, or dried, shredded, crushed, pulverized, pressed, boiled or fermented as necessary, and a solvent is added to contain siphonaxanthin. A method of purifying siphonaxanthin by extracting and subjecting the resulting extract to chromatographic treatment can be mentioned.

  As said solvent, acetone, ethanol, hexane, chloroform etc. are mentioned, for example, Among these, it is preferable to use acetone or ethanol from a viewpoint of extraction efficiency. The amount of the extraction solvent used is usually about 2 to 100 parts by weight, and more preferably about 5 to 10 parts by weight with respect to 1 part by weight of the raw material.

  Specific examples of the extraction method include immersion methods such as cold immersion and digestion; methods of stirring under low temperature, room temperature, or high temperature conditions; percolation methods and the like. After the extraction treatment, the solid phase fraction is removed using a commonly used solid-liquid separation means such as filtration or centrifugation, and an extract containing syphonaxanthin in the liquid fraction can be obtained. The obtained extract can be used as it is to give the siphonaxanthin of the present invention. However, if necessary, the organic solvent component may be removed by performing a treatment such as vacuum distillation.

  In addition, the obtained extract may be further subjected to a purification step to obtain the siphonaxanthin of the present invention. Specifically, a purification method by a known column chromatography method using an HPLC system or the like can be mentioned. Examples of the column used in this method include a silica column, an ODS column, a cyanopropyl column, etc. Among them, an ODS column is preferable.

  The siphonaxanthin obtained by the above-mentioned method may be removed by using a known method, and then dissolved in a more appropriate solvent and stored. Moreover, it is good also as processes, such as drying and concentration, as needed, and it is good also as a dried material and a concentrate. Since siphonaxanthin has a property of being weak against light, it is preferable to keep it in a light-shielding environment when storing.

Fat Precursor Cell Differentiation Inhibitor The fat precursor cell differentiation inhibitor of the present invention comprises the above-mentioned siphonaxanthin as an active ingredient. In the preadipocyte differentiation inhibitor of the present invention, the ratio of siphonaxanthin contained as an active ingredient is usually about 1 to 99% by weight, more preferably about 10 to 90%.

  The effect of the preadipocyte differentiation inhibitor of the present invention can be confirmed by an anti-metabolic syndrome action, a slimming action, a diet action, and the like.

  The mechanism of inhibiting adipogenesis involving the agent for inhibiting differentiation of preadipocytes of the present invention is not particularly limited, but is preferably a mechanism that suppresses differentiation into fat induced by insulin, and a signal cascade via PPARγ is activated. The mechanism etc. which suppress the differentiation to the fat induced by doing can be mentioned.

  The preadipocyte differentiation inhibitor can also be used for animal individuals. The animal to be used is not particularly limited, but has a more preferable inhibitory effect on differentiation of preadipocytes against mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, etc. Show. More preferably, it is a human. Among these, application to humans in a state such as obesity due to an increase in neutral fat is particularly preferable.

  Since the siphonaxanthin of the present invention exerts an effect of suppressing differentiation of adipose precursor cells by being used in a form such as external application that is applied through the skin, internal application that is applied by internal use or ingestion, etc. Siphonaxanthin is used in combination with a composition used as a cosmetic, pharmaceutical, food, or the like.

The amount of the preadipocyte differentiation inhibitor used for an individual animal can be appropriately set depending on the body weight, age, sex, dosage form, degree of desired effect, etc. of the individual animal. As long as the amount used is a form for internal use, the amount of siphonaxanthin, which is an active ingredient, may be usually used in an amount of about 0.1 to 100 mg / kg / day, and may be a form for external use. For example, the amount of siphonaxanthin may be usually used in an amount of about 0.01 to 10 mg per 1 cm ^{2 of} skin.

  In the composition containing the siphonaxanthin of the present invention as an active ingredient, the blending ratio of siphonaxanthin may be an amount that can suppress the differentiation of preadipocytes into adipocytes, and the form and use of the composition The amount of siphonaxanthin is usually about 0.001 to 50% by weight based on the total amount of the composition.

  Below, the usage aspect of the siphonaxanthin of this invention is demonstrated taking cosmetics, a pharmaceutical, and a foodstuff as an example.

In cosmetic cosmetics, a cosmetic agent for inhibiting differentiation of preadipocytes by blending a cosmetically acceptable carrier or additive together with an amount of siphonaxanthin effective for inhibiting differentiation of preadipocytes A composition is provided.

  That is, the cosmetic composition of the present invention contains siphonaxanthin as an active ingredient. The cosmetic composition is useful as an anti-obesity cosmetic because it can effectively exert the action of suppressing the differentiation of preadipocytes and is expected to prevent the increase of adipocytes in the living body. Examples thereof include slimming cosmetics and diet cosmetics.

  The shape of the cosmetic composition is not particularly limited, but for example, paste, lotion, mousse, gel, jelly, liquid, emulsion, suspension, cream, ointment, sheet, aerosol And spray. Further, the form of the cosmetic composition is not limited. For example, makeup cosmetics such as foundation, blusher, and white powder; basic makeup such as lotion, emulsion, cream, lotion, oil, and pack Skin cleansing agents such as facial cleansers, cleansings, body cleansing agents, massage agents, wiping agents, detergents, bathing agents, and the like.

  In addition, the blending ratio of siphonaxanthin in the cosmetic composition may be appropriately set within the above-described ratio range, but preferably, siphonaxanthin is 0. 0 relative to the total amount of the cosmetic composition. The ratio is about 0001 to 50% by weight, preferably about 0.001 to 20% by weight.

In the field of pharmaceutical medicine, a pharmaceutical composition for inhibiting differentiation of preadipocytes is provided by combining pharmaceutically acceptable carriers and additives together with an amount of siphonaxanthin effective for inhibiting differentiation of preadipocytes. Is done. That is, the pharmaceutical composition of the present invention contains siphonaxanthin as an active ingredient.

  Since the pharmaceutical composition effectively exerts an action to suppress differentiation of preadipocytes, it is useful as a preventive and / or therapeutic agent for diseases and symptoms caused by differentiation from preadipocytes into adipocytes. Specific examples include metabolic syndrome and obesity. The pharmaceutical composition includes both pharmaceuticals and quasi drugs.

  The pharmaceutical composition can exert the desired action described above, whether applied internally or applied externally. Therefore, the pharmaceutical composition may be used in a pharmaceutical form such as an internal preparation; an injection such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection; a transmucosal application agent or a transdermal application agent. it can.

  The dosage form of the pharmaceutical composition is appropriately set according to the application form, but as an example, solid preparations such as tablets, powders, powders, granules, capsules; liquids, emulsions, suspensions, etc. Liquid preparations; semi-solid preparations such as ointments and gels.

  The application amount of the pharmaceutical composition can be appropriately set based on the gender and age of the application subject, the application form of the composition, the expected effect, etc., for example, 0.1-100 mg / kg / day Apply in a certain amount.

  Moreover, the blending ratio of siphonaxanthin in the pharmaceutical composition may be appropriately set according to the application form, application amount, etc., but preferably siphonaxanthin is 0 with respect to the total amount of the composition. The ratio is about 0.0001 to 50% by weight, preferably about 0.001 to 20% by weight.

In the field of foods and foods, foods exhibiting the effect of inhibiting differentiation of adipose precursor cells by blending various foods with an amount of siphonaxanthin effective for exerting a differentiation inhibiting action on adipose precursor cells in vivo. A composition can be provided. That is, the present invention can provide food compositions labeled as fat differentiation inhibiting, anti-obesity, dieting, or slimming in the field of food. Examples of the food composition include general health foods, foods for specified health use (including foods for specified health use with conditions), dietary supplements, functional foods, foods for sick people, and the like.

  More specifically, the food composition includes beverages such as soft drinks, carbonated drinks, nutritive drinks, fruit drinks, and lactic acid drinks; frozen confectionery such as ice cream and shaved ice; gum, chocolate, candy, tablet confectionery, snack confectionery, jelly Snacks, jams, creams, etc .; noodles such as buckwheat noodles, instant noodles; processed fishery and livestock products such as kamaboko, ham, sausage; dairy products such as processed milk, fermented milk; salad oil, mayonnaise, whipped cream, dressing Examples include fats and oils and processed foods such as sauces, seasonings such as sauces and sauces, soups, salads, prepared dishes, pickles, breads and cereals. For example, in the case of food for specified health use, dietary supplement, functional food, etc., it may be in the form of powder, granule, capsule, troche, tablet, syrup and the like.

  Conventionally, siphonaxanthin is known to be contained in algae such as seaweed as described above, but the intake in the range of normal eating habits is the differentiation of the above-mentioned preadipocytes into adipocytes The amount is not sufficient to obtain a suppression effect. In addition, taking an excessive amount of siphonaxanthin is not preferable because an effect commensurate with the amount cannot be obtained and there is an economic problem, and there are concerns about unexpected adverse effects on the human body.

  From such a point of view, about 1 to 1000 mg of siphonaxanthin may be ingested with respect to one intake of the food composition. By appropriately setting the intake amount in such a range, the amount of siphonaxanthin to be contained in the food composition can be determined. The amount of the siphonaxanthin is usually about 0.05 to 10% by weight, preferably about 0.1 to 5% by weight, when converted to 1 part by weight of the food composition.

  The siphonaxanthin of the present invention is effective as a food additive for inhibiting the differentiation of preadipocytes because it can effectively exert the effect of inhibiting the differentiation of preadipocytes and is expected to prevent the increase of adipocytes in vivo. It is. The agent can be used for applications such as slimming, anti-metabolic syndrome, and diet.

  The amount of siphonaxanthin added to the food additive for inhibiting preadipocyte differentiation is usually about 0.001 to 10% by weight, preferably about 0.01 to 1% by weight, per 1 part by weight of the agent. is there. Further, the agent can be suitably used specifically when ingesting food or cooking food, etc., and the intake may be about the same as the intake of the above-described food composition.

  Siphonaxanthin used as an active ingredient in the present invention is a component contained in green algae belonging to algae. That is, siphonaxanthin is a kind of natural component that has been ingested as a food from the past, and its safety to the human body has been confirmed. In particular, siphonaxanthin has the performance of lower cytotoxicity than other carotenoids.

  Since siphonaxanthin used as an active ingredient in the present invention has an effect of suppressing differentiation of preadipocytes into adipocytes, siphonaxanthin is used as an inhibitor of differentiation of preadipocytes, a cosmetic composition, and a pharmaceutical composition. It can be made into the active ingredient of a thing, and can be set as the food composition containing a siphonaxanthin. Since these compositions exhibit the effect of suppressing the differentiation of preadipocytes, they can be used for applications such as anti-obesity, slimming, metabolic syndrome, and diet.

The result of having analyzed the preadipocyte differentiation inhibitory effect by the carotenoids containing siphonaxanthin by the amount of neutral fat accumulated in the cells. A microscopic image of neutral fat accumulated in the cells and observed for the preadipocyte differentiation-inhibiting action of carotenoids including siphonaxanthin.

  The present invention is described in further detail below. However, it goes without saying that the present invention is not limited to the following examples.

Manufacture of carotenoids The siphonaxanthin was made from the green algae (Ulva japonica), the fucoxanthin used as a comparative experimental example was made from the dried alga wakame (Undaria pinnatifida), and the neoxanthine was made from the spinach (Spinia oleracea).

  Each sample was freeze-dried, pulverized using a mill mixer, 10 times volume of acetone was added, and extraction was performed overnight at 4 ° C. The solid component was removed from the extract by filtration, and then concentrated by an evaporator and dissolved in methanol. The obtained lysate was subjected to a preparative HPLC system to purify carotenoids.

  In the preparative HPLC system, the column used was LiChroprep RP-18 (manufactured by MERCK) and the pump was LC-9A (manufactured by Shimadzu). As a mobile phase used in the fractionation step, a mixed solution having a volume ratio of acetonitrolyl: water of 90:10 was used. Using the above equipment and conditions, each sample was subjected to a preparative HPLC system, and fractions containing each carotenoid were collected by visual observation, and then subjected to a concentration step using an evaporator and a dissolution step using methanol. A lysate containing was obtained. The obtained lysate was further subjected to an HPLC system.

  In the HPLC system, ODS-80Ts (4.6 cm × 250 mm; manufactured by TOSOH) was used as a column, LC-6AD (manufactured by Shimadzu) as a pump, and SPD-M20A (manufactured by Shimadzu) as a detector. The HPLC conditions used here were a mixed solution of acetonitrile: methanol: water with 0.1% ammonium acetate as the mobile phase and a volume ratio of 75:15:10, and a flow rate of 1.0 ml / min. I went.

  Peaks corresponding to the elution of each carotenoid appeared in about 9 minutes for fucoxanthin and neoxanthine and about 7 minutes for siphonaxanthin, and were collected. To 1 mL of the collected solution, 2 mL of dichloromethane and 0.9 mL of water were added and stirred, and then centrifuged at 1,000 g for 15 minutes to separate the lower layer. The upper layer was stirred after adding an equal amount of dichloromethane, and the lower layer was again collected under the same conditions and combined with the previous lower layer.

  The obtained solutions of various carotenoids were removed with nitrogen, dissolved in a mixed solvent containing equal amounts of dichloromethane and methanol, and stored at −80 ° C. All manufacturing operations were performed under light-shielding conditions as much as possible, and further at low temperatures.

Identification of produced carotenoids Various carotenoids obtained by the above-described method were analyzed by LC-MS method and absorption spectrum method. The results are shown below.
[Fucoxanthin derived from seaweed]
[M + H] ⁺ 659, [M + HH ₂ O] ⁺ 641 Maximum absorption wavelength 447 nm
[Neoxanthine derived from spinach]
[M + H] ⁺ 601, [M + HH ₂ O] ⁺ 583 Maximum absorption wavelength 413, 437, 465 nm
[Siphonaxanthin derived from japonica]
[M + H] ⁺ 601, [M + HH ₂ O] ⁺ 583 Maximum absorption wavelength 449 nm
Effects of inhibiting carotenoids on adipose differentiation Mice-derived adipose precursor cells 3T3-L1 (Human Science Promotion Foundation) were seeded in a 24-well plate at a concentration of 5 × 10 ⁵ cells / mL, and then added to a DMEM medium containing 10% FBS. After cultivating and growing and becoming confluent, maintenance was performed for 2 days. Differentiation of preadipocytes into adipocytes was performed by culturing using a DMEM medium containing insulin having a final concentration of 1 μM, 0.5 mM isobutylmethylxanthine, and 1 μM dexamethasone.

  48 hours after the start of differentiation induction, DMEM to which final concentration of 1 μM of insulin, tetrahydrofuran in which the produced fucoxanthin, neoxanthine, and siphonaxanthin carotenoids were dissolved was added to a final concentration of 0.3%. An experiment for confirming the effect of inhibiting fat differentiation was performed using a medium. Here, various carotenoids are used in the medium at concentrations of 5 μM and 10 μM, respectively, a negative target group is a medium in which carotenoids are not dissolved in tetrahydrofuran, and a medium in which β-carotene is dissolved in tetrahydrofuran as carotenoids. Using. These media were changed twice every 48 hours, and differentiation into adipocytes was confirmed 6 days after the initiation of differentiation induction.

  Confirmation of differentiation into adipocytes was evaluated by using a method of staining with oil red O for neutral fat accumulated in the cells. Distilled water was added to isopropyl alcohol in which 0.3% Oil Red O was dissolved to adjust the isopropyl alcohol to 60%, and then filtered to obtain a staining solution. The cells on the 6th day after the start of differentiation induction were washed with PBS, 60% isopropyl alcohol was added, and the mixture was allowed to stand for 1 minute. Thereafter, 60% isopropyl alcohol was removed, and the staining solution was added and incubated for 20 minutes. After incubation, the cells were washed with PBS and observed with a microscope. In addition, 100% isopropyl alcohol is added to the washed cells, the cells are lysed, and neutral fat stained in the cells is extracted, and then the absorbance at 490 nm is measured using a plate reader, Intracellular neutral fat was quantified.

  FIG. 1 shows the results of confirming the effect of inhibiting the differentiation of various carotenoids into adipocytes by quantifying the amount of neutral fat in the cells. For siphonaxanthin and neoxanthine, the OD value at 490 nm, which is specifically absorbed by oil red O in a concentration-dependent manner, is significantly decreased compared to the negative target group, fucoxanthin and neoxanthine, and into cells. It was found to have an effect of reducing the amount of accumulated triglyceride.

  FIG. 2 shows the results of confirming the effect of various carotenoids on the differentiation of adipocytes by microscopic observation of cells after oil red O staining. This is shown in a microscope observation image when various carotenoids are added at 10 μM. As compared with the negative target group, fucoxanthin, siphonaxanthin can be seen to have fewer red parts that stain fat cells. Furthermore, when neoxanthine was added, the number of cells themselves decreased, and it was found that the cells were toxic to cells.

  From the above results, it was clarified that siphonaxanthin has an effect of significantly suppressing differentiation into adipocytes without showing toxicity to cells.

Claims (3)

A preadipocyte differentiation inhibitor containing siphonaxanthin as an active ingredient. A cosmetic composition comprising siphonaxanthin as an active ingredient. A pharmaceutical composition comprising siphonaxanthin as an active ingredient.

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