JP6201075B1 - PPARγ expression inhibitor, C / EBPα expression inhibitor, food composition for suppressing PPARγ expression, food composition for suppressing C / EBPα expression, cosmetic composition for suppressing PPARγ expression, cosmetic composition for suppressing C / EBPα expression, Method for producing PPARγ expression inhibitor and method for producing C / EBPα expression inhibitor - Google Patents

Abstract

A novel adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, food composition for inhibiting adipocyte differentiation, and a cosmetic composition for inhibiting adipocyte differentiation, which are novel uses of Euglena extract A composition is provided. A novel adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, food composition for inhibiting adipocyte differentiation, and adipocyte differentiation inhibitor containing an Euglena aqueous solvent extract as an active ingredient It is a cosmetic composition. For example, the Euglena aqueous solvent extract is a Euglena water extract or a hot water extract. [Selection] Figure 3

Description

The present invention relates to a PPARγ expression inhibitor, a C / EBPα expression inhibitor, a PPARγ expression- suppressing food composition, a C / EBPα expression-suppressing food composition, a PPARγ expression- suppressing cosmetic composition, and a C / EBPα expression-suppressing agent. The present invention relates to a cosmetic composition, a method for producing a PPARγ expression inhibitor, and a method for producing a C / EBPα expression inhibitor.

  Examples of diseases called lifestyle-related diseases include obesity, hypertension, diabetes, hyperlipidemia, myocardial infarction, and stroke. In recent years, it has been found that these diseases do not develop due to individual causes, but are caused by various causes affecting each other, and such complex lifestyle-related diseases are also called metabolic syndrome.

“Metabolic syndrome” represents a state in which a series of pathological conditions such as type 2 diabetes based on insulin resistance, hyperlipidemia, hypertension, visceral fat obesity, and fatty liver are combined.
Metabolic syndrome is a state in which the action of insulin is reduced due to the accumulation of fat cells, especially fat cells (built-in fat) in the viscera, and risk factors for arteriosclerosis such as hyperglycemia, abnormal lipid metabolism, and hypertension are accumulated. It is.

It has been elucidated that fat cell accumulation can lead to obesity, hypertension, diabetes, hyperlipidemia, myocardial infarction, stroke, etc. Prevention is very important.
As used herein, “obesity” refers to a condition requiring weight loss treatment when there is an associated health disorder due to obesity or excessive accumulation of visceral fat expected to cause the health disorder. .

  In addition, with the recent increase in interest in health and beauty, the number of people who want to maintain a healthy and slim body is increasing, regardless of age or gender. Is very interested in

Conventionally, as an anti-obesity agent for treating or preventing obesity, many of them exhibit an effect of suppressing fat accumulation by decomposing accumulated fat.
Obesity is due to the differentiation of preadipocytes into adipocytes (mature adipocytes), which increase in number and size. Furthermore, transcription factors such as PPARγ and C / EBPα are known as master regulators of adipocyte differentiation in the process of adipocyte differentiation. In order to prevent and treat obesity, it is effective to suppress the differentiation of preadipocytes into adipocytes.
Known adipocyte differentiation inhibitors include hop tissue water extract (Patent Document 1), acidic mucopolysaccharide (Patent Document 2), plant extract (Patent Document 3), and the like. Among them, Patent Document 1 discloses that the water extract of hop tissue suppresses the expression of PPARγ and suppresses the differentiation of adipocytes through this.

On the other hand, Euglena (genus name: Euglena, Japanese name: Euglena) attracts attention as a biological resource that is expected to be used as food, feed, fuel, and the like.
Euglena has 59 kinds of nutrients that correspond to most of the nutrients necessary for humans to live, such as vitamins, minerals, amino acids, and unsaturated fatty acids, and as a supplement to take a variety of nutrients in a balanced manner. The possibility of use as a food supply source in poverty areas where the necessary nutrients cannot be ingested has been proposed.

Euglena is located in the primary producer of the food chain and is cultivated in large quantities because it is preyed by predators and the culture conditions such as light, temperature conditions, and stirring speed are difficult compared to other microorganisms. However, in recent years, due to the diligent research of the present inventors, a large-scale culture technique has been established, which has opened the way for mass supply of Euglena-derived substances such as Euglena and paramylon extracted from Euglena.
Euglena is a unique organism that possesses the animal nature of flagellar movement and at the same time has chloroplasts as a plant and performs photosynthesis, and Euglena itself and substances derived from Euglena are expected to have many functions. ing.
Therefore, it is desired to develop a method for using Euglena and Euglena-derived substances that can be supplied in large quantities.

JP 2010-173942 A JP 2014-9161 A JP 2011-6347 A

The present invention has been made in view of the above problems, and the object of the present invention is a novel adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, and food composition for inhibiting adipocyte differentiation. An object of the present invention is to provide a cosmetic composition for inhibiting adipocyte differentiation.
Another object of the present invention is to provide an adipocyte differentiation inhibitor, a PPARγ expression inhibitor, a C / EBPα expression inhibitor, a food composition for inhibiting adipocyte differentiation, an adipocyte differentiation, which is a novel method of using an Euglena aqueous solvent extract. It is providing the cosmetic composition for suppression.

As a result of intensive studies, the present inventors have found that an Euglena aqueous solvent extract has an action of suppressing differentiation from preadipocytes to adipocytes.
More specifically, in the process of differentiation of preadipocytes into mature adipocytes, the expression of PPARγ and C / EBPα among the key transcription factor groups is suppressed, and the differentiation from preadipocytes to adipocytes is suppressed. It has been clarified that the present invention has been made.

Thus, the object is achieved according to the present invention, P PARganma expression inhibitor you characterized by containing the Euglena water extract or Euglena hot water extract as an active ingredient, C / EBP [alpha] expression inhibitor, PPARy expression inhibition It is solved by a food composition for C / EBPα expression suppression, a cosmetic composition for PPARγ expression suppression, and a cosmetic composition for C / EBPα expression suppression .

Since the present invention contains no Euglena water extract or Euglena hot water extract with safety of a level consistent with the Food Sanitation Law so far as no side effects have been reported, continuous administration and continuation over a long period of time. Ingestion is possible.

The object is achieved, according to the present invention, the Euglena, the dispersion preparation step of obtaining a dispersion by shaking the addition of water, and centrifuged the dispersion, the supernatant, as Mizu抽 extract of Euglena P PARganma expression inhibitor you comprising: the obtaining extraction step, and is achieved by a process for preparing C / EBP [alpha] expression inhibitor.

  At this time, a heating step of heating the dispersion may be performed after the dispersion preparation step and before the extraction step.

According to the present invention, a novel P PARganma expression inhibitor, C / EBP [alpha] expression inhibitor, PPARy expression suppressing food composition, C / EBP [alpha] expression for suppressing food composition, PPARy expression inhibition cosmetic composition, and C / EBPα expression-suppressing cosmetic composition can be provided.
Since the present invention contains no Euglena water extract or hot water extract with a level of safety that meets the food hygiene law so far as no side effects have been reported, continuous administration and continuation over a long period of time. Ingestion is possible.

It is a schematic explanatory drawing which shows the life cycle of an adipocyte and the protein expression level which is a transcription factor. It is explanatory drawing which shows the procedure of the fat differentiation suppression test which verified the fat differentiation inhibitory effect of the Euglena water extract or hot water extract of Example 1 and Examples 2-6 based on one Example of this invention. It is a graph which shows the result of the fat differentiation suppression test of the Euglena hot-water extract of Example 1 of this invention which verified in Test Example 1. FIG. It is a photograph which shows the result of the lipid droplet dyeing | staining after the fat differentiation suppression test of the Euglena hot-water extract of Example 1 of this invention which verified in Test Example 1. FIG. It is a graph which shows the result of the fat differentiation suppression test of the Euglena water extract or hot water extract of Examples 2-6 of this invention which verified in Test example 2. FIG. It is a photograph which shows the result of the lipid droplet dyeing | staining after the fat differentiation suppression test of the Euglena water extract or hot water extract of Examples 2-6 of this invention which verified in Test example 2. FIG. It is a graph which shows the result of the fat differentiation suppression test of the Euglena hot water extract of Example 1 of this invention and the EPA of Comparative Example 1 which was verified in Test Example 3. It is a photograph which shows the result of the lipid droplet dyeing | staining after the fat differentiation suppression test of the EPA of the comparative example 1 verified in Test example 3. FIG. Explanatory drawing which shows the procedure of the fat differentiation suppression test at the time of changing the period which added the Euglena hot-water extract of Example 1 of this invention verified in Test Example 4, and the result of a fat differentiation suppression test are shown. It is a graph. It is a photograph which shows the result of the lipid droplet dyeing | staining after a fat differentiation suppression test at the time of changing the period which adds the Euglena hot-water extract of Example 1 of this invention which verified in Test Example 4. FIG. It is a graph which shows the result of having compared the expression level of the PPARγ gene when the Euglena hot water extract of Example 1 of the present invention, which was verified in Test Example 5, was added. It is a graph which shows the result of having compared C / EBP (alpha) gene expression level when the Euglena hot water extract of Example 1 of this invention which verified in Test Example 5 was added.

Hereinafter, embodiments of the present invention will be described with reference to FIGS.
This embodiment relates to the invention of an adipocyte differentiation inhibitor, a PPARγ expression inhibitor, a C / EBPα expression inhibitor, an anti-obesity agent, and an anti-metabolic syndrome agent comprising Euglena aqueous solvent extract as an active ingredient.

In the present specification, “obesity” is a state in which the body weight is higher than that in a normal state, or a body fat is excessively accumulated. BMI (Body Mass Index, body mass index) is used as a criterion for determining obesity. According to the criteria for determination of obesity by WHO (World Health Organization, World Health Organization), BMI 30 or more is obesity, while in Japan, BMI 25 or more is obesity. The criteria for Japanese judgment are standards defined by the Japanese Society of Obesity and are based on the fact that Japanese people have increased the frequency of complications such as impaired glucose tolerance, dyslipidemia, and hypertension from around BMI 25.
As used herein, “obesity” refers to a condition requiring weight loss treatment when there is an associated health disorder due to obesity or excessive accumulation of visceral fat predicted to cause the health disorder. It is. Obesity is caused by an increase in the number of fat cells and enlargement of fat cells, and the life cycle of fat cells such as proliferation and differentiation of preadipocytes, enlargement, or fat cell death and elimination from the living body. Involved.
In the present specification, “anti-obesity” means prevention or treatment of the above obesity.

In the present specification, “metabolic syndrome” represents a state in which a series of pathological groups such as type 2 diabetes based on insulin resistance, hyperlipidemia, hypertension, visceral fat type obesity, fatty liver and the like are combined, It is also known by names such as syndrome X, insulin resistance syndrome, visceral fat syndrome, and multiple risk factor syndrome.
In the present specification, “antimetabolic syndrome” means prevention or treatment of at least one disease state selected from the above-mentioned disease state group.

<Outline of fat cells>
An adipocyte is a cell having lipid droplets in the cytoplasm, and is classified into a monocystic adipocyte (white adipocyte) and a multivesicular adipocyte (brown adipocyte).
White adipocytes are consumed in excess and convert excess energy that is not consumed into neutral fat and accumulate as an energy source. White adipose cells are widely present in the human body, but are particularly abundant in the lower abdomen, buttocks, thighs, back, upper arm, and viscera, and accumulate as subcutaneous fat and visceral fat.

  It was thought that the number of white adipocytes increased only in a limited period such as infants and adolescence, and the number of white adipocytes in life was determined at that time. However, in recent studies, when white adipocytes already present are filled with neutral fat due to excessive energy intake or lack of exercise, the number of fat cells increases even after puberty, and fat I found out that When the size of the fat cells reaches the upper limit and the fat cannot be stored any more, the surrounding preadipocytes are stimulated by PPARγ and the like to become mature fat cells, which in turn become enlarged, and the mature fat cells also Cell division and fat cell count increase.

The number of white adipocytes in adults around 20 years old is about 40 billion, but the number of white adipocytes in obese people can be as high as 40 to 60 billion.
White fat cells break down the accumulated fat when energy is needed and supply it to the whole body as free fatty acids and glycerol. At that time, the white fat cells that have released the fat return to the preadipocytes, and when the energy becomes excessive again, they become mature fat cells and take in the fat.

  Brown adipocytes, on the other hand, generate heat by breaking down excess fat, and are present in specific parts of the human body, such as behind the neck, around the scapula, under the armpit, around the heart and kidneys. The number of brown adipocytes is greatest immediately after birth, decreases with age as an adult, and particularly decreases after age 40 years. This reduction in the number of brown adipocytes has been reported to be a major cause of middle-age fatness.

<Adipocyte life cycle, transcription factor cascade, and adipocyte differentiation inhibitory action>
As shown in FIG. 1, the life cycle of adipocytes is (1) a process in which mesenchymal stem cells are determined to differentiate into preadipocytes, and (2) proliferation of preadipocytes and differentiation into mature adipocytes. (3) Apoptosis of enlarged fat cells and a process of being excluded from the living body can be considered.
Transcription factors closely related to the adipocyte differentiation process have been clarified by a system using cultured cells or genetically modified individuals.

  PPAR (Peroxisome Proliferator-Activated Receptor, Peroxisome Proliferator-responsive Receptor), C / EBP (CCAAT / Enhancer-Binding Protein, CCAAT / Enhancer Binding Protein), and SREBP-1 / ADDiProte lingleRegulEregElteElegeRegeRegulEgR Determination and Differentiation-Dependent Factor 1) is known as the most important transcription factor for adipocyte differentiation.

  PPAR is known to have families such as PPARα, PPARγ, and PPARδ, among which PPARγ is particularly important for adipocyte differentiation. When PPARγ is forcibly expressed in lipoblasts and preadipocytes, it differentiates into adipocytes.

  C / EBP is known to be a family of C / EBPα, C / EBPβ, C / EBPδ, C / EBPγ, C / EBPε, C / EBPζ, and C / EBPα is similar to PPARγ in adipocyte differentiation. It functions as a master regulator. In addition, C / EBPβ and C / EBPδ are expressed at the early stage of differentiation and control the expression of C / EBPα and PPARγ.

  SREBP1 / ADD1 is known to promote adipocyte differentiation, while being involved in PPARγ ligand generation in the adipocyte differentiation process.

  When these three types of transcription factors (PPAR, C / EBP, SREBP1 / ADD1) cross-talk, the differentiation process of adipocytes proceeds.

<Euglena>
In this embodiment, “Euglena” includes taxonomically classified microorganisms classified as Euglena, variants thereof, variants thereof, and closely related species of Euglenaaceae.
Here, the Euglena genus (Euglena) is a group of organisms belonging to the Excavator, Euglenozoa, Euglena algae, Euglena, Euglena family among eukaryotes.

Specific examples of the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, Euglena viridis, and the like.
As Euglena, Euglena gracilis (E. gracilis), particularly E. gracilis Z strain, can be used. (Chloroplast-deficient strain) and its variant E. gracilis
var. bacillaris, gene mutants such as chloroplast mutants of these species, other Euglenas such as Astaia longa, and substances such as β-1,3-glucanase derived therefrom may be used.

Euglena is widely distributed in fresh water such as ponds and swamps, brackish water, and seawater, and may be used separately from these, or any Euglena that has already been isolated may be used. Good.
Euglena includes all its mutants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like.

In the culture of Euglena cells, examples of the culture solution include a culture solution to which nutrient salts such as a nitrogen source, a phosphorus source, and a mineral are added, for example, a modified Cramer-Myers medium ((NH ₄ ) ₂ HPO ₄ 1.0 g / L. , KH ₂ PO ₄ 1.0 g / L, MgSO ₄ .7H ₂ O 0.2 g / L, CaCl ₂ .2H ₂ O 0.02 g / L, Fe ₂ (SO ₂ ) ₃ 7H ₂ O 3 mg / L, MnCl ₂ · 4H ₂ O 1.8 mg / L, CoSO ₄ · 7H ₂ O 1.5 mg / L, ZnSO ₄ · 7H ₂ O 0.4 mg / L, Na ₂ MoO ₄ · 2H ₂ O 0.2 mg / L, CuSO ₄ .5H ₂ O 0.02 g / L, thiamine hydrochloride (vitamin B ₁ ) 0.1 mg / L, cyanocobalamin (vitamin B ₁₂ , (pH 3.5)) can be used. Note that (NH ₄ ) ₂ HPO ₄ can also be converted to (NH ₄ ) ₂ SO ₄ or NH ₃ aq. In addition, a known Hunter medium or Koren-Hunter medium prepared based on the description of Euglena Physiology and Biochemistry (Edited by Shozaburo Kitaoka, Society of Sciences Publishing Center) may be used.

The pH of the culture solution is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less. By setting the pH to the acidic side, the photosynthetic microorganisms can grow more dominantly than other microorganisms, so that contamination can be suppressed.
Euglena cells may be cultured by an open pond method using sunlight directly, a light collecting method in which sunlight condensed by a light concentrator is sent through an optical fiber, etc., and irradiated in a culture tank and used for photosynthesis.
Euglena cells can be cultured using, for example, a fed-batch method, but flask culture, fermentation using a fermentor, batch culture, semi-batch culture (fed-batch culture), continuous culture (perfusion) Any liquid culture method such as a culture method may be used.

The culture can be performed using a known culture apparatus such as an open pond type, a raceway type, or a tube type, or an experimental culture vessel such as a Sakaguchi flask, an Erlenmeyer flask, or a reagent bottle. Since Euglena to assimilate CO _2, when cultured using the Cramer-Myers medium is autotrophic medium is preferably be passed through into the medium air containing 1 to 5% CO _2. Further, in order to sufficiently develop the chloroplast, about 1 to 5 g of ammonium phosphate may be added per liter of the medium. The culture temperature is usually 20 to 34 ° C., particularly preferably 28 to 30 ° C. Depending on the culture conditions, Euglena usually enters a logarithmic growth phase 2 to 3 days after the start of the culture, and reaches a stationary phase about 4 to 5 days.
Euglena may be cultured under light irradiation (light culture) or non-irradiated (dark culture).

Euglena cells are separated by, for example, centrifugation or simple sedimentation of a culture solution, membrane filtration, or the like.
The dried Euglena alga body is prepared by washing the separated Euglena cells and then freeze-drying them in a known manner. However, adjustment of the dried alga body may be performed by spray drying, heating vacuum drying, or the like.
The adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent of this embodiment were separated from the culture solution as Euglena by centrifugation, sedimentation, membrane filtration, etc. Euglena alga bodies may be used, or dried alga bodies may be used. Moreover, you may use the dry powder of Euglena which grind | pulverized the algal body dried material with the well-known grinder.

<Euglena aqueous solvent extract>
In the present embodiment, “Euglena aqueous solvent extract” means an extract extracted from Euglena using an aqueous solvent, and in particular, water is used as an aqueous solvent at 5 ° C. to 600 ° C. for several seconds to several times. It is preferable to use an Euglena water extract or hot water extract extracted for 10 hours.
The water used for extraction does not necessarily need to be distilled water, pure water, or ultrapure water. For example, it may contain tap water or impurities, but it contains components that hinder the extraction of active ingredients. No water is preferred.

In the present embodiment, the “water extract” means an extract with water of 0 to 50 ° C. (excluding 0 ° C.).
Here, “water” means water at 0 to 50 ° C. (excluding 0 ° C.).
The temperature of water is not particularly limited as long as it is within a range in which the active ingredient can be sufficiently extracted without affecting the active ingredient, but preferably 1 to 40 ° C, more preferably 5 to 35 ° C, Especially preferably, it is 10-30 degreeC.

In the present embodiment, the “hot water extract” means an extract with water having a temperature higher than 50 ° C., and can also be referred to as a “hot water extract”.
Here, “hot water” means water having a temperature higher than 50 ° C., and is a concept including “hot water”, and includes water in a boiling state. Moreover, it is not limited to hot water in a liquid state, and includes hot water in a gas state and a supercritical state.
The temperature of the hot water is not particularly limited as long as it is within a range in which the active ingredient can be sufficiently extracted without affecting the active ingredient, but is preferably higher than 50 ° C. and 120 ° C. or lower, more preferably 50 Higher than 100 ° C. and lower than 100 ° C.

  The pH of water used for extraction is not particularly limited as long as the active ingredient can be sufficiently extracted without affecting the active ingredient, but is preferably 4 to 10, more preferably 5 to 9. Particularly preferably, the pH is 6-8.

  In this embodiment, water is used alone as the aqueous solvent, but the active ingredient can be sufficiently extracted without affecting the active ingredient, and usually one kind of solvent that can be used for extraction is used. Or you may select and use 2 or more types. For example, water, alcohols, glycols and the like can be mentioned, but are not limited thereto. Examples of alcohols include ethanol, methanol, n-propanol, and isopropanol. Examples of glycols include butylene glycol and propylene glycol. Other aqueous solvents include acetone. These solvents may be used alone or as an aqueous solution, and may be used as any two or three or more mixed solvents.

The temperature of the aqueous solvent used for extraction is, for example, 0 ° C. or higher, and is not particularly limited as long as it does not affect the active ingredient. Although an aqueous solvent in a boiling state or a supercritical state can be used, it is preferable to use an aqueous solvent at 5 ° C to 600 ° C, and more preferable to use an aqueous solvent at 10 ° C to 200 ° C.
Therefore, the aqueous solvent for extraction includes an aqueous solvent in a boiling state or a supercritical state. The amount of the aqueous solvent used for the extraction is preferably an amount that can sufficiently dissolve the water-soluble active ingredient contained in Euglena.

The extraction method is also not particularly limited. For example, extraction can be performed by the following method, but the extraction method is not limited to this, and a normal extraction method can be freely selected and used. For example, a method of centrifuging or filtering Euglena alga body dry powder in an aqueous solvent for a predetermined time and then centrifuging or filtering; Euglena alga body dry powder added to an aqueous solvent and shaken to uniformly disperse, and then centrifuged or filtered. Method, etc.
It is also possible to heat the aqueous solvent after the addition of Euglena to facilitate extraction.

Euglena water extraction can be carried out by a conventional method as shown below, but is not limited thereto. For example, the Euglena tissue and water are placed in a container and allowed to stand for a predetermined time with appropriate stirring or shaking, and the resulting extract can be used as a water extract as it is. In addition, for example, a supernatant obtained by centrifuging such an extract can be used as a water extract. Further, such an extract or supernatant can be concentrated and dried to remove water, and this can be used as a water extract. Water extraction may be performed by adding a small amount of alcohol, for example, 10% by mass or less, preferably ethanol, to water in order to increase extraction efficiency and shorten extraction time.
The extraction time in the case of performing water extraction is not particularly limited as long as it is a time during which the active ingredient is extracted, and can be appropriately set in the range of several seconds to several tens of hours according to the extraction temperature.

Extraction with hot water can be performed by a commonly used method as shown below, but is not limited thereto. Euglena is extracted by introducing it with water into a commonly used extractor and then heating. When extracting using boiling water or water in a supercritical state, it is necessary to use an extractor that can withstand the vapor pressure of water. The pressure at the time of extraction can be set to 1 to 5000 atmospheres, and is preferably set to 60 to 400 atmospheres.
When performing extraction under high temperature and high pressure, if the extraction time is too long, the active component may be decomposed or a chemical reaction may occur. Therefore, when extraction is performed under high temperature and high pressure, the extraction time is preferably short, for example, within 3 minutes, more preferably within 1 minute, and particularly preferably within 30 seconds.

The extracted Euglena extract can be used as an active ingredient of the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and metabolic syndrome preventive or therapeutic agent according to this embodiment as it is. However, the extract can be further separated into a fraction having a high activity by an appropriate separation means (for example, partition extraction, gel filtration, silica gel chromatography, reverse phase or normal phase high performance liquid chromatography, etc.). It is also possible to use it.
Further, the Euglena extract or a fraction thereof can be concentrated and dried to remove the aqueous solvent, and this can be used as the aqueous solvent extract.

<Adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor>
The adipocyte differentiation inhibitor, the PPARγ expression inhibitor, and the C / EBPα expression inhibitor according to the present embodiment include a Euglena aqueous solvent extract as an active ingredient.
The adipocyte differentiation inhibitor according to this embodiment suppresses the expression of PPARγ and C / EBPα, which are master regulators of adipocyte differentiation, and suppresses differentiation from preadipocytes to adipocytes in the adipocyte differentiation process. It is.

<Anti-obesity agent / Anti-metabolic syndrome agent>
The anti-obesity agent and anti-metabolic syndrome agent according to the present embodiment contain Euglena aqueous solvent extract as an active ingredient.
Euglena aqueous solvent extract suppresses the expression of PPARγ and C / EBPα, which are master regulators of adipocyte differentiation, and suppresses the differentiation of preadipocytes into adipocytes, thereby suppressing the accumulation of adipocytes, As a result, it exerts preventive or therapeutic effects on anti-obesity and metabolic syndrome.

<Application>
An adipocyte differentiation inhibitor, a PPARγ expression inhibitor, a C / EBPα expression inhibitor, an anti-obesity agent, and an anti-metabolic syndrome agent comprising the Euglena aqueous solvent extract according to the embodiment as an active ingredient are obesity and / or metabolic syndrome It is administered to patients who have received a definite diagnosis.
In addition, the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent of the present embodiment are configured as a pharmaceutical composition, health food, etc. Or those who have developed symptoms of metabolic syndrome, those who have a tendency to increase BMI, those who are likely to suffer from obesity and / or metabolic syndrome genetically or in consideration of the living environment, etc. And prophylactic administration to those with metabolic syndrome.

  Euglena aqueous solvent extract can be ingested as a food and has no side effects, so it can be administered even before a definitive diagnosis of obesity and / or metabolic syndrome. In addition, any time after receiving a definitive diagnosis, for example, when the BMI value decreases to less than 25, when the differentiation of adipocytes seems to become inactive, or other anti-obesity Administration can be continued until the time of switching to the agent.

  In addition, since a person who has completed treatment for obesity and / or metabolic syndrome is likely to relapse obesity and / or metabolic syndrome, the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor of this embodiment Agents, anti-obesity agents, and anti-metabolic syndrome agents may be symptomatic for patients with obesity and / or metabolic syndrome patients who have a BMI value below 25 after treatment. For the purpose of suppressing recurrence, it can be continuously administered as a preventive or inhibitory agent for obesity and / or metabolic syndrome.

In general, obesity and / or metabolic syndrome is known to be caused by various factors such as stress, diet, and genetic factors.
Therefore, the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent of the present embodiment are those who are susceptible to psychological and social stress, For example, it can be administered for a long period of time to a person who is prone to psychogenic stress, a person in a living environment, or a person preparing for a test.

  Further, the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent of the present embodiment may be obesity and / or metabolic depending on diet and genetic factors. Long term for people in environments that are likely to suffer from syndromes, such as those in households with disturbed eating habits, family members such as parents who have obesity and / or metabolic syndrome Can be administered continuously for a period of time.

  Furthermore, the subject to which an adipocyte differentiation inhibitor, a PPARγ expression inhibitor, a C / EBPα expression inhibitor, an antiobesity agent, and an antimetabolic syndrome agent containing the Euglena aqueous solvent extract according to the present embodiment as an active ingredient is It is not limited to persons with the above symptoms or conditions or animals other than humans.

For example, an adipocyte differentiation inhibitor, PPARγ expression inhibitor containing an Euglena aqueous solvent extract as an active ingredient in an obese human with BMI of 25 or more among humans in infancy and puberty, in which white adipocytes are particularly increased A C / EBPα expression inhibitor, an anti-obesity agent, and an anti-metabolic syndrome agent can be administered or ingested.
Although human adolescents and adolescents are prone to increase white adipocytes, white adipocytes are provided by the adipocyte differentiation inhibitory effect, PPARγ expression inhibitory effect, and C / EBPα expression inhibitory effect provided by the Euglena aqueous solvent extract. Can be suppressed.

Here, “infanthood” is a period of less than one year from the age of birth, and “infanthood” is a period of one to six years of age.
The term “puberty” refers to the period from when secondary sexual characteristics begin, to maturity, until height development finally stops. For men, it is generally between 12 and 17 years old, but for women, according to the definition of the Japan Obstetrics and Gynecology Association, “beginning with the development of sexual function (breast development, pubic growth, etc.) The period until the completion of the secondary sexual characteristics and the menstrual cycle are almost smooth. In the case of current Japanese, the average is between 8 and 9 years old and 17 and 18 years old. " According to WHO (World Health Organization), it is a concept having a range depending on gender and individual differences as defined as 10 to 19 years old, but it is a period of 8 to 19 years old.

In addition, an adipocyte differentiation inhibitor, a PPARγ expression inhibitor, a C / EBPα expression inhibitor, an anti-obesity agent, and an anti-metabolic syndrome agent containing Euglena aqueous solvent extract as an active ingredient are administered to humans after the age of 40 can do.
Humans over the age of 40 are losing their ability to generate heat by breaking down excess fat, making it easier to accumulate fat and make middle-aged fat, but the Euglena aqueous solvent extract contains fat. Accumulation of fat cells and middle-aged fat can be suppressed by a cell differentiation inhibitory effect, a PPARγ expression inhibitory effect, and a C / EBPα expression inhibitory effect.

  In addition, an adipocyte differentiation inhibitor, a PPARγ expression inhibitor, a C / EBPα expression inhibitor, an anti-obesity agent, and an anti-metabolic syndrome agent containing the Euglena aqueous solvent extract according to this embodiment as an active ingredient are pharmacologically It can be used as a composition such as a pharmaceutical composition, a cosmetic composition, and a food composition by adding an acceptable additive.

(Pharmaceutical composition)
The adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent comprising the Euglena aqueous solvent extract according to this embodiment as an active ingredient are excellent adipocyte differentiation. The inhibitory effect, the PPARγ expression inhibitory effect, the C / EBPα expression inhibitory effect, the anti-obesity effect and the anti-metabolic syndrome effect can be used suitably for the pharmaceutical composition.
The pharmaceutical composition can be applied to drugs of all dosage forms or quasi drugs. For example, powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, pills, and other oral preparations, external liquids, external gels, creams, ointments, It can be applied to external preparations such as sprays, nasal drops, liniments, lotions, haps, plasters, sprays, aerosols, or injections.

In the pharmaceutical composition according to this embodiment, one or more pharmaceutically acceptable additives can be freely selected and contained.
For example, when the pharmaceutical composition according to the present embodiment is applied to an oral preparation, for example, an excipient, a binder, a disintegrant, a surfactant, a preservative, a coloring agent, a corrigent, a fragrance, a stabilizer, an antiseptic, for example. All additives that can be usually used in the field of pharmaceutical preparations, such as agents and antioxidants, can be contained. In addition, a sustained-release preparation can be obtained using a drug delivery system (DDS).

  In addition, when applying the pharmaceutical composition according to the present embodiment to an external preparation, a base, a surfactant, a preservative, an emulsifier, a colorant, a flavoring agent, a fragrance, a stabilizer, an antiseptic, an antioxidant, an abundance All additives that can be usually used in the field of pharmaceutical preparations, such as an agent, a solubilizing agent, and a suspending agent can be contained.

  Further, when the pharmaceutical composition according to the present embodiment is applied to an injection, for example, a solvent, a stabilizer, a solubilizing agent, a suspending agent, a preservative, an isotonic agent, an antiseptic, an antioxidant, etc. In addition, all additives which can be usually used in the field of pharmaceutical preparations can be contained.

  The adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent according to this embodiment are administered orally, transdermally, intradermally, subcutaneously, intramuscularly The effect is exerted systemically or locally by administration, intravenous administration, or the like, or the effect is exhibited locally at the administration site.

  In the pharmaceutical composition according to the present embodiment, the contents of the adipocyte differentiation inhibitor, the PPARγ expression inhibitor, the C / EBPα expression inhibitor, the antiobesity agent, and the antimetabolic syndrome agent are not particularly limited, depending on the purpose. It is possible to set freely.

(Food composition)
The adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent comprising the Euglena aqueous solvent extract according to this embodiment as an active ingredient are excellent adipocyte differentiation. It is possible to provide a food composition having such an action by blending it into various foods by utilizing the inhibitory effect, the PPARγ expression inhibitory effect, the C / EBPα expression inhibitory effect, the anti-obesity effect and the metabolic syndrome prevention or treatment effect. it can.

  For example, beverages (soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutrition drinks, etc.), seasonings such as soy sauce, soups, creams, various dairy products, processed meat products, agricultural products Processed products, frozen desserts such as ice cream, various powdered foods (including beverages), concentrated beverages, foods for preservation, frozen foods, breads, cereals, confectionery (candy), cookies, biscuits, gum, gummi , Chocolate, etc.), and other food compositions.

Alternatively, it can also be used for health functional foods (specific health functional foods, nutritional functional foods, functional indication foods), so-called health foods (including beverages), concentrated nutrients, liquid foods, infant / infant foods.
Here, food for specified health use is a food containing health functional ingredients that affect physiological functions, etc., and can be used to indicate that it is suitable for specific health use with the permission of the Commissioner of the Consumer Affairs Agency. is there. In the present embodiment, as a specific health use, it is a food that is sold with the label “Make it hard to get fat”, prevention and improvement of fat accumulation, suppression of adipocyte proliferation, prevention and improvement of obesity, etc. .
The nutritional functional food is a food used for supplementing nutritional components (vitamins and minerals) and displays the function of the nutritional components. In order to sell as a functional nutritional food, the amount of nutrients contained in the daily intake standard amount must be within the specified upper and lower limits. You also need to.
In addition, the functional labeling food is a food that displays the functionality based on the scientific basis at the responsibility of the operator. Information on safety and functionality grounds was reported to the Commissioner of the Consumer Affairs Agency before sales.

In the above, the present embodiment includes a Euglena aqueous solvent extract as an active ingredient, a specific health food for preventing fat accumulation / a specific health food for inhibiting adipocyte differentiation, and a nutrition for preventing fat accumulation for obese patients. It can be used as a functional food / nutrient functional food for inhibiting adipocyte differentiation, a functional display food for preventing fat accumulation / a functional display food for inhibiting fat cell differentiation.
In addition, the present embodiment includes a Euglena aqueous solvent extract as an active ingredient, for fat accumulation prevention intended for living bodies, for example, humans before fat accumulation, humans of the metabolic syndrome reserve arm caused by fat accumulation Food for specified health use / Food for specified health use for prevention of fat cell differentiation, Food for nutrition function for preventing fat accumulation / Function food for nutrition function for preventing fat cell differentiation, Function food for preventing fat accumulation / Function display for preventing fat cell differentiation It can be used as food.

  In addition to the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent according to this embodiment, the food composition according to this embodiment is usually a food composition. It is possible to freely select one or more components that can be used in the preparation. For example, all additives that can be usually used in the food field, such as various seasonings, preservatives, emulsifiers, stabilizers, fragrances, colorants, preservatives, and pH adjusters can be contained.

  The content of the adipocyte differentiation inhibitor, the PPARγ expression inhibitor, the C / EBPα expression inhibitor, the anti-obesity agent, and the anti-metabolic syndrome agent in the food composition is not particularly limited and can be freely set according to the purpose. Is possible.

(Cosmetic composition)
The adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent comprising the Euglena aqueous solvent extract according to this embodiment as an active ingredient are excellent adipocyte differentiation. The inhibitory effect, the PPARγ expression inhibitory effect, the C / EBPα expression inhibitory effect, the anti-obesity effect and the metabolic syndrome prevention or improvement effect can be preferably used for the cosmetic composition.
The cosmetic composition can be applied to any form of cosmetic. For example, it can be applied to skin care cosmetics such as lotions, milky lotions, creams, and cosmetics, foundations, concealers, makeup bases, makeup cosmetics such as lipsticks, blushers, eye shadows, and eyeliners, sunscreen cosmetics, etc. .

In addition to the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent according to this embodiment, the cosmetic composition according to this embodiment is usually a cosmetic. One or two or more components that can be used in the composition can be freely selected and blended.
For example, base materials, preservatives, emulsifiers, colorants, preservatives, surfactants, UV absorbers, antioxidants, moisturizers, UV absorbers, perfumes, antiseptics, extenders, colored pigments, alcohol, All additives usually used in the cosmetic field, such as water, can be included.

  In the cosmetic composition according to the present embodiment, the contents of the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent are not particularly limited, depending on the purpose Can be set freely.

<Method for producing adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent>
The adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent of this embodiment are produced by the following method.
First, a dispersion preparation step is performed in which an aqueous solvent is added to Euglena and stirred and / or shaken to obtain a dispersion. In this dispersion preparation step, Euglena powder may be used as Euglena.
Subsequently, the dispersion liquid is centrifuged, and an extraction step is performed to obtain a supernatant as an aqueous solvent extract of Euglena.

In addition, after the dispersion preparation step and before the extraction step, the heating step may be performed by heating the dispersion using heating means such as a high-pressure steam sterilizer.
Specifically, first, a dispersion preparation step is performed in which an aqueous solvent is added to Euglena and stirred and / or shaken to obtain a dispersion.
Next, a heating step is performed in which the dispersion is heated using a heating means and is heated and extracted.
Furthermore, the dispersion liquid is centrifuged, and an extraction step is performed in which the supernatant is obtained as an aqueous solvent extract of Euglena.

  In the heating step, the set temperature of the heating means is room temperature or higher, for example, 20 ° C or higher and 50 ° C or lower, 50 ° C or higher and 80 ° C or lower, 80 ° C or higher and 100 ° C or lower, 100 ° C or higher and 120 ° C or lower, 120 ° C or higher and 150 ° C or lower, It can be set to 150 ° C. or higher and 200 ° C. or lower.

In the heating step, when the Euglena aqueous solvent dispersion is placed in a sealed container, the set temperature of the heating means matches the temperature of the Euglena aqueous solvent dispersion.
In the heating step, when the Euglena aqueous solvent dispersion is placed in an open container, the temperature of the Euglena aqueous solvent dispersion is determined according to the atmospheric pressure around the open container. It becomes about 100 ° C.

  In the heating step, when the Euglena aqueous solvent dispersion is placed in an open container, the heating step and the Euglena aqueous solvent extract concentration step are performed simultaneously.

In addition, instead of centrifuging the dispersion, an aqueous solvent extract of Euglena may be obtained by a general separation method. For example, an aqueous solvent extract of Euglena may be obtained from the filtrate obtained by filtering the dispersion.
Furthermore, a dispersion obtained by adding an aqueous solvent and shaking without performing centrifugation or filtration can be directly used as an Euglena aqueous solvent extract.

The obtained Euglena aqueous solvent extract is further fractionated to a highly active fraction by an appropriate separation means (for example, partition extraction, gel filtration, silica gel chromatography, reversed phase or normal phase high performance liquid chromatography, etc.). A fractionation step obtained in this manner may be performed.
Further, a concentration step of concentrating the obtained Euglena aqueous solvent extract or fraction and / or a drying step of evaporating and drying the aqueous solvent may be performed.

  The aqueous solvent extract of Euglena obtained as described above is used as the adipocyte differentiation inhibitor, PPARγ expression inhibitor, C / EBPα expression inhibitor, anti-obesity agent, and anti-metabolic syndrome agent of this embodiment.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on a specific Example, this invention is not limited to these.
In the following test examples, Euglena hot water extract (Example 1), Euglena water extract or heat extracted at room temperature and different temperatures (25 ° C, 50 ° C, 75 ° C, 95 ° C, 120 ° C). By examining the effect of the water extract (Examples 2 to 6) on differentiation into adipocytes using human subcutaneous adipose-derived stem cells, a method for extracting Euglena that has a higher effect of inhibiting differentiation into adipocytes was examined. It was.
Moreover, the mechanism of adipocyte differentiation suppression was examined by confirming the influence of the Euglena hot water extract on the expression levels of the PPARγ gene and C / EBPα gene, which are master regulators of adipocyte differentiation.

<Example 1>
Euglena powder (Euglena gracilis, EU-1593, manufactured by Euglena Co., Ltd.) 0.5 g was added with 20 ml of ultrapure water, shaken and dispersed uniformly, and then heated and extracted using a dry heat sterilizer (95 ° C. 2 hours). The supernatant obtained by centrifugation (4000 rpm, 3 minutes, 25 ° C.) is collected and filtered through a 0.45 μm sterilizing filter to prepare a Euglena hot water extract (Euglena hot water extract, stock solution). did. The extract was used as an adipocyte differentiation inhibitor.

<Example 2>
20 g of ultrapure water was added to 0.5 g of Euglena powder (Euglena gracilis, EU-1593, manufactured by Euglena Co., Ltd.), shaken and dispersed uniformly, and then extracted at room temperature (25 ° C.) for 2 hours. The supernatant obtained by centrifugation (4000 rpm, 3 minutes, 25 ° C.) was collected and filtered through a 0.45 μm sterilizing filter to prepare a Euglena water extract (Euglena water extract, stock solution). The extract was used as an adipocyte differentiation inhibitor.

<Examples 3 to 5>
Euglena powder (Euglena Gracilis, EU-1593, manufactured by Euglena Co., Ltd.) 0.5 g, added with 20 ml of ultrapure water, shaken and dispersed uniformly, and then each temperature (implemented) using a dry heat sterilizer Example 3: 50 ° C, Example 4: 75 ° C, Example 5: 95 ° C) for 2 hours. The supernatant obtained by centrifugation (4000 rpm, 3 minutes, 25 ° C.) is collected and filtered through a 0.45 μm sterilizing filter to prepare a Euglena hot water extract (Euglena hot water extract, stock solution). did. The extract was used as an adipocyte differentiation inhibitor.

<Example 6>
Euglena powder (Euglena gracilis, EU-1593, manufactured by Euglena Co., Ltd.) 0.5 g was added with 20 ml of ultrapure water, shaken and dispersed uniformly, and then heated and extracted using a high-pressure steam sterilizer (120 ° C. 20 minutes). The supernatant obtained by centrifugation (4000 rpm, 3 minutes, 25 ° C.) is collected and filtered through a 0.45 μm sterilizing filter to prepare a Euglena hot water extract (Euglena hot water extract, stock solution). did. The extract was used as an adipocyte differentiation inhibitor.

<Experimental example 1 Euglena hot water extract using adipose-derived stem cells>
(Culture medium)
As a fat-derived stem cell growth medium (growth medium), a medium containing DMEM, αMEM, 1% FBS, ITS-X, 10 ng / mLb-FGF, and hydrocortisone was used.
As an adipocyte differentiation induction medium, a medium containing DMEM, 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM indomethacin, 10 μg / mL insulin, 33 μM biotin was used.
As the adipocyte differentiation maintenance medium, a medium containing DMEM, 10% FBS, 10 μg / mL insulin, 33 μM biotin was used.

(Preparation of cells)
1) 9 ml of growth medium was placed in a 15 ml tube.
2) A frozen vial of human subcutaneous fat-derived stem cells (Lonza, PT-5006; hereinafter referred to as ASC) was taken out from liquid nitrogen.
3) Placed in a 37 ° C warm bath for 1-2 minutes and melted until the ice pieces were as big as a fingertip.
4) Wipe off with alcohol and place in clean bench.
5) The ice pieces were dissolved in the medium in 1) and all were collected in the 15 ml tube of 1).
6) Centrifuged at 1200 rpm for 3 minutes.
7) The supernatant was removed with an aspirator, and 500 μl to 1 ml of growth medium was added to suspend the cells.
8) 10 μl of trypan blue and cell suspension were mixed and placed in a one-cell counter.
9) The number of cells was counted using a microscope.
10) Cells were seeded at 3-5 × 10 ⁵ cells / dish in a culture dish.
11) The cells were cultured for 3 days in a CO ₂ incubator (37 ° C., 5 vol% CO ₂ ).

  Using the human subcutaneous fat-derived stem cells prepared by the above procedure, a fat differentiation inhibition test of Euglena hot water extract was performed by the procedure shown in FIG. The test procedure is specifically shown below.

(3 days before: cell seeding)
1) The cells were confirmed with a microscope.
2) The cell supernatant was removed with an aspirator, and 5 ml of PBS (−) was added.
3) PBS (-) was removed with an aspirator, and 1 ml of trypsin solution (0.25% (w / v) Trypsin-1 mM EDTA (Wako Pure Chemical Industries, Ltd.) was added dropwise.
4) The mixture was allowed to stand for 2 minutes in a CO ₂ incubator (37 ° C., 5 vol% CO ₂ ).
5) The cells were observed with a microscope, and it was confirmed that the cells were round and scattered.
6) 2 ml of 10% FBS and DMEM were added, and the cells were collected in a 15 ml tube.
7) Centrifugation was performed at 1200 rpm for 3 minutes.
8) The supernatant was removed and resuspended in growth medium, and the number of cells was counted with a hemocytometer.
9) It adjusted with the growth medium so that it might become 25000cells / well, and it seed | inoculated 500 microliters at a time on a 24 well plate.
10) and allowed to stand in a CO ₂ incubator.
11) The cells were cultured for 3 days in a CO ₂ incubator (37 ° C., 5 vol% CO ₂ ).

(Day 0: induction of fat differentiation / addition of Euglena extract)
1) The Euglena extract-added medium solution containing a predetermined concentration of Euglena extract was prepared by adding the Euglena extract of each Example to the adipocyte differentiation induction medium.
2) The culture supernatant was removed with an aspirator, and 500 μl of Euglena extract added medium solution prepared in 1) was added.
3) Adipocyte differentiation induction medium was added to the control.
4) Cultured in a CO ₂ incubator (37 ° C., 5 vol% CO ₂ ) for 7 days.
5) The medium was changed once every 2-3 days.

(Day 7: Fat differentiation induction / Euglena extract added)
1) Euglena extract-added medium solution containing a predetermined concentration of Euglena extract was prepared by adding the Euglena extract of each Example to the adipocyte differentiation maintenance medium.
2) The culture supernatant was removed with an aspirator, and 500 μl of Euglena extract added medium solution prepared in 1) was added.
3) Adipocyte differentiation maintenance medium was added to the control.
4) Cultured in a CO ₂ incubator (37 ° C., 5 vol% CO ₂ ) for 7 days.
5) The medium was changed once every 2-3 days.

(Day 14: Cell measurement and cell fixation)
1) The cells were confirmed with a microscope.
2) A necessary amount of CCK-8 solution was prepared at a ratio of DMEM, P / S medium: Cell Counting Kit-8 = 24: 1. Here, Cell Counting Kit-8 is a living cell number measurement kit (manufactured by Dojindo Laboratories) using a water-soluble tetrazolium salt WST-8 as a coloring reagent.
3) The culture supernatant was removed with an aspirator, and 500 μl of the CCK-8 solution prepared in 2) was added.
4) It was left still for 1 hour in a CO ₂ incubator (37 ° C., 5 vol% CO ₂ ).
5) Each supernatant was transferred to a 96-well plate by 100 μ × 2 wells.
6) The absorbance at each wavelength of 450 nm and 650 nm was measured using a microplate reader.
7) The excess supernatant in 5) was removed with an aspirator, and 500 μl of PBS (−) was added.
8) 7) was carried out twice.
9) With the exception of PBS (−), 4% paraformaldehyde (PFA) was added in an amount of 500 μl / well to fix the cells.
10) Shaded with aluminum foil and stored at 4 ° C. for 24 hours or more.
11) Oil red O staining was performed by the following method to quantify the fat accumulation rate.

(Quantification of staining and fat differentiation)
An Oil Red O stock solution was made in a 50 ml tube at a ratio of Oil Red O: isopropanol = 120 mg: 40 ml and stored at room temperature.
The Oil Red O staining solution was mixed at a ratio of Oil Red O stock solution: ultra pure water = 3: 2, filtered, and immediately used for the following cell staining.
1) The fixing solution in the plate was removed.
2) Washed twice with PBS (−) 500 μl / well.
3) A 60% aqueous solution of isopropanol was added at 500 μl / well and allowed to acclimate for about 2 minutes at room temperature.
4) The 60% isopropanol aqueous solution was removed, and Oil Red O staining solution was added at 250 μl / well.
5) It was allowed to stand at room temperature for 15 minutes.
6) Oil Red O staining solution was removed, and a 60% aqueous isopropanol solution was added at 500 μl / well, and the mixture was gently stirred and washed.
7) A 60% aqueous isopropanol solution was removed, PBS (-) was added at 500 μl / well, and a photograph was taken.
8) Excluding PBS (−), 500 μl / well of 100% isopropanol was added to extract Oil Red O.
9) When extraction was completed, 200 μl / well was transferred to a 96-well plate.
10) Absorbance was measured at 520 nm using a microplate reader.
11) The value of absorbance was defined as the amount of accumulated fat. As the fat accumulation amount per unit cell, a value converted by fat accumulation amount (Oil Red O) / number of cells (CCK-8) was used.
12) The fat accumulation rate was calculated with the fat accumulation rate in the control cultured using the adipocyte differentiation induction medium as 100%.

(Result of Test Example 1)
The graph which compared the fat accumulation rate of each density | concentration (5 vol%, 10 vol%, 20 vol%) by an Euglena hot water extract by analyzing the result of having performed the said test using the Euglena hot water extract of Example 1. Is shown in FIG.
When the fat accumulation rate in the control was 100%, the fat accumulation rate at each concentration was 56.4%, 32.5%, and 7.05% in this order.
The fat accumulation rate decreased as the concentration of Euglena hot water extract increased.
These tests were performed a plurality of times, and similar reproducibility was obtained.

  As shown in FIG. 4, it was found that as the concentration of Euglena hot water extract increased, the number of fat droplets (portion stained with Oil Red O) decreased.

<Test Example 2 Euglena water extract extracted at different temperatures>
Instead of the Euglena hot water extract of Example 1, the water extract or hot water extract of Examples 2-6 (Example 2: 25 ° C., Example 3: 50 ° C., Example 4: 75 ° C. (Example 5: 95 ° C., Example 6: 120 ° C.)

(Result of Test Example 2)
The results of tests using the Euglena water extract or hot water extract of Examples 2 to 6 were analyzed, and each concentration (5 vol%, 10 vol%, Eucalena water extract or hot water extract extracted at different temperatures was analyzed. A graph comparing the fat accumulation rate of 20 vol%) is shown in FIG.
The fat accumulation rate in Euglena water extract or hot water extract at each temperature decreased as the concentration of Euglena water extract or hot water extract increased.
These tests were performed a plurality of times, and similar reproducibility was obtained.

  On the other hand, even when the extraction temperature was increased, the fat accumulation rate did not decrease greatly. From the above results, it was found that the concentration of the extract affects the decrease in the fat accumulation rate rather than the extraction temperature. This indicates that even a hot water extract having a relatively low temperature of about 50 ° C. is effective in reducing the fat accumulation rate.

  As shown in FIG. 6, in the water extract or hot water extract at each temperature, it was found that the number of fat droplets decreased as the concentration of the Euglena water extract or hot water extract increased.

<Test Example 3 Euglena Hot Water Extract and EPA Fat Differentiation Inhibition Test>
A test similar to Test Example 1 was performed using Euglena hot water extract of Example 1 and eicosapentaneenoic acid (EPA), which is known to have a fat differentiation inhibitory effect as Comparative Example 1.

(Result of Test Example 3)
By analyzing the results of the test using the Euglena hot water extract of Example 1 and the test using the EPA of Comparative Example 1, Euglena hot water extract (10 vol% concentration, 20 vol% concentration) and EPA FIG. 7 shows a graph comparing the fat accumulation rate of (10 μM, 50 μM, 100 μM).
In addition, the addition amount of EPA of this test was set so that it might become 6 times more than the amount of EPA contained in Euglena hot water extraction.
The fat accumulation rate in Comparative Example 1 was 80% or more, which was higher than the fat accumulation rate of the Euglena hot water extract of Example 1.
Further, as shown in FIG. 8, it was found that the number of fat droplets (portion stained with Oil Red O) was slightly decreased as compared with the control.
From this result, it was found that among the components contained in the Euglena hot water extract, active components other than EPA, particularly unidentified active components, have the effect of suppressing adipocyte differentiation.

<Test Example 4 Fat Differentiation Inhibition Test with Different Addition Period of Euglena Hot Water Extract>
A test similar to Test Example 1 was conducted except that the Euglena hot water extract of Example 1 was used and the period during which the Euglena hot water extract was added to the medium was changed.
Specifically, when the first half is from 0 to 7 days in which culture is performed using an adipocyte differentiation-inducing medium, and the second half is from 7 to 14 days in which culture is performed using an adipocyte differentiation maintenance medium, i) When Euglena hot water extract is added in the first half and second half, (ii) Euglena hot water extract is added only in the first half, and (iii) Euglena hot water extract is added only in the second half. The fat accumulation rate was compared.

(Result of Test Example 4)
FIG. 9 shows a graph comparing the fat accumulation rate when the result of Test Example 4 was analyzed and the period for adding the Euglena hot water extract was changed.
It was found that the fat accumulation rate was comparable in the case where the period of addition of the Euglena hot water extract was (i) the first half and the second half and (ii) only the first half. On the other hand, it was found that (iii) the fat accumulation rate was high when the addition period was only the latter half. This result has shown that the fat differentiation inhibitory effect of a Euglena hot water extract is exhibiting the effect in the first half of fat differentiation induction.

  As shown in FIG. 10, when the addition period of the Euglena hot water extract is (i) the first half and the second half, and (ii) only the first half, (iii) the added fat is compared with the case where the addition period is only the second half. I found that the drops were low.

<Test Example 5 Gene Expression Analysis by Real-Time PCR>
In order to examine the expression of the PPARγ gene and C / EBPα gene, which are master regulators of adipocyte differentiation, the same test as in Test Example 1 was performed using the Euglena hot water extract of Example 1. Specifically, Euglena hot water extract was added on day 0 and cultured using a differentiation-inducing medium. On day 1, day 2, day 3, day 6, day 13 of culture. The total RNA was extracted from the cells, and the expression levels of the PPARγ gene and the C / EBPα gene were measured using real-time PCR using cDNA obtained by the reverse transcription reaction.

(Recovery and extraction of RNA)
1) The medium was removed and the plate was washed twice with PBS (−).
2) After removing PBS (−), 0.5 ml of total RNA extraction reagent (RNAiso Plus, manufactured by Takara Bio Inc.) was added and transferred to a 1.5 ml microtube (can be stored at −80 ° C.).
3) 100 μl of chloroform (0.2 times the amount of total RNA extraction reagent) was added, and the mixture was stirred with a vortex mixer for 15 seconds and allowed to stand at room temperature for 5 minutes.
4) Centrifugation was performed at 12000 g and 4 ° C. for 15 minutes.
5) During centrifugation, 500 μl of isopropanol was prepared in a new 1.5 ml microtube.
6) After centrifugation, the transparent layer was transferred to a 1.5 ml microtube, stirred with a vortex mixer for 10 seconds, and allowed to stand at room temperature for 10 minutes.
7) Centrifuged at 12000 g, 4 ° C. for 15 minutes.
8) During centrifugation, a 75% aqueous ethanol solution was made and placed on ice.
9) After centrifugation, the supernatant was removed, 500 μl of 75% ethanol was added, and the mixture was slightly stirred with a vortex mixer.
10) Centrifuged at 7500 g, 4 ° C. for 5 minutes.
11) The supernatant was removed as much as possible without sucking the pellet.
12) Dried for 10-30 minutes at room temperature.
13) 20 μl of sterilized water was added, and the pellet was dissolved by pipetting several times and stored at −20 ° C.

(CDNA synthesis)
1) The concentration was measured with a spectrophotometer (NanoDrop ND-2000C, manufactured by Thermo).
2) 5 × PrimeScript RT Master Mix and 2 μl of Total RNA were added to the microtube, and sterilized water was added to 10 μl and mixed.
3) A sample was placed in a thermal cycler (TP600, manufactured by TaKaRa), held at 37 ° C for 15 minutes, held at 85 ° C for 5 seconds, and cooled to 25 ° C to perform a reverse transcription reaction.
4) Diluted 10-fold by adding 90 μl of sterilized water and stored at −20 ° C.

(Gene expression analysis by real-time PCR)
1) Primer SYBR mix having a capacity of +1 number of wells to be used was prepared by mixing reagents in the following ratio.
PowerUp SYBR Green Master Mix (Applied Biosystems): 5 μl × well number Primer: 0.6 μl × well number Sterile water: 1.4 μl × well number 2) 3 μl of each sample was added to a 96-well plate. Sterile water was added to the negative control (NC).
3) 7 μl each of the primer SYBR mix was added, and the plate was spun down with a plate centrifuge (PS-020, manufactured by TOMY).
4) Real-time PCR was performed using a real-time PCR apparatus (Step One Plus, manufactured by Applied Biosystems).
The following primers were used as primers.
-Primer used for analysis of PPARγ gene expression PPARG-F: 5'-GAACGACCAAGTAACTCTCCTCAAAT-3 '(SEQ ID NO: 1)
PPARG-R: 5′-TCTTTATTCATCAAGGAGGCCAGCATT-3 ′ (SEQ ID NO: 2)
Primer used for expression analysis of C / EBPα gene CEBPA-F: 5′-GGGTCTGAGACTCCCTTTCCTT-3 ′ (SEQ ID NO: 3)
CEBPA-R: 5′-CTCATTGGTCCCCCAGGAT-3 ′ (SEQ ID NO: 4)
As an internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which is a housekeeping gene, was similarly amplified.
-Primer GAPDH-F: 5'-TGCACCACCAACTGCTTAGC-3 '(SEQ ID NO: 5) used for expression analysis of House Keeping Gene (GAPDH) gene
GAPDH-R: 5′-TCTTCTGGGTGGCAGTGATG-3 ′ (SEQ ID NO: 6)

(Result of Test Example 5)
FIG. 11 shows the results of comparing the expression levels of the PPARγ genes on the first day, the second day, the third day, the sixth day, and the thirteenth day when the expression level of the PPARγ gene on the 0th day of culture is 1. .
The expression level of the PPARγ gene on the first day, the second day, the third day, the sixth day, and the thirteenth day is as follows. When the Euglena hot water extract was added at 10 vol% and 20 vol%, the PPARγ gene expression level It was found that the gene expression level was significantly reduced.
It was also found that the expression of the PPARγ gene is suppressed as the concentration of Euglena hot water extract increases.

The results of comparing the expression levels of the C / EBPα gene on the first day, the second day, the third day, the sixth day, and the thirteenth day when the C / EBPα gene expression level on the 0th day of culture is 1. As shown in FIG.
When the amount of hot water extract added is 10 vol% and 20 vol%, the C / EBPα gene expression level is the same as the C / EBPα gene expression level compared to the control cultured in a medium that induces adipocyte differentiation. It was found that there was a significant decrease.
It was also found that the expression of the C / EBPα gene is suppressed as the concentration of Euglena hot water extract increases.

  From the results of Test Example 5, it was shown that the expression of the PPARγ gene and the C / EBPα gene was suppressed, so that differentiation into adipocytes was suppressed, leading to a decrease in the amount of accumulated fat.

Claims (10)

A PPARγ expression inhibitor comprising Euglena water extract or Euglena hot water extract as an active ingredient. A C / EBPα expression inhibitor comprising a Euglena water extract or a Euglena hot water extract as an active ingredient. Contains Euglena water extract or Euglena hot water extract as an active ingredient,
A food composition for inhibiting PPARγ expression, which is used for inhibiting PPARγ expression . Contains Euglena water extract or Euglena hot water extract as an active ingredient,
A food composition for suppressing C / EBPα expression, which is used for suppressing C / EBPα expression. Contains Euglena water extract or Euglena hot water extract as an active ingredient,
A cosmetic composition for inhibiting PPARγ expression, which is used for inhibiting PPARγ expression . Contains Euglena water extract or Euglena hot water extract as an active ingredient,
A cosmetic composition for inhibiting C / EBPα expression, which is used for inhibiting C / EBPα expression. Euglena, a dispersion preparation step of adding water and shaking to obtain a dispersion,
Centrifuged said dispersion supernatant method of PPARγ expression inhibitor which comprises an extraction step of obtaining a Mizu抽 extract of Euglena, a. The method for producing a PPARγ expression inhibitor according to claim 7 , wherein a heating step of heating the dispersion is performed after the dispersion preparation step and before the extraction step. Euglena, a dispersion preparation step of adding water and shaking to obtain a dispersion,
And a step of centrifuging the dispersion to obtain a supernatant as a water extract of Euglena. A method for producing a C / EBPα expression inhibitor, comprising: The method for producing a C / EBPα expression inhibitor according to claim 9, wherein a heating step of heating the dispersion is performed after the dispersion preparation step and before the extraction step.