JP2022000030A - Neuronal differentiation promoter, and neuronal differentiation promoting composition - Google Patents

Abstract

To provide a novel functional food composition.SOLUTION: A functional food composition for preventing the loss of functions or improving functions includes astaxanthin, and at least one sesamin compound selected from sesamin, episesamin, and a mixture of sesamin and episesamin as active ingredients. The functions are at least one selected from: the proper using of a machine or tool in human; and the coordinated action of mind and body in human, or at least one selected from: connecting to a skillful action requiring cognitive function in human; and determining and recognizing matter or information in human and quickly and properly handling it.SELECTED DRAWING: None

Description

The present disclosure relates to a nerve differentiation promoting agent and a composition for promoting nerve differentiation.

Techniques for promoting the differentiation of nerve cells have been proposed.
For example, Patent Document 1 discloses that the aniline derivative represented by the formula (I) has a nerve differentiation promoting action.
Further, in recent years, various functions by components having an antioxidant action such as astaxanthin and sesamin have been attracting attention.
As a technique focusing on the function of astaxanthin, for example, Patent Document 2 discloses a cognitive-behavioral ability improving agent containing astaxanthin and / or an ester thereof as a cognitive-behavioral ability improving agent related to sports suitability. There is.
Further, as a technique focusing on the function of a sesamin-based compound, for example, Patent Document 3 uses a dioxabicyclo [3.3.0] octane derivative such as sesamin, which is a component contained in sesame, as an active ingredient. Anti-fatigue agents and the like are disclosed.

Japanese Unexamined Patent Publication No. 2012-229185 Japanese Unexamined Patent Publication No. 2010-27095 Japanese Unexamined Patent Publication No. 2016-147876

An object to be solved by one embodiment of the present invention is to provide a novel neuronal differentiation promoter.
Further, an object to be solved by another embodiment of the present invention is to provide a composition for promoting nerve differentiation containing the above-mentioned nerve differentiation promoting agent.

Specific means for solving the problem include the following aspects.
<1> A nerve differentiation promoting agent containing astaxanthin and a sesamin compound as active ingredients.
<2> The nerve differentiation promoting agent according to <1> above, wherein the sesamin-based compound is sesamin, episesamin, or a mixture of sesamin and episesamin.
<3> The neuronal differentiation promoting agent according to <1> or <2> above, which is used for the purpose of preventing the deterioration of cognitive function or for improving the cognitive function.
<4> The nerve differentiation promoting agent according to <1> or <2> above, which is used for alleviating a feeling of mental fatigue.
<5> A composition for promoting neuronal differentiation, which comprises the neuronal differentiation promoting agent according to any one of <1> to <4> above and a carrier.
<6> The promotion of neural differentiation according to <6> above, wherein the carrier is a powder at 25 ° C. and contains a powder of a sucrose fatty acid ester having an HLB value of 10 or more and a liquid oil agent at 25 ° C. Composition for.

According to one embodiment of the present invention, a novel neuronal differentiation promoter can be provided.
Further, according to another embodiment of the present invention, it is possible to provide a composition for promoting nerve differentiation containing the above-mentioned nerve differentiation promoting agent.

It is a graph which shows the result obtained in the cell metabolism test of an Example. It is a graph which shows one of the results obtained in the gene expression analysis test of an Example. It is a graph which shows the result of the change amount at the 12th week of ingestion in 2 items of the cognitive function rate and the processing rate obtained in the improvement evaluation of the cognitive decline of an example. It is a graph which shows the result of the change amount of the fatigue feeling from before the start of a load obtained in the relaxation evaluation of the mental fatigue feeling of an Example. It is a graph which shows the result of the change amount of the fatigue feeling after 4 hours of a load obtained in the relaxation evaluation of the mental fatigue feeling of an Example.

Hereinafter, the neural differentiation-promoting agent of the present disclosure and its application mode will be described by exemplifying an embodiment. However, the present disclosure is not limited to the following embodiments, and can be carried out with appropriate modifications within the scope of the object of the present invention.

The numerical range indicated by using "~" in the present disclosure means a range including the numerical values before and after "~" as the minimum value and the maximum value, respectively.
In the numerical range described stepwise in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of another numerical range described stepwise. Further, in the numerical range described in the present disclosure, the upper limit value or the lower limit value described in a certain numerical range may be replaced with the value shown in the examples.
In the present disclosure, the amount of each component means, when a plurality of substances corresponding to each component are present in the composition, the total amount of the plurality of substances present in the composition unless otherwise specified. do.

In the present disclosure, the "HLB (Hydrophile-Lipophile Balance) value" refers to a case where a commercially available product is used as an ingredient and the HLB value of the commercially available product is clearly indicated in a document such as a catalog of the commercially available product. , The values shown in the literature such as catalogs are adopted.
If the component to be used is not a commercially available product, or if the HLB value is not clearly shown in a document such as a catalog even if it is commercially available, the HLB value in the present specification is determined by the calculation formula of Griffin. Adopt the value to be. In the Griffin calculation formula, the HLB value is calculated according to the following formula using the value of S (saponification value of the ester) and the value of N (neutralization value of the fatty acid constituting the ester). The closer the HLB value is to 20, the more hydrophilic it is, and the closer it is to 0, the more lipophilic it is.
HLB value = 20 (1-S / N)

In the present disclosure, the expression "glycerin fatty acid ester" includes all of a glycerin fatty acid ester containing one glycerin unit and one fatty acid unit, a glycerin fatty acid ester containing one or more of them, and a glycerin fatty acid ester containing a plurality of both. And are used when these glycerin fatty acid esters are used without distinction.

In the present disclosure, the term "process" is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes.

In the present disclosure, the term "neurite differentiation" includes differentiation of undifferentiated cells into neurons (ie, neurogenesis) and extension of neural stem cells or neurites from neurons (ie, neurite outgrowth). ..

[Neurodifferentiation promoter]
The neuronal differentiation promoting agent according to the present disclosure contains astaxanthin and a sesamin-based compound as active ingredients. The nerve differentiation promoting agent according to the present disclosure is an agent that promotes nerve differentiation in tissues in the brain.

The neuronal differentiation promoting agent according to the present disclosure is based on the findings of the present inventors who have found that a specific combination of astaxanthin and a sesamin-based compound exerts a remarkably excellent promoting effect on neural differentiation.
The present inventors speculate that the reason why the neurodifferentiation promoting agent of the present disclosure exerts the above-mentioned effects is as follows. However, the following speculation does not limitly interpret the effects exerted by the neurodifferentiation promoting agents of the present disclosure, but is described as an example.

The present inventors correlate both the difference in the action of astaxanthin and the sesamin-based compound in the body and the difference in the absorption into the body with the remarkably excellent promoting effect of the nerve differentiation promoting agent of the present disclosure. I think I'm doing it. Regarding the difference in action, the present inventors have confirmed that astaxanthin is an element that can contribute to cell activation. On the other hand, regarding sesamin compounds, the present inventors have found that they are elements that contribute to neural differentiation in the brain. Furthermore, regarding the difference in absorbability, the present inventors have found that sesamin-based compounds have a faster absorption rate into the body than astaxanthin, while astaxanthin has a slower absorption peak than sesamin-based compounds. I have confirmed that. In addition, the present inventors have confirmed that astaxanthin does not have an action of inducing nerve differentiation, such as causing neurites in undifferentiated nerve cells.

By combining astaxanthin and a sesamin-based compound having the above differences, the neurodifferentiation-promoting agent of the present disclosure taken into the body undergoes the following steps 1, 2, and 3 to achieve an excellent promoting effect on neuronal differentiation. It is presumed that it will be exerted.
Step 1: Sesamin compounds are absorbed by cells in the brain and begin to act on the neural differentiation of cells.
Step 2: Astaxanthin is absorbed by the cells in the state of step 1 above by the sesamin compound, and the activity of the cells (that is, intracellular metabolism) is enhanced by the action of astaxanthin. The activation of cells can be confirmed by measuring the amounts of NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) involved in the intracellular energy production and metabolic pathways. Cell activation can also be confirmed by using colorimetric analysis to confirm the presence or absence of increased metabolism of the cells.
Step 3: In step 2, the action of astaxanthin enhances the activity of the cell, thereby promoting the neural differentiation of the cell by the sesamin-based compound.

As will be described later, the nerve differentiation promoting agent of the present disclosure is for promoting nerve differentiation in a specific embodiment including a sucrose fatty acid ester which is powder at 25 ° C. and has an HLB value of 10 or more and an oil agent which is liquid at 25 ° C. When used as a composition, the absorption of astaxanthin in the body can be further enhanced. Therefore, in the composition for promoting neural differentiation in this embodiment, astaxanthin is absorbed by the cells at a higher concentration in the above-mentioned step 2, the activation of the cells is further enhanced, and the neural differentiation in the stage 3 is further promoted. It is thought that it will be done.

Such an effect of the nerve differentiation promoting agent of the present disclosure cannot be obtained by an agent using astaxanthin or a sesamin compound alone, and the nerve differentiation promoting agent of the present disclosure containing astaxanthin and a sesamin compound as an active ingredient can be used. It is a unique synergistic effect. For example, known documents such as Patent Document 1 (Japanese Unexamined Patent Publication No. 2012-229185) described above do not pay attention to the neuronal differentiation promoting action by the combination of astaxanthin and the sesamin compound.

The above-mentioned synergistic effect obtained by the above-mentioned neuronal differentiation-promoting agent of the present disclosure is not particularly limited as long as the above-mentioned method can confirm the above-mentioned neuronal differentiation-promoting effect, but changes in gene expression or cell metabolism, etc. It can be confirmed by changes in proliferation and differentiation. As an example of a specific confirmation method, the following method can be mentioned.
That is, the method illustrated in (1) or (2) below is applied to untreated neurons, astaxanthin alone, sesamin compounds alone, or neurons treated with astaxanthin and sesamin compounds in combination. Can be confirmed by.
(1) RNA is extracted from the cell. The obtained RNA is analyzed by comprehensive gene expression analysis or PCR (Polymerase Chain Reaction) method, and the expression of genes in which fluctuations are observed only with astaxanthin and sesamin compounds is confirmed.
(2) Confirm changes in metabolism, proliferation, differentiation, etc. of the cells.

When the above method (1) or (2) is adopted in the confirmation of the neuronal differentiation promoting effect of the present disclosure, it is preferable to perform both the methods (1) and (2).

As the gene whose expression is confirmed by the method (1) above, a gene whose expression has been confirmed to be associated with neural differentiation is selected. Preferable examples of the above genes include at least one gene selected from the group consisting of PAX6 gene, RAB13 gene, PTBP2 gene, CREBBP gene, PPMA1 gene, MEF2A gene, and CREB3L2 gene.

In the present disclosure, as a method for confirming the expression of a gene related to promotion of neural differentiation, at least selected from the group consisting of PAX6 gene, RAB13 gene, PTBP2 gene, CREBBP gene, PPMA1 gene, MEF2A gene, and CREB3L2 gene. A method for confirming the expression of one gene is used.

Regarding PAX6, RAB13, PTBP2, CREBBP, PPMA1, MEF2A, and CREB3L2, contributions to the following matters have been confirmed regarding neural differentiation. Therefore, the fact that the expression of the above genes is confirmed as a result of treating nerve cells with the nerve differentiation promoter of the present disclosure indicates that the nerve differentiation promoter of the present disclosure contributes to or is involved in the promotion of nerve differentiation. Suggests.
PAX6: Neural stem cell proliferation and differentiation.
RAB13: Elongation of neurites.
PTBP2: RNA splicing regulator. It is deeply involved in the brain formation process during development.
CREBBP: Neurogenesis.
PPMA1: Nerve cell differentiation and survival.
MEF2A: Development and differentiation of nerve cells and regulation of synaptic function in mature nerve cells.

Examples of the method (2) above include a method for confirming the promotion of metabolism of nerve cells. In the present disclosure, the MTT test is used as a method for confirming the promotion of metabolism of nerve cells. The MTT test is a known colorimetric test using MTT (3- (4,5-di-methylthiazol-2-yl) -2,5-diphenyltetrazolium bromide).

Here, the present disclosure comprises at least one gene selected from the group consisting of PAX6 gene, RAB13 gene, PTBP2 gene, CREBBP gene, PPMA1 gene, MEF2A gene, and CREB3L2 gene, including astaxanthin and sesamine-based compounds. Includes a gene expression-promoting agent that promotes expression (hereinafter, also simply referred to as "gene expression-promoting agent").
The present disclosure also includes a nerve cell metabolism-promoting agent (hereinafter, also simply referred to as "metabolism-promoting agent") containing astaxanthin and a sesamin-based compound.
Details of the types, content ratios, etc. of astaxanthin and sesamin compounds contained in the gene expression promoter or metabolism promoter of the present disclosure are the same as those of the matters relating to astaxanthin and sesamin compounds contained in the neurodifferentiation promoter of the present disclosure. The specific contents will be described later.

The subject to which the neurodifferentiation promoting agent of the present disclosure is ingested may be a human or a non-human animal or cell, but is preferably a human.

The neurodifferentiation promoting agent of the present disclosure may be used for medical use or may be used for non-medical use.

The effect of promoting neural differentiation by the neural differentiation promoting agent of the present disclosure contributes to prevention of cognitive decline or improvement of cognitive function. Therefore, preferred application embodiments of the neurodifferentiation-promoting agents of the present disclosure include embodiments used for preventing cognitive decline or improving cognitive function.
Cognitive function is a concept that collectively refers to human intellectual function, and includes understanding, judgment, memory, attention, perception, learning, language, and the like.

In the present disclosure, "preventing a decrease in cognitive function" means to suppress a decrease in cognitive function level in an ingestor who does not have a decrease in cognitive function level due to ingestion of the neurodifferentiation promoting agent of the present disclosure. means.
In the present disclosure, "improving cognitive function" means "improving cognitive function" before ingestion by ingestion of the neuronal differentiation promoter of the present disclosure in an ingestor in which a decrease in cognitive function level is observed by ingestion of the neuronal differentiation promoter of the present disclosure. Ingestors who improve cognitive function levels, maintain cognitive function levels, delay the progression of cognitive function level decline, or do not observe cognitive function level decline. It means that the level of cognitive function is further improved compared to before ingestion.

In addition, for the purpose of preventing cognitive decline or improving cognitive function, when the neurodifferentiation promoter of the present disclosure is applied to so-called functional foods, for example, "People who are concerned about cognitive decline". "For those who are worried about poor memory", "For those who are worried about poor comprehension", "For those who are worried about poor judgment", "For those who are worried about poor responsiveness" For those who are concerned about the decline in processing capacity, for those who are concerned about the decline in cognitive ability, for the prevention of cognitive decline, for the prevention of comprehension decline, and for judgment. It can be indicated by indications such as "to prevent the decline of responsiveness", "to prevent the decline of responsiveness", "to prevent the decline of processing capacity", and "to prevent the decline of cognitive ability".

Ingestion of the neuronal differentiation promoting agent of the present disclosure promotes the neuronal differentiation action in the brain through the above-mentioned steps 1, 2 and 3, thereby causing step 4: prevention of deterioration of cognitive function of the brain or prevention of cognitive decline in the brain. It is thought that cognitive function will be improved.

When the neurodifferentiation promoting agent of the present disclosure is used for the purpose of preventing deterioration of cognitive function or improving cognitive function, the subject to be ingested may be a healthy person or a person with cognitive impairment. The neurodifferentiation promoters of the present disclosure can act more effectively when ingested by persons with mild cognitive impairment (MCI).

The present disclosure also includes a method for improving cognitive decline by using a neuronal differentiation promoting agent (the neuronal differentiation promoting agent of the present disclosure) containing astaxanthin and a sesamin-based compound as active ingredients.

When the neurodifferentiation promoting agent of the present disclosure is applied to the use of suppressing or ameliorating cognitive decline, the effect of suppressing or ameliorating cognitive decline is as a cognitive function test, Cognitrax cognitive function test (hereinafter referred to as “Cognitrax”). , Simply referred to as Cognitrax). Cognitrax is a cognitive function test based on the CNS Vital Signs test developed by CNS Vital Signs of the United States. In this disclosure, the cognitive function test evaluated using "Cognitrax Basic" provided by Health Solutions Co., Ltd. was used as an index.

Brain functions include simple functions such as language memory, visual memory, and learning, as well as higher-order functions as shown in the following examples.
-The ability to accurately understand and respond to recognized or sensed information, and then connect it to dexterous exercises that require cognitive functions such as driving and playing musical instruments.
-The ability to judge and recognize things or information and process them quickly and accurately.
• Attention to simple or complex things.
・ Flexible thinking ability for things.
・ Decision-making ability to judge the situation.

Among the above examples, in particular, "the ability to accurately understand and respond to recognized or sensed information and connect it to dexterous movements that require cognitive functions such as driving and playing musical instruments" and "things or "The ability to judge and recognize information and process it quickly and accurately" can be said to be higher-order cognitive functions, unlike simple memory or learning ability. The neuronal differentiation promoting agent of the present disclosure has an excellent effect in preventing or improving such a decrease in higher cognitive function.
In other words, when the neurodifferentiation promoter of the present disclosure is used for the purpose of preventing or improving the deterioration of cognitive function, the cognitive function is a group consisting of comprehension, judgment, responsiveness and processing ability necessary for dexterous movement. Includes at least one ability more selected.

Cognitrax consists of seven tests: "Language Memory Test", "Visual Memory Test", "Finger Tap Test", "Symbol Digit Coding (SDC) Test", "Stroop Test", "Attention Shift Test" and "Continuous Processing Test". From the results of, "Comprehensive memory", "Language memory", "Visual memory", "Cognitive function speed", "Reaction time", "Comprehensive attention", "Cognitive flexibility", "Processing speed", "Execution function" This is an inspection test that evaluates 11 items of "simple attention" and "exercise speed".
Of the above items, "cognitive function speed" refers to "the ability to accurately understand and respond to recognized or sensed information and to connect it to dexterous movements that require cognitive functions such as driving or playing musical instruments.""Processingspeed" is an item that can be simply evaluated as "the ability to judge and recognize things or information and process things quickly and accurately".
Here, since the cognitive function speed is related to driving, playing a musical instrument, etc., it is considered that the expressive power can be evaluated secondarily.

By using such a multifaceted and advanced test, the ingestion of the neurodifferentiation promoter of the present disclosure correlates with the comprehension, judgment, responsiveness, and processing ability required for dexterous movements. It can be confirmed that the decrease in function is significantly prevented or significantly improved. Ingestion of the neurodifferentiation promoter of the present disclosure has a significant improvement effect, especially in the items of cognitive function rate and processing rate.

Another example of the cognitive function test is CogHealth. CogHealth is a cognitive function test using playing cards on a personal computer screen, and consists of five evaluation items: "simple reaction", "selective reaction", "working memory", "delayed reproduction", and "attention dispersion". There is. Specifically, "press the button when the playing cards on the screen turn over from the back to red", "determine whether the color of the playing cards projected on the screen is red or black and press the button", "screen If the upper playing card is the same as the previous card, press the button "," If the same playing card is displayed on the screen in the same task, press the button "," The playing cards that move up and down are in advance Perform a test such as "press the button when you cross the set line". However, since CogHealth evaluates the average reaction time based on the simple task of pressing / not pressing any button, it is only possible to confirm the cognitive function based on the simple task. It is not possible to confirm multifaceted higher cognitive functions based on complex tasks such as Cognitrax.

The effect of promoting neural differentiation by the neurodifferentiation promoting agent of the present disclosure contributes to a feeling of mental fatigue, that is, alleviation of subjective mental fatigue. Therefore, one of the preferred application embodiments of the nerve differentiation promoting agent of the present disclosure is an embodiment used for alleviating mental fatigue.
In the present disclosure, alleviation of mental fatigue means that the level of subjective mental fatigue is lowered and promoted by ingestion of the neurodifferentiation promoter of the present disclosure as compared with that before ingestion. means.

In addition, when the neurodifferentiation promoter of the present disclosure is applied to so-called functional foods, for example, "for those who are worried about fatigue", "easy to get tired". "For those who are aware of fatigue", "For those who are feeling tired", "For those who are concerned about the transient fatigue that occurs in daily life", etc. can.

By ingesting the neuronal differentiation promoting agent of the present disclosure, the neuronal differentiation action in the brain is promoted through the above-mentioned steps 1, 2 and 3, thereby alleviating the feeling of mental fatigue in stage 4': mental fatigue. it is conceivable that.

The present disclosure also includes a method for alleviating a feeling of mental fatigue by using a nerve differentiation promoting agent (the nerve differentiation promoting agent of the present disclosure) containing astaxanthin and a sesamin-based compound as active ingredients.

Mental fatigue means a feeling of fatigue caused by mental stress. The above-mentioned deterioration of cognitive function causes mental stress in various tasks in daily life. Therefore, cognitive decline is included in the factors that cause mental fatigue.

Specific examples of the feeling of mental fatigue include a feeling of fatigue caused by mental stress in the operation work of visual display terminals (VDT) such as a personal computer (PC).

The daily intake of the neurodifferentiation promoter of the present disclosure can be appropriately set depending on the situation (age, body weight, etc.) of the subject, but astaxanthin is 0.1 mg or more and 30 mg or less (preferably 1 mg or more and 20 mg or less). , More preferably 3 mg or more and 12 mg or less), and the amount of the sesamin compound is 0.1 mg or more and 50 mg or less (preferably 3 mg or more and 30 mg or less, more preferably 5 mg or more and 20 mg or less).

The timing and frequency of ingestion of the neurodifferentiation promoter of the present disclosure are not particularly limited, but from the viewpoint of more efficient absorption rate of astaxanthin, it is preferably taken once a day after meals.
As a method of ingesting the neuronal differentiation promoting agent of the present disclosure, oral ingestion is preferable.

The nerve differentiation promoting agent of the present disclosure is preferably in the form of a composition for promoting nerve differentiation described later. The dosage form applicable to the neurodifferentiation promoting agent of the present disclosure is the same as the matters relating to the dosage form described in the section of the composition for promoting neuronal differentiation.

The astaxanthin and sesamin compounds which are the active ingredients contained in the nerve differentiation promoting agent of the present disclosure will be described in detail.

<Astaxanthin>
The active ingredient in the neurodifferentiation promoter of the present disclosure contains astaxanthin as one of its components.
Astaxanthin includes at least one selected from astaxanthin and its derivatives (esters of astaxanthin, etc.). In the present specification, astaxanthin and its derivatives are collectively referred to as "astaxanthin".

As astaxanthin, astaxanthin derived from natural products such as plants, algae, crustaceans, and bacteria, as well as synthetic astaxanthin obtained by a conventional method can be used.
Astaxanthin can be extracted from cultures of red yeast fafia, green alga Hematococcus, marine bacteria, krill and the like.
From the viewpoint of quality and productivity, astaxanthin is also referred to as an extract from Haematococcus algae (hereinafter referred to as "Haematococcus algae extract") or an extract from Okiami (hereinafter also referred to as "Okiami extract"). Astaxanthin derived from.) Is preferable, and astaxanthin derived from Haematococcus algae extract is particularly preferable.
Astaxanthin is preferably in a state of being dissolved in an oil agent.

Specific examples of Haematococcus algae include Haematococcus pluvialis, Haematococcus lacustris, Haematococcus capensis, Haematococcus capensis, Haematococcus capensis, Haematococcus capensis, and Haematococcus droebagensis. Ensis (Haematococcus zimbabwiensis) and the like can be mentioned.
Among these, as the Haematococcus algae, Haematococcus pluvialis is preferable.

The Haematococcus algae extract is obtained by disrupting the cell wall of the above-mentioned Haematococcus algae by a method disclosed in JP-A-5-68585 and the like, if necessary, and using acetone, ether, chloroform, alcohol (ethanol, methanol). Etc.), or by adding an extraction medium such as carbon dioxide in a supercritical state.

As the Haematococcus algae extract, a commercially available product may be used.
Examples of commercially available products of Haematococcus algae extract include Astaxanthin (registered trademark) -S, ASTOTS (registered trademark) -5O, ASTOTS (registered trademark) -10O, etc. of Fujifilm Healthcare Laboratory Co., Ltd. Examples thereof include Astaxanthin (registered trademark) oil 50F, Astaxanthin (registered trademark) oil 5F, etc., and BioAston SCE7 of Toyo Enzyme Chemistry Co., Ltd. ASTOTS is also called ASTOTS.
As astaxanthin in the present disclosure, a haematococcus algae extract extracted from a natural product can be preferably used by a method that does not use an organic solvent such as alcohol. Examples of such Haematococcus algae extract include the above-mentioned ASTOTS®-10O.

The content of astaxanthin as a pure pigment in the Haematococcus algae extract is preferably 0.001% by mass to 50% by mass, more preferably 0.01% by mass to 25% from the viewpoint of handling at the time of production. It is mass%.
The Haematococcus algae extract may contain astaxanthin or an ester thereof as a pure dye, similar to the dye described in JP-A No. 2-49091.

Examples of commercially available products of krill extract include krill oil, Astax-ST (trade name) of Marine Daio Co., Ltd., and the like.

<Sesamin compounds>
The active ingredient in the neuronal differentiation promoter of the present disclosure contains a sesamin-based compound as one of its components.
Sesamin-based compounds are a general term for a group of compounds including sesamin and its analogs, and the active ingredient in the neuronal differentiation promoting agent of the present disclosure includes at least one selected from these compound groups.

Specific examples of the sesamin-based compound include sesamin, episesamin, sesaminol, episesaminol, sesamolin and the like. The sesamin-based compounds may be stereoisomers or racemates of these compounds. The sesamin-based compound may be sesamin, episesamin, or a mixture of sesamin and episesamin.
Further, as the sesamin-based compound, for example, the dioxabicyclo [3.3.0] octane derivative described in JP-A-4-9331 can also be mentioned as a sesamin analog. Metabolites of sesamin-based compounds (for example, the compounds described in JP-A-2001-139579) are also included in the sesamin analogs in the present disclosure as long as they show that the effect can be obtained.

The method for producing the sesamin-based compound is not particularly limited, and a known method may be used. For example, sesamin may be an extract extracted from sesame oil by a known method.
As the sesamin-based compound, a commercially available product may be used. Examples of commercially available products include Sesavita manufactured by Bioactives Japan Co., Ltd., Sesame extract powder manufactured by Videya Japan Co., Ltd., and the like.

In the nerve differentiation promoting agent of the present disclosure, the content ratio of astaxanthin to the sesamin-based compound (astaxanthin: sesamin-based compound) is preferably 0.1:50 to 50:0.1, preferably 1:20 to 20 on a mass basis. 1 is more preferable, and 3: 12-12: 3 is even more preferable.

[Composition for promoting neural differentiation]
The composition for promoting neuronal differentiation according to the present disclosure contains the above-mentioned neuronal differentiation promoting agent of the present disclosure and a carrier. Examples of the composition for promoting neuronal differentiation include a pharmaceutical composition and a food composition.

The carrier contained in the composition for promoting neural differentiation can be appropriately selected depending on the embodiment of the composition for promoting neural differentiation.
Here, the term "carrier" as used herein refers to a solid or liquid compound that does not react with the active ingredient and that contributes to the maintenance of the form of the composition.

Examples of the carrier include emulsifiers, excipients, binders, disintegrants, lubricants, thickeners, wetting agents, oils, solvents and the like. The carrier may be a component that functions as a dispersion medium for a neuronal differentiation promoter and other components. Further, the sucrose fatty acid ester described later is a component included in one aspect of the carrier.

In addition, the composition for promoting nerve differentiation may contain other additives. Other additives can be appropriately selected depending on the embodiment of the composition. Examples of other additives include preservatives, pH regulators, stabilizers, antioxidants, colorants, defoamers, sweeteners, fragrances and the like.

In addition, examples of other additives include functional ingredients that are expected to bring about beneficial effects on health (effects such as health maintenance, health promotion, prevention or improvement of lifestyle-related diseases) by ingestion.

Such functional components include various vitamins, minerals (zinc, etc.), crocetin, collagen (hydrolyzed collagen, water-soluble collagen, etc.), ornithine, resveratrol, chlorogenic acid, caffeic acid, ubiquinone (coenzyme Q10, etc.). , Flavonoids (flavonols, flavones, flavonols, isoflavones, catechins, anthocyanins, etc.), lignans (does not contain sesamine compounds), curcumin, etc. The functional component may be a plant-derived component, an animal-derived component, a fermentation product of yeast, or a chemically synthesized product suitable for food.

When the composition for promoting neuronal differentiation is applied to a pharmaceutical composition, the components contained in the composition as a carrier or an additive may be selected from pharmaceutically acceptable components. Examples of such ingredients include the ingredients described in the 16th revised Japanese Pharmacopoeia and the like.
In addition, when the composition for promoting neural differentiation is applied to a food composition, the components contained in the composition as a carrier and an additive are from the components generally approved for use in food. Just select

The content of the nerve differentiation promoting agent in the composition for promoting nerve differentiation is not particularly limited as long as the daily intake of the nerve differentiation promoting agent can be set to the above-mentioned amount. For example, the content of the nerve differentiation promoting agent can be 0.01% by mass to less than 100% by mass, and 0.1% by mass to 10% by mass, based on the total mass of the nerve differentiation promoting composition. It may be 0.5% by mass to 5% by mass.

As the dosage form of the composition for promoting neural differentiation, for example, tablets, coated tablets, pills, powders, granules, capsules (soft capsules or hard capsules), liquids, suspensions, emulsions and the like can be orally ingested. Dosage form can be mentioned.
Among these, the dosage form of a soft capsule is preferable. By using a soft capsule as the dosage form, the stability of each component contained in the composition can be maintained. Furthermore, the odor derived from the components contained in the composition can be shielded.

The food composition to which the composition for promoting neural differentiation of the present disclosure is applied can be suitably used as a health food, a functional food, a dietary supplement and the like. The form of these foods may be any form such as solid, semi-solid, liquid, gel and the like. It is also preferable that the food composition is provided as a supplement.

The intake amount of the composition for promoting nerve differentiation may be any amount as long as the daily intake of the nerve differentiation promoting agent is the amount described above.

A preferred embodiment of the composition for promoting neuronal differentiation of the present disclosure is a powder of a sucrose fatty acid ester having an HLB value of 10 or more, which is a powder at 25 ° C. as a carrier and the neuronal differentiation promoting agent of the present disclosure. It is an embodiment of the composition comprising (hereinafter, also simply referred to as “sucrose fatty acid ester”) and an oil agent liquid at 25 ° C. (hereinafter, also referred to as “specific oil agent”).
In the composition of the present embodiment, the sucrose fatty acid ester powder is preferably dispersed in a dispersion medium containing a specific oil agent.
The composition of this embodiment is preferably a food composition.

In the composition of the present embodiment, since the sucrose fatty acid ester is contained in the dispersion medium containing the specific oil agent, the affinity of astaxanthin insoluble in water with water is complemented by the sucrose fatty acid ester. Therefore, it is considered that the absorption of astaxanthin in the body is improved. Therefore, by using the composition of the present embodiment, the absorption of astaxanthin in the body is further improved, and the action of the sesamine-based compound described above is combined with the action of the above-mentioned sesamine-based compound. Therefore, the effect of promoting neural differentiation is further improved.

Hereinafter, suitable components in the case where the composition for promoting neural differentiation of the present disclosure is the above-described embodiment will be described in detail.

<Nervous differentiation promoter>
As the nerve differentiation promoting agent, the above-mentioned matters relating to the nerve differentiation promoting agent are similarly applied, and thus the description thereof will be omitted here.

<Sucrose fatty acid ester powder>
The composition of the present embodiment is a powder at 25 ° C. and contains a powder of a sucrose fatty acid ester having an HLB value of 10 or more. By including the powder of such a specific sucrose fatty acid ester, the absorption of astaxanthin in the body is improved.
The sucrose fatty acid ester powder may be used alone or in combination of two or more.

The sucrose fatty acid ester powder is preferably dispersed in a dispersion medium containing a liquid oil agent (specific oil agent) at 25 ° C. The sucrose fatty acid ester is a powder at 25 ° C. Since the sucrose fatty acid ester is a powder at 25 ° C., it disperses well in a dispersion medium containing a specific oil agent.
The sucrose fatty acid ester powder is preferably sized to pass through a filter with an opening of 180 μm (about 83 mesh), and more preferably sized to pass through a filter with an opening of 150 μm (about 100 mesh). ..

The HLB value of the sucrose fatty acid ester is 10 or more, preferably 12 or more, and more preferably 14 or more, from the viewpoint of absorption of astaxanthin in the body.
Further, the HLB value of the sucrose fatty acid ester is preferably 19 or less from the viewpoint of showing amphipathicity.

The number of carbon atoms of the fatty acid constituting the sucrose fatty acid ester is preferably 8 or more, and more preferably 10 to 18, from the viewpoint of showing amphipathicity, for example.
Specific examples of the sucrose fatty acid ester include sucrose lauric acid ester, sucrose myristic acid ester, sucrose palmitic acid ester, sucrose stearic acid ester, sucrose oleic acid ester and the like.
Among these, the sucrose fatty acid ester may be at least one selected from sucrose lauric acid ester, sucrose palmitic acid ester, and sucrose stearic acid ester from the viewpoint of absorption of astaxanthin in the body. Sucrose lauric acid ester is preferable, and sucrose lauric acid ester is particularly preferable.

Commercially available powdered sucrose fatty acid esters include Ryoto (registered trademark) Sugar Ester S1170 (trade name, sucrose stearic acid ester, HLB value: approx. 11 (catalog value), Mitsubishi Chemical Foods Co., Ltd.), Ryoto ( Registered trademark) Sugar ester S1570 (trade name, sucrose stearic acid ester, HLB value: approx. 15 (catalog value), Mitsubishi Chemical Foods Co., Ltd.), Ryoto (registered trademark) sugar ester S1670 (trade name, sucrose stearic acid) Ester, HLB value: Approx. 16 (catalog value), Mitsubishi Chemical Foods Co., Ltd., Ryoto (registered trademark) Sugar ester P1570 (trade name, sucrose palmitate, HLB value: Approx. 15 (catalog value), Mitsubishi Chemical Foods Co., Ltd., Ryoto (registered trademark) Sugar Ester P1670 (trade name, sucrose palmitate, HLB value: approx. 16 (catalog value), Mitsubishi Chemical Foods Co., Ltd.), Ryoto (registered trademark) Sugar Ester L1695 (Commercial name, sucrose lauric acid ester, HLB value: about 16 (catalog value) and the like can be mentioned.

The content of the sucrose fatty acid ester powder is preferably 0.1% by mass or more based on the total amount of the composition for promoting neuronal differentiation, for example, from the viewpoint of absorption of astaxanthin in the body. It is more preferably 5% by mass or more.
Further, the content of the sucrose fatty acid ester powder is preferably 30% by mass or less, preferably 20% by mass or less, based on the total amount of the composition for promoting neural differentiation, for example, from the viewpoint of ease of ingestion. Is more preferable.

The content of the sucrose fatty acid ester powder is preferably 100 parts by mass or more, preferably 300 parts by mass or more, with respect to 100 parts by mass of astaxanthin, for example, from the viewpoint of absorption of astaxanthin in the body. More preferred.
Further, the content of the sucrose fatty acid ester powder is preferably 10,000 parts by mass or less, and 8,000 parts by mass or less with respect to 100 parts by mass of astaxanthin, for example, from the viewpoint of ease of ingestion. More preferred.

Since the sucrose fatty acid ester having an HLB value of 10 or more is not soluble in oil, it exists in a powder state in a dispersion medium containing a specific oil agent. Therefore, if the presence of the sucrose fatty acid ester powder can be confirmed in the dispersion medium containing the specific oil agent, it can be said that the HLB value of the sucrose fatty acid ester is 10 or more. Examples of the confirmation method include an optical microscope, centrifugation, extraction of a solvent (for example, a solvent that dissolves an oily component without dissolving the sucrose fatty acid ester), a combination thereof, and the like.
In addition, since sucrose fatty acid esters having an HLB value of 10 or more do not dissolve in oil, the water extract of the object can be obtained by liquid chromatography (LC: Liquid Chromatography), mass spectrometry (MS: Mass Spectrometry), or red. By analyzing using external spectroscopy (IR: Infrared Spectroscopy), nuclear magnetic resonance (NMR), a combination of these methods, etc., it is possible to determine that the object contains sucrose fatty acid ester. You can check.

<Liquid oil at 25 ° C>
The composition of this embodiment contains an oil agent (specific oil agent) that is liquid at 25 ° C. The specific oil agent functions as a dispersion medium for the sucrose fatty acid ester powder.
The specific oil agent may be used alone or in combination of two or more.
The specific oil agent is not particularly limited as long as it can be applied to a composition for promoting nerve differentiation and is a liquid oil agent at 25 ° C.
In the present disclosure, the "liquid oil at 25 ° C." means an oil having a melting point of less than 25 ° C.

Examples of the specific oil agent include fats and oils, higher fatty acids and the like.
Oils and fats include olive oil, avocado oil, corn oil, rapeseed oil, egg yolk oil, wheat germ oil, flaxseed oil, safflower oil, cottonseed oil, soybean oil, peanut oil, brown seed oil, kaya oil, rice bran oil, germ oil, Examples thereof include plant-derived fats and oils such as safflower oil (that is, red flower oil), palm oil, coconut oil, almond oil, southern ka oil, camellia oil, evening primrose oil, and grapeseed oil. Moreover, as a specific oil agent, tocopherol, tocotrienol and the like extracted from these plant-derived fats and oils can also be mentioned.
The higher fatty acid is not particularly limited as long as it is a fatty acid having 8 or more carbon atoms, and is not particularly limited. Examples thereof include eicosapentaenoic acid and docosahexaenoic acid.
Among these, as the specific oil agent, each component (astaxanthin, safflower fatty acid ester powder, etc.) contained in the food composition can be stably blended, and as the dosage form of the food composition, a soft capsule is used. When selected, at least one selected from safflower oil and coconut oil is preferable in that liquid physical properties (viscosity, etc.) suitable for soft encapsulation can be obtained, and it is particularly easy to handle industrially. Safflower oil is preferred.

The content of the specific oil agent is preferably 100 parts by mass or more, and more preferably 200 parts by mass or more with respect to 100 parts by mass of astaxanthin, for example, from the viewpoint of handleability at the time of production.
Further, the content of the specific oil agent is preferably 10,000 parts by mass or less, and more preferably 8,000 parts by mass or less with respect to 100 parts by mass of astaxanthin, for example, from the viewpoint of ease of ingestion.

<Polyoxyethylene sorbitan fatty acid ester>
It is preferable that the composition of the present embodiment further contains a polyoxyethylene sorbitan fatty acid ester having an HLB value of 10 or more (hereinafter, also simply referred to as “polyoxyethylene sorbitan fatty acid ester”). By further containing the polyoxyethylene sorbitan fatty acid ester in the composition of the present embodiment, the absorption of astaxanthin in the body is further improved.
When the polyoxyethylene sorbitan fatty acid ester is contained, the polyoxyethylene sorbitan fatty acid ester may be contained alone or in combination of two or more.

The HLB value of the polyoxyethylene sorbitan fatty acid ester is preferably 10 or more, more preferably 12 or more.
The HLB value of the polyoxyethylene sorbitan fatty acid ester is preferably 19 or less from the viewpoint of showing amphipathicity.

The average number of oxyethylene groups added to the polyoxyethylene sorbitan fatty acid ester is, for example, preferably 10 to 30 and preferably 15 to 25 from the viewpoint of the hydrophobicity balance.

Examples of the polyoxyethylene sorbitan fatty acid ester include polyoxyethylene sorbitan monolauric acid ester, polyoxyethylene sorbitan monopalmitic acid ester, polyoxyethylene sorbitan monostearic acid ester, and polyoxyethylene sorbitan monooleic acid ester.
Among these, as the polyoxyethylene sorbitan fatty acid ester, the polyoxyethylene sorbitan monolauric acid ester is preferable from the viewpoint of absorption of astaxanthin in the body.

As a commercial product of polyoxyethylene sorbitan fatty acid ester, Wilsurf (registered trademark) TF-20 (trade name, polyoxyethylene sorbitan monolaurate, HLB value: 16.7 (catalog value), Nichiyu Co., Ltd.) , Willsurf TF-60 (trade name, polyoxyethylene sorbitan monostearic ester, HLB value: 15.7 (catalog value), Nichiyu Co., Ltd.), Wilsurf TF-80 (trade name, polyoxyethylene sorbitan) Examples thereof include monooleic acid ester, HLB value: 15.7 (catalog value), and Nichiyu Co., Ltd.

The content of the polyoxyethylene sorbitan fatty acid ester is preferably 20 parts by mass or more and 50 parts by mass or more with respect to 100 parts by mass of astaxanthin, for example, from the viewpoint of absorption of astaxanthin in the body. More preferred.
Further, the content of the polyoxyethylene sorbitan fatty acid ester is preferably 10,000 parts by mass or less, preferably 8,000 parts by mass or less, based on the total amount of the composition of the present embodiment, for example, from the viewpoint of ease of ingestion. Is more preferable.

<Other ingredients>
The composition of the present embodiment may contain other components other than those described above, if necessary, as long as the effects are not impaired. As the other component, it can be selected from various components exemplified as carriers or additives that can be contained in the composition for promoting neural differentiation.

(Crocetin)
The composition of this embodiment can further contain crocetin.
Crocetin is a kind of carotenoid and can be obtained by hydrolyzing crocin contained in gardenia fruit, stigma of saffron and the like.
By further containing crocetin, the composition of the present embodiment can be expected to have additional effects such as an eye strain improving effect and a sleep improving effect.
When the composition of the present embodiment contains crocetin, the content of crocetin is not particularly limited and can be appropriately set according to the type and degree of the expected effect.

(emulsifier)
The composition of this embodiment may contain an emulsifier. By including the emulsifier, the composition of the present embodiment can more stably contain the sucrose fatty acid ester powder in the dispersion medium containing the specific oil agent.
When the composition of the present embodiment contains an emulsifier, the emulsifier may be contained alone or in combination of two or more.

The emulsifier is not particularly limited, and for example, a nonionic surfactant is preferable.
Examples of the nonionic surfactant include glycerin fatty acid ester and polyglycerin condensed ricinoleic acid ester.
Among these, as the nonionic surfactant, for example, a glycerin fatty acid ester having an HLB value of 15 or less is preferable, and a glycerin fatty acid ester having an HLB value in the range of 1 to 10 is more preferable from the viewpoint of exhibiting amphipathicity. preferable.
The glycerin fatty acid ester includes polyglycerin having an average degree of polymerization of 1 to 10 and fatty acids having 8 to 18 carbon atoms such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, or It is preferably an ester with linolic acid.

Commercially available glycerin fatty acid esters include Emax® BW-36 (trade name, HLB value: 3-5, RIKEN Vitamin Co., Ltd.), SY Glister® MS-3S (trade name, tetraglycerin). Monostearic acid ester, HLB value: 8.4, Sakamoto Yakuhin Kogyo Co., Ltd., SY Glister (registered trademark) MO-3S (trade name, tetraglycerin monooleic acid ester, HLB value: 8.8, Sakamoto Yakuhin Kogyo) ), SY Glister (registered trademark) PO-5S (trade name, hexaglycerin pentaoleic acid ester, HLB value: 4.7, Sakamoto Pharmaceutical Co., Ltd.) and the like.

When the composition of the present embodiment contains an emulsifier, the content of the emulsifier is, for example, relative to 100 parts by mass of the specific oil agent from the viewpoint of dispersion stability of the sucrose fatty acid ester powder in the dispersion medium containing the specific oil agent. It is preferably 1 part by mass to 1000 parts by mass.

[Method for producing a composition for promoting neural differentiation]
The method for producing the composition for promoting neuronal differentiation is not particularly limited, and a known method can be used.
The composition for promoting nerve differentiation is a nerve differentiation promoting agent of the present disclosure, a powder of a sucrose fatty acid ester powdered at 25 ° C. and an HLB value of 10 or more, and an oil agent liquid at 25 ° C. as a carrier. In the case of a composition containing a dispersion medium containing a specific oil agent), it can be produced, for example, by the method described below.

The composition of the above embodiment is a mixture of a nerve differentiation promoting agent, a powder of a sucrose fatty acid ester, a specific oil agent, and, if necessary, other components (hereinafter, also referred to as a “mixing step”). ) Can be produced.
The mixing method is not particularly limited, and for example, all the components may be mixed at once or sequentially. From the viewpoint of sucrose fatty acid ester powder being well dispersed, astaxanthin and a specific oil agent are mixed to obtain an oil solution, and then the sesamine compound, sucrose fatty acid ester powder, etc. are added to the obtained oil solution. It is preferable to mix the components.

The mixing means is not particularly limited, and any commercially available mixing means may be used. Examples of the mixing means include mixing means such as a stirrer and a mixer.
Conditions such as temperature and time at the time of mixing are not particularly limited, and can be appropriately adjusted depending on the type of the component contained in the food composition. For example, the temperature at which the sucrose fatty acid ester powder is contained and mixed is set to a temperature lower than the melting point of the sucrose fatty acid ester because the sucrose fatty acid ester needs to be at a temperature that can maintain the powder morphology. Is preferable.

The above-mentioned production method may include steps other than the above-mentioned mixing step, if necessary. As another step, for example, as a pretreatment step before the mixing step, there is a pulverization step of pulverizing the sucrose fatty acid ester powder.
Examples of the crushing means include means using an apparatus suitable for dry crushing of food raw materials, for example, a fine crusher, a grinder, a crusher, a mill and the like.
The pulverization treatment of the sucrose fatty acid ester powder is carried out in a state where the temperature of the sucrose fatty acid ester powder is preferably maintained at 5 ° C to 25 ° C, more preferably 10 ° C to 20 ° C from the viewpoint of suppressing stickiness. ..
Further, in the pulverization treatment of the sucrose fatty acid ester powder, 90% or more of the pulverized product preferably passes through a filter having an opening of 180 μm (about 83 mesh), and more preferably a filter having an opening of 150 μm (about 100 mesh). Do so as to pass through.

When the dosage form is a soft capsule, the step of encapsulating the mixed solution obtained in the mixing step (hereinafter, also referred to as "content liquid") in the capsule film, and the step of encapsulating the capsule film containing the content liquid are dried. It may include a formulation step such as a step of making it.

The capsule coating preferably contains gelatin, glycerin, and water.
The content of glycerin in the capsule film is preferably, for example, 10 parts by mass to 70 parts by mass with respect to 100 parts by mass of gelatin.
Examples of the method of enclosing the content liquid in the capsule film include known methods such as a rotary type (rotary die type, etc.), a seamless type, and a flat plate type.
The method for drying the capsule film containing the content liquid is not particularly limited, and a known dryer such as a tumbler dryer (that is, a rotary drum type dryer) can be used.
Conditions such as temperature and time at the time of drying are not particularly limited, and can be appropriately adjusted depending on the types of the components contained in the food composition and the components contained in the capsule film.

After drying with a dryer, it is preferable to further dry the soft capsules. In this case, the drying temperature is preferably about 20 ° C. to 30 ° C., the humidity is preferably about 10% RH to 50% RH, and the drying time is preferably about 3 to 10 days.

The shape of the soft capsule is not particularly limited, and may take any form such as an ellipse (OVAL), a rectangle (OBLONG), and a spherical shape (ROUND). Methods or devices well known in the art can be applied to these shapes.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded.

1. 1. Cell metabolism test In the cell metabolism test, a neuroblastoma cell line NB-1 (hereinafter abbreviated as "NB-1 cell") is subjected to a treatment solution (dimethylsulfoxide (DMSO), astaxanthin-containing solution, sesamine-containing solution, etc.). Alternatively, it was treated with a mixed solution of astaxanthin and sesamine), and the presence or absence of improvement in cell metabolic function in the treated cells was observed. The test was carried out by the MTT test using a cell number measurement kit (MTT Cell Count Kit, Nacalai Tesque Co., Ltd.). The details are as follows.

NB-1 cells:
Obtained from JCRB Cell Bank (JCRB0621), National Institutes of Biomedical Innovation, Health and Nutrition.
・ Cell culture:
It was cultured using "RPMI1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) / 10% FBS (Corning) / 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.)".

・ Astaxanthin:
ASTOTS-10O (trade name, astaxanthin oil derived from Haematococcus algae, astaxanthin concentration: 10% by mass, FUJIFILM Corporation)
・ Sesamin:
Sesame extract Sesavita (registered trademark) (trade name, White Semin Extract 90% by mass, sesamin compound, Bioactives Japan Co., Ltd.)

<Preparation of treatment liquid>
As the treatment liquid, the astaxanthin solution, the sesamin solution, the astaxanthin-sesamin mixed solution, and DMSO (control) shown below were used.

-Astaxanthin solution (solution A1 and solution A2)
The above astaxanthin was dissolved in DMSO to prepare two kinds of solutions, 31 μmol / L (solution A1) and 63 μmol / L (solution A2), respectively.

-Sesamin solution (solution S1 and solution S2)
The above sesamin was dissolved in DMSO to prepare two solutions having a sesamin concentration of 52 μmol / L (solution S1) and 105 μmol / L (solution S2), respectively.

-Astaxanthin-sesamin mixed solution (solution AS1 and solution AS2)
The above astaxanthin and sesamine are dissolved in DMSO, and the astaxanthin concentration and the sesamine concentration (astaxanthin + sesamine) are a mixture of 31 μmol / L + 52 μmol / L (solution AS1) and 63 μmol / L + 105 μmol / L (solution AS2). Each solution was prepared. Solution AS1 and solution AS2 contain the neurodifferentiation promoters of the present disclosure.

<MTT test>
NB-1 cells were seeded on 96-well plates (Corning) at a seeding density of ^{4 × 10 5 cells / well using serum-free medium.} After 24 hours, the treatment solution prepared above and MTT (10 μL / well) were added to each well and reacted at 37 ° C. for 3 hours in _{a CO 2 incubator.} Then, 100 μL of the solubilizing solution was added to each well, mixed, and then the absorbance at 570 nm was measured using a microplate reader (FlexStation 3, Molecular Devices). It was carried out at n = 3. Reading with a microplate reader was repeated twice at 10-minute intervals, and a total of 6 data were obtained for each sample solution. The results of rejecting the maximum and minimum values of the obtained data are shown in FIG.

<Result>
As shown in FIG. 1, the results of the test using the astaxanthin-sesamine mixed solution (solution AS1 and solution AS2) as the treatment solution were significantly more neuronal than the results of the test using other treatment solutions. It can be seen that the metabolism is improved.

2. 2. Gene expression analysis test In the gene expression analysis test, NB-1 cells are treated with a treatment solution (DMSO, astaxanthin solution, sesamine solution, or astaxanthin-sesamine mixed solution), and the treated cells are disrupted to generate DNA. This was done by submitting to a macro array. Details are as follows.

<Preparation of treatment liquid>
Among the treatment solutions used in the above cell metabolism test, DMSO, solution A1 (astaxanthin concentration: 31 μmol / L), solution S1 (sesamine concentration: 52 μmol / L), and solution AS1 (astaxanthin concentration 31 μmol / L + sesamine concentration 52 μmol). / L) was used as the treatment solution.

<Preparation of sample>
Samples were prepared by allowing the treatment solution (DMSO, solution A1, solution S1 or solution AS1) to act on NB-1 cells (the same as those used in the cell metabolism test) for 6 hours. Specifically, NB-1 cells were seeded on 96-well plates (Corning) at a seeding density of ^{4 × 10 5 cells / well using serum-free medium.} After 24 hours, the treatment solution prepared above was added to each well and reacted at 37 ° C. for 6 hours in _{a CO 2 incubator.}

<RNA extraction>
RNA was extracted from 500 μL of the sample according to the TRIzol Reagent standard, and 10 μg was purified using the RNeasy mini kit (QIAGEN).
Purification followed the kit's conventions, with some modifications. That is, the amount of 100% ethanol used for aggregation was changed from 250 μL to 950 μL, and 100 μL of RNase-free water was permeated through the spin column membrane by 50 μL to elute RNA.
At the time of RNA elution, RNase-free water was added to the spin column membrane, allowed to stand for 1 minute, and then centrifuged.
The obtained RNA sample was purified and concentrated by the ethanol precipitation method, and then the absorbance was measured with NanoDrop (Thermo fisher scientific) using 1 μL from a 10 μL solution to calculate the concentration. In addition, RNA quality was checked using the Agilent 2100 Bioanalyzer RNA6000 Nanokit (Agilent). It was confirmed that the RNA integrity number (RIN) was 9.4 or higher in all the samples, and there was almost no degradation.

<DNA microarray experiment>
-Acquisition of DNA microarray data Using 500 ng of the purified RNA obtained above, a sample to be used for the DNA microarray was prepared according to the standard method of GeneChip (registered trademark) WT PLUS Reagent Kit (Thermo Fisher scientific). For the synthesized cRNA and single-stranded cDNA, the absorbance was measured by NanoDrop (Thermo fisher scientific), the concentration was calculated, and then electrophoresis was performed with an Agilent 2100 bioanalyzer to confirm the elongation reaction.
The cDNA after confirmation of migration was fragmented, labeled, and hybridized to Caryom (registered trademark) S Array, human (Thermo fisher scientific) to obtain DNA microarray data. The equipment used is as follows.

GeneChip® Fluids Station 450 (cleaning and staining of DNA microarrays)
-GeneChip (registered trademark) Scanner 3000 7G (image scan of DNA microarray)
Affymetrix® GeneChip® Command Console® (AGCC) software program

<Normalization of DNA microarray data and extraction of variable probe set>
Each individual data was normalized by Factor Analysis for Robust Microarray Summarization (qFARMS) in the calculation environment R3.4.
After that, the rank products method was executed in R3.3.1 between the two groups of three patterns: DMSO treatment group vs. astaxanthin treatment group, DMSO treatment group vs. sesamine treatment group, and DMSO treatment group vs. astaxanthin-sesamine treatment group. A comparison was made. FDR <0.05 was defined as a significant expression variation, and the corresponding probe set was extracted as an "expression variation probe set", from which genes related to neuronal differentiation were further extracted.

1) Extraction of expression variation probe set from DNA microarray data The expression variation probe set of each treatment group was extracted with respect to the DMSO treatment group. That is, a probe set in which a significant expression variation of FDR * <0.05 was observed between any of the treatment groups was extracted.
* FDR: False Discovery Rate One of the significance level indicators.

2) Extraction of genes related to neural differentiation using a variable probe set specific to the astaxanthin-sesamine treatment group In 1) above, 128 probe sets whose expression was specifically increased in the astaxanthin-sesamine treatment group were targeted. When genes related to nerve cell differentiation were extracted, PAX6, RAB13, PTBP2, CREBBP, PPM1A, MEF2A, and CREB3L2 were confirmed.

<Normalization and analysis of DNA microarray data>
For each individual data, sst-RMA normalization was performed with the Transcriptome Analysis Console (TAC) software (Thermo Fisher Scientific). Then, after one-way ANOVA with R3.3.2 for genes related to neuronal differentiation, a significant difference was tested with Turkey HSD, and p <0.05 was defined as a significant expression variation.

The results are shown in FIG. In FIG. 2, "Ast" indicates "astaxanthin", "Ses" indicates "sesamin", and "Ast. Ses" indicates "astaxanthin-sesamin".

As shown in FIG. 2, CREBBP and RAB13 were confirmed as genes whose expression was specifically increased in the astaxanthin / sesamin compound treatment group.

As described above, the combination of astaxanthin and the sesamine-based compound improves the metabolism of nerve cells in the "cell metabolism test" as compared with the case of using astaxanthin alone or the case of using the sesamine-based compound alone. It was confirmed, and in the "gene expression analysis test", increased expression of genes related to nerve cell differentiation was confirmed. These results support that the neuronal differentiation-promoting agent of the present disclosure can obtain a remarkable differentiation-promoting effect of nerve cells, which cannot be obtained by an agent using astaxanthin or a sesamin-based compound alone. ..

3. 3. Nerve differentiation promoting agent and composition for promoting nerve differentiation [Manufacturing of composition for promoting nerve differentiation]
A composition containing a neuronal differentiation promoting agent containing astaxanthin and a sesamin-based compound as active ingredients (composition for promoting neuronal differentiation) was produced as a soft capsule preparation shown in Examples below.

<Example 1>
Safflower oil (edible oil, Nisshin Oillio Co., Ltd.) 116.19 parts by mass and Emax (registered trademark) BW-36 (trade name, glycerin fatty acid ester, HLB value: 3-5, RIKEN Vitamin Co., Ltd.) 15 parts by mass were sufficiently mixed with a stirrer under heating at 60 ° C. to obtain a uniform oil solution. It was visually confirmed that the oil solution was uniform.
After cooling this oil solution to 50 ° C, 7 parts by mass of SY Glister CRS-75 (trade name, polyglycerin condensed lysinoleic acid ester, Sakamoto Yakuhin Kogyo Co., Ltd.) and E-MIX-α1000 (trade name, vitamin E). (Tocopherol), Tama Biochemical Co., Ltd. (2.25 parts by mass) and mixed, and further, ester-10O (trade name, ester derived from hematococcus algae, esterin concentration: 10% by mass, Co., Ltd.) Fujifilm Healthcare Laboratory) 34.5 parts by mass, SY Glister MO-3S (trade name, tetraglycerin monooleic acid ester, Sakamoto Yakuhin Kogyo Co., Ltd.) 4 parts by mass, and Wilsurf TF-20 (trade name, Polyoxyethylene sorbitan monolauric acid ester, HLB value: 16.7 (catalog value), 3 parts by mass of Nichiyu Co., Ltd. were added and mixed well to obtain a mixture. A stirrer was used to prepare the mixture.

The obtained mixture contains 6.25 parts by mass of LALMIN Se2000 (trade name, mineral yeast, mineral: Se (selenium), Miwa Pharmaceutical Co., Ltd.) and 10% of beer yeast zinc (trade name, beer yeast, zinc 10). 25 parts by mass of R-α lipoic acid CD (trade name, cyclodextrin inclusion body of α-lipoic acid R compound, Cyclochem Co., Ltd.) and 6.25 parts by mass of vitamin C (trade name, α-lipoic acid R-form cyclodextrin inclusion body) and vitamin C (trade name, α-lipoic acid R-form cyclodextrin inclusion body) Product name, Fuso Chemical Industry Co., Ltd. 25 parts by mass, sesame extract Sesavita (registered trademark) (trade name, White Semin Extract 90% by mass, Sesamine compound, Bioactives Japan Co., Ltd.) 5.56 parts by mass , And thoroughly mixed using a stirrer, and then the obtained mixture was pulverized using a wet mill to obtain a pulverized product.

Next, Ryoto (registered trademark) sugar ester L1695 (trade name, sucrose lauric acid ester, HLB value: about 16, Mitsubishi Chemical Foods Co., Ltd.) was pulverized while maintaining a temperature of 10 ° C to 20 ° C. A pulverized product of Ryoto (registered trademark) sugar ester L1695 (crushed product of sucrose fatty acid ester) was obtained by dry pulverization using a machine. It was confirmed that 90% or more of the obtained pulverized sucrose fatty acid ester passed through a filter having an opening of 150 μm (about 100 mesh).
25 parts by mass of the obtained crushed sucrose fatty acid ester and 25 parts by mass of safflower oil (edible oil and fat, Nisshin Oillio Co., Ltd.) were mixed at room temperature (25 ° C.) using a mixer to obtain the obtained product. The mixture obtained above and the total amount of the pulverized product obtained above were sufficiently mixed at room temperature (25 ° C.) using a stirrer to obtain a soft capsule content liquid.

Next, a gelatin sheet containing 35 parts by mass of glycerin with respect to 100 parts by mass of gelatin was sent to a die roll, and 300 mg of the content liquid was dropped onto the sheet joint to prepare an elliptical (OVAL) -shaped soft capsule. The prepared soft capsule was tumbler-dried and statically dried to prepare a soft capsule preparation. The weight of the soft capsule preparation after drying was measured and found to be 460 ± 30 mg per capsule.
No defects such as scratches and cracks were observed in the obtained soft capsule preparation.

[evaluation]
Using the soft capsule preparation (formulation 1) obtained in Example 1 and the placebo preparation containing no neuronal differentiation promoter, the improvement of cognitive decline and the alleviation of mental fatigue were evaluated.
The neurodifferentiation-promoting agent contained in the preparation 1 which is the example preparation contains 6 mg of astaxanthin and 10 mg of sesamin as active ingredients.

(1) Improvement of cognitive decline The evaluation test was conducted by a randomized, double-blind, parallel-group, placebo-controlled trial in men and women aged 50 to 79 years with mild cognitive impairment (MCI).
Seven of the subjects were ingested with the preparation 1, 7 were in the placebo ingestion group, and a total of 14 subjects were evaluated.
Each subject ingested two capsules for 12 weeks.
The cognitive function test was performed for each subject using the Cognitrax cognitive function test tool (Cognitrax Basic, provided by Health Solution Co., Ltd., http://www.cognitrax.jp/) before ingestion, 6 weeks after ingestion, and ingestion. It was carried out at each time point after 12 weeks.
After that, about each item of Cognitrax test evaluation items, total memory, verbal memory, visual memory, cognitive function speed, reaction time, total attention, cognitive flexibility, processing speed, executive function, simple attention, and motor speed. The standardized score of was analyzed. The comparison between the intake group of the placebo preparation and the intake group of the preparation 1 at the time of each test was statistically analyzed by the unpaired t-test, and the same analysis was performed on the amount of change from before the intake.
The results of changes in the two items of cognitive function rate and processing rate at the 12th week of ingestion are shown in Table 1 and FIG. In FIG. 3, “*” indicates p <0.05.
As a result, a significant improvement was observed in the ingestion group of the pharmaceutical product 1 with respect to the change in the two items of cognitive function rate and processing rate at the 12th week of ingestion.

(2) Evaluation of alleviation of mental fatigue The evaluation test was conducted by a randomized, double-blind, two-test group crossover study in healthy men and women between the ages of 30 and 60 who are tired from daily work. I did it.
The feeling of tiredness in daily work was determined by the fact that the score of the Chandler Fatigue Scale was 21 points or more.
The subjects were divided into two groups of half each, and one group ingested the pharmaceutical product 1 and the placebo preparation in this order, and the other group ingested the pharmaceutical product and the pharmaceutical product 1 in that order, and a total of 22 subjects were evaluated.
Each subject took 2 capsules after every breakfast for 4 weeks.
The evaluation test was conducted using a subjective index according to the Visual Analogue Scale (VAS) shown in the guidelines of the Japanese Society of Fatigue. Specifically, at 4 weeks after ingestion, for each subject, 30 minutes of short-term memory task (2-back task) and 30 minutes of exploration work (Advanced Trail Making Test: ATMT) were alternated as mental workload. 4 sets were performed for a total of 4 hours, and the subjective index (feeling of fatigue) was measured.
The feeling of fatigue is according to the Visual Analogue Scale (VAS) shown in the guidelines of the Japanese Society of Fatigue, before the start of loading, 2 hours after loading, 4 hours after loading, 2 hours after recovery, 4 hours after recovery, and the next morning. It was evaluated with 6 points.
The results are shown in Table 2, FIG. 4 and FIG. In Table 2, FIG. 4 and FIG. 5, “*” indicates p <0.05, and “**” indicates p <0.01.
As a result, the ingestion group of the preparation 1 which is the example preparation showed a significant decrease in the feeling of fatigue at the time of the next morning in the amount of change from before the start of loading as compared with the ingestion group of the placebo preparation.
In addition, it was confirmed that the amount of change from 4 hours after loading showed a significant decrease in fatigue after 2 hours of recovery and 4 hours after recovery.
From this, it was clarified that the example preparation promotes recovery from fatigue (mental fatigue) due to mental work load.

Claims (10)

A functional food composition containing astaxanthin, sesamin, episesamin, and at least one sesamin-based compound selected from a mixture of sesamin and episesamin as an active ingredient for preventing or improving function.
A functional food composition whose function is at least one selected from accurate use of machines or tools in humans and mental and physical emphasized movements in humans. Prevention or improvement of function is suppression or improvement of cognitive decline, and the effect of suppression or improvement of cognitive decline is a significant improvement effect in cognitive function speed and processing speed in Cognitrax cognitive function test. The functional food composition according to claim 1. Further, the functional food composition according to claim 1 or 2, which alleviates the feeling of fatigue due to mental work load in a person who feels tired in daily work. The function according to any one of claims 1 to 3, which comprises 6 mg of astaxanthin, sesamin, episesamin, and 10 mg of at least one sesamin-based compound selected from a mixture of sesamin and episesamin as an active ingredient. Sex food composition. The function according to any one of claims 1 to 4, which comprises a powder of a sucrose fatty acid ester which is a powder at 25 ° C. and has an HLB value of 10 or more, and an oil agent which is liquid at 25 ° C. Sex food composition. A functional food composition containing astaxanthin, sesamin, episesamin, and at least one sesamin-based compound selected from a mixture of sesamin and episesamin as an active ingredient for preventing or improving function.
Functional foods, the function of which is at least one selected from linking to dexterous movements requiring cognitive function in humans and judging and recognizing things or information in humans and processing them quickly and accurately. Composition. Prevention or improvement of function is suppression or improvement of cognitive decline, and the effect of suppression or improvement of cognitive decline is a significant improvement effect in cognitive function speed and processing speed in Cognitrax cognitive function test. The functional food composition according to claim 6. Further, the functional food composition according to claim 6 or 7, which alleviates the feeling of fatigue due to mental work load in a person who feels tired in daily work. The functionality according to any one of claims 6 to 8, which comprises 6 mg of astaxanthin, sesamin, episesamin, and 10 mg of at least one sesamin-based compound selected from a mixture of sesamin and episesamin as an active ingredient. Food composition. The function according to any one of claims 6 to 9, which comprises a powder of a sucrose fatty acid ester which is a powder at 25 ° C. and has an HLB value of 10 or more, and an oil agent which is liquid at 25 ° C. Sex food composition.

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