WO2021167012A1

WO2021167012A1 - Composition for neural function regulation

Info

Publication number: WO2021167012A1
Application number: PCT/JP2021/006166
Authority: WO
Inventors: 礒田　博子; 一憲佐々木; 義信新井; 内田　晴久; 健吾岩田
Original assignee: 国立大学法人筑波大学; ニッポー株式会社
Priority date: 2020-02-18
Filing date: 2021-02-18
Publication date: 2021-08-26
Also published as: JPWO2021167012A1

Abstract

The problem addressed is to provide a novel substance that is derived from natural products and is useful in the regulation of neurological function. The present invention is a method for manufacturing a food substance or a medicinal substance that includes at least one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin, the method including the following step: a step in which a fraction including at least one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin is obtained from the crown portion of a sugarcane plant using a water-based solvent. This composition or the like can be used for any selected from the group consisting of: the promotion of neurogenesis by the protection of nerve cells from amyloid-β, an increase in the secretion of brain neurotransmitters, the promotion of the production of ATP in nerve cells, or the growth of neural stem cells; the treatment of dementia, including senile dementia, Alzheimer's disease, vascular dementia, post-traumatic dementia, dementia resulting from brain tumor, dementia resulting from chronic subdural hematoma, dementia resulting from normal pressure cerebral edema, postmeningitic hydrocephalus, and Parkinson's disease; the treatment of non-dementing cognitive impairment, including mild cognitive impairment (MCI) and reduced cognitive function resulting from aging; the improvement of learning disorders and the improvement of memory disorders; the improvement of learning capability and the improvement of memory capability; the treatment of memory loss; the treatment of cerebral infarction and peripheral nerve injury; the treatment of mental disorders, including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder; the improvement of drive and motivation; and the improvement of disturbances of circadian rhythm.

Description

Composition for regulating nerve function

The present invention is selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, and 3-o-ferloyl quinic acid, and isoorientin (luteolin-6-C-glucoside). Containing a composition useful as a food or pharmaceutical, comprising any of the above.

Polyphenols are natural compounds that are widely present in nature and are known to have various bioactive functions. Among them, chlorogenic acid contained in coffee beans, potatoes and the like has been reported to exhibit physiological activities such as an antioxidant effect, an inhibitory effect on blood glucose elevation, and an anti-obesity effect. In recent years, the effects of chlorogenic acid on nerve function have been studied, and the effects of improving autonomic nerve function, recovering from cerebral fatigue, and improving cognitive function have been reported (Patent Documents 1 to 4).

It is known that sugar cane contains polyphenols such as chlorogenic acid. Sugarcane uses stems with a high sucrose content as a sugar-making raw material, and the head of the treetop (pointing to the tip of the ear above the 5th leaf thickening zone) is removed at the time of harvesting, and most of it is used as feed for cattle. However, based on the finding that sugarcane polyphenols are present in high concentrations in the ear extract, a method for producing sugarcane polyphenol-containing substances from sugarcane ears has been proposed (Patent Document 5). In addition, the antioxidant components of the head of sugar cane were identified as chlorogenic acid (5-o-cafe oil quinic acid) and neochlorogenic acid (3-o-cafe oil quinic acid) (Non-Patent Document 1). It has also been proposed to produce brown sugar rich in polyphenols by adding the head of sugarcane treetop to the brown sugar raw material (Non-Patent Document 2). Isoorientin is known to be contained in cereals and rooibos tea (an extract of Aspalathus linearis leaves), but it has recently been reported that oral administration improved scopolamine-induced cognitive impairment in mice. (Non-Patent Document 3).

On the other hand, the number of patients with dementia is increasing on a global scale, and the number of patients with dementia and its reserves is increasing year by year in Japan as well due to the aging of the population. In the treatment or improvement of lifestyle-related diseases such as dementia, symptomatic treatment with chemically synthesized drugs is generally used. However, medical treatment using these pharmaceutical agents has problems such as the size of the dose and discomfort that burden the patient, and the control of side effects associated therewith, and there are few side effects, and in daily life. It is expected to develop highly safe functional foods derived from natural products and preventive medicines that are suitable for ingestion in the field. In this regard, the present inventors have for the prevention or treatment of Alzheimer-type dementia having at least caffeic oil quinic acid (such as di-o-caffeic oil quinic acid) having a structure ester-bonded with two or more caffeic acids. We have proposed agents (Patent Document 6).

Japanese Unexamined Patent Publication No. 2003-137803 Japanese Unexamined Patent Publication No. 2002-145765 Japanese Unexamined Patent Publication No. 2006-160721 JP-A-2018-039797 Japanese Unexamined Patent Publication No. 2002-161046 (Patent No. 3793779) International release WO2007 / 091613

The present inventors have studied functional analysis and effective utilization of food resources. An object of the present invention is to provide a novel material derived from a natural product and useful for regulating nerve function.

The present invention provides:
[1] At least one selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin, which comprises the following steps. Including, manufacturing method of food material or pharmaceutical material:
At least one selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin from the head of sugar cane using an aqueous solvent. The process of obtaining a fraction containing.
[2] The production method according to 1, which is a method for producing a food material or a pharmaceutical material containing at least isoorientin.
[3] The production method according to 1 or 2, wherein the aqueous solvent is 10 to 90% ethanol, preferably 25 to 40% ethanol.
[4] The production method according to any one of 1 to 3, wherein the food material or the pharmaceutical material is for regulating nerve function.
[5] Food or pharmaceutical materials protect nerve cells from amyloid β, increase the secretion of neural mediators in the brain, promote ATP production in nerve cells, promote neurogenesis by proliferation of neural stem cells, and dormant neural stem cells. Any of 1 to 4 selected from the group consisting of induction of transition from state to activated state, induction of differentiation of neural stem cells into neurons, and promotion of astrocyte development. The manufacturing method described in item 1.
[6] Food or pharmaceutical materials include senile dementia, Alzheimer-type dementia, cerebrovascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, and normal compressed brain. Treatment of dementia, including dementia caused by edema, post-dementia dementia, and Parkinson-type dementia; mild cognitive impairment (MCI), and non-dementia cognitive impairment, including cognitive decline due to aging. Treatment of , And treatment of dementia, including bipolar disorder; for any one selected from the group consisting of improved motivation / motivation and improved circadian rhythm disturbance, any one of 1-4. The manufacturing method described in the section.
[7] A food composition or a pharmaceutical composition for regulating nerve function, which comprises either luteolin or a glycoside thereof.
[8] The composition according to 7, further comprising any of the chlorogenic acids represented by the following formula.

(In the formula, R ¹ , R ² , and R ³ are independently H, cafe oil groups, or ferroyl groups.)
[9] The composition according to 8, which comprises isoorientin, 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, and 3-o-ferloyl quinic acid.
[10] The composition according to any one of 7 to 9, wherein any of luteolin and its glycosides, and any of chlorogenic acids are contained as a sugarcane treetop extract.
[11] Protection of nerve cells from amyloid β, increased secretion of neurotransmitters in the brain, promotion of ATP production in nerve cells, promotion of neurogenesis by proliferation of neural stem cells, from dormant state to activated state of neural stem cells The composition according to any one of 7 to 10 for any one selected from the group consisting of induction of migration, induction of differentiation of neural stem cells into neurons, and promotion of astrocyte development.
[12] Senile dementia, Alzheimer-type dementia, cerebrovascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebral edema, spinal cord Treatment of dementia, including post-dementia dementia and Parkinson-type dementia; Treatment of non-dementia cognitive impairment, including mild cognitive impairment (MCI) and cognitive decline due to aging; Improvement of learning impairment , Improvement of memory impairment; Improvement of learning ability, Improvement of memory ability; Treatment of memory decline; Treatment of cerebral infarction and peripheral neuropathy; Includes attention dementia hyperactivity disorder (ADHD), depression, and bipolar disorder , Treatment of dementia; the composition according to any one of 7 to 10 for any selected from the group consisting of improved motivation / motivation and improved circadian rhythm disorder.

According to the present invention, a food or pharmaceutical material containing a component having a nerve function regulating action can be produced from a natural product.

The nerve function can be regulated by the food material, the pharmaceutical material, the food composition using them, or the pharmaceutical composition obtained by the present invention.

Component analysis of sugar cane treetop extract. As a result of component analysis by HPLC, four main peaks were confirmed, and each peak was (1) 3-o-cafe oil quinic acid, (2) 5-o-cafe oil quinic acid, and (3). It was derived from 3-o-ferloylquinic acid and (4) isoorientin (luteolin-6-C-glucoside). Effect of sugarcane treetop extract on nerve cell death induced by amyloid β. In order to evaluate the neuroprotective effect of the sugar cane treetop extract, experiments were conducted using the human neural model cell SH-SY5Y and the neurotoxin amyloid β (Aβ). The cell viability of the amyloid β-treated group was reduced by about 40% compared with the control group, whereas the cell viability of the amyloid β + sugar cane head extract simultaneous treatment group was compared with that of the amyloid β alone-treated group. A significant concentration-dependent increase was confirmed. Cellular evaluation of four compounds contained in sugarcane treetop extract. A cell viability measurement test and an ATP production test using SH-SY5Y cells were performed. The concentration of each compound was the concentration contained in the treetop extract 50 μg / mL (3-CQA: 0.50 μM, 5-CQA: 0.70 μM, 3-FQA: 0.85 μM, isoorientin: 0.477 μM). .. Compared with Aβ single treatment, simultaneous treatment of each compound and Aβ showed an increasing tendency of cell viability (above). In addition, the increasing tendency of intracellular ATP production was confirmed by the treatment of each compound (bottom). 3-CQA: 3-o-Caffeoylquinic acid, 5-CQA: 5-o-Caffeoylquinic acid, 3-FQA: 3-o-Feruloylquinic acid, ISO: Isoorientin (same in the figure below) Mechanism analysis of neuroprotective action by treetop extract. Treatment with amyloid β resulted in a significant decrease in gene expression of MAP2K4, MAPK14, MAPK8, PI3KCA, AKT1, and PARP1 in SH-SY5Y cells, and treatment with sugar cane shoot head extract significantly increased expression of the above genes. Was done. Furthermore, in CASP3 (Caspase-3), which is a gene related to apoptosis (cell death), a significant increase in gene expression in SH-SY5Y cells was observed by amyloid β treatment, and it was expressed by treatment with sugar cane shoot head extract. It was found to decrease. Evaluation of spatial learning and memory ability by Morris water maze test. In the sugarcane treetop extract-administered group, a significant reduction in the time to reach the platform was observed from the 5th day after the start of the experiment compared with the SAMP8 water-administered group (above). In addition, as a result of the probe test, a significant increase in the number of crossings across the platform installation site was observed in the sugarcane treetop extract administration group compared with the water administration group (middle), and the staying time in the platform installation area increased. It showed a tendency (bottom). A group of genes related to brain function, whose expression changes were observed as a result of comprehensive gene analysis using a microarray. As a result of comprehensive gene analysis by microarray, the expected pathway. Analysis of neurotransmitter concentration in the brain by ELISA. In the SAMP8 + sugar cane head extract administration group, the amounts of dopamine and noradrenaline in the cerebral cortex were significantly increased as compared with the SAMP8 + water administration group. In addition, the amounts of acetylcholine and serotonin showed an increasing tendency. Efficiency of component extraction from sugarcane treetop head. From 1 g of the dried sugarcane treetop head, components were extracted using ethanol having different concentrations of 20%, 40%, 60%, and 80%, respectively, as extraction solvents. As a result of quantitative analysis, the content of each polyphenol component per 100 parts by weight of the extract was the highest among the samples extracted using 40% ethanol as a solvent. Comparison of bioactivity on nerve cells by sugarcane treetop extract. Each sample obtained by the above method was redissolved in 70% ethanol and used for a cell viability measurement test using a human nerve model SH-SY5Y cell. A concentration-dependent and significant increase in cell viability was confirmed in the amyloid β (Aβ) + sugar cane head extract treatment group compared with the Aβ alone treatment group. The cell viability was highest in the group treated with Aβ + sugar cane head extract (solvent 40% ethanol). Effects of four compounds contained in sugarcane treetop extract on nerve cell viability. The n3-CQA, 5-CQA, 3-FQA and ISO dissolved in 70% ethanol were used for the cell viability measurement test using SH-SY5Y cells. 3-CQA, 5-CQA and ISO showed a significant increase in SH-SY5Y cell viability in a concentration-dependent manner. Effect of four compounds contained in sugarcane treetop extract on ATP production in nerve cells. An ATP production test was conducted using SH-SY5Y cells using the above-mentioned four compounds. 3-CQA, 5-CQA and ISO showed a significant increase in the rate of ATP production in SH-SY5Y cells. Effect on gene expression of glycolytic enzymes such as sugar cane treetop extract. To evaluate the effects of four compounds contained in sugar cane head extract or sugar cane head extract on gene expression of enzymes (PGK1, PGAM1, PKM, PC) that catalyze glycolytic reactions. Gene expression analysis in SH-SY5Y cells was performed. ACTB was used as the internal standard. Treatment of sugarcane treetop extract or standard compound for 24 hours significantly increased the expression of PGK1, PGAM1, PKM, and PC as compared with the control group. A mechanism that promotes ATP production in nerve cells, which is suggested by the results of gene expression analysis of glycolytic enzymes. Photograph of neural stem cells (hNSC) derived from human foetation. hNSC floats in a medium for neural stem cell proliferation without adhering to the bottom surface of the flask, and proliferates while forming a spherical cell mass (neurosphere). Gene expression analysis of NES (stem cell marker) at Neurosphere. Using sugar cane shoot head extract, TUBB3, which is a marker for neurons (nerve cells) in human embryo-derived neural stem cells, and astrocytosis or transit amplifying (TA) cells (finite proliferation located between stem cells and differentiated cells) Expression analysis of GFAP, which is a marker for cells), PDGFRA, which is a marker for oligodendrocytes, and NES, which is a marker for neural stem cells, was performed. GAPDH was used as the internal standard. Treatment with a sugarcane treetop sample for 24 hours showed an increase in gene expression depending on the treatment concentration of TUBB3 and GFAP. In addition, a decrease in gene expression was observed depending on the treatment concentration of PDGFRA and NES. Quantitative analysis of BrdU and HuC / D (marker of neural progenitor cells) -positive cells using immunostaining, and gene expression analysis of ASCL1 and HES1 which are neural stem cell activation regulators. A. Fluorescence analysis image diagram, B. Percentage of neural progenitor cells, C. Percentage of newborn cells, D. Percentage of newborn neural progenitor cells E. ASCL1 and HES1 gene expression levels. Effect of sugarcane treetop extract on hNSC differentiation. Treatment with sugar cane shoot head extract did not change the proportion of astrocytes (GFAP-positive cells), but the proportion of neurons (Tuj1-positive cells) showed a treatment concentration-dependent increase. In addition, an extension of the total length of the protrusions of astrocytes (GFAP-positive cells) was observed under the treatment of sugarcane treetop extract. Quantitative results of newborn neurons in mouse brain by immunostaining. In the sugarcane treetop extract-administered group, an increase in the number of nerve neurons (BrdU and DCX-positive cells) (about 1.6 times) was observed as compared with the SAMP8 + water-administered group. Comparison of NTRK2 expression levels in nerve cells with sugarcane treetop extract and compounds. In the sugar cane shoot head extract and compound mixture treatment group, the expression of NTRK2 in SH-SY5Y cells was significantly increased as compared with the control group and the compound simple substance treatment group. In addition, the sugar cane treetop extract-treated group and the compound mixture-treated group showed a large increase in NTRK2 expression even when compared with the ginkgo leaf extract-treated group used as a positive control. Examination of component extraction efficiency by using organic solvents of different concentrations. Using 30-50% ethanol as the extraction solvent, the component extraction efficiency was examined by chemical analysis. As a result of quantitative analysis using HPLC, the content of polyphenol component per 100 parts by weight of the extract was the highest among the samples extracted using 30% ethanol.

The present invention comprises at least one selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin, which comprises the following steps. The present invention relates to a method for producing a food material or a pharmaceutical material, which comprises, as a functional ingredient.
At least one selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin from the head of sugar cane using an aqueous solvent. The process of obtaining a fraction containing.
The present invention also relates to a food composition or a pharmaceutical composition containing either luteolin or a glycoside thereof, and any of the chlorogenic acids described below as an active ingredient.

[Functional ingredients, active ingredients]
Food materials or pharmaceutical materials, or food compositions or pharmaceutical compositions related to the present invention (hereinafter, food materials, pharmaceutical materials, food compositions, and pharmaceutical compositions related to the present invention are collectively referred to as "the present invention." (Sometimes referred to as "composition, etc.") contains chlorogenic acids as a functional ingredient or an active ingredient.

<Chlorogenic acids>
In the present invention, chlorogenic acids refer to compounds represented by the following formulas.

In the formula, R ¹ , R ² , and R ³ are independently H, cafe oil groups, or ferroyl groups, respectively.

Examples of chlorogenic acids include 3-o-cafe oil quinic acid, 4-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, 4-o-ferloyl quina. Acids, 5-o-ferloyl quinicic acid, 3,4-di-o-cafe oil quinic acid, 3,5-di-o-cafe oil quinic acid, and 4,5-di-o-cafe oil quinic acid Is included.

In one of the preferred embodiments, the compositions of the present invention, such as the compositions of the present invention, include 3-o-cafe oil quinic acid (R ¹ = cafe oil group, R ² = R ³ = H), 5- as chlorogenic acids. A group consisting of o-cafe oil quinic acid (R ³ = cafe oil group, R ¹ = R ² = H) and 3-o-ferroyl ^{quinic acid (R 1} = fail group, R ² = R ³ = H). Includes any of the more selected.

The composition and the like of the present invention preferably contain at least one selected from the group consisting of the above three compounds as chlorogenic acids, and more preferably contain at least two selected from the group. It is even more preferable that all three are included.

<Luteolin or its glycoside>
The composition and the like of the present invention contain luteolin or a glycoside thereof as a functional ingredient or an active ingredient. The glycoside of luteolin is a compound in which a sugar is glycosidic bonded to luteolin (3', 4', 5,7-tetrahydroxyflavone). Examples of sugars include glucose, galactose, fructose, glucuronic acid, rhamnose, xylose, arabinose, apiose, rutinose, gentiobiose, primeberose, and dichitoxose. Examples of luteolin glycosides include luteolin 4'-o-glucoside, luteolin 4'-o-glucuronide, luteolin 5-glucuronide, luteolin 5-glucoside, luteolin 5-lucinoside, luteolin 5-o-glucuronide, luteolin 6 -Glucocide (isoorientin), luteolin 6-c-β-d-glucopyranoside 8-c-α-l-arabinopyranoside, luteolin 6-c-arabinoside, luteolin 7- (2-o-apiosil glucoside) ), Luteolin 7- (2-o-glucuronosyl) glucuronide, luteolin 7- (2-sulfoglucoside), luteolin 7-o-glucoside, luteolin 7-o-galactoside, luteolin 7-o-glucuronide, luteolin 7,4' -Di-o-glucuronide, luteolin 7- [6-o- (2-methylbutyryl) -β-glucoside], luteolin 7-o-[2-o- (4-o-acetyl-α-ramnopyranosyl) -β- Glucronopyranoside], luteolin 8-glucoside (orientin) is included.

In one of the preferred embodiments, the composition and the like of the present invention contain a compound represented by the following formula as luteolin or a glycoside thereof.

In the formula, R ⁴ and R ⁵ are H or sugar residues, respectively.

In one particularly preferred embodiment, the compositions and the like of the present invention include isoorientin (R ⁴ = H, R ⁵ = glucose residue) and orientin (R ⁴ = glucose) as luteolin or a glycoside thereof. Includes any residue selected from the group consisting of ^{R 5 = H).}

The composition and the like of the present invention contain at least one selected from the group consisting of the above two compounds as luteolins, and it is more preferable that isoorientin is contained.

<4 types of compounds contained in sugarcane treetop head>
In one particularly preferred embodiment, the compositions and the like of the present invention include 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, and 3-o-ferloyl quinic acid as chlorogenic acids. It contains isoorientin, which is a glycoside of luteolin. The composition or the like containing all of these four kinds of components may be the sugarcane treetop extract itself or a composition containing the same as an active ingredient. This application is the first to report that the head of sugarcane treetop contains these four kinds of compounds.

According to the study by the present inventors, when the extraction is performed under appropriate conditions, the extract containing the four target compounds is dried from 1 g of the dried sugarcane treetop head (including leaves and bark). It can be obtained by weight from 100 mg to 170 mg, more specifically from 130 mg to 140 mg.

The amount of 3-o-cafe oil quinic acid contained in the sugarcane treetop extract provided by the present invention can be 0.20 mg or more per 100 g (dry weight) of the extract, regardless of the content of other components. .. By optimizing the extraction conditions, it can be 0.30 mg or more, 0.35 mg or more, or 0.40 mg or more regardless of the content of other components.

The amount of 5-o-cafe oil quinic acid contained in the sugarcane treetop extract provided by the present invention can be 1.0 mg or more per 100 g (dry weight) of the extract, regardless of the content of other components. .. By optimizing the extraction conditions, the amount can be 1.5 mg or more, 2.0 mg or more, or 2.5 mg or more regardless of the content of other components.

The amount of 3-o-ferloylquinic acid contained in the sugarcane treetop extract provided by the present invention can be 0.10 mg or more per 100 g (dry weight) of the extract, regardless of the content of other components. .. By optimizing the extraction conditions, it can be 0.13 mg or more, 0.16 mg or more, or 0.20 mg or more regardless of the content of other components.

The amount of isoorientin contained in the sugarcane treetop extract provided by the present invention can be 0.80 mg or more per 100 g (dry weight) of the extract, regardless of the content of other components. By optimizing the extraction conditions, the amount can be 1.0 mg or more, 1.2 mg or more, or 1.4 mg or more regardless of the content of other components.

In one of the preferred embodiments, the 3-o-cafe oil quinic acid contained in 100 g (dry weight) of the sugar cane shoot head extract is 0.40 mg or more and 0.60 mg or less, and the 5-o-cafe oil quinic acid is 2.5 mg or more and 3.5 mg or less, 3-o-ferloylquinic acid is 0.20 mg or more and 0.30 mg or less, and isoorientin is 1.4 mg or more and 1.8 mg or less.

In one of the preferred embodiments, the sugarcane shoot head may contain 2.1-2.6 mg / g (dry weight) of isoorientin. Isoorientin is also known to be contained in rooibos tea. Extracts from 10 g of dried green rooibos tea leaves in 500 ml of hot water have been reported to contain 26 mg of isoorientin (Food Chemistry 128: 338-347, 2011).

<Synergistic effect>
In one particularly preferred embodiment, the compositions and the like of the present invention include 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin 4 It contains a type of compound as an active ingredient, or contains a sugar cane head extract as an active ingredient. According to the studies by the present inventors, treatment with 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, and 3-o-ferloyl quinic acid alone causes NTRK2 expression at the experimental concentrations. While showing no change, a mixture of these and isoorientin, and a sugar cane head extract containing all four of these compounds, can synergistically increase NTRK2 expression in neurons. TrkB (Neurotrophic tyrosine kinase receptor type 2, NTRK2) is involved in the development and maturation of the central and peripheral nervous systems, and is activated by in vivo factors such as BDNF (brain-derived neurotrophic factor) to grow and survive nerve cells. It has been reported to activate signal pathways involved in synaptic development and neurogenesis in the hippocampus.

When the composition or the like of the present invention contains four kinds of compounds, their ratio is not particularly limited as long as it has the desired effect, and for example, 3-o-cafe oil quinic acid: 5-o-cafe oil quinic acid: 3-o-ferloylquinic acid: isoorientin can have a molar ratio of 1: 0.10 to 10: 0.15 to 15: 0.080 to 8.0, 1: 0.30 to 3.0: 0.40 to 4.0: 0.20 to 2.0. It can be 1: 0.60 to 1.5: 0.80 to 2.0: 0.40 to 1.0.

[Sugar cane treetop extract, its manufacturing method, etc.]
In the present invention, the sugarcane treetop head refers to the tip portion of the sugarcane (scientific name: Saccharum officinarum L.) above the fifth leaf thickening zone.

The sugarcane treetop head used for producing the composition of the present invention may be the entire sugarcane treetop head, a portion including leaves and bark, and a portion excluding leaves and bark. There may be. The sugarcane treetop head may be raw or dried. Drying can be performed by cold air drying or sun drying. The sugarcane treetop head may be cut, shredded, or crushed to increase extraction efficiency.

The means for extraction from the head of the sugar cane treetop is not particularly limited, and extraction may be performed using a liquid extraction solvent, or supercritical extraction or subcritical extraction may be performed using a supercritical fluid or a subcritical fluid. You may.

The extraction solvent is preferably a solvent that is effective in extracting luteolin or its glycosides and that is effective in extracting chlorogenic acids. Examples of such solvents are water, methanol, ethanol, isopropanol, butanol, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, and methyl ethyl ketone, and mixtures thereof. Preferred examples are aqueous solvents, such as water, or a mixture of water and any of the groups selected from the group consisting of methanol, ethanol, isopropanol, butanol, propylene glycol, butylene glycol, glycerin.

In one of the preferred embodiments, a mixed solvent of water and ethanol is used. The concentration of ethanol can be 5% or more, preferably 10% or more, more preferably 20% or more, and even more preferably 25% or more. The concentration of ethanol can be 95% or less, preferably 90% or less, and more preferably 85% or less. It can be 70% or less. The concentration of ethanol can be 60% or less, preferably 50% or less, more preferably 40% or less, and even more preferably 35% or less. Regarding the present invention, when the ethanol concentration of the extraction solvent containing ethanol is indicated, it is a value based on the volume (v / v) unless otherwise specified, and unless otherwise specified, ethanol is water. It is mixed.

The extraction operation may be performed at room temperature, but preferably when heated under reflux cooling, the components can be extracted efficiently and quickly. The extraction temperature can be 60 ° C. or higher, preferably 70 ° C. or higher, and more preferably 80 ° C. to 100 ° C. When performed under heating, the extract can be obtained efficiently and with high purity.

Further, the extraction may be carried out under pressure and can be carried out at 5 MPa (50 bar) or more, preferably 7.5 MPa (75 bar) or more, and may be 10 MPa (100 bar) or more. In this case as well, heating may be performed in order to efficiently and quickly extract the target component. The temperature can be 25 ° C. or higher, preferably 30 ° C. or higher, more preferably 35 ° C. or higher, and even more preferably 40 ° C. or higher.

The ratio of the raw material to the extraction solvent, the extraction time, and the number of repetitions of the extraction operation can be adjusted appropriately in consideration of the extraction efficiency.

When performing supercritical or subcritical extraction, the fluids include, for example, water, carbon dioxide, ethylene, propylene, ethane, propane, dinitrogen monoxide, chlorodifluoromethane, chlorotrifluoromethane, xenone, ammonia, and methanol and ethanol. Lower alcohols such as can be used. From the viewpoint of safety, it is preferable to use water, ethanol, a mixture thereof, or carbon dioxide.

After extraction, if necessary, the insoluble residue can be removed, concentrated by a conventional method, and dried by means such as spray drying and freeze drying.

Unless otherwise specified, the term "extract" in the present invention includes an extract extracted from a raw material with a solvent, a concentrate of the extract, a dried product, and a crude product.

[Use]
The compositions of the present invention and the like can be used for neural function regulation. Nerve function regulation protects nerve cells from amyloid β, antioxidants (suppression of environmental stress stimulation), anti-inflammatory (improvement of inflammatory state), increased secretion of neurotransmitters in the brain, promotion of ATP production in nerve cells , Promotion of neurogenesis by proliferation of nerve stem cells, treatment of dementia, improvement of non-dementia cognitive disorder, improvement of learning disorder, improvement of memory disorder, improvement of learning ability, improvement of memory ability, treatment of mental illness, Includes improvement of motivation and motivation, and improvement of disturbance of circadian rhythm.

According to the studies by the present inventors, sugarcane shoot head extract was able to reduce the decrease in cell viability due to amyloid β treatment in SH-SY5Y, which is a cell line derived from human neuroblastoma.

Therefore, the composition and the like of the present invention can be expected to have a neuroprotective effect from amyloid β, and are useful for treating diseases or conditions exacerbated by amyloid β. Amyloid β (Aβ) is a peptide consisting of 40 to 42 amino acids, which is toxic to nerve cells and causes cell death. In Alzheimer's disease, Aβ aggregates to form insoluble fibrosis, which becomes amyloid and deposits in the brain. Diseases or conditions exacerbated by amyloid β include Alzheimer's disease.

According to the studies by the present inventors, MAP2K4 (Mitogen-activated protein kinase 4), MAPK14 (Mitogen-activated protein kinase 14), MAPK8 (Mitogen-Activated Protein kinase 8), MAPK8 (Mitogen-Activated Protein kinase 8) in SH-SY5Y cells by treatment with amyloid β, PI3KCA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalyst Subunit Alpha), AKT1 (AKT Serine / Threonine Kinase 1), PARP1 (Poly (ADP-Ribose) Polymerase 1), but significant gene expression reduction is observed. Treatment with the treetop extract showed a significant increase in the expression of these genes. Furthermore, in CASP3 (Caspase-3), which is a gene related to apoptosis (cell death), a significant increase in gene expression in SH-SY5Y cells was observed by amyloid β treatment, and it was expressed by treatment with sugar cane shoot head extract. It was found to decrease.

The above gene clusters respond to stress stimuli on cells and regulate a wide variety of cellular processes such as cell proliferation, growth, differentiation, transformation and apoptosis. Since the expression fluctuations of these amyloid β-induced genes were improved, the extract used in this experiment suppressed environmental stress stimuli such as oxidative stress (antioxidation, etc.) and caused inflammation in nerve cells. It may be useful for improving the condition (neuroinflammation, etc.) (anti-inflammatory).

According to the study by the present inventors, as a result of the Morris water maze test using the aging model mouse (SAMP8), the sugarcane treetop extract-administered group was compared with the SAMP8 water-administered group from the 5th day after the start of the experiment to the platform. A significant reduction in the arrival time of was observed. In addition, as a result of the probe test, a significant increase in the number of crossings across the platform installation site was observed in the sugarcane treetop extract administration group compared with the water administration group, and the staying time in the platform installation area tended to increase. rice field.

From the results of behavioral tests using such aging model mice, the compositions of the present invention have an improving effect on cognitive decline, and for example, dementia (senile dementia, Alzheimer-type dementia, cerebrovascular dementia). Various diseases such as dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebral edema, dementia after meningitis, and Parkinson-type dementia. It may be useful for the treatment of dementia caused by). In addition, the compositions and the like of the present invention may be useful for the treatment of non-dementia cognitive impairment such as mild cognitive impairment (MCI) and deterioration of cognitive function due to aging. Furthermore, the compositions and the like of the present invention may be useful for improving learning or memory disorders (learning and memory disorders associated with brain developmental disorders), improving learning ability, and improving memory ability.

According to the study by the present inventors, the amounts of dopamine and noradrenaline in the cerebral cortex were significantly increased in the sugarcane head extract-administered group of SAMP8 as compared with the SAMP8 water-administered group. In addition, the amounts of acetylcholine and serotonin showed an increasing tendency.

Therefore, the compositions of the present invention containing sugar cane head extract may be useful for the treatment of diseases or conditions that are ameliorated by increased secretion of transmitters in the brain. Furthermore, since an improving effect on the decrease in dopamine and noradrenaline secretion was confirmed, mental illness including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder (manic depression, etc.) It can be useful for the treatment of diseases. In addition, since it was confirmed that it has an improving effect on the decrease in dopamine secretion, it may be useful for improving motivation and motivation.

According to the study by the present inventors, in a comprehensive gene expression analysis using a microarray, a large number of changes in gene expression were observed in the sugarcane head extract-administered group of SAMP8 as compared with the SAMP8 water-administered group. In particular, since it was confirmed that the expression of the clock gene (Per3) was increased, the composition of the present invention and the like may be useful for improving the disorder of the circadian rhythm (biological clock).

In particular, according to the studies by the present inventors, none of the dopamine, noradrenaline, adrenaline, acetylcholine, and serotonin in the sugar cane head extract-administered group of SAMP8 exceeded that of the normal mouse group. The composition can be expected to be safe to use without unnecessarily enhancing the secretion of neurotransmitters in the brain.

Also, since noradrenaline is known to act on arousal-sleep, the compositions of the present invention can improve sleep (eg, improve sleep quality, promote non-rem sleep, improve sleep rhythm), or sleep disorders. It can also be expected for the treatment of (for example, insomnia).

According to the study by the present inventors, sugar cane shoot head extract, and 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin are used. Increased survival rate in SH-SY5Y. In addition, sugar cane shoot head extract, 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, and isoorientin were able to promote the production of ATP. This mechanism also increases the gene expression of phosphoglycerate kinase (PGK1), phosphoglycerate mutase (PGAM1), pyruvate kinase (PKM), and pyruvate carboxylase (PC), which are enzymes involved in glycolysis. It was suggested that this was due to activation of the intracellular glycolysis system.

Therefore, the compositions of the present invention and the like can be used to promote the production of ATP in nerve cells, and are useful for treating diseases or conditions that are improved by promoting the production of ATP in nerve cells. obtain. In addition, it may be useful for the treatment of diseases or conditions that are ameliorated by elevated expression of any gene selected from the group consisting of PGK1, PGAM1, PKM, and PC.

According to the study by the present inventors, by treating human fetal-derived neural stem cell hNSC with a sugar cane treetop head extract, TUBB3 (Tubulin beta III / Tuj1), which is a marker of neurons in neural stem cells, and astrocytes or astrocytes or A processing concentration-dependent increase in gene expression of GFAP (Glial fibrillary acidic protein), which is a marker of transit amplifying (TA) cells (finite proliferating cells located between stem cells and differentiated cells), was observed. That is, it was suggested that the sugarcane treetop head extract induces the transition of neural stem cells from the dormant state to the activated state, and induces the differentiation of neurons and astrocytes from the neural stem cells. Results of cell proliferation determination test using thymidine analog 5-bromo-2'-deoxyuridine (BrdU) and HuC + HuD (HuC / D), which is a marker for neural progenitor cells. The proportion of BrdU-positive cells increased in a treatment concentration-dependent manner by treatment with the head extract of sugar cane. That is, it was suggested that the sugarcane treetop head extract has an effect of promoting the proliferation of neural stem cells. The proportion of both HuC / D / BrdU positive cells showed a treatment concentration-dependent increase. This result suggests that the sugarcane treetop head extract has a promoting effect on the generation of newborn neurons from neural stem cells. HuC refers to ELAV (embryonic lethal, abnormal vision, Drosophila) -like 3 (Hu antigen C; HuC), and HuD refers to ELAV-like 4 (Hu antigen D; HuD).

The promotion of this newborn neuron production is thought to be due to the regulation of ASCL1 and HES1 gene expression. ASCL1 and HES1 are basic helix-loop-helix (bHLH) factors that control fate decisions in cell development and differentiation. In the differentiation of nerve cells, the expression level of HES1 is high and the expression level of ASCL1 is kept low in the state where the stem cell property is maintained (≈ dormant state). When the neural stem cells are activated, the expression level of HES1 decreases and the expression level of ASCL1 increases, so that the neural stem cells differentiate into nerve cells. According to the study by the present inventors, the expression of ASCL1 in hNSC was increased and the expression of HES1 was decreased in a treatment concentration-dependent manner by the treatment with the sugarcane treetop extract.

According to the study by the present inventors, hNSCs having formed spheres were treated with sugar cane shoot head extract, and Tuj1 (Tublin beta III) and GFAP-positive cells were detected by immunostaining. Astrosite (GFAP) was detected. The proportion of neurons (Tuj1-positive cells) did not change, but the proportion of neurons (Tuj1-positive cells) showed a treatment concentration-dependent increase. In addition, an extension of the total length of the protrusions of astrocytes (GFAP-positive cells) was observed under the treatment of sugarcane treetop extract. Therefore, sugar cane headtop extract can induce neuronal differentiation from neural stem cells (induction of differentiation into neurons can be rephrased as neurogenesis and promotion of neuronal production) and promote astrocyte development. can. In addition, in cerebral infarction and peripheral nerve injury, the symptoms can be improved by forming a new neural circuit from the newborn neurons produced by the treatment of the sugar cane headtop extract and recovering the lost neuroplasticity.

Furthermore, in the hippocampal dentate gyrus site in the mouse brain to which the sugar cane shoot head extract was administered, the number of cells in which BrdU, which is a marker for newborn cells, and DCX, which is a marker for immature neurons, are both positive as compared with the target group. An increase (about 1.6 times) was confirmed. From this result, it can be said that the oral administration of sugarcane treetop head extract has a clear effect of promoting neurogenesis in the animal brain.

From the above, the composition and the like of the present invention can be useful for promoting neurogenesis by proliferation of neural stem cells. It may also be useful for the treatment of diseases or conditions that are ameliorated by promoting neurogenesis through the proliferation of neural stem cells.

In order for a component to act on the brain, a therapeutic drug for brain tumors must cross the blood-brain barrier, which protects the brain. Conditions that easily cross the blood-brain barrier include (1) small molecular weight, (2) low protein binding rate, and (3) high lipophilicity. For example, Temodar, a drug applicable to malignant glioma, has an extremely small molecular weight of 194, so it has the advantage of easily passing through the blood-brain barrier and reaching the affected area. The molecular weights of the four components 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin according to the present invention are 354, 354, 368, and 448, respectively. Is. It has been reported that monocafe oil quinic acid crosses the blood-brain barrier and reaches brain tissue after ingestion. (Fitoterapia 99: 139-152, 2014)

In addition, according to the study by the present inventors, a mixture of ginkgo biloba head extract and 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin. Can synergistically increase the expression of NTRK2 in SH-SY5Y cells as compared with single treatment with isoorientin and treatment with ginkgo biloba extract, which has been reported to have a cognitive function improving effect as a positive control. I know. Therefore, the compositions of the present invention can be expected to improve symptoms by effectively improving the TrkB signal, promoting neurogenesis, and suppressing the decrease in nerve cells with aging. More specifically, the compositions of the present invention include diseases in which BDNF-TrkB signal reduction and reduction of nerve cells due to reduction of neurogenesis have been reported, specifically, memory deterioration in aging and Alzheimer's disease. It can be used to treat depression.

In the present invention, the term "treatment" for a disease or condition includes reduction of risk of onset, delay of onset, prevention, treatment, suspension of progression, and delay. Treatment includes actions performed by doctors for the purpose of treating illness, and persons other than doctors, such as dietitians (including registered dietitians and sports nutritionists), public health nurses, midwives, nurses, clinical laboratory technicians, and sports. This includes non-therapeutic acts performed by instructors, beauty staff, estheticians, drug manufacturers, drug sellers, food manufacturers, food sellers, etc. In addition, the treatment includes actions performed by veterinarians for the purpose of treating non-human animal diseases, and persons other than veterinarians, such as veterinarian nurses, pet animal care managers, servants, zookeepers, and veterinary medicines. Includes non-therapeutic acts performed by manufacturers, veterinary drug sellers, pet food manufacturers, pet food sellers, etc. Further treatments include recommendations for administration or intake of specific foods, dietary guidance, health guidance, nutritional guidance (nutrition guidance necessary for medical treatment of the sick and injured, and nutritional guidance for maintaining and improving health. ), Food management, and guidance necessary for improving nutrition related to food.

The target of the treatment in the present invention includes humans (individuals), and preferably, it is desirable to perform any of the above-mentioned treatments, or a human who needs to be subjected to any of the above-mentioned treatments. The target of the treatment in the present invention may be an animal other than human, and examples thereof include pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels (sometimes referred to as pet animals and companion animals). Examples include domestic animals such as cows and pigs, experimental animals such as mice and rats, and animals bred in zoos and the like. The growth stage of the target non-human animal is not particularly limited, and the target of the treatment of the present invention may be, for example, a puppy, an adult dog, an elderly dog, a kitten, an adult cat, or an elderly cat.

[Composition, etc.]
In the present invention, the composition may be the sugar cane head extract itself, and may contain an active ingredient (for example, sugar cane head extract) and other components.

The composition of the present invention can be a food composition or a pharmaceutical composition.

With respect to the present invention, foods include not only solids but also liquids such as soups, beverages and drinks, unless otherwise specified. Further, unless otherwise specified, not only those intended for humans but also those intended for non-human animals, such as feed and pet food, are included. Furthermore, unless otherwise specified, foods include general foods, health foods, supplements, foods with health claims (foods for specified health use (commonly known as Tokuho), foods with nutritional claims, foods with functional claims), and therapeutic foods (foods with functional claims). Those that serve the purpose of treatment. Those that are cooked based on the menu prepared by a nutritionist, etc. after a doctor puts out a meal sheet.), Dietary foods, ingredient-adjusted foods, salt-reduced foods, nursing foods, calorie-reduced foods , Including diet foods.

When the composition of the present invention is in the form of an oral drug, a health food, or a supplement, examples of the dosage form include soft capsules, hard capsules, tablets, pills, powders, granules, and fine granules. , Jelly, tubed, and drinks.

The content of the sugarcane treetop extract, which is an active ingredient, in the composition of the present invention can be designed in consideration of the daily intake and dose. For example, as a daily dose for an adult, the sugarcane treetop extract can be contained as a solid in an amount of 0.2 to 2,000 mg, preferably 0.5 to 1,000 mg, more preferably 1 to 500 mg, and further preferably 2 to 2. It can be 200 mg. The daily dose can be ingested and administered in multiple doses, for example, 2 to 4 doses.

The composition of the present invention may contain components other than the sugarcane treetop extract as long as the desired effect can be exhibited. Other ingredients may be various food-acceptable additives or pharmaceutically acceptable additives. Examples of this include excipients, antioxidants (antioxidants), flavors, seasonings, sweeteners, colorants, thickeners, color formers, bleachers, preservatives, gum bases, bitterness agents, etc. Enzymes, brighteners, acidulants, emulsifiers, fortifiers, manufacturing agents, binders, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, coagulants and the like.

Other components may be functional components other than sugarcane treetop extract. Examples of other functional ingredients include amino acids (eg, branched chain amino acids, astaxanthin), unsaturated fatty acids (eg, EPA, DHA), vitamins, trace metals, polyphenols, egg yolk extract, honey. Processed products, brown sugar, oligosaccharides, dietary fiber, glucosamine, chondroitins, CoQ10, fucoidan, fucoxanthin, astaxanthin, placenta, yeast extract, black vinegar concentrate, plant extract (garlic extract, ginkgo leaf extract, tea extract) Amino acids, bilberry extract, blueberry extract, various carrot extracts, maca extract, bean seed coat extract, St. Jones wort extract, pine bark extract, acai extract, noni extract) and the like.

The composition of the present invention can be ingested and administered with a meal, before or after a meal.

The composition of the present invention can be labeled as being usable for the treatment of the above-mentioned diseases or conditions, and can be labeled as being recommended for ingestion for the above-mentioned subjects. The labeling can be direct or indirect, and examples of direct labeling are descriptions on tangible objects such as the product itself, packages, containers, labels, tags, etc., and examples of indirect labeling are Includes advertising and publicity activities by location or means such as websites, stores, exhibitions, signboards, bulletin boards, newspapers, magazines, television, radio, mailings, e-mails, etc.

[Component analysis of sugar cane treetop extract]
<Preparation of sugarcane treetop extract sample>
From 1 g of dried sugarcane treetop head (including leaves and bark), components were extracted (number of extractions: 4 times) using an automatic extraction device using 80% ethanol as an extraction solvent. The solution after extraction was concentrated using a rotary evaporator, and then lyophilized to obtain a sample. The obtained sample was redissolved in 100% methanol (concentration: 100 mg / mL) and then sterilized by a 0.22 μm filter, which was used for chemical analysis using high performance liquid chromatography (HPLC).

<Component analysis using high performance liquid chromatography (HPLC)>
The conditions for analysis using HPLC were that the column used was ZORBAX (250 x 4.6 mm), the column temperature was 40 ° C, the flow velocity was 1.0 mL / min, the injection volume was 10 μL, the UV wavelength was 328 nm, and the mobile phase was one. A: 10% formic acid; the other was B: acetonitrile: MeOH = 1: 1, and the gradient was 0-100%; 40 min.

<Analysis result>
The chromatogram obtained by HPLC is shown in FIG. As a result of component analysis, four main peaks were confirmed, and each peak was (1) 3-o-cafe oil quinic acid, (2) 5-o-cafe oil quinic acid, and (3) 3 It was derived from -o-ferloylquinic acid and (4) isoorientin (luteolin-6-C-glucoside). The content of each component in the extract was 0.352 ± 0.014 parts by weight of 3-o-cafe oil quinic acid, 0.491 ± 0.006 parts by weight of 5-o-cafe oil quinic acid, and 0.624 per 100 parts by weight of the composition. It consisted of ± 0.051 parts by weight of 3-o-ferroylquinic acid and 0.427 ± 0.001 parts by weight of isoorientin (Table 1).

(1) 3-o-Cafe oil quinic acid

(2) 5-o-cafe oil quinic acid

(3) 3-o-ferloyl quinic acid

(4) Isoorientin (luteolin-6-C-glucoside)

[1. Evaluation of the effect of sugarcane treetop extract on amyloid β-induced neuronal cell death at the cellular level]
1-1. Preparation of sugarcane treetop extract sample A solvent extraction method was used as a method for extracting polyphenol components from sugarcane treetop powder. Specifically, the sugarcane treetop head was soaked in 10 mL of 70% ethanol per 1 g of dried product for 14 days (inverted stirring on the 7th day of extraction), and the components were eluted in the solvent. Then, a sample was obtained by concentrating and drying the extraction solvent sterilized by a 0.22 μm filter. The sample was dissolved in 70% ethanol and used for the cell test described later.

1-2. Experimental method <Culturing human nerve model SH-SY5Y cells>
SH-SY5Y (American Type Culture Collection, Manassas, USA) was used as a human nerve model cell. SH-SY5Y is a cell line derived from human neuroblastoma and is widely used as a model cell for evaluating nerve function. SH-SY5Y cells were seeded in 100 mm sterile petri dishes (BD Falcon, USA) and 15% heat inactivated in 1: 1 (v / v) mixture of Dulbecco's Modified eagle medium and Ham's F-12 medium (Lonza, Japan). fetal bovine serum (Bio West, USA), 1% penicillin (5000 μg / ml)-streptomycin (5000 IU / ml) solution (Lonza), 1% MEM Non-Essential Amino Acids Solution (100x) (Cosmo) It was cultured in a medium having a composition containing Bio, japan) at 37 ° C. in the _{presence of 5% CO 2.} Cells were cultured to reach 60-80% confluence and cells with a passage number of 3-8 were used for each study.

<Evaluation of neuroprotective effect by sugarcane treetop extract>
In order to evaluate the neuroprotective effect of sugarcane treetop extract, an experiment using the neurotoxin amyloid β (Aβ) was conducted. Specifically, (i) control group (ii) Aβ single treatment group (iii) Aβ + sugar cane head extract treatment group was set as the treatment group for cells, and the cells were placed in a 96-well plate together with the medium 2.0 × 10 ⁴ After sowing to cells / well and _{culturing at 37 ° C. in the presence of 5% CO 2} for 24 hours, an amount of amyloid β protein (Aβ1-42) or sugar cane head extract to a final concentration of 5 μM was added to each well. Added. The sugarcane treetop extract was added so that its concentration was 10 μg / mL, 25 μg / mL, or 50 μg / mL. After incubating for 72 hours at 37 ° C in the _{presence of 5% CO 2} , the cell viability was evaluated by measuring the absorbance using the MTT method. Specifically, the medium was removed from the wells, 5 mg / ml MTT solution dissolved in PBS was added (100 μl / well), and the mixture _{was incubated at 37 ° C. in the presence of 5% CO 2} for 6 hours, and then 10%. Sodium dodecyl sulfate (SDS) was added (100 μl / well) and _{incubated at 37 ° C. in the presence of 5% CO 2} for 24 hours to dissolve formazan crystals. Absorbance was measured using a microplate reader (absorbance: 570 nm), and the obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

1-3. Experimental results The cell viability decreased by about 40% in the amyloid β-treated group compared with the control group, whereas the amyloid β + sugar cane head extract simultaneous treatment group was compared with the amyloid β alone-treated group. In comparison, a concentration-dependent significant increase in cell viability was confirmed (Fig. 2). From this result, it was found that the neuronal cell death caused by the treatment with amyloid β was significantly suppressed by the simultaneous treatment of the sugarcane treetop extract, suggesting the neuroprotective effect of the sugarcane treetop extract.

[2. Evaluation of four compounds contained in the head of sugarcane treetops at the cellular level]
2-1. Preparation of compounds In this experiment, 3-O -Caffeoylquinic acid (hereinafter 3-CQA, Nagara Science, Gifu, japan), 5-O -Caffeoylquinic acid (hereinafter 5-CQA, Nagara Science, Gifu, japan), 3-O-Feruloylquinic acid (3-FQA, Nagara Science, Gifu, japan), and Isoorientin (ISO, Sigma-Aldrich, USA) were used as standard compounds. .. In this experiment, the concentration of each compound standard was calculated by calculating the content of 50 μg / mL of sugar cane headtop extract from the quantitative results of HPLC analysis. Specifically, 3-CQA had a concentration of 0.50 μM, 5-CQA had a concentration of 0.70 μM, 3-FQA had a concentration of 0.85 μM, and ISO had a concentration of 0.477 μM. The standard product of each compound was dissolved in 70% ethanol for the cell test.

2-2. Experimental method SH-SY5Y (American Type Culture Collection, Manassas, USA) was used as a human nerve model cell. As the treatment group for cells, (i) control group, (ii) Aβ single treatment group, (iii) Aβ + 3-CQA (0.50 μM), (iv) Aβ + 5-CQA (0.70 μM), (v) Aβ + 3-FQA (0.85 μM), (vi) Aβ + ISO (0.477 μM), (vii) Aβ + compound mixture (3-CQA + 5-CQA + 3-FQA + ISO), (viii) Aβ + sugar cane A group treated with the treetop extract (50 μg / mL) was set and tested in the same manner as in the method 1-2. Above to evaluate the cell viability. In addition, the obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

For each of the above treatment groups, the amount of ATP produced in nerve cells was measured. The experimental method was in accordance with 6-2. <Evaluation of the effect of compound standard products on ATP production in nerve cells>.

2-3. Experimental results In the simultaneous treatment of 3-CQA, 5-CQA, 3-FQA, and ISO compounds with Aβ, the cell viability tended to increase compared to the treatment with Aβ alone. A significant increase in cell viability was observed between the compound mixture and Aβ simultaneous treatment compared to β alone treatment. (Fig. 3 top) From this result, it is suggested that 3-CQA, 5-CQA, 3-FQA and ISO are components involved in the inhibitory effect of amyloid β-induced neuronal cell death by sugarcane treetop extract. Was done.

In addition, the increasing tendency of intracellular ATP production was confirmed by the treatment of each compound. Treatment with a mixture of four polyphenols confirmed a significant increase in intracellular ATP production compared to controls (Fig. 3, bottom).

<Mechanism analysis of neuroprotective action by treetop extract>
Gene expression was analyzed using real-time RT-PCR as a mechanism analysis of the neuroprotective effect of the treetop extract. As the treatment group for SH-SY5Y, (i) control group, (ii) Aβ single treatment group, and (iii) Aβ + sugar cane head extract treatment group were set, and the cells were seeded in a 6-well plate together with the medium, and 37 After culturing for 24 hours at ℃ in the _{presence of 5% CO 2} , amyloid β protein or sugar cane head extract was added in an amount having a final concentration of 5 μM. The sugarcane treetop extract was added so as to have a concentration of 50 μg / mL. After incubation for 24 hours at 37 ° C in the _{presence of 5% CO 2} , mRNA was extracted from the cells in each treatment group using the ISOGEN Kit (Nippon Gene, Japan).

Using the extracted mRNA, reverse transcription polymerase chain reactions (RT-PCR) were performed using Superscript III reverse transcriptase kit (Invitrogen, USA) and 2720 Thermal cycler (Applied Biosystems, USA). TaqMan real time RT PCR amplification reactions using the 7500 Fast Real Time PCR system was carried out to quantify transcripts, and it is a gene involved in the signal pathway involved in the mechanism of action of nerve cell death by amyloid β treatment as a primer. , MAP2K4 (Hs00387426_m1), MAPK14 (Hs01051152_m1), MAPK8 (Hs01548506_m1), PI3KCA (Hs00907957_m1), AKT1 (Hs00178280_m1), PARP1 (Hs00242302_m1), PARP1 (Hs00242302_m1), PARP1 (Hs00242302_m1) .. In addition, GAPDH (Hs02786624_m1) was used as the internal standard. In addition, the obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
Treatment with amyloid β resulted in a significant decrease in gene expression of MAP2K4, MAPK14, MAPK8, PI3KCA, AKT1, and PARP1 in SH-SY5Y cells, and treatment with sugar cane shoot head extract significantly increased expression of the above genes. (Fig. 4). Furthermore, in CASP3, which is a gene related to apoptosis (cell death), amyloid β treatment significantly increased gene expression in SH-SY5Y cells, and treatment with sugar cane shoot head extract decreased expression. (Fig. 4).

[3. Analysis of the effect of sugarcane treetop extract on spatial learning memory ability]
3-1. Preparation of sugarcane treetop extract sample The polyphenol component was extracted from sugarcane treetop powder in the same manner as in 1-1. Above, and used as a sample. The sample dissolved in Mill-Q was used for the animal test described later.

3-2. Experimental method SAMP8 (purchased from Nippon LSC Co., Ltd.) was used as an aging acceleration model. SAMP8 is a model animal of a naturally occurring strain characterized by the onset of early aging-related pathological conditions, and studies on the effects of antioxidant components contained in various foods are being conducted. SAMR1, which indicates normal aging, was used as a control for SAMP8. In this experiment, 16-week-old male mice that began to show aging-like symptoms such as memory impairment were used, and the mice were set in the following three groups: (a) SAMR1 + water-administered group, (b) SAMP8 + water-administered group. (c), SAMP8 + sugar cane head extract administration group.

The dose of sugarcane treetop extract was set to 20 mg / kg per day, and oral administration was used as the administration method. After oral administration of samples and water for 30 days to each group, a behavioral test was conducted to evaluate the spatial learning and memory ability of each group. Ten mice in each group were used in this experiment. During the breeding period, water and feed were freely ingested, and the mice were weighed daily.

After the behavioral test was completed, the brain was isolated and collected from the mice, and comprehensive gene expression was analyzed using a microarray and the concentrations of neurotransmitters in the brain (dopamine, noradrenaline, adrenaline, acetylcholine, serotonin) were analyzed by the ELISA method. ..

<Evaluation of spatial learning memory ability by Morris water maze test>
After 30 days of continuous administration of sugarcane treetop extract, a Morris water maze test was conducted, which is a behavioral test to evaluate the spatial learning and memory ability of animals. The Morris Water Labyrinth Test is an experimental method to evaluate the learning and memory ability of animals. In the test, a transparent escape platform (platform) was installed under the surface of a pool filled with water, and mice dropped into the pool were dropped from the water. Explore the platform motivated by escape behavior. The experimental device consists of a circular water pool (120 cm in diameter and 45 cm in height), the temperature and depth of the water are set to 23 ± 2 ° C and 30 cm, respectively, and there are four virtual quadrants: north, south, east, and west. It was divided into quadrants. At the midpoint of any quadrant, a transparent, invisible escape platform (10 cm in diameter) was installed 1 cm below the surface of the water. When exploring the platform, the mouse uses four visual landmarks on the wall of the pool as clues, allowing the mouse to reach the platform in a short amount of time over time. The spatial learning memory ability of the mouse was evaluated by allowing the mouse to freely search for 60 seconds and measuring the arrival time at the platform. In this test, shortening the arrival time is an index of the learning memory improvement effect. The test was conducted for 8 days, and on the final day of the test, the test was performed with the platform removed, and the mouse stayed in the area where the platform was installed and crossed the position where the platform was installed. The number of times was measured (probe test). By conducting a probe test, we evaluated how much spatial memory can be retained and recalled. The significant difference test between the SAMP8 + water administration group, the SAMP8 + sugar cane head extract administration group and the SAMR1 + water administration group is the two-way ANOVA method (*) for the test results with the 7-day platform installed. According to P <0.05), the test results of the probe test on the final day were performed by the One-way ANOVA method (* P <0.05).

<Experimental results>
As a result of the Morris water maze test, it was observed that the time to reach the platform was significantly shortened in the sugarcane treetop extract-administered group from the 5th day after the start of the experiment as compared with the SAMP8 water-administered group (Fig. 5, top). In addition, as a result of the probe test, a significant increase in the number of crossings across the platform installation site was observed in the sugarcane treetop extract administration group compared with the water administration group (Fig. 5), and the staying time in the platform installation area. Showed an increasing trend (Fig. 5, bottom). From the above results, it was suggested that the sugarcane treetop extract had an effect of improving spatial learning and memory.

<Comprehensive gene analysis by microarray>
After 30 days of continuous administration of sugarcane treetop extract or water and a behavioral test, brain tissue was removed and the cerebral cortex was collected. MRNA was extracted from the separated cerebral cortex using the ISOGEN Kit (Nippon Gene, Japan). Then, using the Gene Atlas 3'IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA), double-stranded cDNA was synthesized from 100 ng mRNA, and then the Gene Chip 3'IVT Express Kit (Affymetrix Inc., Affymetrix Inc., Biotin-labeled amplified RNA (aRNA) was synthesized using Santa Clara, CA, USA). Then, the purified aRNA was fragmented using the Gene Atlas 3'IVT Express Kit, and the Gene Chip MG-430 PM microarray (Affymetrix Inc., Santa Clara, CA, USA) was used at 45 ° C for 16 hours. Hybridization was performed. The chips were then washed and stained with Gene Atlas Fluidics Station 400 (Affymetrix Inc., Santa Clara, CA, USA) and image images scanned by Gene Atlas Imaging Station (Affymetrix Inc., Santa Clara, CA, USA). .. Compared with the SAMP8 + water administration group, the change in gene expression in the SAMP8 + sugar cane head extract administration group was measured by fold-change (magnification change, control signal value as a control) based on the obtained results. It was calculated as a relative ratio obtained by dividing the signal value of. In addition, DAVID (URL: https://david.ncifcrf.gov) was used to analyze the pathway.

<Experimental results>
As a result of microarray analysis, a large number of changes in gene expression were confirmed in the SAMP8 + sugar cane head extract-administered group compared with the SAMP8 + water-administered group (Table 2). Among the fluctuating genes, the total number of genes that were up-regulated (fold-change was 1.15 or more) was 912 (of which 1.5 or more was 6) and down-regulated (fold change was 0.85 or less). The total number of genes was 466 (of which 5 were 0.5 or less). Since the number of genes that were downregulated was larger than the number of genes that were downregulated, we focused on the genes that were downregulated and analyzed using DAVID. As a result of the analysis, the genes that were up-regulated were categorized as a group related to various biological functions, and among them, cholinergic synapses that release acetylcholine as a neurotransmitter, body temperature regulation, etc. Serotonergic synapses that play an important role in sleep, dopaminergic synapses that play a role in controlling cognitive and voluntary movements and motivating behavior associated with rewards, calcium signaling in nerve cells, long-term potentiation in neurotransmission, It was confirmed that the genes related to circadian rhythm, Alzheimer's disease, etc. were elevated (Fig. 6). In addition, as a result of pathway analysis, the expression of genes such as Camk2d, Adcy9, Cacna1a, and Homer1 that responded to the improvement of calcium signal transduction between synapses was increased, and the resulting increase in neuroplasticity and neuroprotective effect, and the clock gene through the increase in expression of Camk2d. Increased expression of Per3 and its resulting improvement in circadian rhythm, increased expression of Cox8b and its resulting improvement in mitochondrial function, etc. were considered as activated gene transmission pathways in the cerebral cortex (Fig. 7).

<Analysis of neurotransmitter concentration in the brain by ELISA>
After 30 days of continuous administration of sugarcane treetop extract or water and a behavioral test, brain tissue was removed and the cerebral cortex was collected. Brain tissue is dissolved in RIPA buffer (Santa Cruz Biotechnology, USA) containing a protease inhibitor, centrifuged (4 ° C, 1,200 g, 20 min), and the supernatant is collected to obtain total protein for neurotransmission. Dopamine, noradrenaline, adrenaline, acetylcholine and serotonin concentrations were measured using the substance ELISA kit (ImmuSmol SAS, France). The measured value was displayed as the concentration per protein amount (g) of the brain tissue calculated by the BCA method. In addition, the obtained values were subjected to a significant difference test (** P <0.01) by the One-way ANOVA method.

<Experimental results>
The levels of dopamine and noradrenaline in the cerebral cortex were significantly increased in the SAMP8 + sugar cane head extract-administered group compared with the SAMP8 + water-administered group (Fig. 8). In addition, the amounts of acetylcholine and serotonin showed an increasing tendency (Fig. 8). That is, from this result, it was considered that sugarcane treetop extract is involved in increasing the amount of neurotransmitters secreted in the brain and improves learning memory.

[4. Examination of component extraction efficiency from sugarcane treetop head by using different concentrations of organic solvent]
<Preparation of sugar cane treetop extraction sample>
Ingredients were extracted from 1 g of dried sugarcane treetop head using ethanol having different concentrations of 20%, 40%, 60%, and 80%, respectively, as extraction solvents. The ethanol concentration is based on the volume (v / v). An automatic extraction device E-916 (Nippon Buch) was used for extraction, and the extraction conditions were set to temperature = 45 ° C, pressure = 100 bar, extraction time = 10 minutes, and number of extractions = 3. The solution after extraction was concentrated using a rotary evaporator, and then freeze-dried to obtain a powdery sample (the color is brown for those using 20% ethanol as a solvent, and green for others. The ethanol concentration is high. The green color of the obtained sample was slightly deeper). The total amount (yield) of each extracted sample is shown in the table below (approximately 130 mg to 140 mg from 1 g of raw material). The obtained sample was redissolved in 100% methanol (concentration: 100 mg / mL), sterilized by a 0.22 μm filter, and then used for quantitative analysis of the components using high performance liquid chromatography (HPLC).

<Quantitative analysis using high performance liquid chromatography (HPLC)>
The analyzer was carried out using Prominence (Shimadzu Corporation). The analysis conditions were ZORBAX (250 x 4.6 mm) column, column temperature 40 ° C, flow rate 1.0 mL / min, injection volume 10 μL, UV wavelength 328 nm, mobile phase A: 10% formic acid. The other was set to B: acetonitrile: MeOH = 1: 1 and the gradient was set to 0-100%; 40 min.

<Analysis result>
The quantitative results obtained by HPLC are shown in FIG. As a result of quantitative analysis, the content of each polyphenol component per 100 parts by weight of the extract was the highest among the samples extracted using 40% ethanol as a solvent.

For 3-CQA, 5-CQA and 3-FQA, 80% ethanol was used as the solvent, and under the extraction conditions of temperature 45 ° C and pressure = 100 bar, two operations were performed to dry the sugarcane treetop head. It is known that 80% or more of the contained amount can be extracted.

[5. Comparative study of bioactivity on nerve cells by sugarcane treetop extract extracted using different concentrations of organic solvent]
5-1. Preparation of sugar cane headtop extract sample For the sugar cane treetop sample, each sample obtained by the above method <Preparation of sugar cane treetop extract sample> is redissolved in 70% ethanol (concentration: 100 mg /). The sample was sterilized with a 0.22 μm filter (mL) and used for the cell test described later.

5-2. Experimental method <Culture of human nerve model SH-SY5Y cells>
Same as 1-2.

<Evaluation of neuroprotective effect by sugarcane treetop extract>
As the treatment group for cells, (i) control group, (ii) Aβ (5 μM) single treatment group, (iii) Aβ + sugar cane head extract (solvent 20% ethanol) treatment group, (iv) Aβ + sugar cane Treetop extract (solvent 40% ethanol) treatment group, (v) Aβ + sugar cane treetop extract (solvent 60% ethanol) treatment group, (vi) Aβ + sugarcane treetop extract (solvent 80% ethanol) treatment group It was set. The test was carried out in the same manner as in the method 1-2. Above, and the cell viability was measured. In addition, the obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

5-3. Experimental results A concentration-dependent significant increase in cell viability was confirmed in the Aβ + sugar cane head extract treatment group compared with the amyloid β alone treatment group (Fig. 10). The cell viability was highest in the group treated with (iv) Aβ + sugar cane head extract (solvent 40% ethanol) (78.6 ± 2.41%, Fig. 10). From this result and the result of the above-mentioned [Examination of component extraction efficiency from sugarcane treetop head by using different concentrations of organic solvent], between the polyphenol component content in the extract and the physiological activity of the extract on nerve cells. Correlation was found.

[6. Evaluation of the effects of four compounds contained in the head of sugarcane treetop on cell viability and ATP production in the neuron model SH-SY5Y]
6-1. Preparation of compounds In this experiment, 3-o -Caffeoylquinic acid (hereinafter 3-CQA, Nagara Science, japan), 5-o -Caffeoylquinic acid (hereinafter 5-CQA, Nagara Science, Japan), 3-o -Feruloylquinic acid (3-FQA, Nagara Science, Japan) and Isoorientin (ISO, Sigma-Aldrich, USA) were used as standard compounds for each compound. As a standard sample of each compound, a sample dissolved in 70% ethanol was used in the test described later.

6-2. Experimental method <Culture of human nerve model SH-SY5Y cells>
Same as 1-2.

<Evaluation of the effect of compound standard products on the survival rate of nerve cells>
As the cell treatment group, (i) control group, (ii) 3-CQA treatment group, (iii) 5-CQA treatment group, (iv) 3-FQA treatment group, and (v) ISO treatment group were set. In addition, the treatment concentrations of the compounds in each treatment group were set to 1 μM, 5 μM, 10 μM, and 20 μM, respectively. The test was carried out in the same manner as in the method 1-2. Above, and the cell viability was measured. The obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Evaluation of the effect of compound standard products on ATP production in nerve cells>
A Cell ATP Assay reagent (Toyo Ink, Japan) was used to measure the amount of ATP produced in nerve cells. ATP is an important indicator of energy metabolism in cells. SH-SY5Y cells were seeded on a 96-well plate so that the number of cells was 2.0 × 10 ⁵ cells / mL, and the cells _{were incubated at 37 ° C. in the presence of 5% CO 2} for 24 hours, and then the cells were treated with the sample. Was done. As the cell treatment group, (i) control group (ii) 3-CQA treatment group (iii) 5-CQA treatment group (iv) 3-FQA treatment group (v) ISO treatment group were set, and the treatment concentration was 10 μM. A sample was added to each well so as to be. Samples were incubated at 37 ° C. in the _{presence of 5% CO 2} for 12, 24, 48 hours and then incubated at room temperature for 30 minutes. Then, 100 μl / well of ATP reagent (Toyo Ink) at room temperature was added, and after incubating in the dark for 1 minute, 150 μL of supernatant from each well was transferred to a 96-well white bottom plate (BD Falcon), and 25 Incubation was performed at ° C for 10 minutes to stabilize luminescence. Then, the ATP production ratio was measured by measuring the emission intensity using a plate reader (Powerscan HT, USA). The obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.
6-3. Experimental results As a result of the cell viability measurement test, for 3-CQA, 5-CQA and ISO compound standards, the survival rate of SH-SY5Y cells showed a significant increase in concentration-dependent by sample treatment. (Fig. 11). In addition, as a result of the ATP production test, the treatment of 3-CQA, 5-CQA and ISO compound standard samples showed a significant increase in the ATP production rate in SH-SY5Y cells (Fig. 12). These results suggest that among the compounds contained in the sugarcane treetop extract, 3-CQA, 5-CQA and ISO activate energy metabolism in SH-SY5Y cells and contribute to the promotion of ATP production. rice field.

[7. Evaluation of the effect of sugarcane treetop extract or four compounds contained in the extract on gene expression of glycolytic enzymes in SH-SY5Y]
7-1. Preparation of sugarcane treetop extraction sample and compound sample The preparation method for each sample is the same as the above method.

7-2. Experimental method <Culturing human nerve model SH-SY5Y cells>
Same as 1-2.

<Evaluation of glycolytic enzyme gene expression in SH-SY5Y cells for the purpose of elucidating the mechanism of promoting ATP production by sugar cane shoot head extract or compound standard>
To elucidate the mechanism of ATP production in nerve cells, to evaluate the effect of sugar cane shoot head extract or compound standard on gene expression of enzymes that catalyze the chemical reaction of glycolysis, which is the energy metabolism system in cells. , Gene expression analysis using real-time RT-PCR was performed. As treatment groups for SH-SY5Y, (i) control group, (ii) sugar cane shoot head extract (50 μg / mL) treatment group, (iii) 3-CQA treatment group, iv) 5-CQA treatment group (v) The ISO treatment group was set, and the treatment concentration of the sugarcane treetop extract sample was set to 50 μg / mL, and the treatment concentration of each compound sample was set to 10 μM. After incubating the cells together with the sample in a 60 mm cell culture dish (BD Falcon) at 37 ° C. in the _{presence of 5% CO 2} for 24 hours, the cells in each treatment group were subjected to ISOGEN kit (Nippon Gene, Japan). mRNA was extracted.

Using the extracted mRNA, reverse transcription polymerase chain reactions (RT-PCR) was performed using Superscript IV reverse transcriptase kit (Invitrogen, USA) and 2720 Thermal cycler (Applied Biosystems, USA). In order to quantify the transcript, TaqMan real time RT PCR amplification reactions using the 7500 Fast Real Time PCR system was performed. As primers, PGK1 (Hs00943178_g1), PGAM1 (Hs01652468_g1), PKM (Hs00761782_s1), and PC (Hs01085875_g1), which are enzymes that catalyze glycolytic metabolism, were used in the reaction, and ACTB (Hs01060665_g1) was used as an internal standard. .. All primers were purchased from Applied Biosystems (USA). The obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
The 24-hour treatment of sugarcane treetop extract or standard compound of each compound significantly increased the expression of PGK1, PGAM1, PKM, and PC in SH-SY5Y cells as compared with the control group (Fig. 13). From this result, the sugar cane headtop extract or each compound standard product activates the glycolytic system, which is an intracellular energy metabolism system, through the increase in gene expression described above, and as a result, promotes ATP production in nerve cells. It was suggested that this should be done (Fig. 14).

[8. Evaluation of self-renewal and differentiation-inducing ability of human neural stem cells (hNSC) by sugarcane treetop extract]
8-1. Preparation of sugarcane treetop sample The sample obtained by the method in 1-1. Above was dissolved in 70% ethanol and used in the test described later.

8-2. Experimental method and results <Culture of human neural stem cells (hNSC)>
Human embryo-derived neural stem cells (Gibco® life technologies, USA) were used for the purpose of evaluating the effects of samples on neural stem cells (hNSC; human Neural Stem Cell). hNSC was seeded in T75 flasks (BD Falcon, USA) and in KnockOut ^TM D-MEM / F-12 with 2% StemPro® Neural Supplement, 20 ng / mL bFGF and 20 ng / mL. EGF, 2 mM GlutaMAX ^TM- I Supplement, 6 units / mL Heparin, and 200 μM Ascorbic acid, a medium for neural stem cell proliferation (all of which are Gibco® life technologies). ), At 37 ° C., in the presence of _{5% CO 2.} In the above medium, hNSC is cultured in a floating state without adhering to the bottom surface of the flask, and proliferates while forming a spherical cell mass (neurosphere) (Fig. 15). Medium exchange was performed every 3-4 days and culture was performed until the diameter of the formed spheres <= 1 mm (9-11 days after sowing).

<Evaluation of the effect of sugarcane treetop extract on gene expression of differentiation markers in hNSC>
As an evaluation of the ability of the sugar cane shoot head extract to induce self-renewal differentiation in neural stem cells, gene expression analysis of differentiation markers in neural stem cells was performed using real-time RT-PCR. As treatment groups for hNSC, (i) control group, (ii) 10 μg / mL sugar cane head extract treatment group, (iii) 25 μg / mL sugar cane head extract treatment group, (iv) 50 μg / mL sugar cane A treetop extract treatment group was set. After culturing hNSC together with the medium in a 6-well plate and confirming the formation of spheres (diameter <= 1 mm), in the sugar cane shoot head extract treatment group, a sample diluted so that the concentration in the medium became the above-mentioned set concentration. Was added, and an equal amount of medium was added in the control group. After incubation for 24 hours at 37 ° C in the _{presence of 5% CO 2} , mRNA was extracted from the cells in each treatment group using the ISOGEN kit (Nippon Gene, Japan).

Using the extracted mRNA, reverse transcription polymerase chain reactions (RT-PCR) was performed using Superscript IV reverse transcriptase kit (Invitrogen, USA) and 2720 Thermal cycler (Applied Biosystems, USA). In order to quantify the transcript, TaqMan real time RT PCR amplification reactions using the 7500 Fast Real Time PCR system was performed. As primers, TUBB3 (Hs00801390_s1), which is a marker for neurons (nerve cells), and GFAP (Hs00909233_m1), which is a marker for astrocytes or transit amplifying (TA) cells (finite proliferating cells located between stem cells and differentiated cells). , PDGFRA (Hs00998018_m1), a marker of oligodendrocytes, and NES (Hs04187831_g1), a marker of neural stem cells, were used in the reaction, and GAPDH (Hs02786624_g1) was used as an internal standard. All primers were purchased from Applied Biosystems (USA). The obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
The 24-hour treatment of sugarcane treetop extract samples increased the expression of TUBB3 and GFAP in hNSC in a treatment concentration-dependent manner (Fig. 16). In addition, the expression of PDGFRA and NES decreased in a treatment concentration-dependent manner (Fig. 16). From this result, it was suggested that the sugarcane treetop head extract induces the transition of neural stem cells from the dormant state to the activated state, and the effect of inducing differentiation into neurons or astrocytes.

<Evaluation of the effect of sugarcane treetop extract on the generation of newborn neurons from hNSC>
Assay using thymidine analog BrdU (5-bromo-2'-deoxyuridine) and neural progenitor cell marker HuC / D to assess the effect of sugar cane head extract on newborn neuron formation from hNSC Was carried out. BrdU is an analog of thymidine, a component of DNA, and BrdU is incorporated into the replicated DNA strand when cells begin to divide in the presence of BrdU. The uptaken BrdU was detected using an antibody, and the cell proliferation rate was evaluated by counting the rate of BrdU-positive cells. HuC / D is a protein that is specifically expressed in the cell body of neurons from the early stage of differentiation, and the proportion of newborn neurons was analyzed by quantifying positive cells together with BrdU. As treatment groups for hNSC, (i) control group, (ii) 10 μg / mL sugar cane head extract treatment group, (iii) 25 μg / mL sugar cane head extract treatment group, (iv) 50 μg / mL sugar cane A treetop extract treatment group was set. After culturing hNSC together with the medium in a 6-well plate and confirming the formation of spheres (diameter <= 1 mm), in the sugar cane shoot head extract treatment group, a sample diluted so that the concentration in the medium became the above-mentioned set concentration. Was added, and an equal amount of medium was added in the control group. After incubating for 24 hours at 37 ° C in the _{presence of 5% CO 2} , 10 μM BrdU (Tokyo Chemical Industry, Japan) was added to each group for 24 hours at _{37 ° C in the presence of 5% CO 2.} BrdU was incorporated into newly proliferated cells by incubation. After incubation, dissociate spheres with Accutase Reagent (Gibco, USA) and dissociate into Geltrex Reagent-coated Nunk Lab-Tek chamber slide 4 well (Thermo Fisher Scientific, USA). The hNSC was seeded at a concentration of ^{5.0 × 10 4} _{cells / mL and incubated at 37 ° C. in the presence of 5% CO 2} for 12 hours to adhere the hNSC to the bottom surface of the slide.

After removing the medium from the slides, BrdU and HuC / D positive cells were detected using immunostaining. The cells were lightly washed once with 1 × PBS, then ice-cooled 4% PFA was added, and the cells were fixed at room temperature for 30 minutes. After fixation, the cells were washed twice with 1 × PBS after removing PFA, and then 2N HCl was added to denature a part of the double-stranded DNA into short-stranded DNA to expose BrdU and bind to the antibody, and the temperature was room temperature for 30 minutes. Incubated in. After that, HCl was removed, 0.1 M Na ₂ B ₄ O ₇ (pH = 8.5) was added, and the acid was neutralized by incubating at room temperature for 15 minutes. After removing Na ₂ B ₄ O ₇ and washing the cells twice with 1 × PBS, add 0.2% Triton X-100 (Sigma-Aldrich, USA) solution and incubate for 5 minutes at room temperature to perform membrane permeation treatment. went. After washing the cells with 1 × PBS three times except for Triton X-100, 5% normal goat serum (Funakoshi, Japan) solution was added and incubated for 1 hour at room temperature to prevent non-specific antibody binding. After removing Normal serum, rabbit monoclonal anti-HuC + HuD (HuC / D, 1: 500, Abcam, Cambridge, UK) and mouse monoclonal anti-BrdU (1: 200, A solution in which Invitrogen, Carlsbad, CA) was suspended was added, and overnight (16 hours) incubation was performed at 4 ° C. After incubation, the cells were washed 3 times with 1 × PBS except for the primary antibody solution, and then the secondary antibody (Alexa Fluor 488 and Alexa Fluor 594 labeled, 1: 500, abcam, UK) was added to the 1% Normal goat serum solution. Suspended solution was added and incubated for 1 hour at room temperature. After removing the secondary antibody solution and washing the cells three times with 1 × PBS, the cells were encapsulated using ProLong (trademark registration) Diamind (Thermo Fisher Scientific, USA) and a cover glass. After encapsulation, the slide glass was stored at 4 ° C and used for analysis using a microscope.

The stained cells were image-analyzed using a confocal microscope TCS SP8 (Leica, Germany) (magnification = 40 ×, zoom = 0.8, scale bar = 100 μm). Images were acquired by randomly selecting 8 to 10 sections for each well, and the acquired images were analyzed using ImageJ. The values obtained by the analysis were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
In the above experiment, treatment with sugarcane shoot head extract did not change the proportion of HuC / D-positive cells, but the proportion of BrdU-positive cells showed an increase in a treatment concentration-dependent manner (Fig. 17). The proportion of both HuC / D / BrdU-positive cells showed a treatment concentration-dependent increase (Fig. 17). From this result, it was suggested that the sugarcane treetop head extract had an effect of promoting the proliferation of neural stem cells and an effect of promoting the generation of newborn neurons from the neural stem cells.

<Mechanism analysis of the effect of inducing newborn neuron generation by sugarcane treetop extract>
In order to analyze the mechanism of the effect of inducing newborn neuron generation by the sugar cane shoot head extract, we performed gene expression analysis of ASCL1 and HES1, which are factors that regulate neural stem cell activation, using real-time RT-PCR. ASCL1 and HES1 are basic helix-loop-helix (bHLH) factors that control fate decisions in cell development and differentiation. In the differentiation of nerve cells, the expression level of HES1 is high and the expression level of ASCL1 is kept low in the state where the stem cell property is maintained (≈ dormant state). When the neural stem cells are activated, the expression level of HES1 decreases and the expression level of ASCL1 increases, so that the neural stem cells differentiate into nerve cells. After performing an experiment similar to the method described in <Evaluation of the effect of sugar cane shoot head extract on the generation of newborn neurons from hNSC>, dissociated hNSC was subjected to 6-well play at a concentration of ^{5.0 × 10 4 cells / mL.} The seeds were seeded and _{incubated at 37 ° C. in the presence of 5% CO 2} for 12 hours to adhere hNSC to the bottom of the slide. After that, mRNA was extracted using the ISOGEN kit (NipponGene, Japan), and gene expression was analyzed in the same manner as described in the above-mentioned <Evaluation of the effect of sugar cane shoot head extract on gene expression of differentiation markers in hNSC>. Was carried out. ASCL1 (Hs00269932_m1) and HES1 (Hs00172878_m1) were used in the reaction as primers, and GAPDH (Hs02786624_g1) was used as an internal standard. All primers were purchased from Applied Biosystems (USA). The obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
In the above experiment, ASCL1 expression showed an increase in the sample treatment group, and HES1 expression showed a treatment concentration-dependent decrease (Fig. 17). This result suggests that sugarcane shoot head extract activates neural stem cells and induces the production of newborn neurons by regulating ASCL1 and HES1 gene expression.

<Evaluation of the effect of sugarcane treetop extract on hNSC differentiation>
The effect of sugar cane head extract on hNSC differentiation was evaluated by inducing differentiation with hNSC adhered to the bottom of the slide and then labeling neurons and astrocytes using immunostaining. As treatment groups for hNSC, (i) control group, (ii) 10 μg / mL sugar cane head extract treatment group, (iii) 25 μg / mL sugar cane head extract treatment group, (iv) 50 μg / mL sugar cane A treetop extract treatment group was set. After culturing hNSC together with the medium in a 6-well plate and confirming the formation of spheres (diameter <= 1 mm), in the sugar cane shoot head extract treatment group, a sample diluted so that the concentration in the medium became the above-mentioned set concentration. Was added, and an equal amount of medium was added in the control group. After a 24-hour incubation at 37 ° C. in the _{presence of 5% CO 2,} the spheres were dissociated with Accutase® Reagent (Gibco, USA) and coated with Geltrex® Reagent. -The dissociated hNSC was seeded in a Tek chamber slide 4 well (Thermo Fisher Scientific, USA) at ^{a concentration of 5.0 × 10 4} cells / mL. Differentiation of hNSC was performed in KnockOut ^TM D-MEM / F-12 with 2% StemPro® Neural Supplement, 2 mM GlutaMAX ^TM- I Supplement, 6 units / mL Heparin, and 200 μM. It was induced by culturing in a medium for neural stem cell differentiation composed of Ascorbic acid (all of which are Gibco® life technologies) at 37 ° C. in the _{presence of 5% CO 2 for 7 days.} Further, even during the induction of differentiation, the sample was added to the medium so as to have the above-mentioned set concentration.

After culturing for 7 days, the medium was removed from the slides, and then Tuj1 (Tublin beta III) and GFAP-positive cells were detected using immunostaining. Immunostaining was performed in the same manner as described in <Evaluation of the effect of sugarcane shoot head extract on the generation of newborn neurons from hNSC> above (no degeneration treatment was performed). Rabbit polyclonal anti-GFAP (1: 1000, Novus Biologicals, Centennial, CO) and mouse monoclonal anti-beta III tubulin (Tuj1, 1: 1000, Abcam, UK) were used as primary antibodies. After staining, images were acquired by randomly selecting 8 to 10 sections for each well using a confocal microscope TCS SP8 (Leica, Germany) (magnification = 40 ×, zoom = 0.9, scale bar = 100 μm). The acquired image was analyzed using Image J. The values obtained by the analysis were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
In the above experiment, treatment with sugar cane shoot head extract did not change the proportion of astrocytes (GFAP-positive cells), but the proportion of neurons (Tuj1-positive cells) showed a treatment concentration-dependent increase ( Figure 19). In addition, an extension of the total length of astrocytes (GFAP-positive cells) was observed under treatment with sugarcane shoot head extract (Fig. 18). From this result, it was suggested that the treatment of sugarcane treetop head extract induces neuronal differentiation from neural stem cells and promotes the development of astrocytes after differentiation.

<Quantification of newborn neurons in mouse brain>
After the behavioral test was completed (see 3-2. Experimental method), the brain was extracted from the mouse for immunohistochemical analysis, fixed at 4 ° C with 4% paraformaldehyde for 14 days, and then 30% sucrose / PBS (see 3-2. Experimental method). Equilibration was performed at 4 ° C for 48 hours at w / v). Brain tissue sections (30 μm) were fractionated using a SM2010R sliding microtome (Leica). Sections were stored at -20 ° C in antifreeze solution (ethylene glycol, glycerol, 0.1 M phosphate buffer, pH 7.4) until use. The tissue section on the slide was washed with PBS, and a part of the double-stranded DNA was denatured into short-stranded DNA to expose BrdU and bound to the antibody. It was conducted. Then, permeabilization was performed with PBS containing 0.03% Triton X-100 (PBS-T) for 15 minutes, and the sections were incubated with 10% Normal donkey serum in PBS-T at room temperature for 1 hour to bind non-specific antibodies. Was prevented. Rat monoclonal anti-BrdU (1: 200, Abcam) and Rabbit polyclonal anti-Doublecortin (DCX, 1: 200, Cell signaling technology) were used as primary antibodies, diluted with blocking buffer, and then incubated overnight. rice field. Then, the sections were washed and incubated with a fluorescent dye-labeled specific secondary antibody at room temperature for 2 hours. The slides were encapsulated using FluorSave (Merck Millipore).

The stained tissue section was used for image acquisition with a Nikon Ti-Eclipse microscope (Nikon). Confocal images for quantification were acquired with a Zeiss LSM710 laser scanning confocal microscope (Leica). The analysis of the hippocampal dentate gyrus, which is the site of neurogenesis, was performed using ImageJ.

<Experimental results>
As a result of the above experiment, in the hippocampal dentate gyrus site in the mouse brain to which the sugar cane shoot head extract was administered, BrdU, which is a marker for newborn cells, and DCX, which is a marker for immature neurons, are both positive as compared with the target group. An increase in the number of cells (about 1.6 times) was confirmed (Fig. 19). From this result, it was suggested that the administration of sugarcane treetop extract promoted neurogenesis in the mouse brain.

<Evaluation of TrkB gene (NTRK2 as gene name) expression in SH-SY5Y cells by sugar cane shoot head extract or compound standard>
The results of microarray analysis suggested that sugarcane shoot head extract activates TrkB (Neurotrophic tyrosine kinase receptor type 2, NTRK2) in mouse brain. TrkB is involved in the development and maturation of the central and peripheral nervous systems, and is activated by in vivo factors such as BDNF (brain-derived neurotrophic factor) for nerve cell growth and survival, synaptic development, and neurogenesis in the hippocampus. It has been reported to activate the signal pathways involved. In order to evaluate the effect of sugar cane shoot head extract or compound standard on TrkB gene expression in neurons, gene expression analysis using real-time RT-PCR was performed. SH-SY5Y was used as a human nerve model cell, and (i) control group, (ii) 3-CQA (0.50 μM), (iii) 5-CQA (0.70 μM), and (iv) 3-FQA (0.85) were used as treatment groups. μM), (v) ISO (0.477 μM), (vi) compound mixture (3-CQA + 5-CQA + 3-FQA + ISO), (vii) sugar cane treetop extract (50 μg / mL) were set. .. In this experiment, Ginkgo biloba extract was used as a positive control. Ginkgo biloba extract (Fujifilm Healthcare Laboratory Co., Ltd.) dissolves powder in 70% ethanol, sterilizes 0.22 μm, prepares a stock solution (100 mg / mL), and then treats group (viii) Ginkgo biloba. It was added to the experimental conditions as an extract (50 μg / mL). After incubating the cells together with the sample in a 60 mm cell culture dish (BD Falcon) at 37 ° C. in the presence of 5% CO2 for 24 hours, mRNA was used from the cells in each treatment group using the ISOGEN kit (Nippon Gene, Japan). Was extracted.
Using the extracted mRNA, reverse transcription polymerase chain reactions (RT-PCR) were performed using the Superscript IV reverse transcriptase kit (Invitrogen, USA) and the 2720 Thermal cycler (Applied Biosystems, USA). TaqMan real time RT PCR amplification reactions were performed using the 7500 Fast Real Time PCR system to quantify transcripts. NTRK2 (Hs00178811_ma) was used as a primer in the reaction, and GAPDH (Hs02786624_g1) was used as an internal standard. All primers were purchased from Applied Biosystems (USA). The obtained values were subjected to a significant difference test (* P <0.05, ** P <0.01) by the One-way ANOVA method.

<Experimental results>
Treatment of sugarcane shooter head extract for 24 hours significantly increased the expression of NTRK2 in SH-SY5Y cells compared to the control group (Fig. 20). In addition, in the test using each compound standard product, only ISO treatment showed a significant increase compared to the control group in the single treatment group, and in the compound mixed treatment group, NTRK2 was larger than that in the single treatment group of each compound. It showed increased expression (Fig. 20). The Ginkgo biloba extract-treated group as a positive control showed a significant increase in NTRK2 expression compared with the control group, but the Ginkgo biloba extract-treated group and the compound mixture-treated group showed a significant increase in NTRK2 expression compared with the Ginkgo biloba extract-treated group. It showed a large increase in NTRK2 expression (Fig. 20).

Under treatment with 3-CQA (0.50 μM), 5-CQA (0.70 μM), and 3-FQA (0.85 μM) alone, no change in NTRK2 expression was shown, and with ISO (0.477 μM) single treatment. In comparison, a large increase in NTRK2 expression (approximately 220% increase) was confirmed under compound mixture treatment. In this result, the NTRK2 expression level is a relative value of the control group, and this suggests that these compounds synergistically increase NTRK2 expression in nerve cells when mixed and treated. ..

In this experiment, the sugar cane treetop extract-treated group and the compound mixture-treated group showed a large increase in NTRK2 expression as compared with the ginkgo biloba extract-treated group. Since signal transduction via TrkB activation plays an important role in promoting synaptic plasticity and neurogenesis (Numakawa et al), sugar cane treetop extract has been reported to have a cognitive function improving effect. Compared with ginkgo biloba extract, which is a plant extract of Synaptic plasticity, it can be expected to improve symptoms by effectively improving TrkB signal, promoting neurogenesis, and suppressing the decrease of nerve cells with aging. Specific diseases include memory loss and depression in aging and Alzheimer's disease (Tapia-Arancibiaet al., 2008; The improvement effect in Erickson et al., 2012) can be expected.

<References>
Numakawa T, Odaka H, Adachi N. “Actions of Brain-Derived Neurotrophin Factor in the Neurogenesis and Neuronal Function, and Its Involvement in the Pathophysiology of Brain Diseases.” Int J Mol Sci. 2018.
Tapia-Arancibia L, Aliaga E, Silhol M, Arancibia S. “New insights into brain BDNF function in normal aging and Alzheimer disease.” Brain Res Rev. 2008.
Erickson KI, Miller DL, Roecklein KA. “The aging hippocampus: interactions between exercise, depression, and BDNF.” Neuroscientist. 2012.

[9. Examination of component extraction efficiency by chemical analysis using 30-50% ethanol as extraction solvent]
In the above test, the content of each polyphenol component per 100 parts by weight of the extract was the highest among the samples extracted using 40% ethanol as a solvent. From this result, in order to examine the extraction conditions using a finer concentration setting, different concentrations of 30%, 35%, 40%, 45%, and 50% as extraction solvents from 1 g of the dried sugar cane headtop, respectively. The components were extracted using ethanol. The ethanol concentration is based on the volume (v / v). An automatic extraction device E-916 (Nippon Buch) was used for extraction, and the extraction conditions were set to temperature = 45 ° C, pressure = 100 bar, extraction time = 10 minutes, and number of extractions = 3. The solution after extraction was concentrated using a rotary evaporator and then freeze-dried to obtain a powdery sample (the color of the sample became deeper green as the ethanol concentration increased). The obtained sample was redissolved in 100% methanol (concentration: 50 mg / mL), sterilized by a 0.22 μm filter, and then used for quantitative analysis of the components using high performance liquid chromatography (HPLC).

<Analysis result>
The quantitative results obtained by HPLC are shown in FIG. As a result of quantitative analysis, the content of each polyphenol component per 100 parts by weight of the extract was the highest among the samples extracted using 30% ethanol as a solvent.

By using the composition of the present invention, the related markets will be expanded and new markets will be expanded through the development of new food and pharmaceutical seeds for the purpose of improving and improving cognitive function and the development of products in the form of supplements and the like. Pioneering is expected. In the medical / health field, further soaring medical costs are a major concern due to the forecast of an increase in the aging population, and since the composition of the present invention is derived from food by-products, it is a major innovation in the medical / health market. Is expected to be.

Claims

A food containing at least one selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin, comprising the following steps: Material or pharmaceutical material manufacturing method:
At least one selected from the group consisting of 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, 3-o-ferloyl quinic acid, and isoorientin from the head of sugar cane using an aqueous solvent. The process of obtaining a fraction containing.
The production method according to claim 1, which is a method for producing a food material or a pharmaceutical material containing at least isoorientin.
The production method according to claim 1 or 2, wherein the aqueous solvent is 25 to 40% ethanol.
The manufacturing method according to any one of claims 1 to 3, wherein the food material or the pharmaceutical material is for regulating nerve function.
Food or pharmaceutical materials protect nerve cells from amyloid β, antioxidant, anti-inflammatory, increase secretion of neurotransmitters in the brain, promote ATP production in nerve cells, promote neurogenesis by proliferation of neural stem cells, nerves It is for any one selected from the group consisting of induction of transition of stem cells from dormant state to activated state, induction of differentiation of neural stem cells into neurons, and promotion of astrocyte development. The manufacturing method according to any one of 4 to 4.
Food or pharmaceutical materials are caused by senile dementia, Alzheimer's dementia, cerebrovascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, normal pressure cerebral edema Treatment of dementia, including dementia, post-dementia dementia, and Parkinson-type dementia; Treatment of non-dementia cognitive impairment, including mild cognitive impairment (MCI) and cognitive decline due to aging; Improvement of learning disorders, improvement of memory disorders; improvement of learning ability, improvement of memory ability; treatment of memory deterioration; treatment of cerebral infarction and peripheral nerve injury; attention defect hyperactivity disorder (ADHD), depression, and bipolar Any one of claims 1-4, for any one selected from the group consisting of treatment of mental disorders, including sexual disorders; increased motivation and motivation; and improved circadian rhythm disturbances. The manufacturing method described in.
A food composition or a pharmaceutical composition for regulating nerve function, which contains either luteolin or a glycoside thereof.
The composition according to claim 7, further comprising any of the chlorogenic acids represented by the following formula.

(In the formula, R 1 , R 2 , and R 3 are independently H, cafe oil groups, or ferroyl groups.)
The composition according to claim 8, which comprises isoorientin, 3-o-cafe oil quinic acid, 5-o-cafe oil quinic acid, and 3-o-ferloyl quinic acid.
The composition according to any one of claims 7 to 9, wherein any of luteolin and its glycosides, and any of chlorogenic acids are contained as a sugarcane treetop head extract.
Protection of nerve cells from amyloid β, increased secretion of neurotransmitters in the brain, promotion of ATP production in nerve cells, promotion of neurogenesis by proliferation of neural stem cells Induction of neural stem cell transition from dormant state to activated state, The composition according to any one of claims 7 to 10, for any one selected from the group consisting of induction of differentiation of neural stem cells into neurons and promotion of astrocyte development.
Senile dementia, Alzheimer-type dementia, cerebrovascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebral edema, after meningitis Treatment of dementia, including dementia and Parkinson-type dementia; Treatment of non-dementia cognitive impairment, including mild cognitive impairment (MCI) and cognitive decline due to aging; Improvement of learning impairment, memory impairment Improvement of learning ability, improvement of memory ability; Treatment of memory loss; Treatment of cerebral infarction and peripheral nerve injury; The composition according to any one of claims 7 to 10, for any one selected from the group consisting of improved motivation / motivation and improved circadian rhythm disorder.