JP7642212B2 - Composition for regulating neural function - Google Patents

Description

The present invention relates to a composition useful as a food or medicine, comprising any one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin (luteolin-6-C-glucoside).

Polyphenols are natural compounds found widely in nature and are known to have various physiologically active functions. Among them, chlorogenic acid, which is found in coffee beans and potatoes, has been reported to exhibit physiological activities such as antioxidant activity, suppression of blood sugar rise, and anti-obesity activity. In recent years, the effect of chlorogenic acid on neurological function has been studied, and it has been reported that it improves autonomic nervous function, recovers from cerebral fatigue, and improves cognitive function (Patent Documents 1 to 4).

It is known that sugarcane contains polyphenols such as chlorogenic acid. The stalks of sugarcane, which contain a high amount of sucrose, are used as raw materials for sugar production, and the tops (the part of the ear above the thickened zone of the fifth leaf) are removed during harvesting and are often used only as cattle feed. However, based on the discovery that sugarcane polyphenols are present in high concentrations in the ear extract, a method for producing a product containing sugarcane polyphenols from sugarcane ears has been proposed (Patent Document 5). In addition, the antioxidant components in the tops of sugarcane have been identified as chlorogenic acid (5-o-caffeoylquinic acid) and neochlorogenic acid (3-o-caffeoylquinic acid) (Non-Patent Document 1). It has also been proposed to produce polyphenol-rich brown sugar by adding the tops of sugarcane to the raw material for brown sugar (Non-Patent Document 2). In addition, isoorientin is known to be contained in millet and rooibos tea (an extract of the leaves of Aspalathus linearis), and it has recently been reported that oral administration of isoorientin improved scopolamine-induced cognitive impairment in mice (Non-Patent Document 3).

On the other hand, the number of dementia patients is increasing worldwide, and in Japan, the number of dementia patients and those at risk of dementia is increasing year by year due to the aging of the population. Symptomatic therapy using chemically synthesized pharmaceutical agents is generally used to treat or improve lifestyle-related diseases such as dementia. However, medical treatment using these pharmaceutical agents poses challenges in terms of the large dosage and discomfort that are a burden to patients, as well as the associated side effects. Therefore, it is expected that functional foods and preventive pharmaceutical agents derived from natural products that have few side effects and are highly safe and suitable for daily intake are developed. In this regard, the present inventors have proposed an agent for preventing or treating Alzheimer's dementia that has at least caffeoylquinic acid (such as di-o-caffeoylquinic acid) having a structure in which two or more caffeic acids are ester-bonded (Patent Document 6).

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

Okinawa Prefectural Agricultural Research Center Research Bulletin 4:52-57, 2010 Okinawa Prefectural Agricultural Research Center Research Bulletin 12:14-20, 2018 Arch. Pharm. Res. 42:722-31, 2019

The present inventors have conducted research into the functional analysis and effective utilization of food resources. The present invention aims to provide a novel material derived from natural products that is useful for regulating neural function.

The present invention provides the following:
[1] A method for producing a food material or pharmaceutical material containing at least one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin, comprising the steps of:
A process for obtaining a fraction containing at least one member selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin from sugarcane tops using an aqueous solvent.
[2] The method according to 1, which is a method for producing a food material or pharmaceutical material containing at least isoorientin.
[3] The method according to 1 or 2, wherein the aqueous solvent is 10 to 90% ethanol, preferably 25 to 40% ethanol.
[4] The manufacturing method described in any one of 1 to 3, wherein the food material or pharmaceutical material is for regulating nerve function.
[5] The method for producing a food material or pharmaceutical material according to any one of items 1 to 4, wherein the food material or pharmaceutical material is for any one of the purposes selected from the group consisting of protecting nerve cells from amyloid beta, increasing the secretion of neurotransmitters in the brain, promoting ATP production in nerve cells, promoting neurogenesis by proliferation of neural stem cells, inducing the transition of neural stem cells from a dormant state to an activated state, inducing differentiation of neural stem cells into neurons, and promoting the development of astrocytes.
[6] The method for producing any one of items 1 to 4, wherein the food material or pharmaceutical material is for any one selected from the group consisting of treatment of dementia, including senile dementia, Alzheimer's dementia, vascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebral edema, post-meningitic dementia, and Parkinson's dementia; treatment of non-dementia cognitive impairment, including mild cognitive impairment (MCI) and decline in cognitive function due to aging; improvement of learning disabilities, improvement of memory impairment; treatment of memory decline; treatment of cerebral infarction and peripheral nerve damage; improvement of learning ability, improvement of memory ability; treatment of mental disorders, including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder; improvement of motivation, and improvement of circadian rhythm disorders.
[7] A food composition or pharmaceutical composition for regulating nerve function, comprising luteolin or one of its glycosides.
[8] The composition according to 7, further comprising any one of chlorogenic acids represented by the following formulas:

(In the formula, R ¹ , R ² , and R ³ are each independently H, a caffeoyl group, or a feruloyl group.)
[9] The composition according to 8, comprising isoorientin, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and 3-o-feruloylquinic acid.
[10] The composition according to any one of items 7 to 9, wherein luteolin or any of its glycosides, and any of chlorogenic acids are contained as an extract from sugarcane tops.
[11] The composition described in any one of items 7 to 10, for any one selected from the group consisting of protecting neurons from amyloid beta, increasing secretion of neurotransmitters in the brain, promoting ATP production in neurons, promoting neurogenesis by proliferation of neural stem cells, inducing the transition of neural stem cells from a dormant state to an activated state, inducing differentiation of neural stem cells into neurons, and promoting the development of astrocytes.
[12] The composition according to any one of items 7 to 10, for any one selected from the group consisting of treatment of dementia, including senile dementia, Alzheimer's dementia, vascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebral edema, post-meningitic dementia, and Parkinson's dementia; treatment of non-dementia cognitive impairment, including mild cognitive impairment (MCI) and decline in cognitive function 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 nerve damage; treatment of mental disorders, including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder; improvement of motivation, and improvement of circadian rhythm disturbance.

The present invention makes it possible to produce food or pharmaceutical materials from natural products that contain ingredients that have a neuromodulatory effect.

The food materials, pharmaceutical materials, and food compositions or pharmaceutical compositions using them obtained by the present invention can regulate nerve functions.

Component analysis of sugarcane top extract. Component analysis by HPLC confirmed four main peaks, each of which was derived from (1) 3-o-caffeoylquinic acid, (2) 5-o-caffeoylquinic acid, (3) 3-o-feruloylquinic acid, and (4) isoorientin (luteolin-6-C-glucoside). Effect of sugarcane top head extract on amyloid beta-induced neuronal cell death. To evaluate the neuroprotective effect of sugarcane top head extract, an experiment was conducted using human neuronal model cells SH-SY5Y and the neurotoxin amyloid beta (Aβ). The amyloid beta-treated group showed a 40% decrease in cell viability compared to the control group, whereas the amyloid beta + sugarcane top head extract co-treated group showed a concentration-dependent significant increase in cell viability compared to the amyloid beta-only treated group. Evaluation of four compounds contained in sugarcane top extract at the cellular level. Cell viability and ATP production tests were performed using SH-SY5Y cells. The concentrations of each compound were the same as those contained in 50 μg/mL of top extract (3-CQA: 0.50 μM, 5-CQA: 0.70 μM, 3-FQA: 0.85 μM, isoorientin: 0.477 μM). Compared to Aβ treatment alone, co-treatment with each compound and Aβ tended to increase cell viability (top). In addition, treatment with each compound tended to increase intracellular ATP production (bottom). 3-CQA: 3-o-Caffeoylquinic acid, 5-CQA: 5-o-Caffeoylquinic acid, 3-FQA: 3-o-Feruloylquinic acid, ISO: Isoorientin (same in the following figures) Analysis of the mechanism of neuroprotection by sugarcane top head extract. Amyloid-β treatment significantly decreased gene expression of MAP2K4, MAPK14, MAPK8, PI3KCA, AKT1, and PARP1 in SH-SY5Y cells, and treatment with sugarcane top head extract significantly increased the expression of the above genes. Furthermore, amyloid-β treatment significantly increased gene expression of CASP3 (Caspase-3), a gene related to apoptosis (cell death), in SH-SY5Y cells, and treatment with sugarcane top head extract decreased the expression. Evaluation of spatial learning and memory ability using the Morris water maze test. In the sugarcane top head extract group, a significant reduction in the time it took to reach the platform was observed from the 5th day of the experiment compared to the SAMP8 water group (top). In addition, as a result of the probe test, the sugarcane top head extract group showed a significant increase in the number of times they crossed the platform area compared to the water group (middle), and they also showed a tendency to spend more time in the platform area (bottom). A group of genes related to brain function that were found to have altered expression as a result of comprehensive genetic analysis using microarrays. Presumed pathways as a result of comprehensive genetic analysis using microarrays. Analysis of brain neurotransmitter concentrations by ELISA. The levels of dopamine and noradrenaline in the cerebral cortex were significantly increased in the SAMP8 + sugarcane top head extract group compared to the SAMP8 + water group. The levels of acetylcholine and serotonin also showed a tendency to increase. Extraction efficiency of components from sugarcane tops. Extraction of components was performed from 1 g of dried sugarcane tops using ethanol of different concentrations, 20%, 40%, 60%, and 80%, as the extraction solvent. Quantitative analysis showed that the content of each polyphenol component per 100 parts by weight of the extract was highest in the sample extracted using 40% ethanol as the solvent. Comparison of physiological activity of sugarcane top head extract on nerve cells. Each sample obtained by the above method was redissolved in 70% ethanol and used in a cell viability test using human nerve model SH-SY5Y cells. A concentration-dependent significant increase in cell viability was confirmed in the amyloid beta (Aβ) + sugarcane top head extract treatment group compared to the Aβ only treatment group. In addition, cell viability was highest in the Aβ + sugarcane top head extract (solvent 40% ethanol) treatment group. Effects of four compounds contained in sugarcane top extract on the viability of nerve cells. n3-CQA, 5-CQA, 3-FQA and ISO were dissolved in 70% ethanol and used in a cell viability test using SH-SY5Y cells. 3-CQA, 5-CQA and ISO showed a significant increase in the viability of SH-SY5Y cells in a concentration-dependent manner. The effect of four compounds contained in sugarcane top extract on ATP production in nerve cells. Using the above four compounds, an ATP production test was performed using SH-SY5Y cells. 3-CQA, 5-CQA and ISO showed a significant increase in the ATP production rate in SH-SY5Y cells. Effect of sugarcane tops extract and other compounds on the gene expression of glycolytic enzymes. To evaluate the effect of sugarcane tops extract or four compounds contained in sugarcane tops extract on the gene expression of enzymes that catalyze chemical reactions in glycolysis (PGK1, PGAM1, PKM, PC), gene expression analysis was performed in SH-SY5Y cells. ACTB was used as an internal standard. After 24 hours of treatment with sugarcane tops extract or standard compounds, the expression of PGK1, PGAM1, PKM, and PC was significantly increased compared to the control group. Mechanism of promoting ATP production in neurons suggested by the results of gene expression analysis of glycolytic enzymes. Photo of human fetal neural stem cells (hNSCs). In neural stem cell proliferation medium, hNSCs float without adhering to the bottom of the flask and proliferate while forming spherical cell clusters (neurospheres). Gene expression analysis of NES (stem cell marker) in neurospheres. Using sugarcane top head extracts, expression analysis was performed on human fetal neural stem cells for TUBB3, a marker for neurons (nerve cells), GFAP, a marker for astrocytes or transit amplifying (TA) cells (limitedly proliferating cells intermediate between stem cells and differentiated cells), PDGFRA, a marker for oligodendrocytes, and NES, a marker for neural stem cells. GAPDH was used as an internal standard. Treatment with sugarcane top head samples for 24 hours resulted in a treatment concentration-dependent increase in gene expression of TUBB3 and GFAP. In addition, a treatment concentration-dependent decrease in gene expression of PDGFRA and NES was observed. Quantitative analysis of BrdU and HuC/D (neural progenitor cell markers) positive cells using immunostaining, and gene expression analysis of ASCL1 and HES1, which are neural stem cell activation regulators. A. Image of fluorescence analysis, B. Percentage of neural progenitor cells, C. Percentage of newly generated cells, D. Percentage of newly generated neural progenitor cells, E. Gene expression levels of ASCL1 and HES1. Effect of sugarcane top extract on hNSC differentiation. Treatment with sugarcane top extract did not change the percentage of astrocytes (GFAP-positive cells), but showed a concentration-dependent increase in the percentage of neurons (Tuj1-positive cells). In addition, the extension of the total length of astrocyte (GFAP-positive cells) processes was observed under treatment with sugarcane top extract. Quantification of newborn neurons in mouse brains using immunostaining. The sugarcane top head extract group showed an increase (approximately 1.6-fold) in the number of neurons (both BrdU and DCX positive cells) compared to the SAMP8 + water group. Comparison of NTRK2 expression in nerve cells by sugarcane top head extract and compounds. NTRK2 expression in SH-SY5Y cells was significantly increased in the sugarcane top head extract and compound mixture treatment group compared to the control group and the compound alone treatment group. Furthermore, the sugarcane top head extract treatment group and compound mixture treatment group showed a large increase in NTRK2 expression compared to the ginkgo biloba leaf extract treatment group used as a positive control. A study on the efficiency of component extraction using organic solvents of different concentrations. Using 30-50% ethanol as the extraction solvent, the efficiency of component extraction was examined by chemical analysis. Quantitative analysis using HPLC showed that the content of polyphenol components per 100 parts by weight of the extract was highest in the sample extracted with 30% ethanol.

The present invention relates to a method for producing a food material or pharmaceutical material containing at least one member selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin as a functional ingredient, the method comprising the steps of:
A process for obtaining a fraction containing at least one member selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin from sugarcane tops using an aqueous solvent.
The present invention also relates to a food composition or pharmaceutical composition comprising, as active ingredients, any one of luteolin and its glycosides, and any one of the chlorogenic acids described below.

[Functional ingredients, active ingredients]
The food materials or pharmaceutical materials, or food compositions or pharmaceutical compositions related to the present invention (hereinafter, the food materials, pharmaceutical materials, food compositions, and pharmaceutical compositions related to the present invention may be collectively referred to as the "compositions, etc. of the present invention") contain chlorogenic acids as functional ingredients or active ingredients.

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

wherein R ¹ , R ² , and R ³ are each independently H, a caffeoyl group, or a feruloyl group.

Examples of chlorogenic acids include 3-o-caffeoylquinic acid, 4-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, 4-o-feruloylquinic acid, 5-o-feruloylquinic acid, 3,4-di-o-caffeoylquinic acid, 3,5-di-o-caffeoylquinic acid, and 4,5-di-o-caffeoylquinic acid.

In one preferred aspect, the composition of the present invention contains, as a chlorogenic acid, any one selected from the group consisting of 3-o-caffeoylquinic acid (R ¹ = caffeoyl group, R ² = R ³ = H), 5-o-caffeoylquinic acid (R ³ = caffeoyl group, R ¹ = R ² = H), and 3-o-feruloylquinic acid (R ¹ = feruloyl group, R ² = R ³ = H).

It is preferable that the compositions of the present invention contain at least one chlorogenic acid selected from the group consisting of the above three compounds, more preferably at least two selected from the group, and even more preferably all three.

<Luteolin or its glycoside>
The composition of the present invention contains luteolin or its glycoside as a functional ingredient or active ingredient. The glycoside of luteolin refers to a compound in which a sugar is glycosidically bonded to luteolin (3',4',5,7-tetrahydroxyflavone). Examples of sugars include glucose, galactose, fructose, glucuronic acid, rhamnose, xylose, arabinose, apiose, rutinose, gentiobiose, primeverose, and diglycerol. Examples of glycosides of luteolin include luteolin 4'-o-glucoside, luteolin 4'-o-glucuronide, luteolin 5-glucuronide, luteolin 5-glucoside, luteolin 5-rutinoside, luteolin 5-o-glucuronide, luteolin 6-glucoside (isoorientin), luteolin 6-c-β-d-glucopyranoside, luteolin 8-c-α-l-arabinopyranoside, luteolin 6-c-arabinoside, luteolin 7-(2-o-apiosylglucoside), ... Luteolin 7-[6-o-(2-methylbutyryl)-β-glucoside], Luteolin 7-o-[2-o-(4-o-acetyl-α-rhamnopyranosyl)-β-glucuronopyranoside], and Luteolin 8-glucoside (orientin).

In one preferred embodiment, the composition of the present invention contains a compound represented by the following formula as luteolin or its glycoside.

In the formula, R ⁴ and R ⁵ are each independently H or a sugar residue.

In one particularly preferred embodiment, the compositions of the present invention contain luteolin or its glycoside selected from the group consisting of isoorientin ( ^R4 = H, ^R5 = glucose residue) and orientin ( ^R4 = glucose residue, ^R5 = H).

The compositions of the present invention contain at least one luteolin selected from the group consisting of the above two compounds, and it is more preferable that they contain isoorientin.

<Four types of compounds contained in sugarcane tops>
In a particularly preferred embodiment, the composition of the present invention contains 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and 3-o-feruloylquinic acid as chlorogenic acids, and also contains isoorientin, a glycoside of luteolin. The composition containing all four of these components may be the sugarcane top extract itself, or a composition containing the extract as an active ingredient. This application is the first to report that sugarcane tops contain these four compounds.

According to the inventors' research, when extraction is performed under appropriate conditions, 100 mg to 170 mg, more specifically 130 mg to 140 mg, of extract containing the four target compounds can be obtained from 1 g of dried sugarcane tops (including leaves and bark) in dry weight.

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

The amount of 5-o-caffeoylquinic acid contained in the sugarcane top extract provided by the present invention can be 1.0 mg or more per 100 g (dry weight) of extract, regardless of the contents 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 contents of other components.

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

The amount of isoorientin contained in the sugarcane top extract provided by the present invention can be 0.80 mg or more per 100 g (dry weight) of extract, regardless of the contents 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 contents of other components.

In one preferred embodiment, the amount of 3-o-caffeoylquinic acid contained in 100 g (dry weight) of sugarcane top extract is 0.40 mg or more and 0.60 mg or less, the amount of 5-o-caffeoylquinic acid is 2.5 mg or more and 3.5 mg or less, the amount of 3-o-feruloylquinic acid is 0.20 mg or more and 0.30 mg or less, and the amount of isoorientin is 1.4 mg or more and 1.8 mg or less.

In one preferred embodiment, sugarcane tops may contain 2.1-2.6 mg/g (dry weight) of isoorientin. It is also known that isoorientin is contained in rooibos tea. It has been reported that an extract of 10 g of dried green rooibos tea leaves in 500 ml of hot water contains 26 mg of isoorientin (Food Chemistry 128:338-347, 2011).

<Synergistic effect>
In a particularly preferred embodiment, the composition of the present invention contains four compounds, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin, as active ingredients, or contains sugarcane top head extract as an active ingredient. According to the study by the present inventors, treatment with 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and 3-o-feruloylquinic acid alone does not show any change in NTRK2 expression at the concentrations tested, while a mixture of these and isoorientin, and a sugarcane top head extract containing all four compounds can synergistically increase NTRK2 expression in nerve cells. TrkB (Neurotrophic tyrosine kinase receptor type 2, NTRK2) is involved in the development and maturation of the central and peripheral nervous systems, and it has been reported that its activation by endogenous factors such as BDNF (brain-derived trophic factor) activates signaling pathways involved in the growth and survival of nerve cells, synaptic development, and neurogenesis in the hippocampus.

When the composition of the present invention contains four types of compounds, the ratio thereof is not particularly limited as long as the desired effect is obtained. For example, the molar ratio of 3-o-caffeoylquinic acid:5-o-caffeoylquinic acid:3-o-feruloylquinic acid:isoorientin can be 1:0.10-10:0.15-15:0.080-8.0, 1:0.30-3.0:0.40-4.0:0.20-2.0, or 1:0.60-1.5:0.80-2.0:0.40-1.0.

[Sugarcane top extract, its manufacturing method, etc.]
In the present invention, the sugarcane top refers to the tip portion of sugarcane (scientific name: Saccharum officinarum L.) above the thickened zone of the fifth leaf.

The sugarcane tops used for producing the compositions of the present invention may be the entire sugarcane tops, or may be a portion including leaves and bark, or may be a portion excluding leaves and bark. The sugarcane tops may be in a raw state or may be dried. Drying may be performed by cold air drying or sun drying. The sugarcane tops may be cut, chopped, or crushed to increase the extraction efficiency.

The means for extraction from sugarcane tops 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 subcritical fluid.

The extraction solvent is preferably a solvent that is effective for extracting luteolin or its glycosides and is also effective for 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, as well as mixtures of any of these. A preferred example is an aqueous solvent, such as water or a mixture of water and any of the group consisting of methanol, ethanol, isopropanol, butanol, propylene glycol, butylene glycol, and glycerin.

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

The extraction procedure may be carried out at room temperature, but it is preferable to heat the extract under reflux cooling, which allows the components to 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 the extraction is carried out under heating, the extract can be obtained efficiently and with high purity.

Extraction may also be carried out under pressure, at 5 MPa (50 bar) or more, preferably 7.5 MPa (75 bar) or more, or even 10 MPa (100 bar) or more. In this case, heating may also be used to efficiently and quickly extract the desired components. The temperature may be 25°C or more, preferably 30°C or more, more preferably 35°C or more, and even more preferably 40°C or more.

The ratio of raw material to extraction solvent, extraction time, and number of times the extraction operation is repeated can be determined appropriately taking into account the extraction efficiency.

When performing supercritical or subcritical extraction, the fluid that can be used is, for example, water, carbon dioxide, ethylene, propylene, ethane, propane, nitrous oxide, chlorodifluoromethane, chlorotrifluoromethane, xenon, ammonia, and lower alcohols such as methanol and ethanol. From the standpoint of safety, it is preferable to use water, ethanol, a mixture of these, or carbon dioxide.

After extraction, if necessary, insoluble residues can be removed, the mixture can be concentrated by conventional methods, and dried by means of spray drying, freeze drying, etc.

In this invention, unless otherwise specified, the term "extract" includes the extract obtained by extraction from the raw material with a solvent, the concentrate of the extract, the dried product, and the crude product.

[Use]
The composition of the present invention can be used for regulating neural function. The regulating neural function includes protecting nerve cells from amyloid beta, antioxidation (suppressing environmental stress stimuli), anti-inflammation (improving inflammatory conditions), increasing the secretion of neurotransmitters in the brain, promoting ATP production in nerve cells, promoting neurogenesis by proliferation of neural stem cells, treating dementia, improving non-dementia cognitive impairment, improving learning disabilities, improving memory impairment, improving learning ability, improving memory ability, treating mental illness, improving motivation, and improving circadian rhythm disorders.

According to the inventors' studies, sugarcane top extract was able to reduce the decrease in cell viability caused by amyloid-β treatment in SH-SY5Y, a cell line derived from human neuroblastoma.

Therefore, the compositions of the present invention are expected to have a neuroprotective effect against amyloid beta, and are useful for treating diseases or conditions aggravated by amyloid beta. Amyloid beta (Aβ) is a peptide consisting of 40-42 amino acids, which is toxic to nerve cells and causes cell death. In Alzheimer's disease, Aβ aggregates to form insoluble fibers that become amyloid and are deposited in the brain. Diseases or conditions aggravated by amyloid beta include Alzheimer's disease.

Furthermore, according to the inventors' study, amyloid-β treatment significantly decreased the gene expression of MAP2K4 (Mitogen activated protein kinase 4), MAPK14 (Mitogen-activated protein kinase 14), MAPK8 (Mitogen-Activated Protein Kinase 8), PI3KCA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha), AKT1 (AKT Serine/Threonine Kinase 1), and PARP1 (Poly(ADP-Ribose) Polymerase 1) in SH-SY5Y cells, but sugarcane top head extract treatment significantly increased the expression of these genes. Furthermore, amyloid-β treatment significantly increased the gene expression of CASP3 (Caspase-3), a gene related to apoptosis (cell death), in SH-SY5Y cells, while sugarcane top head extract treatment decreased the expression.

The above genes respond to stress stimuli on cells and regulate a wide variety of cellular processes, including cell proliferation, growth, differentiation, transformation, and apoptosis. Because the extract used in this experiment improved the amyloid-β-induced changes in the expression of these genes, it may be useful in suppressing environmental stress stimuli, including oxidative stress, in nerve cells (antioxidation, etc.), and improving the associated inflammatory state (neuroinflammation, etc.) (anti-inflammation).

Furthermore, according to the inventors' research, a Morris water maze test using aged model mice (SAMP8) showed that the group administered sugarcane top head extract showed a significant reduction in the time it took to reach the platform from the fifth day of the experiment compared to the group administered SAMP8 water. Furthermore, a probe test showed that the group administered sugarcane top head extract showed a significant increase in the number of times they crossed the platform location compared to the group administered water, and also showed a tendency for the time they spent in the platform location to increase.

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 may be useful for treating, for example, dementia (including senile dementia, Alzheimer's dementia, vascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematomas, dementia caused by normal pressure cerebral edema, postmeningitis dementia, and dementia caused by various diseases such as Parkinson's dementia). The compositions of the present invention may also be useful for treating non-dementia cognitive disorders such as mild cognitive impairment (MCI) and cognitive decline due to aging. Furthermore, the compositions of the present invention may be useful for improving learning or memory disorders (learning and memory disorders associated with brain development disorders), improving learning ability, and improving memory ability.

Furthermore, according to the inventors' research, the amount of dopamine and noradrenaline in the cerebral cortex was significantly increased in the group administered SAMP8 sugarcane top head extract compared to the group administered SAMP8 water. In addition, the amount of acetylcholine and serotonin showed a tendency to increase.

Therefore, the composition of the present invention containing sugarcane top head extract may be useful for treating diseases or conditions that are improved by increasing the secretion of neurotransmitters in the brain. Furthermore, since the improving effect on reduced dopamine and noradrenaline secretion has been confirmed, it may be useful for treating mental disorders including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder (manic depression, etc.). Furthermore, since the improving effect on reduced dopamine secretion has been confirmed, it may also be useful for improving motivation.

Furthermore, according to the inventors' study, comprehensive gene expression analysis using microarrays showed that the group administered SAMP8 sugarcane top extract showed changes in the expression of many genes compared to the group administered SAMP8 water. In particular, increased expression of the clock gene (Per3) was confirmed, so the composition of the present invention may be useful in improving disruptions to circadian rhythms (body clocks).

In particular, according to the inventors' studies, the SAMP8 group administered sugarcane top head extract did not exceed the normal mouse group in terms of dopamine, noradrenaline, adrenaline, acetylcholine, or serotonin, so it can be expected that the composition of the present invention can be used safely without unnecessarily increasing the secretion of neurotransmitters in the brain.

Furthermore, since norepinephrine is known to play a role in wakefulness and sleep, the compositions of the present invention are also expected to be useful for improving sleep (e.g., improving sleep quality, promoting non-REM sleep, improving sleep rhythm) or treating sleep disorders (e.g., insomnia).

According to the present inventors' study, sugarcane top extract, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin increased the survival rate in SH-SY5Y. Sugarcane top extract, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and isoorientin were also able to promote ATP production. It was suggested that this mechanism was due to activation of the intracellular glycolysis via increased gene expression of phosphoglycerate kinase (PGK1), phosphoglycerate mutase (PGAM1), pyruvate kinase (PKM), and pyruvate carboxylase (PC), which are enzymes involved in glycolysis.

Therefore, the compositions of the present invention can be used to promote ATP production in nerve cells, and can be useful for treating diseases or conditions that are improved by promoting ATP production in nerve cells. Furthermore, they can be useful for treating diseases or conditions that are improved by increased expression of any gene selected from the group consisting of PGK1, PGAM1, PKM, and PC.

According to the present inventors' study, treatment of human fetal neural stem cells (hNSCs) with sugarcane top head extract resulted in a treatment concentration-dependent increase in gene expression of TUBB3 (Tubulin beta III/Tuj1), a neuronal marker in neural stem cells, and GFAP (Glial fibrillary acidic protein), a marker of astrocytes or transit amplifying (TA) cells (limitedly proliferating cells intermediate between stem cells and differentiated cells). This suggests that sugarcane top head extract induces the transition of neural stem cells from a dormant state to an activated state, and induces differentiation of neural stem cells into neurons and astrocytes. In addition, a cell proliferation test using the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) and the neural progenitor cell marker HuC+HuD (HuC/D) showed that treatment with sugarcane top head extract increased the proportion of BrdU-positive cells in a treatment concentration-dependent manner. These results suggest that sugarcane top head extract promotes neural stem cell proliferation. Furthermore, the proportion of HuC/D/BrdU positive cells increased in a treatment concentration-dependent manner. These results suggest that sugarcane top head extract promotes the generation of new neurons from neural stem cells. HuC stands for ELAV (embryonic lethal, abnormal vision, Drosophila)-like 3 (Hu antigen C; HuC), and HuD stands for ELAV-like 4 (Hu antigen D; HuD).

This promotion of the generation of new neurons is believed 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 determination during cell development and differentiation. In neural cell differentiation, when stemness is maintained (≒ dormant state), the expression level of HES1 is high and the expression level of ASCL1 is kept low. When neural stem cells become activated, the expression level of HES1 decreases and the expression level of ASCL1 increases, causing the neural stem cells to differentiate into neurons. According to the inventors' studies, treatment with sugarcane top head extract increased the expression of ASCL1 in hNSCs and decreased the expression of HES1 in a treatment concentration-dependent manner.

According to the present inventors' study, when hNSCs that had formed spheres were treated with sugarcane top head extract and Tuj1 (Tublin beta III) and GFAP-positive cells were detected by immunostaining, the percentage of astrocytes (GFAP-positive cells) did not change, but the percentage of neurons (Tuj1-positive cells) showed a treatment concentration-dependent increase. In addition, the extension of the total length of the processes of astrocytes (GFAP-positive cells) was observed under treatment with sugarcane top head extract. Therefore, sugarcane top head extract can induce neuronal differentiation from neural stem cells (induction of differentiation into neurons can also be rephrased as neurogenesis or promotion of neurogenesis), and promote the development of astrocytes. In addition, in the case of cerebral infarction or peripheral nerve injury, new neural circuits are formed from the new neurons produced by treatment with sugarcane top head extract, and symptoms can be improved by restoring lost neural plasticity.

Furthermore, in the hippocampal dentate gyrus region of the brains of mice administered sugarcane top head extract, an increase (approximately 1.6 times) in the number of cells positive for both BrdU, a marker for newborn cells, and DCX, a marker for young neurons, was confirmed compared to the control group. These results demonstrate that oral administration of sugarcane top head extract has a clear effect in promoting neurogenesis in the animal brain.

Based on the above, the compositions of the present invention may be useful for promoting neurogenesis through the proliferation of neural stem cells. They may also be useful for treating diseases or conditions that are improved by promoting neurogenesis through the proliferation of neural stem cells.

In order for a certain ingredient to act on the brain, the drug for treating brain tumors must pass through the blood-brain barrier that protects the brain. The conditions for easily passing through the blood-brain barrier include (1) a small molecular weight, (2) a low protein binding rate, and (3) high lipid solubility. For example, Temodar, a drug used for malignant glioma, has a very small molecular weight of 194, so it is characterized by being able to easily pass through the blood-brain barrier and reach the affected area. The molecular weights of the four ingredients of the present invention, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin, are 354, 354, 368, and 448, respectively. It has been reported that monocaffeoylquinic acid passes through the blood-brain barrier and reaches brain tissue after oral ingestion. (Fitoterapia 99:139-152, 2014)

In addition, according to the study by the present inventors, it has been found that a mixture of sugarcane top extract and 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin synergistically increases the expression of NTRK2 in SH-SY5Y cells, compared to treatment with isoorientin alone and treatment with ginkgo biloba extract, which has been reported to have a positive control effect of improving cognitive function. Therefore, the composition of the present invention can be expected to effectively improve TrkB signaling, promote neurogenesis, and suppress the decrease in nerve cells associated with aging, thereby improving symptoms. More specifically, the composition of the present invention can be used to treat diseases in which the decrease in BDNF-TrkB signaling and the decrease in nerve cells due to the decrease in neurogenesis have been reported, specifically memory loss and depression in aging and Alzheimer's disease.

In the present invention, the term "treatment" of a disease or condition includes reducing the risk of onset, delaying onset, prevention, treatment, and halting or delaying progression. Treatment includes actions performed by physicians with the aim of treating a disease, and non-therapeutic actions performed by persons other than physicians, such as nutritionists (including registered dietitians and sports nutritionists), public health nurses, midwives, nurses, clinical laboratory technicians, sports instructors, beauty consultants, estheticians, pharmaceutical manufacturers, pharmaceutical distributors, food manufacturers, and food distributors. Treatment also includes actions performed by veterinarians with the aim of treating a disease in a non-human animal, and non-therapeutic actions performed by persons other than veterinarians, such as veterinary nurses, pet animal caretakers, stablemen, breeders, animal drug manufacturers, animal drug distributors, pet food manufacturers, and pet food distributors. Further treatment includes the administration or recommendation of the intake of specific foods, dietary guidance, health guidance, nutritional guidance (including nutritional guidance necessary for the recuperation of injured or sick persons, and nutritional guidance for maintaining and promoting health), school lunch management, and guidance necessary for nutritional improvement regarding school lunches.

The subject of the treatment in the present invention includes humans (individuals), and is preferably a human for whom it is desirable to perform any of the above-mentioned treatments or who needs to perform any of the above-mentioned treatments. The subject of the treatment in the present invention may be an animal other than a human, and examples of such animals include pets (also called companion animals) such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels, livestock such as cows and pigs, laboratory animals such as mice and rats, and animals kept in zoos. There are no particular limitations on the growth stage of the target non-human animal, and the subject of the treatment in the present invention may be, for example, a puppy, an adult dog, an elderly dog, a young cat, an adult cat, or an elderly cat.

[Composition, etc.]
The composition of the present invention may be the sugarcane tops extract itself, or may contain an active ingredient (eg, sugarcane tops extract) and other ingredients.

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

In the present invention, unless otherwise specified, food includes not only solids but also liquids, such as soups, beverages, and drinks. Furthermore, unless otherwise specified, food includes not only those intended for humans but also those intended for non-human animals, such as feed and pet food. Furthermore, unless otherwise specified, food includes general foods, health foods, supplements, health functional foods (specified health foods (commonly known as FOSHU), nutrient functional foods, and functional food), as well as therapeutic foods (foods that serve the purpose of treatment; prepared based on a menu prepared by a nutritionist or other such person in accordance with a doctor's dietary prescription), dietary therapy foods, ingredient-adjusted foods, reduced-salt foods, nursing care foods, reduced-calorie foods, and diet foods.

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

The content of the sugarcane tops extract, which is the active ingredient, in the composition of the present invention can be designed taking into consideration the daily intake/administration amount. For example, the daily dose for an adult can be 0.2 to 2,000 mg of sugarcane tops extract in solid form, preferably 0.5 to 1,000 mg, more preferably 1 to 500 mg, and even more preferably 2 to 200 mg. The daily dose can be ingested/administered multiple times, for example, in 2 to 4 divided doses.

The composition of the present invention may contain ingredients other than the sugarcane top extract, so long as the desired effect can be achieved. The other ingredients may be various additives acceptable for use as food or various additives acceptable for use as medicine. Examples include excipients, antioxidants, flavorings, seasonings, sweeteners, coloring agents, thickening stabilizers, colorants, bleaching agents, fungicides, gum bases, bittering agents, enzymes, gloss agents, acidulants, emulsifiers, strengthening agents, manufacturing agents, binders, tonicity agents (isotonicity agents), buffers, solubilizers, preservatives, stabilizers, coagulants, etc.

The other components may be functional components other than sugarcane top extract. Examples of other functional components include amino acids (e.g., branched chain amino acids, ornithine), unsaturated fatty acids (e.g., EPA, DHA), vitamins, trace metals, polyphenols, egg yolk extract, honey products, brown sugar, oligosaccharides, dietary fiber, glucosamine, chondroitins, CoQ10, fucoidan, fucoxanthin, astaxanthin, placenta, yeast extract, black vinegar concentrate, plant extracts (garlic extract, ginkgo leaf extract, tea extract, bilberry extract, blueberry extract, various ginseng extracts, maca extract, bean seed coat extract, St. John's wort extract, pine bark extract, acai extract, noni extract), etc.

The compositions of the present invention can be taken or administered with, before, or after a meal.

The composition of the present invention may be labeled to the effect that it can be used to treat the above-mentioned diseases or conditions, and may also be labeled to recommend ingestion to the above-mentioned subjects. Labeling may be direct or indirect. Examples of direct labeling include descriptions on tangible objects such as the product itself, packaging, containers, labels, and tags, and examples of indirect labeling include advertising and promotional activities in places or by means such as websites, storefronts, exhibitions, signs, bulletin boards, newspapers, magazines, television, radio, mail, and e-mail.

[Component analysis of sugarcane top extract]
<Preparation of sugarcane top extract sample>
Component extraction (four times) was performed from 1 g of dried sugarcane tops (including leaves and bark) using an automatic extraction device with 80% ethanol as the extraction solvent. The extracted solution was concentrated using a rotary evaporator, and then freeze-dried to obtain a sample. The obtained sample was redissolved in 100% methanol (concentration: 100 mg/mL), sterilized through a 0.22 μm filter, and used for chemical analysis using high performance liquid chromatography (HPLC).

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

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

(1) 3-o-Caffeoylquinic acid

(2) 5-o-Caffeoylquinic acid

(3) 3-o-feruloylquinic acid

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

[1. Cellular evaluation of the effect of sugarcane top extract on amyloid-β-induced neuronal cell death]
1-1. Preparation of sugarcane top extract samples A solvent extraction method was used to extract polyphenolic components from sugarcane top powder. Specifically, sugarcane tops were soaked in 10 mL of 70% ethanol per 1 g of dried material for 14 days (inversion and stirring on the 7th day of extraction) to dissolve the components into the solvent. The extraction solvent was then sterilized through a 0.22 μm filter and concentrated to dryness to obtain a sample. The sample was also dissolved in 70% ethanol and used in the cell test described below.

1-2. Experimental method <Cultivation of human nerve model SH-SY5Y cells>
SH-SY5Y (American Type Culture Collection, Manassas, USA) was used as a human neuronal model cell. SH-SY5Y is a cell line derived from human neuroblastoma and is widely used as a model cell for evaluating neuronal function. SH-SY5Y cells were seeded in 100-mm sterile petri dishes (BD Falcon, USA) and cultured in a 1:1 (v/v) mixture of Dulbecco's Modified Eagle medium and Ham's F-12 medium (Lonza, Japan) containing 15% heat inactivated fetal bovine serum (Bio West, USA), 1% penicillin (5000 μg/ml)-streptomycin (5000 IU/ml) solution (Lonza), and 1% MEM Non-Essential Amino Acids Solution (100x) (Cosmo Bio, Japan) at 37°C in the presence of 5% _CO2 . Cells were cultured until they reached 60-80% confluence, and cells at passage numbers 3-8 were used for each study.

<Evaluation of the neuroprotective effect of sugarcane top extract>
To evaluate the neuroprotective effect of sugarcane top head extract, an experiment was carried out using amyloid beta (Aβ), a neurotoxin. Specifically, the following treatment groups were set for the cells: (i) a control group, (ii) a group treated with Aβ alone, and (iii) a group treated with Aβ + sugarcane top head extract. The cells were seeded in a 96-well plate with medium at 2.0 × 10 ⁴ cells/well, and cultured at 37°C and 5% CO ₂ for 24 hours. Amyloid beta protein (Aβ1-42) or sugarcane top head extract was added to each well at a final concentration of 5 μM. Sugarcane top head extract was added at a concentration of 10 μg/mL, 25 μg/mL, or 50 μg/mL. After incubation at 37°C and 5% CO ₂ for 72 hours, cell viability was evaluated by measuring absorbance using the MTT method. Specifically, the medium was removed from the wells, and 5 mg/ml MTT solution dissolved in PBS was added (100 μl/well), and incubated at 37°C and 5% _CO2 for 6 hours. Then, 10% sodium dodecyl sulfate (SDS) was added (100 μl/well), and incubated at 37°C and 5% _CO2 for 24 hours to dissolve the formazan crystals. The absorbance was measured using a microplate reader (absorbance: 570 nm), and the obtained values were subjected to a significance test (*P<0.05, **P<0.01) using the One-way ANOVA method.

1-3. Experimental results The amyloid-β treatment group showed a 40% decrease in cell viability compared to the control group, whereas the amyloid-β + sugarcane top head extract co-treatment group showed a concentration-dependent significant increase in cell viability compared to the amyloid-β only treatment group (Figure 2). From these results, it was found that the neuronal cell death caused by amyloid-β treatment was significantly suppressed by simultaneous treatment with sugarcane top head extract, suggesting the neuroprotective effect of sugarcane top head extract.

[2. Cellular level evaluation of four compounds contained in sugarcane tops]
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 (hereinafter 3-FQA, Nagara Science, Gifu, Japan), and Isoorientin (hereinafter ISO, Sigma-Aldrich, USA), all purchased from Nagara Science Co., Ltd., were used as standard compounds. The concentrations of the standard compounds in this experiment were calculated from the content in 50 μg/mL sugarcane top extract by HPLC. Specifically, 3-CQA was used at a concentration of 0.50 μM, 5-CQA at a concentration of 0.70 μM, 3-FQA at a concentration of 0.85 μM, and ISO at a concentration of 0.477 μM. Standards of each compound were dissolved in 70% ethanol for cell testing.

2-2. Experimental Method SH-SY5Y (American Type Culture Collection, Manassas, USA) was used as a human nerve model cell. The following groups were treated with the cells: (i) control group, (ii) Aβ only 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+sugarcane top extract (50 μg/mL). The same test as in 1-2. above was performed to evaluate cell viability. The obtained values were also subjected to a One-way ANOVA test for significance (*P<0.05, **P<0.01).

The amount of ATP production in neurons was measured for each of the above treatment groups. The experimental method was as described in 6-2. <Evaluation of the effect of standard compounds on ATP production in neurons> below.

2-3. Experimental results
When Aβ was treated simultaneously with 3-CQA, 5-CQA, 3-FQA, and ISO, a trend toward increased cell viability was observed compared to treatment with Aβ alone. When the compound mixture was treated simultaneously with Aβ, a significant increase in cell viability was observed compared to treatment with Aβ alone (Figure 3, top). These results suggest that 3-CQA, 5-CQA, 3-FQA, and ISO are components involved in the inhibitory effect of sugarcane top head extract on amyloid beta-induced neuronal cell death.

In addition, a tendency for intracellular ATP production to increase with treatment with each compound was confirmed. Treatment with the mixture of four polyphenols showed a significant increase in intracellular ATP production compared to the control (Figure 3, bottom).

<Analysis of the mechanism of neuroprotection by the extract from the root of the plant>
To analyze the mechanism of neuroprotection by the sugarcane top head extract, gene expression was analyzed using real-time RT-PCR. SH-SY5Y cells were treated with (i) a control group, (ii) an Aβ-only treatment group, and (iii) an Aβ + sugarcane top head extract treatment group. The cells were seeded in a ₆ -well plate with medium and cultured at 37°C and 5% CO2 for 24 hours, after which amyloid β protein or sugarcane top head extract was added at a final concentration of 5 μM. The sugarcane top head extract was added at a concentration of 50 μg/mL. After incubation at 37°C and 5% _CO2 for 24 hours, mRNA was extracted from the cells in each treatment group using the ISOGEN Kit (NipponGene, Japan).

Reverse transcription polymerase chain reactions (RT-PCR) were performed using the extracted mRNA with the Superscript III reverse transcriptase kit (Invitrogen, USA) and a 2720 Thermal cycler (Applied Biosystems, USA). To quantify the transcripts, TaqMan real time RT PCR amplification reactions were performed using the 7500 Fast Real Time PCR system, with primers for the following genes involved in signaling pathways involved in the mechanism of neuronal death induced by Aβ treatment: MAP2K4 (Hs00387426_m1), MAPK14 (Hs01051152_m1), MAPK8 (Hs01548506_m1), PI3KCA (Hs00907957_m1), AKT1 (Hs00178280_m1), PARP1 (Hs00242302_m1), and CASP3 (Hs00234387_m1) (all from Applied Biosystems, USA), and GAPDH (Hs02786624_m1) as an internal standard. In addition, the obtained values were subjected to a significance test using the One-way ANOVA method (*P<0.05, **P<0.01).

<Experimental Results>
Amyloid-β treatment significantly decreased the gene expression of MAP2K4, MAPK14, MAPK8, PI3KCA, AKT1, and PARP1 in SH-SY5Y cells, and sugarcane top head extract treatment significantly increased the expression of the above genes (Figure 4). Furthermore, amyloid-β treatment significantly increased the gene expression of CASP3, a gene related to apoptosis (cell death), in SH-SY5Y cells, and sugarcane top head extract treatment decreased the expression (Figure 4).

[3. Analysis of the effect of sugarcane top extract on spatial learning and memory ability]
3-1. Preparation of sugarcane top extract sample Polyphenol components were extracted from sugarcane top powder in the same manner as in 1-1. Samples were prepared by dissolving them in Mill-Q and using them in the animal tests described below.

3-2. Experimental Method SAMP8 (purchased from Nippon LSC Co., Ltd.) was used as an accelerated aging model. SAMP8 is a naturally occurring model animal characterized by the early onset of aging-related pathology, and research on the effects of antioxidants contained in various foods is being conducted on it. SAMR1, which shows normal aging, was used as a control for SAMP8. In this experiment, 16-week-old male mice, which begin to show aging-like symptoms such as memory impairment, were used, and the mice were divided into the following three groups: (a) SAMR1 + water administration group, (b) SAMP8 + water administration group, (c) SAMP8 + sugarcane top head extract administration group.

The dosage of sugarcane top extract was set at 20 mg/kg per day, and the administration method was oral. After orally administering the sample and water to each group for 30 days, behavioral tests were conducted to evaluate the spatial learning and memory abilities of each group. Ten mice from each group were used in this experiment. Water and food were available ad libitum during the breeding period, and the mice were weighed daily.

After the behavioral tests were completed, brains were isolated from the mice and subjected to comprehensive gene expression analysis using microarrays and ELISA analysis of brain neurotransmitter concentrations (dopamine, noradrenaline, adrenaline, acetylcholine, and serotonin).

<Evaluation of spatial learning and memory ability using the Morris water maze test>
After 30 days of continuous administration of sugarcane top extract, the Morris water maze test, a behavioral test to evaluate the spatial learning and memory ability of animals, was performed. The Morris water maze test is an experimental method to evaluate the learning and memory ability of animals. In the test, a transparent platform was placed under the surface of a pool filled with water, and mice dropped into the pool explored the platform by escaping from the water. The experimental apparatus consisted of a circular water pool (120 cm in diameter and 45 cm in height), the temperature and depth of the water were set at 23 ± 2°C and 30 cm, respectively, and it was divided into four virtual quadrants: north, south, east, and west. A transparent and invisible platform (10 cm in diameter) was placed 1 cm below the water surface at the midpoint of any quadrant. When exploring the platform, the mice used four visual cues placed on the walls of the pool as cues, and after repeated trials, they were able to reach the platform in a short time. The mice were allowed to explore freely for 60 seconds, and the time it took to reach the platform was measured to evaluate the spatial learning and memory ability of the mice. In this test, the shortening of the reaching time is an index of the improvement of learning and memory. The test was conducted for 8 days, and on the final day of the test, the platform was removed and the test was conducted to measure the time the mouse stayed in the area where the platform was located and the number of times it crossed the platform location (probe test). The probe test was conducted to evaluate the extent to which the spatial memory could be retained and recalled. The significant difference test between the SAMP8 + water group, the SAMP8 + sugarcane top head extract group, and the SAMR1 + water group was conducted by the two-way ANOVA method (*P<0.05) for the test results with the platform installed for 7 days, and the one-way ANOVA method (*P<0.05) for the probe test results on the final day.

<Experimental Results>
The results of the Morris water maze test showed that the group administered the sugarcane top head extract showed a significant reduction in the time it took to reach the platform compared to the group administered SAMP8 water from the 5th day of the experiment (Figure 5, top). The results of the probe test showed that the group administered the sugarcane top head extract showed a significant increase in the number of times they crossed the platform area compared to the group administered water (Figure 5, middle), and also showed a tendency for the time they spent in the platform area to increase (Figure 5, bottom). These results suggest that the sugarcane top head extract has an effect of improving spatial learning and memory.

<Comprehensive genetic analysis using microarrays>
After 30 days of continuous administration of sugarcane top head extract or water and behavioral tests, brain tissue was removed and cerebral cortex was separated. mRNA was extracted from the separated cerebral cortex using ISOGEN Kit (NipponGene, Japan). Then, double-stranded cDNA was synthesized from 100 ng of mRNA using Gene Atlas 3'IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA), and biotin-labeled amplified RNA (aRNA) was synthesized using Gene Chip 3'IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). The purified aRNA was then fragmented using Gene Atlas 3'IVT Express Kit, and hybridization was performed at 45°C for 16 hours using Gene Chip MG-430 PM microarray (Affymetrix Inc., Santa Clara, CA, USA). The chips were then washed and stained using a Gene Atlas Fluidics Station 400 (Affymetrix Inc., Santa Clara, CA, USA), and images were scanned using a Gene Atlas Imaging Station (Affymetrix Inc., Santa Clara, CA, USA). The changes in gene expression in the SAMP8 + sugarcane top extract group compared to the SAMP8 + water group were calculated as fold-change (a relative ratio of the signal value of the target divided by the signal value of the control) based on the results obtained. Pathway analysis was performed using DAVID (URL: https://david.ncifcrf.gov).

<Experimental Results>
Microarray analysis confirmed that many genes in the SAMP8 + sugarcane top extract group were altered in expression compared to the SAMP8 + water group (Table 2). Of the altered genes, a total of 912 genes (including 6 with a fold change of 1.5 or more) were upregulated (fold change of 1.15 or more), and a total of 466 genes (including 5 with a fold change of 0.85 or less) were downregulated (fold change of 0.5 or less). Since the number of upregulated genes was greater than the number of downregulated genes, the upregulated genes were analyzed using DAVID. As a result of the analysis, the genes with increased expression were categorized into groups related to various biological functions, and it was confirmed that genes closely related to brain functions were increased, including cholinergic synapses that release acetylcholine as a neurotransmitter, serotonergic synapses that play an important role in thermoregulation and sleep, dopaminergic synapses that play a role in controlling cognition and voluntary movements and motivating behavior associated with rewards, calcium signaling in neurons, long-term potentiation in neurotransmission, circadian rhythm, Alzheimer's disease, etc. (Fig. 6). In addition, pathway analysis revealed that the increased expression of genes such as Camk2d, Adcy9, Cacna1a, and Homer1 in response to improved calcium signaling between synapses, which leads to improved neuroplasticity and neuroprotection, the increased expression of the clock gene Per3 via increased Camk2d expression, which leads to improved circadian rhythm, and the increased expression of Cox8b, which leads to improved mitochondrial function, were considered to be activated gene transmission pathways in the cerebral cortex (Fig. 7).

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

<Experimental Results>
In the SAMP8 + sugarcane top head extract group, the amount of dopamine and noradrenaline in the cerebral cortex was significantly increased compared to the SAMP8 + water group (Figure 8). In addition, the amount of acetylcholine and serotonin showed a tendency to increase (Figure 8). In other words, from this result, it is considered that the sugarcane top head extract is involved in increasing the amount of neurotransmitters secreted in the brain and improves learning and memory.

[4. Investigation of component extraction efficiency from sugarcane tops using organic solvents of different concentrations]
<Preparation of sugarcane top extract sample>
Extraction of components was performed from 1 g of dried sugarcane tops using ethanol of different concentrations, 20%, 40%, 60%, and 80%, as the extraction solvent. The ethanol concentrations are based on volume (v/v). An automatic extraction device E-916 (Nihon Buchi) was used for extraction, and the extraction conditions were temperature = 45°C, pressure = 100 bar, extraction time = 10 min, and number of extractions = 3. The extracted solution was concentrated using a rotary evaporator, and then freeze-dried to obtain powdered samples (the color was brown for the sample using 20% ethanol as the solvent, and green for the others. The green color of the sample obtained became slightly deeper as the ethanol concentration increased). The total amount (yield) of each sample extracted 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 through 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 analysis was performed using a Prominence (Shimadzu Corporation). The analysis conditions were as follows: column: ZORBAX (250 x 4.6 mm), column temperature: 40°C, flow rate: 1.0 mL/min, injection volume: 10 μL, UV wavelength: 328 nm, mobile phase: A: 10% formic acid; B: acetonitrile: MeOH = 1:1, gradient: 0 - 100%; 40 min.

<Analysis results>
The quantitative results obtained by HPLC are shown in Figure 9. As a result of the quantitative analysis, the content of each polyphenol component per 100 parts by weight of the extract was highest in the sample extracted using 40% ethanol as a solvent.

It has been found that with regard to 3-CQA, 5-CQA and 3-FQA, more than 80% of the amount contained in the dried sugarcane tops can be extracted in two runs using 80% ethanol as the solvent and extraction conditions of a temperature of 45°C and a pressure of 100 bar.

[5. Comparative study of the physiological activity of sugarcane top extracts extracted using different concentrations of organic solvents on nerve cells]
5-1. Preparation of sugarcane tops extract samples The sugarcane tops samples obtained by the method described above in <Preparation of sugarcane tops extract samples> were redissolved in 70% ethanol (concentration: 100 mg/mL) and sterilized through a 0.22 μm filter before being used in the cell tests described below.

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

<Evaluation of the neuroprotective effect of sugarcane top extract>
The following groups were treated with the cells: (i) a control group, (ii) an Aβ (5 μM) single treatment group, (iii) an Aβ + sugarcane tops head extract (solvent 20% ethanol) treatment group, (iv) an Aβ + sugarcane tops head extract (solvent 40% ethanol) treatment group, (v) an Aβ + sugarcane tops head extract (solvent 60% ethanol) treatment group, and (vi) an Aβ + sugarcane tops head extract (solvent 80% ethanol) treatment group. The test was performed in the same manner as in 1-2 above, and cell viability was measured. In addition, the obtained values were subjected to a significance test (*P<0.05, **P<0.01) using the One-way ANOVA method.

5-3. Experimental results
A concentration-dependent significant increase in cell viability was confirmed in the Aβ + sugarcane top head extract treatment group compared to the amyloid β only treatment group (Figure 10). In addition, cell viability was highest in the (iv) Aβ + sugarcane top head extract (solvent 40% ethanol) treatment group (78.6±2.41%, Figure 10). Based on this result and the results of the above [Examination of component extraction efficiency from sugarcane top heads using organic solvents of different concentrations], a correlation was found between the polyphenol content in the extract and the physiological activity of the extract on nerve cells.

[6. Evaluation of the effects of four compounds contained in sugarcane tops on cell viability and ATP production in the SH-SY5Y neuronal cell model]
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 (hereinafter 3-FQA, Nagara Science, Japan), and Isoorientin (hereinafter ISO, Sigma-Aldrich, USA) were used as standard compounds. Standard samples of each compound were dissolved in 70% ethanol and used in the tests described below.

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

<Evaluation of the effect of standard compounds on the survival rate of neural cells>
The treatment groups for cells were (i) a control group, (ii) a 3-CQA treatment group, (iii) a 5-CQA treatment group, (iv) a 3-FQA treatment group, and (v) an ISO treatment group. 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 performed in the same manner as in 1-2 above, and cell viability was measured. The obtained values were subjected to a significance test (*P<0.05, **P<0.01) using the One-way ANOVA method.

<Evaluation of the effect of standard compounds on ATP production in neurons>
To measure the amount of ATP produced in nerve cells, Cell ATP Assay reagent (Toyo Ink, Japan) was used. ATP is an important indicator of cellular energy metabolism. SH-SY5Y cells were seeded in a 96-well plate at a cell density of 2.0 × 10 ⁵ cells/mL and incubated at 37°C and 5% CO ₂ for 24 hours, after which the cells were treated with samples. The treatment groups for the cells were (i) a control group, (ii) a 3-CQA treatment group, (iii) a 5-CQA treatment group, (iv) a 3-FQA treatment group, and (v) an ISO treatment group, and samples were added to each well at a treatment concentration of 10 μM. After incubation at 37°C and 5% CO ₂ for 12, 24, and 48 hours from the sample treatment, the cells were incubated at room temperature for 30 minutes. Then, 100 μl/well of room temperature ATP reagent (Toyo Ink) was added and incubated in the dark for 1 minute, after which 150 μL of the supernatant from each well was transferred to a 96-well white bottom plate (BD Falcon) and incubated at 25°C for 10 minutes to stabilize the luminescence. The luminescence intensity was then measured using a plate reader (Powerscan HT, USA) to determine the rate of ATP production. The obtained values were subjected to a significance test (*P<0.05, **P<0.01) using the One-way ANOVA method.
6-3. Experimental Results The results of the cell viability test showed that the treatment with 3-CQA, 5-CQA and ISO compound standards significantly increased the viability of SH-SY5Y cells in a concentration-dependent manner (Figure 11). The results of the ATP production test showed that the treatment with 3-CQA, 5-CQA and ISO compound standards significantly increased the ATP production rate in SH-SY5Y cells (Figure 12). These results suggest that 3-CQA, 5-CQA and ISO, among the compounds contained in the sugarcane top extract, activate energy metabolism in SH-SY5Y cells and contribute to promoting ATP production.

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

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

<Evaluation of glycolytic enzyme gene expression in SH-SY5Y cells to elucidate the mechanism of ATP production promotion by sugarcane top extract or standard compound>
To clarify the mechanism of ATP production in neurons, we performed gene expression analysis using real-time RT-PCR to evaluate the effect of sugarcane tops extract or compound standards on gene expression of enzymes that catalyze chemical reactions in the glycolysis pathway, which is the energy metabolism pathway in cells. SH-SY5Y cells were treated with (i) a control group, (ii) sugarcane tops extract (50 μg/mL) treatment group, (iii) 3-CQA treatment group, iv) 5-CQA treatment group, and (v) ISO treatment group. The sugarcane tops extract sample was treated at a concentration of 50 μg/mL, and each compound sample was treated at a concentration of 10 μM. The cells were incubated with the samples in a 60 mm cell culture dish (BD Falcon) at 37°C in the presence of 5% _CO2 for 24 hours, and then mRNA was extracted from the cells in each treatment group using the ISOGEN kit (NipponGene, Japan).

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

<Experimental Results>
The expression of PGK1, PGAM1, PKM, and PC in SH-SY5Y cells was significantly increased by 24 hours of treatment with the sugarcane top extract or each compound standard compared to the control group (Figure 13). This result suggests that the sugarcane top extract or each compound standard activates the glycolysis pathway, which is an intracellular energy metabolic pathway, through the increase in the expression of the above genes, and as a result promotes ATP production in nerve cells (Figure 14).

[8. Evaluation of the ability of sugarcane top extract to induce self-renewal and differentiation in human neural stem cells (hNSCs)]
8-1. Preparation of sugarcane top samples The samples obtained by the method in 1-1 above were dissolved in 70% ethanol and used in the tests described below.

8-2. Experimental methods and results <Culture of human neural stem cells (hNSCs)>
Human fetal neural stem cells (Gibco® life technologies, USA) were used to evaluate the effect of the sample on neural stem cells (hNSCs). hNSCs were seeded in a T75 flask (BD Falcon, USA) and cultured at 37°C in the presence of 5% CO2 in a ^neural stem cell proliferation medium (all Gibco® life technologies) containing 2% StemPro® Neural Supplement, 20 ng/mL bFGF, 20 ng/mL EGF, 2 mM GlutaMAX ^™ -I Supplement, 6 units/mL Heparin, and 200 μM Ascorbic acid in KnockOut™ D-MEM/F- ₁₂ . In the above medium, hNSCs were cultured in a floating state without adhering to the bottom of the flask, and proliferated while forming spherical cell clusters (neurospheres) (Figure 15). The medium was replaced every 3 to 4 days, and the culture was continued until the diameter of the formed spheres reached <=1 mm (9 to 11 days after seeding).

<Evaluation of the effect of sugarcane top extract on gene expression of differentiation markers in hNSCs>
To evaluate the ability of sugarcane top 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. The following groups were set as treatment groups for hNSCs: (i) a control group, (ii) a 10 μg/mL sugarcane top head extract treatment group, (iii) a 25 μg/mL sugarcane top head extract treatment group, and (iv) a 50 μg/mL sugarcane top head extract treatment group. hNSCs were cultured in a 6-well plate with medium, and after confirming the formation of spheres (diameter <= 1 mm), a sample diluted to the above-mentioned concentration in the medium was added to the sugarcane top head extract treatment group, and an equal amount of medium was added to the control group. After 24 hours of incubation at 37°C in the presence of 5% _CO2 , mRNA was extracted from the cells in each treatment group using the ISOGEN kit (NipponGene, Japan).

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

<Experimental Results>
Treatment with the sugarcane top head extract for 24 hours increased the expression of TUBB3 and GFAP in hNSCs in a concentration-dependent manner (Figure 16). In addition, the expression of PDGFRA and NES decreased in a concentration-dependent manner (Figure 16). These results suggest that the sugarcane top head extract induces the transition of neural stem cells from a dormant state to an activated state, and induces their differentiation into neurons or astrocytes.

<Evaluation of the effect of sugarcane top extract on the generation of new neurons from hNSCs>
To evaluate the effect of sugarcane top head extract on the generation of new neurons from hNSCs, we performed an assay using thymidine analogue BrdU (5-bromo-2'-deoxyuridine) and neural progenitor cell marker HuC/D. BrdU is an analogue of thymidine, a component of DNA, and when cells begin to divide in the presence of BrdU, BrdU is incorporated into the DNA strand being replicated. The percentage of BrdU-positive cells was counted to evaluate the percentage of cell proliferation. HuC/D is a protein that is specifically expressed in the cell body of neurons from an early stage of differentiation, and the percentage of new neurons was analyzed by quantifying positive cells together with BrdU. The treatment groups for hNSCs were (i) a control group, (ii) a group treated with 10 μg/mL sugarcane top head extract, (iii) a group treated with 25 μg/mL sugarcane top head extract, and (iv) a group treated with 50 μg/mL sugarcane top head extract. hNSCs were cultured in 6-well plates with medium, and after confirming the formation of spheres (diameter <=1 mm), the sugarcane top extract treatment group was diluted with the above-mentioned sample to the medium concentration set above, and the control group was added with an equal amount of medium. After 24 hours of incubation at 37°C and 5% _CO2 , 10 μM BrdU (Tokyo Chemical Industry, Japan) was added to each group, and BrdU was incorporated into the newly proliferated cells by incubation at 37°C and 5% _CO2 for 24 hours. After incubation, the spheres were dissociated using Accutase ® Reagent (Gibco, USA), and the dissociated hNSCs were seeded at a concentration of 5.0 × 10 ⁴ cells/mL onto a Nunk Lab-Tek chamber slide 4 well (Thermo Fisher Scientific, USA) coated with Geltrex ® Reagent. The hNSCs were then incubated at 37°C and 5% CO ₂ for 12 hours to allow them to adhere to the bottom of the slide.

After removing the medium from the slides, BrdU and HuC/D positive cells were detected by immunostaining. The cells were gently washed once with 1×PBS, and then fixed at room temperature for 30 min with ice-cold 4% PFA. After fixation, the PFA was removed and the cells were washed twice with 1×PBS. To denature a part of the double-stranded DNA into short-stranded DNA and expose BrdU for antibody binding, 2N HCl was added and incubated at room temperature for 30 min. HCl was then removed and 0.1 M Na ₂ B ₄ O ₇ (pH=8.5) was added and incubated at room temperature for 15 min to neutralize the acid. _{Na 2} B ₄ O ₇ was removed and the cells were washed twice with 1×PBS, and then 0.2% Triton X-100 (Sigma-Aldrich, USA) solution was added and incubated at room temperature for 5 min to permeabilize the membrane. Triton X-100 was removed, and the cells were washed three times with 1×PBS, and then 5% normal goat serum (Funakoshi, Japan) solution was added and incubated at room temperature for 1 hour to prevent nonspecific antibody binding. After removing the normal serum, a solution containing rabbit monoclonal anti-HuC+HuD (HuC/D, 1:500, Abcam, Cambridge, UK) and mouse monoclonal anti-BrdU (1:200, Invitrogen, Carlsbad, CA) as primary antibodies was added to 1% normal goat serum solution and incubated overnight (16 hours) at 4℃. After incubation, the primary antibody solution was removed, and the cells were washed three times with 1×PBS, and a solution containing secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594 labeled, 1:500, Abcam, UK) was added to 1% normal goat serum solution and incubated at room temperature for 1 hour. After removing the secondary antibody solution and washing the cells three times with 1× PBS, the cells were mounted using ProLong™ Diamind (Thermo Fisher Scientific, USA) and a cover glass. The mounted slide was stored at 4°C and used for microscopic analysis.

After staining, the cells were imaged using a confocal microscope TCS SP8 (Leica, Germany) (magnification = 40x, zoom = 0.8, scale bar = 100 μm). Eight to ten sections were randomly selected from each well, and images were taken. The images were then analyzed using Image J. The values obtained by the analysis were subjected to a significance test using the One-way ANOVA method (*P<0.05, **P<0.01).

<Experimental Results>
In the above experiment, the percentage of HuC/D positive cells did not change with treatment with the sugarcane top extract, but the percentage of BrdU positive cells increased in a treatment concentration-dependent manner (Figure 17). The percentage of both HuC/D and BrdU positive cells also increased in a treatment concentration-dependent manner (Figure 17). These results suggest that the sugarcane top extract has a promoting effect on neural stem cell proliferation and on the generation of new neurons from neural stem cells.

<Mechanism of the induction of new neurogenesis by sugarcane top extract>
To analyze the mechanism of the induction of new neurogenesis by sugarcane top 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 determination in cell development and differentiation. In neural cell differentiation, when stemness is maintained (≒ dormant state), the expression level of HES1 is high and the expression level of ASCL1 is kept low. When neural stem cells become activated, the expression level of HES1 decreases and the expression level of ASCL1 increases, and neural stem cells differentiate into neurons. After performing an experiment similar to that described in the above <Effect of sugarcane top extract on the generation of new neurons from hNSCs>, dissociated hNSCs were seeded at a concentration of 5.0 × 10 ⁴ cells/mL on 6-well plates and incubated at 37℃ and 5% CO ₂ for 12 hours to allow the hNSCs to adhere to the bottom of the slide. Then, mRNA was extracted using ISOGEN kit (NipponGene, Japan), and gene expression was analyzed in the same manner as described in the above <Effect of sugarcane top extract on gene expression of differentiation markers in hNSCs>. ASCL1 (Hs00269932_m1) and HES1 (Hs00172878_m1) were used as primers 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 significance testing (*P<0.05, **P<0.01) using the One-way ANOVA method.

<Experimental Results>
In the above experiment, the expression of ASCL1 was increased in the sample-treated group, and the expression of HES1 was decreased in a concentration-dependent manner (Figure 17). These results suggest that the sugarcane top head extract activates neural stem cells and induces the generation of new neurons by regulating the expression of ASCL1 and HES1 genes.

<Effect of sugarcane top extract on hNSC differentiation>
The effect of sugarcane top head extract on hNSC differentiation was evaluated by inducing differentiation of hNSCs attached to the bottom of the slide, and then labeling neurons and astrocytes using immunostaining. The following groups were set for the treatment of hNSCs: (i) control group, (ii) 10 μg/mL sugarcane top head extract treatment group, (iii) 25 μg/mL sugarcane top head extract treatment group, and (iv) 50 μg/mL sugarcane top head extract treatment group. hNSCs were cultured in a 6-well plate with medium, and after confirming the formation of spheres (diameter <=1 mm), the sample diluted to the above-mentioned concentration in the medium was added to the sugarcane top head extract treatment group, and the same amount of medium was added to the control group. After 24 hours of incubation at 37°C in the presence of 5% _CO2 , the spheres were dissociated using Accutase (registered trademark) Reagent (Gibco, USA), and the dissociated hNSCs were seeded at a concentration of 5.0 x ¹⁰⁴ cells/mL on a Nunk Lab-Tek chamber slide 4 well (Thermo Fisher Scientific, USA) coated with ^Geltrex (registered trademark) Reagent. Differentiation of hNSCs was induced by culturing for 7 days at 37°C in the presence of 5% CO2 in a neural stem cell differentiation medium (all Gibco (registered trademark) life technologies) composed of 2% StemPro (registered trademark) Neural Supplement, 2 mM _GlutaMAX (registered trademark ⁾ -I Supplement, 6 units/mL Heparin, and 200 μM Ascorbic acid in KnockOut TM D-MEM/F-12. Samples were also added to the medium to achieve the above set concentration during differentiation induction.

After 7 days of culture, the medium was removed from the slides, and Tuj1 (Tublin beta III) and GFAP positive cells were detected by immunostaining. Immunostaining was performed as described in the above (no denaturation 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, 8 to 10 randomly selected sections from each well were photographed using a confocal microscope TCS SP8 (Leica, Germany) (magnification = 40 ×, zoom = 0.9, scale bar = 100 μm), and the photographed images were analyzed using Image J. The values obtained by the analysis were subjected to a significance test using the One-way ANOVA method (*P<0.05, **P<0.01).

<Experimental Results>
In the above experiment, the percentage of astrocytes (GFAP-positive cells) did not change with the sugarcane top extract, but the percentage of neurons (Tuj1-positive cells) increased in a concentration-dependent manner (Figure 19). In addition, the extension of the total length of astrocyte (GFAP-positive cells) processes was observed under sugarcane top extract treatment (Figure 18). These results suggest that treatment with sugarcane top extract induces neuronal differentiation from neural stem cells and promotes the development of astrocytes after differentiation.

Quantification of newborn neurons in the mouse brain
After behavioral testing (see 3-2. Experimental Methods), brains were extracted from mice for immunohistochemical analysis and fixed in 4% paraformaldehyde for 14 days at 4°C, followed by equilibration in 30% sucrose/PBS (w/v) for 48 hours at 4°C. Brain tissue sections (30 μm) were prepared using an SM2010R sliding microtome (Leica). Sections were stored at -20°C in a cryoprotectant solution (ethylene glycol, glycerol, 0.1 M phosphate buffer, pH 7.4) until use. The tissue sections on the slides were washed with PBS and denatured with 2N HCl at 42°C for 20 minutes to denature a portion of the double-stranded DNA into short-stranded DNA and expose BrdU for antibody binding. The sections were then permeabilized with PBS containing 0.03% Triton X-100 (PBS-T) for 15 min, and incubated with 10% normal donkey serum in PBS-T for 1 h at room temperature to prevent nonspecific antibody binding. Primary antibodies were Rat monoclonal anti-BrdU (1:200, Abcam) and Rabbit polyclonal anti-Doublecortin (DCX, 1:200, Cell signaling technology), diluted in blocking buffer, and incubated overnight. Sections were then washed and incubated with fluorescent dye-labeled specific secondary antibodies for 2 h at room temperature. Slides were mounted using FluorSave (Merck Millipore).

Stained tissue sections were imaged using a Nikon Ti-Eclipse microscope (Nikon). Confocal images for quantification were acquired using a Zeiss LSM 710 laser scanning confocal microscope (Leica). Analysis of the dentate gyrus, the site of neurogenesis, was performed using Image J.

<Experimental Results>
As a result of the above experiment, it was confirmed that the number of cells positive for both BrdU, a marker for newborn cells, and DCX, a marker for immature neurons, increased (about 1.6 times) in the hippocampal dentate gyrus region of the brain of mice administered the extract from the heads of sugarcane tops, compared to the control group (Figure 19). This result suggests that administration of the extract from the heads of sugarcane tops promotes neurogenesis in the mouse brain.

<Evaluation of TrkB gene (gene name NTRK2) expression in SH-SY5Y cells using sugarcane top extract or compound standard>
The results of microarray analysis suggested that sugarcane top 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 it has been reported that activation by endogenous factors such as BDNF (brain-derived trophic factor) activates signaling pathways related to neuronal growth and survival, synaptic development, and hippocampal neurogenesis. To evaluate the effects of sugarcane top extract or standard compound on TrkB gene expression in neurons, gene expression analysis was performed using real-time RT-PCR. SH-SY5Y cells were used as a human neuronal model, and the following treatment groups were set: (i) control group, (ii) 3-CQA (0.50 μM), (iii) 5-CQA (0.70 μM), (iv) 3-FQA (0.85 μM), (v) ISO (0.477 μM), (vi) compound mixture (3-CQA + 5-CQA + 3-FQA + ISO), and (vii) sugarcane top head extract (50 μg/mL). Ginkgo biloba extract was also used as a positive control in this experiment. Ginkgo biloba extract (FUJIFILM Healthcare Laboratory Co., Ltd.) powder was dissolved in 70% ethanol, sterilized at 0.22 μm, and a stock solution (100 mg/mL) was prepared, and then added to the experimental conditions as treatment group (viii) ginkgo biloba extract (50 μg/mL). The cells were incubated with the samples in a 60 mm cell culture dish (BD Falcon) at 37°C in the presence of 5% CO2 for 24 hours, after which mRNA was extracted from the cells in each treatment group using the ISOGEN kit (NipponGene, Japan).
Reverse transcription polymerase chain reactions (RT-PCR) were performed using the extracted mRNA with the Superscript IV reverse transcriptase kit (Invitrogen, USA) and the 2720 Thermal cycler (Applied Biosystems, USA). To quantify the transcripts, TaqMan real time RT PCR amplification reactions were performed using the 7500 Fast Real Time PCR system. 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 one-way ANOVA test for significance (*P<0.05, **P<0.01).

<Experimental Results>
The expression of NTRK2 in SH-SY5Y cells was significantly increased by 24 hours of treatment with sugarcane tops extract compared to the control group (Figure 20). In addition, in tests using standard compounds, only ISO treatment showed a significant increase in NTRK2 expression compared to the control group, and the compound mixture treatment group showed a large increase in NTRK2 expression compared to the single compound treatment group (Figure 20). In addition, the ginkgo leaf extract treatment group, which was used as a positive control, showed a significant increase in NTRK2 expression compared to the control group, but the sugarcane tops extract treatment group and the compound mixture treatment group showed a large increase in NTRK2 expression compared to the ginkgo leaf extract treatment group (Figure 20).

No change in NTRK2 expression was observed when treated with 3-CQA (0.50 μM), 5-CQA (0.70 μM), or 3-FQA (0.85 μM) alone, and a large increase in NTRK2 expression (about 220% increase) was confirmed when treated with the compound mixture compared to treatment with ISO (0.477 μM) alone. In this result, the NTRK2 expression level is a relative value to the control group, so this suggests that when these compounds are mixed and treated, they synergistically increase NTRK2 expression in neurons.

In this experiment, the sugarcane top head extract treatment group and the compound mixture treatment group showed a significant increase in NTRK2 expression compared to the ginkgo leaf extract treatment group. Signal transduction via TrkB activation plays an important role in promoting synaptic plasticity and neurogenesis (Numakawa et al.). Therefore, sugarcane top head extract is expected to improve symptoms by effectively improving TrkB signaling, promoting neurogenesis, and suppressing the decrease in nerve cells associated with aging, compared to ginkgo leaf extract, a previous plant extract that has been reported to have cognitive function-improving effects. Specific diseases that are expected to improve memory decline and depression in aging and Alzheimer's disease, which have been reported to be involved in the decrease in nerve cells due to the decrease in BDNF-TrkB signaling and the decrease in neurogenesis (Tapia-Arancibia et al., 2008; Erickson et al., 2012).

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 an extraction solvent]
In the above test, the content of each polyphenol component per 100 parts by weight of the extract was highest in the sample extracted using 40% ethanol as a solvent. Based on this result, in order to examine the extraction conditions using more detailed concentration settings, components were extracted from 1 g of dried sugarcane tops using ethanol of different concentrations, 30%, 35%, 40%, 45%, and 50%, as the extraction solvent. The ethanol concentration is based on volume (v/v). An automatic extraction device E-916 (Nihon Buchi) was used for extraction, and the extraction conditions were set as follows: temperature = 45 °C, pressure = 100 bar, extraction time = 10 minutes, and number of extractions = 3 times. The solution after extraction was concentrated using a rotary evaporator and then freeze-dried to obtain a powdered 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 through 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 analysis was performed using a Prominence (Shimadzu Corporation). The analysis conditions were as follows: column: ZORBAX (250 x 4.6 mm), column temperature: 40°C, flow rate: 1.0 mL/min, injection volume: 10 μL, UV wavelength: 328 nm, mobile phase: A: 10% formic acid; B: acetonitrile: MeOH = 1:1, gradient: 0 - 100%; 40 min.

<Analysis results>
The quantitative results obtained by HPLC are shown in Figure 18. As a result of the quantitative analysis, the content of each polyphenol component per 100 parts by weight of the extract was highest in the sample extracted using 30% ethanol as a solvent.

The use of the composition of the present invention is expected to lead to the expansion of related markets and the development of new food and pharmaceutical seeds aimed at enhancing and improving cognitive function, as well as the development of products in the form of supplements, etc. Furthermore, in the medical and health fields, the predicted increase in the elderly population has led to a major concern of further increases in medical costs, and as the composition of the present invention is derived from food by-products, it is expected to be a major innovation in the medical and health markets.

Claims (9)

A method for producing a food material or a pharmaceutical material containing 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin, comprising the steps of:
A process for obtaining a fraction containing 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, 3-o-feruloylquinic acid, and isoorientin from sugarcane tops (which may include any one selected from leaves and bark) using an aqueous solvent. 2. The method according to claim 1, wherein the aqueous solvent is 25 to 40% ethanol, and the step of obtaining the fraction comprises extracting sugarcane tops (which may include any one selected from leaves and bark) using the aqueous solvent under a pressure of 100 bar or more. The manufacturing method according to claim 1 or 2, wherein the food material or pharmaceutical material is for regulating nerve function. 4. The method according to claim 1, wherein the food material or pharmaceutical material is for any one of the purposes selected from the group consisting of protecting nerve cells from amyloid beta , increasing secretion of neurotransmitters in the brain , promoting ATP production in nerve cells, promoting neurogenesis by proliferation of neural stem cells, inducing the transition of neural stem cells from a dormant state to an activated state, inducing differentiation of neural stem cells into neurons, and promoting the development of astrocytes. The method according to any one of claims 1 to 4, wherein the food material or pharmaceutical material is for any one selected from the group consisting of treatment of dementia, including senile dementia, Alzheimer's dementia, vascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebroedema, post-meningitis dementia, and Parkinson's dementia; treatment of non-dementia cognitive impairment, including mild cognitive impairment (MCI) and cognitive decline due to aging; improvement of learning disabilities, improvement of memory impairment; improvement of learning ability, improvement of memory ability; treatment of memory decline; treatment of cerebral infarction and peripheral nerve damage; treatment of mental disorders, including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder; improvement of motivation; and circadian rhythm disturbance; sleep improvement, which is any one of improvement of sleep quality, promotion of non-REM sleep, or improvement of sleep rhythm; treatment of sleep disorders, including insomnia; and improvement of memory decline or depression due to aging or Alzheimer's disease . A food composition or pharmaceutical composition for regulating nerve function, comprising isoorientin, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and 3-o-feruloylquinic acid . The composition according to claim 6, wherein isoorientin, 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and 3-o-feruloylquinic acid are contained as an extract from sugarcane tops (which may include any one selected from leaves and bark) . The composition according to claim 6 or 7, for any one of the following purposes selected from the group consisting of protecting neurons from amyloid beta, increasing secretion of neurotransmitters in the brain, promoting ATP production in neurons, promoting neurogenesis by proliferation of neural stem cells, inducing the transition of neural stem cells from a dormant state to an activated state, inducing differentiation of neural stem cells into neurons, and promoting the development of astrocytes. The composition of claim 6 or 7, for any one selected from the group consisting of: treatment of dementia, including senile dementia, Alzheimer's dementia, vascular dementia, post-traumatic dementia, dementia caused by brain tumors, dementia caused by chronic subdural hematoma, dementia caused by normal pressure cerebroedema, post-meningitic dementia, and Parkinson's 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 nerve damage; treatment of mental disorders, including attention deficit hyperactivity disorder (ADHD), depression, and bipolar disorder ; improvement of motivation; improvement of circadian rhythm disruption; improvement of sleep, either by improving sleep quality, promoting non-REM sleep, or improving sleep rhythm; treatment of sleep disorders, including insomnia; and treatment of memory decline or depression in aging or Alzheimer's disease .