JP7795592B2 - Composition containing isoorientin and at least one of 3-o-caffeoylquinic acid and 5-o-caffeoylquinic acid - Google Patents

Description

The present invention relates to a composition for improving mitochondrial activity, which contains an active ingredient derived from sugarcane tops.

Mitochondria play a central role in energy metabolism, and their activity is essential for the functioning of tissues with high energy demands, such as the brain, liver, and muscles. For this reason, decreased mitochondrial activity can lead to the onset of lifestyle-related diseases and aging-related diseases.

Mitochondrial activity is known to depend on the regulation of metabolic enzyme gene expression by nuclear transcription factors. One energy metabolism regulator is the nuclear receptor coactivator PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α), which is considered an important target for mitochondria, which are responsible for enhancing energy metabolism (Non-Patent Document 1).

Skeletal muscle is one of the tissues in the body in which PGC-1α and mitochondrial energy metabolism are important for its activity. When muscle cells adapt to heavy exercise, increased expression of PGC-1α activates mitochondria, improving the production of ATP, which serves as an energy source for contraction and other activities. It is also known that the glucose required for ATP production is taken up from outside the cell by promoting the migration of glucose transporter GLUT4 (glucose transporter 4) to the cell surface, facilitating the uptake of glucose from the blood into the cell. It is also known that PGC-1α increases in skeletal muscle in cold environments, and therefore is involved in the regulation of heat production. Thus, mitochondrial activation stimulates energy consumption, which in turn stimulates the metabolism of sugars and lipids, which serve as energy sources (Non-Patent Document 2).

Mitochondrial activity is also important in liver metabolism. Disturbances in hepatocyte metabolism induce lipid accumulation in cells, which is thought to be caused by a decrease in the catabolism of long-chain fatty acids via mitochondrial beta-oxidation. Increased expression of PGC-1α in hepatocytes increases the level of CPT1 (Carnitine palmitoyltransferase I), the rate-limiting enzyme in mitochondrial beta-oxidation, promoting mitochondrial fatty acid oxidation and reducing lipid accumulation (Non-Patent Document 3).

In addition, research into mitochondrial biogenesis and energy production in brain neurons has revealed that PGC-1α expression is closely related to neuronal function. For example, in mice in which PGC-1α has been knocked out, neurodegenerative disease-like degeneration and loss of neurons has been observed (Non-Patent Document 4). Furthermore, recent studies have suggested that activation of PGC-1α and the accompanying shift to mitochondrial biogenesis and oxidative metabolism are crucial for the morphological maturation of brain astrocytes (Non-Patent Document 5).

As described above, promoting PGC-1α expression and improving mitochondrial activity are predicted to be highly effective in improving energy metabolism in the body. Therefore, prior art related to promoting PGC-1α expression includes, for example, a mitochondrial function activator containing a benzimidazole derivative as an active ingredient (Patent Document 1), a mitochondrial function decline inhibitor containing catechins as an active ingredient (Patent Document 2), and a mitochondrial function enhancer containing sphingomyelin as an active ingredient (Patent Document 3). Also known is a neurotransmitter that promotes PGC-1α production by containing helipyrone A as an active ingredient (Patent Document 4). Patent Document 1 describes that when a target compound was added to a culture system of L6 myoblasts and hPGC1/L6 cells stably expressing hPGC-1, the transcriptional activation of hPGC-1 was confirmed. Furthermore, it is described that increased PGC-1 expression promotes downstream transcription of the human mitochondrial transcription factor A (hmtTFA) gene, and that when a target compound was added to a culture system of an hmtTFA promoter reporter vector, a cell line stably expressing hmtTFALuc, and hmtTFA/L6 cells, the transcriptional activation of hmtTFA was confirmed. Patent Document 2 describes that in a group of SAM-P1 male mice (senescence-accelerated mouse model) fed a diet containing catechin in combination with exercise, the expression of COX2-4 and HSP72 genes, which are involved in the electron transport chain and mitochondrial differentiation and proliferation associated with aging, was significantly higher than in a control group. Patent Document 3 describes that mice that ingested a test diet containing sphingomyelin had significantly higher PGC-1α gene expression in the gastrocnemius muscle compared to a control diet group, and that mice that ingested a test diet containing sphingomyelin had significantly higher oxygen consumption and lipid combustion compared to a control diet group. Patent Document 4 describes that the addition of Helipyron A to primary neurons obtained from rat fetuses confirmed an increase in PGC1α gene expression in neurons.

The present inventors have invented a method for obtaining a composition containing 3-o-caffeoylquinic acid (hereinafter referred to as 3CQA), 5-o-caffeoylquinic acid (hereinafter referred to as 5CQA), 3-o-feruloylquinic acid (hereinafter referred to as 3FQA), and isoorientin (hereinafter referred to as ISO) from sugarcane tops, and have further discovered that this composition promotes ATP production in nerve cells and astrocyte development (Patent Document 5, Non-Patent Document 6).

3CQA and 5CQA are known to activate lipid metabolism and, in particular, have the effects of preventing and improving obesity, suppressing or lowering blood sugar levels, and preventing and improving hyperleptinemia and hyperinsulinemia (Patent Document 6).

Japanese Patent Application Laid-Open No. 2004-67629 HYPERLINK "javascript:void(0)" Japanese Patent Application Publication No. 2008-63318 JP 2011-157328 A JP 2017-43566 A International Publication WO2021/167012 HYPERLINK "javascript:void(0)" Japanese Patent Application Publication No. 2003-034636

Wu et al., Cell 1999, 98, 115-124 Puigsever et al., Cell 1998, 92, 829-839 Kim et al., Nutr Metab(Lond) 2015, 12, 33 Sun et al., Neuroscience 2020, 440, 39-47 Zehnder et al., Cell Reports 2021, 35, 108952 Iwata et al., Front Cell Dev Biol 2020, 8, 573487

However, currently, no mitochondrial activators or PGC-1α expression promoters using active ingredients derived from sugarcane tops are available.

The present invention provides the following:
[1] Chlorogenic acids represented by the following formula:
(wherein R ¹ , R ² , and R ³ are each independently H, a caffeoyl group, or a feruloyl group), and a food composition or pharmaceutical composition for improving mitochondrial activity, comprising an active ingredient selected from the group consisting of luteolin and its glycosides.
[2] The composition according to 1, wherein the active ingredient is any one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and isoorientin.
[3] The composition according to 1 or 2, wherein the active ingredient is contained as a sugarcane top extract.
[4] A food composition or pharmaceutical composition for treating a disease or condition that can be improved through improving mitochondrial activity or promoting the expression of PGC-1α, comprising the active ingredient defined in 1.
[5] The composition according to claim 4, wherein the active ingredient is contained as a sugarcane top extract.
[6] A food composition or pharmaceutical composition containing the active ingredient defined in 1 for any of the following selected from the group consisting of: non-disease metabolic disorders, including age-related decline in glucose and lipid metabolism; metabolic diseases, including diabetes, hypertension, dyslipidemia, fatty liver, metabolic syndrome, and obesity; and muscle loss (sarcopenia) caused by age or the metabolic disorders; and maintenance and improvement of muscle mass or muscle strength, maintenance and improvement of muscle endurance, and maintenance and improvement of walking ability; and diseases characterized by morphological undermaturity of astrocytes, including Alexander disease.
[7] The composition according to 6, wherein the active ingredient is contained as a sugarcane top extract.
[8] A method for producing a food material or pharmaceutical material for improving mitochondrial activity, comprising any one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and isoorientin, the method comprising the following steps:
A process for obtaining a fraction containing any one selected from the group consisting of 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and isoorientin from sugarcane tops using an aqueous solvent.

Using active ingredients derived from natural products, it is possible to provide compositions for improving mitochondrial activity, compositions for promoting PGC-1α expression, and compositions for treating diseases or conditions that can be improved through improving mitochondrial activity or promoting PGC-1α expression.

Using active ingredients derived from natural products, compositions can be provided for treating metabolic diseases including non-disease metabolic disorders, such as age-related decline in glucose and lipid metabolism; diabetes, hypertension, dyslipidemia, fatty liver, metabolic syndrome, and obesity.

Using active ingredients derived from natural products, a composition can be provided for any of the following purposes selected from the group consisting of maintaining and improving muscle mass or muscle strength, maintaining and improving muscle endurance, and maintaining and improving walking ability.

Using active ingredients derived from natural products, compositions can be provided for treating diseases characterized by morphological maturation defects of astrocytes, including Alexander disease.

Mitochondrial activity in neural stem cell-derived astrocytes. Top: 24-hour treatment: Significant increase in (3)3CQA + 5CQA and (5)5CQA + ISO treatments (*p value < 0.05). Middle: 48-hour treatment: (8)3CQA+5CQA+ISO and 4-mix (All mixed) treatments (p value < 0.1). Bottom: 72-hour treatment: Significant increase in (5)5CQA + ISO, 4-mix (All mixed) and stem cell extract (STEE) treatments (*p value < 0.05), and tendency to increase in (10)5CQA+3FQA+ISO treatment (p value < 0.1). PGC-1α expression in neural stem cell-derived astrocytes *p < 0.05, ****p < 0.0001 Compared to control cells, a significant increase in PGC-1α expression (p < 0.0001) was confirmed in (5)5CQA + ISO, (8)3CQA + 5CQA + ISO, the four-component mixture (All mixed), and stem cell extract (STEE). Mitochondrial activity in myotubes *p < 0.05 Compared to control cells. Rh123 fluorescence intensity was significantly enhanced in treatment groups (3), (8), All mixed, and STEE. Fluorescence intensity also tended to increase in treatment group (5). PGC-1α mRNA and TFAM mRNA expression in myotubes. *p < 0.05, ***p < 0.001. Compared to control cells, PGC-1α mRNA expression was significantly elevated by 6 hours of treatment with 50 μg/mL STEE (p < 0.05, approximately 1.4-fold), and by 6 hours of treatment with the compound mixture (Mix) (p < 0.05, approximately 1.4-fold). Expression levels remained significantly elevated after 24 hours of treatment with the same mixture (p < 0.05, approximately 1.5-fold). TFAM mRNA expression tended to increase after 6 hours of treatment with 50 μg/mL STEE (p = 0.082, approximately 1.25-fold), and remained significantly elevated after 24 hours of treatment with the same mixture (p < 0.05, approximately 1.5-fold). Furthermore, TFAM mRNA expression levels were significantly elevated by 6 hours of treatment with Mix (p < 0.05, approximately 1.25-fold), and were also significantly elevated by 24 hours of treatment with the same sample (p < 0.001, approximately 1.75-fold). Mitochondrial activity in hepatocytes Although not statistically significant, Rh123 fluorescence intensity increased by approximately 120% in STEE after 6 hours of treatment, and by approximately 120% in treatment groups (7), (8), and STEE after 24 hours of treatment. PGC-1α mRNA and TFAM mRNA expression in hepatocytes. *p < 0.05, **p < 0.01. Compared to control cells, PGC-1α mRNA expression was significantly elevated by 24-hour treatment with 30 μg/mL and 50 μg/mL STEE (p < 0.01, approximately 1.4-fold, respectively) and by 24-hour treatment with the compound mixture (Mix) (p < 0.01, approximately 1.4-fold). TFAM mRNA expression was significantly elevated by 24-hour treatment with 30 μg/mL and 50 μg/mL STEE (p < 0.05, approximately 1.2-fold, respectively) and by 24-hour treatment with Mix (p < 0.05, approximately 1.2-fold).

The present invention relates to a food composition or pharmaceutical composition containing an active ingredient selected from the group consisting of chlorogenic acids, luteolin, and its glycosides.

[Active ingredients, functional ingredients]
The composition of the present invention contains, as an active ingredient, any one selected from the group consisting of chlorogenic acids, luteolin, and its glycosides. The active ingredient may also be called a functional ingredient or a component contributing to functionality.

<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.

Specific 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 embodiment, the composition contains a chlorogenic acid 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).

In one embodiment, the composition preferably contains at least one of 3-o-caffeoylquinic acid and 5-o-caffeoylquinic acid selected from the group consisting of the above three compounds as chlorogenic acids, more preferably both, and even more preferably both and 3-o-feruloylquinic acid.

<Luteolin and its glycosides>
In one embodiment, the composition contains, as an active ingredient, any one selected from the group consisting of luteolin and its glycosides (hereinafter, sometimes referred to as luteolins). Luteolin glycosides are compounds 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 digoxose. Examples of luteolin glycosides 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), and luteolin 7-(2 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) are also known.

In one embodiment, the composition contains a compound represented by the following formula as a luteolin:

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

In one embodiment, the composition contains, as the luteolin, either isoorientin (R ⁴ =H, R ⁵ =glucose residue) or orientin (R ⁴ =glucose residue, R ⁵ =H).

In one embodiment, the composition contains isoorientin as a luteolin.

<Combination of active ingredients>
In one embodiment, the composition contains two or more components selected from the group consisting of 3-o-caffeoylquinic acid (3CQA), 5-o-caffeoylquinic acid (5CQA), 3-o-feruloylquinic acid (3FQA), and isoorientin (ISO), from the viewpoint of enhancing the intended effect. The two or more components are preferably a combination containing at least one selected from 3CQA, 5CQA, and ISO. Particularly preferred examples of such combinations include a combination of 3CQA and ISO, a combination of 5CQA and 3FQA, a combination of 5CQA and ISO, a combination of 3FQA and ISO, a combination of 3CQA, 5CQA, and ISO, and a combination of 5CQA, 3FQA, and ISO. In one embodiment, the composition contains a combination of 3CQA, 5CQA, and ISO, and may further contain 3FQA.

When a composition contains the four compounds 3CQA, 5CQA, 3FQA, and ISO, their ratio is not particularly limited as long as the desired effect is achieved. 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>
In one embodiment, the active ingredient of the composition may be a sugarcane top extract. The sugarcane top refers to the top portion of sugarcane (scientific name: Saccharum officinarum L.) above the fifth leaf thickening zone. Unless otherwise specified, the extract includes an extract obtained by extracting a raw material using a solvent, as well as a concentrate, dried product, and a crude product of the extract.

When extraction is performed under appropriate conditions, 100mg to 170mg, more specifically 130mg to 150mg, of extract containing the four compounds 3CQA, 5CQA, 3FQA, and ISO can be obtained from 1g of dried sugarcane tops (including leaves and bark) in dry weight.

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

The amount of 5-o-caffeoylquinic acid contained in sugarcane top extract can be 1.0 mg or more per 100 g (dry weight) of extract, regardless of the content of other components. By optimizing the extraction conditions, the amount can be increased to 1.5 mg or more, 2.0 mg or more, or 2.5 mg or more, regardless of the content of other components.

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

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

In one embodiment, 100 g (dry weight) of sugarcane top extract contains 0.40 mg to 0.60 mg of 3-o-caffeoylquinic acid, 2.5 mg to 3.5 mg of 5-o-caffeoylquinic acid, 0.20 mg to 0.30 mg of 3-o-feruloylquinic acid, and 1.4 mg to 1.8 mg of isoorientin.

In one 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).

<Method for producing sugarcane top extract>
The sugarcane top extract used as an active ingredient can be produced using sugarcane tops as a raw material. The sugarcane tops used as a raw material may be the entire sugarcane top, a portion including leaves and bark, or a portion from which the leaves and bark have been removed. The raw sugarcane tops may be in a raw state or may be dried. Drying can be carried out by cold air drying or sun drying. The sugarcane tops may be cut, shredded, or crushed to increase 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 or subcritical extraction may be performed using a supercritical or subcritical fluid.

The extraction solvent is preferably a solvent that is effective for extracting luteolin or its glycosides and for extracting chlorogenic acids. Examples of such solvents include 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. Preferred examples are aqueous solvents, such as water or mixtures of water with any of the group consisting of methanol, ethanol, isopropanol, butanol, propylene glycol, butylene glycol, and glycerin.

In one embodiment, water is used as the solvent. In another embodiment, a mixed solvent of water and ethanol is used. The ethanol concentration can be 5% or more, 10% or more, 20% or more, or 25% or more. The ethanol concentration can also be 95% or less, 90% or less, 85% or less, 70% or less, or 60% or less. From the perspective of minimizing ethanol use, the ethanol concentration is preferably 50% or less, more preferably 40% or less, and even more preferably 35% or less. In relation to the present invention, when referring to the ethanol concentration of an extraction solvent containing ethanol, unless otherwise specified, the value is based on volume (v/v), and unless otherwise specified, the ethanol is mixed with water.

The extraction procedure can be carried out at room temperature or heated. Heating under reflux cooling allows for efficient and rapid extraction of the components. The extraction temperature can be set to 60°C or higher, preferably 70°C or higher, and more preferably 80°C to 100°C. When carried out under heating, the extract can be obtained efficiently and with high purity.

Extraction can be carried out at normal pressure. Extraction can also be carried out under pressure, at 5 MPa (50 bar) or higher, preferably 7.5 MPa (75 bar) or higher, or even 10 MPa (100 bar) or higher. In this case, heating may also be used to efficiently and quickly extract the target components. The temperature can be set to 25°C or higher, preferably 30°C or higher, more preferably 35°C or higher, and even more preferably 40°C or higher. The ratio of raw material to extraction solvent, extraction time, and number of repetitions of the extraction procedure can be adjusted appropriately, taking into account extraction efficiency.

In one embodiment, the dried sugarcane tops are extracted using hot water at room temperature. In another embodiment, a mixture of water and ethanol is used as the extraction solvent, and automated component extraction is carried out multiple times, for example, 2 to 8 times, for 15 to 30 minutes each, using a high-speed, high-pressure extraction device at a pressure of 100 bar and a temperature of 45°C. The solution obtained by extraction can be concentrated and freeze-dried.

When performing supercritical or subcritical extraction, fluids that can be used include, for example, water, carbon dioxide, ethylene, propylene, ethane, propane, nitrous oxide, chlorodifluoromethane, chlorotrifluoromethane, xenon, ammonia, and lower alcohols such as methanol and ethanol. From a safety standpoint, it is preferable to use water, ethanol, a mixture of these, or carbon dioxide. If necessary, insoluble residues can be removed, the product can be concentrated using conventional methods, and dried by means of spray drying, freeze drying, or other methods.

[Application]
In one aspect, a composition containing an active ingredient selected from the group consisting of chlorogenic acids, luteolin, and its glycosides is used to improve mitochondrial activity. Studies by the present inventors have shown that a composition containing an active ingredient selected from the group consisting of chlorogenic acids, luteolin, and its glycosides can activate mitochondria in immature astrocytes, myotubes, and hepatocytes, and is also expected to activate mitochondria in adipocytes and intestinal epithelial cells.

In one embodiment, a composition containing an active ingredient selected from the group consisting of chlorogenic acids, luteolin, and its glycosides is used to promote the expression of peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α). PGC-1α, a nuclear receptor coactivator, is known as an energy metabolism regulator. PGC-1α is also considered an important target for mitochondria, which are responsible for enhancing energy metabolism (Non-Patent Document 1). Research by the present inventors has shown that a composition containing an active ingredient selected from the group consisting of chlorogenic acids, luteolin, and its glycosides can promote the expression of PGC-1α in immature astrocytes, myotubes, and hepatocytes, and is also expected to activate mitochondria in adipocytes and intestinal epithelial cells. PGC-1α expression refers to the process by which information from the PGC-1α gene is converted into structure and function, and promotion of expression includes promoting the transcription of the gene into mRNA.

In one aspect, a composition containing an active ingredient selected from the group consisting of chlorogenic acids and luteolin and its glycosides can be used to treat a disease or condition that can be improved through improved mitochondrial activity.

In one aspect, a composition comprising chlorogenic acids and an active ingredient selected from the group consisting of luteolin and its glycosides is used for the treatment of a metabolic disease or condition.

In one aspect, a composition containing an active ingredient selected from the group consisting of chlorogenic acids and luteolin and its glycosides can be used to treat a disease or condition that can be improved through the promotion of PGC-1α expression.

In one aspect, a composition containing an active ingredient selected from the group consisting of chlorogenic acids, and luteolin and its glycosides can be used to treat diseases or conditions associated with muscle loss due to aging or the metabolic disorders. Skeletal muscle is one of the tissues in the body in which PGC-1α activity and mitochondrial energy metabolism are important for its activity. When muscle cells adapt to load-bearing exercise, increased expression of PGC-1α activates mitochondria, improving the production of ATP, which serves as an energy source for activities such as contraction.

More specifically, a composition comprising an active ingredient selected from the group consisting of chlorogenic acids, and luteolin and its glycosides can be used for the following diseases or treatments:
Non-disease metabolic disorders, including age-related decline in glucose and lipid metabolism; diabetes, hypertension, dyslipidemia, fatty liver, metabolic syndrome, obesity; sarcopenia, sarcopenia; diseases characterized by morphological maturation defects of astrocytes, including Alexander disease.
Alternatively, a composition containing an active ingredient selected from the group consisting of chlorogenic acids, and luteolin and its glycosides can be used to maintain or improve muscle mass or muscle strength, muscle endurance, and walking ability.

Whether a certain component improves mitochondrial activity can be assessed by, for example, analyzing the amount of rhodamine 123 uptake into mitochondria in the presence or absence of the target component using neural stem cell-derived immature astrocytes (e.g., human fetal neural stem cells), myotube cells (e.g., mouse skeletal muscle-derived myoblasts C2C12), hepatocytes (e.g., human hepatoma-derived cell line HepG2), adipocytes (e.g., mouse preadipocyte cell line 3T3-L1), or intestinal epithelial cells (e.g., human small intestinal epithelial-like cell line Caco-2) based on fluorescence intensity. Whether a certain component promotes PGC-1α expression can be assessed by, for example, analyzing the expression level of PGC-1α mRNA in the presence or absence of the target component using any of the above-mentioned cells. It can also be assessed by analyzing the expression level of TFAM mRNA, whose transcription is promoted downstream of PGC-1α activity.

In this invention, the term "treatment" in reference to a disease or condition includes reducing the risk of onset, delaying onset, prevention, cure, and halting or delaying progression. Treatment includes actions performed by physicians with the purpose of treating a disease, as well as 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 purpose of treating a disease in non-human animals, as well as non-therapeutic actions performed by persons other than veterinarians, such as veterinary nurses, pet animal care managers, stable hands, animal keepers, animal drug manufacturers, animal drug distributors, pet food manufacturers, and pet food distributors. Furthermore, treatment includes the administration or recommendation of specific foods, dietary guidance, health guidance, nutritional guidance (including nutritional guidance necessary for the medical treatment of injured or sick persons, and nutritional guidance for maintaining and promoting health), school meal management, and guidance necessary for improving nutrition in relation to school meals.

Subjects for treatment in the present invention include humans (individuals), and preferably humans for whom any of the above-mentioned treatments is desirable or necessary. Subjects for treatment in the present invention may also be animals other than humans, and examples 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, etc. There are no particular limitations on the developmental stage of the non-human animals, and subjects for treatment in the present invention may be, for example, puppies, adult dogs, senior dogs, kittens, adult cats, and senior cats.

[Composition, etc.]
In one embodiment, the composition may be the sugarcane tops extract itself, or may contain an active ingredient (eg, sugarcane tops extract) and other ingredients.

In one aspect, the composition can be a food composition or a pharmaceutical composition. Unless otherwise specified, food includes not only solids but also liquids, such as soups, beverages, and health 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 (foods for specified health uses (commonly known as FOSHU), nutrient functional foods, and functional food), as well as therapeutic foods (foods that serve therapeutic purposes; prepared based on a menu prepared by a nutritionist or other professional following a doctor's dietary prescription), therapeutic diets, ingredient-modified meals, reduced-salt meals, nursing care meals, reduced-calorie meals, and diet meals.

In one embodiment, the composition is in the form of an oral medicine, health food, or supplement. In such cases, examples of dosage forms include soft capsules, hard capsules, tablets, pills, powders, granules, fine granules, jellies, tube-packed formulations, and drinks.

The content of the active ingredient in the composition can be appropriately determined taking into account the daily intake/administration amount. In one embodiment, the daily dose for an adult contains 0.2 to 2,000 mg of the active ingredient, 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 taken or administered multiple times, for example, 2 to 4 times.

In one embodiment, the composition can contain ingredients other than sugarcane top extract, as long as the desired effect can be achieved. These ingredients may be various additives acceptable for food use or various additives acceptable for pharmaceutical use. Examples of such additives include excipients, antioxidants (antioxidants), flavorings, seasonings, sweeteners, coloring agents, thickening and stabilizing agents, color formers, bleaching agents, fungicides, gum bases, bittering agents, enzymes, glazing agents, acidulants, emulsifiers, strengthening agents, manufacturing agents, binders, tonicity agents (isotonicity agents), buffers, solubilizers, preservatives, stabilizers, and coagulants.

The other ingredients may be functional ingredients. Examples of functional ingredients include amino acids (e.g., branched-chain amino acids, ornithine), unsaturated fatty acids (e.g., EPA, DHA), vitamins, trace metals, polyphenols, egg yolk extract, processed honey, brown sugar, oligosaccharides, dietary fiber, glucosamine, chondroitins, CoQ10, fucoidan, fucoxanthin, astaxanthin, placenta, yeast extract, black vinegar concentrate, plant extracts (garlic extract, ginkgo biloba 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, and noni extract), etc.

In one aspect, the composition can be taken or administered with, before, or after a meal.

In one aspect, the composition can be labeled to indicate that it can be used to treat the aforementioned diseases or conditions, and can be labeled to recommend its intake to the aforementioned subjects. Labeling can be direct or indirect. Examples of direct labeling include inscriptions on tangible objects such as the product itself, packaging, containers, labels, and tags, while examples of indirect labeling include advertising and promotional activities in places or by means such as websites, in-store displays, exhibitions, signs, bulletin boards, newspapers, magazines, television, radio, mail, and email.

Examples of functional claims on compositions include the following:
Maintaining and preventing the decline of muscle strength necessary for independent daily living, maintaining necessary muscle strength (muscle strength needed for standing, walking, etc.), maintaining muscle mass and strength, maintaining walking ability, improving walking function, maintaining muscle that decreases with age, supporting the synthesis of muscle that weakens with age, making it easier to burn fat, reducing abdominal fat, and maintaining healthy liver function.

The following are manufacturing and test examples, but the present invention is not limited to these examples in any way.

[Production Example 1]
-Production of ethanol extract from sugarcane tops-
Four automated extractions of 1 g of dried sugarcane tops (including leaves and bark) were performed using 80% ethanol as the extraction solvent, at a pressure of 100 bar and a temperature of 45°C, using an E-916 high-speed, high-pressure extractor (AG Buchi). Each extraction was performed for approximately 20 minutes. The extracted solution was concentrated using a rotary evaporator and then freeze-dried to obtain 140 mg of sample. The contents of 3CQA, 5CQA, 3FQA, and ISO per 1 g of extract, as determined by component analysis using high-performance liquid chromatography (HPLC), were equal to the analytical values reported in Patent Document 5 (Example [Component Analysis of Sugarcane Tops Extract]) and Non-Patent Document 6 (Chemical Analysis).

Test Example 1
- Mitochondrial activity evaluation test in immature astrocytes derived from neural stem cells -
It has been shown that mitochondrial biogenesis is required for the morphogenesis and maturation of astrocytes. Sugarcane top extract has been suggested to promote the development and maturation of astrocytes. Therefore, we evaluated the effects of sugarcane top extract and its polyphenolic compounds on mitochondrial activity in immature astrocytes.

Human fetal neural stem cells (hNSCs; Cell Applications) were seeded at a density of 10,000 cells/ ^cm² in 96-well plates and then incubated for 48 hours in astrocyte differentiation medium containing Dulbecco's modified Eagle's medium (DMEM), 1% N-2 supplement (Gibco), 1% fetal bovine serum (FBS), and 1% penicillin/streptomycin. The differentiated astrocytes were then switched to astrocyte differentiation medium containing the sample, i.e., sugarcane top extract or polyphenolic compounds (including their combination), and incubated for 24, 48, and 72 hours.

The sugarcane top extract used was the extract obtained in Production Example 1. The final extract concentration in the medium was set to 50 μg/mL. The concentrations of each compound in a 50 μg/mL extract were calculated from the analytical quantitative values described in Patent Document 5 and Non-Patent Document 6. That is, the concentrations in the medium were 0.50 μM for 3CQA, 0.70 μM for 5CQA, 0.85 μM for 3FQA, and 0.48 μM for ISO. In addition to the combination of four compounds, six groups of two-compound combinations and four groups of three-compound combinations were set as treatment groups. The numbering of the two-compound and three-compound combination treatment groups is shown in the table below.

After incubation, mitochondrial activity (content) was assessed using rhodamine 123 (Rh123). Rh123 is readily incorporated into intracellular mitochondria, and its fluorescence intensity can be measured to assess mitochondrial activity. After washing the cells with PBS, a 10 μg/mL Rh123 solution was added to each well and incubated at 37°C for 20 minutes. After washing the cells again, 1% Triton-X solution was added to each well and incubated at room temperature in the dark for 30 minutes to lyse the cells. The cell lysates were transferred to a black, clear-bottom 96-well plate, and fluorescence intensity was measured at λex = 507 nm and λem = 529 nm using a plate reader (Varioskan LUX, Thermo Fisher Scientific). The obtained values were subjected to a one-way ANOVA test for significance (*P<0.05).

-Test Results-
After 24 hours of treatment, mitochondrial Rh123 fluorescence intensity was significantly enhanced in treatment groups (3) and (5). After 48 hours of treatment, fluorescence intensity tended to increase in treatment group (8) and the four-compound mixture (All mixed) (p = 0.097 and p = 0.082, respectively). After 72 hours of treatment, fluorescence intensity was significantly enhanced in treatment groups (5), (8), and sugarcane top head extract (STEE) (Figure 1). These results suggest that sugarcane top head extract activates mitochondria in hNSC-derived immature astrocytes. Furthermore, it is suggested that the polyphenolic components in the extract, 3CQA, 5CQA, and ISO, contribute to this activity.

Test Example 2
- Evaluation of PGC-1α mRNA expression in immature astrocytes derived from neural stem cells -
Mitochondrial activity is dependent on the regulation of metabolic enzyme gene expression by nuclear transcription factors, with the transcription factor PGC-1α playing a key role. It has been shown that activation of PGC-1α, a master regulator of mitochondrial biogenesis, is essential for astrocyte maturation. Since the sugarcane top head extract demonstrated a mitochondrial activation effect in [Test Example 1], we next evaluated the effect of sugarcane top head extract on PGC-1α mRNA expression in immature astrocytes.

First, astrocytes were induced from hNSCs in 24-well plates using the same method as in Test Example 1. The medium for differentiated astrocytes was switched to astrocyte differentiation-inducing medium containing sugarcane top extract or polyphenolic compounds (including combinations thereof), and the cells were further incubated for 48 hours.

The concentrations of sugarcane top extract and each compound in the medium were the same as in [Test Example 1]. The combinations of compounds used as treatment groups were also the same as in [Test Example 1].

After incubation, total RNA was isolated from the cells using the RNeasy micro kit (Qiagen). mRNA expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) using this total RNA as a template on a 7500 Fast Real-Time PCR System (Applied Biosystems). TaqMan primers (Applied Biosystems) for GAPDH (Hs02786624_g1) and PPARGC1 (Hs00173304_m1) were used, and GAPDH was used as an internal standard to determine relative transcript expression levels. One-way ANOVA was used to test for significance (*P<0.05, ****<0.001).

-Test Results-
Figure 2 shows the expression levels of PGC-1α mRNA in hNSC-derived immature astrocytes. Treatment with STEE for 48 hours significantly increased PGC-1α mRNA expression (p < 0.0001, approximately 2.5-fold). Furthermore, among the compound-treated groups at 48 hours, treatment groups (5), (8), and All Mix showed a significant increase in PGC-1α mRNA expression, comparable to that of the STEE-treated group (p < 0.0001).

These results suggest that sugarcane top head extract induces increased PGC-1α mRNA expression in hNSC-derived immature astrocytes. Furthermore, it was suggested that among the polyphenolic components in the extract, 3CQA, 5CQA, and ISO contribute to this activity.

The effects of sugarcane top-derived compositions on mitochondrial activity and PGC-1α mRNA expression can also be evaluated by conducting the tests described below.

Test Example 3
- Mitochondrial activity evaluation test in myotube cells -
In order to enhance energy production for muscle contraction and other activities, it is necessary to improve intracellular mitochondrial activity. We evaluated the effects of sugarcane top extract and its polyphenol compounds on mitochondrial activity in myotube cells.

Mouse skeletal muscle-derived myoblasts, C2C12, were seeded at 30,000 cells/ ^cm² in a 96-well plate and then cultured in a medium containing DMEM, 10% FBS, and 1% penicillin/streptomycin to induce myoblast proliferation. Upon reaching confluence, the medium was replaced with DMEM and 2% horse serum (HS) to induce differentiation into myotubes. After 6 days of culture, the differentiated myotubes were treated with dissolved test samples, i.e., sugarcane top extract or polyphenolic compounds (including their combinations), and then incubated for 6 and 24 hours.

The test samples were the same as those used in Test Example 1. That is, they were a combination of the extract obtained in Production Example 1, each polyphenol compound, and the compounds in Table 1. The final concentration of the extract in the medium was set to 50 μg/mL, and the concentrations of each compound were set to 0.50 μM for 3CQA, 0.70 μM for 5CQA, 0.85 μM for 3FQA, and 0.48 μM for ISO. The concentrations of these compounds were the same when they were treated in combination.

After incubation, mitochondrial activity (content) was evaluated using Rh123. The test method was the same as that described in [Test Example 1]. The obtained values were subjected to a significance test (*P<0.05) using the one-way ANOVA method.

-Test Results-
After 6 hours of treatment, Rh123 fluorescence intensity was significantly enhanced in treatment groups (3), (8), All Mix, and STEE. Furthermore, fluorescence intensity tended to increase in treatment group (5). After 24 hours of treatment, no significant changes in fluorescence intensity were observed in any of the treatment groups (Figure 3). These results suggest that 6 hours of treatment with sugarcane top extract induces mitochondrial activation in myotubes. Furthermore, it was suggested that 3CQA, 5CQA, and ISO, among the polyphenolic components in the extract, contribute to this activity.

Test Example 4
-Examination of PGC-1α mRNA and TFAM mRNA expression in myotube cells-
The transcriptional regulator PGC-1α plays an important role in regulating mitochondrial activity, and downstream of PGC-1α expression, transcription of the mitochondrial transcription factor A (TFAM) gene is promoted, enhancing mitochondrial biogenesis. We evaluated the effects of sugarcane top extract and its polyphenolic compounds on PGC-1α mRNA and TFAM mRNA expression in myotube cells.

First, using the same method as in Test Example 3, differentiation of C2C12 cells into myotubes was induced in a 6-well plate. After 6 days, the test samples, i.e., sugarcane top extract or a polyphenol compound mixture, were added to the differentiated myotubes, and the cells were incubated for an additional 6 or 24 hours.

The sugarcane top extract used was the extract obtained in Production Example 1. The final extract concentrations in the medium were set to 15 μg/mL, 30 μg/mL, and 50 μg/mL. A combination of four compounds was used as the test sample (Mix), and the concentrations of each compound in the medium were set to the content contained in a 50 μg/mL extract, i.e., 0.50 μM for 3CQA, 0.70 μM for 5CQA, 0.85 μM for 3FQA, and 0.48 μM for ISO.

After incubation, mRNA expression levels were measured by qRT-PCR as in [Test Example 2]. The TaqMan primers used were Gapdh (Mm99999915_g1), Ppargc1 (Mm01208835_m1), and Tfam (Mm00447485_m1), and Gapdh was used as an internal standard to determine the relative expression levels of the transcripts. The obtained values were subjected to significance testing using one-way ANOVA (*P<0.05, ***<0.001).

-Test Results-
Figure 4 shows the expression levels of PGC-1α mRNA and TFAM mRNA in myotubes. PGC-1α mRNA expression was significantly elevated by 6 hours of treatment with 50 μg/mL STEE (p < 0.05, approximately 1.4-fold), and no change was observed after 24 hours of treatment with the same compound. PGC-1α mRNA expression was also significantly elevated by 6 hours of treatment with the compound mixture (Mix) (p < 0.05, approximately 1.4-fold), and the expression level remained significantly elevated after 24 hours of treatment with the same compound mixture (p < 0.05, approximately 1.5-fold). TFAM mRNA expression showed an increasing trend after 6 hours of treatment with 50 μg/mL STEE (p = 0.082, approximately 1.25-fold), and the expression level remained significantly elevated after 24 hours of treatment with the same compound mixture (p < 0.05, approximately 1.5-fold). Furthermore, TFAM mRNA expression levels were significantly elevated by 6 hours of treatment with Mix (p < 0.05, approximately 1.25-fold), and the same levels were also significantly elevated after 24 hours of treatment (p < 0.001, approximately 1.75-fold).

These results suggest that sugarcane top extract induces increased PGC-1α mRNA and TFAM mRNA expression in myotube cells. Furthermore, it suggests that 3CQA, 5CQA, 3FQA, or ISO in the extract contributes to this activity.

Test Example 5
- Mitochondrial activity evaluation test in hepatocytes -
In the liver, which is the center of metabolism in the body, intracellular mitochondria play an important role in improving its activity. We evaluated the effects of sugarcane top extract and its polyphenolic compounds on mitochondrial activity in hepatocytes.

First, human hepatoma-derived HepG2 cells were seeded at a density of 30,000 cells/ ^cm² in a 96-well plate and cultured in a medium consisting of DMEM, 10% FBS, and 1% penicillin/streptomycin. After 24 hours of culture, the medium was switched to a test sample, i.e., sugarcane top extract or polyphenolic compounds (including their combinations) dissolved in a low-serum medium containing 1% penicillin/streptomycin (Opti-MEM®), and the cells were incubated for an additional 6 and 24 hours.

The concentrations of sugarcane top extract and each compound in the medium were the same as in [Test Example 3]. The combinations of compounds used as treatment groups were also the same as in [Test Example 3].

After incubation, mitochondrial activity (content) was evaluated using Rh123. The test method was the same as that described in [Test Example 1]. The obtained values were subjected to significance testing using the one-way ANOVA method.

-Test Results-
No significant changes in fluorescence intensity were observed in either treatment group after 6 or 24 hours of treatment (Fig. 5). Although not statistically significant, Rh123 fluorescence intensity increased approximately 120% in STEE after 6 hours of treatment, and Rh123 fluorescence intensity also increased approximately 120% in treatment groups (7), (8), and STEE after 24 hours of treatment (Fig. 5).

Test Example 6
-Examination of PGC-1α mRNA and TFAM mRNA expression in hepatocytes-
As mentioned above, the transcriptional regulator PGC-1α and TFAM, whose transcription is promoted downstream of PGC-1α activity, play important roles in regulating mitochondrial activity. We evaluated the effects of sugarcane top extract and its polyphenolic compounds on PGC-1α mRNA and TFAM mRNA expression in hepatocytes.

First, HepG2 cells were cultured in a 6-well plate using the same method as in Test Example 5. After 24 hours of culture from seeding, the test samples, i.e., sugarcane top extract or polyphenol compound mixture, were added and incubated for an additional 6 or 24 hours.

The test sample was the same as in Test Example 5. The concentration of the test sample in the medium was also the same as in Test Example 5.

After incubation, mRNA expression levels were measured by qRT-PCR as in [Test Example 2]. The TaqMan primers used were GAPDH (Hs02786624_g1), PPARGC1 (Hs00173304_m1), and TFAM (Hs00273372_s1), and GAPDH was used as an internal standard to determine the relative expression levels of the transcripts. The obtained values were subjected to significance testing using one-way ANOVA (*P<0.05, **<0.01).

-Test Results-
The expression levels of PGC-1α mRNA and TFAM mRNA in hepatocytes are shown in Figure 6. PGC-1α mRNA expression was significantly elevated by 24-hour treatment with 30 μg/mL and 50 μg/mL STEE (p < 0.01, approximately 1.4-fold, respectively), and by 24-hour treatment with the compound mixture (Mix) (p < 0.01, approximately 1.4-fold). TFAM mRNA expression was significantly elevated by 24-hour treatment with 30 μg/mL and 50 μg/mL STEE (p < 0.05, approximately 1.2-fold, respectively), and by 24-hour treatment with Mix (p < 0.05, approximately 1.2-fold). Furthermore, 6-hour treatment with either sample did not significantly affect PGC-1α or TFAM mRNA expression.

These results suggest that sugarcane top extract induces increased PGC-1α mRNA and TFAM mRNA expression in hepatocytes. Furthermore, it suggests that 3CQA, 5CQA, 3FQA, or ISO in the extract contributes to this activity.

The effects of sugarcane top-derived components on mitochondrial activity and PGC-1α mRNA expression can also be evaluated by conducting the tests described below.

Example 1
- Evaluation test of mitochondrial activity and PGC-1α mRNA expression in adipocytes -
First, 3T3-L1, a mouse-derived preadipocyte cell line, was grown as preadipocytes in a medium containing DMEM, 10% FBS, and 1% penicillin/streptomycin. Two days after reaching confluence (post-confluence), a differentiation inducer consisting of isobutylmethylxanthine (IBMX), insulin, and dexamethasone (DEX) was added to the medium to induce differentiation into adipocyte-like cells. Simultaneously with the addition of the differentiation inducer, test samples (sugarcane top extract and polyphenolic compounds) were added to achieve appropriate concentrations in the medium. After the addition of the differentiation inducer, differentiation into adipocytes was largely completed after 2–3 days of incubation.

Differentiated adipocytes can be used to assess mitochondrial activity using the aforementioned Rh123, and PGC-1α mRNA expression levels can be assessed using the aforementioned qRT-PCR.

Example 2
- Evaluation test of mitochondrial activity and PGC-1α mRNA expression in intestinal epithelial cells -
First, Caco-2, a human small intestinal epithelial cell line, was cultured in a medium consisting of DMEM, 10% FBS, and 1% penicillin/streptomycin. After the culture reached confluence, the cells were further cultured for 14 days to differentiate into intestinal epithelial cells. Dissolved test samples (sugarcane top extract and polyphenolic compounds) were added to the differentiated intestinal epithelial cells to achieve appropriate concentrations in the medium.

After adding the sample and incubating the intestinal epithelial cells for an appropriate period of time, mitochondrial activity can be assessed using the Rh123 assay described above, and PGC-1α mRNA expression levels can be assessed using the qRT-PCR described above.

Claims (8)

A food composition or pharmaceutical composition for promoting PGC-1α gene expression in myotubes or skeletal muscle, comprising isoorientin and at least one of 3-o-caffeoylquinic acid and 5-o-caffeoylquinic acid. The composition of claim 1, comprising 3-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, and isoorientin. The composition according to claim 1 or 2, wherein the active ingredient is contained as an extract of sugarcane tops (which may include any one selected from leaves and bark). The food composition or pharmaceutical composition according to claim 1 or 2 is intended to support any of the following: maintaining and preventing the decline of muscle strength necessary for independent daily living, maintaining muscle strength necessary for standing and walking, maintaining muscle mass and muscle strength, maintaining walking ability, improving walking function, maintaining muscle that decreases with age, and synthesizing muscle that weakens with age. The composition according to claim 4, wherein the active ingredient is contained as an extract of sugarcane tops (which may include any one selected from leaves and bark). The food composition or pharmaceutical composition according to claim 1 or 2, for any of the following purposes selected from the group consisting of age-related muscle loss (sarcopenia), maintenance or improvement of muscle mass or strength, maintenance or improvement of muscle endurance, and maintenance or improvement of walking ability. The composition according to claim 6, wherein the active ingredient is contained as an extract of sugarcane tops (which may include any one selected from leaves and bark). A method for producing a food material or pharmaceutical material for promoting expression of the PGC-1α gene in myotubes or in skeletal muscles, the food material or pharmaceutical material containing isoorientin and at least one of 3-o-caffeoylquinic acid and 5-o-caffeoylquinic acid, the method comprising the steps of:
A process for obtaining a fraction containing isoorientin and at least one of 3-o-caffeoylquinic acid and 5-o-caffeoylquinic acid from any of sugarcane tops (which may include any of leaves and bark) using an aqueous solvent.