Mitochondrial function-improving agent

Abstract

To provide a mitochondrial function-improving agent that is safe even when taken over a long period of time, can be used as a food or drink, and has excellent mitochondrial function improving effects.SOLUTION: Provided are a mitochondrial function-improving agent containing a young barley leaves-processed product as an active ingredient, and a food or drink containing the mitochondrial function-improving agent. Such a mitochondrial function-improving agent and a food or drink have a comprehensive improving effect on mitochondrial function and are therefore expected to have effects such as activating cell functions such as cell proliferation, metabolism, and repair, anti-aging, and preventing or improving (treating) diseases or conditions (symptoms) caused by decreased mitochondrial function.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for improving mitochondrial function, and more specifically, to an agent for improving mitochondrial function comprising processed young barley leaves as an active ingredient, and a food or beverage comprising the mitochondrial function improver for preventing or ameliorating (treating) a disease or condition (symptom) caused by decreased mitochondrial function. [Background technology]

[0002] Mitochondria, intracellular organelles, play an important role in ATP production and apoptosis (cell death). In addition, most of the energy required by the body when exercising is produced by aerobic energy metabolism by mitochondria. Continuous exercise (training) leads to adaptation of mitochondria in skeletal and cardiac muscles, enabling energy supply and fatigue resistance. Therefore, even at rest, improving mitochondrial function can be expected to activate cell functions such as cell proliferation, metabolism, and repair, as well as have anti-aging effects.

[0003] One of the constituent factors of mitochondria is cardiolipin (hereinafter sometimes referred to as "CL"). CL is a phospholipid that is specifically localized in the inner mitochondrial membrane and accounts for 15 to 20% of the total phospholipid amount in mitochondria (Non-Patent Document 1).

[0004] CL has four acyl chains, and there are multiple CL molecular species depending on the combination of fatty acids bound to it. Among them, for example, CL72:8 (tetralinoleoylcardiolipin) is the most abundant CL in the body, with four acyl chains of linoleic acid (18:2, a fatty acid with 18 carbon atoms and two double bonds), as shown in the chemical formula exemplified below (reprinted from Non-Patent Document 2). This CL molecular species is particularly important for mitochondrial function, and it has been reported that its depletion is the root cause of mitochondrial dysfunction associated with aging, which may ultimately lead to sarcopenia (Non-Patent Document 2).

[0005] [ka]

[0006] CL not only plays a central role in mitochondrial metabolic reactions and processes such as cellular respiration and energy production, but also plays a role in the stability of the cristae structure. CL is also involved in various functions related to mitochondrial dynamics, such as regulating the balance between mitochondrial fusion and fission, mitochondrial biogenesis, protein import into mitochondria, mitophagy, and apoptosis.

[0007] CL is highly susceptible to oxidative modification because it contains a large amount of unsaturated fatty acids and is localized near the electron transport chain of the inner mitochondrial membrane, which is the main site of reactive oxygen species (ROS) production. This modification affects mitochondrial function. Therefore, measurement of the profile of CL molecular species is positioned as a marker for evaluating mitochondrial function or quality.

[0008] On the other hand, it has been reported that young wheat leaves, particularly young barley leaves (Hordeum vulgare L.), have various physiological actions such as antiulcer action, antihypercholesterol action, anti-inflammatory action, hypoglycemic action, antimutagenic action, antithrombotic action, vascular protection action, adipocyte differentiation suppression action, melamine synthesis inhibition action, osteoporosis prevention action, and active oxygen scavenging enzyme (SOD) gene expression promotion action (see, for example, Non-Patent Document 3 and Patent Documents 1 to 4). Also, various methods for producing them have been proposed (see, for example, Patent Documents 1 to 7).

[0009] The present inventors have previously demonstrated the ATP production promoting effect and Keap1-Nrf2 system activating effect of young barley leaves. That is, they demonstrated that the ethyl acetate fraction of young barley leaves contains components that promote ATP production in rat L6 myoblast cells and activate the Keap1-Nrf2 system in cultured human liver cells to produce antioxidant enzymes, and proposed these inventions (Patent Documents 8 and 9).

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0012] If the mitochondrial function in cells can be improved comprehensively, the effects of activating cellular functions and anti-aging can be expected. Therefore, the present invention aims to provide a mitochondrial function improving agent that is highly safe, has a superior mitochondrial function improving effect, and can be taken safely for a long period of time, and a food or drink containing the mitochondrial function improving agent for preventing or improving (treating) a disease or condition (symptom) caused by a decrease in mitochondrial function. [Means for solving the problem]

[0013] In order to achieve the above object, the present inventors investigated the ability of young barley leaves to activate mitochondrial function using cultured human liver cells, and found that processed young barley leaves, which are safe even when taken over a long period of time, have a comprehensive improving effect on mitochondrial function, thereby completing the present invention.

[0014] (1) An agent for improving mitochondrial function, comprising a processed product of young barley leaves as an active ingredient, wherein the improvement in function is one or more effects selected from the group consisting of the following (a) to (c): (a) increasing the expression level of at least one gene associated with mitochondrial respiratory chain complex II, IV or V (b) Improving the profile of cardiolipin molecular species (c) Increasing expression of the cardiolipin synthase 1 gene (2) A food or drink for preventing or improving (treating) a disease or condition (symptom) caused by decreased mitochondrial function, comprising the mitochondrial function improver described in (1) above.

[0015] Further, other embodiments of the present invention include a method for preventing or ameliorating (treating) a disease or condition (symptom) caused by decreased mitochondrial function, comprising a step of administering a processed product of barley leaves to a subject in need of prevention or amelioration (treatment) of a disease or condition (symptom) caused by decreased mitochondrial function; a processed product of barley leaves for use as an agent for preventing or ameliorating (treating) a disease or condition (symptom) caused by decreased mitochondrial function; a processed product of barley leaves for use in the prevention or amelioration (treatment) of a disease or condition (symptom) caused by decreased mitochondrial function; and use of a processed product of barley leaves for producing an agent for preventing or ameliorating (treating) a disease or condition (symptom) caused by decreased mitochondrial function. Effect of the Invention

[0016] According to the present invention, the processed barley leaves, which are the active ingredient, have an effect of improving mitochondrial function, and therefore it is expected to have effects such as activation of cellular functions such as cell proliferation, metabolism, and repair, and anti-aging effects. Furthermore, it is expected that the prevention or improvement (treatment) of a disease or condition (symptom) caused by a decrease in mitochondrial function in a subject who needs such prevention or improvement (treatment) of such disease or condition without side effects.

[0017] The young barley leaves used in the present invention have absolutely no safety issues and can be freely prepared into the form of food, drink, or medicine, so they can be ingested over long periods of time not only by healthy people but also by the elderly, the infirm, and those recovering from illness. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the cell viability of hepatocytes to which BEAE (young barley leaf ethyl acetate extract) was added. [Diagram 2]This is a diagram showing the time-dependent change in OCR (oxygen consumption rate) in hepatocytes in the BEAE group and the control group. In FIG. 2, (A) shows the time-dependent change in OCR in hepatocytes in the BEAE group and the control group, and (B) shows the calculation method from (A) for each parameter indicating mitochondrial function. The area of ​​each item related to mitochondrial function is shown in (B). The terms in the figure have the following meanings (the same applies to the following figures). BEAE (BEAE group); Control; Basal respiration; Maximal respiration; Spare respiratory capacity; Non-mitochondrial respiration; ATP production; Proton leak. [Diagram 3] FIG. 3 shows area under the curve (AUC) calculated based on the OCR in FIG. 2 for each parameter indicating mitochondrial function in hepatocytes in the BEAE group and the control group. [Figure 4] FIG. 1 shows the expression levels of mitochondrial respiratory chain complex-related genes (NDUFS8, SDHD, UQCRC1, COX6B1, and ATP5G1). [Diagram 5] FIG. 1 shows the CL profile of BEAE-added hepatocytes. [Figure 6] FIG. 1 shows the ratios of molecular species having a total carbon number of 68 or less (62-68) and 70 or more (70-74) in four acyl chains relative to the total CL amount. [Figure 7] FIG. 1 shows the expression levels of CL biosynthesis / remodeling-related genes (TAZ, CLS, PNPLA8, and LCLAT1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following describes in detail an embodiment of the present invention, but the following description is merely an example of an embodiment of the present invention, and the present invention is not limited to the following description. Note that in this specification, when the expression "to" is used, it is used as an expression including the numerical values ​​or physical property values ​​before and after it.

[0020] The agent for improving mitochondrial function of the present invention contains processed young barley leaves as an active ingredient. As the young barley leaves, young barley leaves (stems and leaves) harvested from the start of tillering to the start of heading (when the plant height is about 20 to 40 cm) are preferably used.

[0021] Here, the barley may be any species and variety that belongs to the Hordeum vulgare species, such as two-row barley, four-row barley, six-row barley, and naked barley, but two-row barley, six-row barley, and naked barley are preferred, and six-row barley is particularly preferred.

[0022] More specifically, examples of varieties of two-rowed barley (malting barley) include Aominori, Agurimochi, Asaka Gold, Kinuyutaka, Oumiyutaka, Kirinijo, Haruna Nijo, Amagi Nijo, Fuji Nijo, Ishukushirazu, Misato Golden, Kawahonami, Kawamizuki, Kinuka Nijo, Komaki Nijo, Sakitama Nijo, Tone Nijo, Sachiho Golden, Satsukibare, Shunrei, and Mi These include Haru Gold, Sky Golden, Takachiho Golden, Tsuyushirazu, Tone Nijo, Nasu Nijo, Nishino Gold, Nishino Chikara, Nishino Hoshi, Haruka Nijo, Nirasaki Nijo, Nira Nijo, Harushizu, Satsuki Nijo, Houshun, Mikamo Golden, Miho Golden, Myogi Nijo, Yashio Golden, Yachiho Golden, Kitaiku 41, Irodori no Hoshi and Ryofu.

[0023] Examples of six-rowed barley varieties include Asamamugi, Kashimagor, Minorimugi, Katorimugi, Musashinomugi, Drillmugi, Sanadamugi, Haganemugi, Sayakaze, Suzukaze, Shunrai, Silky Snow, Shinjuboshi, Setugenmochi, Natori barley, Hamayutaka, Hayami barley, Fiber Snow, Baitori, and Miyuki barley.

[0024] Examples of naked barley varieties include Ichibanboshi, Senbonhadaka, Sanukihadaka, Kikaihadaka, Sanshu, Nanpuhadaka, Shiratamahadaka, Yunagihadaka, Daishimochi, Toyonokaze, Hayatehadaka, Akakamiriki, Ichiwase, Biwairohadaka, Benihadaka, Hashirihadaka, and Mannenboshi (formerly Mantenboshi), among others.

[0025] These varieties are cultivated by various agricultural experiment stations, agricultural research centers, private companies, etc., and their fruits and young leaves are used for food (including beverages). It is preferable to select and cultivate a variety suitable for the cultivation area from these varieties, and harvest the young leaves (stems and leaves).

[0026] As described above, the young barley leaves in the present invention may be in any form so long as they are young leaves (stem leaves) themselves that have grown to a height of about 20 to 40 cm from the time when tillers begin to appear until the time when heading begins, or processed products thereof.

[0027] Examples of processed products of young barley leaves (stems and leaves) include products that have been juiced, crushed, shredded, dried, roasted, extracted, powdered, and frozen, as well as products that have been juiced and dried into powder, dried, crushed and filtered (puree, juice), shredded and extracted with hot water, and combinations of these. Extracted products also include extracts (including dried extracts) obtained by extracting these products with an aqueous solvent or organic solvent.

[0028] Among these, the most commonly used forms are a powdered product of the juice (squeezed juice) of young barley leaves (stems and leaves) (hereinafter sometimes referred to as "squeezed powder") and a dried product of the young leaves (hereinafter sometimes referred to as "dried powder").

[0029] Here, the squeezed juice powder can be prepared by harvesting (cutting) young barley leaves, squeezing the juice out, mixing it with an appropriate excipient as necessary, and spray drying it in accordance with the methods described in Patent Documents 1 to 5, etc. This squeezed juice powder can be suitably used as the agent for improving mitochondrial function of the present invention as it is, or by mixing it with other excipients, etc.

[0030] Furthermore, the dry powder can be prepared by, for example, harvesting (cutting) young barley leaves, shredding them, and combining drying and pulverizing treatments, and, if necessary, blanching treatments, in accordance with the methods described in Patent Documents 6 and 7. This dry powder can be suitably used as the agent for improving mitochondrial function of the present invention, either as is or in combination with other excipients, etc.

[0031] It has already been confirmed that young barley leaves contain various physiologically active components (see, for example, Non-Patent Document 2). Furthermore, as shown in the Examples below, an ethyl acetate extract of barley leaf juice powder can be suitably used as an active component for improving mitochondrial function. This ethyl acetate extract contains at least the following compounds (a) to (d) (Patent Documents 8 and 9). (a) Composition formula: C 33 H 40 O 19 , Molecular weight: 740, Compound name: Robinin (b) Composition formula: C 27 H 30 O 15 , Molecular weight: 594, Compound name: Saponarin (c) Compositional formula: C 25 H 37 O5N2, molecular weight: 445 (d) Composition formula: C 21 H 20 O 10 , Molecular weight: 432, Compound name: Afzelin

[0032] However, the active ingredient of the mitochondrial function enhancer of the present invention is not limited to these four compounds, and may be any ingredient, so long as it is derived from young barley leaves.

[0033] Here, Robinin, Saponarin, and Afzelin are all compounds in which sugar is glycosidicly bonded to a flavonoid having a Kaempferol skeleton, i.e., flavonol glycosides. These are compounds collectively called polyphenols. Therefore, the mitochondrial function improving agent of the present invention may have polyphenols such as flavonol glycosides as its active ingredient.

[0034] The flavonol glycoside is not limited to the above three compounds, and may be any flavonol glycoside, as long as it has a mitochondrial function improving effect, either alone or in combination. Specifically, in addition to the above three compounds, examples include astragalin, azalein, hyperoside, isoquercitrin, kaempferitrin, quercitrin, rutin, amurensin, and icariin. These specific components derived from young barley leaves are also included in the processed barley leaves of the present invention.

[0035] In the present invention, specific examples of the effect of improving mitochondrial function include (a) the effect of increasing the expression level of at least one associated gene (SDHD, COX6B1, or ATP5G1) of mitochondrial respiratory chain complex II, IV, or V (Complex II, IV, or V), which contributes to ATP production in the electron transport system, and / or the effect of increasing the expression level of at least one of mitochondrial respiratory chain complexes II, IV, or V (Example 4, Figure 4).

[0036] In the present invention, specific examples of the effect of improving mitochondrial function include (b) the effect of improving the profile of cardiolipin molecular species. That is, the effect of improving the profile (composition ratio) of CL molecular species is shown, in which the total amount of CL, which is a mitochondrial constituent factor, is not reduced, CL molecular species with a small carbon number is reduced, and CL molecular species with a large carbon number is increased (Example 4, FIG. 6). Here, examples of CL molecular species with a small carbon number include CL molecular species whose total carbon number of four acyl chains is less than 70, 68 or less, or 62 to 68, and examples of CL molecular species with a large carbon number include CL molecular species whose total carbon number of four acyl chains is 70 or more, or 70 to 74.

[0037] Furthermore, in the present invention, a specific example of the effect of improving mitochondrial function is (c) the effect of increasing the expression level of the cardiolipin synthase 1 gene (Example 4, FIG. 7).

[0038] As described in detail in the Examples, it is presumed that in the present invention, mitochondrial function is improved and ATP production is promoted due to these effects of the processed barley leaves.

[0039] In the present invention, as described above, the processed barley leaves have an effect of improving mitochondrial function, and therefore, by using the mitochondrial function improving agent of the present invention, effects such as activation of cell functions such as cell proliferation, metabolism, and repair, and anti-aging can be expected. In addition, for subjects who need to prevent or improve (treat) a disease or condition (symptom) caused by a decrease in mitochondrial function, the prevention or improvement (treatment) of such a disease or condition is expected without side effects. Examples of such diseases or conditions caused by a decrease in mitochondrial function include neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, etc.), chronic kidney disease, diabetes, heart disease, and liver dysfunction.

[0040] In addition, the mitochondrial function improving agent of the present invention is expected to be used in a method for preventing or ameliorating (treating) a disease or condition (symptom) caused by decreased mitochondrial function, which comprises a step of administering a processed product of young barley leaves to a subject in need of such prevention or amelioration (treatment) of a disease or condition (symptom) caused by decreased mitochondrial function.

[0041] In this specification, the term "prevention or amelioration" of a disease or condition is used to mean regulation, delay of progression, alleviation, prevention of onset, prevention of recurrence, suppression, and the like of a disease or condition.

[0042] The processed barley leaves of the present invention have high affinity with water, can be dissolved or suspended, and have excellent miscibility with ingredients that can be normally added to foods, beverages, and medicines. Furthermore, barley leaves are an ingredient that has already been used for many years as foods and beverages, and are therefore highly safe. Therefore, the mitochondrial function improving agent containing the processed barley leaves as an active ingredient can be appropriately incorporated into daily dietary life and ingested without difficulty and with peace of mind.

[0043] The mitochondrial function improving agent of the present invention is an agent containing processed barley leaves as an active ingredient for improving mitochondrial function, with the limited use of "use for improving mitochondrial function", and can be used alone as a food or drink or a pharmaceutical product (formulation). It can also be contained as an active ingredient for improving mitochondrial function in various compositions such as food or drink or pharmaceutical products. This makes it possible to obtain a food or drink composition or pharmaceutical product composition for improving mitochondrial function. The obtained food or drink composition or pharmaceutical product composition for improving mitochondrial function can be effectively used for the diseases or conditions (symptoms) caused by the above-mentioned decrease in mitochondrial function.

[0044] The mitochondrial function improving agent of the present invention is not particularly limited as long as it is in a form that can be orally ingested, such as a solution, suspension, emulsion, powder, solid molding, etc. Specific examples include powders, capsules, tablets, granules, powders, as well as forms used in ordinary foods such as beverages and foods and pharmaceuticals. In addition, suitable examples of the formulation form include powders, capsules, tablets, granules, drinks, etc., which are easy to adjust the intake amount.

[0045] In the present invention, the processed barley leaves may be used as they are, or may be used after preparing a desired preparation by a conventional method. For example, it may be mixed with various additives permitted for preparation and molded into a composition. The additives may be any additives that are added within a range that does not impair the effects of the present invention, and examples of such additives include herbal medicines, vitamins, minerals, excipients, surfactants, coating agents, oils and fats, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, pH adjusters, acidulants such as citric acid, malic acid, and gluconic acid, etc.

[0046] Examples of the excipients include lactose, starch, cellulose, maltitol, dextrin, etc. Examples of the surfactants include glycerin fatty acid esters, sucrose fatty acid esters, etc. Examples of the coating agents include gelatin, pullulan, shellac, zein, etc. Examples of the oils and fats include wheat germ oil, rice germ oil, safflower oil, etc. Examples of the waxes include beeswax, rice bran wax, carnauba wax, etc. Examples of the sweeteners include sucrose, glucose, fructose, stevia, saccharin, sucralose, etc.

[0047] Examples of the herbal medicines include Korean ginseng, American ginseng, Panax notoginseng, Ganoderma lucidum, propolis, agaricus, blueberries, ginkgo leaves and extracts thereof, etc. Examples of the vitamins include oil-soluble vitamins such as vitamin D and K, water-soluble vitamins such as vitamins B1, B2, B6, B12, C, niacin, pantothenic acid, folic acid, biotin, etc.

[0048] The present invention also provides a food, drink, or pharmaceutical product comprising an agent for improving mitochondrial function, the agent having processed young barley leaves as an active ingredient. Here, "comprising" may mean that a physiologically acceptable carrier is included depending on the desired product form, and also means that other auxiliary ingredients that can be used in combination are included.

[0049] In the present invention, the term "food and drink" refers to any food and drink other than pharmaceuticals, and is not particularly limited as long as it is in a form that can be orally ingested as described above. Specific examples of such food and drink include instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups, miso soups, freeze-dried foods, and other instant foods; beverages such as soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, and alcoholic drinks; flour products such as bread, pasta, noodles, cake mixes, fried chicken powder, and bread crumbs; sweets such as candy, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, and dessert sweets; and soybeans. These include condiments such as sauces, tomato processed seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew bases; processed oils and fats, butter, margarine, mayonnaise, etc.; dairy products such as milk drinks, yogurt, lactic acid bacteria drinks, ice cream, cream, etc.; processed eggs, fish ham and sausages, fish paste products, etc.; processed livestock products such as meat ham and sausages; processed agricultural products such as canned agricultural goods, jams, marmalades, pickles, boiled beans, cereals, etc.; frozen foods, etc.

[0050] Food and beverages also include categories such as health foods (e.g., functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally regulated foods, supplements, etc.), health functional foods (e.g., foods for specified health uses, nutritionally functional foods, foods with functional claims, etc.), and special purpose foods (e.g., foods for the sick, infant formula, milk powder for pregnant or breastfeeding women, etc.), as well as foods and beverages labeled for risk reduction, prevention, or improvement of diseases or conditions (symptoms) caused by increased lipid absorption or decreased mitochondrial function.

[0051] According to another aspect of the present invention, a food or beverage can be provided that contains an agent for improving mitochondrial function and is labeled with the function of preventing or improving a disease or condition that can be prevented or improved by improving mitochondrial function.

[0052] In the present invention, a pharmaceutical product is prepared as an oral or parenteral formulation in accordance with a conventional method by combining additives acceptable for formulation. In the case of an oral formulation, as described above, there is no particular limitation as long as it is in a form that can be taken orally. In the case of a parenteral formulation, it can be in the form of an injection or suppository. From the viewpoint of simplicity, an oral formulation is preferable. Examples of additives acceptable for formulation include those similar to those described above.

[0053] In the present invention, the content of the active ingredient in the mitochondrial function improving agent is difficult to uniformly define due to the type and form of the preparation, the purpose of prevention or improvement, etc., but for example, the content is usually about 15 mg to 15 g, preferably about 50 mg to 9 g, per day for an adult (body weight 60 kg) as a squeezed powder of young barley leaves, and usually about 50 mg to 15 g, preferably about 100 mg to 9 g, per day as a dried powder of young barley leaves. Furthermore, the content in the preparation may be appropriately set in consideration of the daily intake so that the required daily intake of the active ingredient can be ingested (taken). Furthermore, the content of the specific ingredient may be about the amount contained in the squeezed powder or dried powder of the young barley leaves. EXAMPLES

[0054] The present invention will be described in more detail below with reference to examples (experimental examples), but the technical scope of the present invention is not limited to these examples.

[0055] [Example 1] Production of barley leaf juice powder According to the method described in JP 2009-148213 A (Patent Document 5), 10 kg of young leaves (stems and leaves) of barley grown in Hokkaido were squeezed to obtain 8 kg of juice. The obtained juice was spray-dried to obtain a juice powder. Hereinafter, this may be referred to as "100% Hokkaido young barley leaf extract powder."

[0056] [Example 2] Preparation of ethyl acetate extract from young barley leaves 10.05 g of the above "Hokkaido Young Barley Leaf Extract Powder 100%" was dissolved in 90 mL of deionized water and left to stand at room temperature for 1 hour to extract the water-soluble components. During the extraction, the solution was mixed every 15 minutes.

[0057] The extracted solution was centrifuged at 8,000 rpm and 20°C for 15 minutes, and the supernatant was suction filtered through filter paper (No. 2, ADVANTEC). The residue after filtration was redissolved in 90 mL of deionized water, and the same procedure was repeated with an extraction time of 30 minutes to obtain a water extract of "Hokkaido Young Barley Leaf Extract Powder 100%" again.

[0058] The barley leaf water extract obtained from the second extraction was added to a separatory funnel and thoroughly mixed with an equal amount of ethyl acetate. The mixture was left to stand for 1 hour for liquid-liquid extraction, and the ethyl acetate fraction was collected.

[0059] This procedure was repeated three times, and the collected ethyl acetate fraction was dried to obtain the barley green young leaves ethyl acetate extract (hereinafter sometimes referred to as "BEAE"). The amount of BEAE obtained from the barley green young leaves extract powder (10.05 g) was 0.054 g, and the recovery rate was 0.54%.

[0060] The collected BEAE was dissolved in 50% ethanol to a concentration 50 times the target concentration for each experiment, and the BEAE solution dissolved in ethanol was stored at -80°C in the dark until use for functional evaluation. When BEAE was added to the cells, the final concentration of ethanol in the culture medium was controlled to be 1%.

[0061] [Example 3] Measurement of total polyphenol content in ethyl acetate extract from young barley leaves The total polyphenol content of the obtained BEAE was measured using the Folin-Ciocalteu method (Reference 1). The collected BEAE was dissolved in 50% ethanol to a concentration of 5,000 μg / mL, stirred with an ultrasonic device for 1 minute, and then allowed to stand for 90 minutes to prepare a sample for measuring the total polyphenol content.

[0062] A 50% ethanol solution of catechin hydrate (Cayman Chemical Company) was used as the standard solution. Five concentrations of standard solutions were prepared: 62.5, 125, 250, 500, and 1,000 μg / mL. 4 mL of deionized water was placed in a 15 mL centrifuge tube, and 50 μL of the sample or standard solution, 1 mL of 5-fold diluted Folin & Ciocalteu's phenol reagent (Sigma-Aldrich Japan LLC), and 1 mL of 10% (w / v) aqueous sodium carbonate solution (Kanto Chemical Co., Ltd.) were added in that order and stirred. Three replicates were prepared for each sample and standard solution.

[0063] In addition, a blank was prepared by adding 50% ethanol instead of the sample or standard solution in the above procedure. All reaction solutions were left to stand at room temperature in the dark for 1 hour, and then the absorbance was measured (dominant wavelength: 760 nm). The total amount of polyphenols contained in the sample was expressed in μg catechin equivalents.

[0064] The total polyphenol content of BEAE (0.054 g) was 4.43 mg, and the total polyphenol concentration (catechin equivalent) was 8.24% (w / w).

[0065] [Example 4] Functional evaluation of ethyl acetate extract from young barley leaves 1. Method 1.1. Cell culture Human liver-derived cell line C3A was purchased from the American Type Culture Collection. Fetal bovine serum (FBS), Penicillin-Streptomycin-Neomycin, and GlutaMAX were used. TM C3A was subcultured in Minimum Essential Medium (MEM, glutamine-free, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with supplements at final concentrations of 10% (v / v), 1% (v / v), and 1 mM, respectively. All cell cultures were performed at 37°C in 5% CO2.

[0066] 1.2. Cell viability test To investigate the cell viability 24 hours after adding BEAE to C3A, a cell viability test was performed using the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) method. C3A was added to a 96-well plate at 1.0 × 10 5 BEAE was dissolved in FBS-free MEM to prepare culture media with final concentrations of 0, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL, and these were replaced with the culture media during the culture (BEAE group except for 0 μg / mL BEAE).

[0067] After 22 hours, 10 μL of WST-1 reagent (a mixture of 6.3 mg / mL WST-1 (Dojindo Laboratories) and 0.7 mg / mL 1-Methory PMS (Dojindo Laboratories) in a volume ratio of 9:1) was added, and the cells were incubated for another 2 hours. TMThe absorbance at 450 nm was measured using a microplate reader. Measurements were performed in n = 6 for each group, and cell viability was calculated as a relative value, with the average absorbance of the control group (0 μg / mL BEAE) set to 1. The figures are shown as mean ± standard deviation (SD).

[0068] 1.3. Analysis of mitochondrial function using the extracellular flux analyzer XFp The mitochondrial function of hepatocytes treated with BEAE was investigated using an extracellular flux analyzer. C3A was added to an XFp Cell Culture Miniplate (Agilent Technologies, Inc.) at 1.25 × 10 5 80 μL / well at a concentration of 1.0 × 10 cells / mL 4 After culturing for 2 hours, 120 μL of MEM was added to bring the total volume to 200 μL. The cells were further cultured for 22 hours. After that, BEAE was dissolved in FBS-free MEM to a final concentration of 12.5 μg / mL, and the culture medium was replaced with this medium (BEAE group).

[0069] Sterile water was used instead of BEAE for the control group. After 24 hours of stimulation, the supernatant in the wells of the XFp Cell Culture Miniplate was replaced with assay medium {Seahorse XF DMEM Medium, pH 7.4 (Agilent Technologies, Inc.), 9.8 mL; 100x GlutaMAX (gibco), 100 μL; 100 mM Sodium Pryuvate (gibco), 100 μL; Glucose (Nacalai Tesques, Inc.), 0.01 g} and left to stand in an incubator (37°C, no CO2 control) for 1 hour. During this time, the XFp sensor cartridge, which had been hydrated for 48 hours, was calibrated using the extracellular flux analyzer XFp (Agilent Technologies). After the incubation of the XFp Cell Culture Miniplate was completed, the oxygen consumption rate (OCR) was measured using the extracellular flux analyzer XFp.

[0070] The final concentrations of the reagents used in the measurement were as follows: Oligomycin (final concentration: 4 μM), FCCP (final concentration: 1 μM), and Rotenone / Antimycin A (final concentration: 0.5 μM). After the reagents were added, the mix-wait-measurement (2-0-3 min) process was repeated four times before the next reagent was added.

[0071] The data was analyzed using the accompanying software, Wave ver 2.3.0.19 (Seahorse Bioscience). The area under the curve (AUC) was calculated based on a graph showing the time-dependent change in OCR from the measurement results, and parameters indicating mitochondrial function, such as basal mitochondrial respiration, maximum respiration, spare respiratory capacity, non-mitochondrial respiration, ATP production, and proton leak, were calculated. Each item was expressed as a relative value with the value of the control group set at 1, and the figures were expressed as mean ± SD (n = 6 per group).

[0072] 1.4. Analysis of gene expression levels To investigate the gene expression level in hepatocytes treated with BEAE, RNA was extracted from the cells. 5 The cells were seeded at a concentration of 1000 cells / mL at 1 mL / well and precultured for 24 hours. The cells were stimulated with FBS-free MEM containing BEAE at a final concentration of 25 μg / mL (BEAE group) or sterile water (control group).

[0073] After 24 hours of culture, the cells were washed once with 300 μL / well of PBS. RNA was extracted from the cells using an RNA extraction kit (PureLink TM The procedure was carried out according to the instructions attached to the RNA Mini Kit (Ambion, Life Technologies). The RNA concentration was determined using a NanoDrop One (Thermo Fisher Scientific Inc.). TM The RNA (500 ng) was synthesized into cDNA using Ace qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd.) according to the manufacturer's instructions. The resulting cDNA was stored at -80°C until analysis.

[0074] The expression level of the target gene was measured using the cDNA. TM Next SYBR TM qPCR Mix, 5.0 μL; 0.3 μM Forward primer, 0.5 μL; 0.3 μM Reverse primer, 0.5 μL; Nuclease-free water, 2.0 μL of the master mix was mixed with 2.0 μL of cDNA, and the mixture was analyzed using a real-time PCR analysis system (CFX Connect TM The analysis was performed using a real-time system.

[0075] The PCR reaction was performed at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 60°C for 5 seconds, and 72°C for 10 seconds. The expression level of each gene was calculated by the ΔΔCq method.

[0076] The target genes were as follows: NADH: Ubiquinone Oxidoreductase Core Subunit S8 (NDUFS8), SuccinateDehydrogenase Complex Subunit D (SDHD), Ubiquinol-Cytochrome C Reductase Core Protein 1 (UQCRC1), Cytochrome C Oxidase Subunit 6B1 (COX6B1), ATP Synthase, H+ Transporting, Mitochondrial F0 Complex, Subunit C1 (Subunit 9) (ATP5G1), and Tafazzin, Phospholipid-Lysophospholipid Transacylase (TAZ), Cardiolipin Synthase 1 (CLS), Patatin LikePhospholipase Domain Containing 8 (PNPLA8), and Lysocardiolipin Acyltransferase 1 (LCLAT1), which are genes related to CL biosynthesis and remodeling.

[0077] The expression level of each target gene was corrected by the expression level of the housekeeping gene β-actin (ACTB). The primers used are shown in Table 1. Measurements were performed in n = 8 for each group, and gene expression levels were calculated as relative values ​​with the control group set as 1, and are expressed as mean ± SD.

[0078] [Table 1]

[0079] 1.5. Cellular lipid extraction To investigate the lipid composition of hepatocytes to which BEAE had been added, cells stimulated under the same conditions as those used for RNA extraction were harvested with 500 μL of PBS. n = 1 for 3 wells. To measure the protein concentration of the cell suspension, 50 μL of the harvested cell suspension (500 μL) was transferred to another tube and stored at -80°C. The remaining cell suspension (450 μL) was centrifuged at 10,000 rpm for 1 minute, the supernatant was discarded, and the cells were resuspended in 1 mL / tube of PBS to wash the cells. The cells were centrifuged again at 10,000 rpm for 1 minute, the supernatant was discarded, and the cell mass was collected.

[0080] Lipid extraction was performed using the following procedure, which is a modification of the Folch method. An internal standard (IS, SPLASH) dissolved in methanol was added to the tube containing the cell mass. TM LIPID MIX TM 200 μL of Quantitative Mass Spec Internal Standard (Avanti Polar Lipids, Inc.) and 400 μL of chloroform were added, and the mixture was vortexed at 3,500 rpm for 10 minutes. The mixture was then centrifuged at 15,000 rpm at 4°C for 10 minutes, and the organic solvent layer was collected. The residue was re-extracted with 400 μL of chloroform.

[0081] The extract was dried and redissolved in 100 μL of methanol, vortexed at 3,500 rpm for 3 min, and then centrifuged at 15,000 rpm at 4°C for 10 min. The supernatant was transferred to a measurement vial and used as a sample for liquid chromatography / mass spectrometry (LC / MS) (n = 6 per group).

[0082] The composition of CL in the lipid-extracted samples was measured by LC / MS according to a previous report (Reference 2). Peak areas were calculated using Xcalibur 2.2 (Thermo Fisher Scientific Inc.), and the calculated peak areas were corrected for the protein amount (μg protein) of each sample. The area / μg protein of each detected molecular species was expressed as the mean ± SD. The total amount of all detected molecular species was also compared.

[0083] The protein concentration in the harvested cells was measured using Pierce TM Measurements were performed using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc.) according to the instructions.

[0084] 1.6. Statistical analysis All data were statistically analyzed using GraphPad Prism V7.0 software (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA was used for statistical analysis, and multiple group comparison tests were performed using Tukey's multiple comparison test. The significance level was set at 5%.

[0085] 2. Results 2.1. Cell viability In C3A, BEAE increased cell viability at concentrations of 25 μg / mL or less, whereas it decreased cell viability at concentrations of 50 μg / mL or more (Figure 1).

[0086] In Fig. 1, n = 6, One-way ANOVA with Dunnett's multiple comparisons test (vs BEAE 0 μg / mL), * P < 0.05, *** P < 0.001, **** P < 0.0001.

[0087] Mitochondrial function The time course of the oxygen consumption rate (OCR) in human hepatocytes in the BEAE and control groups is shown in Fig. 2. In Fig. 2, (A) shows the time course of the OCR in hepatocytes in the BEAE and control groups, and (B) shows the method for calculating the parameters of mitochondrial function from (A). In Fig. 3B, the areas related to mitochondrial function are shown (Basal respiration; Maximal respiration; Spare respiratory capacity; Non-mitochondrial respiration; ATP production; Proton leak).

[0088] The above six items related to mitochondrial function were calculated from the time-dependent changes in OCR. The five parameters of mitochondrial basal respiration, maximum respiration, spare respiratory capacity, ATP production, and proton leak were significantly increased in the BEAE group compared to the control group (Figure 3). No significant difference was observed in non-mitochondrial respiration.

[0089] In Figure 3, n = 6, Student's t-test, * P < 0.05, ** P < 0.01, ns: not significant.

[0090] 2.3. Expression levels of mitochondrial respiratory chain complex-related genes BEAE increased the expression levels of SDHD, COX6B1, and ATP5G1, which are complex II-, complex IV-, and complex V-associated genes, respectively, in hepatocytes (Fig. 4), whereas BEAE decreased the expression levels of NDUFS8 and UQCRC1, which are complex I-, and complex III-associated genes, respectively.

[0091] In Figure 4, n = 8, Student's t-test, ** P < 0.01, *** P < 0.001, ns: not significant.

[0092] 2.4. CL Profile Twenty-nine molecular species of CL were detected in BEAE-treated hepatocytes (Figure 5). Twelve molecular species of CL, including CL72:8 (CL62:2, CL64:2, CL64:5, CL66:2, CL66:3, CL66:6, CL68:2, CL68:3, CL70:4, CL70:7, CL72:7, and CL72:8), were significantly increased in the BEAE group compared to the control group. In addition, CL64:4 and CL66:5 were significantly decreased in the BEAE group. On the other hand, there was no significant difference between the control group and the BEAE group in the total amount of CL, which is the sum of all 29 molecular species.

[0093] In addition, the 29 detected molecular species were divided into those with a total carbon number of 68 or less (62-68) in the four acyl chains and those with a total carbon number of 70 or more (70-74), and the ratio was calculated by dividing the total amount of each by the total amount of CL, expressed as a percentage. This ratio significantly decreased in the BEAE group for those with a total carbon number of 68 or less in the four acyl chains, and significantly increased in those with a total carbon number of 70 or more in the four acyl chains (Figure 6).

[0094] In Figures 5 and 6, n = 6, Student's t-test, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant.

[0095] 2.5. Expression of genes related to CL biosynthesis and remodeling BEAE increased the expression level of the CLS gene in hepatocytes (Fig. 7), whereas no significant difference was observed in the expression levels of the three genes TAZ, PNPLA8, and LCLAT1.

[0096] In Figure 7, n = 8, Student's t-test, *P < 0.05, ns: not significant.

[0097] 3. Discussion In this experiment, the viability of the human liver-derived cell line C3A was not decreased by the addition of BEAE at a concentration of 25 μg / mL or less (Figure 1). 5 It was shown that the maximum concentration that could be added was 25 μg / mL per cells / mL, and this condition was adopted in subsequent experiments.

[0098] To explore the mitochondrial activation function of young barley leaves, ATP production and mitochondrial respiration were evaluated using an extracellular flux analyzer (Figure 2). In a previous study by the present inventors, the total amount of ATP production was reported to be increased by the addition of young barley leaf extract, although the experiment was conducted using rat L6 myoblast cells and an extract made by extracting barley leaves with ethyl acetate without powdering them (Patent Document 8).

[0099] The results of the ATP production amount measured by the extracellular flux analyzer used in this experiment showed that the amount of ATP production derived from glycolysis was negligible and showed the amount of ATP production derived from mitochondria (Reference 3).

[0100] Therefore, our results demonstrated that BEAE increases mitochondrial ATP production in hepatocytes.

[0101] Basal respiration is the net sum of all processes that can consume oxygen in the cell. Basal respiration increases due to increased ATP production, increased proton leak, or increased respiration not via mitochondria (Reference 4).

[0102] In this experiment, it is possible that both increased ATP production and proton leak contributed to the increase in basal respiration. Maximal respiration indicates the maximum amount of oxygen consumption that the electron transport chain can perform. In addition, spare respiratory capacity is the difference between maximum respiration and basal respiration, and it plays an important role in protecting cells against oxidative stress (Reference 5). A decrease in spare respiratory capacity is associated with various pathologies, such as heart disease, neurodegenerative diseases, and smooth muscle cell death (Reference 6).

[0103] Therefore, it was suggested that the increase in maximum and spare respiratory capacity by BEAE in this experiment contributed to the activation of mitochondrial function.

[0104] Non-mitochondrial respiration refers to oxygen consumption by pathways other than mitochondria. In this experiment, BEAE did not change this parameter, which is considered to support the effect of BEAE on mitochondrial respiratory function.

[0105] Proton leak refers to the phenomenon in which some protons flow back into the mitochondrial matrix, and is the rate at which oxygen is consumed during heat production, not during ATP synthesis. There are many factors that may increase proton leak. For example, increased uncoupling protein (UCP) activity and H2O due to damage to the mitochondrial inner membrane and / or ETC complexes. + leakage into the matrix (Reference 4).

[0106] Another reason for the increased proton leak is that oligomycin increases the mitochondrial membrane potential, which increases proton leakage through the mitochondrial membrane, and as a result, ATP production may be underestimated and proton leak may be overestimated.

[0107] In this case, an increase in ATP production was observed in hepatocytes treated with BEAE, even if it was an underestimate, providing strong evidence that BEAE promotes an increase in ATP production.

[0108] As mentioned above, the addition of BEAE to hepatocytes improved mitochondrial function, suggesting that BEAE may have affected the mitochondrial respiratory chain complexes that control respiratory function.

[0109] The addition of BEAE increased the expression of genes related to Complexes II, IV, and V (by approximately 40%-60%) (Figure 4). Complex II is an important enzyme complex in both the citric acid cycle and the electron transport chain, and animals lacking Complex II activity are associated with increased reactive oxygen species and premature aging (Reference 7).

[0110] Complex IV also regulates ATP production and is the main determinant of maximum and reserve respiratory capacity (Reference 6). Complex V is the ATP synthase itself. Therefore, these enzyme complexes, whose gene expression levels were increased by BEAE, play an important role in respiration within the electron transport chain. Although the addition of BEAE caused a decrease of about 10% in Complexes I and III, the change was small.

[0111] These findings suggest that BEAE induces the expression of some mitochondrial respiratory chain complex-related genes in hepatocytes, which may be involved in improving mitochondrial functionality.

[0112] In addition, the total amount of CL did not decrease, and the profile of CL molecular species was improved, with a decrease in CL molecular species with small carbon numbers and an increase in CL molecular species with large carbon numbers (Figures 5 and 6). CL72:8 is also called mature CL and is the CL species that contributes most to ATP production. CL72:8 also contributes to maintaining mitochondrial structure. In addition, a relationship between mitochondrial morphological abnormalities and an increase in CL species with small carbon numbers (e.g. CL66-68) has been reported in various pathological conditions (References 8-11).

[0113] These findings suggest that the improvement of mitochondrial function by BEAE may be accompanied by an improvement in the CL profile. BEAE also increased the expression of the CLS (Cardiolipin Synthase 1) gene (Figure 7). CLS is an enzyme that plays a role in synthesizing immature CL from phosphatidylglycerol and is involved in the starting point of CL remodeling. It is considered that the increase in CLS by BEAE may have been the initiation of CL remodeling, and it is also speculated that it may have supported the increase in CL72:8 and CL molecular species with similar carbon numbers observed in this experiment. Therefore, it is speculated that BEAE induces the expression of CLS, which may activate the remodeling of CL molecular species, improving the profile of CL molecular species.

[0114] 4. References References 1. Singleton VL, et al.

[14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology 299:152-178, 1999. Reference 2. Toshihiro Sakurai et al. A mouse model of short-term, diet-induced fatty liver with abnormal cardiolipin remodeling via downregulated Tafazzin gene expression. J Sci Food Agric. 2021 Sep;101(12):4995-5001 Reference 3. Tan B, et al. The profiles of mitochondrial respiration and glycolysis using extracellular flux analysis in porcine enterocyte IPEC-J2. Animal Nutrition 1:239-243, 2015. Reference 4. Hill BG, et al. Integration of cellular bioenergetics with mitochondrial quality control and autophagy. Biol. Chem 393(12):148, 2012. Reference 5. Zelickson BR, et al. Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes. Biochimica et Biophysica Acta 1807:1573-158, 2011. Reference 6. Desler C, et al. Is There a Link between Mitochondrial Reserve Respiratory Capacity and Aging? Journal of Aging Research Article ID 192503, 9 pages, 2012. Reference 7. Gimenez-Roqueplo AP, et al. The R22X Mutation of the SDHD Gene in Hereditary Paraganglioma Abolishes the Enzymatic Activity of Complex II in the Mitochondrial Respiratory Chain and Activates the Hypoxia Pathway. Am. J. Hum. Genet. 69:1186-1197, 2001. Reference 8. Wortmann SB, et al. Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness. Nat. Genet. 44:797-802, 2012. Reference 9. Angelini R, et al. Cardiolipin fingerprinting of leukocytes by MALDI-TOF / MS as a screening tool for Barth syndrome. J. Lipid Res. 56:1787-94, 2015. Reference 10. Mancuso DJ, et al. Genetic ablation of calcium-independent phospholipase A2{gamma} leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction. J. Biol. Chem., 284:35632-44, 2009. Reference 11. Sakurai T, et al. A mouse model of short‐term, diet‐induced fatty liver with abnormal cardiolipin remodeling via downregulated Tafazzin gene expression. J. Sci. Food Res. 101:4995-5001, 2021.

Claims

1. A mitochondrial function improving agent comprising a processed product of young barley leaves as an active ingredient, wherein the improvement in function is one or more effects selected from the group consisting of the following (a) to (c): (a) increasing the expression level of at least one gene associated with mitochondrial respiratory chain complex II, IV, or V (b) Improving the profile of cardiolipin molecular species (c) Increasing the expression level of the cardiolipin synthase 1 gene

2. A food or drink for preventing or improving (treating) a disease or condition (symptom) caused by decreased mitochondrial function, comprising the mitochondrial function improver according to claim 1.

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