JP2015096494A - In vivo redox status-improving agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an in vivo redox status-improving agent (redox modulator) under proteasomal activity-inhibiting conditions.SOLUTION: The invention provides an in vivo redox status-improving agent and the improvement method under proteasomal activity-reducing conditions by containing at least one kind of sesame lignan selected from sesamin, episesamin, sesaminol, and sesaminol glycoside. An improvement of the in vivo redox status is to decrease mitochondrial internal reactive oxygen species, or to maintain mitochondrial activity.

Description

  The present invention relates to an in vivo redox state improving agent (redox modulator). More specifically, it relates to the improvement of the redox state in vivo by sesamin under conditions where proteasome activity is reduced. By providing mitochondrial activity maintenance function when proteasome activity is reduced, aging and Alzheimer's are provided. In vivo redox state improvement that is useful for inhibiting the progression of Parkinson's disease, Lewy body disease, triplet repeat disease, amyotrophic lateral sclerosis, cataract, arteriosclerosis, diabetic nephropathy, photoaging of the skin, etc. The present invention relates to an agent (redox modulator).

  Recently, various injuries observed in living cells and tissues due to active oxygen have attracted attention. Reactive oxygen is very reactive and is involved in various diseases such as stroke, myocardial infarction, cataracts, rheumatism, cancer, stomach ulcers, skin wrinkles and blemishes by destroying various components and tissues of the body. It is becoming clear. Factors that increase active oxygen include aging, UV exposure, and mental stress. When active oxygen increases, oxidized proteins, so-called abnormal proteins, accumulate in the body and cause various diseases. Has been. In the past, in order to prevent oxidative damage due to active oxygen, attempts have been made to suppress oxidation of proteins by reducing the active oxygen in the living body by ingesting an antioxidant. As typical antioxidants, tocopherols, carotenoids, flavonoids, sesame lignans and the like are known, and some of these are used in foods and cosmetics.

  For example, sesame seeds are used as food materials, cosmetics, and medical materials that are effective in improving skin roughness and preventing skin aging by using natural ingredients as active ingredients. Sesame extract containing sesame lignans, which contain the antioxidants sesamin, sesamorin, episesamin, sesamol, sesaminol, sesamorinol, and the like, and active ingredients of saturated fatty acids, unsaturated fatty acids, and enzymes have been proposed (patents) Reference 1). Moreover, the food composition (Patent Document 2) having an enhanced physiological activity of sesamin comprising a composition containing sesamin and gamma oryzanol, which have known physiological activities such as an antioxidant action, an antihypertensive action, an anti-inflammatory action, etc. In addition, a complex of sesamin has been proposed in which various physiological activities of sesamin are dramatically increased by inclusion of sesamin with an inclusion compound or inclusion in a liposome (Patent Document 3).

  In addition, accumulation of abnormal proteins in vivo increases with the oxidizing action of active oxygen and age, and it has become clear that they are involved in the pathogenesis common to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Prevention and improvement of diseases caused by abnormal protein accumulation in the body has become a major issue, but with regard to the protection of abnormal protein accumulation, active oxygen generated in the body due to oxidative stress is eliminated by ingesting antioxidant substances. Attempts have been made to suppress protein oxidation.

  Proteasome is known as an enzyme that removes abnormal proteins in a living body. The proteasome consists of a large multi-component complex with a complex molecular structure that removes abnormal proteins, plays a role in protein quality control, and removes proteins that have been mutated or damaged by ultraviolet rays or oxidation. Is closely related to. For example, a composition has been proposed that promotes the activity of proteasome abnormalities in the living body and prevents and ameliorates various diseases. For example, a proteasome activity promoter containing an extract of Mannentake (Patent Document 4), an abnormal protein removing agent containing soybean saponin having a proteasome activity promoting action (Patent Document 5), a kale having a proteasome activity promoting action and / or its An abnormal protein removing agent containing an extract (Patent Document 6) has been proposed.

  Conventionally, as a method for separating sesamins from sesame oil, sesame seeds and sesame seeds, for example, various organic solvents (acetone) which are substantially immiscible with sesame oil and which can extract and dissolve sesamins. , Methyl ethyl ketone, diethyl ketone, methanol, ethanol, etc.) are extracted and concentrated, and a method for obtaining sesamin by evaporating and removing the solvent from the solvent fraction (extraction separation method) is known (Patent Document 7, 8). There are also various methods for separating sesamin and the like from a distillate containing sesamin obtained by steam distillation of sesame oil under reduced pressure, such as deodorized scum, which is a deodorized distillate produced in the purification (deodorization) step of sesame oil. Are known. For example, in Patent Document 9, in the method for separating and producing sesamin, the deodorizing scum generated in the deodorizing step of sesame oil production is allowed to stand, and the solid part obtained by removing the liquid part of the upper layer is used as a raw material. Separating and producing sesamin by performing a cleaning / purification process consisting of only two or more treatments at different temperatures with a solvent of the first, a first treatment in which at least two treatments at different temperatures are performed at room temperature, and 50 It is disclosed that the method comprises a second treatment performed at a temperature of not lower than ° C., and the solvent is ethanol containing 90% by volume or more of ethanol.

  For the production of sesaminol glycoside, sesame-derived raw material containing sesaminol glycoside and water are mixed, and this is put into a pressure vessel and held at a temperature range of 100 to 155 ° C. for 5 to 60 minutes. A method for producing a hydrothermally-treated extract containing a sesaminol glycoside characterized by this fact has been proposed, and the sesaminol glycoside can be efficiently and economically extracted (Patent Document 10). Also, a novel food material derived from sesame seeds that contains sesaminol trisaccharide at a high concentration and contains an oil component in an appropriate amount, and has excellent color, flavor, and texture as food. Has also been proposed (Patent Document 11).

JP 2010-1267 A JP 2010-177873 A JP 2010-24240 A JP 2002-29996 A Japanese Patent Application No. 2002-179592 JP 2004-91398 A Japanese Patent Publication No. 7-25564 JP-A-3-27319 Japanese Patent No. 4863532 JP 2014-97932 A Japanese Patent Laid-Open No. 10-234342

Nakai et al., J. Agric. Food Chem. (2003) 51, pp. 1666-1670. Ide et al., Biochmica Acta (2001) 1534, pp. 1-13 Hirose et al., J. of lipid Research (1991) 32, pp. 629-638. Chondrogannini and Gonos IUBMB Life (2008) 60.pp.651-655 Mitsuo Namiki ・ Sadasaku Kobayashi, "Science of Sesame", Asakura Shoten, October 10, 1989, 62, 160 Namiki, Mitsuo, “Sesame-Its Science and Functionality” Maruzen Co., Ltd., Maruzen Publishing Co., Ltd., issued November 1, 1998, page 26

It has been reported as a phenomenological theory that changes in intracellular redox state due to feeding have various effects on the living body. The health promotion effect of sesame has been widely known since ancient times, and high physiological activity of sesame lignan has been reported from recent studies. The main ones are discovery of radical scavenging activity by biochemical assays (Non-patent Document 1), regulation of lipid metabolism including cholesterol found at the individual level (Non-patent Documents 2 and 3), etc. Is given.
The present inventors pay attention to a decrease in proteasome activity as a change at the cellular level accompanying aging (Non-patent Document 4). The proteasome is a proteolytic enzyme complex that degrades denatured proteins in cells and is one of the important mechanisms for suppressing abnormal protein accumulation. So far, the present inventors have detected that addition of a proteasome inhibitor, that is, a decrease in the ability of the proteasome causes intracellular oxidation in experiments targeting cultured cell lines using the fluorescent redox sensor protein redoxflor. Assuming that this result mimics cellular aging, etc., we believe that by searching for compounds that have the effect of suppressing intracellular oxidation at that time, we will contribute to the discovery of new components with anti-aging effects, We decided to start research and development with the goal of finding compounds that exert antioxidative effects in cells with reduced proteasome ability.
That is, an object of the present invention is to provide an in vivo redox state improving agent (redox modulator). More specifically, it aims to improve the redox state in vivo under conditions that reduce proteasome activity, and aims to provide a substance that can impart survival activity to cells by maintaining mitochondrial activity under reduced proteasome activity. . Diseases that develop due to inhibition of the proteasome include aging, Alzheimer's disease, Parkinson's disease, Lewy body disease, triplet repeat disease, amyotrophic lateral sclerosis, cataract, arteriosclerosis, diabetic nephropathy, skin photoaging And the present invention intends to provide a substance for alleviating symptoms caused by these diseases.

The present invention provides 1) the physiological effects of sesamin, including the effects on the mitochondrial state known as the source of reactive oxygen species, by finding the antioxidant action of sesame lignans, especially sesamin, under proteasome inhibition conditions Based on the result of testing. 2) Of the hot water extraction samples from products and by-products at each stage of the sesame oil production process, especially the extraction sample from the primary sesame seeds also has an oxidation-inhibiting effect and physiological activity during proteasome inhibition. I found it to have.
Abnormal proteins accumulate in the living body where the proteasome is inhibited by the degradation of abnormal proteins being stopped. Abnormal protein accumulation is considered to be a common pathogenesis mechanism of neurodegenerative diseases such as aging, Parkinson's disease, and Alzheimer's disease. Under the inhibition of the proteasome, intracellular oxidation is further triggered. The inventors first made an effort to find a substance capable of preventing intracellular oxidation under proteasome inhibition, and first, under proteasome inhibition by sesame lignan consisting of sesamin, episesamin and sesaminol. Antioxidant action was found.
Sesame lignans such as sesamin, episesamin, sesaminol, sesamol, sesamol, sesaminol glycosides are known as antioxidants, but among sesame lignans known as antioxidants, The inventors have found that sesamin, episesamin, sesaminol and sesaminol glycosides inhibit intracellular oxidation under proteasome inhibition, and have reached the present invention.

The gist of the present invention is the in vivo redox state improving agent under the following proteasome inhibition conditions (1) to (4).
(1) An in-vivo redox state ameliorating agent under reduced proteasome activity characterized by containing at least one sesame lignan selected from sesamin, episesamin, sesaminol and sesaminol glycoside.
(2) The in-vivo redox state ameliorating agent according to (1) above, wherein the improvement of the in-vivo redox state is to reduce the mitochondrial reactive oxygen species.
(3) The in-vivo redox state improving agent according to (1) above, wherein the improvement of the in-vivo redox state is to maintain mitochondrial activity.
(4) The in-vivo redox state improving agent according to (1) above, wherein the improvement of the redox state in the living body is to increase cell survival activity.

In addition, the gist of the present invention is a pharmaceutical composition for improving the in vivo redox state under the following proteasome activity lowering condition (5).
(5) Conditions for reducing proteasome activity, comprising the in vivo redox state improving agent or pharmaceutically acceptable salt according to any one of (1) to (4) above and a pharmaceutically acceptable excipient A pharmaceutical composition for improving the underlying in vivo redox state.

  According to the present invention, it is possible to provide an in vivo redox state ameliorating agent under proteasome inhibition conditions (under reduced proteasome activity conditions). Symptoms caused by accumulation of abnormal proteins due to oxidative action in the living body with reduced proteasome activity due to the redox state improving agent in the living body under proteasome inhibition conditions (under reduced proteasome activity conditions) of the present invention Can be suppressed. For example, prevention or prevention of diseases caused by abnormal protein degradation such as aging, Alzheimer's disease, Parkinson's disease, Lewy body disease, triplet repeat disease, amyotrophic lateral sclerosis, cataract, arteriosclerosis, diabetic nephropathy, skin photoaging Effective for treatment. In addition to the in vivo antioxidant action with reduced proteasome activity, it is possible to exert the effect of reducing the reactive oxygen species in the mitochondria and the effect of maintaining the mitochondrial membrane potential. By these, effects such as an increase in survival activity can be imparted to the cells. That is, according to the present invention, it is possible to provide a pharmaceutical composition for improving the in vivo redox state under the condition of reducing proteasome activity.

2 shows the effect of suppressing intracellular oxidation of sesamin and its analogs during proteasome inhibition. It shows the effect of reducing mitochondrial reactive oxygen species caused by sesamin under proteasome inhibition conditions. The effect of maintaining mitochondrial membrane potential caused by sesamin under proteasome inhibition conditions is shown. The effect of sesamin on cell survival activity under proteasome inhibition conditions is shown. The physiological activity at the time of proteasome inhibition which each fraction of the sesame pressing burning extract by XAD-2000 column shows. In the figure, None: medium only, bortezomib: bortezomib 0.5 μM The physiological activity at the time of proteasome inhibition by sesaminol 3-glycoside is shown. In the figure, None: medium only, bortezomib: bortezomib 0.5 μM, SG: sesaminol triglyceride The numbers (2.5, 5, 10) all indicate the added concentration (μM).

  The present invention relates to proteasome-inhibiting conditions simulating aging characterized by containing at least one sesame lignan selected from sesamin, episesamin, sesaminol and sesaminol glycosides (that is, conditions for reducing proteasome activity) The present invention relates to an in vivo redox state improving agent under a reduced proteasome ability and a pharmaceutical composition for improving the in vivo redox state, reducing mitochondrial reactive oxygen species, maintaining mitochondrial activity, It has effects such as an increase in cell survival activity.

Abnormal proteins accumulate in the living body where the proteasome is inhibited by the degradation of abnormal proteins being stopped. Abnormal protein accumulation is considered to be a common pathogenesis mechanism of neurodegenerative diseases such as aging, Parkinson's disease, and Alzheimer's disease. In addition, under the inhibition of the proteasome, intracellular oxidation is further caused. The present inventors conducted experiments by adding proteasome inhibitors to create proteasome inhibition conditions. To find a substance capable of preventing intracellular oxidation under proteasome inhibition under the assumption that proteasome inhibition conditions, ie, reduced proteasome activity, or reduced proteasome ability, mimic cell aging As a result, the antioxidative action under proteasome inhibition by sesame lignan consisting of sesamin, episesamin, sesaminol and sesaminol glycoside was found.
Sesame lignans such as sesamin, episesamin, sesaminol, sesaminol glycosides, sesamolin, sesamol and the like are known as antioxidants, but among sesame lignans known as antioxidants, sesamin, The present inventors have found that episesamin, sesaminol and sesaminol glycosides suppress intracellular oxidation under proteasome inhibition.

[Proteasome]
The proteasome is a huge enzyme complex that performs proteolysis, and is distributed both in the cytoplasm and in the nucleus in cells of eukaryotic microorganisms. A system that degrades a protein labeled with ubiquitin with a proteasome is called a ubiquitin-proteasome system, and is a mechanism related to various functions in the cell such as cell cycle, cell cycle control, immune response, and signal transduction. Proteasome specifically degrades the target protein and removes it from the cell. This proteolytic system is universally involved in important life phenomena such as metabolism, cell cycle, stress response, endoplasmic reticulum quality control, nerve function and immune response. For this reason, if proper degradation is disrupted due to abnormal proteasomes, protein excess or deficiency occurs, resulting in various neurological disorders or individual abnormalities.
Proteosome abnormality breaks down when appropriate degradation occurs, excess protein is generated, and as a result, abnormal protein accumulates, resulting in extensive cell damage and individual abnormalities, aging, Alzheimer's disease, Parkinson's disease, Lewy body disease, It is known that triplet repeat disease, amyotrophic lateral sclerosis, cataract, arteriosclerosis, diabetic nephropathy, photoaging of the skin, and the like occur.

The sesame lignan used in the present invention is at least one sesame lignan selected from sesamin, episesamin, sesaminol, and sesaminol glycoside. There are various types of “sesame lignans” such as sesamin, sesamorin, and sesaminol (see Non-Patent Document 5, page 160). Sesame oil contains 0.340 to 11.3% (see page 62 of Non-Patent Document 5). The sesame defatted koji contains 0,868% of sesaminol glycoside (see Table 2.1 on page 26 of Non-Patent Document 6). Sesaminol glycosides are sesaminol with a sugar molecule attached, and when it enters the body, it is converted to sesaminol by the action of intestinal bacteria. It is absorbed from the intestinal tract. Sesaminol glycosides are isolated from sesame plant extracts, but plants other than sesame, for example, the same sesame plant and pepper family as sesame, citrus, scorpionaceae, urchinaceae, oleaceae, It may also be present in plants such as the urchinaceae family. Among these raw materials for extraction, sesame (Sesamum indicum L.) is easily available, and sesame seeds have been used as edible seeds such as washed sesame seeds, roasted sesame seeds, ground sesame seeds, and kneaded sesame seeds since ancient times. It is also widely used as sesame oil. In addition, sesame is most preferable as an extraction raw material because it has advantageous properties such as easy extraction and subsequent purification of sesaminol glycosides. Sesame as an extraction raw material can be used in any of seeds, above-ground parts, and underground parts, regardless of the type and production area. The extraction raw material is pulverized to an appropriate size from the viewpoint of improving the extraction efficiency, and then degreased with hexane or the like as necessary, and the degreased extraction raw material is mixed with water or a hydrophilic organic solvent and a mixed solvent thereof. Extraction is performed at a temperature from room temperature to the boiling point of the solvent. In addition, as a method for obtaining a sesaminol glycoside-containing extract regardless of extraction, a method of squeezing juice from a fresh plant can also be employed.
Although the sesaminol glycoside used in the Examples was produced by the following method, it is not limited to that method. Sesame pressing lees and water were mixed and held in an oil bath at 100 ± 5 ° C. for 5 to 60 minutes, and then the sesame pressing lees aqueous solution was filtered to obtain a hot water extract of sesame pressing lees as a filtrate. This hot water extract was freeze-dried, and the column was eluted with distilled water, 20%, 60%, and 99% methanol in this order using an adsorptive resin column, and finally with acetone. The liquid of each fraction was removed by lyophilization, suspended in PBS (phosphate buffered saline), and subjected to MTT assay (Thiazolyl Blue Tetrazoline Bromide method). Although the extraction amount of sesaminol glycoside varies depending on the amount of water, temperature, and time, extraction is possible.

INDUSTRIAL APPLICABILITY The present invention can provide a method for improving a redox state in a living body under reduced proteasome activity conditions for an animal that needs to improve the redox state in a living body under reduced proteasome activity conditions. In order to improve the redox state in vivo under reduced proteasome activity, an effective amount can be obtained by parenteral or enteral route for animals that need to improve the redox state in vivo under reduced proteasome activity. This is carried out by administering an in vivo redox state improving agent under reduced proteasome activity containing at least one sesame lignan selected from sesamin, episesamin, sesaminol and sesaminol glycoside.
And it is characterized by including the following steps (A) and (I).
(A) ingesting at least one sesame lignan selected from sesamin, episesamin, sesaminol and sesaminol glycoside,
(A) Detectable in a redox state in a living body under reduced proteasome activity conditions (a) FRET value indicated by intracellular redox flor under reduced proteasome activity conditions,
(B) mitochondria reactive oxygen species level under reduced proteasome activity conditions,
(C) A step of confirming that the intracellular redox has been normally restored by the mitochondrial potential under the condition of reduced proteasome activity, or (d) the cell survival activity value under the condition of reduced proteasome activity.
The animal is a mammal, preferably a human.

  In addition, an in vivo redox state-improving agent under reduced proteasome activity containing at least one sesame lignan selected from sesamin, episesamin, sesaminol, and sesaminol glycosides, is sesamin, episesamin, sesaminol, and It is characterized in that it is ingested at a rate of 3 to 60 mg / day as the content of at least one sesame lignan selected from sesaminol glycosides.

  Furthermore, according to the present invention, it is possible to provide a food or drink for improving the redox state in a living body under a condition that reduces proteasome activity. That is, the redox state-improving agent in vivo under the above-mentioned conditions for reducing the proteasome activity is added to the food as a content of at least one sesame lignan selected from sesamin, episesamin, sesaminol, and sesaminol glycoside. 1 to at least one sesame lignan selected from sesamin, episesamin, sesaminol, and sesaminol glycoside for improving the redox state in vivo under conditions of reduced proteasome activity, in the range of 01 to 100% by weight It can be provided as a food / drink for improving the redox state in a living body under conditions of reduced proteasome activity for continuous ingestion at a daily dose of 3 to 60 mg / day.

[Fluorescence resonance energy transfer (FRET)]
The present invention is based on the result of quantitatively examining the intracellular antioxidant activity under proteasome inhibition using FRET probe groups that visualize fluorescence reactions in vivo using fluorescence resonance energy transfer.
When a signal is input from the outside of the cell, a large number of molecules interact with each other, and the interaction varies in time and space. These various interactions are finally output as life phenomena such as cell differentiation, cytoskeleton reorganization, and gene expression. Identification and functional analysis of molecules involved in such intracellular signal transduction have been performed by genetic, biochemical and molecular biological techniques. These existing methods are effective to know the position of the target molecule in the signal cascade and the enzyme activity in the test tube, but it is not possible to know spatiotemporal information such as “where and when” in the cell. could not. In order to solve this problem, fluorescence reaction energy transfer (FRET) developed in recent years was used to visualize the reaction in the living body, which was employed in the measurement of the antioxidant action in the examples of the present invention.

  Next, an experimental method performed to demonstrate the antioxidant effect of sesamin and the like under proteasome inhibition conditions is shown. In addition to substances such as sesamin, the main substances used were bortezomib as a proteasome inhibitor, DMSO (dimethyl sulfoxide) as a solvent, resveratrol as a antioxidant (for positive control), mitochondrial electron Rotenone is a transmission system inhibitor.

[Experiment Method 1]
Quantification of the FRET value indicated by intracellular redox flor during proteasome inhibition was performed by the following method.
1. The trypsin-treated redox flore-expressing CHO cells were transferred to a glass bottom culture dish with a diameter of 3.5 cm and placed in a CO ₂ incubator (37 ° C., CO ₂ concentration: 7%) in an F-12 medium containing bovine serum (10%). And until the area ratio becomes 70%.
2. Simultaneously with the addition of bortezomib (1 μM), sesamin or a similar compound (both dissolved in DMSO to a concentration of 10 mM and stored at −80 ° C. as a stock solution) is cultured in the target cell line so that the final concentration becomes 10 μM. F-12 medium), and allowed to stand at 37 in a CO ₂ incubator for 8 hours.
3. Data acquisition was performed using an Olympus fluorescence microscope IX70 and a Photometrics CoolSnap HQ2 CCD camera to collect fluorescence signals from the intracellular redox flor through a CFP filter and a FRET filter. Signal acquisition was performed by selecting 3 fields of view for each sample.
4). For each field of the obtained observation image, at least 40 points located in the cell are selected, and the CFP intensity and FRET intensity at each point are measured using the area measurement program of Metamorph 7.0 software manufactured by Molecular Devices. did. In addition, the intensity of the cell-free portion was also measured as background. After subtracting the background value from the extracted intensity value, the FRET intensity value divided by the CFP intensity value was calculated as the FRET ratio.

[Experiment Method 2]
Visualization of mitochondrial reactive oxygen species level during proteasome inhibition was performed by the following method.
1. 2 × 10 ⁵ CHO-KI cells were transferred to a glass bottom culture dish having a diameter of 3.5 cm, and cultured in an F-12 medium containing bovine serum (10%) in a CO ₂ incubator for 24 hours.
2. Simultaneously with the addition of bortezomib (1 μM), sesamin (10 μM) or resveratrol (10 μM) was added, and the cells were further cultured for 8 hours.
3. The medium was removed, 5 μM MitoSOX ^TR Red (manufactured by Molecular Probes) was added to a 2 μl dish and allowed to stand at 37 ° C. for 10 minutes.
4). After the additive solution was removed, it was washed twice with Hank's balanced salt solution (Life Technologies).
5). 2 mL of a new Hank's balanced salt solution was added to the dish, and an observation image was obtained using a confocal microscope LSM510 META manufactured by Carl Zeiss.

[Experiment Method 3]
Detection of the mitochondrial potential during proteasome inhibition was performed by the following method.
1. 2 × 10 ⁵ CHO-KI cells were transferred to a glass bottom culture dish having a diameter of 3.5 cm, and cultured in an F-12 medium containing bovine serum (10%) in a CO ₂ incubator for 24 hours.
2. Simultaneously with the addition of bortezomib (1 μM), sesamin (10 μM) or resveratrol (10 μM) was added, and the cells were further cultured for 8 hours.
3. After removing the medium, 400 nM MitoTracker ^™ Red CMXRos (Molecular Probes) staining solution preheated to 37 ° C. was added, and the mixture was allowed to stand at 37 ° C. for 20-30 minutes.
4). After removing the staining solution, preheated bovine serum (10%)-containing F-12 medium was added, and an observation image was obtained using a confocal microscope LSM510META manufactured by Carl Zeiss.
5). The fluorescence intensity in each cell was calculated using ImageJ software.

[Experiment Method 4]
Cell activity measurement using MTT [3- (4,5-di-methylthiazol-2-yl) -2,5-diphenyltetrazole bromide] during proteasome inhibition was performed by the following method.
1. 100 μL of bovine serum-containing F-12 medium in each well was added to a 96-well (well) microplate, where CHO-K1 cells were mixed with a CO ₂ incubator (37 ° C., CO ₂ concentration: 70% by area ratio). 7%).
2. Bortezomib (dimethyl sulfoxide solution) was added to the cell culture to a final concentration of 1 μM. At the same time, a sample was prepared by adding sesamin or resveratrol (DMSO solution) to a final concentration of 10 μM.
3. The cells were cultured for 8 hours in a CO ₂ incubator.
4). 10 μL of MTT solution (5 mg / ml) was added, and the mixture was allowed to stand in a CO ₂ chamber for 4 hours.
5). 100 μL of a dissolution buffer (isopropanol added with 0.04 N hydrochloric acid) was added and suspended by vipetting.
6). The microplate was sealed and left at room temperature for 4 hours.
7). The absorbance at 570 nm of each well was measured with a microplate reader.
The relative value was calculated with the absorbance of a sample (control) to which bortezomib and extract were not added at all as 100%.

  Of at least one sesame lignan selected from the sesamin, episesamin, sesaminol and sesaminol glycosides of the present invention to be added to an in-vivo redox state improver, pharmaceutical composition or food or drink under conditions of reduced proteasome activity The ratio is not particularly limited as long as it has the function, but it can be appropriately selected from the range of 0.01 to 100% by weight. In addition, the content of at least one sesame lignan selected from sesamin, episesamin and sesaminol contained in the food or drink or pharmaceutical composition is 0.5 to 100 mg, preferably 1 to 1 per oral intake. 60 mg, more preferably 3 to 60 mg.

When the sesame lignan of the present invention is used as a pharmaceutical product (pharmaceutical composition), the dosage form may be any dosage form that can be conveniently administered orally or parenterally, for example, injection. Liquid, ring solution, powder, granule, tablet, capsule, enteric solvent, troche, liquid for internal use, suspension, emulsion, syrup, liquid for external use, poultice, nasal drop, ear drops, eye drops, Inhalants, ointments, lotions, suppositories and the like can be mentioned.
These various preparations are known according to conventional methods and can be used normally in the pharmaceutical formulation technical fields such as excipients, binders, preservatives, stabilizers, disintegrants, lubricants, and corrigents depending on the purpose. It can be formulated using adjuvants. Further, as an external preparation, vaseline, paraffin, fats and oils, lanolin, macrogol and the like can be used as a base, and ointments, creams and the like can be prepared by ordinary methods.
The dose varies depending on the purpose of administration and the situation (gender, age, body weight, etc.) of the administration subject, but in general, in the case of oral administration to adults, the total amount of sesame lignan of the present invention per day 0.5-100 mg, preferably 1-60 mg, more preferably 3-60 mg, and can be administered in one or more divided doses.

When the sesame lignan of the present invention is used as a food or drink, it may be in the form of the above-mentioned pharmaceutical preparation, or a solid or liquid food or a favorite product such as bread, noodles, rice, confectionery (biscuits, cakes, candy). , Chocolate, Japanese confectionery), agricultural products such as tofu and processed products thereof, sake, medicinal liquor, fermented foods such as mirin, vinegar, soy sauce, miso, dressing, mayonnaise, margarine, shortening, edible fats and oils such as edible fat, yogurt, Processing forms such as livestock foods such as ham, bacon and sausage, marine foods such as kamaboko, fried tempura, hampen, fruit juice drinks, soft drinks, sports drinks, alcoholic drinks, tea drinks and the like may be used. It can also be used as animal feed or pet food.
When used as a health food or functional food, the form thereof may be the above-mentioned pharmaceutical preparation or food and drink. For example, protein (a protein source is a highly nutritious milk protein with balanced amino acid, soy protein, Proteins such as egg albumin are the most widely used, but in addition to these degradation products, egg white oligopeptides, soybean hydrolysates, etc., mixtures of amino acids alone are also used), sugars, fats, trace elements, vitamins Further, it may be processed forms such as natural liquid foods, semi-digested nutritional foods, component nutritional foods, drinks, etc., in which emulsifiers, flavors and the like are blended.
When used as a health food or drink, for example, it can be added to dried foods, supplements, soft drinks, mineral water, alcoholic beverages, etc., but is not limited thereto.
The food and drink of the present invention can be processed and produced by a general production method by adding a required amount of the sesame lignan of the present invention to a food material substantially free of the sesame lignan of the present invention. Examples of the food and drink that does not substantially contain the sesame lignan of the present invention include food and drink that does not use sesame or the like as a raw material. The content of the sesame lignan of the invention is extremely small, and the total content of the sesame lignan of the present invention is less than 0.1 mg, preferably 0.8 mg or less per daily intake of the food or drink, or The amount of sesame lignan of the present invention of less than 0.1 mg, preferably 0.8 mg or less per daily intake of food or drink is included in food or drink substantially free of the sesame lignan of the present invention. The blending amount of the sesame lignan of the present invention varies depending on the dosage form and the morphological properties of the food, but is generally 0.001 to 50%, but is not particularly limited. Ingestion as a health food or functional food is used to improve the redox state of living organisms under conditions of reduced proteasome activity, and the preparation of hospital meals under the supervision of a dietitian based on a doctor's meal. In this case, the sesame lignan of the present invention can be added to any food that substantially does not contain the sesame lignan of the present invention, and can be given to the patient in the form of a functional food prepared on the spot. In order to improve the redox state in the living body under the condition that the proteasome activity is lowered, the food and drink of the present invention should be taken orally in a range of 3 to 60 mg of the sesame lignan of the present invention per day as a guide. Is desirable.
Next, the present invention will be described more specifically with reference to examples.

[Inhibition of intracellular oxidation by sesamin and its analogs during proteasome inhibition]
In this example, the effect of suppressing intracellular oxidation of sesamin and its analogs during proteasome inhibition was examined, and the following results were obtained.
In order to investigate whether the antioxidant activity of sesamin is also observed under proteasome inhibition conditions simulating aging, the proteasome inhibitor bortezomib (Bort) and sesamin and its similar compounds Co-addition was performed to visualize the intracellular redox state. Resveratrol, which has also been reported to have anti-aging and antioxidant effects, was used as a positive control for this experiment.
The results are shown in Figure 1. When bortezomib is added alone, the FRET efficiency shown by redox floe decreases, and when visualized, the inside of the cell is displayed in a warm color (red to orange) to a cold color (green to blue) (in the drawing, it is displayed in black and white) ), Indicating intracellular oxidation upon addition of bortezomib. Under such conditions, it was found that sesamin exhibits an oxidation-inhibiting effect equivalent to or higher than that of resveratrol at the concentration of 10 μM (FIGS. 1A and B).
When a similar experiment was conducted with respect to sesamin analog compounds, episesamin and sesaminol showed similar oxidation-inhibiting effects, but sesamolin and sesamol showed the same effect. There was no (FIG. 1C).

FIG. 1 shows the oxidation-suppressing effect of sesamin and its analogs when proteasome is inhibited.
(A) is a visualization of the intracellular redox state by the intracellular redox flow, and is color-coded according to the index according to the FRET efficiency indicated by the redox flow (shown in black and white in the drawing).
DMSO: Add solvent only.
Bort: Bortezomib (proteasome inhibitor) 1 μM was added.
Reservatorol (resveratrol) and Sesamine (sesamin) are both added at 10 μM.
(B) quantifies the FRET efficiency exhibited by redox fleur, and the bar indicates standard error.
(C) shows the oxidation-inhibiting effect of proteasome-inhibited wrinkles by sesamin analog compounds. Bars indicate standard error.

[Visualization of reduction effect of reactive oxygen species in mitochondria caused by sesamin under proteasome inhibition conditions]
In this example, the effect of sesamin on reducing mitochondrial reactive oxygen species under proteasome inhibition conditions was examined, and the following results were obtained.
In general, intracellular oxidation is thought to be largely due to reactive oxygen species (ROS) derived from mitochondria, one of the organelles. In order to investigate whether or not mitochondria-derived reactive oxygen species are involved in the mechanism by which sesamin suppresses intracellular oxidation, proteasome-inhibited cells were stained with MitoSOX ^™ Red reagent. This dye is localized to the mitochondria depending on the concentration of reactive oxygen species inside the mitochondria, especially super peroxide.
The results are shown in FIG. The addition of bortezomib was found to increase the amount of reactive oxygen species in mitochondria, similar to the addition of rotenone, an inhibitor of the mitochondrial electron transport system. Co-addition of sesamin under these conditions attenuated the mitochondrial localization of this pigment and decreased the amount of reactive oxygen species in the mitochondria. The effect was similar to resveratrol.
Rotenone (mitochondrial electron transport system inhibitor) (Rotenone) was added at a concentration of 1 μM, and the other substances were the same as in Example 1.

[Effect of sesamin on mitochondrial activity under proteasome inhibition conditions]
In this example, the test was conducted for maintaining mitochondrial activity of sesamin under proteasome inhibition conditions, and the following results were obtained.
In order to investigate whether the reduction effect of reactive oxygen species in mitochondria during proteasome inhibition by sesamin leads to the maintenance of mitochondria's own functionality, the mitochondrial membrane potential produced by proton emission from the mitochondrion is an indicator of its function It was. For visualization of the mitochondrial membrane potential, cell staining was performed with MitoTracker ^™ Red CMXRos, which shows mitochondrial localization depending on this potential.
The results are shown in FIG. The addition of bortezomib alone weakened the staining with this dye, indicating that the mitochondrial membrane localization decreased, that is, the function decreased. On the other hand, when co-added with sesamin, the intensity of this dye staining was higher than that with bortezomib alone, indicating that sesamin is responsible for maintaining mitochondrial function. This effect was comparable to resveratrol at the same concentration.

In this example, the effect of sesamin on cell survival activity under proteasome inhibition conditions was tested.
The above results indicate that sesamin suppresses intracellular oxidation and contributes to the maintenance of mitochondrial function. Therefore, it was investigated whether these effects affect cell viability. As a method, FIG. 4 shows the results of using an MTT assay in which the MTT conversion activity by reductase in living cells is measured. It was found that the decrease in cell (survival) activity by bortezomib was significantly suppressed by co-addition of sesamin. This revealed an increase in cell activity corresponding to the oxidation-suppressing effect of sesamin.

[Sesaminol glycoside bioactivity assay]
Experimental method 1. Income and analysis of sesaminol glycoside concentrated fraction from hot sesame extract of sesame pressed rice cake 100 g of sesame pressed rice cake and 570 ml of water were mixed and heated to reflux in an oil bath at 103 ° C. for 60 minutes. After 60 minutes, the sesame squeezed rice bran aqueous solution was filtered to obtain a hot water extract of sesame squeezed squeeze as a filtrate. This hot water extract was freeze-dried, and the column was eluted with distilled water, 20%, 60%, and 99% methanol in this order using an XAD-2000 column as an adsorbent resin, and finally eluted with acetone. . The liquid of each fraction was removed by lyophilization and suspended in PBS to obtain a sample for use in the MTT assay.
2. Analysis using purified sesaminol glycoside A commercially available sesaminol triglycoside was dissolved in PBS and subjected to the same MTT assay as described above.

[Experimental results and discussion]
1. Income and analysis of sesaminol glycoside concentrate fraction from sesame pressed hot water extract Sesaminol 3 which is a kind of lignan from sesame pressed hot water extract using XAD-2000 column as adsorbent resin It was revealed that glycosides can be concentrated. In order to clarify whether sesaminol 3-glycoside exhibits physiological activity during proteasome inhibition, the fraction of each step of adsorption and elution using XAD-2000 is the same as in the above experiment (Experimental method 1). Was added together with the proteasome inhibitor Bortezomib, and then cell activity was assayed by MTT assay. As a result, it was found that 60% and 99% methanol elution fractions enriched with sesaminol triglyceride increased the cell viability (FIG. 5).
2. Analysis using purified sesaminol triglyceride In order to further clarify the physiological activity of sesaminol glycoside, purified sesaminol triglyceride was dissolved in PBS, and the same level of sesamin as mentioned above during proteasome inhibition. The effect on cell activity by adding at a concentration (μM order) was examined. As a result, sesaminol 3-glycoside caused a significant increase in the survival rate at 10 μM, revealing its physiological activity (FIG. 6).

The present invention has found an antioxidant action under proteasome inhibition conditions simulating aging of sesame lignans, particularly sesamin, and includes the physiological effects of sesamin, including the effect on the mitochondrial state known as the source of reactive oxygen species. Clarified the effect. Diseases that develop due to decreased proteasome activity include aging, Alzheimer's disease, Parkinson's disease, Lewy body disease, triplet repeat disease, amyotrophic lateral sclerosis, cataract, arteriosclerosis, diabetic nephropathy, light of the skin Aging and the like are known, and it is expected that the ingested antioxidant clarified by the present invention imparts a survival activity to cells by the function of maintaining mitochondrial activity, and alleviates symptoms due to these diseases.

Claims (5)

  An in-vivo redox state improving agent under proteasome activity-reducing conditions, comprising at least one sesame lignan selected from sesamin, episesamin, sesaminol and sesaminol glycosides.   The in-vivo redox state-improving agent according to claim 1, wherein the improvement of the in-vivo redox state is to reduce the reactive oxygen species in mitochondria.   The in vivo redox state improving agent according to claim 1, wherein the improvement of the in vivo redox state is to maintain mitochondrial activity.   The in vivo redox state improving agent according to claim 1, wherein the improvement of the in vivo redox state is to increase cell survival activity. 5. In vivo redox conditions under reduced proteasome activity comprising the in vivo redox state improving agent or pharmaceutically acceptable salt according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient. A pharmaceutical composition for improving the condition.