JP2020037549A - Composition for protection of neuron from reactive oxygen species, prevention of denaturation of brain tissue and/or repair of brain tissue - Google Patents

Abstract

To provide a composition for protection of neurons from reactive oxygen species, prevention of denaturation of brain tissue and/or repair of brain tissue.SOLUTION: The present invention provides a composition for protection of neurons from reactive oxygen species, prevention of denaturation of brain tissue and/or repair of brain tissue, the composition containing sesaminol as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue, comprising sesaminol as an active ingredient. The composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue of the present invention can be suitably used for neurodegenerative diseases such as Parkinson's disease.

  A neurodegenerative disease is a general term for a disease in which a specific group of nerve cells is affected by some kind of disorder and causes a movement disorder, a cognitive disorder and the like. Among them, Parkinson's disease is a neurodegenerative disease in which the production of dopamine in the brain is reduced due to degeneration / dropout of dopaminergic neurons in the substantia nigra. Parkinson's disease occurs at a rate of 100 to 150 per 100,000 people, and its frequency of occurrence is second only to Alzheimer's disease among neurodegenerative diseases. Since the incidence rate increases with the age of the elderly, there is a concern that the number of patients will further increase in Japan, which has entered an aging society.

  The symptoms of Parkinson's disease vary from patient to patient. The most common symptoms are lack of motion and stiffness, characterized by increased voluntary skeletal muscle stiffness. Additional symptoms include resting tremor, sluggishness, rigidity, postural reflex and gait disorders. Common secondary symptoms include depression, sleep problems, balance problems, forward bending, dementia, speech problems, breathing problems, and dysphagia. Symptoms gradually worsen over time, eventually leading to death. It is said that the onset of motor disorder in Parkinson's disease begins with a disorder of the substantia nigra of the midbrain, and as the disease progresses, the lesion extends from the midbrain to the cerebral cortex. In a normal human brain, dopamine is produced by dopaminergic neurons present in the substantia nigra, and the dopaminergic neurons that extend to the striatum of the basal ganglia release dopamine in the striatum. It communicates information by delivering dopamine to cells involved in exercise. The direct cause of the movement disorders described above has been found to be a deficiency of the neurotransmitter dopamine produced in the brain.

In recent years, various injuries seen in cells and tissues in a living body due to reactive oxygen species (Reactive Oxygen Species: ROS) have attracted attention. The active oxygen species is a general term for compounds in which oxygen molecules have been changed to compounds having higher reactivity, and includes superoxide anion radicals, hydrogen peroxide, hydroxy radicals, singlet oxygen and the like, and acts as an oxidizing agent in a living body.
When reactive oxygen species increase, oxidized proteins accumulate in the living body as so-called abnormal proteins, which cause various diseases.

  Accumulation of abnormal proteins in vivo increases with the oxidizing action of reactive oxygen species and age, and it has become clear that they are involved in the pathogenesis mechanism common to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The prevention and improvement of diseases caused by the accumulation of abnormal proteins in living organisms is a major issue.However, regarding the prevention of accumulation of abnormal proteins, reactive oxygen species generated in living organisms due to oxidative stress can be reduced by ingestion of antioxidants. Attempts have been made to eliminate the protein by eliminating it. For example, Patent Document 1 discloses that sesame lignans are used as a redox condition improving agent (redox modulator) in vivo. Specifically, it is disclosed that intracellular oxidation induced by inhibition of the proteasome is prevented by sesame lignans.

JP-A-2015-96494

The above-mentioned literature describes that in vitro sesamin and the like show an effect of suppressing intracellular oxidation under conditions of reduced proteasome activity, but it has been demonstrated that in vivo, degeneration of nerve tissue was prevented or repaired. Not.
In neurodegenerative diseases such as Parkinson's disease, it is important to establish fundamental treatment and prevention methods that are not symptomatic. In particular, it is an important task to find components useful for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue.

  The present inventors have conducted intensive studies and found that sesaminol, a kind of sesame lignans, has the effect of protecting nerve cells from reactive oxygen species in vitro, preventing degeneration of brain tissue in vivo, The present inventors have found that the present invention has a tissue repairing action, and have completed the present invention.

Thus, according to the present invention, there is provided a composition comprising sesaminol as an active ingredient for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue.
According to the present invention, there is provided a food, a quasi-drug, or a medicine containing the above composition.
According to the present invention, there is provided a food composition containing sesaminol as an active ingredient for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue.
According to the present invention there is provided the use of sesaminol for the manufacture of a composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue.

  According to the present invention, a composition containing sesaminol as an active ingredient is expected to provide protection of nerve cells from reactive oxygen species, prevention of degeneration of brain tissue, and / or repair of brain tissue.

It is a difference in cell viability by adding sesaminol to a human neuroblastoma-derived cell line (SH-SY5Y). This is a difference in cell viability due to the addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y. It is a difference in cell viability when 6-OHDA is added after adding sesaminol to SH-SY5Y. FIG. 4 is a diagram showing, by a cell fluorescence image, a difference in the amount of intracellular ROS production between cells to which 6-OHDA was added to SH-SY5Y or cells to which 6-OHDA and sesaminol were added. 5 is a graph in which the fluorescence intensity of the fluorescence image of FIG. It is a figure showing the flow of a rotor rod test to a mouse for establishment of a Parkinson's disease model animal. Figure 7 shows the effect of rotenone on the dwell time on rods of mice during the rotor rod test. It is a figure showing the flow of a rotor rod test to a mouse after administration of rotenone. Figure 4 shows the effect of sesaminol on the dwell time on rods of mice during the rotor rod test. It is a weight change of the mouse | mouth during the experiment of adding rotenone and sesaminol. It is a change of the food intake of the mouse during the experiment of adding rotenone and sesaminol. It is a hematoxylin-eosin (HE) -stained histological image (x4.2) around the substantia nigra in the brain tissue collected from the mouse after the experiment of adding rotenone and sesaminol. It is an HE-stained tissue image (x20.0) around the substantia nigra in the brain tissue collected by dissecting the mouse after the experiment of adding rotenone and sesaminol. It is a stained image showing a difference in morphology of a nucleus between a cell obtained by adding 6-OHDA to SH-SY5Y or a cell obtained by adding 6-OHDA and sesaminol. It is a change of the colon length of the mouse after the experiment of adding rotenone and sesaminol.

  The composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue (hereinafter, also simply referred to as “composition”) of the present embodiment contains sesaminol as an active ingredient. Including. Sesaminol is known as lignans contained in sesame seeds and is a compound represented by the following structural formula. As used herein, the term "sesaminol" also includes epimers of sesaminol. An epimer of sesaminol includes, for example, 2-episesaminol.

In the present embodiment, protection of nerve cells from reactive oxygen species refers to an action of eliminating reactive oxygen species to prevent nerve cells from being damaged by reactive oxygen species.
In the present embodiment, the brain tissue includes, for example, tissues in the brain such as cerebral cortex, basal ganglia, amygdala, hippocampus, thalamus, hypothalamus, midbrain, pons, medulla oblongata, or cerebellum, and brain including central nervous system. Means any organization that exists within.

In the present embodiment, prevention of degeneration of brain tissue from reactive oxygen species and / or repair of brain tissue refers to the action of preventing spongy degeneration in brain tissue and the prevention or alleviation of disturbance of nerve bundles in brain tissue. Refers to action. Cavernous degeneration refers to vacuoles formed by swelling of synaptic terminals and inclusion bodies formed by abnormal proteins in nerve tissue.
The brain tissue in the present embodiment is preferably the midbrain, and more preferably the substantia nigra of the midbrain.
Since the composition of the present embodiment has these effects, it is expected to have an effect of treating, preventing and / or alleviating neurodegenerative diseases, particularly Parkinson's disease.

  The origin of sesaminol used in the present invention is not particularly limited, and may be sesaminol derived from plants such as sesame seeds, or may be sesaminol obtained by synthesis or semi-synthesis. The method itself of obtaining sesaminol from sesame seeds and the like is known, for example, a method of producing sesaminol by the action of an enzyme possessed by a predetermined microorganism using sesame seeds and defatted cake of the seeds as a raw material ( See JP-A-2006-61115 and JP-A-2008-167712).

  In the present embodiment, it is preferable to use sesaminol obtained from sesaminol glycosides contained in defatted sesame seeds according to the method described in JP-A-2008-167712. When a purified liquid of sesaminol obtained from defatted sesame seeds by this method is measured by high-performance liquid chromatography (HPLC) under the analytical conditions shown in the preparation examples described below, a large amount of sesaminol and 2-episesaminol are obtained. While sesamin, sesamolin and sesaminol glycosides were not detected, indicating that they were substantially not contained. In the composition containing sesaminol obtained by the above method, it can be said that the active ingredient is substantially only sesaminol. That is, in the present embodiment, the active ingredient may be substantially only sesaminol. The sesaminol obtained by the above method can be used as it is in the composition of the present embodiment, but if necessary, concentrated, diluted, filtered, deodorized, decolorized, subjected to a treatment such as drying. Is also good.

  In the present embodiment, the composition may be substantially composed of only sesaminol, but as long as the action of sesaminol is not impaired, quasi-drugs, pharmaceuticals, and the like are usually used in the technical field. The additives used may be appropriately compounded.

  Such additives can be appropriately selected from pharmaceutically and / or food-hygienic acceptable additives. For example, binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sugar, corn starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene) Glycols), disintegrants (starch, crystalline cellulose, etc.), wetting agents (sodium lauryl sulfate, etc.), suspending agents (sorbitol, syrup, methylcellulose, glucose syrup, gelatin, edible fat with water, etc.), emulsifiers (lecithin Sorbitan monooleate), non-aqueous excipients (almond oil, fractionated coconut oil or oily esters such as glycerin, propylene glycol, ethyl alcohol), preservatives (methyl or propyl p-hydroxybenzoate, sorbin) Acid, etc.), incense (Synthetic flavors, natural flavors, etc.), chelating agents (disodium edetate, sodium citrate, sodium metaphosphate, etc.), sweeteners (sucrose, stevia, etc.), pH adjusters (sodium hydrogen carbonate, potassium carbonate, etc.), Powders (pigments, pigments, resins, etc.), thickeners (gum arabic, methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.) and the like can be mentioned.

  The method of administration of the composition of the present embodiment is not particularly limited, and may be oral administration, injection administration (subcutaneous, intradermal, intramuscular, intravenous, transarterial), and administration onto the skin (application of an ointment, etc.). The component can be appropriately selected from, for example, delivery of the component to a locally affected area) or transdermal administration (the active ingredient is delivered to the systemic circulating blood stream through the skin), and preferably oral administration or injection administration.

  The dose of the composition of the present embodiment is not particularly limited, and can be appropriately determined depending on the weight, health condition, and the like of the administration target. For example, in the case of oral administration, the dose of the composition is in the range of 2 to 400 mg, preferably 4 to 80 mg, more preferably 2 to 40 mg as sesaminol per adult day. . In the case of injection administration, the amount of sesaminol per adult day is in the range of 0.2 to 40 mg, preferably 0.4 to 8 mg, more preferably 0.2 to 4 mg. The frequency of administration of the composition of the present embodiment is not particularly limited, and the composition can be administered once or multiple times a day.

  The composition of the present embodiment can be provided as a composition for preventing, treating or ameliorating Parkinson's disease. The composition of the present embodiment is suitable for symptoms that develop and progress due to Parkinson's disease. Such symptoms include, for example, lack of movement and stiffness, increased stiffness of voluntary skeletal muscle, tremor at rest, sluggishness, rigidity, postural reflex disorder, gait disorder, depression, sleep disorder, balance disorder, These include forward bending posture, dementia, speech problems, breathing problems, and dysphagia. Since the symptoms of Parkinson's disease vary from patient to patient, one or more of the above symptoms may be combined depending on the patient.

  The composition of the present embodiment can prevent apoptosis of nerve cells. For example, administration of sesaminol can prevent morphological abnormalities such as nucleus shrinkage due to reactive oxygen species. By preventing apoptosis of nerve cells, it is possible to prevent, for example, symptoms that develop and progress due to Parkinson's disease as described above.

  The composition of the present embodiment can be administered to mammals including humans, and is particularly suitable for healthy persons and patients with Parkinson's disease. The composition of the present embodiment can be administered to a healthy subject for the prevention of Parkinson's disease, and to a patient with Parkinson's disease for treatment of the disease or improvement of the disease state.

  The composition of the present embodiment may be in any form of food, quasi-drugs, pharmaceuticals, research reagents, and the like, as long as administration or ingestion of sesaminol to mammals including humans is possible. But preferably in the form of food, quasi-drugs or pharmaceuticals. That is, the scope of the present invention also includes foods, quasi-drugs, or pharmaceuticals containing a composition for preventing, treating, and / or improving Parkinson's disease, which contains sesaminol as an active ingredient. Further, within the scope of the present invention, a food containing a composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue, comprising sesaminol as an active ingredient, Quasi-drugs or pharmaceuticals are also included.

  More specifically, the form of quasi-drugs or pharmaceuticals includes, for example, tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, injections, infusions, and the like. Examples of the form of the food include, for example, beverages (drinks, etc.), nutritional functional foods, foods for specified health use, supplements (tablets, capsules, granules, etc.), foods for patients (hospital foods, nursing foods, etc.), etc. Is mentioned. Alternatively, the composition of the present embodiment may be in the form of a food additive (liquid, powder, paste, etc.). Further, the composition of the present embodiment may be added as a food additive to an existing seasoning or the like. The production itself of the composition in these forms can be performed by a method known in the art.

  The food containing the composition of the present embodiment is obtained, for example, by adding the composition of the present embodiment to a food or food raw material substantially free of sesaminol so that sesaminol is contained in a sufficient amount. be able to. However, plants containing sesaminol (eg, sesame seeds) are not included in the composition of the present embodiment in the form of food. In the present embodiment, the food composition refers to a composition suitable for eating and drinking or a composition that can be added to food and drink.

  The sesaminol content in the composition of the present embodiment is not particularly limited and can be appropriately set according to the form of the composition. In one example, the sesaminol content is from 0.0001 to 50% by weight, preferably from 0.0005 to 40% by weight, more preferably from 0.001 to 35% by weight, based on the total weight of the composition.

  The scope of the present invention also includes the use of sesaminol for the preparation of a composition for protecting neurons from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue. Sesaminol and a composition containing the same as an active ingredient are the same as those described above. The administration method and dosage of this composition are the same as those described above.

  Furthermore, the scope of the present invention also includes a method for protecting nerve cells from reactive oxygen species, which comprises administering a composition containing sesaminol as an active ingredient. Sesaminol and a composition containing the same as an active ingredient are the same as those described above. The administration method and dosage of this composition are the same as those described above.

  In one embodiment, a quasi-drug composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue, comprising sesaminol as an active ingredient, is provided. In still another embodiment, there is provided a pharmaceutical composition comprising sesaminol as an active ingredient for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Preparation Example: Preparation of Sesaminol In this Example, Paenibacillus sp. Sesameol obtained from sesameol glycosides contained in sesame defatted lees by culturing in a medium containing sesame. Specifically, sesaminol was prepared as follows.

  Strain KB0549 was grown in a medium obtained by adding 1.0% tryptone, 0.5% yeast extract and 0.89% NaCl to a hot water extract of sesame defatted lees (manufactured by Takemoto Yushi Co., Ltd.) to obtain a culture of KB0549 strain. The obtained culture solution is added to sesame defatted lees (10.0 kg; heat-sterilized to a water content of 70%, adjusted to pH 6.0), and intermittently stirred and aerated at 37 ° C. for 6 days using a solid fermenter. Fermentation treatment was performed.

  The fermented sesame defatted lees were dried until the water content became 8.5%. To the obtained dried product, 100 L of 95% ethanol was added to 10.0 kg of the dried product, and the mixture was stirred while heating to 50 ° C. to extract sesaminol. The obtained extract was subjected to diatomaceous earth filtration using a filter press to remove solids, thereby obtaining a filtrate (82 L). The obtained filtrate was concentrated to 4.1 L using a vacuum concentrator. To the obtained concentrated solution, 4 times or more of 99.5% ethanol was added, and insoluble materials were removed by filtration with filter paper. Then, the obtained liquid was concentrated by an evaporator to obtain a highly concentrated liquid of sesaminol (4.05 L).

As a result of subjecting sesaminol and the sesaminol-related compound in the obtained highly concentrated solution to HPLC, the high concentration solution (4.05 L) contained 18.4 g of sesaminol. The analytical conditions in HPLC are as follows:
HPLC: HITACHI LaChrom
Column: Wakosil-II 5C18HG (φ4.6 * 250 mm, Wako Pure Chemical Industries, Ltd.)
Developing solvent: A; 10% acetonitrile + 0.1% trifluoroacetic acid, B: 80% acetonitrile + 0.1%
Trifluoroacetic acid and B were developed with a 10-100% linear gradient (40 minutes).
Flow rate: 0.8 ml / min
Analysis wavelength: 280 nm

  From the obtained highly concentrated solution, a purified solution was obtained using a column purification technique such as silica gel or gel filtration purification in order to increase the sesaminol content. The purified liquid of sesaminol contains almost no sesamin, sesamolin and sesaminol glycoside. The sesame defatted lees used in this preparation method contain almost no lipophilic sesamin and sesamolin because they migrate to sesame oil, and also because sesaminol glycosides are converted to sesaminol by fermentation treatment.

  The purified sesaminol solution was dissolved in DMSO to prepare a sesaminol solution (8.56 mg / ml) used in this example. It has been confirmed by the present inventors that the obtained sesaminol solution is not toxic to various cell lines used in this example and does not affect the survival.

Example 1: Protective effect of sesaminol on neurons-Examination in in vitro experimental system-

Experimental Samples / Reagents / Equipment Sesaminol solution (8.56 mg / ml) was obtained as described above.
6-Hydroxydopamine (6-OHDA) was purchased from R & D System. 6-OHDA was used in suspension in DMSO.
The cell line SH-SY5Y derived from human neuroblastoma was purchased from Public Health England. SH-SY5Y has a tyrosine hydroxylase and dopamine β-hydroxylase characteristic of dopaminergic neurons, and is widely used in in vitro studies on Parkinson's disease.
Fetal bovine serum (FBS) was purchased from Sigma. FBS was allowed to stand in water at 56 ° C. for 30 minutes to inactivate complement components in serum.
For sterile aqueous sodium bicarbonate, 5 ml of 8% NaHCO ₃ solution (40 g / 500 ml H ₂ O) was dispensed into each ampule tube that had been heated (200 ° C, 30 minutes), and the ampule tube was sealed using a burner. After that, it was produced by autoclaving.
Penicillin was purchased from Meiji Seika Pharma Co., Ltd.
Streptomycin was purchased from Meiji Seika Pharma.
Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical Co., Ltd.
After 0.1% penicillin, 0.1% streptomycin, and 2% L-glutamine (584 mg / l) sterilized by filtration were added to DMEM medium sterilized by an autoclave, the pH was adjusted to 7.5 using sterilized sodium bicarbonate water.
Phosphate-buffered saline (PBS (-)) is, 0.8% NaCl, 0.02% KCl , 0.02% KH 2 PO 4, pH-solution containing _{0.29% Na 2 HPO 4 · 12H} 2 O becomes 7.2-7.4 It was prepared by adjusting as follows.
The trypsin / EDTA solution was prepared by adding 0.025 g of trypsin and 0.010 g of EDTA · 2Na to 100 ml of PBS, mixing, and sterilizing with a filter. The solution after preparation was stored frozen.
MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazolium bromide) was purchased from Dojin Chemical Laboratory Co., Ltd. MTT was dissolved in PBS (-) to a concentration of 5 mg / ml.
DMSO (dimethyl sulfoxide) was purchased from Wako Pure Chemical Industries, Ltd.
The membrane permeable probe 2 ', 7'-dichlorofluorescin-diacetate (DCFH-DA) was purchased from Sigma.
96-well culture plates were purchased from Greiner-Bio-One.
The plate mixer was manufactured by Biotech, the microplate reader was Wallac-1420-ARVO-sx, and the fluorescence microscope was OLYMPUS-LSC101.

Cell culture method
SH-SY5Y was seeded in a φ100 mm cell culture dish containing 10% FBS-containing DMEM medium, and cultured at 37 ° C. in a 5% CO 2 incubator. The culture medium was changed every 2-3 days. When the cell concentration becomes 70 to 80% confluent visually, after washing with PBS (-), the cells are detached from the culture dish with a trypsin / EDTA solution, an equal amount of culture medium is added, and 1500 rpm 20 ° C for 5 minutes. And centrifuged under the following conditions. After removing the supernatant, a culture medium was added, and cells having a constant cell concentration were seeded in various culture vessels as cells for culture and cells for experiments.

6-OHDA was used for preparing an in vitro Parkinson's disease model. 6-OHDA is a dopamine analog, a neurotoxin that destroys catecholaminergic nerve endings. It is rapidly oxidized when taken up into cells via the dopamine transporter, and simultaneously reacts with reactive oxygen species (ROS). Since it is known that one kind of H ₂ O ₂ is produced, it is widely used for preparing a Parkinson's disease model.

Measurement of viable cell number The MTT method was used for measuring the viable cell number. By measuring the number of living cells by the MTT method, the cytotoxicity of sesaminol or 6-OHDA on SH-SY5Y and the effect of sesaminol on 6-OHDA-induced neuronal injury were evaluated.

Measurement of intracellular reactive oxygen species (ROS) production (DCFH-DA method)
DCFH-DA is deacetylated by intracellular esterase to form 2 ′, 7′-dichlorodihydro-fluorescin (DCFH) in a non-fluorescent form. Furthermore, it is rapidly oxidized by ROS and changes to 2 ′, 7′-dichlorodihydro-fluorescin (DCF) which shows strong fluorescence. Using this principle, the intracellular ROS production was evaluated by measuring the fluorescence intensity of each group.

(1-1) Effect of sesaminol on cell viability of SH-SY5Y
The effect of sesaminol on the cell viability of SH-SY5Y was determined by the following method.
SH-SY5Y was seeded on a 96-well culture plate at 2.0 × 10 ⁵ cells / ml and left overnight to adhere to the dish. For the main culture, the medium was changed, sesaminol was added to the culture medium so that the final concentration was 0.25, 0.5, 1.0, 5.0, 7.5 or 10.0 μg / ml, and the cells were cultured in an incubator for 24 hours. After completion of the main culture, the medium was removed, and 100 μl of a culture medium containing a 10% 5 mg / ml MTT solution was added to each well, and the mixture was allowed to stand in an incubator for 2 to 4 hours. After removing the medium, 200 μl of DMSO was added to each well. The culture plate was stirred with a plate mixer for 5 minutes to dissolve the formed formazan dye. The absorbance at a wavelength of 535 nm was measured with a microplate reader.

(1-2) Effect of 6-OHDA on cell viability of SH-SY5Y
The effect of 6-OHDA on the cell viability of SH-SY5Y was changed to a final concentration of 5, 10, 20, 30, 40 μM by changing the addition of sesaminol in the experimental method described in (1-1). 6-OHDA was added to the medium to determine

(1-3) Effect of sesaminol on neuronal damage induced by 6-OHDA
The effect of sesaminol on neuronal damage induced by 6-OHDA was examined based on the experimental method described in (1-1). Sesaminol was added to the medium to a final concentration of 0.25, 0.5, 1.0, 5.0, or 10.0 μg / ml and incubated for 2 hours. Thereafter, 6-OHDA was added to a final concentration of 20 μM, and the mixture was incubated for 24 hours. And the MTT method was performed.

(1-4) Effect of sesaminol on 6-OHDA-induced intracellular ROS production
The effect of sesaminol on the intracellular ROS production induced by 6-OHDA was determined by the following method.
SH-SY5Y cells were seeded on a φ35 mm dish at 1.0 × 10 ⁵ cells / ml and left overnight to adhere to the dish. For the main culture, a final concentration of 1 μg / ml sesaminol was added to the culture medium, and two hours later, 6 μH-OHDA at a final concentration of 20 μM was added, followed by culturing for 7.5 hours. Thirty minutes before the end of the culture, 5 μl of a 2.4 mM DCFH-DA solution was added to the medium (2 ml). After completion of the culture, the medium was removed on ice, and washed twice with PBS (-). Next, a cover glass (24 × 24 mm) was affixed to the dish, excess water was absorbed with a filter paper, and observed with a fluorescence microscope. As a control, a medium containing no sesaminol solution and a sample containing neither sesaminol nor 6-OHDA were prepared and observed together.

(1-5) Effect of sesaminol on nuclear morphology of SH-SY5Y to which 6-OHDA was added In order to confirm whether the decrease in cell viability was due to apoptotic cell death, PI using Propidium Iodide (PI) Nuclear staining was performed by a staining method.
SH-SY5Y was seeded on a φ35 mm dish at 1.0 × 10 ⁵ cells / ml and left overnight to adhere to the dish. For the main culture, a final concentration of 1 μg / ml sesaminol was added to the culture medium, and two hours later, 6 μH-OHDA at a final concentration of 20 μM was added, followed by culturing for 7.5 hours.
500 μl of a PI solution (Propidium Iodide 5 μg / ml) was added to the cultured cells, and the mixture was incubated for 15 minutes. After the incubation, the cells were washed three times with PBS (−). mm) was applied to a dish, excess water was absorbed with a filter paper, and observed with a fluorescence microscope.As a control, a medium containing no sesaminol solution and cells containing no sesaminol and 6-OHDA were used as controls. Was observed in the same manner.

result
FIG. 1 shows the change in cell viability due to the addition of sesaminol to SH-SY5Y. Sesaminol had no effect on the cell growth rate of SH-SY5Y at a concentration of 10 μg / ml or less, indicating that no cytotoxicity was observed at a concentration of 10 μg / ml or less.

  FIG. 2 shows the change in cell viability due to the addition of 6-OHDA to SH-SY5Y. “**” in the figure indicates that the p value is less than 0.01 with respect to the control. 6-OHDA significantly reduced the cell viability at 20 μM or more, depending on the added concentration. From these results, 20 μM of 6-OHDA was used in subsequent experiments as a neuropathy model.

  FIG. 3 shows the change in cell viability due to the addition of sesaminol to SH-SY5Y with 6-OHDA. “A” and “b” in the figure indicate that the p value is less than 0.01 between different characters. Cell viability significantly reduced by the addition of SH-SY5Y was restored to control levels by the addition of 1 μg / ml or 5 μg / ml sesaminol. From this, it was clarified that sesaminol used in the present test has a neuroprotective effect. In the subsequent experiments, the effective concentration was 1 μg / ml of sesaminol concentration.

  FIGS. 4 and 5 show the effect of sesaminol addition on SH-SY5Y in which intracellular ROS was induced by 6-OHDA. FIG. 4A shows a fluorescence image of the cell, and FIG. 4B shows a bright field of the cell. “A” and “b” in FIG. 5 indicate that the p value is less than 0.01 between different characters. The fluorescence intensity depends on the amount of ROS produced. From FIG. 4, it was observed that ROS was remarkably increased in the cells to which 6-OHDA was added as compared with the control, and it can be seen from FIG. In addition, in the cells to which 6-OHDA and sesaminol were added, it was found that the fluorescence intensity derived from ROS was reduced to the control level. Intracellular ROS production was significantly increased by 6-OHDA, but was suppressed by the addition of sesaminol. Therefore, it was suggested that sesaminol suppresses 6-OHDA-induced neuronal injury by decreasing intracellular ROS production.

  FIG. 14 shows the effect of sesaminol on the nuclear morphology of SH-SY5Y to which 6-OHDA was added. As shown in FIG. 14, when 6-OHDA was added, morphological abnormalities such as nuclear shrinkage (white arrows in the center image) were observed, and cell death due to apoptosis was observed. On the other hand, when sesaminol was added, almost no nuclear morphological abnormality was observed even when 6-OHDA was added. Therefore, sesaminol was found to suppress nuclear morphological abnormalities caused by 6-OHDA, suggesting that it has an effect of preventing apoptosis of nerve cells.

Example 2: Effect of sesaminol on Parkinson's disease model animal-Examination with in vivo experimental system-

(2-1) Establishment of Parkinson's disease model animal For production of Parkinson's disease model animal, rotenone represented by the following structural formula was used. Rotenone is a compound whose solution is used as a pesticide or insecticide, and is known to increase ROS by inhibiting mitochondrial complex I and lead to nerve cell death by its oxidative stress. It induces apoptosis in rat midbrain substantia nigra dopaminergic neurons by ROS production.

(I) Animal breeding
Ten-week-old male C57BL6 / J mice (13 mice) were divided into two groups: (1) control group (n = 6) and (2) rotenone group (n = 7).
To prepare a Parkinson's disease model, a solution of rotenone in 3% carboxymethylcellulose (CMC) + 1.25% chloroform solution was orally administered to the rotenone group once a day for 28 days using a gastric tube. The amount of rotenone to be administered was 10 mg / kg body weight based on the body weight of the mouse. Feed (solid food) and water were available ad libitum.

(Ii) Athletic ability test (rotor rod test)
The mouse was placed on a rotating rod, and the time until the mouse fell (residence time) was measured. Mice that develop motor dysfunction, a symptom of Parkinson's disease, are more likely to fall off the accelerating rod. A preliminary test was performed to familiarize the mouse with the rod, and then the test was performed the day after the final oral administration of rotenone. The rotation speed of the rod was accelerated from 6 rpm to 33 rpm for the first 30 seconds, and then rotated at a constant speed (33 rpm). The measurement time was up to 5 minutes. This test was performed three times (FIG. 6).

  FIG. 7 shows the results of a rotor rod test for establishing a Parkinson's disease model animal. In the figure, “**” indicates that the p-value is less than 0.01 for the control, and “*” indicates that the p-value is less than 0.05 for the control. The dwell time on the rod in the rotenone group was significantly reduced compared to the control group. Therefore, it was shown that a Parkinson's disease model animal could be established under these experimental conditions. In the following experiments, the effects of sesaminol were examined using model animals created under the same conditions.

(2-2) Effect of sesaminol feeding on Parkinson's disease model animals (i) Animal breeding
Twenty-five 7-week-old male C57BL6 / J mice were preliminarily reared on a solid diet for 4 days and on a control diet for 4 days. Table 1 shows the compositions of the control diet and the sesaminol diet described below. Feed composition was prepared according to AIN-93M. Feed and water were freely available.

After a control diet for 4 days, 25 mice were divided into four groups: a control group, a rotenone group, a sesaminol (L) group, and a sesaminol (H) group. Of these four groups, the sesaminol (L) group was given a 0.0008% sesaminol diet, and the sesaminol (H) group was given a 0.008% sesaminol diet for 7 days instead of the control diet. During the same period, the remaining control group and rotenone group were given a control diet. Thereafter, rotenone was orally administered to the group other than the control for 28 days. The amount of rotenone to be administered was dissolved in a 3% CMC + 1.25% chloroform solution and administered so as to be 10 mg / kg body weight based on the body weight of the mouse. The control group was orally administered a solvent without rotenone (3% CMC + 1.25% chloroform) for 28 days. Table 2 shows combinations of test meals and oral administration substances during the experiment.

(Ii) Exercise ability test A rotor rod test was performed on the four groups of mice immediately after the start of the administration of rotenone or a solvent and every week after the start of the administration. FIG. 8 is a schematic diagram of the flow of the experiment. Preliminary experiments revealed that the mouse became accustomed to the rod speed during the test, so the rod speed was gradually increased in this test. For each number in FIG. 8, a test was performed once as a preliminary test at 6-10 rpm and max 3 minutes, and once at 6-20 rpm and max 3 minutes. This test, 6-25 rpm immediately after the start of administration, 3 times of max 5 minutes test, 6-30 rpm on day 7, 3 times of max 5 minutes test, 6-33 rpm on days 14-28, A max 5 minute test was performed three times, and the dwell time was measured.

(Iii) Dissection test Dissection was performed 2 days after the end of rotenone administration. The weight of the brain, liver, spleen, kidney, and jejunum and colon in each group were measured.

(2-3) Influence of Sesaminol Ingestion on the Colon of Parkinson's Disease Model Animals Among the above four mice, the sesaminol group (H) was fed a 0.00008% sesaminol diet having the composition shown in Table 3 below. Except for changing to the sesaminol group (LL), 25 C57BL6 / J mice were bred in the same manner as in the experiment in (2-2), and rotenone was administered except for the control, and the mice were dissected. The colon was removed from each dissected mouse and its length was measured.

Results The effect of sesaminol on the staying time on the rod during the rotor rod test of rotenone-administered mice during an experiment to confirm the feeding effect of sesaminol on Parkinson's disease model animals is shown in FIG. In the figure, “**” indicates that the p value is less than 0.01 for the control, and “##” indicates that the p value is less than 0.01 for the rotenone administration group. “#” Indicates that the p value is less than 0.05 for the rotenone administration group. On the 21st day of oral administration, the stay time of the rod in the rotenone group was significantly shortened compared to the control group, but the stay time of the rod in the sesaminol (L) group was significantly prolonged compared to the rotenone group. did. On the 28th day of oral administration, the dwell time of the rod in the rotenone group did not change from the 21st day,
The rod stay time was significantly prolonged in the sesaminol (L) group compared to the rotenone group. Although no significant difference was observed in the sesaminol (H) group, the staying time was longer than that in the rotenone group. FIGS. 10 and 11 show the results of measurement of body weight change and food consumption during the same experiment. No significant difference was observed in body weight and food intake during the breeding period in each group.

Table 3 shows the dissection results in each group after the experiment in which the effects of sesaminol intake on the Parkinson's disease model animals were observed.
There were no significant differences in brain, liver, spleen, kidney weight and jejunum / colon length in each group, but colon shortened in the rotenone group and recovered in the sesaminol group compared to the control group A trend was observed.

  FIGS. 12 (x4.2) and 13 (x20.0) show HE-stained tissue images of the brain tissue obtained in the dissection experiment. In the rotenone group, many vacuoles (sponge-like degeneration) were observed in and around glial cells. In addition, vacuoles were also observed in the nerve bundle, and disturbance of the nerve bundle was observed. However, in the sesaminol (L) and (H) groups that ate sesaminol, spongiform degeneration and disturbance of nerve bundles in the brain were remarkably reduced. This suggests that sesaminol restores motor function by preventing or repairing rotenone-induced spongiform degeneration in the brain and disruption of nerve bundles, revealing that sesaminol has an antiparkinsonian effect. became.

  FIG. 15 shows the results of examining the effect of sesaminol consumption on the colon of a Parkinson's disease model animal in the experiment of (2-3). It has been reported that in Parkinson's disease model mice, the length of the colon becomes shorter. In the present rotenone group, the colon was shortened in the rotenone group compared to the control group. On the other hand, in the sesaminol group, the colon length tended to recover.

Formulation Examples Hereinafter, formulation examples of the composition of the present embodiment will be described. As sesaminol, one obtained by the method described in the above Preparation Example is used. In the following formulation examples, the mixing ratio of each component is shown by weight%.

Tablets Tablets are manufactured as follows. After mixing and homogenizing the following components, they are formed by a tableting machine to obtain tablets.
Sesaminol 5.00
DL-α-tocopherol acetate 1.33
Silicic anhydride 1.50
Calcium stearate 1.33
Lactose 45.67
Hydroxypropyl cellulose 2.67
Crystalline cellulose
(Total 100.00)

Claims (6)

  A composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue, comprising sesaminol as an active ingredient.   The composition according to claim 1, wherein the brain tissue is a midbrain.   3. The composition according to claim 1, wherein the brain tissue is substantia nigra of the midbrain.   A food, a quasi-drug or a pharmaceutical comprising the composition according to claim 1.   A food composition comprising sesaminol as an active ingredient for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue, and / or repairing brain tissue.   Use of sesaminol for the manufacture of a composition for protecting nerve cells from reactive oxygen species, preventing degeneration of brain tissue and / or repairing brain tissue.