Composition for inhibiting neuronal degeneration

Abstract

To provide means for inhibiting neuron degeneration.SOLUTION: A composition for inhibiting neuron degeneration contains effusol and / or dehydroeffusol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a composition for inhibiting neuronal degeneration, etc. The contents of all documents described in this specification are incorporated herein by reference. [Background technology]

[0002] It is known that degeneration of nerve cells causes movement disorders. For example, Parkinson's disease is a progressive neurodegenerative disease characterized by the degeneration of dopaminergic neurons in the substantia nigra-striatum of the midbrain, resulting in motor dysfunction such as tremors in the limbs and muscle stiffness. However, the cause of this degeneration and loss remains unknown. Dopamine is unstable and produces reactive oxygen species through autoxidation, so it is thought that reactive oxygen species are involved in the degeneration of dopaminergic neurons. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Singhuber J, Baburin L, Khom S et al.: GABAA receptor modulators from the Chinese herbal drug junci medulla-the pith of juncus effuses. Planta med 78: 455-458 (2012) [Non-patent document 2] Wang YG, Wang YL, Zhai HF et al. : Phenanthrens from juncus effusus with anxiolytic and sedative activities. Nat Prod Res. 26: 1234-1239 (2012) [Non-patent document 3] Liao YJ, Zhai HF, Zhang B et al. : Anxiolytic and sedative effects of dehydroeffusol from juncus effusus in mice. Planta med. 77: 416-420 (2011) [Non-patent document 4] Haruna Tamano, Ryusuke Nishio, Hiroki Morioka, Atsushi Takeda: Mol. Neurobiol., 56, 435-443 (2019). [Non-Patent Document 5] Haruna Tamano, Hiroki Morioka, Ryusuke Nishio, Azusa Takeuchi, Atsushi Takeda: Mol. Neurobiol., 56, 4539-4548 (2019). [Non-patent document 6] Haruna Tamano, Ryusuke Nishio, Hiroki Morioka, Ryo Furuhata, Yuuma Komata, Atsushi Takeda: Mol. Neurobiol., 56, 7789-7799 (2019). Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have demonstrated that intracellular Zn2+ is reduced by the production of hydrogen peroxide, a reactive oxygen species (ROS). 2+ We hypothesized that disruption of dopaminergic homeostasis is the cause of dopaminergic neurodegeneration and have tested this hypothesis using a Parkinson's disease model in which the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was administered to unilateral substantia nigra compacta in rats. We have reported that 6-OHDA excessively increases Zn2+ levels in dopaminergic neurons in the substantia nigra compacta, inducing cell death and causing movement disorders (Non-Patent Documents 4-6).

[0005] Therefore, the inventors believed that by developing a means to suppress neuronal degeneration, it may be possible to treat pathologies caused by such neuronal degeneration, and conducted extensive research with the aim of developing such a means. [Means for solving the problem]

[0006] The present inventors have found that efsol and dehydroefsol can exert an inhibitory effect on neuronal degeneration, and have made further improvements.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A composition for inhibiting neuronal degeneration, comprising efsol and / or dehydroefsol. Section 2. The composition of claim 1, wherein the neuronal cells are dopaminergic neurons. Section 3. The composition according to claim 2, which is used for a subject suffering from dopaminergic neuron degeneration. Section 4. 4. The composition according to claim 1, which is used to improve movement disorders caused by neuronal degeneration. Section 5. 5. The composition according to claim 1, which is for treating Parkinson's disease. [Effects of the Invention]

[0008] The compositions and methods encompassed by the present disclosure can suppress neuronal degeneration, which is expected to lead to recovery from pathological conditions (e.g., movement disorders) caused by neuronal degeneration. [Brief explanation of the drawings]

[0009] [Figure 1] The figure shows the results of measuring the amount of intracellular reactive oxygen species (ROS) using a fluorescent reagent when 6-OHDA, which is used to create a Parkinson's disease model, was administered together with efsol or dehydroefsol to the substantia nigra compacta of one side of a rat. [Figure 2]6-OHDA, used to create a Parkinson's disease model, was administered together with efsol or dehydroefsol to the substantia nigra pars compacta of one side of a rat, and the amount of intracellular hydrogen peroxide was measured using a fluorescent reagent. [Figure 3] The results of measuring the amount of intracellular Zn2+ using a fluorescent reagent when 6-OHDA, which is used to create a Parkinson's disease model, was administered together with efsol or dehydroefsol to the substantia nigra compacta of one side of a rat. [Figure 4] The figure shows the results of measuring the amount of degenerated neurons by tyrosine hydroxylase (TH) immunostaining when 6-OHDA, which is used to create a Parkinson's disease model, was administered together with efsol or dehydroefsol to the substantia nigra pars compacta of a rat. [Figure 5] The chromatograms obtained by HPLC analysis of a 50% aqueous ethanol extract of the whole rush plant or a 50% aqueous ethanol extract of the rush core are shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a composition for inhibiting neuronal degeneration, and the like. The present disclosure includes all of the compositions disclosed herein and recognizable by a person skilled in the art.

[0011] The composition for suppressing neuronal degeneration included in the present disclosure contains at least one compound selected from the group consisting of efsol and dehydroefsol. Such a composition may also be referred to as the composition of the present disclosure.

[0012] Efsol is a compound represented by the following structural formula:

[0013] [ka]

[0014] The efsole used in the composition of the present disclosure may be a synthetic product or one extracted and purified from a naturally occurring substance. It can be synthesized by known methods or methods easily derived from known methods. It can also be purchased as a commercially available product. When obtained from nature, efsole is known to exist in rush (wick grass), for example, and can be obtained by extraction from rush and purification as needed. For example, efsole can be extracted with water, methanol, ethanol, or a mixture of at least two of these, followed by purification as needed. The extract is not particularly limited, but methanol or a 40-60% aqueous ethanol solution is preferred. The resulting extract may be further fractionated and / or purified as needed. Fractionation can be performed, for example, by liquid-liquid partition extraction. The partition can be performed using, for example, water, n-hexane, ethyl acetate, or the like. Purification can be performed, for example, by silica gel column chromatography and / or high-performance liquid chromatography (HPLC). More specifically, for example, a method may be used in which a methanol extract of rush is concentrated under reduced pressure, followed by liquid-liquid partition extraction with water and n-hexane, ethyl acetate is further added to the resulting aqueous layer to perform liquid-liquid partition extraction, and the ethyl acetate layer is purified by chromatography (silica gel column chromatography and high performance liquid chromatography).The part of the rush used for extraction is not particularly limited; for example, the whole rush plant or the core of the rush may be used for extraction.

[0015] Dehydroefsol is a compound represented by the following structural formula:

[0016] [ka]

[0017] The dehydroefsol used in the composition of the present disclosure may be a synthetic product or may be obtained by extracting and purifying a product found in nature. It can be synthesized by known methods or methods easily derived from known methods. When obtained from nature, for example, dehydroefsol is known to exist in rush (wick grass) and can be obtained by extracting (and purifying, if necessary) from rush. More specifically, it can be obtained by extracting rush with water, ethanol, or a mixture thereof. A 40% to 60% aqueous ethanol solution is particularly preferred, with a 50% aqueous ethanol solution being particularly preferred. If necessary, the resulting extract may be further fractionated and / or purified. Fractionation can be performed, for example, by liquid-liquid partition extraction. The partition can be performed, for example, using water and ethyl acetate. Purification can be performed, for example, by high-performance liquid chromatography (HPLC). The part of the rush used for extraction is not particularly limited; for example, the whole plant or the core of the rush may be used for extraction. The use of rush core is particularly preferred because it contains a large amount of dehydroefsol.

[0018] The composition of the present disclosure is used to inhibit the degeneration of neurons (particularly brain neurons). In particular, it is preferably used to inhibit the degeneration of dopaminergic neurons. Furthermore, the degeneration of neurons encompasses all cases in which neurons are damaged, including, for example, neuronal loss and neuronal death.

[0019] Furthermore, as described above, since motor dysfunction occurs due to neuronal degeneration, it is expected that the composition of the present disclosure can suppress neuronal degeneration and thereby improve motor dysfunction. Therefore, the composition of the present disclosure can also be used to improve motor dysfunction caused by neuronal degeneration. In particular, it can be preferably used for the treatment of Parkinson's disease.

[0020] The subject for use of the composition of the present disclosure is preferably a subject suffering from neuronal degeneration (particularly dopaminergic neurons), and more preferably a subject suffering from motor dysfunction. The subject is not limited to humans. For example, the composition can also be used in non-human mammals. Mammals kept as pets or livestock are particularly preferred. Specific examples include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, and llamas.

[0021] The biological extract (preferably rush extract) itself is also included in the composition of the present disclosure. In other words, the biological extract containing efsol and / or dehydroefsol (and if necessary, purified ones, or ones further blended with other components if necessary) is also included in the composition of the present disclosure as long as it is effective.

[0022] Furthermore, the composition of the present disclosure is preferably an oral composition or an injectable composition, although not particularly limited thereto. When the composition is an injectable composition, its administration method can be exemplified by intravascular administration (e.g., intravenous administration or intraarterial administration) or direct administration to the brain or spinal cord. When the composition is an oral composition, it can be used, for example, as an oral pharmaceutical composition or a food composition, as described below.

[0023] Efsol and dehydroefsol are compounds with a phenanthrene skeleton, and it has been reported that morphine, a compound with a phenanthrene skeleton, passes through the blood-brain barrier, and that dehydroefsol may also pass through the blood-brain barrier (Non-Patent Document 3, supra). Furthermore, efsol is a fat-soluble component that is insoluble in water, but its fat-solubility is not as high as that of fatty acids, and it can be said to have suitable fat-solubility for passing through the blood-brain barrier. From the above, it is thought that efsol and / or dehydroefsol can reach the brain and exert their effects, for example, when administered orally or intravascularly.

[0024] The composition of the present disclosure can be preferably used as a pharmaceutical composition or a food composition. When used as a pharmaceutical composition, the composition (sometimes referred to as the "pharmaceutical composition of the present disclosure") may contain efsol and / or dehydroefsol (or a biological extract containing efsol and / or dehydroefsol) and, if necessary, pharmaceutically acceptable bases, carriers, additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavoring agents, odorants, surfactants, humectants, preservatives, pH adjusters, thickeners, etc.). Such bases, carriers, additives, etc. are specifically described, for example, in the Pharmaceutical Additives Dictionary 2016 (Yakuji Nippo Co., Ltd.), and those described therein can be used. The formulation form is also not particularly limited; the active ingredient and other ingredients can be mixed using conventional methods to prepare formulations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewable tablets, and soft tablets. For example, tablets can be produced by tableting. Either direct tableting, in which the mixed raw materials are directly tableted, or granule tableting, in which the mixed raw materials are granulated and then tableted, can be used. For example, capsules can be either soft capsules or hard capsules.

[0025] The amount of efsol and / or dehydroefsol in the pharmaceutical composition according to the present disclosure is not particularly limited as long as it exhibits the effect of preventing or improving memory impairment, and can be appropriately determined depending on the subject. It is preferably 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass. The lower limit may be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by mass.

[0026] The timing of administration of the pharmaceutical composition according to the present disclosure is not particularly limited, and can be appropriately selected taking into consideration, for example, the formulation, the age of the subject, the severity of the symptoms of the subject, etc. The administration form is not particularly limited, but oral administration and intravascular administration (intravenous administration, intraarterial administration) are preferred.

[0027] The dosage of the pharmaceutical composition according to the present disclosure can be appropriately selected depending on the age of the subject, the severity of the symptoms of the subject, and other conditions. It can be appropriately set based on the amount of efsol and / or dehydroefsol contained, as long as the effect is not impaired. Although not particularly limited, for example, the amount of efsol and / or dehydroefsol administered per day to an adult is preferably about 0.5 to 100 mg, more preferably about 1 to 50 mg, and even more preferably about 5 to 30 mg, about 6 to 24 mg, about 10 to 24 mg, or about 12 to 24 mg. It can be administered once a day or multiple times (preferably 2 to 3 times a day). In the case of non-human mammals, the dosage can also be appropriately set with reference to that for humans.

[0028] When the composition of the present disclosure is used as a food composition (e.g., a food or beverage or a food additive), the composition (hereinafter sometimes referred to as the "food composition of the present disclosure") may be blended with efsol and / or dehydroefsol (or a biological extract containing efsol and / or dehydroefsol), as well as hygienically acceptable bases, carriers, additives, and other ingredients and materials that can be used in foods and beverages. Examples of such food compositions include processed foods, beverages, health foods, functional foods, dietary supplements, supplements, health foods, foods for specified health uses, nutritional functional foods, foods with functional claims, and foods for patients (hospital foods, sick foods, nursing care foods, etc.) that contain efsol and / or dehydroefsol for preventing or improving memory impairment. Without being particularly limited, when the efsol and / or dehydroefsol blended in the food composition is a biological extract (preferably rush extract), the food composition may be, for example, a processed food, health food, nutritional functional foods, foods for specified health uses, supplements, or foods for patients that contain the extract. Furthermore, efsol and / or dehydroefsol may be powdered and added to various foods and beverages such as beverages (juice, etc.), confectioneries, breads, soups (including powdered soups, etc.), and processed foods. Hospital food is food provided when a patient is admitted to a hospital, sick food is food for sick people, and nursing care food is food for people receiving care. The food composition according to the present disclosure can be preferably used as hospital food, sick food, or nursing care food, particularly for patients who are hospitalized, recuperating at home, or receiving care and who are suffering from memory disorders.

[0029] When preparing the food composition according to the present disclosure as a health food (nutritional functional food, food for specified health uses, etc.) or supplement, it is preferable to prepare it in the form of, for example, granules, capsules, tablets (including chewable tablets), beverages (drinkable preparations), etc. to facilitate continuous intake, and capsules, tablets, and pills are particularly preferred from the standpoint of ease of intake. However, the present disclosure is not particularly limited to these. The food composition according to the present disclosure in the form of granules, capsules, tablets, etc. can be appropriately prepared according to conventional methods using pharmaceutically and / or food hygienically acceptable carriers, etc. Furthermore, even when preparing it in other forms, conventional methods can be followed.

[0030] The amount of efsol and / or dehydroefsol in the food composition according to the present disclosure is not particularly limited as long as the effect can be exhibited. It is preferably 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass. The lower limit may be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by mass.

[0031] The intake amount, intake target, etc. of the food composition according to the present disclosure are preferably the same as, for example, the pharmaceutical composition according to the present disclosure described above.

[0032] The present disclosure also encompasses a method for producing a composition for inhibiting neuronal degeneration, which comprises a step of compounding efsol and / or dehydroefsol, as well as a method for inhibiting the degeneration of neurons (particularly brain neurons, particularly dopaminergic neurons) by administering efsol and / or dehydroefsol (preferably the above-mentioned composition for inhibiting neuronal degeneration containing efsol and / or dehydroefsol), and thereby a method for improving motor dysfunction. The various conditions for these methods are as described above.

[0033] It should be noted that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0034] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0035] Hereinafter, embodiments of the present disclosure will be described in more detail with reference to examples, but the embodiments of the present disclosure are not limited to the following examples. Wistar rats were purchased from Japan SLC Co., Ltd.

[0036] (i) Saline containing 800 μM 6-hydroxydopamine (6-OHDA; purchased from SIGMA-ALDRICH), (ii) saline containing 800 μM 6-OHDA and 200 μM efsol, or (iii) saline containing 800 μM 6-OHDA and 200 μM dehydroefsol were prepared and used in the following studies.

[0037] (Experiment 1) 6-OHDA (800 μM) was co-administered with either efsol (200 μM) or dehydroefsol (200 μM) in a 1 μL volume into the substantia nigra compacta of a unilateral rat brain over a 50-minute period. Administration was performed via a drilled hole in the skull of an anesthetized rat and an injection cannula (inner diameter: 0.15 mm, outer diameter: 0.35 mm) inserted into the substantia nigra compacta. Ten minutes after administration, intracellular ROS levels in the substantia nigra were measured using APF (aminophenyl fluorescein) fluorescence. Administration of 6-OHDA alone significantly increased intracellular ROS levels compared with saline (control), and this increase was suppressed by co-administration of efsol or dehydroefsol (Figure 1). AFP exhibits strong fluorescence upon reaction with activated enzymes. The graph in Figure 1 represents the relative fluorescence intensity of the area enclosed by the white dotted line in the micrograph. The area surrounded by the white dotted line is the “substantia nigra pars compacta.” This also applies to the following figures.

[0038] (Experiment 1-2) As in Experiment 1, 1 μL of 6-OHDA (800 μM) was administered to the substantia nigra compacta of rats unilaterally over a 50-minute period, along with either efsol (200 μM) or dehydroefsol (200 μM). Ten minutes after administration, the intracellular hydrogen peroxide levels in the substantia nigra were measured using HYDROP fluorescence. Administration of 6-OHDA alone significantly increased intracellular hydrogen peroxide levels compared with administration of saline (control), and this increase was suppressed by simultaneous administration of efsol or dehydroefsol (Figure 2). HYDROP is a fluorescent probe that specifically detects H2O2 (hydrogen peroxide).

[0039] (Experiment 2) Hydrogen peroxide-induced Zn in the substantia nigra 2+ As in Experiment 1, 1 μL of 6-OHDA (800 μM) was administered together with efsol (200 μM) or dehydroefsol (200 μM) into the substantia nigra pars compacta of rats over a 50-minute period. Ten minutes after the end of administration, 6-OHDA administration alone increased Zn levels in the substantia nigra. 2+The zinc ion level was significantly increased, but the increase was completely suppressed by simultaneous administration of efsol or dehydroefsol (Fig. 3). 2+ The amount was measured.

[0040] (Experiment 2-2) Furthermore, the effects of efsol or dehydroefsol on neuronal cell death were examined by tyrosine hydroxylase (TH) immunostaining. As in Experiment 1, 1 μL of 6-OHDA (800 μM) was administered unilaterally to the substantia nigra pars compacta of rats together with efsol (200 μM) or dehydroefsol (200 μM) for 50 minutes. Two weeks after the end of administration, rats were anesthetized, perfused with ice-cold 4% paraformaldehyde, and their brains were removed. Slices were prepared from the fixed brains at -20°C, and the slices were treated with an anti-tyrosine hydroxylase antibody (Abcam) and an Alexa Fluor 633 goat anti-rabbit secondary antibody (ThermoFisher) and immunostained for tyrosine hydroxylase (TH), a dopaminergic neuronal marker. Administration of 6-OHDA alone reduced the number of TH-positive cells in the substantia nigra pars compacta to approximately 50%, and dopaminergic neurodegeneration was observed in approximately half of the cells. This neurodegeneration was completely suppressed by co-administration of efsol or dehydroefsol (Figure 4).

[0041] From the above results, it was inferred that efsole and dehydroefsole are sufficiently effective (capable of improving movement disorders) even in experimental model animals that exhibit movement disorders.

[0042] The efsol and dehydroefsol used were obtained by further purifying a 50% ethanol aqueous extract of rush (whole plant or core). Figure 5 shows the chromatograms obtained by HPLC analysis of a 50% ethanol aqueous extract of whole rush or a 50% ethanol aqueous extract of rush core. The extraction, fractionation, and purification of rush core are described in detail below. Five kilograms of commercially available wick (dried stem pith of rush, a member of the Juncaceae family) was extracted twice by refluxing in a 20-fold volume of 50% aqueous ethanol at 80°C for two hours, followed by filtration. The filtrate was concentrated under reduced pressure, and 1.3 liters of distilled water was added to the concentrate, forming a suspension. 1.3 liters of ethyl acetate was added, and liquid-liquid partition extraction was performed using a separatory funnel. This process was repeated three times, resulting in separation into an aqueous layer and an ethyl acetate layer. The ethyl acetate was evaporated under reduced pressure, followed by drying, yielding 26.2 g of ethyl acetate extract. This ethyl acetate extract was fractionated into 13 fractions, A to M, using silica gel column chromatography (510 mL) (eluent: chloroform containing 2% methanol to chloroform containing 40% methanol). Fractions F and G were purified by preparative HPLC to obtain Compound 1 (0.218 g) and Compound 2 (1.725 g). Compounds 1 and 2 were identified as efsole and dehydroefsole, respectively, based on NMR spectroscopy.

Claims

1. A composition for inhibiting dopamine neuron degeneration, comprising efsol and / or dehydroefsol.

2. The composition according to claim 1, which is used for a subject suffering from dopaminergic neuron degeneration.

3. The composition according to claim 1 or 2, which is for improving movement disorders caused by neuronal degeneration.

4. The composition according to any one of claims 1 to 3, which is for treating Parkinson's disease.

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