JP2023019487A - Composition for inhibiting neuron degeneration - Google Patents

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 The present disclosure relates to compositions for suppressing nerve cell degeneration and the like. It should be noted that the contents of all documents mentioned herein are hereby incorporated by reference.

It is known that degeneration of nerve cells causes dyskinesia. For example, Parkinson's disease is a progressive neurodegenerative disease characterized by degeneration of the mesolanistriatal dopaminergic neurons, resulting in motor dysfunction such as limb tremors and muscle stiffness. However, the cause of the degeneration and dropout has not been elucidated. Since dopamine is unstable and produces reactive oxygen species through autoxidation, it is believed that reactive oxygen species are involved in the degeneration of dopaminergic neurons.

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) Wang YG, Wang YL, Zhai HF et al. : Phenanthrens from juncus effusus with anxiolytic and sedative activities. Nat Prod Res. 26: 1234-1239 (2012) 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) Haruna Tamano, Ryusuke Nishio, Hiroki Morioka, Atsushi Takeda: Mol. Neurobiol., 56, 435-443 (2019). Haruna Tamano, Hiroki Morioka, Ryusuke Nishio, Azusa Takeuchi, Atsushi Takeda: Mol. Neurobiol., 56, 4539-4548 (2019). Haruna Tamano, Ryusuke Nishio, Hiroki, Morioka, Ryo Furuhata, Yuuma Komata, Atsushi Takeda: Mol. Neurobiol., 56, 7789-7799 (2019).

The present inventors hypothesized that disruption of intracellular Zn ²⁺ homeostasis due to the production of hydrogen peroxide, which is reactive oxygen species (ROS), is the cause of dopaminergic neurodegeneration. - has been validated using a Parkinson's disease model in which 6-hydroxydopamine (6-OHDA) was administered to the unilateral substantia nigra compacta in rats. It has been reported that 6-OHDA excessively increases Zn2+ levels in dopaminergic neurons in the substantia nigra pars compacta, induces cell death, and causes movement disorders (Non-Patent Documents 4-6).

Therefore, the present inventors believed that the development of a means for suppressing neuronal degeneration would enable the treatment of conditions caused by such neuronal degeneration, and aimed to develop such a means. I considered it.

The present inventors have found that efusol and dehydroefsol can exert an inhibitory effect on nerve cell degeneration, and made further improvements.

The disclosure includes, for example, subject matter described in the following sections.
Section 1.
A composition for inhibiting nerve cell degeneration, containing efsol and/or dehydroefsol.
Section 2.
2. The composition of claim 1, wherein the neurons are dopaminergic neurons.
Item 3.
3. The composition of claim 2 for use in subjects suffering from dopaminergic neuronal degeneration.
Section 4.
The composition according to any one of claims 1 to 3, which is used for improving movement disorders caused by neuronal degeneration.
Item 5.
The composition according to any one of claims 1 to 4, which is used for treating Parkinson's disease.

Nerve cell degeneration can be suppressed by the compositions, methods, and the like included in the present disclosure. This is expected to help recover from disease states (such as movement disorders) caused by neuronal degeneration.

6 shows the results of measuring the amount of intracellular reactive oxygen (ROS) with a fluorescent reagent when 6-OHDA used for creating a Parkinson's disease model was administered together with efsol or dehydroefsol to the unilateral substantia nigra pars compacta of rats. 6 shows the results of measuring intracellular hydrogen peroxide levels with 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 unilateral substantia nigra pars compacta of rats. 6 shows the results of measuring intracellular Zn ²⁺ levels with a fluorescent reagent when 6-OHDA used for Parkinson's disease model preparation was administered together with efsol or dehydroefsol to the unilateral substantia nigra pars compacta of rats. Tyrosine hydroxylase (TH) immunostaining results of degenerated nerve cell mass when 6-OHDA used for Parkinson's disease model creation was administered together with efsol or dehydroefsol to the unilateral substantia nigra pars compacta of rats. indicates A chromatogram obtained by HPLC analysis of the rush whole grass 50% ethanol aqueous solution extract or the rush core 50% ethanol aqueous solution extract is shown.

Each embodiment included in the present disclosure will be described in further detail below. The present disclosure preferably includes, but is not limited to, compositions for suppressing nerve cell degeneration and the like, and includes everything disclosed herein and recognized by a person skilled in the art.

The nerve cell degeneration suppression composition included in the present disclosure contains at least one compound selected from the group consisting of efsol and dehydroefsol. Such compositions are sometimes referred to as compositions of the present disclosure.

Efsol is a compound represented by the following structural formula.

Efsol used in the composition of the present disclosure may be a synthetic product or an extracted and purified product existing in nature. It can be synthesized by a known method or a method that can be easily conceived from a known method. Moreover, a commercial item can also be purchased and used. When obtained from the natural world, for example, Efsol is known to exist in rush (wicker plant), and can be obtained by extracting and, if necessary, purifying from rush. For example, it can be obtained by extraction with water, methanol, ethanol, a mixture of at least two of these, or the like, and purification if necessary. The extract is not particularly limited, but methanol or a 40-60% ethanol aqueous solution is preferable. In addition, the obtained extract may be further fractionated and/or purified as necessary. Fractionation can be performed, for example, by liquid-liquid partition extraction. The distribution can be performed using, for example, water, n-hexane, ethyl acetate, and the like. Further, purification can be performed by, for example, silica gel column chromatography and/or high performance liquid chromatography (HPLC). More specifically, for example, the rush methanol extract is concentrated under reduced pressure, liquid-liquid partition extraction is performed with water and n-hexane, ethyl acetate is further added to the obtained aqueous layer, and liquid-liquid partition extraction is performed. and purifying the ethyl acetate layer by chromatography (silica gel column chromatography and high performance liquid chromatography). The portion of rush to be subjected to extraction is not particularly limited, and for example, the whole rush may be subjected to extraction, or the core of rush may be subjected to extraction.

Dehydroefsol is a compound represented by the following structural formula.

The dehydroefsol used in the composition of the present disclosure may be a synthetic product or an extracted and purified product found in nature. It can be synthesized by a known method or a method that can be easily conceived from a known method. When obtained from the natural world, for example, dehydroefsol is known to exist in rush (wicker plant), and can be obtained by extraction (and, if necessary, purification) from rush. More specifically, it can be obtained by extracting, for example, rush with water, ethanol, or a mixture thereof. As the extraction liquid, a 40 to 60% ethanol aqueous solution is particularly preferable, and a 50% ethanol aqueous solution is particularly preferable. In addition, the obtained extract may be further fractionated and/or purified as necessary. Fractionation can be performed, for example, by liquid-liquid partition extraction. The partitioning can be done, for example, with water and ethyl acetate. Alternatively, purification can be performed, for example, by high performance liquid chromatography (HPLC). The part of the rush to be extracted is not particularly limited, and for example, the whole rush or the core of the rush may be extracted. It is particularly preferable to use the core of rush because a large amount of dehydroefsol is present in the core of rush.

The composition of the present disclosure is used to suppress degeneration of nerve cells (especially brain nerve cells). Among others, it is preferably used for suppressing degeneration of dopaminergic neurons. Further, degeneration of nerve cells includes general damage to nerve cells, such as loss of nerve cells and death of nerve cells.

In addition, as described above, motor dysfunction is caused by degeneration of nerve cells. Therefore, it is expected that the composition of the present disclosure suppresses nerve cell degeneration, thereby improving motor dysfunction. Therefore, the composition of the present disclosure can also be used for improving movement disorders caused by neuronal degeneration. In particular, it can be preferably used for treatment of Parkinson's disease.

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

The biological extract (preferably rush extract) itself is also included in the composition of the present disclosure. In other words, as long as the biological extract containing Efsol and/or dehydroefsol (and optionally purified or optionally further blended with other ingredients) is effective, Included in the compositions of the present disclosure.

Also, the composition of the present disclosure is preferably an oral composition or an injectable composition, although not particularly limited. In the case of an injection composition, examples of administration methods include transvascular administration (eg, intravenous administration or transarterial administration) and direct administration to the brain or spinal cord. Moreover, when it is an oral composition, it can be used, for example, as an oral pharmaceutical composition or a food composition as described below.

Efsol and dehydroefsol are compounds having a phenanthrene skeleton, and it has been reported that morphine, a compound having a phenanthrene skeleton, passes through the blood-brain barrier, and dehydroefsol may also add to the blood-brain barrier. (Non-Patent Document 3 above). Furthermore, while Efsol is a fat-soluble component that does not dissolve in water, its fat-solubility is not as high as that of fatty acids, and it can be said that it has a fat-solubility suitable for passing through the blood-brain barrier. From the above, it is considered that efsol and/or dehydroefsol can reach the brain and exert its effect by, for example, oral administration or transvascular administration.

The composition of the present disclosure can preferably be used as a pharmaceutical composition or food composition. When used as a pharmaceutical composition, the composition (sometimes referred to as a "pharmaceutical composition according to the present disclosure") contains efsol and/or dehydroefsol (or a biological extract containing efsol and/or dehydroefsol) ), pharmaceutically acceptable bases, carriers, additives (for example, excipients, binders, disintegrants, lubricants, solvents, sweeteners, coloring agents, flavoring agents, flavoring agents, Surfactants, moisturizing agents, preservatives, pH adjusters, thickening agents, etc.) may be added. Such base materials, carriers, additives and the like are specifically described, for example, in Dictionary of Pharmaceutical Excipients 2016 (Yakuji Nippo Co., Ltd.), and for example, those described therein can be used. The formulation form is also not particularly limited, and the active ingredient and other ingredients are mixed by a conventional method, such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, and emulsions. , jellies, chewables, soft tablets and the like. For example, tablets may be manufactured by a tableting method. Either a direct compression method in which the mixed raw materials are tableted as they are, or a granule compression method in which the mixed raw materials are granulated and then tableted can be used. For example, capsules may be soft capsules or hard capsules.

The amount of Efsol and/or Dehydroefsol in the pharmaceutical composition according to the present disclosure is not particularly limited as long as the effect of preventing or improving memory impairment is exhibited, and can be appropriately set according to the subject. It is preferably 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, still more preferably 0.05 to 80% by mass. The lower limit may be approximately 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, or 80% by mass.

The timing of administration of the pharmaceutical composition according to the present disclosure is not particularly limited, and the timing of administration can be appropriately selected in consideration of, for example, the formulation form, the age of the subject of administration, the degree of symptoms of the subject of administration, and the like. The mode of administration is not particularly limited, but oral administration and transvascular administration (intravenous administration, transarterial administration) are preferred.

The dosage of the pharmaceutical composition according to the present disclosure can be appropriately selected according to the age of the subject of administration, the degree of symptoms of the subject of administration, other conditions, and the like. Based on the amount of Efsol and/or dehydroefsol contained in particular, it can be appropriately set within a range in which the effect is not impaired. Although not particularly limited, for example, the amount of efsol and/or dehydroefsol administered per adult per day is preferably about 0.5 to 100 mg, more preferably about 1 to 50 mg, about 5 to 30 mg, 6 About 24 mg, about 10 to 24 mg, or about 12 to 24 mg is more preferable. It can be administered once a day or in multiple doses (preferably 2 to 3 times). In the case of non-human mammals as well, the dosage can be appropriately set with reference to the case of the subject.

When the composition of the present disclosure is used as a food composition (for example, food and drink or food additives), the composition (hereinafter sometimes referred to as the "food composition according to the present disclosure") contains Efsol and / or Formulated with dehydroefsol (or bioextracts containing dehydroefsol and/or dehydroefsol), food hygienically acceptable bases, carriers, additives, and other ingredients and materials that can be used as foods and drinks. obtain. For example, processed foods, beverages, health foods, functional foods, dietary supplements, supplements, foods with health claims, foods for specified health uses, nutritional functional foods containing efsol and/or dehydroefsol for preventing or improving memory impairment , food with functional claims, or food for sick people (hospital food, sick food, nursing care food, etc.). Although not particularly limited, when the Efsol and/or dehydroefsol blended in the food composition is a biological extract (preferably rush extract), for example, processed foods containing the extract, health It may be a food, a food with nutrient function claims, a food for specified health uses, a supplement, a food for the sick, and the like. In addition, efsol and/or dehydroefsol can be powdered, for example, and used in various foods and drinks such as beverages (juices, etc.), confectionery, breads, soups (including powdered soups, etc.), and processed foods. It may be contained. Hospital food is food provided when a person is admitted to a hospital, sick food is food for a sick person, and nursing care food is food for a person being cared for. The food composition according to the present disclosure is particularly useful as hospital food, sick food, or nursing care food for patients who are hospitalized, treated at home, or who are receiving nursing care and have memory impairment. It can be preferably used.

When preparing the food composition according to the present disclosure as a health food (food with nutrient function claims, food for specified health use, etc.) or supplement, for example, granules, capsules, tablets (chewable formulations, etc.) are used to facilitate continuous intake. including), beverages (drinks), etc. Among them, capsules, tablets, and tablet forms are preferable from the viewpoint of ease of ingestion. However, it 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. Moreover, even when preparing other forms, conventional methods may be followed.

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, still more preferably 0.05 to 80% by mass. The lower limit may be approximately 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, or 80% by mass.

The amount of intake, target of intake, 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.

The present disclosure includes a method for producing a composition for suppressing nerve cell degeneration, including a step of blending efsol and/or dehydroefsol, and efsol and/or dehydroefsol (preferably the above-described efsol and/or dehydroefsol A composition for suppressing neuronal degeneration containing (a composition for suppressing neuronal degeneration containing Various conditions in these methods are as described above.

In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Also, the present disclosure encompasses any and all combinations of the constituent elements described herein.

Also, the various characteristics (property, structure, function, etc.) described for each of the embodiments of the disclosure described above may be combined in any way to identify subject matter encompassed by the disclosure. That is, the present disclosure encompasses all subject matter consisting of any and all possible combinations of the features described herein.

Hereinafter, the embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples. The rats used were Wistar rats purchased from Japan SLC Co., Ltd.

(i) saline containing 800 μM 6-hydroxydopamine (6-OHDA; purchased from SIGMA-ALDRICH), (ii) saline containing 800 μM 6-OHDA and 200 μM efusol, or (iii) 6-OHDA Physiological saline containing 800 μM and 200 μM dehydroefsol was prepared and used for the following studies.

(Experiment 1) 1 μL of 6-OHDA (800 μM) was administered to the unilateral substantia nigra pars compacta of the rat brain together with Efsol (200 μM) or Dehydroefsol (200 μM) for 50 minutes. Specifically, administration was performed by opening a hole in the skull of an anesthetized rat and inserting an injection cannula (inner diameter: 0.15 mm, outer diameter: 0.35 mm) into the substantia nigra pars compacta. Ten minutes after the end of the administration, the intracellular ROS level in the substantia nigra was measured by fluorescence of APF (Aminophenyl Fluorescein). It increased significantly, and the increase was suppressed by co-administration of efsol or dehydroefsol (Fig. 1). AFP shows strong fluorescence when it reacts with an active enzyme. The graph in FIG. 1 quantifies the fluorescent hair intensity (relative value) in the portion surrounded by the white dotted line in the micrograph. In addition, the portion surrounded by the white dotted line is the “substantia substantia nigra dense part”. The same applies to the following figures.

(Experiment 1-2) As in Experiment 1, 1 μL of 6-OHDA (800 μM) was administered to the unilateral substantia nigra compacta of rats together with efsol (200 μM) or dehydroefsol (200 μM) for 50 minutes. Ten minutes after the end of the administration, the substantia nigra cell hydrogen peroxide level was measured by HYDROP fluorescence. The increase was suppressed by co-administration of efsol or dehydroefsol (Fig. 2). HYDROP is a fluorescent probe that specifically detects H ₂ O ₂ (hydrogen peroxide).

(Experiment 2) The hydrogen peroxide-induced increase in substantia nigra Zn ²⁺ levels was measured. As in Experiment 1, 1 μL of 6-OHDA (800 μM) was administered to the unilateral substantia nigra pars compacta together with efsol (200 μM) or dehydroefsol (200 μM) for 50 minutes. Ten minutes after the end of the administration, 6-OHDA alone significantly increased the substantia nigra Zn ²⁺ level, but the increase was completely suppressed by the co-administration of efsol or dehydroefsol (Fig. 3). The amount of Zn ²⁺ was measured using ZnAF-2DA (Goryo Chemical Co., Ltd.), which is a fluorescent probe for detecting zinc ions.

(Experiment 2-2) Furthermore, the effect of efsol or dehydroefsol on nerve cell death was examined by tyrosine hydroxylase (TH) immunostaining. As in Experiment 1, 1 μL of 6-OHDA (800 μM) was administered to the unilateral substantia nigra pars compacta together with efsol (200 μM) or dehydroefsol (200 μM) for 50 minutes. Two weeks after the end of administration, the rats were anesthetized, perfused with ice-cold 4% paraformaldehyde, and the brain was excised. Slices were made from fixed brains at -20°, and slices were treated with anti-tyrosine hydroxylase antibody (Abcam), Alexa Fluor 633 goat anti-rabbit secondary antibody (ThermoFisher), to detect dopaminergic marker Tyrosine hydroxylase (TH ) was immunostained. 6-OHDA alone reduced TH-positive cells in the substantia nigra pars compacta to about 50%, and dopaminergic neurodegeneration was observed in about half of them. This neurodegeneration was completely suppressed by co-administration of efsol or dehydroefsol (Fig. 4).

From the above results, it was speculated that Efsol and Dehydroefsol are sufficiently effective (can improve movement disorders) even in experimental model animals in which movement disorders appear.

Efsol and dehydroefsol were obtained by further purifying a 50% ethanol aqueous solution extract of rush (whole plant or core). FIG. 5 shows a chromatogram obtained by HPLC analysis of the rush whole grass 50% ethanol aqueous solution extract or the rush core 50% ethanol aqueous solution extract. Extraction, fractionation and purification of rush core are described in detail below. 5 kg of commercially available wick (a product obtained by drying the pith of rush stems of the Rutaceae family) was extracted twice in 20 volumes of 50% hydrous ethanol under heating at 80° C. for 2 hours under reflux, followed by filtration. The filtrate was concentrated under reduced pressure, 1.3 liters of distilled water was added to the concentrate to suspend it, 1.3 liters of ethyl acetate was added, and liquid-liquid partition extraction was performed using a separating funnel. This was repeated three times and separated into an aqueous layer and an ethyl acetate layer. Ethyl acetate was distilled off under reduced pressure and dried to obtain 26.2 g of ethyl acetate extract. This ethyl acetate extract was fractionated into 13 fractions from A to M by silica gel column chromatography (510 ml) (eluate: chloroform containing 2% methanol to chloroform containing 40% methanol). Fractions F and G were purified by preparative HPLC to give compound 1 (0.218 grams), compound 2 (1.725 grams). Compounds 1 and 2 were identified as efsol and dehydroefsol, respectively, by measuring their NMR spectra.

Claims (5)

A composition for inhibiting nerve cell degeneration, containing efsol and/or dehydroefsol. 2. The composition of claim 1, wherein the neurons are dopaminergic neurons. 3. The composition of claim 2 for use in subjects suffering from dopaminergic neuronal degeneration. The composition according to any one of claims 1 to 3, which is used for improving movement disorders caused by neuronal degeneration. The composition according to any one of claims 1 to 4, which is used for treating Parkinson's disease.

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