Composition for inhibiting amyloid beta toxicity

Abstract

To provide a novel method for suppressing the toxicity of amyloid beta protein.SOLUTION: A composition for suppressing amyloid beta toxicity comprises dehydroeffusol.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a composition for suppressing amyloid beta toxicity, etc. [Background technology]

[0002] Amyloid beta protein (sometimes referred to as "Aβ" in this specification) is one of the candidates for the causative agent of Alzheimer's disease. It is well known that Aβ aggregates and forms amyloid fibrils, which induces neuronal death, leading to Alzheimer's disease. It is also known that Aβ has the ability to self-aggregate, and that zinc ions promote this. [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 pit 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) Summary of the Invention [Problem to be solved by the invention]

[0004] The main cause of Aβ toxicity is thought to be zinc ions themselves. Even if the self-aggregation ability of Aβ is not inhibited, by suppressing the toxicity of zinc ions, which are thought to be the main cause of toxicity, the result is that Aβ (especially Aβ 1-42 ) is believed to be able to suppress the neurotoxicity of Aβ (especially Aβ 1-42 Although Aβ is considered to be one of the causes of Alzheimer's disease, its toxicity is due to zinc toxicity, and it is thought that zinc promotes the entry of Aβ into neurons, ultimately resulting in zinc toxicity within the neurons. Therefore, it is possible that neurotoxicity can be suppressed by reducing the amount of zinc ions within neurons.

[0005] Therefore, the present inventors considered the possibility that the neurotoxicity of Aβ could be suppressed by reducing the amount of zinc ions in nerve cells in the presence of Aβ, and proceeded with their investigations. [Means for solving the problem]

[0006] The present inventors found that dehydroefsol may be able to reduce the amount of zinc ions in nerve cells, 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 suppressing amyloid beta toxicity containing dehydroefsol. Section 2. A composition for inhibiting hippocampal neuronal cell death, comprising dehydroefsol. Section 3. A composition for inducing metallothionein in nerve cells, comprising dehydroefsol. Section 4. The composition of claim 3, for a subject having an intracellular zinc ion concentration at which zinc ions can be released from amyloid beta aggregates. Section 5. The composition according to any one of claims 1 to 4, for a subject having amyloid beta aggregates in the hippocampus. Effect of the Invention

[0008] The present disclosure provides a method that is useful for reducing the amount of zinc ions in neuronal cells and suppressing neuronal toxicity in the presence of Aβ. [Brief description of the drawings]

[0009] [Figure 1] The results of investigating neuronal cell death in the dentate gyrus of the hippocampus when Aβ1-42 was administered intravenously to mice are shown below. It can be seen that dehydroefsol suppressed the cell death marker PI in the dentate gyrus of the hippocampus, which was increased by human Aβ1-42, to the control level. [Diagram 2] The results of investigating neuronal cell death in the dentate gyrus of the hippocampus when Aβ1-42 was administered intracerebroventricularly in mice are shown below. It can be seen that dehydroefsol suppressed the neurodegeneration marker FJB in the dentate gyrus of the hippocampus, which was increased by human Aβ1-42, to the control level. [Diagram 3] The results of immunostaining the dentate gyrus of the hippocampus with anti-Aβ antibody after Aβ1-42 was administered into the lateral ventricle of mice are shown. It can be seen that dehydroefsol does not inhibit Aβ uptake into neurons. [Figure 4] The results show that zinc ions in the dentate gyrus of the hippocampus when Aβ1-42 was administered into the lateral ventricle of mice were detected using a fluorescent probe for detecting zinc ions, ZnAF-2DA (Goryo Chemical Co., Ltd.). It can be seen that dehydroefsol inhibits the Aβ1-42-induced increase in zinc ions in nerve cells. [Diagram 5] The results of immunostaining with anti-metallothionein antibody in the hippocampal dentate gyrus of mice administered Aβ1-42 into the lateral ventricle are shown. It can be seen that dehydroefsol increases metallothionein expression in neurons. [Figure 6]This is a model diagram showing that dehydroefsol increases metallothionein (MT) expression in neurons and suppresses Aβ1-42-induced increase in zinc ions (Zn2+). [Figure 7] 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 PREFERRED EMBODIMENTS

[0010] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes a composition containing dehydroefsole, and its uses and users, but is not limited thereto, and the present disclosure includes all that is disclosed in the present specification and that can be recognized by a person skilled in the art.

[0011] The composition included in the present disclosure (hereinafter, sometimes referred to as the "composition of the present disclosure") contains dehydroefsole. Dehydroefsole is a compound represented by the following structural formula:

[0012] [ka]

[0013] Dehydroefsol may be a synthetic product or may be obtained by extracting and purifying a product that exists in nature. It can be obtained by synthesis using a known method or a method that can be easily conceived from a known method. When it is obtained from nature, for example, it is known that dehydroefsol exists in rush (wick grass), and it can be obtained by extracting (and purifying as necessary) from rush. More specifically, it can be obtained by extracting, for example, rush with water, ethanol, or a mixture thereof. As the extract, a 40 to 60% ethanol aqueous solution is particularly preferred, and a 50% ethanol aqueous solution is particularly preferred. Furthermore, if necessary, the obtained 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. Furthermore, purification can be performed, for example, by high performance liquid chromatography (HPLC). Furthermore, the part of the rush used for extraction is not particularly limited, and for example, the whole plant of the rush may be used for extraction, or the core of the rush may be used for extraction. Since a large amount of dehydroefsol exists in the core of the rush, it is particularly preferred to use the core of the rush.

[0014] It is believed that Aβ aggregates and forms amyloid fibrils, which cause neuronal death. Furthermore, the aggregation of Aβ is thought to be mediated by zinc ions (Zn 2+ ), the present inventors have discovered that dehydroefsol induces metallothionein in neurons (preferably hippocampal neurons), thereby reducing the zinc ion concentration within neurons and suppressing zinc toxicity (and thus suppressing Aβ toxicity).

[0015] For this reason, the composition of the present disclosure is particularly useful, for example, for suppressing amyloid beta toxicity, for suppressing neuronal cell (particularly hippocampal neuronal cell) death (particularly for suppressing neuronal cell death in the presence of Aβ), or for inducing metallothionein (particularly for inducing metallothionein in neuronal cells, etc.). Although not particularly limited, when used for inducing metallothionein in neuronal cells, it is preferable to use it for a subject whose intracellular zinc ion concentration is equal to or higher than the concentration that induces amyloid beta-induced zinc ion toxicity. In addition, zinc ions are thought to promote the formation of Aβ aggregates, but zinc ions are released from Aβ aggregates taken up into cells and neurons, and it is thought that the zinc ions cause neuronal toxicity (see FIG. 6). For this reason, when used for inducing metallothionein in neuronal cells, it is preferable that the zinc ion concentration in the neuronal cells is a concentration at which zinc ions can be released from the taken-up Aβ aggregates (i.e., the zinc ion concentration in the neuronal cells is relatively low). Aβ in the Aβ aggregates is Aβ 1-42 In addition, the Aβ aggregate is preferably Aβ 1-42 and zinc ions. As the nerve cells, hippocampal nerve cells are more preferable.

[0016] Although not wishing to be bound by theory, the extracellular zinc ion concentration is estimated to be about 10 nM, and Aβ levels are about 100 to 500 pM. 1-42 In the presence of Zn-Aβ 1-42 Aggregates can be formed and rapidly taken up by hippocampal neurons. Since the zinc ion concentration in cells is usually low (approximately 100 nM), Aβ 1-42 From Zn 2+ is released and becomes toxic.

[0017] Metallothionein is a protein that is rich in cysteine ​​thiol groups and binds to metals such as zinc via the thiol groups. It is believed to play a role in maintaining zinc homeostasis, removing heavy metal toxicity, and eliminating radicals derived from reactive oxygen species.

[0018] In addition, the subject for which the composition of the present disclosure is used is not particularly limited, but is preferably a subject having amyloid beta aggregates (and thus amyloid fibers) in the hippocampus, such as Alzheimer's disease patients.

[0019] The composition of the present disclosure can be used not only for humans but also for non-human mammals. In particular, mammals kept as pets and livestock are preferred. Specific examples include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

[0020] The composition of the present disclosure is preferably used, for example, in the pharmaceutical and food fields. As described in detail below, the composition may consist of only dehydroefsole, or may contain dehydroefsole and other components (various bases, carriers, additives, etc.). In addition, the biological extract (preferably rush extract) itself is also included in the composition. In other words, the biological extract containing dehydroefsole (and further containing other components as necessary) is also included in the composition containing dehydroefsole of the present disclosure. Note that, since a composition is usually composed of multiple components, it may not be appropriate to refer to the composition of the present disclosure as being composed of only dehydroefsole, but in this specification, even if the composition consists of only dehydroefsole, it is referred to as a composition. (In other words, the term "composition" is synonymous with the term "agent.")

[0021] When the composition of the present disclosure is used in the pharmaceutical field (including drugs and quasi-drugs), the composition (hereinafter sometimes referred to as the "pharmaceutical composition of the present disclosure") may consist of dehydroefsole alone, or may be a composition containing other ingredients. For example, in the pharmaceutical composition of the present disclosure, the active ingredient dehydroefsole may be mixed with pharma- ceutical acceptable bases, carriers, additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavorings, odorants, surfactants, moisturizers, preservatives, pH adjusters, thickeners, etc.) as necessary. Such bases, carriers, additives, etc. are specifically described, for example, in the Pharmaceutical Additives Dictionary 2016 (Yakuji Nipposha Co., Ltd.), and for example, those described therein can be used. The formulation form is not particularly limited, and the active ingredient and other ingredients can be mixed by a conventional method to prepare a formulation such as a tablet, coated tablet, powder, granule, fine granule, capsule, pill, liquid, suspension, emulsion, jelly, chewable agent, soft tablet, etc. For example, the tablet can be produced by a tableting method. Either a direct tableting method in which the mixed raw materials are directly tableted, or a granule tableting method in which the mixed raw materials are granulated and then tableted, can be used. For example, in the case of a capsule, either a soft capsule or a hard capsule can be used.

[0022] The amount of dehydroefsole in the pharmaceutical composition according to the present disclosure is not particularly limited as long as the effect 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, and even more preferably 0.05 to 80% by mass. The lower limit may be about 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.

[0023] The administration time of the pharmaceutical composition according to the present disclosure is not particularly limited, and can be appropriately selected in consideration of, for example, the formulation form, 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, and oral administration is more preferred.

[0024] 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 symptoms of the subject, and other conditions. In particular, the amount of dehydroefsole contained can be appropriately set within a range that does not impair the effects of the present disclosure. Although not particularly limited, for example, the amount of dehydroefsole administered per day to an adult is preferably about 1 to 50 mg, more preferably about 5 to 30 mg, about 6 to 24 mg, about 10 to 24 mg, or about 12 to 24 mg. In addition, it can be administered once a day or in multiple divided doses (preferably 2 to 3 times). In the case of a non-human mammal, the dosage can be appropriately set with reference to the case of the person.

[0025] When the composition of the present disclosure is used as a food composition (e.g., food and beverage products or food additives), the composition (hereinafter sometimes referred to as the "food composition of the present disclosure") is a composition in which dehydroefsol and a base, carrier, additive, and other ingredients and materials that can be used as food and beverage products that are acceptable from the viewpoint of food hygiene are appropriately blended. Examples of such food compositions that contain dehydroefsol include processed foods, beverages, health foods, functional foods, nutritional supplements, supplements, health functional foods, foods for specified health uses, nutritional functional foods, foods with functional claims, and foods for sick people (hospital foods, sick people's foods, or nursing care foods, etc.) for enhancing learning and memory ability. Although not particularly limited, when the dehydroefsol blended in the food composition is a biological extract (preferably a rush grass extract), the food composition may be, for example, a processed food, health food, nutritional functional foods, foods for specified health uses, supplements, foods for sick people, etc., in which the extract is blended. In addition, dehydroefsol may be powdered and added to various foods and beverages such as beverages (juices, etc.), confectioneries, breads, soups (including powdered soups, etc.), and processed foods. The food composition according to the present disclosure can also be used preventively when Aβ aggregation (particularly Aβ aggregation in the hippocampus) is predicted to occur in the near future. In addition, hospital food is food provided when hospitalized, sick food is food for sick people, and nursing 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 food for patients who are hospitalized, recuperating at home, or receiving care and who need to prevent and / or inhibit Aβ aggregation.

[0026] 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 agents, etc.), beverages (drinks), etc., so that it is easy to take it continuously, and among them, capsules, tablets, and tablets are preferable from the viewpoint of ease of intake. 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 pharmaceutical and / or food hygienically acceptable carriers, etc. Furthermore, even when preparing it in other forms, it is sufficient to follow conventional methods.

[0027] The amount of 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 about 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.

[0028] The subjects of ingestion of the food composition according to the present disclosure are preferably the same as those of the pharmaceutical composition according to the present disclosure described above.

[0029] The present disclosure also includes a method for producing the composition of the present disclosure, which includes a step of compounding dehydroefsole, as well as a method for suppressing amyloid beta toxicity, a method for suppressing hippocampal neuronal cell death, a method for inducing metallothionein in the hippocampus, etc., by administering dehydroefsole (preferably the composition of the present disclosure containing the above-mentioned dehydroefsole). The various conditions in these methods are as described above.

[0030] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes all arbitrary combinations of the constituent features described in this specification.

[0031] In addition, 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 included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0032] The present invention will be described in more detail below, but the present invention is not limited to the following examples.

[0033] In the following study, human amyloid beta protein (1-42) was purchased and used as the amyloid beta protein (Synthetic human Aβ1-42, ChinaPeptides, Shanghai, China). The amino acid sequence of the human amyloid beta protein is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. The human amyloid beta protein was also referred to as Aβ 1-42 It is sometimes written as:

[0034] Aβ in the lateral ventricles of mice 1-42 (25μM, 20μl) or saline as a control was administered. Two weeks later, frozen brain sections or whole brain slices were prepared, and hippocampal dentate gyrus granule cell death was evaluated using a confocal laser microscope with Fluoro-Jade-B (FJB), a neurodegeneration marker, and propidium iodide (PI), a cell death marker. Mice were orally administered dehydroefsol (15mg / kg, 10mL / kg / day) once a day for a total of six times, and Aβ was injected into the lateral ventricle.1-42 Or saline was administered to evaluate Aβ 1-42 Neurodegeneration and cell death in the hippocampal dentate gyrus induced by the treatment were suppressed to control levels (Fig. 1 and Fig. 2; in these figures, vehicle refers to 0.5% carboxymethyl cellulose sodium).

[0035] Pretreatment with dehydroefsol did not inhibit Aβ uptake into neurons as assessed by anti-Aβ antibody (Figure 3). 1-42 Induced intracellular Zn 2+ The increase in the zinc ion level was suppressed to the control level (Figure 4). Note that Figure 4 is a photograph taken using ZnAF-2DA (Goryo Chemical Co., Ltd.), a fluorescent probe for detecting zinc ions.

[0036] Next, we investigated the induction of metallothionein (MT), an intracellular zinc-binding protein, by dehydroefsol. Immunostaining of MT revealed that MT levels in the hippocampal dentate gyrus were significantly increased by six doses of dehydroefsol (Figure 5). MT induced by dehydroefsol was not associated with Aβ expression. 1-42 Induced intracellular Zn 2+ Suppresses the increase in Aβ levels 1-42 It was found that it prevented induced hippocampal neuronal cell death (Figure 6).

[0037] The dehydroefsol used was obtained by further purifying the 50% ethanol aqueous solution extract of rush grass (whole plant or core). Figure 7 shows the chromatogram obtained by HPLC analysis of the 50% ethanol aqueous solution extract of the whole plant or the 50% ethanol aqueous solution extract of the rush grass core. The extraction, fractionation and purification of the rush grass core are explained in detail below. 5 kg of commercially available wick grass (dried pith of the stem of rush grass of the Juncaceae family) was extracted twice by refluxing at 80°C for 2 hours in 20 times the amount of 50% aqueous ethanol and filtered. The filtrate was concentrated under reduced pressure, 1.3 liters of distilled water was added to the concentrated liquid to suspend it, and 1.3 liters of ethyl acetate was added and liquid-liquid partition extraction was performed using a separatory funnel. This was repeated three times, and the water layer and ethyl acetate layer were separated. The 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, 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, by measuring the NMR spectrum.

Claims

1. A composition containing dehydroefsol for use in a subject having an intracellular zinc ion concentration of 100 nM or less, the concentration at which zinc ions can be released from amyloid beta aggregates, for inducing metallothionein in the neurons of the subject.

2. The composition of claim 1 for a subject having amyloid beta aggregates in the hippocampus.

3. The composition according to claim 1 or 2, for suppressing hippocampal neurodegeneration and / or hippocampal neuronal cell death.

4. The composition according to claim 1 or 2, for suppressing the toxicity of zinc ions released from amyloid beta aggregates in neuronal cells.

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