An alpha-synuclein aggregation inhibitor containing roasted mucuna beans as the active ingredient.

Abstract

This invention provides an α-synuclein aggregation inhibitor with improved efficacy. [Solution] An α-synuclein agglutination inhibitor containing roasted mucuna beans roasted at 70-250°C for 3-60 minutes as the active ingredient. The α-synuclein agglutination inhibitor is provided as a food or beverage containing it.

Description

Technical Field

[0001] The present invention relates to an α-synuclein aggregation inhibitor containing roasted mukuna beans as an active ingredient.

Background Art

[0002] Lewy body disease is a group of diseases that develop as a result of Lewy bodies being formed in the nerve cells of the brain, causing damage to the nerve cells. There are Lewy body dementia and Parkinson's disease. The main constituent component of Lewy bodies is the protein α-synuclein (αSyn), which is considered to be one of the main causes of Lewy body disease. α-Synuclein is a protein with unknown function and exists by binding to the cell membrane or being free in the cytoplasm. It is also widely distributed in nerve cells. In the brains of patients, α-synuclein undergoes phosphorylation and ubiquitination and then fibroticates, aggregates, and accumulates. α-Synuclein accumulates in the nerve cells of the brain (due to aging, heredity, etc.). When accumulation occurs in the substantia nigra, nerve cell dropout occurs, leading to Parkinson's disease where dopamine is not produced sufficiently. When α-synuclein accumulates in the cerebral cortex, it leads to Lewy body dementia.

[0003] Lewy body dementia has a higher mortality rate and lower QOL (visual hallucinations, Parkinsonian symptoms) compared to Alzheimer's dementia. An antibody therapeutic agent, Lecanemab, has been developed for Alzheimer's disease, but there is currently no effective treatment method for Lewy body dementia. To prevent the onset and progression of Lewy body disease, it is sufficient if Lewy bodies are not formed, and it is considered that inhibiting the formation of aggregates of α-synuclein or removing aggregates is an effective countermeasure.

[0004] On the other hand, mucuna beans (also known as levodopa or L-3,4-dihydroxyphenylalanine) are important precursors to the neurotransmitters dopamine and adrenaline, and are known to be effective against human brain diseases such as Parkinson's disease. They contain a very high amount of L-DOPA, approximately 3-9% by dry weight. For this reason, there is a lot of activity in developing health foods and processed foods using mucuna beans (for example, Patent Documents 1-3, Non-Patent Document 1). While various processing conditions and product forms for mucuna beans have been investigated, the main objectives have been to investigate methods effective in preventing a decrease in levodopa content and preventing other diseases. There have been no studies that have focused on inhibiting or removing α-synuclein aggregate formation.

[0005] Furthermore, it has been reported that mucuna beans may have preventative effects against dementia and other related conditions. In the search for natural materials effective in treating Parkinson's disease, studies using the Parkinson's model nematode have reported that mucuna bean extract exhibits protective effects against dopamine neurodegeneration and inhibits α-synuclein aggregation (Non-Patent Literature 2). The mucuna bean extract used here is obtained by low-temperature immersion methanol extraction of dried, coarsely ground material. Studies using mice have reported that mucuna beans are effective in preventing Alzheimer's symptoms and the accumulation of beta-amyloid (Aβ) (Non-patent Literature 3). The mucuna beans used are those that have been soaked in distilled water, sterilized by high-pressure steam, freeze-dried, and then ground.

[0006] However, no reports have been found indicating that mucuna beans roasted under specific conditions inhibit α-synuclein amyloid aggregation. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2021-119754 [Patent Document 2] Japanese Patent Publication No. 2008-110922 [Patent Document 3] Japanese Patent Publication No. 2008-81478 [Non-patent literature]

[0008] [Non-Patent Document 1] Industrial Technology, No. 45 (2022), pp. 66-69 [Non-Patent Document 2] Journal of Ayurveda and Integrative Medicine 11 (2020) 440-447 [Non-Patent Document 3] Scientific Reports (2022) 12:996 (https: / / doi.org / 10.1038 / s41598-022-04777-z) [Overview of the project] [Problems that the invention aims to solve]

[0009] The problem that this invention aims to solve is to provide an α-synuclein aggregation inhibitor with improved efficacy. [Means for solving the problem]

[0010] The α-synuclein aggregation inhibitor according to the present invention contains roasted mucuna beans roasted at 70-250°C for 3-60 minutes as an active ingredient. [Effects of the Invention]

[0011] According to the present invention, an α-synuclein aggregation inhibitor with improved efficacy is provided. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the appearance of mucuna beans roasted at different temperatures (roasting time 15 minutes). (Example 1) [Figure 2] Figure 2 is a graph showing the relationship between roasting temperature and the inhibition rate of α-synuclein aggregation. (Example 1) [Figure 3] Figure 3 is a graph showing the relationship between roasting temperature and the amount of levodopa (roasting time: 15 minutes). (Example 3)

Mode for Carrying Out the Invention

[0013] The Mucuna bean used in the present invention is a vine leguminous plant that inhabits various parts of the world such as India, Asia, and Africa. In Japan, it is also called "Hashiou bean" (Phaseolus vulgaris), and it is said to have been cultivated as a food until the Edo period, but its cultivation has declined since then. In recent years, the health effects of Mucuna beans have been noticed, and cultivation in Japan has resumed. It is preferable to use Mucuna beans that have been harvested firmly after full ripening on the tree and naturally dried in a well-ventilated place. After natural drying, the beans are taken out of the pods and used as Mucuna beans for roasting. The moisture content of the dried Mucuna beans (dried Mucuna beans) is preferably 5 - 20%, more preferably 10 - 15%.

[0014] In the present invention, from the point of improving the action as an α-synuclein aggregation inhibitor, Mucuna beans are roasted at a temperature of 70 - 250°C for 3 - 60 minutes. Although the mechanism of action is not clear, it is推测 that roasting Mucuna beans increases some component that inhibits the aggregation of α-synuclein. In the present invention, α-synuclein aggregation inhibition includes inhibiting the formation of α-synuclein aggregates and removing α-synuclein aggregates. The roasting conditions are a temperature of 70 - 250°C for a time of 3 - 60 minutes, but the preferable roasting time changes depending on the temperature. The preferable roasting time at each temperature is (a) at a temperature of 70 - 100°C for 10 - 60 minutes, more preferably 30 - 50 minutes, (i) at a temperature of 100 - 140°C for 10 - 50 minutes, more preferably 25 - 40 minutes, (u) at a temperature of 140 - 180°C for 3 - 40 minutes, more preferably 10 - 35 minutes, (e) at a temperature of 180 - 220°C for 3 - 25 minutes, more preferably 10 - 20 minutes, (o) at a temperature of 220 - 250°C for 3 - 25 minutes, more preferably 5 - 10 minutes, It is as follows.

[0015] The roasting method is not particularly limited. For example, it can be carried out by heating the Mucuna beans using electric heat, hot air, a burner, microwaves, etc. from the outside. Also, the roasting machine to be used is not particularly limited. For example, a rotary fluidized bed type, a rotary drum type, a rotary kiln type, etc. can be used.

[0016] The α-synuclein aggregation inhibitor containing the roasted Mucuna beans thus obtained as an active ingredient can be provided as a food or drink. The form when provided as a food or drink is not limited. The roasted Mucuna beans may be powdered and provided as a dried food product sealed in a container, or may be provided by being contained as an additive in various foods and beverages such as tea. For example, when blending roasted Mucuna bean powder and Tamba black soybeans, a fragrant and flavorful tea is produced. By making it into a tea bag type, it can be easily drunk even when out. From the viewpoints of ease of intake as a food or drink and improving quality stability, the average particle size of the powder is preferably 300 μm or less, 80 μm or less, and the moisture is preferably 10% or less. From the viewpoint of the action of α-synuclein aggregation inhibition, the content of roasted Mucuna beans in the food or drink is preferably at least 0.1% or more, and can be 1% or more, 10% or more, 20% or more, 40% or more, 80% or more, 98% or more, 100%.

Example

[0017] Since a newly developed method was used for exploring the roasting conditions of Mucuna beans, the method will be described. The exploration of the roasting conditions was carried out by adding samples obtained from Mucuna beans with changed roasting conditions to clone cells that stably overexpress α-synuclein aggregation and measuring the degree of inhibition of α-synuclein aggregation (Roasting condition exploration method 1). Also, as a search method according thereto, a method using the size of the target peak in HPLC of each sample (Roasting condition exploration method 2) was also used in combination. Each method will be described below.

[0018] (Method 1 for exploring roasting conditions: A method using clonal cells that stably overexpress α-synuclein aggregation) The scheme for this method is as follows: Since this method observes aggregation behavior at the intracellular level within human nerve cells, it is considered a more realistic and effective evaluation system than the method using the Parkinson's model nematode described in Non-Patent Document 2 mentioned above. (1) Prepare a Mucuna bean MeOH extract at a concentration of 100 mg / mL (DMSO) and use it as a sample. (2) Establishment of a stable αSyn overexpression cell line: To overexpress αSyn in human neuroblast cells SH-SY5Y, the pCMV-SNCA plasmid vector was introduced into the cells by lipofection. Cells in which the target gene was incorporated into the genomic DNA and stably expressed were then selected using antibiotics. After obtaining several cell lines, the αSyn expression level was confirmed by Western blotting, and the cell line with the highest expression (SH clone) was used for subsequent experiments. (3) Evaluation of amyloid formation inhibitory ability: Intracellular aggregate formation was promoted by seeding, in which α-Syn amyloid fragments were added as seeds to the culture medium of SH clones. Three hours after seeding, mucuna bean extract dissolved in DMSO was added to the cell culture medium and cultured for 48 hours. After culturing, cells were fixed with 4% paraformaldehyde, and the aggregates were stained with the β-sheet structure-specific binding fluorescent dye FSB, and the nuclei were stained with the DNA-specific binding fluorescent dye DCS-1. Fluorescence staining images were obtained using a fluorescence microscope. The aggregate staining area was determined from the images using the image analysis software ImageJ, and the number of nuclei was directly counted to determine the number of cells. Finally, the aggregate area per cell was determined and the intracellular aggregation inhibitory ability of mucuna bean extract was evaluated by comparing it with the control (DMSO treatment). (4) The α-synuclein aggregation inhibition rate was calculated using the following formula (Formula 1).

[0019] α-synuclein aggregation inhibition rate = (AB) / A × 100 (Equation 1) However, in Equation 1, A is the aggregate area per control cell, and B is the aggregate area per cell treated with mucuna bean extract.

[0020] (Method 2 for exploring roasting conditions: Using the size of the target peak in HPLC) The scheme for this method is as follows: (1) After adjusting the Mucuna bean MeOH extract to 1 mg / mL, it was analyzed by HPLC under the following conditions. UV detector: wavelength 280 nm, Column: Nacalai Tesque COSMOSIL 5C18-AR-II Packed Column 4.6 mm I.D. × 250 mm, Mobile phase: (A) 0.1% acetic acid (B) MeOH, Column temperature: 40°C, Flow rate: 1 ml / min [Mobile phase conditions] min B concentration 0-5 2% 5-35 98% 35-39 98% 39-40 2% (2) Focus on HPLC peaks that increase during roasting and correlate with the data from "Method 1 for Exploring Roasting Conditions," and consider the magnitude of these peaks to correspond to the amount of α-synuclein aggregation inhibitors. Evaluation Criteria: ○: Large peak = Contains a large amount of inhibitory components. △: Moderately sized peak = Contains a certain amount of inhibitory components. ×: Small peak = Contains a small amount of inhibitory components.

[0021] (Measuring levodopa content by HPLC) The scheme for this method is as follows: (1) After adjusting the Mucuna bean MeOH extract to 1 mg / mL, it was analyzed by HPLC under the following conditions. Fluorescence detection wavelength: ex282 / em630nm, Column: Nacalai Tesque COSMOSIL 5C18-AR-II Packed Column 4.6 mm I.D. × 250 mm, Mobile phase: (A) 0.1% acetic acid (B) MeOH, Column temperature: 40℃, Flow rate: 1 ml / min [Mobile phase conditions] min B concentration 0-10 2% 10-15 98% 15-25 98% 25-35 2% (2) Levodopa purchased from Tokyo Chemical Industry Co., Ltd. was used as a standard sample for the calibration curve, and levodopa was quantified. [Examples]

[0022] (Examination using "Exploration Method for Roasting Conditions 1") The mucuna beans were harvested when fully ripe on the vine, naturally dried in a well-ventilated area, and then removed from their pods to be used as dried mucuna beans for roasting. The moisture content of the dried mucuna beans was 13.8%. 100g of dried mucuna beans were roasted using a roasting machine (KAKACOO KAKA-G400) at the specified temperature (roasting temperature) for a roasting time of 15 minutes. For each roasted mucuna bean, the α-synuclein aggregation inhibition rate was determined using "Exploratory Method for Roasting Conditions 1".

[0023] Figure 1 shows the appearance of mucuna beans roasted at different temperatures (15 minutes roasting time). The higher the roasting temperature, the darker the color becomes, indicating that the components of mucuna beans change during roasting. Table 1 shows the relationship between roasting temperature and the inhibition rate of α-synuclein aggregation. Figure 2 is a graph of this relationship.

[0024] [Table 1] Compared to unroasted (dried mucuna beans), increasing the roasting temperature from 80°C to 160°C significantly increased the inhibition rate of α-synuclein aggregation. At temperatures above 160°C, the inhibition rate exceeded 90%. It was found that roasting mucuna beans significantly improves the inhibition rate of alpha-synuclein aggregation. This suggests that consuming roasted mucuna beans may help prevent the progression and onset of Lewy body dementia. [Examples]

[0025] (Examination using "Exploration Method for Roasting Conditions 2") Next, we examined the effect of roasting time at each roasting temperature. Using the same roasting machine as in Example 1, we roasted dried mucuna beans by changing the roasting temperature and roasting time. For each roasted mucuna bean, we evaluated the amount of α-synuclein aggregation inhibitory component using "Roasting Condition Exploration Method 2". Table 2 shows the relationship between roasting temperature, roasting time, and the amount of α-synuclein aggregation inhibitory component.

[0026] [Table 2] It was observed that α-synuclein aggregation inhibitors were generated to some extent at 120°C for 30 minutes or more, and at 160°C for 5 minutes or more. In particular, 15 to 30 minutes at 160°C and 5 to 15 minutes at 200°C were frequently observed. These results indicate that there are suitable roasting conditions for mucuna beans. [Examples]

[0027] (Levodopa content) In mucuna beans, the influence of roasting conditions on levodopa content, which has been studied conventionally, was also investigated. The roasting of mucuna beans was the same as in Example 1 (roasting time 15 minutes). Table 3 shows the relationship between roasting temperature and levodopa amount. Figure 3 is a graph of this relationship.

[0028] [Table 3] When the roasting temperature exceeds 120°C, the levodopa content decreases, and at temperatures above 200°C, it almost disappears. From this, it is thought that the functional component that inhibits α-synuclein aggregation, which increases during the roasting of mucuna beans, is not levodopa. Some people experience adverse health effects from levodopa intake, or wish to avoid it due to interactions with prescribed medications or other health reasons. Since mucuna beans roasted at temperatures above 200°C contain almost no levodopa, providing such high-temperature roasted mucuna beans to such individuals can be beneficial. Thus, the α-synuclein aggregation inhibitor containing roasted mucuna beans of the present invention possesses characteristics not found in conventional mucuna beans or mucuna bean extracts, making it useful for a wide range of people. [Industrial applicability]

[0029] According to the present invention, an α-synuclein aggregation inhibitor with improved efficacy is provided, which may be able to prevent the progression and onset of Lewy body disease. In addition, roasting mucuna beans improves their taste and ease of consumption, making them easily available as a food and beverage, thus contributing to improving people's quality of life.

Claims

1. An α-synuclein aggregation inhibitor containing roasted mucuna beans roasted at 70-250°C for 3-60 minutes as its active ingredient.

2. Food and beverages containing the α-synuclein aggregation inhibitor described in claim 1.

3. A method for searching for roasting conditions for mucuna beans that are effective against the α-synuclein aggregation inhibitor described in claim 1, using clonal cells that stably overexpress α-synuclein aggregation.

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