Alzheimer-type dementia inhibiting agent and method for producing the same

Abstract

To provide: an Alzheimer-type dementia inhibiting agent that is safe and can be taken at a daily dosage or used as a functional food, and that makes it possible to inhibit Alzheimer-type dementia; and a method for producing the Alzheimer-type dementia inhibiting agent.SOLUTION: An Alzheimer-type dementia inhibiting agent according to the present invention contains an above-ground portion of sesame (Sesamum indicum) or an extract thereof as an active ingredient. A method for producing the Alzheimer-type dementia inhibiting agent according to the present invention includes the steps of sowing and growing seeds of sesame (Sesamum indicum) and collecting the above-ground portion of the grown sesame.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for inhibiting Alzheimer's dementia, which is safe, can be taken daily, and can be used as a health food, and is capable of inhibiting Alzheimer's dementia, and a method for producing the same. [Background technology]

[0002] According to the "Dementia Prevalence by Age Group of a Cohort of 10,000 People" published by the Ministry of Health, Labor and Welfare, the prevalence of dementia in the 65-69 age group is 1.5%, while the prevalence of dementia in the 85-89 age group is as high as 44.3% (Non-Patent Document 1). A similar trend can be seen in the report in Non-Patent Document 2.

[0003] Among dementias, the number of patients with Alzheimer's dementia was 57 million worldwide as of 2019, and is estimated to reach 150 million by 2050. Alzheimer's dementia is induced by the long-term accumulation of amyloid beta (hereinafter abbreviated as "Aβ") in the brain. Therefore, it is considered important to eliminate the accumulation of Aβ precursors early and inhibit the aggregation of Aβ in order to suppress Alzheimer's dementia. It has also been said that human islet amyloid polypeptide (hereinafter abbreviated as "hIAPP"), which is similar to Aβ, accumulates in human pancreatic beta cells and is associated with the onset of Alzheimer's disease and type II diabetes (Non-Patent Document 2).

[0004] As described above, Alzheimer's dementia is induced by the accumulation of Aβ in the brain over many years, and therefore there is a demand for a drug that can be easily taken daily for many years, is highly safe, and is inexpensive. For example, Patent Documents 1 to 5 describe grape seed extract, turmeric extract, Uncaria tomentosa extract, green tea extract, and perilla extract as natural product-derived extracts having Aβ aggregation inhibitory activity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2009 / 137818 Brochure [Patent Document 2] International Publication No. 2007 / 109210 Brochure [Patent Document 3] International Publication No. 2002 / 042429 Brochure [Patent Document 4] International Publication No. 2001 / 049307 Brochure [Patent Document 5] Patent No. 6707251 [Non-patent literature]

[0006] [Non-Patent Document 1] Ministry of Health, Labor and Welfare, "Trends in Dementia Measures", [online], [Retrieved August 23, 2022], Internet<https: / / kouseikyoku.mhlw.go.jp / kantoshinetsu / houkatsu / 000237803.pdf> [Non-Patent Document 2] Moeko Shinohara, Pharmacia, 2022, Vol. 58, No. 8, pp. 768-771 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, plant-derived Aβ aggregation inhibitors are known. However, only donepezil hydrochloride tablets are available in Japan for treating Alzheimer's disease. Donepezil hydrochloride tablets can have serious side effects, and they can only suppress the progression of symptoms rather than fundamentally treating Alzheimer's disease. Therefore, there is a demand for safe drugs that can fundamentally treat Alzheimer's disease, such as by inhibiting the production of Aβ precursors and the aggregation of Aβ. Therefore, an object of the present invention is to provide an agent for inhibiting Alzheimer's dementia that is safe, can be taken daily, and can be used as a health food, and can inhibit Alzheimer's dementia, and a method for producing the same. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems, and as a result, have discovered that the above-ground parts of sesame, which are safe for consumption, contain a component capable of fundamentally suppressing Alzheimer's disease, thereby completing the present invention. The present invention will now be described.

[0009] [1] An agent for preventing Alzheimer's disease, comprising the above-ground parts of sesame (Sesamum indicum) or an extract thereof as an active ingredient. [2] The agent for inhibiting Alzheimer's disease according to [1], wherein the above-ground parts of sesame are leaves. [3] The agent for suppressing Alzheimer's disease according to [1] or [2], wherein the aerial parts or an extract thereof contain pedaliin. [4] A method for producing an agent for suppressing Alzheimer's disease, comprising the steps of: Sowing and growing sesame (Sesamum indicum) seeds; A method comprising the step of harvesting the above-ground parts of grown sesame. [5] The method according to [4] above, in which the seeds are harvested when one or more buds are visible and before they become ripe. [6] The method according to [4] or [5], further comprising a step of extracting active ingredients from the above-ground parts of sesame using a solvent to obtain an extract solution. [7] The method according to [6], wherein the solvent is an alcohol-based solvent. [8] The method according to [7], further comprising a step of extracting the alcohol-based solvent extract with a hydrocarbon solvent to obtain an extraction residue. Effect of the Invention

[0010] The active ingredient for Alzheimer's dementia according to the present invention is contained in the aboveground part of sesame, which is also used for food, so it is safe and can be taken every day.In addition, it is considered that the Alzheimer's dementia according to the present invention can fundamentally prevent and / or treat Alzheimer's dementia by directly removing the cause of Alzheimer's dementia, such as reducing Aβ precursors and inhibiting Aβ aggregation.Therefore, the present invention is industrially very excellent as a preventive or therapeutic drug for Alzheimer's dementia. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a PCA diagram showing the difference in genomic expression between normal aging mouse SAM-R and senescence accelerated model mouse SAM-P8. [Diagram 2] FIG. 2 is a PCA diagram showing the difference in genome expression between normal aged mice SAM-R and accelerated aging model mice SAM-P8 that were orally administered powder of Goma Kurohachi (registered trademark) leaf extract (SSa) for 7 days. [Diagram 3] FIG. 3(1) shows the results of PCA analysis at the gene level between the control group and the donepezil-administered group, and FIG. 3(2) shows the results of PCA analysis at the exon level. [Figure 4] FIG. 4(1) shows the results of PCA analysis at the gene level between the control group and the actioside-treated group, and FIG. 4(2) shows the results of PCA analysis at the exon level. [Diagram 5] FIG. 5(1) shows the results of PCA analysis between the control group and the SSa extract powder-administered group, and FIG. 5(2) shows the results of PCA analysis at the exon level. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The Alzheimer's dementia inhibitor of the present invention contains the above-ground parts of sesame (Sesamum indicum) or an extract thereof as an active ingredient. In the present disclosure, the active ingredient refers to a component that has an inhibitory effect on Alzheimer's dementia among the components contained in the Alzheimer's dementia inhibitor of the present invention. In other words, the Alzheimer's dementia inhibitor of the present invention contains the above-ground parts of sesame or an extract thereof in an amount that exhibits an inhibitory effect on Alzheimer's dementia. Specifically, without being particularly limited, for example, the ratio of the above-ground parts of sesame or an extract thereof in the Alzheimer's dementia inhibitor of the present invention can be 10% by mass or more and 100% by mass or less. In the present invention, inhibition includes the concept of prevention, which inhibits the onset of Alzheimer's dementia, and the concept of treatment, which alleviates and / or tolerates the symptoms of Alzheimer's dementia.

[0013] Sesame (Sesamum indicum) is an annual plant of the Sesamum genus in the Pedaliaceae family, and it is said that there are about 3,000 species, based on the differences in the size, color, and shape of the seeds. There is no particular limitation on the type of sesame, but the inventors have experimentally confirmed that the leaves of Goma Kurohachi (registered trademark) have an excellent inhibitory effect on Alzheimer's disease.

[0014] Goma Kurohachi (registered trademark) is small black sesame that has been cultivated since ancient times in a specific region of Myanmar under the name Sa Mong Nan Net Thae. Its seeds are very similar in weight and appearance to the seeds of the registered lignan sesame (Registration No. 19697, Application No. 20790, Type of agricultural, forestry and fishery plant: Sesamum indicum L., Variety name: ITCFA2001; Registration No. 19698, Application No. 20791, Type of agricultural, forestry and fishery plant: Sesamum indicum L., Variety name: ITCFA2002). However, the inventors have clarified through genome analysis that the two genomes have large insertions and deletions, and that they are evolutionarily different varieties. The difference in the genomes of Goma Kurohachi and lignan sesame can be verified by PCR.

[0015] The above-ground part refers to the part that is not underground and can be seen above ground when sesame seeds are sown and grown. The above-ground part of sesame can be obtained by cutting and removing the underground part from grown sesame.

[0016] The above-ground part of sesame is preferably from a plant having, for example, two or more true leaves. The above-ground part of sesame is preferably harvested until fully ripe seeds are produced, for example, from plants having immature fruits. Also, plants with a plant height of 50 cm or more, plants having one or more buds but not yet flowering, plants having one or more flowers, etc. can be harvested. The period from sowing to harvesting can be appropriately adjusted, and can be, for example, one month or more, preferably two months or more or 2.5 months or more, and can be five months or less, preferably four months or less, and more preferably 3.5 months or less.

[0017] As the above-ground part of sesame, leaves are preferred. The leaves may or may not include the petiole. Leaves without the petiole have a higher content of active ingredients per weight, but leaves with the petiole have the advantage that they are less laborious to collect.

[0018] The above-ground parts of sesame may be used as is, or may be subjected to one or more treatments selected from washing with water, drying, crushing, hydrolysis, and extraction. Examples of hydrolysis treatment include treatment at 25±5°C and humidity of 90% or more for 1 hour to 5 hours, or rotation in a drum followed by heating to stop hydrolysis. Hydrolysis treatment may reduce bitterness.

[0019] The form of the active ingredient of the Alzheimer's dementia inhibitor according to the present invention is not particularly limited, and examples thereof include liquid, paste, and powder. Powder includes coarse powder, micro powder, and nano-micro powder.

[0020] The form of the Alzheimer's dementia inhibitor according to the present invention is not particularly limited, and examples thereof include solid, gel, and liquid forms. More specifically, examples thereof include powders, fine granules, granules, tablets, coated tablets, capsules, lozenges, and liquids.

[0021] The Alzheimer's disease inhibitor according to the present invention may contain additives other than the active ingredient depending on the dosage form. Examples of additives include excipients, binders, lubricants, disintegrants, emulsifiers, stabilizers, absorption promoters, differentiation agents, and preservatives, and the amount of each additive is not particularly limited and may be appropriately adjusted.

[0022] The Alzheimer's disease inhibitor according to the present invention can be produced, for example, by a method comprising the following steps.

[0023] 1. Sesame seeding and growing process In this process, sesame seeds are sown and grown. This process may be carried out in accordance with the usual method of growing sesame. For example, sesame seeds may be sown from mid-May to mid-June in Japan after the maximum temperature exceeds 20°C. Specifically, holes are made about 1 cm deep, 5-6 seeds are placed in each hole, then the holes are covered with soil and watered thoroughly. When the plants have 1-2 true leaves, poorly growing plants are thinned out so that there are about 3 plants in each hole, when there are 3-4 true leaves, there are 2 plants, and when there are 5-6 true leaves, there is 1 plant.

[0024] 2. Sesame seed harvesting process In this step, the above-ground parts of the grown sesame seeds are harvested. Specifically, the above-ground parts are separated from the underground parts of the grown sesame seeds. The harvesting time and post-processing are as described above.

[0025] 3.Extraction process In this process, the active ingredient is extracted from the above-ground part of sesame using a solvent. This process may or may not be performed, but by extracting it, it is possible to further enhance the inhibitory effect of the active ingredient on Alzheimer's disease.

[0026] The solvent used for extraction in this step is not particularly limited and may be appropriately selected, but from the viewpoint of safety, for example, an aqueous solvent may be mentioned. An aqueous solvent refers to water or a mixed solvent of water and a water-soluble organic solvent. The ratio of the water-soluble organic solvent in the mixed solvent may be appropriately adjusted, but may be, for example, 20% by mass or more, preferably 30% by mass or more or 40% by mass or more, and more preferably 50% by mass or more. The upper limit of the ratio is not particularly limited, but for example, the ratio is preferably 98% by mass or less or 95% by mass or less, and more preferably 90% by mass or less or 80% by mass or less. Examples of the water-soluble organic solvent include alcohol-based solvents such as methanol and ethanol; ether-based solvents such as tetrahydrofuran; amide-based solvents such as dimethylformamide and dimethylacetamide; and sulfoxide-based solvents such as dimethylsulfoxide. From the viewpoint of relatively little harm to the human body, ethanol is suitable as the water-soluble organic solvent. In addition, the type of water is not particularly limited, and purified water, distilled water, pure water, tap water, etc. may be used without any particular restriction.

[0027] The extraction conditions in this step are not particularly limited, and a conventional method can be used. For example, about 5 mL to 100 mL of solvent is added per 1 g of the aboveground part of sesame, and extraction is performed for about 30 minutes to 10 hours. The extraction temperature is also not particularly limited, and extraction may be performed at room temperature, extraction may be performed at or below the boiling point of the solvent, or heating to reflux. For example, when water is used as the solvent, heating may be performed at 60°C to 100°C.

[0028] After extraction, general post-treatment may be carried out. For example, solids may be removed from the mixture after extraction by filtration or centrifugation. Furthermore, the obtained solution may be distilled, concentrated, or dried. Examples of drying methods include heat drying, vacuum drying, freeze drying, and spray drying. In addition, the active ingredient may be further purified by chromatography or the like.

[0029] The amount of the Alzheimer's dementia inhibitor of the present invention to be used should be appropriately adjusted depending on whether it is preventive use or therapeutic use, the severity of the patient, other conditions, age, sex, etc., and is not particularly limited. For example, the Alzheimer's dementia inhibitor of the present invention can be used at 10 mg or more per day in solid content when taken orally, and at 100 mg or more when used externally. Since the Alzheimer's dementia inhibitor of the present invention is very safe, the upper limit of the amount to be used is not particularly limited, but for example, it can be 2 g or less per day in solid content when taken or 5 g or less when used externally. In addition, the number of times per day can be 1 time or more and 5 times or less.

[0030] The Alzheimer's disease inhibitor according to the present invention can be administered not only to humans but also to animals other than humans. Examples of animals to which the agent is administered include livestock such as horses, cows, pigs, sheep, goats, camels, and llamas; sports animals such as racehorses; pet animals such as dogs and cats; laboratory animals such as mice, rats, guinea pigs, and rabbits; poultry such as chickens, ducks, turkeys, and ostriches; and seafood such as farmed fish.

[0031] The Alzheimer's dementia inhibitor of the present invention has an extremely excellent Aβ aggregation inhibitory effect. Specifically, it exhibits the effects of reducing Aβ precursors, inhibiting the formation of Aβ aggregates, and normalizing gene expression. Therefore, the Alzheimer's dementia inhibitor of the present invention is useful as a preventive and / or therapeutic drug for Alzheimer's dementia. EXAMPLES

[0032] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the present invention by making appropriate modifications within the scope of the above and below-mentioned aims, and all of these modifications are included in the technical scope of the present invention.

[0033] Example 1: Preparation of SSa extract powder Goma Kurohachi (registered trademark) seeds were sown in April 2019, and about three months later, leaves were harvested when buds were visible and dried at 60°C for 12 hours. The dried leaves (100 g) were added to 60% ethanol water (1 L), shaken well, and left at room temperature overnight. The solid matter was then removed by filtration to obtain an extract. The obtained extract was concentrated under reduced pressure at 60°C or less to obtain an extract powder (yield: 15.5 g). Hereinafter, the obtained powder is referred to as "SSa extract powder."

[0034] Example 2: Animal studies Seven-week-old male normal aging mice SAM-R and male aging accelerated model mice SAM-P8 (both obtained from Japan SLC) were randomly divided into two groups of 10 mice each and habituated for one week. Next, the SAM-R SSa extract powder group and the SAM-P8 SSa extract powder group were orally administered 500 mg / kg of SSa extract powder for seven days. All mice in each group were allowed to freely consume normal feed and water. After 30 days of breeding, the mice were euthanized by anesthesia, and the whole brain was removed and stored at -80°C until genome analysis. A lipid and tissue dissolving reagent (QIAzol Lysis Reagent, QIAGEN) was added to the brain sample (100-150 mg), and the sample was ground using a homogenizer (Polytron, Central Science Trading Co., Ltd.) for 20-40 seconds, and then left at room temperature for 5 minutes to dissolve the brain sample. Chloroform (0.2 mL) was then added, and the sample was stirred well for 15 seconds, and then left to stand for 2-3 minutes. The sample was centrifuged at 13,000 g at 4°C for 15 minutes, and separated into an aqueous layer, an intermediate layer, and a phenol-chloroform layer. Since RNA is contained in the aqueous layer, the aqueous layer was collected in a separate tube, and isopropyl alcohol (0.5 mL) was added and stirred, and the sample was left to stand at room temperature for 10 minutes. The sample was then centrifuged at 13,000 g at 4°C for 15 minutes, and the supernatant was removed. 70% ethanol (1 mL) was added to the precipitate, stirred well, and centrifuged at 13,000 g at 4°C for 5 minutes, and the supernatant was removed. The pieces were air-dried for 5 to 10 minutes to evaporate the alcohol, and then purified water (50 μL) was added and the pieces were stored at -80°C until RNA sequencing. Using an RNA sample preparation kit ("TruSeq Standard mRNA Library Prep kit" manufactured by Illumina), specific mRNA was enriched from the total RNA obtained, and cDNA was synthesized using these mRNAs as templates to create a library for sequencing. Next, the cDNA library was sequenced using a next-generation sequencer ("NoVaSeq6000" manufactured by Illumina) with 100 bp, paired-end, and 40 million reads per sample. To compare the analysis results, a STAR>cufflinks>cuffdif analysis was performed. The mouse genome database used was GRCm38.92, and gene expression levels at the gene level and its component exon level were analyzed. Specifically, a mapping tool called STAR was used to map to the reference mouse sequence (GRCm38.92), and then Cufflinks was used to assemble the reads. The mapped reads were counted per gene and per exon. Furthermore, cuffdiff was used to quantify gene expression levels and identify gene / exon sites whose expression levels were changed between the two groups. Next, PCA (Principal Component Analysis) was performed, and the differences in genome expression levels were plotted on the principal component axes (PC1 and PC2 axes). In this case, the contribution rate (eigenvector) shown under the PC1 and PC2 axes is important, and the axis with the highest contribution rate can be used as the basis for comparison between the two groups.

[0035] FIG. 1 shows a PCA diagram showing the difference in gene expression levels between normal aging mouse SAM-R and senescence accelerated model mouse SAM-P8. As shown in Figure 1, even on the horizontal axis (PC1 axis) where the contribution rate (eigenvector) is larger, it can be seen that there are significant differences in gene expression between the normal aging mouse SAM-R and the senescence accelerated model mouse SAM-P8.

[0036] Figure 2 shows a PCA diagram showing the difference in genome expression between normal aging mice SAM-R and aging-accelerated mouse model SAM-P8 that was orally administered SSa extract powder for 7 days. As shown in Figure 2, on the horizontal axis (PC1 axis) where the contribution rate (eigenvector) is larger, it was found that when SSa extract powder was administered to the senescence-accelerated mouse model SAM-P8, genome expression was close to that of the normal aged mouse SAM-R.

[0037] Example 3: Animal studies Seven-week-old male aging-accelerated mouse model SAM-P8 (obtained from Japan SLC Co., Ltd.) were randomly divided into four groups of 10 mice each: a control group, a group administered the commercially available Alzheimer's disease drug donepezil (trade name: Aricept, manufactured by Eisai Co., Ltd.), a group administered Actioside (manufactured by Nanjing Spring and Autumn Biological Co., Ltd.), which is known to have Aβ aggregation inhibitory effects, and a group administered SSa extract powder. Each group was orally administered 5mg / kg donepezil, 100mg / kg actioside, and 500mg / kg SSa extract powder for 7 consecutive days.Otherwise, each group of mice was allowed to freely consume normal feed and water.After 30 days of breeding, the mice were euthanized by anesthesia, and the whole brain was removed and stored at -80°C until genome analysis.Next, PCA analysis was performed at the gene level and exon level in the same manner as in Example 2. Figures 3 to 5 show the results of PCA analysis between the control group and the donepezil-administered group, the actioside-administered group, and the SSa extract powder-administered group, respectively. In Figures 3 to 5, the upper row (1) shows the results of PCA analysis at the gene level, and the lower row (2) shows the results of PCA analysis at the exon level.

[0038] As shown in Figures 3 and 4, on the horizontal axis (PC1 axis) where the contribution rate (eigenvector) is larger, no significant differences were observed in the expression patterns between the control group and the donepezil- and actioside-administered groups. On the other hand, as shown in Figure 5, a significant difference in expression pattern was observed between the control group and the SSa extract powder administration group on the horizontal axis (PC1 axis) where the contribution rate (eigenvector) was larger. Moreover, the expression pattern was close to the expression pattern of normal aging mice shown in Figure 2. In other words, while donepezil and actioside probably reduce Aβ aggregation and alleviate symptoms after Aβ aggregation and the onset of dementia, it is possible that some components are absorbed from the SSa extract powder of the present invention and have some effect on genome expression in the brain, normalizing the expression pattern.

[0039] Example 4: Clinical Trials A single-center, placebo-controlled, randomized, single-blind, parallel-group comparative study on the effect of SSa to reduce plasma amyloid-β precursors was conducted with the approval of the ethical committee as follows. Twelve apparently healthy elderly people aged 65 years or older (average age: 68.75 years) were divided into a placebo group, 3 men and 3 women, and a placebo group, 3 groups, administered with molokheiya powder and 3 groups administered with SSa extract powder. Note that, since the administration of 500 mg / kg of SSa extract powder showed a useful effect on intracerebral genome expression in the senescence-accelerated mouse model SAM-P8, it was considered that humans should take 3 to 4 g / day. The SSa extract powder group was given 1.5 g of SSa extract powder twice a day, morning and evening, for two months, and the placebo group was given 1.5 g of molokheiya powder twice a day, morning and evening, for two months. Blood was collected twice, in the morning of the day before the start of the study and 4 to 5 hours after the end of the final administration, and the amount of amyloid beta (Aβ) precursor in the blood was measured by IP-MS (Patent No. 6410810; Akinori Nakamura et al., Nature, 554, 249-254, 2018), which combines immunoprecipitation and mass spectrometry. Specifically, the collected blood was treated with EDTA, centrifuged at 3,000 rpm, and the separated plasma was stored at -77 to -79 °C and used to measure the amyloid beta precursor. Table 1 shows the biomarker index that indicates the amount of amyloid beta precursor accumulation.

[0040] [Table 1]

[0041] As shown in the results in Table 1, among both the SSa extract powder administration group and the placebo group, the subjects with a biomarker index of 1 or more before sample intake accounted for 2 / 6, which was 33.3%. The average value of the difference in biomarker values between "before intake in the SSa extract powder administration group" and "after intake in the SSa extract powder administration group" was -1.207, and the average value of the difference in biomarker values between "before intake in the placebo group" and "after intake in the placebo group" was -0.4925. According to the t-test, there was a significant tendency in the difference in the amount of amyloid-β precursor substance between groups at a significance level of 0.05 < p < 0.1. That is, it became clear that the administration of SSa extract powder significantly reduced the amyloid-β precursor substance in the blood compared to the placebo group.

[0042] Example 5: Component Analysis (1) Fractionation and Aβ aggregation inhibitory activity In 2019, dried SSa leaves produced in Hanagoe, Kyoto (500 g) were roughly cut and immersed in n-hexane (1 L) at room temperature for 2 hours, and the solid content was separated by filtration. The immersion in n-hexane and the separation of the solid content were repeated. The obtained filtrate was used as the n-hexane fraction. Then, 80% ethanol water (1 L) was added to the separated solid content, and it was immersed at room temperature for 2 hours while stirring occasionally, and the solid content was separated by filtration. The immersion in 80% ethanol water and the separation of the solid content were repeated. The obtained filtrate was concentrated under reduced pressure to 150 mL. The obtained concentrated 80% ethanol SSa extract (150 mL) was applied to an adsorption column ("Diaion (registered trademark) HP-20", manufactured by Mitsubishi Chemical Corporation), and chlorophyll was removed by eluting with methanol. The methanol eluate was concentrated under reduced pressure, ethyl acetate was added to the residue, filtered, and the filtrate was concentrated under reduced pressure to obtain an ethyl acetate fraction. Using n-butanol solution and water, an n-butanol fraction and a water fraction were obtained in the same manner. Each fraction was concentrated under reduced pressure. The Aβ aggregation inhibitory activity of each fraction was tested using Thioflavin-T as follows. Human Aβ ("Human, 1-42" manufactured by Peptide Institute) was dissolved in 0.1% ammonia water to a concentration of 250 μM. Each sample of each fraction was added to a phosphate buffer solution (0.05 M sodium phosphate + 0.1 M sodium chloride, pH 7.4) (hereinafter abbreviated as "PBS") so that the final concentrations were 0.01 g / L, 0.05 g / L, and 0.1 g / L. Aβ solution was added thereto so that the final concentration was 25 μM, and the reaction was allowed to proceed at 37°C. After the start of the reaction, a portion of the reaction solution was collected at 0, 4, 8, and 24 hours, and 2.5 μL was dispensed into each well of a 96-well plate for fluorescence measurement ("Nunc Fluoro-Nunc Plate" manufactured by Thermo Fisher Science). Then, 1 mM Thioflavin-T solution (250 μL) dissolved in 50 mM glycine-sodium hydroxide buffer (pH 8.5) was added to each well. The fluorescence intensity emitted when Thioflavin-T labeled the β-sheet structure of Aβ fibrils was measured. The fluorescence intensity was measured using a microplate reader ("Wallac 1420 ARVO MX" manufactured by Perkin Elmer) under the wavelength conditions of Excitation: 420 nm, Emission: 485 nm. Based on the fluorescence intensity after 24 hours, the inhibition rates of each sample addition amount of 0.01, 0.05, and 0.1 g / L were calculated using statistical analysis software PriProbit, with the Aβ fluorescence intensity set at 100. 50 The results are shown in Table 2.

[0043] [Table 2]

[0044] As shown in the results in Table 2, it was revealed that the Aβ protein aggregation inhibitory activity was concentrated in the n-butanol fraction of SSa.

[0045] (2) Ingredients with Aβ aggregation inhibitory activity and ingredients with hIAPP aggregation inhibitory activity Actioside, which is contained in the mint family plant Chorogi, whose tuber part is used in New Year's dishes, is known to have an Aβ aggregation inhibitory effect (Patent No. 6424757; Manami Kurisu et al., Bioscience, Biotechnology, and Biochemistry, 77, 1329-1332, 2013). Therefore, the Aβ aggregation inhibitory activity of the flavonoids Pedaliin and Actioside was compared in the same manner as in Example 5(1). In addition, human islet amyloid polypeptide (hIAPP) is a component of amyloidogenic peptide deposits in the islets of Langerhans, is derived from amyloid precursor protein, and has high sequence similarity to Aβ. Therefore, the hIAPP aggregation inhibitory activity was also tested. Specifically, each compound was added to PBS (0.05 M NaH2Po4, 0.1 M NaCl, pH 7.4) to final concentrations of 1 μM, 10 μM, and 100 μM, and hIAPP aggregation inhibitory activity was measured in the same manner as in Example 5(1), except that hIAPP (Karebay Biochem) was used instead of Aβ. The results are shown in Table 3.

[0046] [Table 3]

[0047] As shown in Table 3, it was revealed that pedaliin has stronger Aβ aggregation inhibitory activity than actioside, which is known to have Aβ aggregation inhibitory activity. In addition, pedaliin also had stronger hIAPP aggregation inhibitory activity than actioside.

[0048] (3) Consideration of the optimal collection time for SSa Goma Kurohachi (registered trademark) from Hanase was harvested when A: the plant height was 50-60 cm, B: when buds began to form, or C: when immature fruits formed, and the leaves including the petioles were dried and finely ground. Each ground dried leaf (0.4 g) was added to methanol (500 mL) and extracted for 12 hours with occasional stirring. After filtering the mixture, the filtrate was concentrated to dryness using a rotary evaporator. The resulting residue was dissolved in methanol (1 mL) and analyzed by LC / MS under the following conditions. LC conditions Column: ODS ACQUITY UPLC BEH c18 1.7 m 2.1 × 100 mm (Waters) Mobile phase: A (acetonitrile containing 0.1% formic acid): B (H2O containing 0.1% formic acid) = 5:95 initial → A:B = 65:35 8min → B = 100 10min → A:B = 5:95 15min Flow rate: 0.3mL / min MS conditions ESI TOF ms Positive ion mode Capillary voltage: 3.2 kV Cone voltage: 20 eV Source temperature: 120℃ 4 μL of each sample was injected, and the MS ions of pedaliin were detected to determine the relative percent composition. The results are shown in Table 4.

[0049] [Table 4]

[0050] As shown in Table 4, the leaves, including the petioles, from the time when the buds start to form to the time when the immature fruit forms contain a lot of pedaliin, which shows excellent Aβ aggregation inhibitory activity and hIAPP aggregation inhibitory activity. Therefore, for the Alzheimer's disease inhibitor, it was decided that Goma Kurohachi (registered trademark) should be harvested from the time when the buds start to form to the time when the immature seeds start to form.

[0051] Example 6: Genomic Analysis The seeds of Goma Kurohachi (registered trademark) are very similar to so-called lignan sesame seeds in terms of the number of seeds per gram and color tone. Therefore, DNA was prepared from both and genome analysis was performed. The number of bases in the sesame genome is known to be about 270 million bases, and in our genome analysis, we obtained sequence data that was about 55 times the number of bases in the sesame genome per variety, which means that we analyzed any genome region 55 times. Therefore, probabilistically, all genome regions were covered, and it can be determined that misreadings were corrected by the analysis program. As a result of genome analysis, it was found that there were large insertions and deletions of sequences between the genomes of Gomakurohachi (registered trademark) and lignan sesame, and it became clear that Gomakurohachi (registered trademark) and lignan sesame are evolutionarily different varieties. From the obtained analysis data, it is possible to verify the genome differences between Gomakurohachi and lignan sesame by PCR.

Claims

1. A therapeutic agent for Alzheimer's disease, comprising the above-ground parts of Sa Mong Nan Net Thae or an extract thereof as an active ingredient.

2. The therapeutic agent for Alzheimer's disease according to claim 1, wherein the above-ground part is a leaf.

3. The therapeutic agent for Alzheimer's disease according to claim 1, wherein the aerial parts or an extract thereof contain pedaliin.

4. A method for producing a therapeutic agent for Alzheimer's disease, comprising the steps of: sowing and growing seeds of Sa Mong Nan Net Thae; and A method comprising the step of harvesting the above-ground parts of grown Sa Mong Nan Net Thae.

5. 5. The method of claim 4, wherein the seeds are harvested when one or more buds are visible and the seeds are ripe.

6. 5. The method according to claim 4, further comprising the step of extracting the effective ingredient from the aerial part using a solvent to obtain an extract solution.

7. The method according to claim 6, wherein the solvent is an alcohol-based solvent.

8. 8. The method of claim 7, further comprising the step of extracting the alcohol-based solvent extract with a hydrocarbon solvent to obtain an extraction residue.

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