Alzheimer's disease inhibitor 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 disease, which is safe, can be taken daily, and can be used as a health food, and is capable of inhibiting Alzheimer's disease, 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 people aged 65-69 is 1.5%, but the prevalence rate in people aged 85-89 is as high as 44.3% (Non-Patent Document 1). A similar trend can also be seen in the report in Non-Patent Document 2.

[0003] Among dementias, the number of patients with Alzheimer's disease was estimated to be 57 million worldwide as of 2019, reaching 150 million by 2050. Alzheimer's disease is triggered by the long-term accumulation of amyloid beta (hereinafter abbreviated as "Aβ") in the brain. Therefore, early elimination of Aβ precursor accumulation and inhibition of Aβ aggregation are thought to be important for preventing Alzheimer's disease. It has also been reported 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 disease is induced by the accumulation of Aβ in the brain over many years, and therefore there is a need for safe, inexpensive drugs that can be taken easily and daily over many years. For example, Patent Documents 1 to 5 describe grape seed extract, turmeric extract, Uncaria tomentosa extract, green tea extract, and perilla extract, respectively, as extracts derived from natural products that have 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 Pamphlet [Patent Document 4] International Publication No. 2001 / 049307 Pamphlet [Patent Document 5] Patent No. 6707251 [Non-patent literature]

[0006] [Non-Patent Document 1] Ministry of Health, Labour 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, the only Alzheimer's disease treatment currently available in Japan is donepezil hydrochloride tablets. However, donepezil hydrochloride tablets can have serious side effects and cannot fundamentally treat Alzheimer's disease; they can only suppress the progression of symptoms. Therefore, there is a need for safe drugs that can fundamentally treat Alzheimer's disease, such as by inhibiting the production of Aβ precursors and Aβ aggregation. Therefore, an object of the present invention is to provide an Alzheimer's disease inhibitor that is safe, can be taken daily, and can be used as a health food, and can suppress Alzheimer's disease, as well as a method for producing the same. [Means for solving the problem]

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

[0009] [1] An agent for suppressing Alzheimer's disease, characterized by containing the above-ground parts of sesame (Sesamum indicum) or an extract thereof as an active ingredient. [2] The agent for suppressing Alzheimer's disease according to [1], wherein the above-ground parts of sesame are leaves. [3] The Alzheimer's disease suppressant 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: 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, wherein the seeds are harvested when one or more buds are visible and before the seeds ripen. [6] The method according to [4] or [5], further comprising a step of extracting the active ingredient from the aerial parts of sesame using a solvent to obtain an extract solution. [7] The method according to [6], wherein an alcohol-based solvent is used as the solvent. [8] The method according to [7], further comprising the step of extracting the alcohol-based solvent extract with a hydrocarbon solvent to obtain an extraction residue. [Effects of the Invention]

[0010] The active ingredient for Alzheimer's disease according to the present invention is contained in the above-ground parts of sesame, which are also used for food, and is therefore safe and can be taken regularly every day. Furthermore, it is believed that the Alzheimer's disease according to the present invention can fundamentally prevent and / or treat Alzheimer's disease by directly eliminating the cause of Alzheimer's disease, such as by reducing Aβ precursors or inhibiting Aβ aggregation. Therefore, the present invention is industrially very advantageous as a preventive or therapeutic agent for Alzheimer's disease. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a PCA diagram showing the differences in genomic expression between normal aging mouse SAM-R and senescence-accelerated mouse model SAM-P8. [Figure 2] Figure 2 is a PCA diagram showing the difference in genome expression between normal aging mice SAM-R and senescence-accelerated mouse model SAM-P8, which was orally administered powder of extract of Gomakurohachi (registered trademark) leaves (SSa) for 7 days. [Figure 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. [Figure 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 INVENTION

[0012] The Alzheimer's disease inhibitor of the present invention contains the aerial parts of sesame (Sesamum indicum) or an extract thereof as an active ingredient. In the present disclosure, the term "active ingredient" refers to a component contained in the Alzheimer's disease inhibitor of the present invention that has the effect of inhibiting Alzheimer's disease. In other words, the Alzheimer's disease inhibitor of the present invention contains the aerial parts of sesame or an extract thereof in an amount sufficient to exert the effect of inhibiting Alzheimer's disease. Specifically, without particular limitation, for example, the proportion of the aerial parts of sesame or an extract thereof in the Alzheimer's disease inhibitor of the present invention can be 10% by mass or more and 100% by mass or less. Note that, in the present invention, "inhibition" encompasses both the concept of prevention, which inhibits the onset of Alzheimer's disease, and the concept of treatment, which alleviates and / or alleviates the symptoms of Alzheimer's disease.

[0013] Sesame (Sesamum indicum) is an annual plant of the Pedaliaceae family, and is said to have approximately 3,000 species, based on differences in seed shape, size, color, etc. There are no particular restrictions 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 a small black sesame seed 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 registered lignan sesame (Registration No. 19697, Application No. 20790, Agricultural, Forestry, and Fisheries Plant Type: Sesamum indicum L., Variety Name: ITCFA2001; Registration No. 19698, Application No. 20791, Agricultural, Forestry, and Fisheries Plant Type: Sesamum indicum L., Variety Name: ITCFA2002). However, the inventors have demonstrated through genome analysis that the two species are evolutionarily distinct, with large insertions and deletions of sequences between the two genomes. Genomic differences between 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 seeds.

[0016] The above-ground parts of sesame are preferably those from plants with two or more true leaves. Furthermore, it is preferable to harvest the above-ground parts of sesame until fully ripe seeds have set, for example, those with immature fruits. Also, plants with a height of 50 cm or more, those with one or more buds but before flowering, and those before one or more flowers have been seen can also be harvested. The period from sowing to harvest can be adjusted as appropriate, but can be, for example, one month or more, preferably two months or more or 2.5 months or more, or 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 contain petioles. Leaves without petioles contain a higher amount of active ingredients per weight, but leaves with petioles have the advantage of being easier to collect.

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

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

[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 powder, fine granules, granules, tablets, coated tablets, capsules, lozenges, and liquid forms.

[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 enhancers, differentiating agents, and preservatives. The amounts of these additives are not particularly limited and may be adjusted as appropriate.

[0022] The Alzheimer's disease suppressant 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 can be carried out in accordance with the usual method for growing sesame. For example, sesame seeds can be sown in Japan from mid-May to mid-June once the maximum temperature exceeds 20°C. Specifically, holes about 1 cm deep are dug, 5 to 6 seeds are placed in each hole, then the holes are covered with soil and watered thoroughly. When the seeds have 1 to 2 true leaves, poorly growing seeds are thinned out to about 3 seeds per hole, and when the seeds have 3 to 4 true leaves, they are thinned out to 2 seeds, and when the seeds have 5 to 6 true leaves, they are thinned out to 1 seed.

[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 aerial parts of sesame using a solvent. This process may or may not be performed, but extraction may 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 selected as appropriate. From the perspective of safety, examples include aqueous solvents. An aqueous solvent refers to water or a mixed solvent of water and a water-soluble organic solvent. The proportion of the water-soluble organic solvent in the mixed solvent may be adjusted as appropriate, but can 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. There is no particular upper limit to this proportion, but for example, the proportion is preferably 98% by mass or less or 95% by mass or less, more preferably 90% by mass or less or 80% by mass or less. Examples of water-soluble organic solvents 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 dimethyl sulfoxide. Ethanol is a preferred water-soluble organic solvent due to its relatively low toxicity to the human body. Furthermore, the type of water is not particularly limited; purified water, distilled water, pure water, tap water, and the like can be used without particular restrictions.

[0027] The extraction conditions for this step are not particularly limited, and conventional methods can be used. For example, approximately 5 mL to 100 mL of solvent is added per gram of sesame aerial parts, and extraction is carried out for approximately 30 minutes to 10 hours. The extraction temperature is also not particularly limited, and extraction may be carried out at room temperature, below the boiling point of the solvent, or by heating under reflux. For example, when water is used as the solvent, heating may be carried out at a temperature of 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. Furthermore, the active ingredient may be further purified by chromatography or the like.

[0029] The amount of the Alzheimer's disease inhibitor of the present invention to be used is not particularly limited, and should be adjusted appropriately depending on whether the use is preventive or therapeutic, the severity of the patient's condition, other conditions, age, sex, etc. For example, the Alzheimer's disease inhibitor of the present invention can be used in an amount of 10 mg or more per day in solid content terms when administered orally, and 100 mg or more per day when administered topically. Because the Alzheimer's disease inhibitor of the present invention is very safe, there are no particular upper limits to the amount used, but for example, it can be 2 g or less per day in solid content terms when administered or 5 g or less per day when administered topically. Furthermore, the number of times per day can be from 1 to 5 times.

[0030] The Alzheimer's disease inhibitor of the present invention can be administered not only to humans but also to animals other than humans. Examples of animals to which the agent can be administered include livestock such as horses, cows, pigs, sheep, goats, camels, and llamas; sports animals such as racehorses; pets 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 disease inhibitor of the present invention has an extremely excellent Aβ aggregation inhibitory effect. Specifically, it exhibits effects such as reducing Aβ precursors, thereby inhibiting the formation of Aβ aggregates, and normalizing gene expression. Therefore, the Alzheimer's disease inhibitor of the present invention is useful as a preventive and / or therapeutic agent for Alzheimer's disease. [Example]

[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 invention by making appropriate modifications within the scope of the above and below-described aims, and all such 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 approximately three months later, leaves were harvested when buds appeared and dried at 60°C for 12 hours. Dried leaves (100 g) were added to 60% ethanol water (1 L), shaken well, and left overnight at room temperature. The solids were then removed by filtration to obtain an extract. The resulting extract was concentrated under reduced pressure at 60°C or below to obtain an extract powder (yield: 15.5 g). Hereinafter, the obtained powder will be referred to as "SSa extract powder."

[0034] Example 2: Animal experiments Seven-week-old male normal aging mice, SAM-R, and male senescence-accelerated model mice, SAM-P8 (both obtained from Japan SLC), were randomly divided into two groups of 10 mice each and allowed to acclimate for one week. The SAM-R SSa extract powder group and the SAM-P8 SSa extract powder group were then orally administered 500 mg / kg of SSa extract powder for seven days. All mice in each group were allowed free access to normal chow and water. After 30 days of breeding, the mice were euthanized by anesthesia, and their whole brains were removed and stored at -80°C until genome analysis. Brain samples (100–150 mg) were lysed by adding a lipid and tissue lysis reagent (QIAzol Lysis Reagent, QIAGEN) to the sample. The mixture was homogenized for 20–40 seconds using a homogenizer (Polytron, Central Scientific Trading Co., Ltd.) and then left at room temperature for 5 minutes. Chloroform (0.2 mL) was then added, vortexed for 15 seconds, and then allowed to stand for 2–3 minutes. The mixture was centrifuged at 13,000 g at 4°C for 15 minutes, resulting in separation into an aqueous layer, an interphase, and a phenol-chloroform layer. Since the aqueous layer contains RNA, the aqueous layer was collected in a separate tube, and isopropyl alcohol (0.5 mL) was added. The mixture was then vortexed and allowed to stand at room temperature for 10 minutes. The mixture was then centrifuged at 13,000 g at 4°C for 15 minutes, after which the supernatant was removed. 70% ethanol (1 mL) was added to the precipitate, vortexed, and centrifuged at 13,000 g at 4°C for 5 minutes, and the supernatant was removed. The mixture was air-dried for 5 to 10 minutes to evaporate the alcohol, and then purified water (50 μL) was added and the mixture was stored at -80°C until RNA sequencing. Specific mRNAs were enriched from the total RNA using an RNA sample preparation kit (Illumina's "TruSeq Standard mRNA Library Prep kit"), and cDNA was synthesized using these mRNAs as templates to create a sequencing library. The cDNA library was then sequenced using a next-generation sequencer (Illumina's "NoVaSeq6000") with 100 bp, paired-end, and 40 million reads per sample. To compare the analysis results, we performed a STAR>cufflinks>cuffdif analysis. The mouse genome database used was GRCm38.92, and we analyzed gene expression levels at the gene and exon levels. Specifically, we used a mapping tool called STAR to map to the reference mouse sequence (GRCm38.92), and then assembled the reads using Cufflinks. The mapped reads were counted at the gene and exon level. Furthermore, we used cuffdiff to quantify gene expression levels and identify gene / exon regions whose expression levels varied 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). In this case, the contribution rate (eigenvector) shown below the PC1 and PC2 axes is important, and the axis with the highest contribution rate can be used as the basis for comparison between two groups.

[0035] Figure 1 shows a PCA diagram showing the difference in gene expression levels between normal aging mice SAM-R and senescence-accelerated mouse model SAM-P8. As shown in Figure 1, even on the horizontal axis (PC1 axis) where the contribution rate (eigenvector) is larger, 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 senescence-accelerated mouse model SAM-P8, which 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, when SSa extract powder was administered to the senescence-accelerated mouse model SAM-P8, genome expression was found to be close to that of the normal aging mouse SAM-R.

[0037] Example 3: Animal Experiments Seven-week-old male senescence-accelerated model mice SAM-P8 (obtained from Japan SLC) 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 inhibitory effects on Aβ aggregation; and a group administered SSa extract powder. Each group was orally administered 5 mg / kg of donepezil, 100 mg / kg of actioside, and 500 mg / kg of SSa extract powder for 7 consecutive days. Mice in each group were allowed free access to normal feed and water. After 30 days of breeding, the mice were euthanized by anesthesia, and the whole brains were removed and stored at -80°C until genome analysis. PCA analysis was then performed at the gene and exon levels 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 the 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. Furthermore, the expression pattern was similar to the expression pattern of normal aging mice shown in Figure 2. In other words, donepezil and actioside probably reduce Aβ aggregation and alleviate symptoms after Aβ aggregation and the onset of dementia, whereas some components may be absorbed from the SSa extract powder of the present invention and have some effect on genome expression in the brain, normalizing its expression pattern.

[0039] Example 4: Clinical Trials A single-center, placebo-controlled, randomized, single-blind, parallel-group comparative study on the effect of SSa on reducing plasma amyloid-β precursors was conducted with the approval of the ethics committee as follows. Twelve apparently healthy elderly people aged 65 years or older (mean age: 68.75 years), three men and three women, were divided into a placebo group administered with molokheiya powder and a placebo group administered with SSa extract powder. Note that, since the administration of 500 mg / kg of SSa extract powder showed a beneficial effect on the brain genome expression of SAM-P8, a senescence-accelerated mouse model, in Example 3, it was considered that a daily intake of 3 to 4 g of SSa extract powder is necessary for humans. The SSa extract powder group received 1.5g of SSa extract powder twice a day, morning and evening, for two months, while the placebo group received 1.5g of molokheiya powder twice a day, morning and evening, for two months. Blood samples were collected twice: once in the morning before the start of the study and again 4–5 hours after the final dose. The amount of amyloid-β (Aβ) precursor in the blood was measured using IP-MS (Patent Publication No. 6410810; Akinori Nakamura et al., Nature, 554, 249–254, 2018), a combination of immunoprecipitation and mass spectrometry. Specifically, the collected blood was treated with EDTA and centrifuged at 3,000 rpm. The separated plasma was stored at −77 to −79°C and then subjected to amyloid-β precursor measurement. Table 1 shows biomarker indices representing the amount of Aβ precursor accumulation.

[0040] [Table 1]

[0041] As shown in the results in Table 1, in both the SSa extract powder administration group and the placebo group, among the subjects with a biomarker index of 1 or more before sample intake, the number was 2 / 6, accounting for 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 between groups at a significance level of 0.05 < p < 0.1. That is, it was revealed that the administration of SSa extract powder significantly reduced the amyloid-β precursor in the blood compared to the placebo group.

[0042] Example 5: Component Analysis (1) Fractionation and Aβ aggregation inhibition activity In 2019, 500 g of dried SSa leaves produced in Hanazoe, Kyoto 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," Peptide Institute) was dissolved in 0.1% aqueous ammonia to a concentration of 250 μM. Each fraction was added to phosphate buffer (0.05 M sodium phosphate + 0.1 M sodium chloride, pH 7.4) (hereinafter abbreviated as "PBS") to final concentrations of 0.01 g / L, 0.05 g / L, and 0.1 g / L. Aβ solution was added to the mixture to a final concentration of 25 μM, and the mixture was incubated at 37°C. Aliquots of the reaction mixture were taken at 0, 4, 8, and 24 hours after the start of the reaction, and 2.5 μL aliquots were dispensed into each well of a 96-well plate for fluorescence measurement ("Nunc FluoroNunc Plate," Thermo Fisher Science). Then, 250 μL of 1 mM Thioflavin-T solution 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. Fluorescence intensity was measured using a microplate reader (Wallac 1420 ARVO MX, Perkin Elmer) under 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 Table 2, it was revealed that the Aβ protein aggregation inhibitory activity was concentrated in the n-butanol fraction of SSa.

[0045] (2) Aβ aggregation inhibitory component and hIAPP aggregation inhibitory component Actioside, contained in the mint family plant, Chinese artichoke, whose tubers are used in New Year's dishes, is known to have Aβ aggregation inhibitory activity (Patent No. 6424757; Manami Kurisu et al., Bioscience, Biotechnology, and Biochemistry, 77, 1329-1332, 2013). Therefore, the Aβ aggregation inhibitory activity of the flavonoid pedaliin and actioside was compared as described in Example 5(1). Human islet amyloid polypeptide (hIAPP) is a component of amyloidogenic peptide deposits in the pancreatic islets of Langerhans. It is derived from the amyloid precursor protein and shares high sequence similarity with 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 the 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, pedaliin was found to have stronger Aβ aggregation inhibitory activity than actioside, which is known to have Aβ aggregation inhibitory activity. Furthermore, 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 fruit formed. The leaves, including the petioles, were dried and finely ground. Each dried, ground leaf (0.4 g) was added to methanol (500 mL) and extracted for 12 hours with occasional stirring. The mixture was filtered, and 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 (HO containing 0.1% formic acid) = 5:95 initial → A:B = 65:35 8 min → B = 100 10 min → A:B = 5:95 15 min 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 ion of pedaliin was detected to determine the relative percent composition. The results are shown in Table 4.

[0049] [Table 4]

[0050] As shown in Table 4, leaves containing petioles from the time when buds first appear until immature fruit formation are found to contain large amounts of pedaliin, which exhibits excellent Aβ aggregation inhibitory activity and hIAPP aggregation inhibitory activity. Therefore, we decided to harvest Goma Kurohachi (registered trademark) from the time when buds appear until immature seeds appear for the purpose of developing an Alzheimer's disease inhibitor.

[0051] Example 6: Genomic analysis The seeds of Gomakurohachi (registered trademark) are very similar to the so-called lignan sesame seeds in terms of the number of seeds per gram and color tone. Therefore, DNA was prepared from both seeds and genome analysis was performed. The sesame genome is known to have approximately 270 million bases, and our genome analysis yielded sequence data that was approximately 55 times the number of bases in the sesame genome per variety, meaning that any given genome region was analyzed 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 revealed that Gomakurohachi (registered trademark) and lignan sesame have large insertions and deletions of sequences between their genomes, and that they are evolutionarily different varieties. From the analysis data obtained, it is possible to verify the differences in the genomes of Gomakurohachi and lignan sesame by PCR.

Claims

1. A therapeutic agent for Alzheimer's disease, comprising the aerial 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: sowing and growing seeds of Sa Mong Nan Net Thae; Harvesting the above-ground parts of the grown Sa Mong Nan Net Thae; and The method comprises the step of subjecting the collected aerial parts to one or more treatments selected from washing with water, drying, pulverization, hydrolysis, and extraction to obtain an active ingredient of a therapeutic agent for Alzheimer's disease in a liquid, paste, or powder form.

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. The method according to claim 4, wherein the active ingredient is extracted from the above-ground parts using a solvent to obtain an extract solution.

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

8. The method according to 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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