Agents and methods for maintaining or improving brain function using crude drugs

Abstract

The present invention provides an agent for maintaining or improving brain function, which contains a crude drug selected from the group consisting of leaves, fruits, and seeds of Laphania tetrandra.

Description

TECHNICAL FIELD

[0001] The present application relates to an agent and a method for maintaining or improving brain function, promoting repair of nerve cells, or inducing neurogenesis, removing a neurodegenerative disease-causing protein accumulated in the brain, or treating or preventing a neurodegenerative disease, using crude drugs, and a food and a drug containing the agent. BACKGROUND

[0002] With the aging of society and the westernization of living and eating habits, the number of patients with cognitive disorders is rapidly increasing worldwide. The social cost of medical care and nursing, and the economic loss caused by the reduction of employment of patients and their families are enormous, and it has become a serious social problem.

[0003] As representative cognitive disorders, Alzheimer's disease, frontotemporal lobar cognitive disorder, Lewy body-type cognitive disorder, and the like are known. Any of these is a neurodegenerative disease, and cognitive disorders caused by these are called degenerative cognitive disorders. In these diseases, it is believed that a specific protein is condensed and accumulated in the nervous system, which causes nerve cells to die, and thus induces cognitive disorders. Specifically, for Alzheimer's disease, Aβ and Tau are accumulated in the brain; for frontotemporal lobar cognitive disorder, Tau and TDP-43 are accumulated in the brain; and for Lewy body-type cognitive disorder, alpha synuclein is accumulated in the brain (Non-Patent Literature 1: Spires-Jones et al., Acta Neuropathol., (2017), 134 [2]: 187-205).

[0004] As an anti-cognitive disorder drug, drugs that inhibit the production of these pathogenic proteins or remove pathogenic proteins from the brain are being developed. As a candidate drug for the anti-cognitive disorder drug, an inhibitor for the enzyme that produces Aβ (β-secretase or γ-secretase) for Alzheimer's disease (non-patent literature 2: Luo et al., Cell & Biosci., (2022), 12:2), an Aβ vaccine (non-patent literature 3: Valiukas et al., Vaccines, (2022), 10[9]: 1527), or an Aβ antibody (non-patent literature 4: Song et al., Transl. Neurodegener., (2022), 11:18), and the like are being studied; a Tau vaccine (non-patent literature 5: Medina, Int. J. Mol. Sci., (2018), 19[4]: 1160) or a Tau antibody (non-patent literature 6: Ji et al., Drugs, (2021), 81

[10] : 1135-1152) for frontotemporal dementia, and the like; an alpha synuclein vaccine or an alpha synuclein antibody for Lewy body dementia (non-patent literature 7: Alzforum website, FBRI LLC, search results for alpha synuclein targets, https: / / www.alzforum.org / therapeutics / search?fda_statuses=&target_types%5B%5D=33416&therapy_types=&conditions=&keywords-entry=&keywords=, searched on December 2022) and the like.

[0005] However, most of the anti-cognitive disorder drug candidates developed so far have not been confirmed to have the expected efficacy in clinical trials with cognitive disorder patients as subjects, and have ultimately ended in failure (non-patent literature 8: Asher et al., Life Sciences, (2022), 306: 120861).

[0006] As the leaves of Pipturus albidus, a herbaceous plant native to Hawaii, it is used as a material for a popular herbal tea in Hawaii. In Hawaii, Pipturus albidus, particularly its fruit, has been used historically as a natural folk remedy for regulating blood sugar levels, blood pressure, cholesterol levels, relieving stress or fatigue, and reducing inflammation (non-patent literature 9: Rafii et al., Alzheimers Dement., (2022), 1-7). However, there is no known effect on cognitive disorders.

[0007] Prior Art Documents

[0008] Non-patent literature

[0009] Non-patent literature 1. Spires-Jones et al., Acta Neuropathol., (2017), 134[2]:187-205

[0010] Non-patent literature 2. Luo et al., Cell & Biosci., (2022), 12:2

[0011] Non-patent literature 3. Valiukas et al., Vaccines, (2022), 10[9]:1527

[0012] Non-patent literature 4. Song et al., Transl. Neurodegener., (2022), 11:18

[0013] Non-patent literature 5. Medina, Int. J. Mol. Sci., (2018), 19[4]:1160

[0014] Non-patent literature 6. Ji et al., Drugs, (2021), 81

[10] :1135-1152

[0015] Non-patent literature 7. Alzforum website, FBRI LLC, Search results for alpha synuclein targets, https: / / www.alzforum.org / therapeutics / search?fda_statuses=&target_types%5B%5D=33416&therapy_types=&conditions=&keywords-entry=&keywords=, accessed December 2022

[0016] Non-patent literature 8. Asher et al., Life Sciences, (2022), 306:120861

[0017] Non-patent literature 9. Rafii et al., Alzheimers Dement., (2022), 1-7

[0018] Non-patent literature 10. Chun et al., Native Hawaiian medicines, First People's Productions Honolulu, (1994), 216-217

[0019] Non-patent document 11. Afzal et al., Molecules, (2022), 27

[21] :7604

[0020] Non-patent document 12. Payne et al., Biomolecules, (2022), 12[3]:371

[0021] Non-patent document 13. Li et al., Neuroscience, (2009), 159[4]:1208-15

[0022] Non-patent document 14. Yoo et al., Phytother Res., (2010), 24[7]:1065-70

[0023] Non-patent document 15. Colucci-D'Amato et al., Int. J. Mol. Sci., (2020), 21

[20] :7777

[0024] Non-patent document 16. Horgusluoglu et al., Am. J. Med. Gene. B. Neuropsychiatr. Genet., (2017), 174[1]:93-112

[0025] Non-patent document 17. Socala et al., Int. J. Mol. Sci., (2020), 22[1]:107

[0026] Non-patent document 18. Gao et al., Drug Des. Devel. Ther., (2020), 14:1705-1716

[0027] Non-patent document 19. Singh et al., Oxid. Med. Cell Longev., (2020), 2020:6571484

[0028] Non-patent document 20. Liu et al., Drug Des. Devel. Ther., (2020), 14:51-60

[0029] Non-patent document 21. Tahir et al., Biomed. Pharmacother., (2021), 137:111253

[0030] Non-patent document 22. Xu et al., Behav. Brain Res., (2014), 264:173-80

[0031] Non-patent literature 23. Sun et al., J. Neuroinflammation, (2021), 18[1]:131

[0032] Non-patent literature 24. Moghbelinejad et al., Toxicol Lett., (2014), 224[1]:108-13

[0033] Non-patent literature 25. Umeda et al., Am. J. Pathol., (2013), 183[1]:211-25

[0034] Non-patent literature 26. Umeda et al., Ann. Clin. Transl. Neurol., (2015), 2[3]:241-55

[0035] Non-patent literature 27. Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. U.S.A., (1997), 94

[24] :13287-92

[0036] Non-patent literature 28. Van Dam et al., Eur. J. Neurosci., (2003), 17[2]:388-96

[0037] Non-patent literature 29. Umeda et al., Front. Neurosci., (2021), 15:763476

[0038] Non-patent literature 30. Lee et al., Proc. Natl. Acad. Sci. U.S.A., (2002), 99

[13] :8968-73

[0039] Non-patent literature 31. Umeda et al., Int. J. Mol. Sci., (2021), 22:8453

[0040] Non-patent literature 32. Liu et al., Neuron, (2016), 90[3]:521-534

[0041] Non-patent literature 33. Hatanaka et al., Biomedicines, (2022), 10[5]:1080 SUMMARY

[0042] PROBLEMS TO BE SOLVED BY THE INVENTION

[0043] The present application should solve the problem of providing a novel means for maintaining or improving brain function, promoting repair of nerve cells or inducing neurogenesis, removing a neurodegenerative disease pathogenic protein accumulated in the brain, or treating or preventing a neurodegenerative disease.

[0044] Means for solving the problem

[0045] As a result of intensive studies by the present inventors, it has been found that crude drugs selected from the leaves, fruits, and seeds of Grevillea robusta have various effects such as maintaining or improving brain function, promoting repair of nerve cells or inducing neurogenesis, removing a neurodegenerative disease pathogenic protein accumulated in the brain, or treating or preventing a neurodegenerative disease in each specific form, thereby completing the present application.

[0046] That is, the gist of the present application relates to, for example, the following.

[0047] Item 1. An agent for maintaining or improving brain function, which contains a crude drug selected from the leaves, fruits, and seeds of Grevillea robusta.

[0048] Item 2. The agent for maintaining or improving brain function according to item 1, wherein the brain function is cognitive function.

[0049] Item 3. An agent for promoting repair of nerve cells or inducing neurogenesis, which contains a crude drug selected from the leaves, fruits, and seeds of Grevillea robusta.

[0050] Item 4. An agent for removing a neurodegenerative disease pathogenic protein accumulated in the brain, which contains a crude drug selected from the leaves, fruits, and seeds of Grevillea robusta.

[0051] Item 5. The agent according to item 4, wherein the neurodegenerative disease pathogenic protein is one or two or more proteins selected from the group consisting of β-amyloid protein (Aβ), Tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein produced by RAN (repeat-associated non-ATG) translation, prion, and SOD-1.

[0052] Item 6. An agent for treating or preventing a neurodegenerative disease, which contains a crude drug selected from the leaves, fruits, and seeds of Grevillea robusta.

[0053] Item 7. The agent according to item 6, wherein the neurodegenerative disease is degenerative cognitive disorder.

[0054] Item 8. The agent according to item 7, wherein the degenerative cognitive disorder is one or two or more cognitive disorders selected from the group consisting of Alzheimer's disease, frontotemporal lobar cognitive disorder, Lewy body cognitive disorder, and cognitive disorder caused by Parkinson's disease or amyotrophic lateral sclerosis.

[0055] Item 9. The agent according to any one of items 1 to 8, wherein the aforesaid crude drug selected from the leaf, fruit and seed of Melia toosendan is a broken product and / or extract of the leaf and / or fruit and / or seed of Melia toosendan.

[0056] Item 10. The agent according to any one of items 1 to 9, wherein the aforesaid crude drug is administered to a subject in an amount of 0.01 mg to 10 g per day.

[0057] Item 11. A food comprising the agent according to any one of items 1 to 10.

[0058] Item 12. A medicament comprising the agent according to any one of items 1 to 10.

[0059] Item 13. A method for maintaining or improving brain function in a subject, comprising administering a crude drug selected from the leaf, fruit and seed of Melia toosendan to a subject in need thereof.

[0060] Item 14. The method according to item 13, wherein the brain function is cognitive function.

[0061] Item 15. A method for promoting repair of neural cells or inducing neurogenesis in a subject, comprising administering a crude drug selected from the leaf, fruit and seed of Melia toosendan to a subject in need thereof.

[0062] Item 16. A method for removing a neurodegenerative disease pathogenic protein accumulated in the brain of a subject, comprising administering a crude drug selected from the leaf, fruit and seed of Melia toosendan to a subject in need thereof.

[0063] Item 17. The method according to item 16, wherein the neurodegenerative disease pathogenic protein is one or two or more proteins selected from the group consisting of β-amyloid (Aβ), Tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein produced by RAN (repeat-associated non-ATG) translation, prion protein and SOD-1.

[0064] Item 18. A method for treating or preventing a neurodegenerative disease in a subject, comprising administering a crude drug selected from the leaf, fruit and seed of Melia toosendan to a subject in need thereof.

[0065] Item 19. The method according to item 18, wherein the neurodegenerative disease is degenerative cognitive disorder.

[0066] Item 20. The method according to item 19, wherein the degenerative cognitive disorder is one or two or more cognitive disorders selected from the group consisting of Alzheimer's disease, frontotemporal lobar cognitive disorder, Lewy body cognitive disorder and cognitive disorder resulting from Parkinson's disease or amyotrophic lateral sclerosis.

[0067] Item 21. The method according to any one of items 13 to 20, wherein the aforesaid crude drug selected from the leaf, fruit and seed of Distylium racemosum is a broken product and / or extract of the leaf and / or fruit and / or seed of Distylium racemosum.

[0068] Item 22. The method according to any one of items 13 to 21, wherein the aforesaid crude drug is administered to the subject in an amount of 0.01 mg to 10 g per day.

[0069] Effects of the Invention

[0070] According to the present application, by using a crude drug selected from the leaf, fruit and seed of Distylium racemosum in each specific form, a novel means for maintaining or improving brain function, promoting repair of nerve cells or inducing neurogenesis, removing a neurodegenerative disease-causing protein accumulated in the brain, or treating or preventing a neurodegenerative disease can be provided. BRIEF DESCRIPTION OF DRAWINGS

[0071] [ Figure 1 ] Figure 1 A graph showing the results of the Morris water maze test of Tau784 mice (in the table, "Tg+Leaf-ext") to which a hot water extract (Leaf-ext) of the leaf of Distylium racemosum was orally administered at a dose of 100 μg per day for 1 month, in comparison with Tau784 mice (in the table, "Tau784") to which water was administered as a subject.

[0072] [ Figure 2 ] Figure 2 A graph showing the Tau pathology in the entorhinal cortex of Tau784 mice (in the table, "Tau784+Leaf-ext") to which a hot water extract (Leaf-ext) of the leaf of Distylium racemosum was orally administered at a dose of 100 μg per day for 1 month, in comparison with Tau784 mice (in the table, "Tau784") to which water was administered as a subject and non-gene recombinant mice (in the table, "non-Tg"). Figure 2 A is a photograph showing the results of staining of phosphorylated Tau and Tau oligomers, Figure 2 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0073] [ Figure 3 ] Figure 3 A graph showing the synaptophysin pathology in the CA2 / 3 region of the hippocampus of Tau784 mice (in the table, "Tau784+Leaf-ext") to which a hot water extract (Leaf-ext) of the leaf of Distylium racemosum was orally administered at a dose of 100 μg per day for 1 month, in comparison with Tau784 mice (in the table, "Tau784") to which water was administered as a subject and non-gene recombinant mice (in the table, "non-Tg"). Figure 3 A is a photograph showing the results of staining of synaptophysin,Figure 3 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0074] [ Figure 4 ] Figure 4 is a graph showing the microglial cell pathology in the hippocampus (HC) and cerebral cortex (CTX) of Tau784 mice to which a hot water extract of the leaves of Distylium racemosum (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with Tau784 mice to which water was administered (Table "Tau784") and non-genetically recombinant mice (Table "non Tg") as subjects. Figure 4 A is a photograph showing the staining results of activated microglial cells, Figure 4 B is a graph showing the number of positive cells of each photograph.

[0075] [ Figure 5 ] Figure 5 is a graph showing the results of the Morris water maze test of APP23 mice to which a hot water extract of the leaves of Distylium racemosum (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with APP23 mice to which water was administered (Table "Tg") and non-genetically recombinant mice (Table "non Tg") as subjects.

[0076] [ Figure 6 ] Figure 6 is a graph showing the amyloid pathology in the cerebral cortex and hippocampus of APP23 mice to which a hot water extract of the leaves of Distylium racemosum (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with APP23 mice to which water was administered (Table "APP23") as subjects. Figure 6 A is a photograph showing the staining results of amyloid deposits and Aβ oligomers, Figure 6 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0077] [ Figure 7 ] Figure 7 is a graph showing the synaptophysin pathology in the hippocampal CA2 / 3 region of APP23 mice to which a hot water extract of the leaves of Distylium racemosum (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with APP23 mice to which water was administered (Table "APP23") and non-genetically recombinant mice (Table "non Tg") as subjects. Figure 7 A is a photograph showing the staining results of synaptophysin, Figure 7B is a graph showing the results of quantifying the staining intensity of each photograph.

[0078] [ Figure 8 ] Figure 8 A is a graph showing the results of quantifying the staining intensity of each photograph. Figure 8 A is a photograph showing the results of staining of activated microglia, Figure 8 B is a graph showing the number of positive cells of each photograph.

[0079] [ Figure 9 ] Figure 9 A is a graph showing the results of quantifying the staining intensity of each photograph.

[0080] [ Figure 10 ] Figure 10 A is a graph showing the results of quantifying the staining intensity of each photograph. Figure 10 A is a photograph showing the results of staining of phosphorylated α-synuclein, Figure 10 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0081] [ Figure 11 ] Figure 11 A is a graph showing the results of quantifying the staining intensity of each photograph. Figure 11A is a photograph showing the results of staining of α-synuclein oligomers, Figure 11 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0082] [ Figure 12 ] Figure 12 A graph showing synucleinopathy pathology in the hippocampal CA2 / 3 region of Huα-Syn(A53T) mice ("αSyn-Tg" in the table) to which a hot water extract of the leaves of Melia toosendan (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with Huα-Syn(A53T) mice ("αSyn-Tg" in the table) to which water was administered and non-genetically recombinant mice ("non Tg" in the table). Figure 12 A is a photograph showing the results of staining of synuclein, Figure 12 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0083] [ Figure 13 ] Figure 13 A graph showing microglial cell pathology in the hippocampus (HC) and cerebral cortex (CTX) of Huα-Syn(A53T) mice ("αSyn-Tg" in the table) to which a hot water extract of the leaves of Melia toosendan (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with Huα-Syn(A53T) mice ("αSyn-Tg" in the table) to which water was administered and non-genetically recombinant mice ("non Tg" in the table). Figure 13 A is a photograph showing the results of staining of activated microglial cells, Figure 13 B is a graph showing the number of positive cells of each photograph.

[0084] [ Figure 14 ] Figure 14 A graph showing the results of the Morris water maze test of C9-500 mice ("Tg" in the table) to which a hot water extract of the leaves of Melia toosendan (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with C9-500 mice ("Tg" in the table) to which water was administered and non-genetically recombinant mice ("non Tg" in the table).

[0085] [ Figure 15 ] Figure 15A diagram illustrating the pathology caused by the C9orf72 gene mutation in the prefrontal cortex (PFC) of C9-500 mice (“C9-500+Leaf-ext”) that were orally administered a hot water extract of the leaves of *Alternanthera philoxeroides* (Leaf-ext) at a dose of 100 μg / day for one month, compared with that of C9-500 mice treated with water (“C9-500” in the table). Figure 15 Photo A shows the staining results of RNA G-tetramer, poly-GA, poly-GP, and phosphorylated TDP-43. Figure 15 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0086] [ Figure 16 ] Figure 16 A diagram illustrating the pathology of double-stranded RNA-dependent protein kinase (PKR) in the prefrontal cortex (PFC) of C9-500 mice (“C9-500” in the table) who were orally administered a hot water extract of the leaves of *Alternanthera philoxeroides* (Leaf-ext) for one month at a dose of 100 μg / day, compared with water-treated C9-500 mice (“C9-500” in the table) and non-recombinant mice (“Non-Tg” in the table). Figure 16 Photograph A shows the staining results of phosphorylated PKR. Figure 16 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0087] [ Figure 17 ] Figure 17 A diagram showing the pathology of synaptic proteins in the CA2 / 3 region of the hippocampus in C9-500 mice ("C9-500+Leaf-ext") that were orally administered with a hot water extract of the leaves of *Alternanthera philoxeroides* (Leaf-ext) for one month at a dose of 100 μg / day, compared with water-treated C9-500 mice ("C9-500" in the table) and non-recombinant mice ("non-Tg" in the table). Figure 17 Photo A shows the staining results of synaptic vesicle proteins. Figure 17 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0088] [ Figure 18 ] Figure 18 A diagram showing the microglial cell pathology in the prefrontal cortex (PFC) of C9-500 mice ("C9-500+Leaf-ext") that were orally administered a hot water extract of the leaves of *Alternanthera philoxeroides* (Leaf-ext) for one month at a dose of 100 μg / day for comparison with water-treated C9-500 mice ("C9-500" in the table) and non-recombinant mice ("non-Tg" in the table). Figure 18A is a photograph showing the staining results of activated microglia cells, Figure 18 B is a graph showing the number of positive cells of each photograph.

[0089] [ Figure 19 ] Figure 19 A graph showing the results of the Morris water maze test of Tau784 mice to which hot water extract of leaves (Leaf-ext), simple crushed powder of leaves (Leaf-pwd), and simple crushed powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium were orally administered at a dose of 30 μg / day for 1 month, in comparison with Tau784 mice to which water was administered (Tg in the table), and non-gene recombined mice (non-Tg in the table).

[0090] [ Figure 20 ] Figure 20 A graph showing the Tau pathology in the olfactory cortex of Tau784 mice to which hot water extract of leaves (Leaf-ext), simple crushed powder of leaves (Leaf-pwd), and simple crushed powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium were orally administered at a dose of 30 μg / day for 1 month, in comparison with Tau784 mice to which water was administered (Tau784 in the table), and non-gene recombined mice (non-Tg in the table). Figure 20 A is a photograph showing the staining results of phosphorylated Tau and Tau oligomers, Figure 20 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0091] [ Figure 21 ] Figure 21 A graph showing the synaptophysin pathology in the CA2 / 3 region of the hippocampus of Tau784 mice to which hot water extract of leaves (Leaf-ext), simple crushed powder of leaves (Leaf-pwd), and simple crushed powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium were orally administered at a dose of 30 μg / day for 1 month, in comparison with Tau784 mice to which water was administered (Tau784 in the table), and non-gene recombined mice (non-Tg in the table). Figure 21 A is a photograph showing the staining results of synaptophysin, Figure 21 B is a graph showing the results of quantifying the staining intensity of each photograph.

[0092] [ Figure 22 ] Figure 22 A graph showing BDNF expression in the cerebral cortex (CTX) of Tau784 mice (respectively "Tg+Leaf-ext", "Tg+Leaf-pwd" and "Tg+Fruit-pwd" in the table) orally administered with white tupelo leaf hot water extract (Leaf-ext), leaf simple crushed powder (Leaf-pwd) and fruit (including seeds) simple crushed powder (Fruit-pwd) at a dose of 30 μg / day for 1 month in comparison with water-administered Tau784 mice ( "Tau784" in the table) and non-genetically recombinant mice ( "non Tg" in the table) as the subject. Figure 22 A is a photograph showing the staining result of BDNF, Figure 22 B is a graph showing the result of quantifying the staining intensity of each photograph.

[0093] [ Figure 23 ] Figure 23 A graph showing the level of neurogenesis in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn(A53T) mice ( "αSyn-Tg+Fruit-pwd" in the table) orally administered with white tupelo fruit (including seeds) simple crushed powder (Fruit-pwd) at a dose of 30 μg / day for 1 month in comparison with water-administered Huα-Syn(A53T) mice ( "αSyn-Tg" in the table) and non-genetically recombinant mice ( "non Tg" in the table) as the subject. Figure 23 A is a photograph of immunofluorescence staining of BrdU (red) and doublecortin (DCX) (green). Double-positive cells (yellow) positive for both BrdU (red) and DCX (green) are considered as regenerated neurons. Figure 23 B is a graph showing the result of quantifying the number of double-positive cells (yellow) of each photograph.

[0094] [ Figure 24 ] Figure 24 A graph showing the result of Morris water maze test of Tau784 mice orally administered with a mixture of catechin 0.087 μg, chlorogenic acid 0.036 μg and rutin 0.123 μg ( "Tg+3 polyphenol mixture" in the table), or only catechin 0.087 μg ( "Tg+catechin" in the table) for 1 month in comparison with water-administered Tau784 mice ( "Tg" in the table) and non-genetically recombinant mice ( "non Tg" in the table) as the subject. DETAILED DESCRIPTION

[0095] The present application will be described in detail below according to specific embodiments. However, the present application is not limited to the following embodiments, and can be implemented in any manner without departing from the spirit of the present application.

[0096] [Summary]

[0097] As described above, the reason why the anti-cognitive disorder drugs of the prior art researches almost end in failure, in addition to the problem of side effects, is considered to be that the drug is administered too late and the wrong molecule is targeted. It has been shown that the accumulation of Aβ in the brain begins more than 20 years before the onset of Alzheimer's disease, and the accumulation of Tau also begins about 10 years before. Aβ accumulates, Tau accumulates, and nerve cells begin to die, and thereafter eventually lead to the onset of cognitive disorders. In other words, at the time of the onset of cognitive disorders, a large number of nerve cells have already died. If Aβ or Tau is to be removed, the action needs to be performed before the nerve cells begin to die. That is, the drug targeting Aβ or Tau acts to prevent rather than treat. In addition, it has been generally believed that the insoluble aggregates of proteins such as senile plaques or neurofibrillary changes (accumulation of aggregated Tau) cause the disease by killing nerve cells. However, it has recently been considered that the soluble oligomers formed in the early stages cause cognitive disorders by impairing the function of nerve cells. Therefore, to prevent cognitive disorders, it is necessary to remove the oligomers of the disease-causing proteins.

[0098] From the viewpoint of so-called prevention, it is desirable that it is not Aβ-specific, Tau-specific, or α-synuclein-specific, but acts on the oligomers of various disease-causing proteins with one dose. Further, it is also necessary to repair the nerve cells damaged by the oligomers and restore the function of the brain. In addition, prevention of cognitive disorders is long-term, and it is desirable that the preventive drug is safe and inexpensive, and as much as possible, can be taken by oneself without the help of a medical doctor. Thus, since the requirements for the preventive drug of cognitive disorders are many, it is not easy to achieve with a single component of a pharmaceutical. In addition, in order to prevent cognitive disorders, all the elderly people take it for a long period, and in the field of pharmaceuticals, there is a risk that the medical economy will eventually collapse.

[0099] To solve the problem, the present inventors and the like focused on crude drugs used in natural therapies or traditional Chinese medicine and Han medicine with a long history. If there is a crude drug that effectively improves cognitive function, the elderly can judge and obtain it by themselves without going to a medical doctor, and by taking it by adding it to meals and the like, it is possible to strive to prevent cognitive disorders from daily life.

[0100] Therefore, as a result of intensive research by the present inventors and the like, it was found that a crude drug selected from the leaves, fruits, and seeds of Grewia asiatica has various effects of maintaining or improving brain function, promoting repair of nerve cells or inducing neurogenesis, removing neurodegenerative disease-causing proteins accumulated in the brain, or treating or preventing neurodegenerative diseases. The present application is based on the insight.

[0101] That is, according to an aspect of the present application, there is provided a brain function maintaining or improving agent, a nerve cell repair promoting or nerve regeneration inducing agent, a brain-accumulated neurodegenerative disease pathogenic protein removing agent, or a neurodegenerative disease treating or preventing agent (these are sometimes collectively referred to as "the agent of the present application") containing crude drugs selected from the group consisting of leaves, fruits, and seeds of Pipturus albidus.

[0102] In addition, according to an aspect of the present application, there is provided a food containing the agent of the present application (this is sometimes referred to as "the food of the present application" as needed).

[0103] In addition, according to an aspect of the present application, there is provided a medicine containing the agent of the present application (this is sometimes referred to as "the medicine of the present application" as needed).

[0104] In addition, according to an aspect of the present application, there is provided a method of maintaining or improving brain function, a method of promoting nerve cell repair or inducing nerve regeneration, a method of removing brain-accumulated neurodegenerative disease pathogenic protein, or a method of treating or preventing neurodegenerative disease, which comprises administering one or two or more of crude drugs selected from the group consisting of leaves, fruits, and seeds of Pipturus albidus, the agent of the present application, the food of the present application, and the medicine of the present application to a subject (these are sometimes collectively referred to as "the method of the present application" as needed).

[0105] [Pipturus albidus]

[0106] According to one embodiment, the agent of the present application contains crude drugs selected from the group consisting of leaves, fruits, and seeds of Pipturus albidus. As described above, the leaves of Pipturus albidus, which is a herbaceous plant native to Hawaii, are used as a material of a popular herb tea in Hawaii. In Hawaii, Pipturus albidus, particularly its fruits, have been historically used as a natural folk medicine for regulating blood sugar value, blood pressure, cholesterol value, alleviating stress or fatigue, or reducing inflammation (Non-patent literature 9: Rafii et al., Alzheimers Dement., (2022), 1-7). However, there is no known effect on cognitive disorders.

[0107] In addition, as the main components of the leaves of Pipturus albidus, three kinds of polyphenols, (+)-catechin, chlorogenic acid, and rutin, have been identified (Non-patent literature 10: Chun et al., Native Hawaiian medicines, First People's Productions Honolulu, (1994), 216-217). The contents of catechin and rutin in the leaves of Pipturus albidus are significantly higher than those in other commercially available tea leaves (the above Non-patent literature 10). It is suggested that these polyphenols have a beneficial effect on model mice or humans of neurodegenerative diseases.

[0108] Catechins are a kind of flavonoids, consisting of catechin itself and its derivatives, and are contained in a large amount in tea leaves. They exhibit anti-inflammatory and antioxidant effects by blocking the production of cytokines and inflammatory pathways, chelating metal ions, and removing free radicals (Non-patent Literature 11: Afzal et al., Molecules, (2022), 27

[21] :7604; and Non-patent Literature 12: Payne et al., Biomolecules, (2022), 12[3]:371). Catechins, particularly epigallocatechin-3-gallate, exhibit inhibition of the production of Aβ, Tau phosphorylation, and protein aggregation of amyloid, thereby preventing the decrease in cognitive function of AD and PD (Non-patent Literatures 11 and 12). Further, it has also been reported that the expression of brain-derived neurotrophic factor BDNF and neurogenesis are increased if administered orally to mice (Non-patent Literature 13: Li et al., Neuroscience, (2009), 159[4]:1208-15; and Non-patent Literature 14: Yoo et al., Phytother Res., (2010), 24[7]:1065-70). BDNF or neurogenesis is important for the repair or regeneration of the brain (Non-patent Literature 15: Colucci-D'Amato et al., Int. J. Mol. Sci., (2020), 21

[20] :7777; and Non-patent Literature 16: Horgusluoglu et al., Am. J. Med. Gene. B. Neuropsychiatr. Genet., (2017), 174[1]:93-112).

[0109] Green acid, also called 5-O-caffeoylquinic acid, is contained in a large amount in coffee beans. It has a variety of effects on health, such as anti-inflammatory effect, antioxidant effect, neuroprotective effect, liver-protective effect, heart-protective effect, chemopreventive effect, anti-diabetic effect, anti-obesity effect, suggesting that it reduces the risk of neurodegenerative diseases and improves cognitive function by regular intake (Non-patent Literature 17: Socala et al., Int. J. Mol. Sci., (2020), 22[1]:107). If it is orally administered to a model mouse of AD and PD, the memory and motor function of the mouse are improved (Non-patent Literature 18: Gao et al., Drug Des. Devel. Ther., (2020), 14: 1705-1716; and Non-patent Literature 19: Singh et al., Oxid. Med. Cell Longev., (2020), 2020:6571484), and if it is orally administered to a model rat of cerebral ischemia / reperfusion, it shows promotion of the expression of BDNF and nerve growth factor (NGF) (Non-patent Literature 20: Liu et al., Drug Des. Devel. Ther., (2020), 14:51-60).

[0110] Eriodictyol is a member of flavonols, which is a subtype of flavonoids, and is contained in various plants. It shows anti-inflammatory effect, antioxidant effect, and neuroprotective effect (Non-patent Literature 21: Tahir et al., Biomed. Pharmacother., (2021), 137:111253). If it is orally administered to a model mouse of AD, the Aβ and Tau oligomers are reduced, and the cognitive function is improved (Non-patent Literature 22: Xu et al., Behav. Brain Res., (2014), 264:173-80; and Non-patent Literature 23: Sun et al., J. Neuroinflammation, (2021), 18[1]:131). Further, it is also reported that if it is intraperitoneally administered to a rat into which Aβ is injected intracerebrally, the expression of BDNF is increased (Non-patent Literature 24: Moghbelinejad et al., Toxicol Lett., (2014), 224[1]:108-13).

[0111] As described above, regarding the three polyphenols contained in the leaves of Vatica astrocoronata, namely, catechin, green acid, and eriodictyol, effects such as improvement of cognitive function are suggested. However, effects such as improvement of cognitive function are completely unknown with respect to the leaves or fruits of Vatica astrocoronata.

[0112] Thus, the present inventors, as detailed in the Examples described later, fractionated leaves and fruits (containing seeds) from commercially available dried leaves of Camellia japonica, prepared respective hot water extracts and simple crushed powders thereof, and investigated effects thereof on cognitive function and neuropathology using four types of dementia model mice.

[0113] First, as a model of frontotemporal dementia-Tau (FTD-Tau), Tau784 mice (Non-patent Literature 25: Umeda et al., Am. J. Pathol., (2013), 183[1]:211-25; and Non-patent Literature 26: Umeda et al., Ann. Clin. Transl. Neurol., (2015), 2[3]:241-55) were used, and hot water extract of leaves of Camellia japonica (Leaf-ext) was administered at a dose of 1000 pg / day, 100 pg / day, and 30 pg / day for one month (Example 1). As a result, any one of cognitive function ( Figure 1 ), Tau pathology ( Figure 2 ), synaptophysin pathology ( Figure 3 ), and microglial cell pathology ( Figure 4 ) was significantly improved at all doses. Among them, in the case of administration at a dose of 1000 pg / day and 100 pg / day, each pathology was improved to a level equivalent to that of non-gene recombined mice of the same litter.

[0114] In addition, as a model of Alzheimer's dementia (AD), APP23 mice (Non-patent Literature 27: Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. U.S.A., (1997), 94

[24] :13287-92; Non-patent Literature 28: Van Dam et al., Eur. J. Neurosci., (2003), 17[2]:388-96; and Non-patent Literature 29: Umeda et al., Front. Neurosci., (2021), 15:763476) were used, and hot water extract of leaves of Camellia japonica (Leaf-ext) was administered at a dose of 100 pg / day for one month (Example 2). As a result, any one of cognitive function ( Figure 5 ), amyloid pathology ( Figure 6 ), synaptophysin pathology ( Figure 7 ), and microglial cell pathology ( Figure 8 ) was improved to a level equivalent to or close to that of non-gene recombined mice of the same litter.

[0115] In addition, as a model of Lewy body dementia (DLB), Huα-Syn (A53T) mice (Non-patent Literature 30: Lee et al., Proc. Natl. Acad. Sci. U.S.A., (2002), 99

[13] : 8968-73; and Non-patent Literature 31: Umeda et al., Int. J. Mol. Sci., (2021), 22:8453) were used, and hot water extract of the leaves of Alangium salicifolium (Leaf-ext) was administered at a dose of 100 μg / day for one month (Example 3). As a result, any one of cognitive function ( Figure 9 ), synuclein pathology ( Figure 10 and 11 ), synaptophysin pathology ( Figure 12 ), and microglial cell pathology ( Figure 13 ) was improved to the same level as or close to that of the non-gene recombined littermates.

[0116] In addition, as a model of frontotemporal lobar dementia-TDP (FTD-TDP), C9-500 mice (Non-patent Literature 32: Liu et al., Neuron, (2016), 90[3]:521-534; and Non-patent Literature 33: Hatanaka et al., Biomedicines, (2022), 10[5]:1080) were used, and hot water extract of the leaves of Alangium salicifolium (Leaf-ext) was administered at a dose of 100 μg / day for one month (Example 4). As a result, any one of cognitive function ( Figure 14 ), pathology caused by C9orf72 gene variation ( Figure 15 ), double-stranded RNA-dependent protein kinase (PKR) pathology ( Figure 16 ), synaptophysin pathology ( Figure 17 ), and microglial cell pathology ( Figure 18 ) was improved to the same level as or close to that of the non-gene recombined littermates. The above results suggest that the leaves of Alangium salicifolium have an improving effect on cognitive function or various pathologies related thereto.

[0117] In addition, as a model of frontotemporal lobar dementia-Tau (FTD-Tau), Tau784 mice (Non-patent Literatures 25 and 26 described above) were used, and hot water extract of the leaves of Alangium salicifolium (Leaf-ext), simple crushed powder of the leaves (Leaf-pwd), and simple crushed powder of the fruits (including seeds) (Fruit-pwd) were administered at a dose of 30 μg / day for one month (Example 5). As a result, any one of cognitive function ( Figure 19 ), Tau pathology ( Figure 20), synaptophysin pathology ( Figure 21 ), and BDNF expression ( Figure 22 ) were observed. Among them, in the simple pulverized powder of the leaves (Leaf-pwd) and the simple pulverized powder of the fruits (Fruit-pwd) of the Alstonia scholaris, each pathology was improved to the level equivalent to or close to that of the non-gene recombination mice. In particular, according to the simple pulverized powder of the fruits (Fruit-pwd) of the Alstonia scholaris, surprisingly, the cognitive function of the Tau784 mice was significantly enhanced to a higher level than that of the non-gene recombination mice. The above results suggest that the pulverized material of the leaves or the fruits (Fruit-pwd) of the Alstonia scholaris has a much superior effect on the improvement of cognitive function or the improvement of various pathologies compared to the extract.

[0118] In addition, as a model of Lewy body-type cognitive disorder (DLB), using the Huα-Syn(A53T) mice (non-patent documents 30 and 31 described above), the simple pulverized powder of the fruits (Fruit-pwd) of the Alstonia scholaris was administered at a dose of 100 μg / day for one month (Example 6). As a result, surprisingly, the level of neurogenesis in the dentate gyrus (DG) and the substantia nigra (SN) of the Huα-Syn(A53T) mice was improved to a level far exceeding that of the non-gene recombination mice. The above results suggest that the pulverized material of the fruits (Fruit-pwd) of the Alstonia scholaris has an effect of promoting the youth of the brain through the repair or regeneration of neurons.

[0119] In addition, in order to verify whether the above-mentioned effect on the improvement of cognitive function or the improvement of various pathologies possessed by the leaves or the fruits (Fruit-pwd) of the Alstonia scholaris is due to the effect of the three polyphenols contained in the leaves of the Alstonia scholaris, namely catechin, chlorogenic acid, and rutin, the following experiment was conducted. That is, as a model of frontotemporal lobe cognitive disorder-Tau (FTD-Tau), using the Tau784 mice (non-patent documents 25 and 26 described above), a mixture of catechin, chlorogenic acid, and rutin contained in an amount equivalent to 30 μg of the simple pulverized powder of the fruits (Fruit-pwd) of the Alstonia scholaris, or a catechin single product, was administered for one month. As a result, the cognitive function improvement effect of these catechin / chlorogenic acid / rutin mixtures and the catechin single product was much worse than that of the non-gene recombination mice, and the effect was incomplete. Therefore, it was found that the above-mentioned various effects possessed by the hot water extract of the leaves (Leaf-ext), the simple pulverized powder of the leaves (Leaf-pwd), and the simple pulverized powder of the fruits (Fruit-pwd) of the Alstonia scholaris are not due to only the aforementioned three polyphenols, but rather, are largely attributed to other unknown components contained in the Alstonia scholaris.

[0120] [Form of crude drug]

[0121] According to one embodiment, the leaves of Alangium salicifolium, the fruits of Alangium salicifolium, and the seeds of Alangium salicifolium can be used alone, the leaves of Alangium salicifolium can be used alone, the fruits of Alangium salicifolium can be used alone, the seeds of Alangium salicifolium can be used alone, or two or three of the leaves, the fruits, and the seeds of Alangium salicifolium can be used in combination.

[0122] The leaves and / or the fruits and / or the seeds of Alangium salicifolium can be used in any form. As examples of such forms, without limitation, the leaves and / or the fruits and / or the seeds of Alangium salicifolium can be used as they are, or can be used in the form of a crushed product and / or an extract.

[0123] Specifically, when the crude drug selected from the leaves, the fruits, and the seeds of Alangium salicifolium is made into the form of a crushed product such as a crushed powder, the processing conditions thereof are not particularly limited, and are exemplified as follows. First, the crude drug is subjected to drying treatment. The conditions of the drying treatment are not limited, and any of the publicly known various conditions can be used, and as examples, drying at a temperature of 0 to 100°C in the form of natural drying or heat drying, and the like can be cited. Then, the dried crude drug is subjected to crushing treatment. The means of the crushing treatment are not limited, and any of the publicly known various means can be used, and crushing by manual work, or crushing using a pulverizer in the form of wet-type or dry-type pulverization, and the like can be cited. The crushed product of the crude drug thus obtained can be used as it is, or a powder having a particle diameter controlled to be below a predetermined value can be obtained by sieving. For example, a sieve having a mesh width of 0.02 mm to 20 mm can be used to adjust the particle diameter.

[0124] In addition, when the crude drug selected from the leaves, the fruits, and the seeds of Alangium salicifolium is made into the form of an extract, the processing conditions thereof are not particularly limited, and for example, after the aforementioned crude drug is subjected to drying treatment and / or crushing treatment as appropriate, an extraction solvent is added to perform extraction. Specifically, 1 part by mass of the dried and crushed product of the crude drug is added with, for example, 1 to 100 parts by mass of an extraction solvent, and extraction is performed by keeping it for, for example, 0.1 to 100 hours. The extraction solvent is not particularly limited, and any of the publicly known various solvents can be used, and examples thereof can include water or a hydrophilic organic solvent such as ethanol. When extraction is performed using an extraction solvent, it is performed at ordinary temperature or with heating. For example, when water is used with heating, it can be heated at 30 to 100°C in a state where hot water is added to the crushed product. In addition, stirring treatment, ultrasonic treatment, heating reflux treatment, and the like can be used as necessary to improve the extraction efficiency. After the extraction is completed, the liquid component is separated from the solid component by manual work or a means such as filtration. The separated liquid component can be used as an extract. They can be used after the solvent is evaporated and concentrated as necessary.

[0125] [Effects of the agent]

[0126] According to one embodiment, the agent of the present application is an agent for maintaining or improving brain function. The "maintenance or improvement of brain function" as referred to in the present application means the maintenance or improvement of cognitive function and / or motor function, without limitation.

[0127] The "maintenance or improvement of cognitive function" as referred to in the present application means the prevention of or improvement in abnormalities such as memory impairment, disturbance of orientation, disturbance of judgment and understanding, disturbance of executive function, disturbance of behavior, disturbance of cognition, aphasia, and the like, which are observed in mild forgetfulness or cognitive disorders, without limitation.

[0128] The "maintenance or improvement of motor function" as referred to in the present application means the prevention of or improvement in abnormalities such as a decrease in motor function due to aging, Parkinson's symptoms such as tremor, muscle rigidity, disturbance of postural reflex, akinesia / hypokinesia, which are observed in Parkinson's disease, involuntary movement, which is observed in Huntington's disease, disturbance of coordinated movement, which is observed in spinocerebellar degeneration, muscle weakness, which is observed in amyotrophic lateral sclerosis, and the like, without limitation.

[0129] The maintenance or improvement of brain function by the agent of the present application can be evaluated, for example, as shown in the examples described later, by providing a cognitive function test such as a Morris water maze test or a motor function test such as a rotarod test, or observing a brain section after staining, after administration of the agent to a model animal in which a brain pathology is developing.

[0130] According to one embodiment, the agent of the present application is an agent for promoting repair of neural cells or inducing neurogenesis. The "promotion of repair of neural cells" as referred to in the present application can be exemplified by restoration of the number or function of synapses, restoration of the function of neural cells, promotion of maintenance and restoration of the function of synapses or neural cells, and enhancement of expression of brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF) or the like, which protect neural cells from various stresses, without limitation. The "induction of neurogenesis" as referred to in the present application means the induction of reappearance of immature neural cells actively performing DNA synthesis, without limitation. As an index of DNA synthesis, active uptake of a nucleic acid analogue such as BrdU or the like can be exemplified. As a marker of immature neural cells, expression of doublecortin or the like can be exemplified. The promotion of repair of neural cells or the induction of neurogenesis by the agent of the present application can be evaluated, for example, as shown in the examples described later, by observing induction of expression of brain-derived neurotrophic factor (BDNF) or observing newly born neurons by staining a brain section, after administration of the agent to a model animal in which a cognitive disorder or a brain pathology is developing.

[0131] According to one embodiment, the agent of the present application is an agent for removing a neurodegenerative disease-causing protein accumulated in the brain. The "neurodegenerative disease-causing protein" referred to in the present application means a protein identified or presumed to be a causative substance of cognitive disorder. Examples thereof include, but are not limited to, one or two or more proteins selected from the group consisting of β-amyloid (Aβ), Tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein produced by RAN (repeat-associated non-ATG) translation, prion protein, SOD-1, and the like. The effect of the agent of the present application to maintain or improve brain function can be evaluated, for example, as shown in the examples described later, by observing the brain sections after staining following administration of the agent to a model animal in which cognitive disorder is induced.

[0132] According to one embodiment, the agent of the present application is an agent for treating or preventing a neurodegenerative disease. The "neurodegenerative disease" referred to in the present application means a neurodegenerative disease in which any of the aforementioned neurodegenerative disease-causing proteins is accumulated in the brain. Examples of the neurodegenerative disease are not limited, and include Alzheimer's disease (Aβ, Tau), frontotemporal lobar cognitive disorder (Tau, TDP-43, FUS / TLS), Lewy body cognitive disorder (α-synuclein), Parkinson's disease (α-synuclein), multiple system atrophy (α-synuclein), Huntington's disease (polyglutamine), amyotrophic lateral sclerosis (TDP-43, FUS / TLS, RAN protein, SOD-1), spinocerebellar degeneration (polyglutamine, RAN protein), Creutzfeldt-Jakob disease (prion protein), and the like (in addition, examples of the neurodegenerative disease-causing protein that can be seen to be accumulated in each neurodegenerative disease are shown in parentheses). Among them, the agent for treating or preventing a neurodegenerative disease of the present application is preferably an agent for treating or preventing degenerative cognitive disorder. Examples of the degenerative cognitive disorder are not limited, and include Alzheimer's disease, frontotemporal lobar cognitive disorder, Lewy body cognitive disorder, cognitive disorder caused by Parkinson's disease or amyotrophic lateral sclerosis, and the like. Among them, if the frontotemporal lobar cognitive disorder caused by tau protein accumulation is further subdivided, Pick's disease, corticobasal degeneration, progressive supranuclear palsy, and the like can be exemplified. The effect of the agent of the present application to treat or prevent a neurodegenerative disease can be evaluated, for example, as shown in the examples described later, by providing a cognitive function test such as Morris water maze test or a motor function test such as rotarod test, or observing the brain sections after staining following administration of the agent to a model animal in which a neurodegenerative disease such as degenerative cognitive disorder is induced.

[0133] [Formulation, usage and dosage of the agent]

[0134] Although the dosage form of the agent of the present application is not particularly limited, it can be used, for example, in the form of crude drug of leaves, fruits and seeds of Grevillea robusta, in the form of a crushed material, in the form of an extract of the crushed material, in the form of an extract residue of the crushed material, or the like, directly or in combination with other ingredients such as a desired excipient and / or carrier. In addition, when used in the form of a food (food of the present application) or a medicine (medicine of the present application) containing the agent of the present application, the leaves and / or fruits and / or seeds of Grevillea robusta as the effective ingredient of the agent of the present application can be used in combination with other ingredients in the form of the corresponding food or medicine. Details will be described later.

[0135] Although the use of the agent of the present application is not particularly limited, it is usually administered orally. In particular, the agent of the present application is preferably used in the form of a food (food of the present application) or an oral medicine (medicine of the present application). Details will be described later.

[0136] Although the amount of the agent of the present application is not particularly limited, the leaves and / or fruits and / or seeds of Grevillea robusta as the effective ingredient of the agent of the present application can be administered to a subject in an amount of usually 0.01 mg / day or more, or 0.1 mg / day or more, further 0.5 mg / day or more, and still further 1.0 mg / day or more, and in an amount of usually 10 g / day or less, further 5 g / day or less, and still further 1 g / day or less.

[0137] [Food]

[0138] According to one aspect of the present application, there is provided a food (food of the present application) containing the agent of the present application. The food of the present application is characterized by containing any one of the agents of the present application, and can be used for the use of the agent.

[0139] The food of the present application can be formed in any form that can be taken orally, such as a solution, a suspension, an emulsion, a powder, a solid shaped product, or the like. In addition, it can be shaped into a dosage form such as a capsule, a tablet, a syrup, a granule, or the like, in the same manner as the medicine of the present application described later.

[0140] The food of the present application can be manufactured in the form of, for example, a beverage such as tea, black tea, coffee, a cold drink, an alcoholic beverage, a carbonated beverage, a milk beverage, a juice beverage, a nutritional beverage, a concentrated beverage, a powdered beverage (juice powder, soup powder, or the like), or the like; a supplement; a confectionery such as a candy, a gummy candy, chewing gum, chocolate, a cookie, a biscuit, or the like; an ice product such as ice cream; a dairy product such as yogurt, processed milk, or the like; a wheat flour product such as a cereal, a bread, a cake mix, or the like; a noodle such as buckwheat noodles; an oil processed product such as mayonnaise, fresh cream, salad dressing, or the like; a marine processed product; a livestock processed product; an agricultural processed product. In manufacturing these foods, the agent of the present application can be contained by adding the agent of the present application, and the food of the present application can be manufactured.

[0141] In the food of the present application, in addition to other food materials, a sweetening agent, a coloring agent, a preservative, a thickening agent, a stabilizer, a gelling agent or a paste, an antioxidant such as ascorbic acid, a color-developing agent, a bleaching agent, a mold-preventing agent or an insect-preventing agent, a yeast food, a gum base, an alkali water, a bittering agent, an enzyme, a gloss agent, a flavoring agent, a souring agent, a chewing gum softener, a seasoning, a coagulating agent for tofu, an emulsifying agent, a pH adjusting agent, a leavening agent, a nutritional supplement such as a vitamin, a mineral, an amino acid, and the like, and a manufacturing agent, or the like can be added as needed.

[0142] The food of the present application can also be provided as a food in which the effects, the advantages, the functions, or the uses of the agent of the present application are indicated under the systems of each country. For example, in Japan, the food of the present application can also be produced as a health food (a specific health food, a functionally labeled food, a nutritionally functional food).

[0143] The content of the leaves and / or the fruits and / or the seeds of Gomphna leaf and / or Gomphna fruit and / or Gomphna seed as the effective ingredient of the agent of the present application in the food of the present application can be appropriately set in the range in which the effects of the present application can be obtained, similarly to the agent of the present application. Specifically, the content of the leaves and / or the fruits and / or the seeds of Gomphna leaf and / or Gomphna fruit and / or Gomphna seed as the effective ingredient of the agent of the present application can be adjusted to be generally 0.01 mg / day or more, or 0.1 mg / day or more, wherein 0.5 mg / day or more, further 1.0 mg / day or more, and in addition, generally 10 g / day or less, wherein 5 g / day or less, further 1 g / day or less.

[0144] The number of times and the frequency of ingestion of the food of the present application are arbitrary, and can be appropriately set to be any number of times and frequency of 1 to several times a day, daily, every other day, every 2 days, or 1 to 7 days a week. The food of the present application can be provided by admixing the amount of the leaves and / or the fruits and / or the seeds of Gomphna leaf and / or Gomphna fruit and / or Gomphna seed required to obtain the effects of the agent of the present application in the food according to the number of times and the frequency of ingestion required, and the food of the present application can be expected to obtain the desired effects according to the kind of the desired effects.

[0145] The food of the present application is extremely useful because it is characterized by containing the agent of the present application, and shares various advantages of the agent of the present application. In addition, it is a food, and can be safely and easily used not only for patients suffering from a specific disease, but also for healthy persons.

[0146] In addition, the food of the present application can be used for animals other than humans who can use the agent of the present application.

[0147] [Pharmaceuticals]

[0148] According to one aspect of the present application, a pharmaceutical (a pharmaceutical of the present application) containing the agent of the present application is provided. The pharmaceutical of the present application is characterized by containing any one of the agents of the present application, and can be used for the uses of the agent.

[0149] The route of administration of the medicament of the present application is not limited, and is usually oral administration of a medicine for internal use (oral agent).

[0150] The medicament of the present application is a medicine to which the agent of the present application is added as an effective ingredient, and can be formulated into any dosage form such as a capsule, a tablet, a granule, a powder, a syrup, a suspension, a liquid for internal use, and the like. In this case, the medicament of the present application can be formulated into a pharmaceutical composition in which the agent of the present application is combined with other additives usually used in the manufacture of oral agents, by a method for manufacturing a medicine known to those skilled in the art.

[0151] The additives used in the manufacture of the medicament of the present application can be exemplified by, for example, excipients, disintegrants, binders, lubricants, coating agents, dispersants, flowing agents, stabilizers, preservatives, buffers, corrigents, suspending agents, emulsifying agents, odorants, solubilizers, colorants, thickening agents, and the like. In addition, by further combining a pharmaceutically acceptable carrier, a medicament of the present application can be provided which can further improve the effects and advantages of the agent of the present application.

[0152] The medicament of the present application can also contain a product which is in conformity with the standards for pharmaceutical products under the regulations of each country. Examples thereof can be exemplified by, for example, a quasi-drug in Japan.

[0153] The content of the leaves and / or fruits and / or seeds of Gomphna leaf and / or fruit and / or seed as the effective ingredient of the agent of the present application in the medicament of the present application can be appropriately set within a range in which the effects of the present application can be obtained, similarly to the agent of the present application. Specifically, the content of the leaves and / or fruits and / or seeds of Gomphna leaf and / or fruit and / or seed as the effective ingredient of the agent of the present application can be adjusted to be usually 0.01 mg / day or more, or 0.1 mg / day or more, and more specifically 0.5 mg / day or more, and further 1.0 mg / day or more, and also usually 10 g / day or less, and more specifically 5 g / day or less, and further 1 g / day or less.

[0154] The number of times and frequency of taking the medicament of the present application are arbitrary, and can be appropriately set to be any number of times and frequency of 1 to several times a day, daily, every other day, every 2 days, or 1 to 7 days a week. According to the number of times and frequency of taking desired, by formulating the leaves and / or fruits and / or seeds of Gomphna leaf and / or fruit and / or seed in an amount required to obtain the effects of the agent of the present application, the administration of the present application can be provided which is expected to obtain the desired effects according to the kind of the effects desired.

[0155] The medicament of the present application is extremely useful because it is characterized by containing the agent of the present application, and shares various advantages of the agent of the present application.

[0156] In addition, the medicament of the present application can be used for animals other than humans, in addition to humans, to which the agent of the present application can be applied.

[0157] [Method]

[0158] According to an aspect of the present application, there is provided a method for maintaining or improving brain function, a method for promoting repair of neural cells or inducing neurogenesis, a method for removing a neurodegenerative disease-causing protein accumulated in the brain, or a method for treating or preventing a neurodegenerative disease (a method of the present application) by administering to a subject one or two or more selected from crude drugs of leaves, fruits, and seeds of Grevillea robusta, a preparation of the present application, a food of the present application, and a medicament of the present application.

[0159] The method of the present application is to administer to a subject leaves and / or fruits and / or seeds of Grevillea robusta as an effective ingredient of a preparation of the present application, in the form of a preparation of the present application, a food of the present application, and / or a medicament of the present application. Details thereof are described above in the description of the preparation of the present application, the food of the present application, and the medicament of the present application.

[0160] Examples

[0161] The present application is described in more detail below according to examples, which are merely illustrative examples for convenience of illustration, and the present application is not limited in any sense to these examples.

[0162] Materials and methods

[0163] Preparation of hot water extract and non-extraction simple pulverized powder of leaves and fruits of Grevillea robusta

[0164] Dried tea leaves of Grevillea robusta were purchased from Nakihalani Farm, LLC (Hawaii, USA). Hot water extract of leaves of Grevillea robusta was prepared at TechnoPro L&D Co., Ltd. (Tokyo, Japan). 250 g of tea leaves of Grevillea robusta was added to 3 L of water and soaked for 1 hour. After boiling for 15 minutes while stirring, the mixture was left to stand for 24 hours. After removing the tea leaves, suction filtration was performed using filter paper with a pore size of 8 μm. The filtrate was concentrated to 500 mL using an evaporator while heating at 40°C. The concentrated solution was freeze-dried to obtain 66 g of extract powder. Non-extraction simple pulverized powder of leaves and fruits of Grevillea robusta was prepared in the laboratory. Since a small amount of fruits (including seeds) was mixed in the package of commercially available tea leaves of Grevillea robusta, the tea leaves and fruits were separated by hand work and pulverized to particles of 20-50 μm using a grinder (Fine Powder Mill FM-100, Labonect, Sakai, Japan), respectively. These particles were collected as non-extraction simple pulverized powder.

[0165] Component analysis of Grevillea robusta

[0166] It is known that the leaves of the whiteleaf acronychia contain catechin, chlorogenic acid, and rutin as main components (Non-patent Literature 10: Chun et al., Native Hawaiian medicines, First People's Productions Honolulu, (1994), 216-217). The whiteleaf acronychia specimen prepared as above was sent to the Japan Food Research Laboratories (Tokyo, Japan) to measure the contents of these polyphenols.

[0167] In the case of catechin, 0.4 g of the material was suspended in 30 mL of a mixture of methanol and oxalic acid (8:2) and extracted by shaking for 10 minutes. After centrifugal separation, the supernatant was recovered and the precipitate was further extracted twice with methanol. The supernatants of the three extractions were combined to a total of 100 mL. The extract was diluted and separated by high-performance liquid chromatography (HPLC) using a reverse-phase Inert Sustain C18 column (GL Sciences, Tokyo, Japan) with a mobile phase of a mixture of 0.1% acetic acid and acetonitrile (89:11). The elution fractions were continuously analyzed by electrospray ionization (ESI)-mass spectrometry (MS) using an Xevo TQ MS (Waters Corporation, Milford, Massachusetts).

[0168] In the case of chlorogenic acid, 0.2 g of the material was suspended in 80 mL of a mixture of methanol and 0.02 M perchloric acid (1:9) and extracted by shaking for 10 minutes. After centrifugal separation, the supernatant was recovered and the precipitate was further extracted twice with methanol. The supernatants of the three extractions were combined to a total of 250 mL. The extract was separated by HPLC using a reverse-phase CAPCELL PAK C18 ACR column (Osaka Soda, Osaka, Japan) with a mobile phase of a mixture of water, acetonitrile, and phosphoric acid (920:80:2). The absorbance of the elution fractions at 325 nm was measured.

[0169] Finally, in the case of rutin, 0.4 g of the material was suspended in 60 mL of a mixture of methanol and 2.5% acetic acid (8:2) and extracted by shaking for 10 minutes. After centrifugal separation, the supernatant was recovered and the precipitate was further extracted twice with methanol. The supernatants of the three extractions were combined to a total of 200 mL. The extract was diluted and separated by HPLC using a reverse-phase Unison UK-C18 column (Imtakt USA, Portland, Oregon, USA) with a mobile phase of a mixture of water containing 0.4% citric acid, acetonitrile, and 2-propanol (200:38:2). The absorbance of the elution fractions at 360 nm was measured.

[0170] • Mice

[0171] Four different mouse models of neurodegenerative cognitive disorders were used.

[0172] Tau784 mice are a model of FTD in which both 3-repeat and 4-repeat human Tau are expressed at adult age due to the presence of a Tau intron mutation, with 4-repeat human Tau being predominantly expressed (Non-patent Literature 25: Umeda et al., Am. J. Pathol., (2013), 183[1]:211-25; and Non-patent Literature 26: Umeda et al., Ann. Clin. Transl. Neurol., (2015), 2[3]:241-55). According to previous studies by the present inventors et al., these mice show Tau hyperphosphorylation, Tau oligomer formation, synapse loss, and memory impairment at 6 months after birth, show microglial cell activation at 12 months after birth, and show neurofibrillary tangle formation and neuronal loss at 15 months after birth due to the imbalance of Tau isoforms.

[0173] APP23 mice are a model of AD in which human APP having a Swedish (KM670 / 671NL) mutation is expressed (Non-patent Literature 27: Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. U.S.A., (1997), 94

[24] :13287-92; Non-patent Literature 28: Van Dam et al., Eur. J. Neurosci., (2003), 17[2]:388-96; and Non-patent Literature 29: Umeda et al., Front. Neurosci., (2021), 15:763476). These mice show memory impairment at 3 months after birth. According to previous studies by the present inventors et al., these mice show Aβ oligomer accumulation, synapse loss, and amyloid deposition at 15 months after birth.

[0174] Huα-Syn(A53T) system G2-3 mice are made as a model of PD in which human α-synuclein originally having an A53T mutation is expressed (Non-patent Literature 30: Lee et al., Proc. Natl. Acad. Sci. U.S.A., (2002), 99

[13] :8968-73; and Non-patent Literature 31: Umeda et al., Int. J. Mol. Sci., (2021), 22:8453). According to previous studies by the present inventors et al., these mice show α-synuclein oligomer accumulation from 4 months after birth, show cognitive impairment at 6 months after birth, and show motor dysfunction at 9 months after birth. Thus, up to 9 months after birth can be considered a model of DLB.

[0175] C9-500 mice are FTD / ALS model mice into which a human full-length C9orf72 gene is introduced (Non-patent Literature 32: Liu et al., Neuron, (2016), 90[3]:521-534; and Non-patent Literature 33: Hatanaka et al., Biomedicines, (2022), 10[5]:1080). They have a variation of about 500 times abnormal elongation of the GGGGCC sequence of intron 1a (hexanucleotide repeat expansion, HRE), which is reported to exhibit various pathologies including TDP-43 (Non-patent Literature 32 above). We confirmed that the mice showed accumulation of RNA G-quadruplex, dipeptide repeat protein (DPR) such as RNA foci-forming dipeptide repeat proteins (polyGA or polyGP), phosphorylated TDP-43, and the like at 3 months of age, and showed synapse loss, neuron loss, microglial cell activation at 6 months of age (Non-patent Literature 33 above). Incidentally, DPR is produced by repeat-associated ATG-independent translation (RAN translation) controlled by double-stranded RNA-dependent protein kinase (PKR). The cognitive function of the mice started to decrease at 4.5 months of age, but the motor function remained normal at 12 months of age (Non-patent Literature 33 above). That is, the mice can be considered as a model of FTD-TDP up to 12 months of age.

[0176] All transgenic (Tg) mice were maintained as heterozygous animals and used. All animal experiments were approved by the Ethics Committee of Osaka Prefecture University (Osaka, Japan) and conducted in accordance with the Guidelines for Animal Experiments of Osaka Prefecture University.

[0177] • Handling of mice

[0178] To investigate the effects of the hot water extract of the leaves of A. alniphyllus, the extract powder was suspended in water by ultrasonic treatment at concentrations of 3.33, 0.33, and 0.10 mg / mL. Each 300 μL of the suspensions (containing 1,000, 100, and 30 μg of the powder) was orally administered to male and female Tau784 mice for 1 month, 5 days a week (Monday to Friday). As a control group, the same amount of water was administered to Tg and non-Tg littermates of the same age. For APP23, Huα-Syn (A53T), and C9-500 mice, 0.33 mg / mL of the suspension (100 μg of the powder / 300 μL) was orally administered to male and female mice for 1 month.

[0179] To compare the effects of the hot water extract of the leaves of A. alniphyllus, the simple crushed powder of the leaves of A. alniphyllus, and the simple crushed powder of the fruits of A. alniphyllus, each powder was suspended in water by ultrasonic treatment at a concentration of 0.10 mg / mL. Each 300 μL of the suspensions (containing 30 μg of the powder) was orally administered to Tau784 mice for 1 month.

[0180] To investigate the effects of polyphenols contained in the white bark of the Japanese pagodatree, catechin, chlorogenic acid, and rutin (all from Fujifilm-Wako, Osaka, Japan) were placed in a tube and dissolved in water to make a mixture. The concentrations were prepared to be 0.29, 0.12, and 0.41 μg / mL, respectively. 300 μL of the mixture (containing 0.087 μg of catechin, 0.036 μg of chlorogenic acid, and 0.123 μg of rutin) was orally administered to Tau784 mice for 1 month. The administration amount of these polyphenols was equivalent to the amount contained in 30 μg of simple crushed powder of the fruit. A solution containing only catechin (0.087 μg / 300 μL) was also administered to Tau784 mice.

[0181] • Behavioral test

[0182] The spatial reference memory of the mice was evaluated by the Morris water maze test (non-patent literature 34: Kelley Bromley-Brits et al., J. Vis. Exp., (2011), 53e2920). In one place of a circular pool with a diameter of 1 m, a platform with a diameter of 10 cm was set at a depth of 1 cm below the water surface in such a manner that it could not be seen from the side of the mouse. The mouse was placed in the pool and allowed to swim for 60 seconds, and the time until it swam to the platform as hard as it could (expressed as escape latency) was measured. If the platform was not reached within 60 seconds, the experimenter placed the mouse on the platform and allowed it to rest for a while. This trial was performed for each mouse 5 times a day with an interval of 5 minutes, and was performed continuously for 4 days. The ability to acquire memory was thus determined.

[0183] • Histological analysis of neuropathology

[0184] After the behavioral test, the mice in each group were divided into two groups, one group for histological analysis and the other group for future biochemical analysis. The mice for histological analysis were fixed by perfusion with 4% paraformaldehyde, and brain sections were prepared according to the literature (Non-patent literature 27: Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. U.S.A., (1997), 94

[24] : 13287-92; and Non-patent literature 33: Hatanaka et al., Biomedicines, (2022), 10[5]: 1080), and neuropathology was performed by immunohistochemical staining. The antibodies used in immunohistochemistry are shown below. The evaluation of pathology was performed by quantifying the staining intensity or staining area in a certain range of brain regions using NIH ImageJ software. Neuroinflammation (Non-patent literature 35: Muzio et al., Front Neurosci., (2021), 24

[15] : 742065) was evaluated by counting the number of Iba-1-positive activated microglial cells in a certain range of brain regions. The correspondence of each neuropathology and the antibody used for detection is shown in the following table.

[0185] Table 1

[0186] · Histological analysis of BDNF expression and neurogenesis

[0187] Tau784 mice were evaluated for BDNF expression by administering three samples of white oak bark at 30 μg / day for one month. Brain sections were stained with an anti-BDNF antibody (GTX132621; GeneTex, Irvine, CA), and the staining intensity in a certain range of brain regions was quantified using NIH ImageJ software.

[0188] Neurogenesis was evaluated using aged Huα-Syn(A53T) mice. A simple suspension of crushed powder of white oak bark fruit was administered to the mice orally at 30 μg / day for one month. A thymidine analog, 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich), which is selectively incorporated into the DNA of proliferating cells, was dissolved in Tris-buffered physiological saline, pH 7.6, at 5 mg / mL, and 300 μL (containing 1.5 mg) of this solution was intraperitoneally administered to the mice daily for the last 5 days of white oak bark administration. Brain sections were double-stained with an anti-BrdU mouse monoclonal antibody (IBL) and an anti-doublecortin rabbit polyclonal antibody (Abeam). Cells positive for both of these antibodies were considered to be newly generated neurons, and their number was counted in a certain range of brain regions.

[0189] · Statistical analysis

[0190] For comparison of the average among 3 or more groups, ANOVA or 2-factor repeated measures ANOVA (for the locomotor test) was used, followed by Fisher's PLSD test. When p value < 0.05, it was judged to have a significant difference.

[0191] Example 1. Investigation of the effect of hot water extract of leaves of Alangium salicifolium (Leaf-ext) on Tau784 mice

[0192] The effect of hot water extract of leaves of Alangium salicifolium (Leaf-ext) on Tau784 mice was investigated. To 12-14 month-old Tau784 mice (average body weight 31.8 g), hot water extract of leaves of Alangium salicifolium (Leaf-ext) powder was orally administered at 1000 μg / day, 100 μg / day, or 30 μg / day for 1 month. To Tau784 mice and littermate non-gene recombinant mice of the same age, the same amount of water was administered as a control.

[0193] Figure 1 A graph showing the results of the Morris water maze test of Tau784 mice (in the table, "Tg + Leaf-ext") to which hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered for 1 month in comparison with Tau784 mice (in the table, "Tau784") to which water was administered as a subject. Figure 1 A is a graph showing the results of the group with the dose of 1000 μg / day and 100 μg / day, Figure 1 B is a graph showing the results of the group with the dose of 30 μg / day. From these results, it is clear that the memory of Tau784 mice was dose-dependently improved by the administration of hot water extract of leaves of Alangium salicifolium (Leaf-ext). In particular, in the group with the dose of 1000 μg / day and 100 μg / day, the memory of Tau784 mice was improved to the same level as that of littermate non-gene recombinant mice.

[0194] Figure 2 A graph showing the Tau pathology in the olfactory cortex of Tau784 mice (in the table, "Tau784 + Leaf-ext") to which hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered for 1 month with the dose of 100 μg / day in comparison with Tau784 mice (in the table, "Tau784") to which water was administered as a subject. Figure 2 A is a photograph showing the staining results of phosphorylated Tau and Tau oligomers, Figure 2 B is a graph showing the results of quantifying the staining intensity of each photograph. From these results, it is clear that the levels of phosphorylated Tau and Tau oligomers in Tau784 mice were significantly reduced by the administration of hot water extract of leaves of Alangium salicifolium (Leaf-ext).

[0195] Figure 3 A graph showing synaptophysin pathology in the hippocampal CA2 / 3 region of Tau784 mice ("Tau784" in the table) to which a hot water extract of the leaves of Distylium racemosum (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with Tau784 mice ("Tau784") to which water was administered and non-gene recombinant mice ("non-Tg") in the table. Figure 3 A is a photograph showing the results of staining of synaptophysin, Figure 3 B is a graph showing the results of quantifying the staining intensity of each photograph. As is apparent from these results, the synaptophysin level in the hippocampal CA2 / 3 region of Tau784 mice was improved to the same level as that of non-gene recombinant mice of the same litter by administration of the hot water extract of the leaves of Distylium racemosum (Leaf-ext).

[0196] Figure 4 A graph showing microglial cell pathology in the hippocampus (HC) and cerebral cortex (CTX) of Tau784 mice ("Tau784" in the table) to which a hot water extract of the leaves of Distylium racemosum (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month in comparison with Tau784 mice ("Tau784") to which water was administered and non-gene recombinant mice ("non-Tg") in the table. Figure 4 A is a photograph showing the results of staining of activated microglial cells, Figure 4 B is a graph showing the number of positive cells in each photograph. As is apparent from these results, the level of activated microglial cells in the hippocampus (HC) and cerebral cortex (CTX) of Tau784 mice was improved to the same level as that of non-gene recombinant mice of the same litter by administration of the hot water extract of the leaves of Distylium racemosum (Leaf-ext).

[0197] Example 2. Investigation of the effect of a hot water extract of the leaves of Distylium racemosum (Leaf-ext) on APP23 mice

[0198] The effect of a hot water extract of the leaves of Distylium racemosum (Leaf-ext) on APP23 mice was investigated. A hot water extract of the leaves of Distylium racemosum (Leaf-ext) powder was orally administered at a dose of 100 μg / day to 14-17 month-old APP23 mice (average body weight 30.1 g) for 1 month. The same amount of water was administered to APP23 mice and non-gene recombinant mice of the same litter of the same age as controls.

[0199] Figure 5This graph illustrates the results of the Morris water maze test in APP23 mice ("Tg+Leaf-ext") administered orally at a dose of 100 μg / day for one month, compared to APP23 mice administered water ("Tg" in the table) and non-recombinant mice ("Non-Tg" in the table). These results clearly demonstrate that administration of the hot water extract of *L. leucantha* leaves (Leaf-ext) improved the memory of APP23 mice to a level comparable to that of their littermate non-recombinant mice (Non-Tg).

[0200] Figure 6 The figure shows the pathology of amyloid protein in the cerebral cortex and hippocampus of APP23 mice (“APP23+Leaf-ext”) that were orally administered a hot water extract of the leaf of *Alternanthera philoxeroides* (Leaf-ext) at a dose of 100 μg / day for one month, compared with APP23 mice administered water (“APP23” in the table). Figure 6 Photograph A shows the staining results of amyloid protein deposition and Aβ oligomers. Figure 6 Figure B shows the results of quantifying the staining intensity of each photograph. These results clearly demonstrate that application of the hot water extract of *Leaf-ext* leaves significantly reduced amyloid deposition and Aβ oligomer levels in APP23 mice.

[0201] Figure 7 A diagram showing the pathology of synaptic proteins in the CA2 / 3 region of the hippocampus in APP23 mice (“APP23+Leaf-ext”) that were orally administered a hot water extract of the leaves of *Alternanthera philoxeroides* (Leaf-ext) for one month at a dose of 100 μg / day, compared with APP23 mice administered water (“APP23” in the table) and non-recombinant mice (“non-Tg” in the table). Figure 7 Photo A shows the staining results of synaptic vesicle proteins. Figure 7 Figure B shows the results of quantifying the staining intensity of each photograph. These results clearly demonstrate that application of the hot water extract of *Leaf-ext* leaves improved the synaptic vesicle protein levels in the CA2 / 3 region of the hippocampus in APP23 mice to levels comparable to those in their littermates (non-recombinant mice).

[0202] Figure 8A is a photograph showing the staining results of activated microglia cells, B is a graph showing the number of positive cells in each photograph. From these results, it is clear that the number of activated microglia cells in the hippocampus (HC) and cerebral cortex (CTX) of the APP23 mice was improved to the same level as the littermates of the non-gene recombination mice by administration of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext). Figure 8 A is a photograph showing the staining results of activated microglia cells, Figure 8 B is a graph showing the number of positive cells in each photograph. From these results, it is clear that the number of activated microglia cells in the hippocampus (HC) and cerebral cortex (CTX) of the APP23 mice was improved to the same level as the littermates of the non-gene recombination mice by administration of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext).

[0203] Example 3. Investigation of the effect of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext) on Huα-Syn(A53T) mice

[0204] The effect of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext) on Huα-Syn(A53T) mice was investigated. The hot water extract of the leaves of Alangium salicifolium (Leaf-ext) powder was administered orally at a dose of 100 μg / day for 1 month to 7-8 month-old Huα-Syn(A53T) mice (average body weight 28.4 g). The same amount of water was administered to the same age Huα-Syn(A53T) mice and littermates of the non-gene recombination mice as a control.

[0205] Figure 9 A is a photograph showing the staining results of activated microglia cells, B is a graph showing the number of positive cells in each photograph. From these results, it is clear that the number of activated microglia cells in the hippocampus (HC) and cerebral cortex (CTX) of the APP23 mice was improved to the same level as the littermates of the non-gene recombination mice by administration of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext).

[0206] Figure 10 and 11Figure 1 is a graph showing the pathology of alpha-synuclein in the hippocampus (HC) and entorhinal cortex (EC) of Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which a hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which water was administered. Figure 10 A is a photograph showing the results of staining of phosphorylated alpha-synuclein, Figure 11 A is a photograph showing the results of staining of alpha-synuclein oligomers, Figure 10 B and Figure 11 B is a graph showing the results of quantification of the staining intensity of each photograph. As is apparent from these results, the levels of phosphorylated alpha-synuclein and alpha-synuclein oligomers in Huα-Syn(A53T) mice were significantly reduced by administration of the hot water extract of leaves of Alangium salicifolium (Leaf-ext).

[0207] Figure 12 Figure 2 is a graph showing the pathology of synaptophysin in the hippocampal CA2 / 3 region of Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which a hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which water was administered and non-genetically recombinant mice (in the table, "non-Tg"). Figure 12 A is a photograph showing the results of staining of synaptophysin, Figure 12 B is a graph showing the results of quantification of the staining intensity of each photograph. As is apparent from these results, the level of synaptophysin in the hippocampal CA2 / 3 region of Huα-Syn(A53T) mice was improved to the same level as that of littermates of non-genetically recombinant mice by administration of the hot water extract of leaves of Alangium salicifolium (Leaf-ext).

[0208] Figure 13 Figure 3 is a graph showing the pathology of microglial cells in the hippocampus (HC) and cerebral cortex (CTX) of Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which a hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which water was administered and non-genetically recombinant mice (in the table, "non-Tg"). Figure 13 A is a photograph showing the results of staining of activated microglial cells, Figure 13B is a graph showing the number of positive cells of each photograph. From these results, it is apparent that the activated microglial cells in the hippocampus (HC) and cerebral cortex (CTX) of Huα-Syn(A53T) mice were improved to the same level as that of the littermates non-gene recombinant mice by administration of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext).

[0209] Example 4. Investigation of the effect of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext) on C9-500 mice

[0210] The effect of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext) on C9-500 mice was investigated. The hot water extract of the leaves of Alangium salicifolium (Leaf-ext) powder was orally administered at 100 μg / day to 9-10 month-old C9-500 mice (average body weight 29.2 g) for 1 month. The same amount of water was administered to C9-500 mice and littermates non-gene recombinant mice of the same age as controls.

[0211] Figure 14 A graph showing the results of the Morris water maze test of C9-500 mice (Table "Tg + Leaf-ext") to which the hot water extract of the leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with C9-500 mice (Table "C9-500") to which water was administered as a subject. From these results, it is apparent that the memory of C9-500 mice was improved to a level close to that of littermates non-gene recombinant mice (non-Tg) by administration of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext).

[0212] Figure 15 A graph showing the pathology caused by C9orf72 gene variation in the prefrontal cortex (PFC) of C9-500 mice to which the hot water extract of the leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with C9-500 mice (Table "C9-500") to which water was administered as a subject. Figure 15 A is a photograph showing the results of staining of RNA G-quadruplex, polyGA, polyGP, and phosphorylated TDP-43, Figure 15 B is a graph showing the results of quantifying the staining intensity of each photograph. From these results, it is apparent that the level of pathology caused by C9orf72 gene variation in the prefrontal cortex (PFC) of C9-500 mice was significantly reduced by administration of the hot water extract of the leaves of Alangium salicifolium (Leaf-ext).

[0213] Figure 16A graph showing the pathology of double-stranded RNA-dependent protein kinase (PKR) in the prefrontal cortex (PFC) of C9-500 mice ("C9-500") to which a hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with C9-500 mice ("C9-500") to which water was administered and non-genetically recombinant mice ("Non-Tg") to which water was administered. Figure 16 A is a photograph showing the results of staining of phosphorylated PKR, Figure 16 B is a graph showing the results of quantifying the staining intensity of each photograph. As is apparent from these results, by administering the hot water extract of leaves of Alangium salicifolium (Leaf-ext), the PKR level of the prefrontal cortex (PFC) of C9-500 mice was reduced to a level equivalent to that of littermate non-genetically recombinant mice.

[0214] Figure 17 A graph showing the pathology of synaptophysin in the hippocampal CA2 / 3 region of C9-500 mice ("C9-500") to which a hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with C9-500 mice ("C9-500") to which water was administered and non-genetically recombinant mice ("Non-Tg") to which water was administered. Figure 17 A is a photograph showing the results of staining of synaptophysin, Figure 17 B is a graph showing the results of quantifying the staining intensity of each photograph. As is apparent from these results, by administering the hot water extract of leaves of Alangium salicifolium (Leaf-ext), the synaptophysin level of the hippocampal CA2 / 3 region of C9-500 mice was improved to a level equivalent to that of littermate non-genetically recombinant mice.

[0215] Figure 18 A graph showing the pathology of microglial cells in the prefrontal cortex (PFC) of C9-500 mice ("C9-500") to which a hot water extract of leaves of Alangium salicifolium (Leaf-ext) was orally administered at a dose of 100 μg / day for 1 month, in comparison with C9-500 mice ("C9-500") to which water was administered and non-genetically recombinant mice ("Non-Tg") to which water was administered. Figure 18 A is a photograph showing the results of staining of activated microglial cells, Figure 18 B is a graph showing the number of positive cells of each photograph. As is apparent from these results, by administering the hot water extract of leaves of Alangium salicifolium (Leaf-ext), the activated microglial cell level of the prefrontal cortex (PFC) of C9-500 mice was improved to a level equivalent to that of littermate non-genetically recombinant mice.

[0216] Example 5. Investigation of effects of hot water extract of leaves of Alangium salicifolium (Leaf-ext), simple pulverization of leaves of Alangium salicifolium (Leaf-pwd), and simple pulverization of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) on Tau784 mice

[0217] Effects of hot water extract of leaves of Alangium salicifolium (Leaf-ext), simple pulverization of leaves of Alangium salicifolium (Leaf-pwd), and simple pulverization of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) on Tau784 mice were investigated. Hot water extract of leaves of Alangium salicifolium (Leaf-ext), simple pulverization of leaves of Alangium salicifolium (Leaf-pwd), and simple pulverization of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) were orally administered to 7-9 months old Tau784 mice (average body weight 28.9 g) at 30 μg / day for 1 month, respectively. The same amount of water was administered to Tau784 mice and littermate non-gene recombinant mice of the same age as controls.

[0218] Figure 19 A graph showing the results of Morris water maze test of Tau784 mice (in the table, "Tg+Leaf-ext", "Tg+Leaf-pwd", and "Tg+Fruit-pwd", respectively) to which hot water extract of leaves of Alangium salicifolium (Leaf-ext), simple pulverization of leaves of Alangium salicifolium (Leaf-pwd), and simple pulverization of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) were orally administered at a dose of 30 μg / day for 1 month, in comparison with Tau784 mice (in the table, "Tg") and non-gene recombinant mice (in the table, "non-Tg") to which water was administered as a control. From these results, it is apparent that the memory of Tau784 mice was improved by administration of hot water extract of leaves of Alangium salicifolium (Leaf-ext), but not as much as that of littermate non-gene recombinant mice. The memory of Tau784 mice was improved to the same level as that of littermate non-gene recombinant mice by administration of simple pulverization of leaves of Alangium salicifolium (Leaf-pwd). Surprisingly, the memory of Tau784 mice was significantly enhanced to a higher level than that of littermate non-gene recombinant mice by administration of simple pulverization of fruits (including seeds) of Alangium salicifolium (Fruit-pwd).

[0219] Figure 20Figures showing Tau pathology in the entorhinal cortex of Tau784 mice to which hot water extract of leaves (Leaf-ext), simple pulverized powder of leaves (Leaf-pwd) and simple pulverized powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium were orally administered at a dose of 30 μg / day for 1 month in comparison with Tau784 mice to which water was administered (Tg in the table). Figure 20 A is a photograph showing the results of staining of phosphorylated Tau and Tau oligomers, Figure 20 B is a graph showing the results of quantifying the staining intensity of each photograph. As is apparent from these results, the level of phosphorylated Tau in the entorhinal cortex (EC) was significantly reduced by administration of simple pulverized powder of leaves (Leaf-pwd) and simple pulverized powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium, but the effect was stronger with simple pulverized powder of fruits (including seeds) (Fruit-pwd). On the other hand, hot water extract of leaves (Leaf-ext) showed only a little effect. On the other hand, the level of Tau oligomers was significantly reduced regardless of what was administered, but the effect was highest with simple pulverized powder of fruits (including seeds) (Fruit-pwd) and weakest with hot water extract of leaves (Leaf-ext).

[0220] Figure 21 Figures showing synaptophysin pathology in the hippocampal CA2 / 3 region of Tau784 mice to which hot water extract of leaves (Leaf-ext), simple pulverized powder of leaves (Leaf-pwd) and simple pulverized powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium were orally administered at a dose of 30 μg / day for 1 month in comparison with Tau784 mice to which water was administered (Tg in the table) and non-gene recombinant mice (non-Tg in the table). Figure 21 A is a photograph showing the results of staining of synaptophysin, Figure 21 B is a graph showing the results of quantifying the staining intensity of each photograph. As is apparent from these results, the level of synaptophysin in the hippocampal CA2 / 3 region of Tau784 mice was significantly restored by administration of simple pulverized powder of leaves (Leaf-pwd) and simple pulverized powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium, but the effect was stronger with simple pulverized powder of fruits (including seeds) (Fruit-pwd). On the other hand, hot water extract of leaves (Leaf-ext) only incompletely restored.

[0221] Figure 22 A graph showing BDNF expression in the cerebral cortex (CTX) of Tau784 mice (in the table, "Tg") compared with non-genetically recombinant mice (in the table, "non-Tg") to which hot water extract of leaves (Leaf-ext), simple crushed powder of leaves (Leaf-pwd), and simple crushed powder of fruits (including seeds) (Fruit-pwd) of Alangium salicifolium were orally administered at a dose of 30 μg / day for 1 month. Figure 22 A is a photograph showing the results of staining of BDNF, Figure 22 B is a graph showing the results of quantifying the staining intensity of each photograph. From these results, it is apparent that the BDNF level of the cerebral cortex (CTX) of Tau784 mice was significantly increased to a higher level than that of non-genetically recombinant littermates for the simple crushed powder of fruits (including seeds) of Alangium salicifolium (Fruit-pwd), significantly increased to the same level as that of non-genetically recombinant littermates for the simple crushed powder of leaves (Leaf-pwd), but only showed a little effect for the hot water extract of leaves (Leaf-ext).

[0222] Example 6. Investigation of the effect of simple crushed powder of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) on Huα-Syn(A53T) mice

[0223] The effect of simple crushed powder of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) on Huα-Syn(A53T) mice was investigated. Simple crushed powder of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) was orally administered to 10-11 month-old Huα-Syn(A53T) mice (average body weight 28.9 g) at a dose of 30 μg / day for 1 month. The same amount of water was administered to Huα-Syn(A53T) mice of the same age and non-genetically recombinant littermates as controls.

[0224] Figure 23 A graph showing the level of neurogenesis in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn(A53T) mice (in the table, "αSyn-Tg+Fruit-pwd") to which simple crushed powder of fruits (including seeds) of Alangium salicifolium (Fruit-pwd) was orally administered at a dose of 30 μg / day for 1 month, compared with Huα-Syn(A53T) mice (in the table, "αSyn-Tg") to which water was administered and non-genetically recombinant mice (in the table, "non-Tg"). Figure 23A is a photograph of immunofluorescence staining of BrdU (red) and doublecortin (DCX) (green). Double-positive cells (yellow) positive for both BrdU (red) and DCX (green) are regarded as regenerated neurons. Figure 23 B is a graph showing the results of quantifying the number of double-positive cells (yellow) in each photograph. It is apparent from these results that the level of neurogenesis in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn (A53T) mice was surprisingly improved to a level far exceeding that of the littermate non-gene recombined mice by administration of the simple crushed powder of the fruit (including seeds) of Artocarpus communis. Such results suggest that the fruit (including seeds) of Artocarpus communis has an effect of promoting the rejuvenation of the brain through the repair or regeneration of neurons.

[0225] Example 7. Investigation of the effects of 3 polyphenols on Tau784 mice

[0226] The 3 polyphenols contained in Artocarpus communis, namely catechin, chlorogenic acid, and rutin, have all been reported to have an anti-cognitive disease effect. Thus, there is a possibility that the various effects described above contained in the hot water extract of the leaves of Artocarpus communis (Leaf-ext), the simple crushed powder of the leaves (Leaf-pwd), and the simple crushed powder of the fruit (including seeds) (Fruit-pwd) are due to these polyphenols. Thus, the effects of the 3 polyphenols, namely catechin, chlorogenic acid, and rutin, on Tau784 mice were investigated.

[0227] First, the contents of the 3 polyphenols, namely catechin, chlorogenic acid, and rutin, contained in 100 g each of the hot water extract of the leaves of Artocarpus communis (Leaf-ext), the simple crushed powder of the leaves (Leaf-pwd), and the simple crushed powder of the fruit (including seeds) (Fruit-pwd) were quantified. The results are shown in the following table.

[0228] Table 2

[0229] Next, to 8- to 10-month-old Tau784 mice (average body weight 32.0 g), a mixture of catechin 0.087 μg, chlorogenic acid 0.036 μg, and rutin 0.123 μg, or only catechin 0.087 μg was orally administered for 1 month. The administration amounts of these polyphenols were equivalent to the amounts contained in 30 μg of the simple crushed powder of the fruit (including seeds) of Artocarpus communis. The same amount of water was administered to 8- to 10-month-old Huα-Syn (A53T) mice and littermate non-gene recombined mice as controls.

[0230] Figure 24A graph showing the results of the Morris water maze test of Tau784 mice to which a mixture of catechin 0.087 μg, chlorogenic acid 0.036 μg and rutin 0.123 μg (Table "Tg + 3 polyphenols mixture") or only catechin 0.087 μg (Table "Tg + catechin") was administered orally for 1 month in comparison with non-gene recombinant mice (Table "non-Tg") of the administration water as the object. From these results, it is apparent that administration of the mixture of 3 polyphenols improved the memory of Tau784 mice, but the effect was far inferior to that of the littermates, non-gene recombinant mice, and the effect was incomplete. In addition, catechin alone showed a weaker effect than the mixture of polyphenols. If these results are compared with the respective effects of the preceding Examples 1 to 6, it is apparent that the above-mentioned various effects possessed by the hot water extract of the leaves of Distylium racemosum (Leaf-ext), the simple crushed powder of the leaves (Leaf-pwd) and the simple crushed powder of the fruits (including seeds) (Fruit-pwd) are not due to the above-mentioned 3 polyphenols alone, but rather, the contribution of other unknown components contained in Distylium racemosum is greater.

[0231] Industrial applicability

[0232] The present application can be widely used in the field of functional foods or drugs, etc. which are required to maintain or improve cognitive function, etc., and has a great utility value.

Claims

1. An agent for maintaining or improving brain function, comprising a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta.

2. The agent for maintaining or improving brain function according to claim 1, wherein the brain function is cognitive function.

3. An agent for promoting repair of nerve cells or inducing neurogenesis, comprising a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta.

4. An agent for removing a neurodegenerative disease pathogenic protein accumulated in the brain, comprising a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta.

5. The agent according to claim 4, wherein the neurodegenerative disease pathogenic protein is one or two or more proteins selected from the group consisting of β-amyloid protein (Aβ), Tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein produced by RAN (repeat-associated non-ATG) translation, prion protein, and SOD-1.

6. An agent for treating or preventing a neurodegenerative disease, comprising a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta.

7. The agent according to claim 6, wherein the neurodegenerative disease is a degenerative cognitive disorder.

8. The agent according to claim 7, wherein the degenerative cognitive disorder is one or two or more cognitive disorders selected from the group consisting of Alzheimer's disease, frontotemporal lobar cognitive disorder, Lewy body cognitive disorder, and cognitive disorder due to Parkinson's disease or amyotrophic lateral sclerosis.

9. The agent according to any one of claims 1 to 8, wherein the crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta is a crushed product and / or an extract of leaves and / or fruits and / or seeds of Grevillea robusta.

10. The agent according to any one of claims 1 to 9, wherein the crude drug is administered to a subject in an amount of 0.01 mg to 10 g per day.

11. A food product comprising the agent according to any one of claims 1 to 10.

12. A medicament comprising the agent according to any one of claims 1 to 10.

13. A method for maintaining or improving brain function in a subject, the method comprising administering a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta to a subject in need thereof.

14. The method according to claim 13, wherein the brain function is cognitive function.

15. A method for promoting repair of nerve cells or inducing neurogenesis in a subject, comprising administering a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta to a subject in need thereof.

16. A method for removing a neurodegenerative disease pathogenic protein accumulated in the brain of a subject, comprising administering a crude drug selected from the group consisting of leaves, fruits, and seeds of Grevillea robusta to a subject in need thereof.

17. The method according to claim 16, wherein the neurodegenerative disease pathogenic protein is one or two or more proteins selected from the group consisting of β-amyloid protein (Aβ), Tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein produced by RAN (repeat-associated non-ATG) translation, prion protein, and SOD-1.

18. A method for treating or preventing neurodegenerative disease in a subject, comprising administering to a subject in need thereof a crude drug selected from the group consisting of leaves, fruits and seeds of Distylium racemosum to the subject in need thereof.

19. The method according to claim 18, wherein the neurodegenerative disease is degenerative cognitive disorder.

20. The method according to claim 19, wherein the degenerative cognitive disorder is one or more than two cognitive disorders selected from the group consisting of Alzheimer's disease, frontotemporal lobar cognitive disorder, Lewy body type cognitive disorder and cognitive disorder resulting from Parkinson's disease or amyotrophic lateral sclerosis.

21. The method according to any one of claims 13 to 20, wherein the crude drug selected from the group consisting of leaves, fruits and seeds of Distylium racemosum is a crushed product and / or extract of leaves and / or fruits and / or seeds of Distylium racemosum.

22. The method according to any one of claims 13 to 21, wherein the crude drug is administered to the subject in an amount of 0.01 mg to 10 g per day.

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