Agents and methods for maintaining or improving brain function using herbal medicines

Abstract

Provided is an agent for maintaining or improving cerebral function, the agent containing a crude drug selected from the leaf, fruit, or seed of Pipturus albidus.

Description

[Technical Field]

[0001] The present invention relates to agents and methods for using herbal medicines to maintain or improve brain function, promote the repair of nerve cells or induce neurogenesis, remove proteins that cause neurodegenerative diseases and accumulate in the brain, or treat or prevent neurodegenerative diseases, as well as foods and medicines containing such agents. [Background technology]

[0002] With the aging of society and the westernization of lifestyles and eating habits, the number of dementia patients is rapidly increasing worldwide. The social costs required for medical care and nursing care, as well as the economic losses due to the decline in the workforce of patients and their families, are enormous, and this has become a major social problem.

[0003] Representative forms of dementia include Alzheimer's disease, frontotemporal dementia, and dementia with Lewy bodies. All of these are neurodegenerative diseases, and dementia caused by them is called degenerative dementia. In these diseases, specific proteins aggregate and accumulate in the nervous system, which is thought to cause the death of neurons, resulting in the onset of dementia. Specifically, Aβ and tau accumulate in the brain in Alzheimer's disease, tau and TDP-43 in frontotemporal dementia, and α-synuclein in dementia with Lewy bodies (Non-Patent Document 1: Spires-Jones et al., Acta Neuropathol., (2017), 134[2]:187-205).

[0004] Development is underway to develop anti-dementia drugs that suppress the production of these causative proteins or remove them from the brain. Candidates for such anti-dementia drugs include, for Alzheimer's disease, inhibitors of Aβ-producing enzymes (β-secretase and γ-secretase) (Non-Patent Document 2: Luo et al., Cell & Biosci., (2022), 12:2), Aβ vaccines (Non-Patent Document 3: Valiukas et al., Vaccines, (2022), 10[9]:1527), and Aβ antibodies (Non-Patent Document 4: Song et al., Transl. Neurodegener., (2022), 11:18), and for frontotemporal dementia, tau vaccines (Non-Patent Document 5: Medina, Int. J. Mol. Sci., (2018), 19[4]:1160) and tau antibodies (Non-Patent Document 6: Ji et al., Drugs, (2021), 81

[10] :1135-1152), and for dementia with Lewy bodies, alpha-synuclein vaccines and alpha-synuclein antibodies (Non-patent Document 7: Alzforum website, FBRI LLC, search results for alpha-synuclein Target, https: / / www.alzforum.org / therapeutics / search?fda_statuses=&target_types%5B%5D=33416&therapy_types=&conditions=&keywords-entry=&keywords=, searched December 2022) have been investigated.

[0005] However, most of the anti-dementia drug candidates developed to date have failed to demonstrate the expected efficacy in clinical trials targeting dementia patients (Non-patent document 8: Asher et al., Life Sciences, (2022), 306:120861).

[0006] The leaves of the Hawaiian herb Mamaki (Pipturus albidus) are used as an ingredient in a popular herbal tea in Hawaii. In Hawaii, Mamaki, especially its fruit, has a history of being used as a natural folk medicine to regulate blood sugar, blood pressure, and cholesterol levels, relieve stress and fatigue, and reduce inflammation (Non-Patent Document 9: Rafii et al., Alzheimers Dement., (2022), 1-7). However, it is unknown whether it is effective against dementia. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Spires-Jones et al., Acta Neuropathol., (2017), 134[2]:187-205 [Non-patent document 2] Luo et al., Cell & Biosci., (2022), 12:2 [Non-patent document 3] Valiukas et al., Vaccines, (2022), 10[9]:1527 [Non-patent document 4] Song et al., Transl. Neurodegener., (2022), 11:18 [Non-Patent Document 5] Medina, Int. J. Mol. Sci., (2018), 19[4]:1160 [Non-patent document 6] Ji et al., Drugs, (2021), 81

[10] :1135-1152 [Non-Patent Document 7] Alzforumウェブサイト, FBRI LLC, alpha-synuclein Target's search results, https: / / www.alzforum.org / therapeutics / search?fda_statuses=&target_types%5B%5D=33416&therapy_types=&conditions=&keywords-entry=&keywords=, December 2022 search [Non-licensed Document 8] Asher et al., Life Sciences, (2022), 306:120861 [Non-licensed Document 9] Rafii et al., Alzheimers Dement., (2022), 1-7 [Non-licensed Document 10] Chun et al., Native Hawaiian medicines, First People's Productions Honolulu, (1994), 216-217 [Non-licensed Document 11] Afzal et al., Molecules, (2022), 27

[21] :7604 [Non-licensed Document 12] Payne et al., Biomolecules, (2022), 12[3]:371 [Non-licensed Document 13] Li et al., Neuroscience, (2009), 159[4]:1208-15 [Non-licensed Document 14] Yoo et al., Phytother Res., (2010), 24[7]:1065-70 [Non-licensed Document 15] Colucci-D'Amato et al., Int. J. Mol. Sci., (2020), 21

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

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[24] :13287-92 [Non-patent document 28] Van Dam et al., Eur. J. Neurosci., (2003), 17[2]:388-96 [Non-Patent Document 29] Umeda et al., Front. Neurosci., (2021), 15:763476 [Non-Patent Document 30] Lee et al., Proc. Natl. Acad. Sci. USA, (2002), 99

[13] :8968-73 [Non-Patent Document 31] Umeda et al., Int. J. Mol. Sci., (2021), 22:8453 [Non-Patent Document 32] Liu et al., Neuron, (2016), 90[3]:521-534 [Non-Patent Document 33] Hatanaka et al., Biomedicines, (2022), 10[5]:1080 Summary of the Invention [Problem to be solved by the invention]

[0008] The problem to be solved by the present invention is to provide a new means for maintaining or improving brain function, promoting the repair of nerve cells or inducing neurogenesis, removing proteins that cause neurodegenerative diseases and accumulate in the brain, or treating or preventing neurodegenerative diseases. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have discovered that herbal medicines selected from the leaves, fruits, and seeds of Mamaki, each in a specific form, have various effects such as maintaining or improving brain function, promoting the repair of nerve cells or inducing neurogenesis, removing proteins that cause neurodegenerative diseases that accumulate in the brain, or treating or preventing neurodegenerative diseases, and have completed the present invention.

[0010] That is, the gist of the present invention relates to, for example, the following. [Item 1] An agent for maintaining or improving brain function, comprising a medicinal herb selected from the leaves, fruits, and seeds of Mamaki. [Item 2] The agent for maintaining or improving brain function according to Item 1, wherein the brain function is a cognitive function. [Item 3] An agent for promoting the repair of nerve cells or inducing neurogenesis, comprising a medicinal herb selected from the leaves, fruits, and seeds of Mamaki. [Item 4] An agent for removing proteins that cause neurodegenerative diseases and accumulate in the brain, comprising a medicinal herb selected from the leaves, fruits, and seeds of Mamaki. [Item 5] The agent according to Item 4, wherein the neurodegenerative disease-causing protein is one or more proteins selected from amyloid beta (Aβ), tau, alpha-synuclein, TDP-43, FUS / TLS, polyglutamine, RAN (repeat-associated non-ATG) translated proteins, prion, and SOD-1. [Item 6] A therapeutic or preventive agent for neurodegenerative diseases, comprising a medicinal herb selected from the leaves, fruits, and seeds of Mamaki. [Item 7] The agent according to Item 6, wherein the neurodegenerative disease is degenerative dementia. [Item 8] The agent according to Item 7, wherein the degenerative dementia is one or more types of dementia selected from Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, and dementia caused by Parkinson's disease or amyotrophic lateral sclerosis. [Item 9] The agent according to any one of Items 1 to 8, wherein the herbal medicine selected from the leaves, fruits, and seeds of Mamaki is crushed material and / or extract of the leaves and / or fruits and / or seeds of Mamaki. [Item 10] The agent according to any one of Items 1 to 9, wherein the herbal medicine is administered to a subject in an amount of 0.01 mg to 10 g per day. [Item 11] A food product comprising the agent according to any one of items 1 to 10. [Item 12] A medicine comprising the agent according to any one of items 1 to 10. [Item 13] A method for maintaining or improving brain function in a subject, comprising administering to a subject in need thereof a herbal medicine selected from the leaves, fruits, and seeds of Mamaki. [Item 14] The method according to Item 13, wherein the brain function is a cognitive function. [Item 15] A method for promoting the repair of nerve cells or inducing neurogenesis in a subject, the method comprising administering a herbal medicine selected from the leaves, fruits, and seeds of Mamaki to a subject in need thereof. [Item 16] A method for removing a neurodegenerative disease-causing protein that accumulates in the brain of a subject, the method comprising administering to a subject in need thereof a herbal medicine selected from the leaves, fruits, and seeds of Mamaki. [Item 17] The method described in Item 16, wherein the neurodegenerative disease-causing protein is one or more proteins selected from amyloid beta (Aβ), tau, alpha-synuclein, TDP-43, FUS / TLS, polyglutamine, RAN (repeat-associated non-ATG) translated proteins, prion, and SOD-1. [Item 18] A method for treating or preventing a neurodegenerative disease in a subject, comprising administering to a subject in need thereof a herbal medicine selected from the leaves, fruits, and seeds of Mamaki. [Item 19] The method according to Item 18, wherein the neurodegenerative disease is degenerative dementia. [Item 20] The method according to Item 19, wherein the degenerative dementia is one or more types of dementia selected from Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, and dementia caused by Parkinson's disease or amyotrophic lateral sclerosis. [Item 21] The method according to any one of Items 13 to 20, wherein the herbal medicine selected from the leaves, fruits, and seeds of Mamaki is crushed material and / or extract of Mamaki leaves and / or fruits and / or seeds. [Item 22] The method according to any one of Items 13 to 21, wherein the herbal medicine is administered to the subject in an amount of 0.01 mg to 10 g per day. [Effects of the Invention]

[0011] According to the present invention, by using herbal medicines selected from the leaves, fruits, and seeds of Mamaki, each in a specified form, a new means is provided for maintaining or improving brain function, promoting the repair of nerve cells or inducing neurogenesis, removing proteins that cause neurodegenerative diseases and accumulate in the brain, or treating or preventing neurodegenerative diseases. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows the results of the Morris water maze test in Tau784 mice (Tg+Leaf-ext) orally administered a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (Tg) and non-transgenic mice (Non-Tg). Figure 1A shows the results for the 1000 μg / day and 100 μg / day groups, and Figure 1B shows the results for the 30 μg / day group. [Figure 2] Figure 2 shows tau pathology in the entorhinal cortex of Tau784 mice (labeled "Tau784+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (labeled "Tau784"). Figure 2A shows photographs of staining for phosphorylated tau and tau oligomers, and Figure 2B shows a graph quantifying the staining intensity of each photograph. [Figure 3] Figure 3 shows synaptophysin pathology in the hippocampal CA2 / 3 region of Tau784 mice (labeled "Tau784+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (labeled "Tau784") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 3A is a photograph showing the results of synaptophysin staining, and Figure 3B is a graph showing the quantification of staining intensity in each photograph. [Figure 4] Figure 4 shows microglial pathology in the hippocampus (HC) and cerebral cortex (CTX) of Tau784 mice (labeled "Tau784+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (labeled "Tau784") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 4A shows photographs of the staining of activated microglia, and Figure 4B shows graphs showing the number of positive cells in each photograph. [Figure 5] Figure 5 is a graph showing the results of a Morris water maze test in APP23 mice ("Tg+Leaf-ext" in the table) that were orally administered a hot water extract of Mamaki leaves (Leaf-ext) at a dose of 100 μg / day for one month, in comparison with APP23 mice ("Tg" in the table) that were administered water as controls, and non-genetically modified mice ("Non-Tg" in the table). [Figure 6] Figure 6 shows amyloid pathology in the cerebral cortex and hippocampus of APP23 mice (labeled "APP23+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control APP23 mice (labeled "APP23"). Figure 6A shows photographs of staining for amyloid deposits and Aβ oligomers, and Figure 6B shows a graph quantifying the staining intensity of each photograph. [Figure 7] Figure 7 shows synaptophysin pathology in the hippocampal CA2 / 3 region of APP23 mice (labeled "APP23+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control APP23 mice (labeled "APP23") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 7A is a photograph showing the results of synaptophysin staining, and Figure 7B is a graph showing the quantification of staining intensity in each photograph. [Figure 8]Figure 8 shows microglial pathology in the hippocampus (HC) and cerebral cortex (CTX) of APP23 mice (labeled "APP23+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control APP23 mice (labeled "APP23") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 8A is a photograph showing the staining results for activated microglia, and Figure 8B is a graph showing the number of positive cells in each photograph. [Figure 9] Figure 9 is a graph showing the results of a Morris water maze test in Huα-Syn(A53T) mice ("Tg+Leaf-ext" in the table) that were orally administered a hot water extract of mamaki leaves (Leaf-ext) at a dose of 100 μg / day for one month, compared with Huα-Syn(A53T) mice ("Tg" in the table) and non-genetically modified mice ("Non-Tg" in the table) that were administered water as controls. [Figure 10] Figure 10 shows α-synuclein pathology in the hippocampus (HC) and entorhinal cortex (EC) of Huα-Syn(A53T) mice treated orally with 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for 1 month ("αSyn-Tg+Leaf-ext" in the table), compared with control Huα-Syn(A53T) mice treated with water ("αSyn-Tg" in the table). Figure 10A shows photographs of phosphorylated α-synuclein staining, and Figure 10B shows the quantification of staining intensity in each photograph. [Figure 11] Figure 11 shows α-synuclein pathology in the hippocampus (HC) and entorhinal cortex (EC) of Huα-Syn(A53T) mice treated orally with 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for 1 month ("αSyn-Tg+Leaf-ext" in the table), compared with control Huα-Syn(A53T) mice treated with water ("αSyn-Tg" in the table). Figure 11A shows photographs of staining for α-synuclein oligomers, and Figure 11B shows the quantification of staining intensity in each photograph. [Figure 12]Figure 12 shows synaptophysin pathology in the hippocampal CA2 / 3 region of Huα-Syn(A53T) mice (labeled "αSyn-Tg+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Huα-Syn(A53T) mice (labeled "αSyn-Tg") and non-transgenic mice (labeled "Non-Tg"). Figure 12A shows photographs of synaptophysin staining, and Figure 12B shows the quantification of staining intensity in each photograph. [Figure 13] Figure 13 shows microglial pathology in the hippocampus (HC) and cerebral cortex (CTX) of Huα-Syn(A53T) mice (labeled "αSyn-Tg+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Huα-Syn(A53T) mice (labeled "αSyn-Tg") and non-transgenic mice (labeled "Non-Tg"). Figure 13A shows photographs of activated microglia staining, and Figure 13B shows graphs showing the number of positive cells in each photograph. [Figure 14] Figure 14 is a graph showing the results of a Morris water maze test in C9-500 mice ("Tg+Leaf-ext" in the table) that were orally administered a hot water extract of Mamaki leaves (Leaf-ext) at a dose of 100 μg / day for one month, in comparison with C9-500 mice ("Tg" in the table) that were administered water as controls, and non-genetically modified mice ("Non-Tg" in the table). [Figure 15] Figure 15 shows the pathology caused by mutations in the C9orf72 gene in the prefrontal cortex (PFC) of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered a hot water extract of mamaki leaves (Leaf-ext) at a dose of 100 μg / day for one month, compared with control C9-500 mice (labeled "C9-500") administered water. Figure 15A shows photographs of staining for RNA G-quadruplex, poly-GA, poly-GP, and phosphorylated TDP-43, and Figure 15B shows a graph quantifying the staining intensity of each photograph. [Figure 16] Figure 16 shows double-stranded RNA-dependent protein kinase (PKR) pathology in the prefrontal cortex (PFC) of the hippocampus in C9-500 mice (labeled "C9-500+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control C9-500 mice (labeled "C9-500") and non-transgenic mice (labeled "Non-Tg"). Figure 16A shows the results of phosphorylated PKR staining, and Figure 16B shows the results of quantifying the staining intensity in each photograph. [Figure 17] Figure 17 shows synaptophysin pathology in the hippocampal CA2 / 3 region of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered 100 μg / day of a hot water extract of Mamaki leaves (Leaf-ext) for one month, compared with control C9-500 mice (labeled "C9-500") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 17A is a photograph showing the results of synaptophysin staining, and Figure 17B is a graph showing the quantification of staining intensity in each photograph. [Figure 18] Figure 18 shows microglial pathology in the prefrontal cortex (PFC) of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control C9-500 mice (labeled "C9-500") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 18A shows photographs of activated microglia staining, and Figure 18B shows graphs showing the number of positive cells in each photograph. [Figure 19]Figure 19 shows the results of the Morris water maze test in Tau784 mice (represented by "Tg+Leaf-ext," "Tg+Leaf-pwd," and "Tg+Fruit-pwd" in the table) orally administered hot water extract of Mamaki leaves (Leaf-ext), simply crushed powder of leaves (Leaf-pwd), and simply crushed powder of fruit (including seeds) (Fruit-pwd) at a dose of 30 μg / day for one month, compared with Tau784 mice (represented by "Tg" in the table) and non-transgenic mice (represented by "Non-Tg" in the table) administered with water as control. [Figure 20] Figure 20 shows tau pathology in the entorhinal cortex of Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered hot water extract of mamaki leaves (Leaf-ext), crushed leaf powder (Leaf-pwd), and crushed fruit powder (including seeds) at a dose of 30 μg / day for one month, compared with control Tau784 mice (Tau784). Figure 20A shows photographs of staining for phosphorylated tau and tau oligomers, and Figure 20B shows a graph quantifying the staining intensity of each photograph. [Figure 21] Figure 21 shows synaptophysin pathology in the hippocampal CA2 / 3 region of Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered hot water extract of mamaki leaves (Leaf-ext), crushed leaf powder (Leaf-pwd), and crushed fruit powder (including seeds) at a dose of 30 μg / day for 1 month, compared with control Tau784 mice (Tau784) and non-transgenic mice (Non-Tg). Figure 21A shows photographs of synaptophysin staining, and Figure 21B shows graphs quantifying the staining intensity of each photograph. [Figure 22]Figure 22 shows BDNF expression in the cerebral cortex (CTX) of Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered 30 μg / day of hot water extract of mamaki leaves (Leaf-ext), crushed leaf powder (Leaf-pwd), and crushed fruit powder (including seeds) (Fruit-pwd) for one month, compared with control Tau784 mice (Tau784) and non-transgenic mice (Non-Tg). Figure 22A shows photographs of BDNF staining, and Figure 22B shows the quantification of staining intensity for each photograph. [Figure 23] Figure 23 shows the neurogenesis levels in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn(A53T) mice ("αSyn-Tg+Fruit-pwd") orally administered 30 μg / day of crushed mamaki fruit (including seeds) powder (Fruit-pwd) for one month, compared with control Huα-Syn(A53T) mice ("αSyn-Tg") and non-transgenic mice ("Non-Tg"). Figure 23A shows immunofluorescent staining for BrdU (red) and doublecortin (DCX) (green). Double-positive cells (yellow), which were both BrdU (red) and DCX (green), were considered to be newborn neurons. Figure 23B shows the results of quantifying the number of double-positive cells (yellow) in each photograph. [Figure 24] Figure 24 is a graph showing the results of the Morris water maze test in Tau784 mice that were orally administered a mixture of 0.087 μg of catechin, 0.036 μg of chlorogenic acid, and 0.123 μg of rutin ("Tg+3 polyphenol mixture" in the table) or 0.087 μg of catechin alone ("Tg+chatechin" in the table) for one month, in comparison with Tau784 mice that were administered water as controls ("Tg" in the table) and non-genetically modified mice ("Non-Tg" in the table). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments and can be embodied in any form without departing from the spirit of the present invention.

[0014] [overview] As mentioned above, the failure of most anti-dementia drugs, aside from side effects, is thought to be due to the drug being administered too late or targeting the wrong molecule. It has been shown that Aβ accumulation in the brain begins more than 20 years before the onset of Alzheimer's disease, and tau accumulation begins approximately 10 years before. Aβ and tau accumulate, causing neuronal death, and only then does dementia develop. In other words, by the time dementia develops, many neurons have already died. Removing Aβ and tau is only meaningful if it is done before neuronal death begins. In other words, the role of drugs targeting Aβ and tau is prevention, not treatment. Previously, it was thought that disease developed when insoluble protein aggregates, such as senile plaques and neurofibrillary tangles (accumulations of aggregated tau), killed neurons. However, it has recently been suggested that soluble oligomers, formed in the precursor stage, impair neuronal function, resulting in dementia. Therefore, in order to prevent dementia, it is necessary to remove oligomers of the causative protein.

[0015] From a prevention perspective, it is desirable for a single drug to act on oligomers of various causative proteins, rather than being specific to Aβ, tau, or α-synuclein. Furthermore, it is necessary for the drug to repair nerve cells damaged by oligomers and restore brain function. Because dementia prevention is a long-term process, it is desirable for the preventive drug to be safe, inexpensive, and, ideally, self-administered non-invasively, without the assistance of a doctor. Given the numerous requirements for a dementia preventive drug, it is difficult to achieve them with a single-ingredient drug. Furthermore, if all middle-aged and elderly people were to take a drug for the long term to prevent dementia, the medical economy would eventually collapse.

[0016] To solve this problem, the inventors focused on herbal medicines that have a long history of use in natural medicine and traditional Chinese medicine. If there were herbal medicines that are effective in improving cognitive function, middle-aged and elderly people could obtain them at their own discretion without seeing a doctor, and by ingesting them in addition to meals, they could work to prevent dementia as part of their everyday lives.

[0017] Therefore, the present inventors have conducted extensive research and found that herbal medicines selected from the leaves, fruits, and seeds of Mamaki have various effects, such as maintaining or improving brain function, promoting the repair of nerve cells or inducing neurogenesis, removing proteins that cause neurodegenerative diseases and accumulate in the brain, or treating or preventing neurodegenerative diseases. The present invention is based on these findings.

[0018] That is, according to one aspect of the present invention, there is provided an agent for maintaining or improving brain function, an agent for promoting the repair of nerve cells or inducing neurogenesis, an agent for removing proteins that cause neurodegenerative diseases that accumulate in the brain, or an agent for treating or preventing neurodegenerative diseases (these may be collectively referred to as "agents of the present invention" as appropriate), which contains a herbal medicine selected from the leaves, fruits, and seeds of Mamaki.

[0019] Furthermore, according to one aspect of the present invention, a food product containing the agent of the present invention is provided (this may be appropriately referred to as the "food product of the present invention").

[0020] Furthermore, according to one aspect of the present invention, there is provided a medicament comprising the agent of the present invention (which may be appropriately referred to as the "medicament of the present invention").

[0021] Furthermore, according to one aspect of the present invention, there is provided a method for maintaining or improving brain function, a method for promoting the repair of nerve cells or inducing neurogenesis, a method for removing a protein that causes a neurodegenerative disease and accumulates in the brain, or a method for treating or preventing a neurodegenerative disease (these may be collectively referred to as the "method of the present invention" as appropriate), which comprises administering to a subject one or more of a herbal medicine selected from the leaves, fruits, and seeds of Mamaki, the agent of the present invention, the food of the present invention, and the pharmaceutical of the present invention.

[0022] [Mamaki] According to one embodiment, the agent of the present invention contains a herbal medicine selected from the leaves, fruit, and seeds of mamaki. As mentioned above, the leaves of mamaki (Pipturus albidus), a herb native to Hawaii, are used as an ingredient in a popular herbal tea in Hawaii. In Hawaii, mamaki, especially its fruit, has a history of being used as a natural folk medicine to regulate blood sugar, blood pressure, and cholesterol levels, relieve stress and fatigue, and reduce inflammation (Non-Patent Document 9: Rafii et al., Alzheimers Dement., (2022), 1-7). However, it is unknown whether it is effective against dementia.

[0023] Three polyphenols, (+)-catechin, chlorogenic acid, and rutin, have been identified as major components of mamaki leaves (Non-Patent Document 10: Chun et al., Native Hawaiian medicines, First People's Productions Honolulu, (1994), 216-217). The catechin and rutin content of mamaki leaves is significantly higher than that of other commercially available tea leaves (Non-Patent Document 10). These polyphenols have been suggested to have beneficial effects on mouse models of neurodegenerative diseases and humans.

[0024] Catechins, a type of flavonoid, are found in abundance in tea leaves. They exhibit anti-inflammatory and antioxidant properties by blocking cytokine production and inflammatory pathways, chelating metal ions, and scavenging free radicals (Afzal et al., Molecules, (2022), 27

[21] :7604; and Payne et al., Biomolecules, (2022), 12[3]:371). Catechins, particularly epigallocatechin-3-gallate, have been shown to inhibit Aβ production, tau phosphorylation, and amyloid protein aggregation, thereby preventing cognitive decline in AD and PD (Non-Patent Documents 11 and 12). Furthermore, oral administration of BDNF to mice has been reported to increase brain-derived neurotrophic factor (BDNF) expression and neurogenesis (Non-Patent Document 13: Li et al., Neuroscience, (2009), 159[4]:1208-15; and Non-Patent Document 14: Yoo et al., Phytother Res., (2010), 24[7]:1065-70). BDNF and neurogenesis are important for brain repair and regeneration (Non-Patent Document 15: Colucci-D'Amato et al., Int. J. Mol. Sci., (2020), 21

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

[0025] Chlorogenic acid, also known as 5-O-caffeoylquinic acid, is found in large amounts in coffee beans. It has a variety of beneficial health effects, including anti-inflammatory, antioxidant, neuroprotective, hepatoprotective, cardioprotective, chemopreventive, antidiabetic, and anti-obesity effects. Regular intake of chlorogenic acid has been suggested to reduce the risk of neurodegenerative diseases and improve cognitive function (Non-Patent Document 17: Socala et al., Int. J. Mol. Sci., (2020), 22[1]:107). Oral administration of benzodiazepine to AD and PD model mice improved memory and motor function (Non-Patent Document 18: Gao et al., Drug Des. Devel. Ther., (2020), 14:1705-1716; and Non-Patent Document 19: Singh et al., Oxid. Med. Cell Longev., (2020), 2020:6571484), and oral administration to a rat model of cerebral ischemia / reperfusion has been shown to promote the expression of BDNF and nerve growth factor (NGF) (Non-Patent Document 20: Liu et al., Drug Des. Devel. Ther., (2020), 14:51-60).

[0026] Rutin, a member of the flavonol subtype of flavonoids, is found in various plants. It exhibits anti-inflammatory, antioxidant, and neuroprotective properties (Non-Patent Document 21: Tahir et al., Biomed. Pharmacother., (2021), 137:111-253). Oral administration of rutin to AD model mice reduced Aβ and tau oligomers and improved cognitive function (Non-Patent Document 22: Xu et al., Behav. Brain Res., (2014), 264:173-80; and Non-Patent Document 23: Sun et al., J. Neuroinflammation, (2021), 18[1]:131). Furthermore, intraperitoneal administration of rutin to rats intracerebrally infused with Aβ has been reported to increase BDNF expression (Non-Patent Document 24: Moghbelinejad et al., Toxicol Lett., (2014), 224[1]:108-13).

[0027] As described above, the three polyphenols contained in mamaki leaves, namely catechin, chlorogenic acid, and rutin, have been suggested to have effects such as improving cognitive function. However, the effects of mamaki leaves and fruit on improving cognitive function are completely unknown.

[0028] Therefore, as will be described in detail in the Examples below, the inventors separated the leaves and fruits (including seeds) from commercially available dried Mamaki tea leaves, prepared hot water extracts and simply crushed powders of each, and examined their effects on cognitive function and neuropathology using four types of degenerative dementia model mice.

[0029] First, we used Tau784 mice as a model of frontotemporal dementia-tau (FTD-Tau) (Non-Patent Document 25: Umeda et al., Am. J. Pathol., (2013), 183[1]:211-25; and Non-Patent Document 26: Umeda et al., Ann. Clin. Transl. Neurol., (2015), 2[3]:241-55). We administered a hot water extract of mamaki leaves (Leaf-ext) at doses of 1000 μg / day, 100 μg / day, and 30 μg / day for one month (Example 1). Significant improvements were observed at all doses in cognitive function (Figure 1), tau pathology (Figure 2), synaptophysin pathology (Figure 3), and microglial pathology (Figure 4). In particular, when administered at doses of 1000 μg / day and 100 μg / day, each pathology was improved to the same level as that of non-transgenic littermates.

[0030] In addition, APP23 mice (Non-Patent Document 27: Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA, (1997), 94

[24] :13287-92; Non-Patent Document 28: Van Dam et al., Eur. J. Neurosci., (2003), 17[2]:388-96; and Non-Patent Document 29: Umeda et al., Front. Neurosci., (2021), 15:763476) were used as a model of Alzheimer's disease (AD), and a hot water extract of mamaki leaves (Leaf-ext) was administered at a dose of 100 μg / day for one month (Example 2). As a result, cognitive function (Figure 5), amyloid pathology (Figure 6), synaptophysin pathology (Figure 7), and microglial pathology (Figure 8) were all improved to levels comparable to or close to those of non-transgenic littermates.

[0031] In addition, Huα-Syn(A53T) mice (Non-Patent Document 30: Lee et al., Proc. Natl. Acad. Sci. USA, (2002), 99

[13] :8968-73; and Non-Patent Document 31: Umeda et al., Int. J. Mol. Sci., (2021), 22:8453) were used as a model of dementia with Lewy bodies (DLB). A hot water extract of mamaki leaves (Leaf-ext) was administered at a dose of 100 μg / day for one month (Example 3). As a result, cognitive function (Figure 9), synuclein pathology (Figures 10 and 11), synaptophysin pathology (Figure 12), and microglial pathology (Figure 13) were all improved to levels comparable to or close to those of non-transgenic littermates.

[0032] In addition, we used C9-500 mice as a model of frontotemporal dementia-TDP (FTD-TDP) (Non-Patent Document 32: Liu et al., Neuron, (2016), 90[3]:521-534; and Non-Patent Document 33: Hatanaka et al., Biomedicines, (2022), 10[5]:1080) and administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month (Example 4). As a result, cognitive function (Figure 14), pathology caused by mutations in the C9orf72 gene (Figure 15), double-stranded RNA-dependent protein kinase (PKR) pathology (Figure 16), synaptophysin pathology (Figure 17), and microglial pathology (Figure 18) were all improved to levels comparable to or close to those of non-transgenic littermates. These results suggest that mamaki leaves have the effect of improving cognitive function and various related pathologies.

[0033] In addition, Tau784 mice (see Non-Patent Documents 25 and 26) were used as a model of frontotemporal dementia-tau (FTD-Tau) and administered 30 μg / day of mamaki leaf hot water extract (Leaf-ext), simply crushed leaf powder (Leaf-pwd), and simply crushed fruit powder (including seeds) (Fruit-pwd) for one month (Example 5). Significant improvements were observed for all preparations in cognitive function (Figure 19), tau pathology (Figure 20), synaptophysin pathology (Figure 21), and BDNF expression (Figure 22). In particular, simply crushed mamaki leaf powder (Leaf-pwd) and simply crushed fruit powder (including seeds) (Fruit-pwd) improved each pathology to levels comparable to or close to those of non-transgenic littermates. In particular, the administration of crushed powder of mamaki fruit (including seeds) (Fruit-pwd) surprisingly enhanced the cognitive function of Tau784 mice to a level even higher than that of their non-transgenic littermates. These results suggest that crushed mamaki leaves and fruit (including seeds) have far superior effects on improving cognitive function and various pathologies than extracts.

[0034] In addition, we used Huα-Syn(A53T) mice (see Non-Patent Documents 30 and 31) as a model of dementia with Lewy bodies (DLB) and administered 100 μg / day of crushed mamaki fruit (including seeds) powder (Fruit-pwd) for one month (Example 6). Surprisingly, the neurogenesis levels in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn(A53T) mice were significantly improved, far exceeding those of non-transgenic littermates. These results suggest that crushed mamaki fruit (including seeds) promotes brain rejuvenation through neuronal repair and regeneration.

[0035] Furthermore, to verify whether the cognitive function-improving effects of mamaki leaves and fruit (including seeds) and the ameliorative effects on various pathologies described above are attributable to the three polyphenols contained in mamaki leaves, namely, catechin, chlorogenic acid, and rutin, the following experiment was conducted. Specifically, using Tau784 mice (see Non-Patent Documents 25 and 26) as a model of frontotemporal dementia-tau (FTD-Tau), mice were administered a mixture of catechin, chlorogenic acid, and rutin equivalent to the amount contained in 30 μg of simply crushed mamaki fruit (including seeds) powder, or catechin alone, for one month. The results showed that the cognitive function-improving effects of the catechin / chlorogenic acid / rutin mixture and catechin alone were far inferior to those of non-transgenic littermates and were incomplete. Therefore, it is clear that the various effects of the hot water extract of mamaki leaves (Leaf-ext), simply crushed powder of leaves (Leaf-pwd), and simply crushed powder of fruit (including seeds) (Fruit-pwd) described above are not solely due to the three polyphenols mentioned above, but rather are largely due to the contribution of other unknown components contained in mamaki.

[0036] [Form of herbal medicine] According to one embodiment, the mamaki leaves, fruits, and seeds may be mamaki leaves alone, mamaki fruits alone, mamaki seeds alone, or a combination of two or three of the mamaki leaves, fruits, and seeds.

[0037] The leaves and / or fruits and / or seeds of Mamaki may be used in any form, including, but not limited to, the leaves and / or fruits and / or seeds of Mamaki as they are, or in the form of crushed material and / or extract.

[0038] Specifically, when a medicinal herb selected from the leaves, fruits, and seeds of the mamaki tree is crushed into a crushed powder or other crushed form, the processing conditions are not particularly limited, but are, for example, as follows. First, the medicinal herb is dried. The drying conditions are not limited, and various known conditions may be used. Examples include drying at temperatures of 0 to 100°C, such as natural drying or heat drying. Next, the dried medicinal herb is crushed. The crushing method is also not limited, and various known methods may be used. Examples include manual crushing and crushing using a crusher, such as wet crushing or dry crushing. The crushed medicinal herb obtained may be used as is, or it may be passed through a sieve to obtain a powder with a particle size controlled to a predetermined value or less. For example, the particle size may be adjusted using a sieve with openings of 0.02 mm to 20 mm.

[0039] Furthermore, when a medicinal herb selected from the leaves, fruits, and seeds of Mamaki is prepared in the form of an extract, the processing conditions are not particularly limited. For example, the medicinal herb may be optionally dried and / or crushed, followed by the addition of an extraction solvent. Specifically, extraction can be performed by adding, for example, 1 to 100 parts by mass of extraction solvent to 1 part by mass of dried and crushed medicinal herb and incubating for, for example, 0.1 to 100 hours. The extraction solvent is also not particularly limited, and various known solvents can be used, including water and hydrophilic organic solvents such as ethanol. Extraction using an extraction solvent can be performed at room temperature or with heating. For example, when heating with water, the crushed herb can be heated to 30 to 100°C after adding hot water. Furthermore, if necessary, stirring, ultrasonic treatment, heating under reflux, or the like can be used in combination to enhance extraction efficiency. After extraction is complete, the liquid and solid components are separated manually or by filtration. The separated liquid component can be used as the extract. In either case, the solvent may be evaporated and concentrated before use, if necessary.

[0040] [Action of the agent] According to one embodiment, the agent of the present invention is an agent for maintaining or improving brain function. In the present invention, "maintaining or improving brain function" means, but is not limited to, maintaining or improving cognitive function and / or motor function.

[0041] In the present invention, maintaining or improving "cognitive function" means, but is not limited to, preventing or improving abnormalities such as mild forgetfulness, memory impairment seen in dementia, disorientation, impaired judgment and comprehension, impaired executive function, apraxia, agnosia, and aphasia.

[0042] In the present invention, maintaining or improving "motor function" means, but is not limited to, preventing or improving abnormalities such as a decline in motor function due to aging, parkinsonism such as tremor, muscle rigidity, impaired postural reflexes, akinesia / hypokinesia seen in Parkinson's disease, involuntary movements seen in Huntington's disease, coordination disorders seen in spinocerebellar degeneration, and muscle weakness seen in amyotrophic lateral sclerosis.

[0043] The effect of the agent of the present invention on maintaining or improving brain function can be evaluated by administering the agent to a model animal that develops brain pathology, and then subjecting the animal to a cognitive function test such as a Morris water maze test or a motor function test such as a rotarod test, or by staining and observing brain slices, as shown in the Examples below, for example.

[0044] According to one embodiment, the agent of the present invention is an agent for promoting neuronal repair or inducing neurogenesis. In the present invention, "promoting neuronal repair" includes, but is not limited to, restoring the number and function of decreased synapses, restoring decreased neuronal function, and enhancing the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which promote the maintenance and recovery of synapse and neuronal function and protect neurons from various stresses. In the present invention, "inducing neurogenesis" includes, but is not limited to, inducing the emergence of immature neurons that actively synthesize DNA. Indicators of DNA synthesis include active incorporation of exogenously administered nucleic acid analogs such as BrdU and expression of doublecortin as a marker of immature neurons. The neuronal repair-promoting effect or neurogenesis-inducing effect of the agent of the present invention can be evaluated by administering the agent to model animals with dementia or brain pathology and then observing the induction of brain-derived neurotrophic factor (BDNF) expression, or by staining brain sections and observing newly generated neurons, as shown in the Examples below.

[0045] According to one embodiment, the agent of the present invention is an agent for removing a neurodegenerative disease-causing protein that accumulates in the brain. In the present invention, "neurodegenerative disease-causing protein" refers to a protein that has been identified or suspected as a causative agent of dementia. Examples include, but are not limited to, one or more proteins selected from amyloid beta (Aβ), tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, proteins produced by RAN (repeat-associated non-ATG) translation, prion, SOD-1, etc. The effect of the agent of the present invention on maintaining or improving brain function can be evaluated by administering the agent to an animal model that develops dementia and then staining and observing brain sections, as shown in the Examples below.

[0046] According to one embodiment, the agent of the present invention is a therapeutic or preventive agent for neurodegenerative diseases. In the present invention, the term "neurodegenerative disease" refers to, but is not limited to, a neurodegenerative disease in which any of the aforementioned proteins responsible for neurodegenerative diseases accumulates in the brain. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease (Aβ, tau), frontotemporal dementia (tau, TDP-43, FUS / TLS), dementia with Lewy bodies (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), and Creutzfeldt-Jakob disease (prion). (Note that examples of proteins responsible for neurodegenerative diseases whose accumulation is observed in each neurodegenerative disease are shown in parentheses.) In particular, the therapeutic or preventive agent for neurodegenerative disease of the present invention is preferably a therapeutic or preventive agent for degenerative dementia. Examples of degenerative dementia include, but are not limited to, Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, Parkinson's disease, and dementia caused by amyotrophic lateral sclerosis. Frontotemporal dementia, in which tau accumulates, can be further classified into Pick's disease, corticobasal degeneration, and progressive supranuclear palsy. The therapeutic or preventive effect of the agent of the present invention on neurodegenerative disease can be evaluated, for example, as shown in the Examples below, by administering the agent to an animal model that develops a neurodegenerative disease (e.g., degenerative dementia), followed by a cognitive function test such as a Morris water maze test or a motor function test such as a rotarod test, or by staining and observing brain sections.

[0047] [Dosage form, dosage and administration] The dosage form of the agent of the present invention is not particularly limited, and for example, a herbal medicine selected from mamaki leaves, fruits, and seeds can be used as is in any form, such as crushed material, an extract of the crushed material, or an extraction residue of the crushed material, or can be formulated together with other ingredients such as desired excipients and / or carriers. Furthermore, when used in the form of a food (food of the present invention) or medicine (medicine of the present invention) containing the agent of the present invention, the mamaki leaves and / or fruits and / or seeds, which are the active ingredients of the agent of the present invention, can be mixed with other ingredients appropriate for the form of the food or medicine, respectively, and used. Details will be described below.

[0048] The method of use of the agent of the present invention is not particularly limited, but it is usually administered orally. In particular, the agent of the present invention is preferably used in the form of a food (the food of the present invention) or an oral medicine (the medicine of the present invention). Details will be described later.

[0049] The dosage of the agent of the present invention is not particularly limited, but for example, the agent of the present invention can be administered to a subject so that the active ingredient, mamaki leaves and / or fruits and / or seeds, is usually 0.01 mg / day or more, or 0.1 mg / day or more, particularly 0.5 mg / day or more, or even 1.0 mg / day or more, and usually 10 g / day or less, particularly 5 g / day or less, or even 1 g / day or less.

[0050] [Food] According to one aspect of the present invention, there is provided a food product containing the agent of the present invention (food product of the present invention). The food product of the present invention is characterized by containing any one of the agents of the present invention and is used for the intended purpose of the agent.

[0051] The food of the present invention can be in any form that can be orally ingested, such as a solution, suspension, emulsion, powder, solid molding, etc. Furthermore, it can be molded into dosage forms such as capsules, troches, syrups, granules, etc. in the same manner as the medicine of the present invention described below.

[0052] The food of the present invention can be produced as, for example, beverages such as tea, black tea, coffee, soft drinks, alcoholic drinks, carbonated drinks, dairy drinks, fruit juice drinks, nutritional drinks, concentrated drinks, powdered drinks (powdered juice, powdered soup, etc.); supplements; confectioneries such as candy, gummies, chewing gum, chocolate, cookies, biscuits; frozen desserts such as ice cream; dairy products such as yogurt and processed milk; flour products such as cereals, bread, cake mix; noodles such as buckwheat; oil and fat processed products such as mayonnaise, whipped cream, dressing; processed seafood products; processed livestock products; processed agricultural products.The food of the present invention can be produced by adding and containing the agent of the present invention during the production of these foods.

[0053] In addition to other food ingredients, additives such as sweeteners, coloring agents, preservatives, thickeners, stabilizers, gelling agents or thickening agents, antioxidants such as ascorbic acid, color formers, bleaching agents, anti-mold or anti-fungal agents, yeast food, gum base, alkaline water, bittering agents, enzymes, glazing agents, flavorings, acidulants, chewing gum softeners, seasonings, tofu coagulants, emulsifiers, pH adjusters, leavening agents, nutritional fortifiers such as vitamins, minerals and amino acids, and manufacturing agents can be added to the food of the present invention, as needed.

[0054] The food of the present invention may be provided as a food that displays the action, effect, function, or use of the agent of the present invention under the system of each country. For example, in Japan, the food of the present invention may be produced as a food with health claims (specified health claims, functional food, food with nutrient claims).

[0055] The content of the mamaki leaves and / or fruits and / or seeds, which are the active ingredients of the agent of the present invention, in the food of the present invention can be appropriately set within a range that achieves the effects of the present invention, in the same manner as the agent of the present invention. Specifically, the content of the mamaki leaves and / or fruits and / or seeds, which are the active ingredients of the agent of the present invention, can be adjusted so that the amount is usually 0.01 mg / day or more, or 0.1 mg / day or more, preferably 0.5 mg / day or more, or even 1.0 mg / day or more, and usually 10 g / day or less, preferably 5 g / day or less, or even 1 g / day or less.

[0056] The number and frequency of ingestion of the food of the present invention are arbitrary and can be set as appropriate, such as once to several times a day, every day, every other day, every two days, or 1 to 7 days a week. By incorporating the leaves and / or fruits and / or seeds of mamaki in an amount necessary to obtain the effects of the agent of the present invention into the food according to the desired number and frequency of ingestion, it is possible to provide a food of the present invention that can be expected to have the desired effect depending on the type of desired action and effect.

[0057] The food of the present invention is characterized by containing the agent of the present invention, and therefore shares the advantages of the various agents of the present invention and is extremely useful. In addition, as a food, it can be safely and easily used not only by patients suffering from a specific disease but also by healthy individuals.

[0058] Furthermore, the food of the present invention can be used not only for humans but also for animals other than humans to which the use of the agent of the present invention can be applied.

[0059] [Pharmaceuticals] According to one aspect of the present invention, there is provided a medicament comprising the agent of the present invention (the medicament of the present invention). The medicament of the present invention is characterized by comprising any one of the agents of the present invention and is used for the intended use of the agent.

[0060] The administration route of the pharmaceutical of the present invention is not limited, but it is usually an internal medicine (oral agent) that is administered orally.

[0061] The medicament of the present invention can be manufactured by adding the agent of the present invention as an active ingredient and in any dosage form, such as tablets, capsules such as soft capsules and hard capsules, solid preparations such as powders, granules, drops and pills, semisolid preparations such as jellies, and liquid preparations such as syrups, suspensions and oral liquids. In this case, the medicament of the present invention can be formulated as a pharmaceutical composition combining the agent of the present invention with other additives commonly used in the production of oral preparations, by a pharmaceutical manufacturing method known to those skilled in the art.

[0062] Examples of additives used in the production of the medicament of the present invention include excipients, disintegrants, binders, lubricants, coating agents, dispersants, fluidizing agents, stabilizers, preservatives, buffers, flavoring agents, suspending agents, emulsifiers, flavoring agents, solubilizing agents, colorants, thickeners, etc. Furthermore, by further combining with a pharmaceutically acceptable carrier, it is possible to provide the medicament of the present invention with further enhanced actions and effects of the agent of the present invention.

[0063] The pharmaceuticals of the present invention also include products that are equivalent to pharmaceuticals under the systems of each country, such as quasi-drugs in Japan.

[0064] The content of the leaves and / or fruits and / or seeds of mamaki, which are the active ingredients of the agent of the present invention, in the pharmaceutical of the present invention can be appropriately set within a range that achieves the effects of the present invention, in the same manner as the agent of the present invention. Specifically, the content of the leaves and / or fruits and / or seeds of mamaki, which are the active ingredients of the agent of the present invention, can be adjusted so that the amount is usually 0.01 mg / day or more, or 0.1 mg / day or more, preferably 0.5 mg / day or more, or even 1.0 mg / day or more, and usually 10 g / day or less, preferably 5 g / day or less, or even 1 g / day or less.

[0065] The number and frequency of administration of the pharmaceutical of the present invention are optional and can be set as appropriate, such as once to several times a day, every day, every other day, every two days, or 1 to 7 days a week. By incorporating the amount of mamaki leaves and / or fruits and / or seeds necessary to obtain the effects of the agent of the present invention according to the desired number and frequency of administration, it is possible to provide an administration of the present invention that can be expected to produce the desired effect depending on the type of desired action and effect.

[0066] The pharmaceutical of the present invention is characterized by containing the agent of the present invention, and therefore shares the advantages of the agent and is extremely useful.

[0067] Furthermore, the pharmaceutical of the present invention can be used not only for humans but also for animals other than humans to which the use of the agent of the present invention can be applied.

[0068] [method] According to one aspect of the present invention, there is provided a method for maintaining or improving brain function, a method for promoting the repair of nerve cells or inducing neurogenesis, a method for removing a protein that causes a neurodegenerative disease and accumulates in the brain, or a method for treating or preventing a neurodegenerative disease (the method of the present invention), which comprises administering to a subject one or more of a herbal medicine selected from the leaves, fruits, and seeds of Mamaki, an agent of the present invention, a food of the present invention, and a pharmaceutical of the present invention.

[0069] The method of the present invention may be carried out by administering to a subject the leaves and / or fruits and / or seeds of Mamaki, which are the active ingredients of the agent of the present invention, in the form of the agent of the present invention, the food of the present invention, and / or the medicine of the present invention. Details thereof are as described above in the explanation of the agent of the present invention, the food of the present invention, and the medicine of the present invention. [Example]

[0070] The present invention will be described in more detail below with reference to examples. However, these examples are merely examples shown for the convenience of explanation, and the present invention is not limited to these examples in any sense.

[0071] Materials and Methods Preparation of hot water extracts and non-extracted simple crushed powders of Mamaki leaves and fruits Dried mamaki tea leaves were purchased from Nakihalani Farm, LLC (Hawaii, USA). A hot water extract of mamaki leaves was prepared at Technopro L&D (Tokyo, Japan). 250 g of mamaki tea leaves were added to 3 L of water and steeped for 1 hour. After boiling for 15 minutes with stirring, the mixture was left to stand for 24 hours. After removing the tea leaves, the extract was filtered under suction using 8 μm pore size filter paper. The filtrate was concentrated to 500 mL using an evaporator at 40 °C. The concentrate was freeze-dried to obtain 66 g of extract powder. A simple crushed powder of mamaki leaves and fruit without extract was prepared in our laboratory. Because a small amount of fruit (including seeds) was mixed in with the commercially available mamaki tea package, the tea leaves and fruit were manually separated and crushed into 20–50 μm particles using a grinder (Fine Powder Mill FM-100, Labonect, Sakai, Japan). These particles were collected and used as an unextracted, simply crushed powder.

[0072] ·Mamaki ingredient analysis Mamaki leaves are known to contain catechin, chlorogenic acid, and rutin as major components (Non-Patent Document 10: Chun et al., Native Hawaiian medicines, First People's Productions Honolulu, (1994), pp. 216-217). The mamaki samples prepared above were sent to the Japan Food Research Laboratories (Tokyo, Japan) to measure the content of these polyphenols.

[0073] For catechin, 0.4 g of material was suspended in 30 mL of a mixture of methanol and oxalic acid (8:2) and extracted by shaking for 10 minutes. After centrifugation, the supernatant was collected, and the sediment was subjected to two additional methanol extractions. The supernatants from the three extractions were combined to a total volume of 100 mL. The extract was diluted and separated by high-performance liquid chromatography (HPLC) using a reversed-phase Inert Sustain C18 column (GL Sciences, Tokyo, Japan) with a 0.1% acetic acid and acetonitrile mixture (89:11) as the mobile phase. The eluted fractions were subsequently analyzed by electrospray ionization (ESI)-mass spectrometry (MS) using a XevoTQMS (Waters Corporation, Milford, MA).

[0074] For chlorogenic acid, 0.2 g of 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 centrifugation, the supernatant was collected, and the sediment was subjected to two additional methanol extractions. The supernatants from the three extractions were combined to a total volume of 250 mL. The extract was separated by HPLC using a reversed-phase CAPCELL PAKC 18 ACR column (Osaka Soda, Osaka, Japan) with a mobile phase of water, acetonitrile, and phosphoric acid (920:80:2). The absorbance of the eluted fractions at 325 nm was measured.

[0075] Finally, for rutin, 0.4 g of 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 centrifugation, the supernatant was collected, and the sediment was subjected to two additional methanol extractions. The supernatants from the three extractions were combined to a total volume of 200 mL. The extract was diluted and separated by HPLC using a reversed-phase Unison UK-C18 column (Imtakt USA, Portland, OR, USA) with a mobile phase of water, acetonitrile, and 2-propanol (200:38:2) containing 0.4% citric acid. The absorbance of the eluted fractions was measured at 360 nm.

[0076] ·mouse Four different neurodegenerative dementia mouse models were used.

[0077] Tau784 mice are an FTD model that express both three- and four-repeat human tau in adulthood due to the presence of a tau intronic mutation (Umeda et al., Am. J. Pathol., (2013), 183[1]:211-25; and Umeda et al., Ann. Clin. Transl. Neurol., (2015), 2[3]:241-55). Previous studies by the present inventors have shown that these mice exhibit tau hyperphosphorylation, tau oligomerization, synaptic loss, and memory impairment at 6 months of age, microglial activation at 12 months of age, and neurofibrillary tangle formation and neuronal loss at 15 months of age due to the imbalanced expression of tau isoforms.

[0078] APP23 mice are an AD model expressing human APP with the Swedish (KM670 / 671NL) mutation (Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA, (1997), 94

[24] :13287-92; Van Dam et al., Eur. J. Neurosci., (2003), 17[2]:388-96; and Umeda et al., Front. Neurosci., (2021), 15:763-476). These mice exhibit memory impairment at 3 months of age. Previous studies by the present inventors have shown that these mice exhibit accumulation of Aβ oligomers, synaptic loss, and amyloid deposition at 15 months of age.

[0079] The Huα-Syn(A53T) strain G2-3 mice were originally generated as a PD model expressing human α-synuclein with the A53T mutation (Non-Patent Document 30: Lee et al., Proc. Natl. Acad. Sci. USA, (2002), 99

[13] :8968-73; and Non-Patent Document 31: Umeda et al., Int. J. Mol. Sci., (2021), 22:8453). Previous studies by the present inventors have shown that these mice exhibit accumulation of α-synuclein oligomers from 4 months of age, cognitive impairment at 6 months of age, and motor dysfunction at 9 months of age. Therefore, these mice can be considered a model of DLB ​​up to 9 months of age.

[0080] The C9-500 mouse is a model of FTD / ALS introduced with the full-length human C9orf72 gene (Non-Patent Document 32: Liu et al., Neuron, (2016), 90[3]:521-534; and Non-Patent Document 33: Hatanaka et al., Biomedicines, (2022), 10[5]:1080). It harbors a mutation in intron 1a, resulting in an abnormal expansion of the GGGGCC sequence to approximately 500 repeats (hexanucleotide repeat expansion, HRE), and has been reported to exhibit various pathologies, including TDP-43 (Non-Patent Document 32). We confirmed that these mice exhibited accumulation of RNAG-quadruplexes, dipeptide repeat proteins (DPRs) such as poly-GA and poly-GP, and phosphorylated TDP-43 at 3 months of age, and showed synapse loss, neuron loss, and microglial activation at 6 months of age (Non-Patent Document 33). Incidentally, DPR is produced by repeat-associated ATG-independent translation (RAN translation) controlled by double-stranded RNA-dependent protein kinase (PKR). Although cognitive function in these mice began to decline at 4.5 months of age, motor function remained normal even at 12 months of age (Non-Patent Document 33). Thus, these mice can be considered a model of FTD-TDP up to 12 months of age.

[0081] All transgenic (Tg) mice were maintained and used as heterozygous animals. All animal experiments were approved by the Ethics Committee of Osaka Municipal University (Osaka, Japan) and were performed in accordance with the Osaka Municipal University Guide for Animal Experiments.

[0082] Mouse treatment To investigate the effects of hot water extract of mamaki leaves, the extract powder was sonicated and suspended in water at concentrations of 3.33, 0.33, and 0.10 mg / mL. 300 μL of each suspension (containing 1,000, 100, and 30 μg powder) was orally administered to male and female Tau784 mice, 5 days a week (Monday through Friday) for one month. Age-matched transgenic and non-transgenic littermates received the same volume of water as controls. APP23, Huα-Syn(A53T), and C9-500 mice were orally administered a 0.33 mg / mL suspension (100 μg powder / 300 μL) for one month.

[0083] To compare the effects of three mamaki preparations (hot water extract of mamaki leaves, crushed powder of mamaki leaves, and crushed powder of mamaki fruit), each powder was sonicated and suspended in water at a concentration of 0.10 mg / mL. 300 μL of each suspension (containing 30 μg powder) was orally administered to Tau784 mice for one month.

[0084] To investigate the effects of polyphenols contained in mamaki, catechin, chlorogenic acid, and rutin (all from Fujifilm-Wako, Osaka, Japan) were placed in a tube and dissolved in water to prepare a mixture. The concentrations were adjusted to 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 one month. The dose of these polyphenols was equivalent to the amount contained in 30 μg of simply crushed fruit powder. A solution containing only catechin (0.087 μg / 300 μL) was also administered to Tau784 mice.

[0085] Behavioral testing Spatial reference memory in mice was assessed using the Morris water maze test (Non-Patent Document 34: Kelley Bromley-Brits et al., J. Vis. Exp., (2011), 53e2920). A 10-cm-diameter platform was placed 1 cm below the surface of a circular pool with a diameter of 1 m, so that it was not visible to the mouse. Mice were placed in the pool and allowed to swim for 60 seconds, and the time it took to reach the platform was measured (represented as escape latency). If the mouse did not reach the platform within 60 seconds, the experimenter placed the mouse on the platform and allowed it to rest for a while. This trial was conducted for each mouse five times a day at 5-minute intervals for four consecutive days. This measured memory acquisition ability.

[0086] Histological analysis of neuropathology After behavioral testing, mice from each group were divided into two groups: one for histological analysis and the other for future biochemical analysis. Mice for histological analysis were perfusion-fixed with 4% paraformaldehyde and then brain sections were prepared and immunohistochemically stained for neuropathology as previously described (Non-Patent Document 27: Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA, (1997), 94

[24] :13287-92; and Non-Patent Document 33: Hatanaka et al., Biomedicines, (2022), 10[5]:1080). The antibodies used for immunohistochemistry are listed below. Pathology was assessed by quantifying staining intensity or staining area in a defined brain region using NIH ImageJ software. Neuroinflammation (Muzio et al., Front Neurosci., (2021), 24

[15] :742065) was assessed by counting the number of activated Iba-1-positive microglia in a specific brain region. The correspondence between each neuropathology and the corresponding detection antibody is shown in the table below.

[0087] [Table 1]

[0088] Histological analysis of BDNF expression and neurogenesis BDNF expression was assessed in Tau784 mice treated with three different mamaki preparations at 30 μg / day for 1 month. Brain sections were stained with an anti-BDNF antibody (GTX132621; GeneTex, Irvine, CA), and staining intensity in a range of brain regions was quantified using NIH ImageJ software.

[0089] Neurogenesis was assessed using aged Huα-Syn(A53T) mice. Mice were orally administered a powder suspension of crushed mamaki fruit at 30 μg / day for one month. 5-Bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich), a thymidine analogue that selectively incorporates into the DNA of proliferating cells, was dissolved in Tris-buffered saline, pH 7.6, at 5 mg / mL. 300 μL of this solution (containing 1.5 mg) was administered intraperitoneally to mice daily for the final five days of mamaki administration. Brain sections were double-stained with anti-BrdU mouse monoclonal antibody (IBL) and anti-doublecortin rabbit polyclonal antibody (Abcam). Cells positive for both antibodies were considered to be newly generated neurons, and their numbers were counted in a defined brain region.

[0090] ·Statistical analysis Comparisons of means between three or more groups were performed using ANOVA or two-way repeated measures ANOVA (for behavioral tests) followed by Fisher's PLSD test. Differences were considered significant when p-values ​​<0.05.

[0091] [Example 1] Examination of the effect of hot water extract of Mamaki leaves (Leaf-ext) on Tau784 mice The effects of a hot water extract of mamaki leaves (Leaf-ext) on Tau784 mice were examined. 12-14 month-old Tau784 mice (average weight 31.8g) were orally administered 1000μg / day, 100μg / day, or 30μg / day of powdered hot water extract of mamaki leaves (Leaf-ext) for one month. As controls, Tau784 mice and non-transgenic littermates of the same age were given the same amount of water.

[0092] Figure 1 shows the results of the Morris water maze test in Tau784 mice (Tg+Leaf-ext) orally administered a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (Tg) and non-transgenic mice (Non-Tg). Figure 1A shows the results for the 1000 μg / day and 100 μg / day groups, and Figure 1B shows the results for the 30 μg / day group. These results clearly show that administration of a hot water extract of mamaki leaves (Leaf-ext) improved the memory of Tau784 mice in a dose-dependent manner. In particular, the memory of Tau784 mice administered the 1000 μg / day and 100 μg / day groups improved to a level comparable to that of their non-transgenic littermates.

[0093] Figure 2 shows tau pathology in the entorhinal cortex of Tau784 mice (labeled "Tau784+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (labeled "Tau784"). Figure 2A shows photographs depicting the staining results for phosphorylated tau and tau oligomers, and Figure 2B shows a graph depicting the quantification of staining intensity in each photograph. These results clearly demonstrate that administration of a hot water extract of mamaki leaves (Leaf-ext) significantly reduced phosphorylated tau and tau oligomer levels in Tau784 mice.

[0094] Figure 3 shows synaptophysin pathology in the hippocampal CA2 / 3 region of Tau784 mice (Tau784+Leaf-ext in the table) orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (Tau784 in the table) and non-transgenic mice (Non-Tg in the table). Figure 3A shows photographs of synaptophysin staining, and Figure 3B shows a graph showing the quantification of staining intensity in each photograph. These results clearly show that administration of a hot water extract of mamaki leaves (Leaf-ext) improved synaptophysin levels in the hippocampal CA2 / 3 region of Tau784 mice to a level comparable to that of their non-transgenic littermates.

[0095] Figure 4 shows microglial pathology in the hippocampus (HC) and cerebral cortex (CTX) of Tau784 mice (Tau784+Leaf-ext) orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Tau784 mice (Tau784) and non-transgenic mice (Non-Tg). Figure 4A shows photographs of activated microglia staining, and Figure 4B shows a graph showing the number of positive cells in each photograph. These results clearly demonstrate that administration of a hot water extract of mamaki leaves (Leaf-ext) improved the levels of activated microglia in both the hippocampus (HC) and cerebral cortex (CTX) of Tau784 mice to levels comparable to those of non-transgenic littermates.

[0096] [Example 2] Examination of the effect of hot water extract of mamaki leaves (Leaf-ext) on APP23 mice The effects of a hot water extract of mamaki leaves (Leaf-ext) on APP23 mice were examined. 14-17 month-old APP23 mice (average weight 30.1g) were orally administered 100μg / day of powdered hot water extract of mamaki leaves (Leaf-ext) for one month. As controls, APP23 mice of the same age and non-transgenic littermates were given the same amount of water.

[0097] Figure 5 is a graph showing the results of the Morris water maze test in APP23 mice ("Tg+Leaf-ext" in the table) that were orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with APP23 mice ("Tg" in the table) and non-transgenic mice ("Non-Tg" in the table) that were administered water as a control. As is clear from these results, administration of the hot water extract of mamaki leaves (Leaf-ext) improved the memory of the APP23 mice to a level nearly equivalent to that of their non-transgenic littermates (non-Tg).

[0098] Figure 6 shows amyloid pathology in the cerebral cortex and hippocampus of APP23 mice (labeled "APP23+Leaf-ext") orally administered a hot water extract of mamaki leaves (Leaf-ext) at a dose of 100 μg / day for one month, compared with control APP23 mice (labeled "APP23"). Figure 6A shows photographs depicting staining results for amyloid deposits and Aβ oligomers, and Figure 6B shows a graph depicting the quantification of staining intensity in each photograph. As is clear from these results, administration of a hot water extract of mamaki leaves (Leaf-ext) significantly reduced amyloid deposits and Aβ oligomer levels in APP23 mice.

[0099] Figure 7 shows synaptophysin pathology in the hippocampal CA2 / 3 region of APP23 mice (labeled "APP23+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control APP23 mice (labeled "APP23") administered water and non-transgenic mice (labeled "Non-Tg"). Figure 7A shows photographs depicting synaptophysin staining results, and Figure 7B is a graph showing the quantification of staining intensity in each photograph. These results clearly demonstrate that administration of a hot water extract of mamaki leaves (Leaf-ext) improved synaptophysin levels in the hippocampal CA2 / 3 region of APP23 mice to a level comparable to that of their non-transgenic littermates.

[0100] Figure 8 shows microglial pathology in the hippocampus (HC) and cerebral cortex (CTX) of APP23 mice (labeled "APP23+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control APP23 mice (labeled "APP23") and non-transgenic mice (labeled "Non-Tg"). Figure 8A shows photographs depicting the staining results for activated microglia, and Figure 8B is a graph showing the number of positive cells in each photograph. These results clearly demonstrate that administration of a hot water extract of mamaki leaves (Leaf-ext) improved the levels of activated microglia in both the hippocampus (HC) and cerebral cortex (CTX) of APP23 mice to levels comparable to those of non-transgenic littermates.

[0101] [Example 3] Examination of the effect of hot water extract of Mamaki leaves (Leaf-ext) on Huα-Syn(A53T) mice The effects of a hot water extract of mamaki leaves (Leaf-ext) on Huα-Syn(A53T) mice were examined. Seven- to eight-month-old Huα-Syn(A53T) mice (average body weight 28.4 g) were orally administered 100 μg / day of powdered hot water extract of mamaki leaves (Leaf-ext) for one month. As controls, Huα-Syn(A53T) mice of the same age and non-transgenic littermates were given the same amount of water.

[0102] Figure 9 shows the results of the Morris water maze test in Huα-Syn(A53T) mice ("Tg+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with Huα-Syn(A53T) mice ("Tg") and non-transgenic mice ("Non-Tg") administered water as controls. These results clearly show that administration of the hot water extract of mamaki leaves (Leaf-ext) improved the memory of Huα-Syn(A53T) mice to a level nearly equivalent to that of their non-transgenic littermates (non-Tg).

[0103] Figures 10 and 11 show α-synuclein pathology in the hippocampus (HC) and entorhinal cortex (EC) of Huα-Syn(A53T) mice (labeled "αSyn-Tg+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for 1 month, compared with control Huα-Syn(A53T) mice (labeled "αSyn-Tg"). Figure 10A shows a photograph of phosphorylated α-synuclein staining, and Figure 11A shows a photograph of α-synuclein oligomer staining. Figures 10B and 11B are graphs showing the quantification of staining intensity in each photograph. These results clearly show that administration of the hot water extract of mamaki leaves (Leaf-ext) significantly reduced the levels of phosphorylated α-synuclein and α-synuclein oligomers in Huα-Syn(A53T) mice.

[0104] Figure 12 shows synaptophysin pathology in the hippocampal CA2 / 3 region of Huα-Syn(A53T) mice ("αSyn-Tg+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control Huα-Syn(A53T) mice ("αSyn-Tg") and non-transgenic mice ("Non-Tg"). Figure 12A shows photographs of synaptophysin staining, and Figure 12B shows a graph quantifying the staining intensity in each photograph. These results clearly demonstrate that administration of a hot water extract of mamaki leaves (Leaf-ext) improved synaptophysin levels in the hippocampal CA2 / 3 region of Huα-Syn(A53T) mice to levels comparable to those of their non-transgenic littermates.

[0105] Figure 13 shows microglial pathology in the hippocampus (HC) and cerebral cortex (CTX) of Huα-Syn(A53T) mice (labeled "αSyn-Tg+Leaf-ext") orally administered 100 μg / day of mamaki leaf extract (Leaf-ext) for one month, compared with control Huα-Syn(A53T) mice (labeled "αSyn-Tg") and non-transgenic mice (labeled "Non-Tg"). Figure 13A shows photographs of activated microglia staining, and Figure 13B shows graphs showing the number of positive cells in each photograph. These results clearly demonstrate that administration of mamaki leaf extract (Leaf-ext) improved the levels of activated microglia in the hippocampus (HC) and cerebral cortex (CTX) of Huα-Syn(A53T) mice to levels comparable to those of non-transgenic littermates.

[0106] [Example 4] Examination of the effect of hot water extract of mamaki leaves (Leaf-ext) on C9-500 mice The effects of a hot water extract of mamaki leaves (Leaf-ext) on C9-500 mice were examined. Nine to ten-month-old C9-500 mice (average weight 29.2 g) were orally administered 100 μg / day of powdered hot water extract of mamaki leaves (Leaf-ext) for one month. As controls, C9-500 mice of the same age and non-transgenic littermates were given the same amount of water.

[0107] Figure 14 is a graph showing the results of the Morris water maze test for C9-500 mice ("Tg+Leaf-ext" in the table) that were orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with C9-500 mice ("Tg" in the table) and non-transgenic mice ("Non-Tg" in the table) that were administered water as a control. As is clear from these results, administration of the hot water extract of mamaki leaves (Leaf-ext) improved the memory of the C9-500 mice to a level nearly equivalent to that of their non-transgenic littermates (non-Tg).

[0108] Figure 15 shows the pathology caused by C9orf72 gene mutations in the prefrontal cortex (PFC) of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered a hot water extract of mamaki leaves (Leaf-ext) at a dose of 100 μg / day for one month, compared with control C9-500 mice (labeled "C9-500"). Figure 15A shows photographs showing the staining results for RNA G-quadruplex, poly-GA, poly-GP, and phosphorylated TDP-43, and Figure 15B shows a graph showing the quantification of staining intensity for each photograph. These results clearly show that administration of the hot water extract of mamaki leaves (Leaf-ext) significantly reduced the level of pathology caused by C9orf72 gene mutations in the prefrontal cortex (PFC) of C9-500 mice.

[0109] Figure 16 shows double-stranded RNA-dependent protein kinase (PKR) pathology in the prefrontal cortex (PFC) of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control C9-500 mice (labeled "C9-500") and non-transgenic mice (labeled "Non-Tg"). Figure 16A shows photographs of phosphorylated PKR staining, and Figure 16B shows a graph quantifying the staining intensity in each photograph. These results clearly demonstrate that administration of the hot water extract of mamaki leaves (Leaf-ext) reduced PKR levels in the prefrontal cortex (PFC) of C9-500 mice to levels comparable to those of non-transgenic littermates.

[0110] Figure 17 shows synaptophysin pathology in the hippocampal CA2 / 3 region of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control C9-500 mice (labeled "C9-500") and non-transgenic mice (labeled "Non-Tg"). Figure 17A shows photographs of synaptophysin staining, and Figure 17B is a graph showing the quantification of staining intensity in each photograph. These results clearly show that administration of a hot water extract of mamaki leaves (Leaf-ext) improved synaptophysin levels in the hippocampal CA2 / 3 region of C9-500 mice to a level comparable to that of their non-transgenic littermates.

[0111] Figure 18 shows the microglial pathology in the prefrontal cortex (PFC) of C9-500 mice (labeled "C9-500+Leaf-ext") orally administered 100 μg / day of a hot water extract of mamaki leaves (Leaf-ext) for one month, compared with control C9-500 mice (labeled "C9-500") and non-transgenic mice (labeled "Non-Tg"). Figure 18A shows photographs depicting the staining results for activated microglia, and Figure 18B shows a graph depicting the number of positive cells in each photograph. These results clearly demonstrate that administration of the hot water extract of mamaki leaves (Leaf-ext) improved the level of activated microglia in the prefrontal cortex (PFC) of C9-500 mice to a level comparable to that of their non-transgenic littermates.

[0112] [Example 5] Examination of the effects of hot water extract of Mamaki leaves (Leaf-ext), simply crushed leaf powder (Leaf-pwd), and simply crushed fruit (including seeds) powder (Fruit-pwd) on Tau784 mice The effects of hot water extract of mamaki leaves (Leaf-ext), simply crushed leaf powder (Leaf-pwd), and simply crushed fruit (including seeds) powder (Fruit-pwd) on Tau784 mice were examined. Seven- to nine-month-old Tau784 mice (average weight 28.9 g) were orally administered 30 μg / day of hot water extract of mamaki leaves (Leaf-ext), simply crushed leaf powder (Leaf-pwd), and simply crushed fruit (including seeds) powder (Fruit-pwd) for one month. As controls, Tau784 mice and non-transgenic littermates of the same age were given the same amount of water.

[0113] Figure 19 shows the results of the Morris water maze test in Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered 30 μg / day of a hot water extract of mamaki leaves (Leaf-ext), a simple crushed leaf powder (Leaf-pwd), and a simple crushed fruit powder (including seeds) (Fruit-pwd) for one month, compared with Tau784 mice (Tg) and non-transgenic mice (Non-Tg) administered water as controls. These results clearly show that administration of the hot water extract of mamaki leaves (Leaf-ext) improved memory in Tau784 mice, but not as much as in their non-transgenic littermates. Administration of crushed mamaki leaves (Leaf-pwd) improved the memory of Tau784 mice to the same level as that of their non-transgenic littermates. Surprisingly, administration of crushed mamaki fruit (including seeds) (Fruit-pwd) significantly enhanced the memory of Tau784 mice, even to a higher level than that of their non-transgenic littermates.

[0114] Figure 20 shows tau pathology in the entorhinal cortex of Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered hot water extract of mamaki leaves (Leaf-ext), crushed leaf powder (Leaf-pwd), and crushed fruit powder (including seeds) at a dose of 30 μg / day for one month, compared with control Tau784 mice (Tau784). Figure 20A shows photographs of staining for phosphorylated tau and tau oligomers, and Figure 20B shows a graph quantifying the staining intensity of each photograph. These results clearly show that the levels of phosphorylated tau in the entorhinal cortex (EC) were significantly reduced by administration of the powdered leaves (Leaf-pwd) and the powdered fruit (including seeds) of Mamaki (Maki quince). However, the effect of the powdered fruit (including seeds) was stronger. The hot water extract of the leaves (Leaf-ext) had only a slight effect. Meanwhile, the levels of tau oligomers were significantly reduced by both administrations. However, the effect was strongest for the powdered fruit (including seeds) and weakest for the hot water extract of the leaves (Leaf-ext).

[0115] Figure 21 shows synaptophysin pathology in the hippocampal CA2 / 3 region of Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered hot water extract of mamaki leaves (Leaf-ext), crushed leaf powder (Leaf-pwd), and crushed fruit powder (including seeds) at a dose of 30 μg / day for 1 month, compared with control Tau784 mice (Tau784) and non-transgenic mice (Non-Tg). Figure 21A shows photographs of synaptophysin staining, and Figure 21B shows graphs quantifying the staining intensity of each photograph. These results clearly show that the synaptophysin levels in the hippocampal CA2 / 3 region of Tau784 mice were significantly restored by administration of the powdered leaves (Leaf-pwd) and the powdered fruits (including seeds) (Fruit-pwd), but the effect of the powdered fruits (including seeds) (Fruit-pwd) was stronger. On the other hand, the hot water extract of the leaves (Leaf-ext) only incompletely restored the synaptophysin levels.

[0116] Figure 22 shows BDNF expression in the cerebral cortex (CTX) of Tau784 mice (Tg+Leaf-ext, Tg+Leaf-pwd, and Tg+Fruit-pwd) orally administered 30 μg / day of hot water extract of mamaki leaves (Leaf-ext), crushed leaf powder (Leaf-pwd), and crushed fruit powder (including seeds) (Fruit-pwd) for one month, compared with control Tau784 mice (Tau784) and non-transgenic mice (Non-Tg). Figure 22A shows photographs of BDNF staining, and Figure 22B shows the quantification of staining intensity for each photograph. As is clear from these results, the BDNF levels in the cerebral cortex (CTX) of Tau784 mice were significantly increased by the simple crushed powder of mamaki fruit (including seeds) (Fruit-pwd) to a level higher than that of non-transgenic littermates, and by the simple crushed powder of leaves (Leaf-pwd) to a level equivalent to that of non-transgenic littermates, whereas the hot water extract of leaves (Leaf-ext) had only a slight effect.

[0117] [Example 6] Examination of the effect of simply crushed powder of mamaki fruit (including seeds) (Fruit-pwd) on Huα-Syn(A53T) mice The effects of crushed mamaki fruit (including seeds) powder (Fruit-pwd) on Huα-Syn(A53T) mice were examined. Ten- to eleven-month-old Huα-Syn(A53T) mice (average weight 28.9 g) were orally administered 30 μg / day of crushed mamaki fruit (including seeds) powder (Fruit-pwd) for one month. As controls, Huα-Syn(A53T) mice and non-transgenic littermates of the same age were given the same amount of water.

[0118] Figure 23 shows the neurogenesis levels in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn(A53T) mice ("αSyn-Tg+Fruit-pwd") orally administered 30 μg / day of crushed mamaki fruit (including seeds) powder (Fruit-pwd) for one month, compared with control Huα-Syn(A53T) mice ("αSyn-Tg") and non-transgenic mice ("Non-Tg"). Figure 23A shows immunofluorescent staining for BrdU (red) and doublecortin (DCX) (green). Double-positive cells (yellow), which were both BrdU (red) and DCX (green), were considered to be newborn neurons. Figure 23B shows the results of quantifying the number of double-positive cells (yellow) in each photograph. These results clearly show that administration of a simple crushed powder of mamaki fruit (including seeds) (Fruit-pwd) significantly improved neurogenesis in the dentate gyrus (DG) and substantia nigra (SN) of Huα-Syn(A53T) mice to levels far exceeding those of non-transgenic littermates. These results suggest that mamaki fruit (including seeds) has the ability to promote brain rejuvenation through neuronal repair and regeneration.

[0119] [Example 7] Examination of the effects of three types of polyphenols on Tau784 mice Three polyphenols contained in mamaki, namely, catechin, chlorogenic acid, and rutin, have been reported to have anti-cognitive effects. Therefore, the various effects of the hot water extract of mamaki leaves (Leaf-ext), simply crushed leaf powder (Leaf-pwd), and simply crushed fruit (including seeds) powder (Fruit-pwd) may be due to these polyphenols. Therefore, we investigated the effects of these three polyphenols, namely, catechin, chlorogenic acid, and rutin, on Tau784 mice.

[0120] First, the contents of three polyphenols, namely catechin, chlorogenic acid, and rutin, contained in 100g of each of the hot water extract of Mamaki leaves (Leaf-ext), the powder of simply crushed leaves (Leaf-pwd), and the powder of simply crushed fruit (including seeds) (Fruit-pwd) were quantified. The results are shown in the table below.

[0121] [Table 2]

[0122] Next, 8- to 10-month-old Tau784 mice (average weight 32.0 g) were orally administered a mixture of 0.087 μg of catechin, 0.036 μg of chlorogenic acid, and 0.123 μg of rutin, or 0.087 μg of catechin alone for one month. The doses of these polyphenols were equivalent to the amounts contained in 30 μg of simply crushed mamaki fruit (including seeds) powder. As controls, Huα-Syn(A53T) mice and non-transgenic littermates of the same age were given the same amount of water.

[0123] Figure 24 shows the results of the Morris water maze test in Tau784 mice orally administered a mixture of 0.087 μg of catechin, 0.036 μg of chlorogenic acid, and 0.123 μg of rutin ("Tg+3 polyphenol mixture") or 0.087 μg of catechin alone ("Tg+chatechin") for one month, compared with control Tau784 mice ("Tg") and non-transgenic mice ("Non-Tg") administered water. These results clearly show that administration of the three polyphenol mixture improved memory in Tau784 mice, but the effect was incomplete and far inferior to that of non-transgenic littermates. Furthermore, administration of catechin alone showed a weaker effect than the polyphenol mixture. Comparing these results with the effects of each of the above Examples 1 to 6, it is clear that the various effects of the hot water extract of mamaki leaves (Leaf-ext), simply crushed leaf powder (Leaf-pwd), and simply crushed fruit (including seeds) powder (Fruit-pwd) are not solely due to the three polyphenols mentioned above, but rather are largely due to the contribution of other unknown components contained in mamaki. [Industrial Applicability]

[0124] The present invention can be widely applied to fields such as functional foods and pharmaceuticals where maintenance or improvement of cognitive function is desired, and its utility value is extremely great.

Claims

1. An agent for maintaining or improving brain function, comprising a herbal medicine selected from the leaves, fruits, and seeds of Mamaki.

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

3. An agent for promoting the repair of nerve cells or inducing neurogenesis, comprising a medicinal herb selected from the leaves, fruits, and seeds of Mamaki.

4. An agent for removing proteins that cause neurodegenerative diseases and accumulate in the brain, comprising a herbal medicine selected from the leaves, fruits, and seeds of Mamaki.

5. 5. The agent according to claim 4, wherein the neurodegenerative disease-causing protein is one or more proteins selected from amyloid beta (Aβ), tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein derived from RAN (repeat-associated non-ATG) translation, prion, and SOD-1.

6. A therapeutic or preventive agent for neurodegenerative diseases, comprising a medicinal herb selected from the leaves, fruits, and seeds of Mamaki.

7. The agent according to claim 6, wherein the neurodegenerative disease is degenerative dementia.

8. The agent according to claim 7, wherein the degenerative dementia is one or more types of dementia selected from Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, and dementia caused by Parkinson's disease or amyotrophic lateral sclerosis.

9. The agent according to claim 1, wherein the herbal medicine selected from the leaves, fruits, and seeds of Mamaki is crushed and / or extract of Mamaki leaves and / or fruits and / or seeds.

10. The agent according to claim 1, wherein the herbal medicine is administered to a subject in an amount of 0.01 mg to 10 g per day.

11. The agent according to any one of claims 1 to 10, (1) Maintenance or improvement of brain function, (2) promoting repair of nerve cells or inducing neurogenesis; (3) Removal of neurodegenerative disease-causing proteins that accumulate in the brain, and / or (4) Treatment or prevention of neurodegenerative diseases Food for.

12. The agent according to any one of claims 1 to 10, (1) Maintenance or improvement of brain function, (2) promoting repair of nerve cells or inducing neurogenesis; (3) Removal of neurodegenerative disease-causing proteins that accumulate in the brain, and / or (4) Treatment or prevention of neurodegenerative diseases Medicine for.

13. 1. Use of a herbal medicine selected from the leaves, fruits, and seeds of Mamaki in the manufacture of a food or medicine for maintaining or improving brain function in a subject.

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

15. 1. Use of a herbal medicine selected from the leaves, fruits, and seeds of Mamaki in the manufacture of a food or medicine for promoting the repair of nerve cells or inducing neurogenesis in a subject.

16. Use of a herbal medicine selected from the leaves, fruits, and seeds of Mamaki in the manufacture of a food or medicine for removing a neurodegenerative disease-causing protein that accumulates in the brain of a subject.

17. The use according to claim 16, wherein the neurodegenerative disease-causing protein is one or more proteins selected from amyloid beta (Aβ), tau, α-synuclein, TDP-43, FUS / TLS, polyglutamine, a protein derived from repeat-associated non-ATG (RAN) translation, prion, and SOD-1.

18. 1. Use of a herbal medicine selected from the leaves, fruits, and seeds of Mamaki in the manufacture of a food or medicine for treating or preventing a neurodegenerative disease in a subject.

19. 19. The use according to claim 18, wherein the neurodegenerative disease is degenerative dementia.

20. 20. The use according to claim 19, wherein the degenerative dementia is one or more types of dementia selected from Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, and dementia caused by Parkinson's disease or amyotrophic lateral sclerosis.

21. The use according to any one of claims 13 to 20, wherein the herbal medicine selected from the leaves, fruits, and seeds of Mamaki is crushed material and / or extract of Mamaki leaves and / or fruits and / or seeds.

22. The use according to any one of claims 13 to 220, wherein the herbal medicine is administered to a subject in an amount of 0.01 mg to 10 g per day.

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