Cognitive function improving agent, neurite outgrowth agent, and therapeutic composition containing the same

Abstract

An object of the present invention is to provide a new cognitive function improving agent containing a component that was not previously known to improve cognitive function, and to clarify the mechanism of action of the component's cognitive function improving effect. A further object of the present invention is to provide a new neurite outgrowth agent and to use the neurite outgrowth agent as a composition for treating neurodegenerative diseases. The present invention provides a cognitive function improving agent or a neurite outgrowth agent containing 2-O-trans caffeoylhydroxycitric acid, which preferably contains 2-O-trans caffeoylhydroxycitric acid derived from coriander.

Description

[Technical Field]

[0001] The present invention relates to a cognitive function improving agent and a neurite outgrowth agent, specifically to a cognitive function improving agent containing 2-O-trans caffeoylhydroxycitric acid, as well as a neurite outgrowth agent and a therapeutic composition containing the same. [Background technology]

[0002] In recent years, the number of patients developing cognitive impairment has been increasing, becoming a social problem. Known types of cognitive impairment include Alzheimer's disease, vascular dementia, dementia with Lewy bodies, and frontotemporal lobar degeneration (FTLD), but the majority of cases are Alzheimer's disease. Several agents for improving cognitive function have been proposed, including those containing plant extracts. For example, a cognitive function improver containing ginkgo biloba extract as active ingredients, along with propolis and curcumin, has been proposed (Patent Document 1).

[0003] It has also been reported that coriander seed extract may improve age-related memory impairment in a senescence-accelerated mouse model (Non-Patent Document 1), and that coriander leaves may improve memory impairment in mice (Non-Patent Document 2). SAMP mice are known as an animal model of cognitive dysfunction. Among them, SAMP8 mice are a model that exhibits a decline in learning and memory characteristic of aging (Non-Patent Document 3).

[0004] Furthermore, the relationship between the effect of improving neurite outgrowth in nerve cells and the therapeutic effect on cognitive dysfunction and neurodegenerative diseases has also been discussed (Non-Patent Document 3, Patent Document 3, etc.).

[0005] On the other hand, 2-O-trans-caffeoylhydroxycitric acid is a compound that can be extracted from various plants using an aqueous extraction solvent such as water. For example, it has been reported to be extracted from the leaves and stems of Yellow Mombin (Anacardiaceae) (Non-Patent Document 4), corn seedlings (Non-Patent Document 5), Knautia arvensis (Caprifoliaceae) (Non-Patent Document 6), orchard grass (Poaceae) (Non-Patent Document 7), hibiscus (Non-Patent Document 8), and coriander (Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2021 / 085496 publication [Patent Document 2] WO2024 / 071421 publication [Patent Document 3] Japanese Patent Application Laid-Open No. 2024-69369 [Non-patent literature]

[0007] [Non-Patent Document 1] Nutrients 2020, 12, 455, “Effects of Coriandrum sativum Seed Extract on Aging-Induced Memory Impairment in Samp8 Mice” [Non-patent document 2] J Sci Food Agric 2011; 91: 186-192, Reversal of memory deficits by Coriandrum sativum leaves in mice [Non-patent document 3] Experimental Gerontology 40 (2005) 774-783, The senescence-accelerated prone mouse (SAMP8): A model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in Alzheimer's disease [Non-patent document 4] Phytochemistry, Vol. 31, No. 6, pp. 1979-1981, 1992, ANTIVIRAL CAFFEOYL ESTERS FROM SPONDIAS MOMBIN [Non-Patent Document 5] Agric. Biol. Chem., 41 (2), 359-367, 1977, Three New Substituted Cinnamoyl Hydroxycitric Acids from Corn Plant [Non-patent document 6] Natural Product Communications Vol. 6 (11) 2011, 1627-1630, Phenolic Constituents of Knautia arvensis Aerial Parts [Non-Patent Document 7] Phytochemistry Volume 69, Issue 16, November 2008, Pages 2799-2806, Extraction, structural characterization and evaluation of hydroxycinnamate esters of orchard grass (Dactylis glomerata) as substrates for polyphenol oxidase [Non-patent document 8] Nat Prod Res. 2017 December; 31(24): 2885-2892., Comparison of metabolic profiles and bioactivities of the leaves of three edible Congolese Hibiscus species Summary of the Invention [Problem to be solved by the invention]

[0008] There is still a need for compositions that improve cognitive impairment, and searches are being conducted to find active ingredients for this purpose. An objective of the present invention is to provide a new cognitive function improver containing an ingredient that has not been known to improve cognitive function. Another objective of the present invention is to clarify the mechanism of action of the ingredient's cognitive function improving effect.

[0009] Another object of the present invention is to provide a novel neurite outgrowth agent and to use the neurite outgrowth agent as a composition for treating neurodegenerative diseases. [Means for solving the problem]

[0010] A first aspect of the present invention relates to a cognitive function improver shown below. <1> A cognitive enhancer containing 2-O-trans caffeoylhydroxycitric acid. <2> The 2-O-trans caffeoylhydroxycitric acid is derived from a plant of the genus Coriander in the family Umbelliferae. <1> The cognitive function improving agent according to claim 1. <3> The 2-O-trans caffeoylhydroxycitric acid is derived from coriander. <1> The cognitive function improving agent according to claim 1. <4> The 2-O-trans caffeoyl hydroxycitric acid is derived from coriander leaves. <1> or <2> The cognitive function improving agent according to claim 1. <5> For oral ingestion, <1> ~ <4> The cognitive function improving agent according to any one of the above.

[0011] The second aspect of the present invention relates to the following neurite outgrowth agent and a therapeutic composition containing the same. <6> A neurite outgrowth agent comprising 2-O-trans caffeoylhydroxycitric acid. <7> The 2-O-trans caffeoylhydroxycitric acid is derived from a plant of the genus Coriander in the family Umbelliferae. <6> The neurite outgrowth agent according to claim 1. <8> The 2-O-trans caffeoylhydroxycitric acid is derived from coriander. <6> The neurite outgrowth agent according to claim 1. <9> The 2-O-trans caffeoyl hydroxycitric acid is derived from coriander leaves. <6> or <7> The neurite outgrowth agent according to claim 1. <10> For oral ingestion, <6> ~ <9> The neurite outgrowth agent according to any one of the preceding claims. <11> The aforementioned <6> ~ <10> 10. A composition for treating a neurodegenerative disease, comprising the neurite outgrowth agent according to any one of the preceding items. <12> The aforementioned <6> ~ <10> 10. A composition for treating a neurodegenerative disease comprising the neurite outgrowth agent according to any one of claims 1 to 9. <13> The neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal dementia, and amyotrophic lateral sclerosis. <12> A therapeutic composition for a neurodegenerative disease described in the above. <14> Improving NGF-induced neurite outgrowth, <6> ~ <10> The neurite outgrowth agent according to any one of the preceding claims. [Effects of the Invention]

[0012] First, the present invention provides a new cognitive function improver. Furthermore, since the cognitive function improver provided by the present invention can contain an extract of a plant that has been consumed by humans as an active ingredient, it is likely to be highly safe and can be used not only as a pharmaceutical product but also as a food product such as a health food. Second, the present invention provides a new neurite outgrowth agent. The neurite outgrowth agent of the present invention can be used as a therapeutic composition for treating neurodegenerative diseases. [Brief explanation of the drawings]

[0013] [Figure 1] This is a chromatogram of HPLC analysis of coriander extract (leaf extract powder) and fractions 1 to 5 obtained using an open column. [Figure 2] This is an HMBC spectrum obtained by NMR measurement of the purified fraction C. [Figure 3] Chromatograms from HPLC analysis of coriander extract (leaf extract powder), coriander extract (seed extract powder), and 2-O-trans-caffeoylhydroxycitric acid (isolated purified product). [Figure 4] 1 is a graph showing the neuronal differentiation promoting effect (neurite outgrowth) of coriander extract (leaf extract powder and seed extract powder). [Figure 5] 1 is a graph showing the neuronal differentiation promoting effect (neurite outgrowth) of each fraction of coriander extract (leaf extract powder). [Figure 6] 1 is a graph showing the effect of coriander extract (leaf extract powder and seed extract powder) on gene expression of neurofilament light chain (Nefl) in nerve cells. [Figure 7] 1 is a graph showing the differentiation-promoting effect of purified fractions A to C on nerve cells (neurite outgrowth). [Figure 8] 1 is a graph showing the results of a repetition learning test in the Barnes maze test. [Figure 9] FIG. 1 is a schematic diagram showing a Barnes maze divided into four sections in a probe test in the Barnes maze test. [Figure 10] 1 is a graph showing the results of a probe test in the Barnes maze test. DETAILED DESCRIPTION OF THE INVENTION

[0014] [1. Cognitive function improver] The cognitive function improver of the present invention contains 2-O-trans caffeoylhydroxycitric acid, and typically contains 2-O-trans caffeoylhydroxycitric acid as an active ingredient.

[0015] [1-1,2-O-trans caffeoyl hydroxycitric acid] 2-O-trans caffeoylhydroxycitric acid is a phenolic compound having the following structure. 2-O-trans caffeoylhydroxycitric acid may be chemically synthesized or extracted from various plants. Whether or not 2-O-trans caffeoylhydroxycitric acid is contained in an extract from various plants can be confirmed by checking whether or not a specific peak is present in the chromatogram of HPLC analysis (see Examples for detailed conditions). [ka]

[0016] [1-2. Plant extracts] Plants that can be used as raw materials for extracting 2-O-trans-caffeoylhydroxycitric acid are not particularly limited as long as they contain 2-O-trans-caffeoylhydroxycitric acid, and examples include coriander, hibiscus, yellow mombin, corn, orchardgrass, Knautia arvensis, and other plants. Preferred plants are those of the genus Coriander in the family Umbelliferae, and more preferably coriander.

[0017] The part of each plant used as a raw material for extraction is not particularly limited, and any part containing 2-O-trans caffeoylhydroxycitric acid may be selected. Examples of such parts include the whole plant, flowers, inflorescences, spikes, pericarp, fruit, stems, branches, leaves, rhizomes, root bark, roots, and seeds. The plant used as a raw material may be a fresh plant or a dried plant. Plant drying may be by heat drying, such as hot air drying, or freeze-drying. The plant used as a raw material for extraction may be crushed, pulverized, ground, or powdered. These may be sieved to adjust the size. The plant used as a raw material for extraction may be pretreated, such as by hot water treatment or steam treatment, prior to extraction. Specifically, it may be blanched (immersed in hot water). These pretreatments may improve the extraction efficiency of 2-O-trans caffeoylhydroxycitric acid.

[0018] The method for extracting 2-O-trans caffeoylhydroxycitric acid from each plant is not particularly limited, but is usually carried out by solvent extraction. 2-O-trans caffeoylhydroxycitric acid is considered to be soluble in aqueous solvents, and the extraction solvent can be water or an aqueous solvent containing water, preferably water. The temperature of the extraction solvent during extraction is not particularly limited, but is not particularly limited as long as it is in the range of 0°C to 100°C. If extraction is performed under pressure, the temperature may be above 100°C, but is preferably 40°C or higher, more preferably 60°C or higher, and even more preferably 80°C or higher. By appropriately setting the extraction temperature, the extraction efficiency of 2-O-trans caffeoylhydroxycitric acid can be improved. The extraction time is also not particularly limited as long as 2-O-trans caffeoylhydroxycitric acid can be extracted, and may be, for example, 1 minute or more (10 minutes or more, 30 minutes or more) or 3 days or less (1 day or less, 10 hours or less).

[0019] The extract may be the solvent extracted from each plant itself (extract), or may be a concentrated extract, or the extract or its concentrate dried and solidified (powder), etc. The extract or its concentrate can be dried and solidified by heat drying or freeze-drying, preferably freeze-drying.

[0020] [1-3. Coriander extract] As mentioned above, 2-O-trans-caffeoylhydroxycitric acid can be extracted from coriander. Coriander, also known as cilantro, is a plant of the Coriander genus in the Apiaceae family and is commonly used as a seasoning or medicinal herb. Coriander is believed to have medicinal properties, and in traditional medicine it is used to treat a variety of ailments, including cramps, neuralgia, stomach upset, dysentery, indigestion, and dizziness. Coriander extracts have also been reported to have antiallergic, antibacterial, anticancer, antioxidant, and antidiabetic effects (see, e.g., Industrial Crops and Products, Volume 87, September 2016, Pages 54-63).

[0021] The coriander extract may be an extract from any part of the coriander plant, such as the whole plant, fruits, seeds, stems, leaves, roots, or a combination thereof, as long as 2-O-trans-caffeoylhydroxycitric acid can be extracted from it. However, extracts from stems, leaves, flowers, fruits, seeds, or a combination thereof are preferred, and extracts from leaves, seeds, or a combination thereof are more preferred, and extracts from leaves are even more preferred. The conditions for extraction from coriander are also as described above in the section [1-2. Extracts from Plants].

[0022] [1-4. Other optional ingredients] The cognitive function improver of the present invention may be composed solely of 2-O-trans caffeoylhydroxycitric acid, or may be composed solely of a plant extract containing 2-O-trans caffeoylhydroxycitric acid, but may also contain optional additives (optional formulation aids such as excipients, binders, disintegrants, lubricants, flavorings, solubilizers, suspending agents, coating agents, etc.) depending on the respective delivery form.

[0023] Furthermore, the cognitive function improver of the present invention may contain a physiologically active substance (for example, an active ingredient for improving cognitive function) other than 2-O-trans caffeoylhydroxycitric acid.

[0024] [1-5. Uses of cognitive function improvers] The term "cognitive function" in the cognitive function improving agent of the present invention refers to a collective term for memory, trial, comprehension, judgment, and other activities performed in the brain. Cognitive function can be objectively measured, for example, using the Neurocognitive Index (NCI) standardized score in the Cognitrax test as an index (see CNS Vital Signs LLC. CNS Vital Signs Interpretation Guide. 2019). Furthermore, cognitive function improvement refers to an improvement in intellectual function that integrates a wide range of functional areas, such as memory, processing speed, executive function, attention, and cognitive flexibility. Cognitive function improvement can be measured, for example, by an increase in the Neurocognitive Index (NCI) standardized score in the Cognitrax test.

[0025] Cognitive function is known to decline not only with age but also due to diseases and disorders. Examples of diseases that impair (cause impairment of) cognitive function include the following: [Table 1]

[0026] The cognitive function improving agent of the present invention may be orally ingested or administered to healthy individuals (those not suffering from the above-mentioned diseases that impair cognitive function) or to patients suffering from cognitive impairment. In other words, subjects to which the cognitive function improving agent of the present invention is administered may be, for example, dementia patients such as Alzheimer's disease patients, individuals with mild cognitive impairment, which is a precursor to dementia, individuals at risk of cognitive decline, individuals who are aware of their forgetfulness, individuals who have been told by others that they are forgetful, individuals with sleep disorders, or individuals in need of liver function improvement. Preferably, the cognitive function improving agent of the present invention is orally ingested or administered to healthy individuals. The subject may be, for example, 45 years of age or older, or may be, for example, an elderly person aged 60 years of age or older, 65 years of age or older, 70 years of age or older, or 75 years of age or older.

[0027] The cognitive function improver of the present invention can be taken into the body by oral ingestion or administration to a human. The oral ingestion or administration route may be oral or parenteral, but is preferably oral. The daily intake or administration amount of the cognitive function improver of the present invention is preferably 0.01 mg / day to 100 mg / day in terms of 2-O-trans caffeoylhydroxycitric acid. Furthermore, the daily intake or administration amount of the cognitive function improver of the present invention is preferably 1 mg / day to 2000 mg / day in terms of coriander extract. The cognitive function improver of the present invention may be orally taken or administered in multiple doses, such as once a day, twice a day, or three times a day.

[0028] [1-6. Preparations of cognitive function improvers] The cognitive function improving agent of the present invention can be formulated into any form. The formulation may be in any form, such as a solid, liquid, or paste, and may be in the form of tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, or injections (including those prepared as liquids by mixing with distilled water or infusions such as amino acid infusions or electrolyte infusions). These various formulations can be prepared, for example, by mixing the above-mentioned cognitive function improving agent with other ingredients as necessary and formulating the formulation into the above-mentioned dosage form.

[0029] The content of 2-O-trans caffeoylhydroxycitric acid in the formulation of the cognitive function improver of the present invention is not particularly limited as long as it is 0.1 to 99% by mass, and more specifically, it may be in the range of 0.1 to 50% by mass, or 0.1 to 10% by mass, or 0.1 to 1% by mass.

[0030] The cognitive function improver of the present invention can be used as a food composition or as one of its components. Preferably, the food composition emphasizes the tertiary function of food, i.e., the function of regulating physical condition. Examples of products that emphasize the tertiary function of food include health foods, functional foods, nutritional functional foods, nutritional supplements, supplements, and foods for specified health uses.

[0031] When the cognitive function improver of the present invention is provided as a food composition, the cognitive function improver can be appropriately added to known foods and beverages to produce a food composition having a cognitive function improving effect. Examples of the food composition include milk and dairy products, seasonings, beverages, confectioneries, breads, noodles, oils and fats, processed meat products, processed seafood products, processed agricultural products, frozen foods, and instant foods.

[0032] Food compositions containing the cognitive function improver of the present invention may be offered or sold as food compositions labeled with the health use of improving cognitive function. "Labeling" includes all acts intended to inform consumers of the intended use. Any expression that can conjure up or infer the intended use falls under the "labeling" category of the present invention, regardless of the purpose, content, object, or medium of the labeling. Preferably, the "labeling" is carried out in a manner that directly identifies the intended use to consumers. Specifically, this includes acts such as transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging that lists the intended use; displaying or distributing product advertisements, price lists, or transaction documents that list the intended use; or providing information containing the intended use via electromagnetic means (e.g., the Internet).

[0033] The content of the labeling is preferably one approved by the government, etc. (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval.) It is also preferable that such content of the labeling be attached to promotional materials at the point of sale, such as packaging, containers, catalogs, pamphlets, and POP displays, as well as other documents.

[0034] [2. Neurite outgrowth agents] The neurite outgrowth agent of the present invention contains 2-O-trans caffeoylhydroxycitric acid, which is as described in the section [1. Cognitive function improving agent].

[0035] The neurite outgrowth agent of the present invention can improve neurite outgrowth of nerve cells. Here, the neurite may be an axon or a dendrite. The neurite outgrowth may be NGF (nerve growth factor)-induced neurite outgrowth. Here, NGF may be orally ingested or administered together with the neurite outgrowth agent of the present invention, or may be produced by the subject to which the neurite outgrowth agent is orally ingested or administered. In this way, NGF can improve neurite outgrowth together with the neurite outgrowth agent of the present invention.

[0036] The neurite outgrowth-improving effect of the neurite outgrowth agent of the present invention can be confirmed, for example, by measuring an increase in the number of neurite-bearing cells in a sample. That is, the neurite outgrowth-improving effect can be confirmed, for example, by the fact that the number of neurite-bearing cells in a sample after oral ingestion or administration of the neurite outgrowth agent is greater than the number of neurite-bearing cells before oral ingestion or administration of the neurite outgrowth agent. "Neurite-bearing cells" may be defined as cells that present a protrusion at least one times the length of the cell body. That is, "neurite-bearing cells" are particularly nerve cells (i.e., neurons) that bear a protrusion at least one times the length of the cell body.

[0037] The "amount of neurite-bearing cells" can refer to the absolute amount of neurite-bearing cells or the relative amount of neurite-bearing cells. The relative amount of neurite-bearing cells can be calculated, for example, as the ratio of the number of neurite-bearing cells to the total number of cells in a sample. In particular, the relative amount of neurite-bearing cells can be calculated as the ratio of the number of neurite-bearing cells after use of a neurite outgrowth agent to the total number of cells in a sample, and the neurite outgrowth improvement effect can be confirmed by comparing the relative amount of neurite-bearing cells before and after use of a neurite outgrowth agent.

[0038] Improvement in neurite outgrowth can be an indicator of neuronal differentiation. Therefore, the neurite outgrowth agent of the present invention can promote neuronal differentiation, i.e., have the effect of promoting neuronal differentiation (neuron differentiation-promoting effect). In other words, the neurite outgrowth agent of the present invention can be considered to improve neurite outgrowth by promoting neuronal differentiation.

[0039] Promotion of neuronal differentiation can be confirmed, for example, by analyzing the gene expression profile of neurons. For example, neurofilament light chain (Nefl), known as a neuronal marker (neuron differentiation marker), is a gene expressed during neuronal differentiation. Therefore, the expression level of neurofilament light chain expressed by neurons can be compared before and after application of the neurite outgrowth agent of the present invention to confirm whether the expression level is higher after application of the neurite outgrowth agent.

[0040] Furthermore, oral ingestion or administration of the neurite outgrowth agent of the present invention to a subject may exhibit pharmacological effects based on improved neurite outgrowth (or neuronal differentiation-promoting effects). More specifically, the neurite outgrowth agent of the present invention may protect or improve neural function through its neurite outgrowth-improving effect. That is, the improvement in neurite outgrowth may prevent, improve, or treat symptoms associated with, for example, nerve disorders.

[0041] Symptoms associated with neurological disorders that can be prevented, ameliorated, or treated by the neurite outgrowth agent of the present invention may be neurodegenerative diseases. Furthermore, symptoms associated with neurological disorders that can be prevented, ameliorated, or treated by the neurite outgrowth agent of the present invention may be neurodegenerative diseases. Examples of neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), dementia (dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), etc.), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Huntington's disease, dystonia, and transmissible spongiform encephalopathies (TSEs). encephalopathy (TSE), chorea-acanthocytosis (ChAc), adrenoleukodystrophy (ALD), multiple system atrophy (MSA), spinocerebellar degeneration (SCD), amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal and bulbar muscular atrophy (SBMA), spinal muscular atrophy (SMA), Charcot-Marie-Tooth disease (CMT), Batten disease, and the like are included; preferably, Alzheimer's disease;AD), Parkinson's disease (PD), dementia (dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), etc.), multiple system atrophy (MSA), and amyotrophic lateral sclerosis (ALS). ;

[0042] The neurite outgrowth agent of the present invention can be formulated into a pharmaceutical composition for treating neurodegenerative diseases. The formulation may be in the same form as described in the above section [1-6.]. Furthermore, the neurite outgrowth agent of the present invention or a therapeutic composition containing the same can be orally ingested or administered to animals, including humans. [Example]

[0043] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention should not be construed as being limited by the descriptions of these examples.

[0044] A. Preparation of Coriander Extract Dried coriander leaf chips and dried crushed coriander seeds were mixed with 20 times the mass of water and stirred for 1 hour in a water bath. After cooling, the extract was suction filtered through filter paper (ADVANTECH No. 2). The filtrate was freeze-dried for 3 days in a dipping freeze dryer (TAITEC, VD-800R) to obtain coriander extracts (leaf extract powder and seed extract powder).

[0045] B. Analysis of Coriander Leaf Extract Powder B-1. Fractionation using an open column Column fractionation resin (DIAION TMThe coriander leaf extract powder was applied to an open column (φ6 × 50 cm) packed with HP20 (Mitsubishi Chemical), and then eluted with water and aqueous methanol to obtain five fractions (fractions 1 to 5). The coriander leaf extract powder and each fraction were subjected to HPLC analysis under the analytical conditions shown in Table 2 to obtain chromatograms. The chromatograms are shown in Figure 1. [Table 2]

[0046] B-2. Purification from fraction 1 using preparative HPLC Fraction 1 obtained in B-1 above was analyzed using a preparative HPLC (Thermo Fischer Scientific, Ultimate 3000) under the analytical conditions shown in Table 3. A chromatogram was obtained (not shown) using a photodiode array (PDA, detection wavelengths 220 nm and 330 nm) detector and a corona-charged particle (CAD) detector. Three major regions with peaks detected on the chromatogram were collected (preparative purified fractions A to C). Preparative purified fraction A contains components with retention times of approximately 11 to 17 minutes, preparative purified fraction B contains components with retention times of approximately 17 to 22 minutes, and preparative purified fraction C contains a component with a retention time of approximately 27 minutes. Preparative purified fractions A and B were collected as a mixture of multiple peaks, while preparative purified fraction C was isolated as a single peak. [Table 3]

[0047] B-3. ​​NMR measurement of purified product C The purified product C obtained in B-2 above was dissolved in deuterated methanol and analyzed using a nuclear magnetic resonance spectrometer (NMR, Bruker Biospin AVANCEIII 600, 600 MHz). 1 H-NMR measurement, 13C-NMR measurement and HMBC measurement were carried out. Figure 2 shows the results of HMBC measurement by NMR. From the results shown in Figure 2, it was estimated that the component of the purified fraction C has a molecular planar structure of 2-O-trans caffeoylhydroxycitric acid. 1 H-NMR and 13 The C-NMR measurement results were compared with the results of the existing literature (Natural Product Communications. 2023;18(7).1-11, Isolation and Quantification of 2-O-Caffeoyl-hydroxycitric Acid, an Active Component in Hot Water Extracts of Coriander (Coriandrum sativum L.), Which Inhibits Degranulation of RBL-2H3 Cells). 1 H-NMR and 13 Comparison with the C-NMR measurements showed good agreement (see Tables 4 and 5). [Table 4] [Table 5]

[0048] C. Qualitative analysis of coriander seed extract powder (confirmation of the presence of 2-O-trans-caffeoylhydroxycitric acid) A chromatogram was obtained by performing HPLC analysis on coriander seed extract powder under the conditions shown in Table 2. Similarly, chromatograms were obtained by performing HPLC analysis on coriander leaf extract powder and an isolated and purified product of caffeoylhydroxycitric acid under the conditions shown in Table 2. These chromatograms are shown for comparison in Figure 3. As shown in Figure 3, a peak corresponding to caffeoylhydroxycitric acid was observed at a retention time of 5.3 minutes in both the chromatograms of coriander seed extract powder and coriander leaf extract powder.

[0049] D. The neuronal differentiation promoting effect of coriander extract powder (leaves and seeds) and its fractions The neuronal differentiation-promoting effect of coriander seed extract, coriander leaf extract, and their fractions was evaluated using neurite outgrowth as an indicator of PC12 cell (a cell line isolated from a rat adrenal medulla pheochromocytoma). In the presence of NGF (nerve growth factor), PC12 cells cease proliferation and extend neurites, becoming sympathetic ganglion neuron-like. We investigated whether coriander seed extract, coriander leaf extract, and their fractions promote neurite outgrowth.

[0050] D-1. Passaging of PC12 cells PC12 cells (RIKEN Cell Bank, #RCB0009) were cultured in the DMEM medium shown below, and passaged when the cell density reached 60-70% confluence. DMEM medium: 39.5 ml of DMEM was mixed with 0.5 ml of 100x P / S, 5 ml of FBS, and 5 ml of HS. DMEM: DMEM (Low Glucose) with L-Glutamine and Phenol Red (Fujifilm Wako Pure Chemical Industries, Ltd., #041-29775) P / S: Penicillin-Streptomycin Mixed Solution (Nacalai Tesque, #26253-84) FBS:Fetal Bovine Serum(COSMO BIO, #CCP-FBS-BR500) HS:Horse Serum(Gibco, #16050-122)

[0051] D-2. Evaluation of neurite-bearing cell rate Study Group: (1) Control group: Denoted as Control in Figure 4 (2) NGF alone group (50 ng / ml): Denoted as NGF in Figures 4 and 5 (3) Coriander extract (leaf extract powder and seed extract powder) alone (100 μg / ml): shown as leaves and seeds in Figure 4 (4) NGF (50 ng / ml) + coriander extract (leaf extract powder and seed extract powder) (100 μg / ml): In Figures 4 and 5, these are referred to as NGF + leaf and NGF + seed. (5) NGF (50 ng / ml) + Fractions 1 to 5 of coriander leaf extract powder (Fraction 1 is 100 μg / ml, Fractions 2 to 5 are 10 μg / ml): shown as Fraction 1, Fraction 2, Fraction 3, Fraction 4, and Fraction 5 in Figure 5.

[0052] 4.0 x 10 in a 12-well plate 3 PC12 cells were seeded at 1000 μL / well and cultured in a CO2 incubator for 24 hours. The culture supernatant was removed, and the test sample (differentiation medium shown below with NGF, extract, or NGF and extract added) was added at 1000 μL / well. The cells were cultured in a CO2 incubator for 72 hours to induce neuronal differentiation. Differentiation medium: 1 ml of 5% BSA and 49 ml of DMEM(-) were added to a 50 ml centrifuge tube and mixed. DMEM(-) was prepared by adding 0.5 ml of 100x P / S to 49.5 ml of DMEM.

[0053] After 72 hours of culture, the cells were observed under a microscope, and five fields of view per well were photographed using an attached camera. The number of cells and the number of neurite-bearing cells were counted visually. Neurite-bearing cells were defined as cells with processes longer than the cell body. Approximately 150-200 cells were observed per well. The rate of neurite-bearing cells for each well was calculated using the following formula: Formula: Percentage of neurite-bearing cells (%) = (Total number of neurite-bearing cells / Total number of cells) × 100

[0054] The results are shown in Figure 4. When NGF alone was added to the culture medium of PC12 cells (2), neurite outgrowth was confirmed compared to the control group (1). On the other hand, when coriander extract (leaf extract powder and seed extract powder) alone was added to the culture medium of PC12 cells (3), no neurite outgrowth was observed in either case, as with the control group (1). In contrast, when coriander extract (leaf extract powder and seed extract powder) was added together with NGF to the culture medium of PC12 cells (4), neurite outgrowth was significantly enhanced in both cases compared to when NGF alone was added (2).

[0055] Furthermore, when fractions 1 to 5 of coriander leaf extract powder were added together with NGF to the culture medium of PC12 cells (5), it was confirmed that the addition of fraction 1 or fraction 3 significantly enhanced neurite outgrowth compared to the addition of NGF alone (2). The results are shown in Figure 5.

[0056] As shown in Figures 4 and 5, coriander extracts, leaf extract powder and seed extract powder, were found to have the effect of promoting the differentiation of nerve cells, and it was further found that this effect was due to fractions 1 and 3 of the leaf extract powder.

[0057] E. Gene expression analysis using coriander extract (leaf extract powder and seed extract powder) Neurofilament light chain (Nefl), a neuronal marker, is a protein expressed during neuronal differentiation and can be evaluated as a neuronal differentiation marker. In this experiment, we analyzed the promoting effect of coriander extract on Nefl gene expression in PC12 cells.

[0058] Study Group: NGF alone group (50 ng / ml): shown as NGF in Figure 6 NGF (50 ng / ml) + coriander extract (leaf extract powder) (10 μg / ml or 100 μg / ml): shown as NGF + leaf (10) and NGF + leaf (100) in Figure 6 NGF (50 ng / ml) + coriander extract (seed extract powder) (10 μg / ml or 100 μg / ml): shown as NGF + Seed (10) and NGF + Seed (100) in Figure 6

[0059] 1.2 x 10 cells in a 12-well plate 5 PC12 cells were seeded at 1000 μL / well and cultured in a CO2 incubator for 24 hours. The culture supernatant was removed, and the test sample (differentiation medium shown below supplemented with NGF or NGF and coriander extract) was added at 1000 μL / well, followed by 24 hours of culture in a CO2 incubator. The culture supernatant was removed, and Isogen II was added at 500 μL / well to lyse the cells. RNA lysates were prepared from the collected cell lysate according to the method described for Isogen II (Nippon Gene, #311-07361). Differentiation medium: 1 ml of 5% BSA (Albumin from Bovine Serum, fatty acid-free, low endotoxin, suitable for cell culture (Sigma, #A8806)) and 49 ml of DMEM(-) were added to a 50 ml centrifuge tube and mixed. DMEM(-) was prepared by adding 0.5 ml of 100x P / S to 49.5 ml of DMEM.

[0060] Reverse transcription (RT-PCR) was performed using the ReverTra Ace qPCR RT Master Mix (TOYOBO, #FSQ-201) according to the method described. RT-qPCR was performed using primers according to the method described in the THUNDERBIRD Next SYBR qPCR / UNG Set (TOYOBO, #QPX201 / UNG101). Actb was used as the internal standard. Test samples were evaluated in duplicate. Data were analyzed by setting a threshold for each gene to obtain linearity of the calibration curve, and calculating the relative quantification value (Qty) from the Ct value of each sample. These values ​​were corrected by the relative quantification value of the internal standard, and the relative expression level was calculated by setting the mean value of the sample (NGF) without the test sample as 1.

[0061] The results are shown in Figure 6. As shown in Figure 6, compared to the addition of NGF alone, the addition of coriander extract (leaf extract powder and seed extract powder) increased the level of Nefl gene expression, and this was significant when the added concentration was 100 μg / ml.

[0062] F. Evaluation of the neuronal differentiation promoting effect of purified fractions A to C The purified fractions A to C described in "B-2." above were evaluated for their neuronal differentiation promoting activity in the same manner as in D. above, using neurite outgrowth of PC12 cells as an index.

[0063] Study Group: NGF only group (50 ng / ml): indicated as NGF in Figure 7 NGF (50 ng / ml) + coriander extract (leaf extract powder) (100 μg / ml): shown as NGF + leaf (100) in Figure 7 NGF (50 ng / ml) + purified product A (10 μg / ml): shown as NGF+A(10) in Figure 7 NGF (50 ng / ml) + purified product A (100 μg / ml): shown as NGF+A(100) in Figure 7 NGF (50 ng / ml) + purified product B (3 μg / ml): shown as NGF+B(3) in Figure 7 NGF (50 ng / ml) + purified product B (30 μg / ml): shown as NGF+B(30) in Figure 7 NGF (50 ng / ml) + purified product C (3 μg / ml): shown as NGF+C(3) in Figure 7 NGF (50 ng / ml) + purified C (30 μg / ml): shown as NGF+C(30) in Figure 7

[0064] PC12 cells were cultured and passaged under the conditions described in D-1 above. Furthermore, the test substance was added and incubated to induce neuronal differentiation as described in D-2 above. The cells were observed under a microscope, and the number of cells and the number of neurite-bearing cells were counted to calculate the neurite-bearing cell rate.

[0065] The results are shown in Figure 7. As shown in Figure 7, the addition of coriander extract (leaf extract powder) increased the percentage of neurite-bearing cells compared to the addition of NGF alone. The addition of purified fractions A and B did not significantly affect the percentage of neurite-bearing cells, but the addition of purified fraction C significantly increased the percentage of neurite-bearing cells. Furthermore, even at a low concentration of 3 μg / ml, the percentage of neurite-bearing cells significantly increased. This indicates that the neuronal differentiation-promoting effect of coriander extract (leaf extract powder) is at least partially due to purified fraction C (2-O-trans-caffeoylhydroxycitric acid).

[0066] G. Animal model study of cognitive impairment using coriander leaf extract powder G-1. Preparation of Coriander Leaf Extract Powder: Coriander leaf extract powder was obtained in the same manner as described in A. above, and dextrin (excipient) was mixed with it in a ratio of extract powder:dextrin = 4:1 (weight ratio) to obtain a leaf extract powder preparation.

[0067] G-2. Test group (n=10): (1) Control group: SAMR1 mice were administered with the solvent (water), and are referred to as SAMR1 in Figures 8 and 10. (2) SAMP8(-) group: SAMP8 mice were administered with the solvent (water), and are shown as SAMP8(-) in Figures 8 and 10. (3) SAMP8(+) low dose group: SAMP8 mice were administered 100 mg / kg of leaf extract powder as an aqueous solution, shown as SAMP8(100) in Figures 8 and 10. (4) SAMP8(+) high dose group: 300 mg / kg of leaf extract powder was administered as an aqueous solution to SAMP8 mice, and is shown as SAMP8(300) in Figures 8 and 10.

[0068] Ten-week-old mice were acclimated for one week and then orally administered with each sample. The oral administration volume of water or aqueous solution was adjusted to 10 ml per kg of mouse body weight.

[0069] G-3. Test system SAMP8 mice are a member of the SAMP strain of mice, a model of accelerated aging that exhibits symptoms such as learning and memory impairment, emotional disorders, and sarcopenia. Spatial learning and memory were assessed using the SAMP8 model through behavioral assessment using the Barnes maze test. The conditions for the Barnes maze test were established based on the test conditions described in the aforementioned "Nutrients 2020, 12, 455." Briefly, a maze was prepared with 20 circular holes (25 mm diameter) along the circumference of a circular table (455 mm diameter). An escape box (130 mm wide, 95 mm deep, 50 mm high) was placed under one of the holes. The maze was surrounded by a partition with three visible patterns, such as a triangle, double circle, or star. The table was illuminated at 750 Lx, and the mouse was placed in the center of the table.

[0070] G-3-1. Repetitive learning test Ten-week-old mice (SAMR1 or SAMP8 mice) underwent a repetitive learning test (spatial memory test) on days 1, 2, 3, 4, and 2 weeks. Specifically, mice were placed in a starter tube cylinder (38 mm diameter, 73 mm height, gray), which was placed in the center of a table, and the starter tube was removed. After two minutes, the mice were given two minutes to reach the escape box, and the time it took for the mice to reach the escape box was measured. (If the mice did not reach the escape box within two minutes, they were forcibly placed in the escape box.) Four trials were conducted on each test day, and the average time for the four trials was calculated.

[0071] The results of the repeated learning test are shown in Figure 8. As shown in Figure 8, it was suggested that the time to find the escape box was shorter in the control group (SAMR1 mice), the low-dose SAMP8(+) group, and the high-dose SAMP8(+) group compared with the SAMP8(-) group. In particular, the high-dose SAMP8(+) group showed a significantly shorter time to find the escape box compared with the SAMP8(-) group.

[0072] G-3-2. Probe test A probe test was conducted on mice that had completed the second-week repetitive learning test. Specifically, the escape box of the maze apparatus was removed. After placing the mouse in the cylinder for 20 seconds, the cylinder was removed and the mouse was allowed to search for the target hole for 2 minutes, after which the mouse was returned to its cage. For the probe test, the Barnes maze was divided into four zones: the target zone (the zone where the escape box was located), the left zone, the right zone, and the opposite zone (see Figure 9). The mouse was allowed to explore the divided maze for 1 minute (60 seconds), and the time spent in each zone and the distance traveled within each zone were measured.

[0073] The results of the probe test of the Barnes maze test are shown in Figure 10. Figure 10A shows the time spent by mice in each zone (target zone, left zone, right zone, and opposite zone). Figure 10B shows the distance traveled by mice within each zone. As shown in Figure 10A, the time spent in the target zone in the SAMP8(-) group was significantly reduced compared to the control group, and the time spent in the target zone in the SAMP8(+) group (both the low-dose group and the high-dose group) was longer compared to the SAMP8(-) group. Furthermore, as shown in Figure 10B, the distance traveled in the target zone in the SAMP8(-) group was significantly reduced compared to the control group, and the distance traveled in the SAMP8(+) group (both the low-dose group and the high-dose group) was significantly longer compared to the control group.

[0074] H. Discussion of Experimental Results SAMP8 mice are an animal model that exhibits age-related cognitive decline, and it has been reported that gene expression in the brain changes. Specifically, the aforementioned Non-Patent Document 3 (Experimental Gerontology 40 (2005) 774-783) reports that the brains of SAMP8 mice exhibit reduced expression of the Nerve Growth Factor (NGF) gene and reduced levels of Neurofilament triple L protein (NEFL), which are considered to be associated with neurodegenerative diseases and contribute to the decline in cognitive function.

[0075] It is also believed that neurodegeneration in Alzheimer's disease is caused by the deposition of amyloid beta peptides in plaques in brain tissue (the amyloid hypothesis). When amyloid beta peptides are deposited, soluble oligomers are produced, which inhibit long-term potentiation in hippocampal cells (damaging synapses and neurites of neurons), thereby interfering with memory formation (Science_297_353 to 356 (2002)).

[0076] Furthermore, it has been proposed to use agents that promote the outgrowth of neurites in nerve cells (neurite outgrowth promoters) as therapeutic agents for Alzheimer's disease (for example, JP 2018-203722 A). That is, JP 2018-203722 A describes attempts to use NGF as a therapeutic agent for Alzheimer's disease, but also describes the problem that NGF, being a high molecular weight protein, does not reach the brain when orally ingested and cannot pass through the blood-brain barrier when administered intravenously, and describes the provision of a drug that has neuronal differentiation-promoting activity and neurite formation activity, is resistant to degradation by digestive enzymes, and can pass through the blood-brain barrier.

[0077] Based on the above, it can be assumed that the main reason why SAMP8 mice orally administered coriander extract powder showed improved memory and learning is that the components contained in the coriander extract powder reached the mouse brain and promoted neurite outgrowth of brain neurons. Furthermore, of the fractions of coriander extract powder, fractions 1 and 3 showed the effect of promoting neurite outgrowth. Since fraction 1 contains 2-O-trans-caffeoylhydroxycitric acid (prepared purified product C) as its main component, it can be concluded that 2-O-trans-caffeoylhydroxycitric acid has the effect of improving cognitive function. [Industrial Applicability]

[0078] The cognitive function improver of the present invention is expected to improve the cognitive function of animals, including humans, when used as a food or pharmaceutical (preferably an oral pharmaceutical). Furthermore, the cognitive function improver of the present invention may be highly safe because it contains an extract of a plant that has been consumed by humans as an active ingredient. Furthermore, the neurite outgrowth agent of the present invention is also expected to be applicable as a food or pharmaceutical, and may be used as a therapeutic composition for neurodegenerative diseases.

Claims

1. A cognitive function improving agent for oral administration, containing 2-O-trans caffeoyl hydroxycitric acid as an active ingredient for improving cognitive function.

2. The cognitive function improving agent according to claim 1, wherein the 2-O-trans caffeoylhydroxycitric acid is derived from a plant of the genus Coriander in the family Umbelliferae.

3. The cognitive function improving agent according to claim 1, wherein the 2-O-trans caffeoyl hydroxycitric acid is derived from coriander.

4. The cognitive function improving agent according to claim 1, wherein the 2-O-trans caffeoyl hydroxycitric acid is derived from coriander leaves.

5. A neurite outgrowth agent comprising 2-O-transcaffeoylhydroxycitric acid as an active ingredient for neurite outgrowth.

6. The neurite outgrowth agent according to claim 5, wherein the 2-O-trans caffeoylhydroxycitric acid is derived from a plant of the genus Coriander in the family Umbelliferae.

7. The neurite outgrowth agent according to claim 5, wherein the 2-O-trans caffeoylhydroxycitric acid is derived from coriander.

8. The neurite outgrowth agent according to claim 5, wherein the 2-O-trans caffeoylhydroxycitric acid is derived from coriander leaves.

9. The neurite outgrowth agent according to any one of claims 5 to 8, for oral administration.

10. A therapeutic composition for a neurodegenerative disease, comprising the neurite outgrowth agent according to any one of claims 5 to 8.

11. A therapeutic composition for a neurodegenerative disease comprising the neurite outgrowth agent according to any one of claims 5 to 8.

12. The composition for treating a neurodegenerative disease according to claim 11, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal dementia, and amyotrophic lateral sclerosis.

13. The neurite outgrowth agent according to any one of claims 5 to 8, which improves NGF-induced neurite outgrowth.

14. Use of 2-O-trans caffeoylhydroxycitric acid in the manufacture of a cognitive function improver.

15. Use of 2-O-trans caffeoylhydroxycitric acid in the manufacture of a neurite outgrowth agent.

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