Unshiu mandarin extract having effect of preventing cognitive decline or improving cognitive function, and method for producing the same

Abstract

To provide a naturally derived material that is effective in preventing cognitive decline or improving cognitive function, and a method for producing the same.SOLUTION: The present invention provides an Unshiu mandarin extract having an effect of preventing cognitive decline or improving cognitive function, which is prepared by subjecting unripe Unshiu mandarins to subcritical water extraction treatment; and a method for producing the Unshiu mandarin extract having an effect of preventing cognitive decline or improving cognitive function by subjecting unripe Unshiu mandarins to subcritical water extraction treatment.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a material, food or medicine that is effective for preventing cognitive decline associated with aging or for improving cognitive function, and in particular to a material derived from natural ingredients that is effective for preventing or improving cognitive dysfunction. [Background technology]

[0002] As the aging society progresses, the number of dementia patients is rapidly increasing worldwide. According to the Institute for Health Metrics and Evaluation in the United States, 57 million people worldwide suffered from dementia in 2019, and this number is expected to increase to 153 million by 2050. In Japan, the number of dementia patients is estimated to be approximately 6 million as of 2020, and is expected to increase to around 7 million by 2025, accounting for approximately one in five people aged 65 or older. In particular, Alzheimer's disease, the most common form of dementia, is a major social problem, and there is a demand for the development of effective treatments. However, currently approved treatments can only reduce the progression of cognitive function, they cannot stop it.

[0003] Alzheimer's disease begins with the accumulation of amyloid beta (Aβ) protein in nerve cells in the brain, followed by the accumulation of tau protein, which leads to neurofibrillary changes and the loss and disappearance of nerve cells, resulting in the appearance of symptoms such as cognitive decline. However, the problem is that it takes 20 to 30 years from the start of Aβ accumulation for the symptoms of cognitive decline to appear.

[0004] As mentioned above, at present, once the disease develops, cognitive decline can only be delayed, but it cannot be reversed. Therefore, it is extremely important to find natural products and food components that have the physiological activity to inhibit the accumulation of Aβ and prevent the onset of the disease.

[0005] "Chinpi" is a general term for the dried peel of Unshu mandarin oranges and related species, and has been known since ancient times as a traditional Chinese medicine, used in East Asia, including Japan, China, and Korea, for stomach health, expectorant, cough suppressant, etc. The main components of this chimpi are flavonoids such as hesperidin, narirutin, and nobiletin.

[0006] In Non-Patent Document 1, patients with mild to moderate Alzheimer's disease who were already taking the dementia treatment drug donepezil hydrochloride were divided into two groups, an intervention group and a control group, and cognitive function was evaluated in the intervention group by taking Citrus unshiu peel, which contains a high amount of nobiletin. As a result, no significant change was observed in cognitive function one year later in the intervention group, but the control group showed a significant deterioration, indicating the possibility that Citrus unshiu peel, which contains a high amount of nobiletin, can halt the deterioration of cognitive function.

[0007] Furthermore, Non-Patent Document 2 reports that when nobiletin was administered daily for four months starting from nine months into APPTg mice, which are recognized as useful as a model animal for chronic Alzheimer's disease, context-dependent fear dependence disorder was significantly improved, and immunohistochemical analysis using an anti-Aβ antibody showed that Aβ deposition in the hippocampus was significantly reduced by administration of nobiletin.

[0008] Non-Patent Document 4 reports that when nobiletin was administered daily for one month to 4-6 month old senescence accelerated model mice SAMP8, both object recognition dysfunction and hippocampus-dependent context-dependent fear memory impairment were improved.

[0009] Patent Document 1 claims that a composition containing glycerophosphocholine and compounds thereof as a first active ingredient and hesperidin, narirutin, and compounds thereof as a second active ingredient is effective in treating and / or preventing Alzheimer's disease, and in the examples, it confirms the synergistic effect of combining glycerophosphocholine with an extract of Citrus unshiu peel containing hesperidin and narirutin.

[0010] In addition, Non-Patent Document 4 reports that hesperidin maintains the cognitive function and Hippocampus histological structure of stressed albino Wistar rats through the enhancement of brain-derived neurotrophic factor, and in this report, it is reported that hesperidin reduces hippocampal amyloid levels, preserves hippocampal histological structure, and preserves cognitive function. Non-Patent Document 5 reports that narirutin has multifunctional activity in the treatment of Alzheimer's disease, and also describes that it has strong Aβ aggregation inhibitory ability.

[0011] As mentioned above, dried orange peel and the flavonoids contained in it, such as hesperidin and narirutin, are known to be effective in suppressing the accumulation of Aβ and improving cognitive function, but there have been no reports of the effect of unripe Satsuma mandarin extract in preventing or improving cognitive decline.

[0012] Incidentally, N-acetylneuraminic acid (Neu5Ac), a type of sialic acid, is abundant in the brain and is known to play an important role in cognitive function.

[0013] On the other hand, the inventors have found that another sialic acid, N-glycolylneuraminic acid (Neu5Gc), accumulates in the brain, inhibiting the formation of neural circuits and severely impairing cognitive function (Non-Patent Document 6), and promoting the aggregation of Aβ, the causative agent of Alzheimer's disease (Non-Patent Document 7).

[0014] The enzyme involved in the synthesis of Neu5Gc (CMAH) is not expressed in the brains of any mammals, so Neu5Gc cannot be synthesized in the brain. In addition, humans lack the CMAH gene, so they cannot synthesize Neu5Gc throughout the body.

[0015] However, it has been discovered that trace amounts of Neu5Gc exist in the human brain, and that it is particularly abundant in the hippocampus, which is involved in memory formation.

[0016] Neu5Gc is found in red meat such as beef and horse meat, cheese, etc. The inventors have confirmed that most of the Neu5Gc derived from food is excreted in urine, but a portion of it migrates into human blood and then into the brain (Non-Patent Document 8).

[0017] The inventors have reported that Neu5Gc accumulates with aging in the hippocampus region of the brain, which is involved in memory, in experiments using rats (Non-Patent Document 6). In addition, when Neu5Gc is forcibly expressed in the hippocampus of rats, memory function evaluated in the Morris water maze and long-term potentiation (LTP), a fundamental process of memory measured by extracellular potential recording, are significantly attenuated. Based on this, the inventors have reported that Neu5Gc accumulated in the brain with aging is associated with the attenuation of cognitive function (Non-Patent Document 9).

[0018] The inventors also found that the brains of senescence-accelerated model mice had more Neu5Gc accumulated than the brains of normal mice, and found that the hippocampus of senescence-accelerated model mice overexpressing Neu5Gc had higher levels of highly neurotoxic amyloid beta 42 (Aβ 42 ) accumulation was confirmed in the hippocampus of normal mice, which showed higher Aβ aggregation tendency than that in the hippocampus of normal mice. Neu5Gc accumulates in the brain with aging, and accumulation of Neu5Gc in the brain is a mechanism by which highly neurotoxic Aβ 42 At the 33rd Academic Conference of the Senescence Accelerated Mouse (SAM) Society, it was reported that Neu5Gc is strongly associated with the onset and progression of Alzheimer's disease because it promotes the aggregation of .

[0019] Therefore, if safe and reliable foods and other natural ingredients that suppress the accumulation of Aβ and / or Neu5Gc in the brain can be consumed from adolescence onwards, it is expected that the decline in cognitive function and the onset of Alzheimer's disease can be prevented. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] International Publication No. 2018 / 190146 [Patent Document 2] Patent Publication No. 2021-165257 [Non-patent literature]

[0021] [Non-Patent Document 1] Japanese Journal of Pharmacology, 145, 234-236, 2015 [Non-Patent Document 2] J. Pharmacol. Exp. Ther., 326, 739-744, 2008 [Non-Patent Document 3] Behav. Brain Res., 250, 351-360, 2013 [Non-Patent Document 4] Neurotoxicity Research, 40, 179-185, 2022 [Non-Patent Document 5] International Journal of Biological Macromolecules, 103, 733-743, 2017 [Non-Patent Document 6] The 137th Annual Meeting of the Pharmaceutical Society of Japan, 27F-am04S, Sendai, March 27, 2017 [Non-Patent Document 7] The 38th Annual Meeting of the Japanese Society of Carbohydrate Research, P-095, Nagoya, August 20, 2019 [Non-Patent Document 8] PLos One, 10(6), e0131061, 2015 [Non-Patent Document 9] (Japan Society of Hospital Pharmacists Tokai Block / Japan Pharmaceutical Society Tokai Branch Joint Academic Conference 2014, P118, Shizuoka, November 9, 2014) [Non-Patent Document 10] The 33rd Academic Conference of the Senescence Accelerated Mouse (SAM) Society, Research Report 14: "Elucidation of the effect of rare sugar species accumulated in the aging brain on Alzheimer's disease" Shizuoka, July 15, 2018 Summary of the Invention [Problem to be solved by the invention]

[0022] The object of the present invention is to provide a material derived from natural ingredients that is effective in preventing cognitive decline or improving cognitive function, and a method for producing the same. [Means for solving the problem]

[0023] The inventors of the present application have discovered that an extract obtained by subjecting unripe Unshu mandarins, including the peel, to a subcritical water extraction process can exhibit excellent anti-allergic and / or anti-stress effects, that the components involved in this anti-allergic and / or anti-stress effect are the poorly soluble components hesperidin and narirutin, and that the extract containing hesperidin and narirutin can be produced by subjecting the mandarins to a subcritical water extraction process, and have filed a patent application (Patent Document 2).

[0024] In addition, the inventors of the present application further studied the above-mentioned Satsuma mandarin extract and discovered that the above-mentioned extract containing hesperidin and narirutin, which is produced by subjecting the entire unripe Satsuma mandarin, including the peel, to subcritical water extraction processing, is an effective ingredient in preventing or improving cognitive function decline caused by a decrease in estrogen, and have filed a patent application (Patent Application No. 2023-097219).

[0025] The decline in cognitive function due to a decrease in estrogen is a symptom specific to menopausal women, and is brought about by a decrease in the activity of estrogen receptors.

[0026] On the other hand, the decline in cognitive function due to the accumulation of Aβ and / or Neu5Gc in the brain during aging is a symptom that is not related to gender and is brought about by neurofibrillary tangles, neuronal loss / loss, and / or inhibition of the formation of neural circuits, and its mechanism of action is completely different.

[0027] The inventors fed senescence-accelerated model mice with food containing Satsuma mandarin extract treated with subcritical water for six months, and confirmed that the effect of preventing and improving cognitive decline was greater than that of senescence-accelerated model mice fed with a normal diet. As a result of analyzing Aβ and Neu5Gc contained in the brain, it was found that both were significantly reduced in senescence-accelerated model mice fed with a special diet containing Satsuma mandarin extract compared to senescence-accelerated model mice fed with a normal diet, and it was confirmed that the effect of preventing and improving cognitive decline is brought about by the inhibition of accumulation of Aβ and / or Neu5Gc.

[0028] It has also been reported that hesperidin and narirutin reduce the accumulation of Aβ and alleviate Alzheimer's dementia (Non-Patent Documents 4 and 5). Subcritical water-treated Unshu mandarin extract contains more hesperidin and narirutin than extracts extracted with hot water or ethanol (Patent Document 2), suggesting that the hesperidin and narirutin contained in the extract are involved in the effect of subcritical water-treated Unshu mandarin extract in preventing and improving cognitive decline.

[0029] The present invention relates to an extract of Unshu mandarin orange having an effect of preventing or improving cognitive decline, which is obtained by subjecting unripe Unshu mandarin orange to subcritical water extraction, and contains hesperidin and narirutin as indicator components of the effect of preventing or improving cognitive decline. Furthermore, the effect of preventing or improving cognitive decline is brought about by suppressing the accumulation of Aβ and / or Neu5Gc.

[0030] The present invention can be exemplified as follows.

[0031] [1] An Unshu mandarin extract having an effect of preventing or improving decline in cognitive function, which is obtained by subjecting unripe Unshu mandarins to subcritical water extraction treatment.

[0032] [2] [1] An Unshu mandarin extract having hesperidin and narirutin as indicator components of the Unshu mandarin extract's effect of preventing or improving the decline in cognitive function.

[0033] [3] [2] The Unshu mandarin extract contains 30 mg / g or more of the hesperidin and 10 mg / g or more of the narirutin.

[0034] [4] An extract of any of the Unshu mandarin oranges [1] to [3] that inhibits the accumulation of amyloid beta (Aβ) and / or N-glycolylneuraminic acid (Neu5GC) in the brain.

[0035] [5] A cognitive function improver having the effect of preventing or improving cognitive decline, the compounding ingredient of which is a Satsuma mandarin extract of any one of [1] to [4].

[0036] [6] A method for producing a Satsuma mandarin extract having an effect of preventing or improving decline in cognitive function, comprising subjecting unripe Satsuma mandarins to a subcritical water extraction treatment.

[0037] [7] A method for producing a Satsuma mandarin extract having an effect of preventing or improving cognitive decline, comprising [6] containing hesperidin and narirutin as indicator components of the Satsuma mandarin extract's effect of preventing or improving cognitive decline.

[0038] [8] [7] A method for producing a Satsuma mandarin extract having an effect of preventing or improving cognitive decline, the method comprising the steps of: (a) containing 30 mg / g or more of the hesperidin and 10 mg / g or more of the narirutin.

[0039] [9] A method for producing a Satsuma mandarin extract according to any one of [6] to [8], wherein the Satsuma mandarin extract inhibits accumulation of amyloid beta (Aβ) and / or N-glycolylneuraminic acid (Neu5GC) in the brain. Effect of the Invention

[0040] According to the present invention, it is possible to provide a material derived from natural ingredients that is effective in preventing or improving cognitive decline, and a method for producing the same. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 shows the body weights of mice immediately after starting the special diet (day 0), after 3 months (day 92), and after 6 months (day 167) in a feeding test of Unshu mandarin extract powder using senescence-accelerated mouse model SAMP8. [Diagram 2] FIG. 13 is a graph showing the amount of food consumed per day on days 2, 4, and 6, starting 24 weeks after the start of feeding the special diet in a feeding test of Unshu mandarin orange extract powder using the senescence-accelerated mouse model SAMP8. [Diagram 3] FIG. 1 shows the results of a novel object recognition test in a Satsuma mandarin extract powder feeding test using senescence-accelerated mouse model SAMP8, where (a) shows the percentage of exploration time for the novel object, and (b) shows the discrimination index for the novel object. [Figure 4] FIG. 1 shows the results of immobility times on the first and second days of a forced swimming test in a Satsuma mandarin extract powder feeding test using senescence-accelerated mouse model SAMP8. [Diagram 5] This figure shows the sialic acid content in the brain (hippocampus) of mice 6 months after starting the special diet in a feeding test of Unshu mandarin orange extract powder using the aging-accelerated mouse model SAMP8, where (a) shows the content of N-glycolylneuraminic acid (Neu5Gc) and (b) shows the content of N-acetylneuraminic acid (Neu5Ac). [Figure 6] FIG. 1 shows the amount of amyloid beta 42 (Aβ42) in the brain (hippocampus) of mice 6 months after starting the special diet in a feeding test of Unshu mandarin orange extract powder using the senescence-accelerated mouse model SAMP8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The Unshu mandarin extract having the effect of preventing or improving cognitive decline and the method for producing the same according to this embodiment are obtained by subjecting unripe Unshu mandarins to a subcritical water extraction treatment.

[0043] The above-mentioned Unshu mandarin extract, which has the effect of preventing or improving cognitive decline, contains hesperidin and narirutin, and according to the research of the present inventors, it has been found that these are indicator components for the above-mentioned Unshu mandarin extract's effect of preventing or improving cognitive decline.

[0044] In addition, the above-mentioned Unshu mandarin extract, which has the effect of preventing or improving cognitive decline, was found to contain 30 mg / g or more of hesperidin and 10 mg / g or more of narirutin, which are indicator components of the effect of preventing or improving cognitive decline.

[0045] In the present invention, cognitive function refers to higher brain functions including judgment, calculation, understanding, learning, thinking, language, spatial recognition, object recognition, and memory (short-term memory, long-term memory).

[0046] In the present invention, a decline in cognitive function means a decline in any one or more of the cognitive functions exemplified above, and means a decline in cognitive function such as a decline in memory associated with Alzheimer's dementia.

[0047] In the present invention, prevention or improvement of decline in cognitive function means preventing or suppressing the decline in cognitive function exemplified above, and maintaining or improving the function.

[0048] In addition, the cognitive function improving effect in this specification refers to the effect of preventing or improving the decline in cognitive function described above, and this particularly includes the effect of reducing memory decline in a novel object recognition test, as performed in the examples described below.

[0049] The Unshu mandarin extract having the effect of preventing or improving the decline in cognitive function can be produced as follows in accordance with the production method described in Patent Document 2.

[0050] In producing the above-mentioned Satsuma mandarin extract, the unripe Satsuma mandarins to be subjected to the subcritical water extraction treatment are used in their entirety, including the peel.

[0051] Here, the unripe Unshu mandarins are those harvested between late June and late August, preferably between early July and late July, with a diameter of 2 cm to 6 cm, and are generally called "green mandarins." Hereinafter, in this specification, the above-mentioned "Unshu mandarins" may be referred to as "green mandarins."

[0052] Types of unripe Unshu mandarins include very early Unshu, early Unshu, mid-ripening Unshu, and normal Unshu, and cultivars include Aoshima, Aoshima No. 4, Katayama, Kotobukitaro, Takabayashi Wase, Nichinan No. 1, Miyagawa Wase, and Okitsu Wase.

[0053] When the unripe unshu mandarin orange is subjected to the subcritical water treatment, the unripe unshu mandarin orange as the raw material is appropriately crushed and ground to form a paste. The method of the treatment is not particularly limited, but examples thereof include a grinder, a comitroller, a colloid mill, a food processor, a juicer mixer, a pin mill, a ball cutter, a ball mill, a hammer mill, a hammer crusher, a line mill, a cutter mill, a roll mill, a roll crusher, an impact mill, a rotary mill, a vibration mill, a planetary mill, a bead mill, etc.

[0054] In addition, when carrying out the subcritical water treatment, water can be added as necessary. There is no particular restriction on the amount of water to be added, but when water is added, the amount is 2 to 10 times, preferably 3 to 7 times, the mass ratio of the unripe Satsuma mandarin used as the raw material.

[0055] There are no particular limitations on the subcritical water treatment equipment, and both batch and continuous systems are possible. From the standpoint of production efficiency, it is preferable to use a continuous subcritical water extractor.

[0056] Subcritical water refers to the state of water in the region below its critical point (374°C, 22.1 MPa). The definitions of the lower limit temperature and pressure of subcritical water are not clearly defined, but in the present invention, for comparison with heat treatment at normal pressure, reactions in a subcritical water state are performed at 100°C or higher, and the pressure conforms to the conditions described below.

[0057] That is, in producing the above-mentioned Unshu mandarin extract of the present invention, the maximum temperature reached in the subcritical water treatment is 130 to 180°C, preferably 160 to 180°C, and there is no particular restriction on the pressure, but the pressure is such that water can remain liquid (pressure that does not become gaseous), for example, 0.24 MPa or more if the maximum temperature reached is 130°C, and 1.01 MPa or more if it is 180°C. Regarding the treatment time, the reaction in the subcritical water state is 10 to 60 seconds, preferably 10 to 20 seconds. In the case of a batch-type subcritical water treatment, it is easy to extend the reaction time, but in the case of a continuous type, there are restrictions on the equipment, and it is not easy to extend the reaction time.

[0058] The extract after the subcritical water treatment is subjected to solid-liquid separation to recover the unshu mandarin extract from which impurities have been removed. The treatment method is not particularly limited, but includes filtration, centrifugation, etc. For example, decanter type centrifuge (screw decanter), screw press, Westfalia centrifuge, de-cone type separator, filter press, belt press, vacuum dehydrator, sifter (vibrating sieve), strainer, non-woven filter, screw discharge type continuous centrifuge, filter cloth reversal type centrifuge, roller press, belt screen, bag filter separator, drum screen, basket type centrifuge, multi-plate wave filter, multi-disk type dehydrator, membrane separation device, etc.

[0059] The extract after solid-liquid separation may be concentrated and powdered as necessary. There are no particular limitations on the concentration method, but examples include boiling concentration, vacuum concentration, freeze concentration, membrane concentration, and ultrasonic atomization separation. For example, there are plate concentrators, flash concentrators, global concentrators, single-can vacuum concentrators, centrifugal thin-film vacuum evaporators, high-speed rotary vacuum evaporators, heat pump high-speed rotary vacuum evaporators, UF membrane concentrators, multi-stage thin-film evaporators, VFC type evaporative concentrators, FFC type evaporative concentrators, coil concentrators, scraper concentrators, and external loop concentrators.

[0060] The method for drying the extract is not particularly limited, and examples thereof include spray drying, freeze drying, vacuum reduced pressure drying, drum drying, hot air drying, etc. Examples of the method include spray dryers, reduced pressure spray drying devices, nano spray drying devices, electrostatic spray drying devices, vacuum freeze dryers, microwave reduced pressure dryers, vacuum belt dryers, single drum dryers, double drum dryers, vacuum drum dryers, air flow dryers, and pulse combustion spray dryers.

[0061] As described in Patent Document 2, as an example of the production of a Satsuma mandarin extract by the above-mentioned subcritical water treatment, the inventors of the present application used unripe Satsuma mandarins with a diameter of 2 cm to 6 cm harvested in mid-July from Shizuoka Prefecture, added 600 kg of water to 100 kg of these Satsuma mandarins, and performed continuous extraction under subcritical water treatment conditions of 180°C and 3 MPa, followed by solid-liquid separation in a separator to produce a Satsuma mandarin extract.

[0062] The hesperidin and narirutin contents contained in the unripe Satsuma mandarins themselves used in the production of this Satsuma mandarin extract were 0.38 g / 100 mL and 0.110 g / 100 mL.

[0063] In contrast, the hesperidin and narirutin contents in the Unshu mandarin extract produced as described above after subcritical water treatment were measured in the same manner, and the hesperidin content was 0.36 g / 100 mL and the narirutin content was 0.096 g / 100 mL.

[0064] In addition, the Unshu mandarin extract produced as described above was concentrated in a vacuum concentrator and powdered using a spray dryer with a formulation containing 50% excipients to produce Unshu mandarin extract powder.The hesperidin and narirutin contents in this Unshu mandarin extract powder were measured using high performance liquid chromatography, and the hesperidin content was 4.8 g / 100 g and the narirutin content was 1.9 g / 100 g.

[0065] In Patent Document 2, in order to confirm the extraction effect of subcritical water extraction, as a comparison control for the production of Satsuma mandarin extract by the above-mentioned subcritical water treatment, 600 kg of water was added to 100 kg of the above-mentioned unripe Satsuma mandarins, and an Satsuma mandarin extract (hot water extraction) was extracted under hot water treatment conditions of 90°C and 0.1 MPa (normal pressure), and 600 kg of 100% ethanol was added to 100 kg of the above-mentioned unripe Satsuma mandarins, and an Satsuma mandarin extract (ethanol extraction) was extracted under room temperature treatment conditions, and the hesperidin content and narirutin content of each obtained Satsuma mandarin extract were measured in the same manner to verify the extraction efficiency.

[0066] The results showed that the extraction rate of hesperidin from unripe Satsuma mandarins was 25% with hot water extraction, 42% with ethanol extraction, and 95% with subcritical water extraction, and the extraction rate of narirutin was 28% with hot water extraction, 35% with ethanol extraction, and 87% with subcritical water extraction. While it is not possible to efficiently extract hesperidin and narirutin with normal hot water extraction or conventional ethanol extraction, this is improved by subcritical water extraction, and it has been confirmed that it is possible to produce a Satsuma mandarin extract containing high concentrations of hesperidin and narirutin.

[0067] According to what the present inventors have confirmed in Patent Document 2, by performing subcritical water extraction processing, it is possible to produce an Unshu mandarin extract having at least three times the hesperidin content and at least twice the narirutin content compared to an extract produced by performing hot water extraction on the same starting material, unripe Unshu mandarins, and it has been confirmed that it is possible to produce an Unshu mandarin extract having at least twice the hesperidin content and at least twice the narirutin content compared to an extract produced by performing ethanol extraction on the same starting material, unripe Unshu mandarins.

[0068] The Satsuma mandarin extract of the present invention having the effect of preventing or improving cognitive decline, as described in Patent Document 2, is an Satsuma mandarin extract obtained by subjecting unripe Satsuma mandarins to subcritical water extraction treatment, so that the hesperidin content is at least three times and the narirutin content is at least twice as high as that of an extract produced by subjecting the same starting material, unripe Satsuma mandarins, to hot water extraction, and that the hesperidin content is at least twice as high and the narirutin content is at least twice as high as that of an extract produced by subjecting the same starting material, unripe Satsuma mandarins, to ethanol extraction.

[0069] The Unshu mandarin extract according to the present invention, which has the effect of preventing or improving cognitive decline, can be used to prepare and manufacture various foods, quasi-drugs, and pharmaceuticals that have the above-mentioned effect of preventing or improving cognitive decline.

[0070] In this case, by appropriately adopting a process of blending the Unshu mandarin extract of the present invention in the process of preparing and manufacturing various foods, quasi-drugs, and medicines, it is possible to produce various foods, quasi-drugs, and medicines.

[0071] In addition, after producing Unshu mandarin extract powder as described above from the Unshu mandarin extract having the effect of preventing or improving cognitive function according to the present invention, it is also possible to combine this with materials used in foods, quasi-drugs, and medicines described below to produce various foods, quasi-drugs, and medicines having the effect of preventing or improving the decline in cognitive function described above.

[0072] When the Unshu mandarin extract having the effect of preventing or improving cognitive decline according to the present invention is used in food, the form of the food may include various food compositions such as bread, cakes, noodles, confectioneries, jellies, frozen foods, ice cream, dairy products, beverages, as well as forms similar to those of the oral preparations described below (tablets, capsules, syrups, etc.).

[0073] Such various forms of food can be prepared and manufactured by using the Satsuma mandarin extract or its extract powder of the present invention alone, or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, aromatics, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc.

[0074] The above-mentioned foods include foods that are based on the concept of preventing or improving cognitive decline and, if necessary, are labeled as such, functional foods, foods for specified health uses, foods for the sick, supplements, etc.

[0075] In addition, by preparing the above-mentioned Unshu mandarin extract having the effect of preventing or improving cognitive decline according to the present invention as a blended component, a cognitive function improver having the effect of preventing or improving cognitive decline can be manufactured. That is, one embodiment of the present invention is a cognitive function improver having the effect of preventing or improving cognitive decline, in which the above-mentioned Unshu mandarin extract having the effect of preventing or improving cognitive decline according to the present invention is included as a blended component.

[0076] A cognitive function improving agent having the effect of preventing or improving cognitive decline, which contains as an ingredient the Unshu mandarin extract of the present invention having the effect of preventing or improving cognitive decline as described above, can be manufactured by blending the Unshu mandarin extract of the present invention itself or the extract powder of the Unshu mandarin extract of the present invention itself with materials known in the art that are used in foods, quasi-drugs, and pharmaceuticals, using techniques known in the art.

[0077] As mentioned above, Alzheimer's disease begins with the accumulation of amyloid beta (Aβ) protein in neurons in the brain. In addition, Neu5Gc accumulates in the brain, inhibiting the formation of neural circuits and strongly impairing cognitive function (Non-Patent Documents 6 and 9), and also promoting the aggregation of Aβ, the causative agent of Alzheimer's disease (Non-Patent Documents 7 and 10).

[0078] As shown in the examples described below, the Unshu mandarin extract of the present invention improves memory function and reduces the amount of Neu5Gc and amyloid beta in the brain. 42 (Aβ 42 ) and has been shown to reduce the amount of

[0079] Therefore, a cognitive function improver containing the Unshu mandarin extract of the present invention as an ingredient can be used to prevent or improve decline in cognitive function.

[0080] A cognitive function improving agent containing the Unshu mandarin extract of the present invention, which is obtained by subjecting unripe Unshu mandarins to subcritical water extraction treatment and is useful for preventing or improving cognitive decline, can be useful for preventing or improving cognitive decline, as verified in the examples described below.

[0081] Furthermore, when a cognitive function improving agent prepared by including in its compounded ingredients the Unshu mandarin extract having the effect of preventing or improving cognitive decline according to the present invention is used to prevent or improve cognitive decline, such use may be in humans or non-human animals, or in samples derived therefrom.

[0082] This use may be either therapeutic or non-therapeutic, where "non-therapeutic" refers to a concept that does not include medical procedures, i.e., a concept that does not include a method of surgery, therapy, or diagnosis of a human being, and more specifically, a concept that does not include a method of surgery, therapy, or diagnosis performed on a human being by a physician or a person under the instruction of a physician.

[0083] The cognitive function improving agent prepared by including the Unshu mandarin extract having the effect of preventing or improving cognitive decline according to the present invention in the compounding ingredients can be a medicine, quasi-drug, or food for humans or animals that exerts the effect of preventing or improving cognitive decline. It can also be a material or preparation to be used by being compounded in the medicine, quasi-drug, or food.

[0084] As mentioned above, foods in this case include foods that are based on the concept of preventing or improving cognitive decline and, as necessary, labelled as such, functional foods, foods for specified health uses, foods for the sick, supplements, etc.

[0085] When preparing a medicine by including the Satsuma mandarin extract having the cognitive function decline prevention or improvement effect according to the present invention in the compounding ingredients, the medicine can be administered in any dosage form.The dosage form includes, for example, oral administration by tablets, capsules, granules, powders, syrups, etc., or parenteral administration by injections, suppositories, inhalants, transdermal absorbents, external preparations, etc.The preferred form is oral administration.

[0086] Such various pharmaceutical formulations can be prepared using the Unshu mandarin extract of the present invention alone or in appropriate combination with other pharma- ceutically acceptable excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavorings, fragrances, coating agents, carriers, diluents, etc.

[0087] Target subjects for preventing or improving cognitive decline using the Unshu mandarin extract having the effect of preventing or improving cognitive decline according to the present invention, and cognitive function improvers, foods, quasi-drugs and pharmaceuticals containing this as an ingredient, include people whose memory and learning abilities have declined due to the onset of Alzheimer's disease, and people who wish to maintain or improve their cognitive function. EXAMPLES

[0088] (1) Preparation of Unshu mandarin orange extract powder Shizuoka Prefecture Green Mandarin Extract PSW (manufactured by Nikken Foods Co., Ltd.) was used. This Shizuoka Prefecture Green Mandarin Extract PSW is a powdered product produced by adding water to unripe Unshu mandarins harvested in mid-July to make a paste, extracting it with subcritical water at 180°C and 3 MPa, and adding an excipient to the extract that has been separated into solid and liquid, in accordance with the production method described in Patent Document 2, and spray-drying the powdered product, which is a functional food material (Unshu mandarin extract powder) standardized to contain 30 mg / g or more of hesperidin and 10 mg / g or more of narirutin.

[0089] (2) Animal testing Forty-eight 4-week-old male senescence-accelerated model mice, SAMP8, were purchased (Japan SLC) and housed in cages with four mice per cage, with littermates in each cage whenever possible. After one month of acclimatization, feeding of solid special food was started. Experimental animals were housed in an environment with a temperature of 23±1°C and humidity of 55±5%, with a 12-hour light-dark cycle with the light period from 8:00 to 20:00. The research plan for animal experiments was approved in advance by the Animal Experiment Committee of Shizuoka University. In addition, all animal experiments were conducted in compliance with the Shizuoka University Animal Experiment Regulations.

[0090] The tests were conducted on three groups of accelerated aging model mice (SAMP8 group) fed a normal diet, three groups (SAMP8 group) fed a special diet containing 0.5% by mass of Satsuma mandarin extract PSW powder, and three groups (SAMP8 group) fed a special diet containing 5% by mass of Satsuma mandarin extract PSW powder. The mice were divided into three groups (16 mice per group).

[0091] (3) Body weight and food intake Body weight was measured before the start of the special diet, and then again at 3 and 6 months. The results showed that there were no differences between the groups before the start of the special diet, and after 3 and 6 months, although there was a slight tendency for weight to increase in the special diet group that received 0.5% Satsuma mandarin extract, there was no significant difference between them and the normal diet group (Figure 1).

[0092] In addition, 24 weeks after starting the special diet, food intake was measured for each cage three times over six days over a 48-hour period, and the amount of food consumed per animal per 24 hours was calculated. As a result, no significant difference was observed in food intake compared to normal food on any of the days examined (Figure 2).

[0093] From the above results, there were no significant differences in body weight and food intake between all special diet groups and the SAMP8 normal diet group (control), and therefore it is believed that the effects of each food can be appropriately examined by comparing them with the SAMP8 normal diet group in this study.

[0094] (4) Novel object recognition test A novel object recognition test was conducted 25 weeks after the start of the special diet. The special diet was continued during the test. The test was recorded with a video camera, and the behavior of the mice was analyzed by video using ANY-maze (registered trademark) software (Stoelting, USA).

[0095] The novel object recognition test is a test to evaluate the cognitive function of mice based on their response to novel objects. First, two identical objects were placed 20 cm apart in an open field (diameter 57 cm x height 33 cm), and the mouse was allowed to explore for 10 minutes. The mouse was then placed at a point equidistant from the two objects with its head facing the wall. Next, 24 hours later, one of the objects was replaced with an object (novel object) with a different texture, shape, and color, and the mouse was placed in the field and allowed to explore in the same way. The discrimination index and recognition index were calculated according to the following formula from the data analysis and used as an index of the mouse's cognitive function.

[0096] a: Exploration time for familiar objects on the second day b: Exploration time for novel objects

[0097] Search time (%)={b / (a+b)}×100 A value of 50% or more indicates that the rat explored the novel object more (memory was established). A value of 50% indicates that the rat explored both objects for the same amount of time.

[0098] Discrimination index = (ba) / (a+b) A positive value indicates more exploration time for the novel object (memory established), while a zero value indicates equal exploration time for both objects.

[0099] The novel object recognition test is a test that utilizes the rodent's characteristic of preferring novelty, and can evaluate memory and learning ability. In particular, since it does not use reinforcing factors such as rewards or punishments, it can detect memories formed under conditions of low emotional levels. The exploration time (Figure 3(a)) and discrimination time (Figure 3(b)) were significantly higher in the special diet group containing 5% Satsuma mandarin extract by mass than in the SAMP8 normal diet group, suggesting that the special diet containing 5% Satsuma mandarin extract by mass improves memory ability.

[0100] (5) Forced swimming test Depressive-like behavior (immobility) was evaluated using a forced swimming test. On the first day, mice were placed in an acrylic cylindrical cylinder (20 cm in diameter, 45 cm in height, 22 cm in depth) filled with water, and the immobility time was measured for 15 minutes. On the second day, 24 hours later, mice were similarly placed in the cylinder, and the immobility time was measured for 5 minutes. The immobility time is an index that reflects depression tendency.

[0101] The percentage of immobility time of the senescence-accelerated model mouse SAMP8 fed a special diet containing Unshu mandarin extract was not significantly different from the percentage of immobility time of the senescence-accelerated model mouse SAMP8 fed a normal diet on both days 1 and 2 (Figure 4). From this, no effect on depression-like behavior was observed in this test.

[0102] (6) Blood collection and hippocampal removal After anesthetizing the mouse with isoflurane, the chest was opened and blood was collected. The blood was transferred to a Cabiject II (registered trademark) (Terumo Corporation) and centrifuged at 3000g for 10 minutes at 20°C. The supernatant (plasma) was transferred to another tube and stored at -80°C. Next, the blood was removed by cardiac perfusion with PBS, and the brain was then excised. The brain was cut into left and right halves, and the hippocampus was excised from the right brain. The excised left brain and right hippocampus were measured for wet weight and then stored at -80°C.

[0103] (7) Sialic acid analysis The right hippocampus was removed and placed in an Eppendorf tube. After measuring the wet weight, 10-fold amount of TBS was added and homogenized under ice cooling. 180 μL of 40 mM sulfuric acid was added to 20 μL of homogenization solution, and hydrolysis was performed by heating in 80°C hot water for 15 hours to liberate sialic acid. After ice cooling, the mixture was centrifuged at 4°C and 15,000 rpm for 5 minutes, and the resulting supernatant was passed through a filter (Millex-GV (registered trademark), 0.22 μm) to remove impurities. An equal amount of DMB solution was added to this, and the mixture was fluorescently derivatized in a 60°C water bath for 2.5 hours under light shielding. After ice cooling, 100 μL of the solution was injected into a high performance liquid chromatography (HPLC) to quantify the amount of sialic acid contained. The concentration was calculated using an absolute calibration curve method in which the concentration in a sample is calculated based on the peak area of ​​a known amount.

[0104] HPLC analysis conditions Mobile phase: methanol (Wako Pure Chemical Industries, Ltd.) / water = 25 / 75 (w / w) ·Flow rate: 1.2mL / min (100kg / cm 2 ) Column temperature: 25℃ Detector: Fluorescence detector (excited at 373 nm, measured at 448 nm) Column: TSKgel ODS-100V 5μm (4.6mm x 15mm) (Tosoh Corporation)

[0105] Neu5Ac plays an important role in memory. On the other hand, Neu5Gc gradually accumulates in the brain with aging, and it is known that Neu5Gc accumulates to a high degree in the brains of Alzheimer's disease patients in particular. It is known that Neu5Gc accumulated in the brain reduces memory learning ability by attenuating synaptic plasticity, and promotes the aggregation of amyloid, which causes the onset of Alzheimer's disease. For this reason, the accumulation of Neu5Gc in the brain is strongly associated with the onset and progression of dementia.

[0106] We analyzed the amount of Neu5Gc in the brain and found that the amount of Neu5Gc in the brain of the senescence accelerated mouse model SAMP8 fed a special diet containing 0.5% or 5% by mass of Satsuma mandarin extract was significantly reduced compared to the normal diet (Figure 5(a)). On the other hand, the amount of Neu5Ac in the brain, which is essential for memory, was not affected by the addition of Satsuma mandarin extract (Figure 5(b)).

[0107] These results suggest that Unshu mandarin extract may be effective in improving the decline in memory function associated with aging by reducing the amount of Neu5Gc in the brain, as well as in preventing the onset of Alzheimer's disease.

[0108] (6) Amyloid analysis The right hippocampus of a mouse was placed in an Eppendorf tube, and after measuring the wet weight, 10 volumes of TBS were added and homogenized under ice cooling. Next, 80 μL of the hippocampal homogenized solution was added to 176 μL of formic acid (4°C, 99%, Wako Pure Chemical Industries, Ltd.) and suspended, and incubated at 37°C for 15 minutes. After that, while cooling the tube, ultrasonic disruption was performed for about 60 seconds until sufficient dissolution. 256 μL was taken into an ultracentrifuge tube (HITACHI), and centrifuged at 4°C, 50,000 rpm, for 60 minutes, and then 105 μL of the supernatant was added to 2 mL of FA Neutraization Solution and gently mixed. After dispensing the amount required for measurement, it was stored at -80°C until use. Amyloid β was measured using the Human / Rat βAmyloid (42) ELISA Kit Wako High-sensitive. 42 (Aβ42 ) was quantified. Quantification was performed according to the instructions in the kit.

[0109] As a result, Aβ in the brain of the senescence-accelerated mouse model SAMP8 42 The amount of Aβ in the brain decreased in a concentration-dependent manner with the administered Unshu mandarin extract (Figure 6). 42 It has been suggested that reducing the amount of nicotinamide may be effective in improving the decline in memory function associated with aging and in preventing the onset of Alzheimer's disease.

[0110] (7) Statistical analysis Significant differences compared to the normal diet group were tested using one-way analysis of variance followed by Dunnett's multiple comparisons.

Claims

1. An Unshu mandarin extract having an effect of preventing or improving decline in cognitive function, which is obtained by subjecting unripe Unshu mandarins to subcritical water extraction treatment.

2. The Unshu mandarin extract according to claim 1, which contains hesperidin and narirutin as indicator components of the Unshu mandarin extract's effect of preventing or improving the decline in cognitive function.

3. 3. The Unshu mandarin extract according to claim 2, which contains 30 mg / g or more of hesperidin and 10 mg / g or more of narirutin.

4. The Satsuma mandarin extract according to claim 1, which inhibits the accumulation of amyloid beta (Aβ) and / or N-glycolylneuraminic acid (Neu5GC) in the brain.

5. A cognitive function improving agent having an effect of preventing or improving cognitive decline, comprising the Unshu mandarin extract according to any one of claims 1 to 4 as a compounding ingredient.

6. A method for producing a Satsuma mandarin extract having an effect of preventing or improving decline in cognitive function, comprising subjecting unripe Satsuma mandarins to a subcritical water extraction treatment.

7. A method for producing a Satsuma mandarin extract having an effect of preventing or improving cognitive decline as described in claim 6, which contains hesperidin and narirutin as indicator components of the Satsuma mandarin extract's effect of preventing or improving cognitive decline.

8. 8. A method for producing a Satsuma mandarin extract having the effect of preventing or improving cognitive decline as described in claim 7, which contains 30 mg / g or more of hesperidin and 10 mg / g or more of narirutin.

9. 7. The method for producing a Satsuma mandarin extract according to claim 6, wherein the Satsuma mandarin extract inhibits accumulation of amyloid beta (Aβ) and / or N-glycolylneuraminic acid (Neu5GC) in the brain.

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