Composition for inhibiting amyloid-beta-induced neuronal cell death, and food, pharmaceuticals, and feed containing the composition.

Abstract

To provide a novel composition for suppressing neurocyte death by amyloid β, a food product, a medicine, and a livestock feed containing the composition.SOLUTION: The present invention provides a composition for suppressing amyloid β-inducing neurocyte death which contains phosphatidylserine and sphingomyelin. Further, the invention provides a composition for suppressing amyloid β-inducing neurocyte death in which the total of phosphatidylserine and the sphingomyelin is 1 wt.% or more and 100 wt.% or less. Further, the invention provides a composition for suppressing amyloid β-inducing neurocyte death in which a value obtained by dividing a value of wt.% of phosphatidylserine by a value of wt.% of the sphingomyelin is 0.1 or more and 10 or less. Furthermore, the invention provides a food product, a medicine, and a livestock feed containing the composition.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for suppressing amyloid-β-induced neuronal cell death, and foods, pharmaceuticals, and feeds containing the composition.

Background Art

[0002] Dementia presents core symptoms such as memory impairment, disorientation, and aphasia, as well as peripheral symptoms such as wandering, restlessness, and sleep disorders. As the symptoms progress, it becomes difficult for patients to carry out their daily lives, which also places a heavy burden on the caregivers. In the future, in Japan, with the aging of the population, the number of dementia patients is expected to increase rapidly. In the "Study on Future Projections of the Elderly Population with Dementia in Japan" by Professor Ninomiya of Kyushu University, the number of dementia patients in Japan as of 2012 was estimated to be 4.62 million, and it is projected to reach 7 million in 2025 and 9.5 million in 2040.

[0003] Alzheimer's type dementia is the most common type of dementia, and one of its causes is amyloid-β (Aβ) peptide. This peptide is produced from amyloid precursor protein by proteolytic enzymes, causes neuronal cell death, and leads to brain atrophy and decline in cognitive ability.

[0004] Therapeutic agents for dementia, including Alzheimer's type, include donepezil, galantamine, rivastigmine, memantine, etc. However, these administrations are symptomatic treatments and do not cure dementia. Also, because they are therapeutic drugs, healthy people cannot take them for preventive purposes. Therefore, it is important to suppress neuronal cell death caused by amyloid-β with foods that can be taken daily and prevent dementia.

[0005] Phosphatidylserine is one of the components that make up cell membranes and is found in large quantities in brain and nerve cells. It has been reported that phosphatidylserine intake suppresses cognitive decline in patients with Alzheimer's disease and in the elderly (Non-Patent Literature 1, 2). It has also been suggested that phosphatidylserine suppresses neuronal cell death in the hippocampus (Non-Patent Literature 3).

[0006] Sphingomyelin is a major component of the myelin sheath of nerve cells. Previous studies have reported that the amount of sphingomyelin in the brain decreases with age, and that cognitive processing speed correlates with the health of the myelin sheath (Non-Patent Literature 4). It has also been suggested that sphingomyelin intake may be effective in improving learning ability (Patent Literature 1).

[0007] However, there are no studies or other materials that suggest that combining phosphatidylserine and sphingomyelin enhances the effectiveness against amyloid-beta-induced neuronal cell death. Furthermore, there are no studies or other materials that disclose foods, pharmaceuticals, or animal feeds containing this composition. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2003-146883 [Non-patent literature]

[0009] [Non-Patent Document 1] Rolf RE, et al, Eur Neuropsychopharmacol., 2, 2, 149-55, 1992 [Non-Patent Document 2] T. Cenacchi, et al, Aging Clin. Exp. Res., 5, 2, 123-133, 1993 [Non-Patent Document 3] S. Suzuki, et al, Jpn. J. Pharmacol. 81, 237-289, 1999 [Non-Patent Document 4] Po H. Lu, et al, J Clin Exp Neuropsychol., 33, 10, 1059-1068, 2011. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a novel composition that suppresses amyloid-beta-induced neuronal cell death (hereinafter sometimes referred to as "amyloid-beta-induced neuronal cell death"), and a food, pharmaceutical, and feed containing the composition. [Means for solving the problem]

[0011] To solve the above problems, the inventors investigated useful methods for suppressing amyloid-beta-induced neuronal cell death and found that combining phosphatidylserine and sphingomyelin can synergistically suppress neuronal cell death, thus completing the present invention. In other words, the present invention includes the following components. (1) A composition for inhibiting amyloid-beta-induced neuronal cell death, comprising phosphatidylserine and sphingomyelin as active ingredients. (2) The composition for inhibiting amyloid-beta-induced neuronal cell death according to (1), wherein the total amount of phosphatidylserine and sphingomyelin is 1% by weight or more and 100% by weight or less. (3) The amyloid-beta-induced neuronal cell death inhibitory composition according to (1), wherein the value obtained by dividing the weight percentage of phosphatidylserine by the weight percentage of sphingomyelin is 0.1 or more and 10 or less. (4) Food and beverages containing a composition for suppressing amyloid-beta-induced neuronal cell death as described in any of (1) to (3). (5) A pharmaceutical product comprising a composition for inhibiting amyloid-beta-induced neuronal cell death as described in any of (1) to (3). (6) A feed comprising a composition for suppressing amyloid-beta-induced neuronal cell death as described in any of (1) to (3). [Effects of the Invention]

[0012] The present invention provides a composition containing phosphatidylserine and sphingomyelin as a composition for suppressing amyloid-beta-induced neuronal cell death, and a food, pharmaceutical, and animal feed containing said composition. Ingestion of the composition of the present invention makes it possible to safely and easily suppress amyloid-beta-induced neuronal cell death. [Brief explanation of the drawing]

[0013] [Figure 1] This graph shows the effect (%) on improving cell viability (=(cell viability of the group treated with amyloid-beta and the sample)-(survival rate of the group treated with amyloid-beta)) when human neuroblastoma (SH-SY5Y) is treated with each medium containing amyloid-beta. From left to right: phosphatidylserine (80 μg / ml) (SPM 0 / PS 80), sphingomyelin (40 μg / ml) (SPM 40 / PS 0), sphingomyelin (40 μg / ml) + phosphatidylserine (80 μg / ml) (SPM 40 / PS 80), sphingomyelin (80 μg / ml) (SPM 80 / PS 0), sphingomyelin (80 μg / ml) + phosphatidylserine (80 μg / ml) (SPM 80 / PS 80), sphingomyelin (160 μg / ml) (SPM 160 / PS 0), sphingomyelin (160 μg / ml) + phosphatidylserine (80 μg / ml) (SPM 160 / PS 80). [Modes for carrying out the invention]

[0014] The amyloid-beta-induced neuronal cell death composition of the present invention will be described in detail below.

[0015] (Composition for inhibiting amyloid-beta-induced neuronal cell death) The composition for suppressing amyloid-β-induced neuronal cell death of the present invention contains phosphatidylserine and sphingomyelin. The content of phosphatidylserine and sphingomyelin in the composition for suppressing amyloid-β-induced neuronal cell death only needs to be such that the total of phosphatidylserine and sphingomyelin is 1% by weight or more and 100% by weight or less of the composition for suppressing amyloid-β-induced neuronal cell death, preferably 10% by weight or more and 100% by weight or less, more preferably 20% by weight or more and 100% by weight or less, and most preferably 30% by weight or more and 100% by weight or less. Also, the weight ratio of phosphatidylserine to sphingomyelin contained in the composition for suppressing amyloid-β-induced neuronal cell death only needs to be such that the value obtained by dividing the weight percentage of phosphatidylserine in the composition for suppressing amyloid-β-induced neuronal cell death by the weight percentage of sphingomyelin in the composition for suppressing amyloid-β-induced neuronal cell death is 0.1 or more and 10 or less, preferably 0.2 or more and 2 or less, and most preferably 0.5 or more and 2 or less. The composition for suppressing amyloid-β-induced neuronal cell death of the present invention can be prepared using a material containing phosphatidylserine and a material containing sphingomyelin described below to prepare the above composition. Also, a composition prepared from milk or the like to have the above composition may be used as it is.

[0016] (Phosphatidylserine) The phosphatidylserine used in the composition for suppressing amyloid-β-induced neuronal cell death of the present invention will be described. The phosphatidylserine used in the composition for suppressing amyloid-β-induced neuronal cell death of the present invention is not only reagent-grade phosphatidylserine with few impurities, but also a material containing phosphatidylserine can be used as it is. The phosphatidylserine used in the composition for suppressing amyloid-β-induced neuronal cell death of the present invention is preferably derived from soybeans, and more preferably phosphatidylserine derived from mammalian milk.

[0017] (Sphingomyelin) The sphingomyelin used in the composition for suppressing amyloid-β-induced neuronal cell death of the present invention is not only of reagent grade, but also sphingomyelin prepared from chicken eggs, milk, etc. can be used as it is. The sphingomyelin used in the composition for suppressing amyloid-β-induced neuronal cell death of the present invention is preferably derived from chicken eggs, and more preferably sphingomyelin derived from mammalian milk.

[0018] (Intake amount of the composition for suppressing amyloid-β-induced neuronal cell death) The effective intake amount of the composition for suppressing amyloid-β-induced neuronal cell death of the present invention may be ingested so that the total amount of phosphatidylserine and sphingomyelin is 2 mg / day or more. In addition, the composition for suppressing amyloid-β-induced neuronal cell death of the present invention can be added to foods and drinks, pharmaceuticals, and feeds as described below. The addition amount of the composition for suppressing amyloid-β-induced neuronal cell death at that time may be appropriately adjusted so that the above-mentioned effective amount can be ingested.

[0019] (Foods and drinks, pharmaceuticals, and feeds containing the composition for suppressing amyloid-β-induced neuronal cell death) The composition for suppressing amyloid-β-induced neuronal cell death of the present invention may be used as it is, but since it can be used together with other raw materials usually contained in foods, pharmaceuticals, and feeds, it can also be used in powders, granules, tablets, capsules, drink agents, etc. according to conventional methods. It is also possible to mix it into foods and drinks such as yogurt, milk drinks, wafers, and feeds.

[0020] (Quantification method of phosphatidylserine and sphingomyelin in the composition for suppressing amyloid-β-induced neuronal cell death) The content of sphingomyelin in the composition for suppressing amyloid-β-induced neuronal cell death of the present invention can be measured by the method of Haruta et al. (Bioscience, Biotechnology, & Biochemistry (2008) 72, 8, 2151-2157).

[0021] (Evaluation of the effect of the composition for suppressing amyloid-β-induced neuronal cell death on suppressing amyloid-β-induced neuronal cell death) The inhibitory effect of the present invention's composition on amyloid-beta-induced neuronal cell death can be evaluated, for example, by comparing neuronal cell death in animal brains or cultured cells with and without the composition, after administering or adding amyloid-beta. Evaluation indicators for neuronal cell death include cell viability and the expression of genes and proteins related to amyloid-beta-induced neuronal cell death response. More specifically, evaluation can be performed using the in vitro methods shown in the examples. [Examples]

[0022] The following describes some embodiments of the present invention in detail, but the present invention is not limited to these embodiments. [Test Example 1] Test to confirm the effect of suppressing amyloid-beta-induced neuronal cell death We conducted a study to confirm that when cultured nerve cells were exposed to amyloid-beta, combining phosphatidylserine or sphingomyelin more strongly suppressed the decrease in cell viability caused by amyloid-beta than adding either phosphatidylserine or sphingomyelin alone.

[0023] 1. Test Method Human neuroblastoma (SH-SY5Y) 1 × 10 4 Seeds were seeded in a 96-well plate at a density of cells / well, incubated at 37°C for 24 hours, (i) Medium supplemented with 10% serum mixed with amyloid-beta (Peptide Institute, 10 μM) (Control) (ii) A medium supplemented with 10% serum containing a mixture of amyloid-beta (10 μM) and phosphatidylserine (Nagara Science; 80 μg / ml), (iii) A medium supplemented with 10% serum containing a mixture of amyloid-beta (10 μM), phosphatidylserine (0, 80 μg / ml), and sphingomyelin (Nagara Science; 40, 80, 160, 800 μg / ml), The culture medium was changed for each sample. Subsequently, the cells were incubated at 37°C for 24 hours and subjected to Cell Titer 96® Non-Radioactive Cell Proliferation Assay (Promega) to measure cell viability (%). The percentage of enhanced cell viability was calculated by subtracting the percentage of cell viability (i) in the group treated with amyloid-beta alone from the percentage of cell viability (ii or iii) in the group treated with amyloid-beta and the sample (N=3 for each group). The experimental results are shown in Figure 1.

[0024] 2. Test Results In Figure 1, the cell viability enhancement rate was 8.4% with phosphatidylserine 80 μg / ml alone and 2.7% with sphingomyelin 40 μg / ml alone. However, when phosphatidylserine 80 μg / ml and sphingomyelin 40 μg / ml (phosphatidylserine / sphingomyelin = 2) were added together, the rate increased to 23.1%, indicating a synergistic effect stronger than that of each phospholipid alone. While sphingomyelin at 80 μg / ml alone yielded a 14.6% effect, the co-addition of phosphatidylserine at 80 μg / ml and sphingomyelin at 80 μg / ml (phosphatidylserine / sphingomyelin = 1) resulted in a 40.7% effect, demonstrating a synergistically stronger effect than each phospholipid alone. While sphingomyelin at 160 μg / ml alone yielded an effect of 11.5%, the co-addition of phosphatidylserine at 80 μg / ml and sphingomyelin at 160 μg / ml (phosphatidylserine / sphingomyelin = 0.5) resulted in an effect of 40%, indicating a synergistically stronger effect than each phospholipid alone. Furthermore, a similar effect was observed when phosphatidylserine 80 μg / ml and sphingomyelin 800 μg / ml (phosphatidylserine / sphingomyelin = 0.1) were added together.

[0025] [Example 1] Method for preparing sphingomyelin concentrate Sphingomyelin was extracted and purified from buttermilk powder (manufactured by Snow Brand Megmilk Co., Ltd.) using the method described in Japanese Patent Publication No. 5-132490. As a result, sphingomyelin with a purity of 98.1% was obtained.

[0026] [Example 2] Method for preparing phosphatidylserine concentrate The phosphatidylserine was extracted and purified from buttermilk powder (manufactured by Snow Brand Megmilk Co., Ltd.) using the method described in Japanese Patent Publication No. 5-132490. As a result, phosphatidylserine with a purity of 97.5% was obtained.

[0027] [Example 3] Preparation of capsules for inhibiting amyloid-beta-induced neuronal cell death The raw materials were mixed according to the formulation shown in Table 1, granulated by a conventional method, and filled into capsules to produce the amyloid-beta-induced neuronal cell death inhibitory capsule formulation of the present invention.

[0028] [Table 1]

[0029] [Example 4] (Preparation of tablets for inhibiting amyloid-beta-induced neuronal cell death) After mixing the raw materials according to the formulation shown in Table 2, the mixture was molded into 1g tablets using a conventional method and compressed to produce the amyloid-beta-induced neuronal cell death inhibitory tablets of the present invention.

[0030] [Table 2]

[0031] [Example 5] Preparation of a gel-like food for suppressing amyloid-beta-induced neuronal cell death 2.5 g of sphingomyelin concentrate (Example 1) and 7.5 g of phosphatidylserine concentrate (Example 2) were dissolved in 700 g of deionized water, heated to 50°C, and then stirred and mixed for 30 minutes at 9500 rpm using an Ultra-Turrax T-25 (manufactured by IKA Japan). To this solution, 40 g of sorbitol, 2 g of acidulant, 2 g of flavoring, 5 g of pectin, 5 g of whey protein concentrate, 1 g of calcium lactate, and 235 g of deionized water were added and stirred and mixed. The mixture was then filled into 200 ml Chia Packs, sterilized at 85°C for 20 minutes, and then sealed to prepare 5 bags (200 g each) of the gel-like food for inhibiting amyloid-beta-induced neuronal cell death according to the present invention. None of the gel-like food for inhibiting amyloid-beta-induced neuronal cell death obtained in this way showed any precipitation, and no abnormalities were detected in flavor. This gel-like food product for inhibiting amyloid-beta-induced neuronal cell death contained 245.3 mg of sphingomyelin and 731.3 mg of phosphatidylserine per 100 g.

[0032] [Example 6] Preparation of a beverage for suppressing amyloid-beta-induced neuronal cell death After dissolving 2g of acidulant in 700g of deionized water, 1.0g of sphingomyelin concentrate (Example 1) and 9.0g of phosphatidylserine concentrate (Example 2) were dissolved, and the mixture was heated to 50°C and then stirred and mixed for 30 minutes at 9500rpm using an ultra-disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan). After adding 100g of maltitol, 20g of reduced starch syrup, 2g of flavoring, and 166g of deionized water, the mixture was filled into 100ml glass bottles, sterilized at 90°C for 15 minutes, and then sealed to prepare 10 bottles (100ml each) of amyloid-beta-induced neuronal cell death suppression beverage. None of the amyloid-beta-induced neuronal cell death suppression beverages obtained in this way showed any precipitation, and no abnormalities in flavor were detected. This beverage for suppressing amyloid-beta-induced neuronal cell death contained 98.1 mg of sphingomyelin and 877.5 mg of phosphatidylserine per 100 g.

[0033] [Example 7] Preparation of feed for suppressing amyloid-beta-induced neuronal cell death 1.8 kg of sphingomyelin concentrate (Example 1) and 0.2 kg of phosphatidylserine concentrate (Example 2) were dissolved in 98 kg of deionized water, heated to 50°C, and then stirred and mixed for 40 minutes at 3600 rpm using a TK homomixer (MARKII 160 model; manufactured by Tokushu Kika Kogyo Co., Ltd.) to obtain a mixed solution containing 1757 mg / 100g of sphingomyelin and 200 mg / ml of phosphatidylserine. 10 kg of this solution was mixed with 12 kg of soybean meal, 14 kg of soybean protein, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, and 2 kg of mineral mixture. The mixture was then sterilized at 120°C for 4 minutes to produce 100 kg of the dog feed for suppressing amyloid-beta-induced neuronal cell death according to the present invention. Furthermore, this dog feed for suppressing amyloid-beta-induced neuronal cell death contained 176.6 mg of sphingomyelin and 19.5 mg of phosphatidylserine per 100 g. [Industrial applicability]

[0034] This invention provides a novel composition for suppressing amyloid-beta-induced neuronal cell death, comprising phosphatidylserine and sphingomyelin, as well as a food, pharmaceutical, and animal feed containing the composition. It is expected that ingesting the composition of this invention can safely and easily suppress amyloid-beta-induced neuronal cell death.

Claims

1. A composition for inhibiting amyloid-beta-induced neuronal cell death, comprising phosphatidylserine and sphingomyelin as active ingredients.

2. The amyloid-beta-induced neuronal cell death inhibitory composition according to claim 1, wherein the total amount of phosphatidylserine and sphingomyelin is 1% by weight or more and 100% by weight or less.

3. The amyloid-beta-induced neuronal cell death inhibitory composition according to claim 1, wherein the value obtained by dividing the weight percentage of phosphatidylserine by the weight percentage of sphingomyelin is 0.1 or more and 10 or less.

4. The composition for inhibiting amyloid-beta-induced neuronal cell death according to any one of claims 1 to 3, wherein the composition is a food product.

5. The composition for inhibiting amyloid-beta-induced neuronal cell death according to any one of claims 1 to 3, wherein the composition is a pharmaceutical product.

6. The composition for suppressing amyloid-beta-induced neuronal cell death according to any one of claims 1 to 3, wherein the composition is a feed.

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