Functional food containing bonito-derived extract for preventing or improving neuropsychiatric disease

Abstract

To provide a functional food having usage for rationally preventing or improving neuropsychiatric disease.SOLUTION: According to an embodiment of the present invention, a functional food for the prevention or improvement of neuropsychiatric diseases characterized by containing a bonito-derived extract extracted from dried bonito flakes through hot water or water extraction, is provided. Here, the aforementioned neuropsychiatric diseases are those caused by brain inflammation and blood-brain barrier disruption, and the functional food possesses anti-inflammatory effects, effects to improve blood-brain barrier integrity, and effects to activate the cardiac acetylcholine production system.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a functional food containing an extract derived from skipjack tuna for preventing or improving psychoneurological diseases.

Background Art

[0002] With the increase in the elderly population, the number of patients suffering from psychoneurological diseases such as Alzheimer's dementia, Parkinson's disease, and schizophrenia is on the rise. However, no radical treatment methods have been established yet, and finding a method for prevention or delaying the onset has become an important issue.

[0003] Based on such a social background, in recent years, it has gradually become clear that food components affect brain function, and interest in the preventive effect by daily intake of functional foods has been increasing.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention has been made in view of the above circumstances, and an object thereof is to provide a functional food having a use for rationally preventing or improving psychoneurological diseases.

Means for Solving the Problems

[0006] The present inventors have obtained the following findings regarding functional foods for preventing or improving psychoneurological diseases, and have conducted intensive experiments and the like to complete the present invention.

[0007] That is, in recent years, it has been reported that in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and schizophrenia, abnormal functions of the blood-brain barrier (BBB) are involved. The blood-brain barrier plays an important role in isolating the brain from substances flowing into the brain parenchyma through the blood, controlling the substance circulation inside and outside the brain, and maintaining a constant brain internal environment. However, if its function fails, direct contact between harmful substances and nerve cells occurs in the brain, leading to nerve cell death accompanied by brain inflammation and decreased nerve activity.

[0008] Here, the present inventors have found that cardiomyocytes of the heart itself have a system (NNCCS) that produces acetylcholine (ACh). And the physiological function of NNCCS provided in cardiomyocytes has been clarified to modify central nervous system functions via the vagus nerve in addition to the circulatory system. As an example, it has been found that this NNCCS is involved in maintaining the blood-brain barrier (BBB), and new findings regarding a novel function as a mediator of organ cross-talk by this system have been obtained. There has been no report of a system that indirectly enhances the BBB function via nerves in a specific manner, and it is considered to be highly novel. In addition, activation of this system is also suggested to lead to enhancement of circulatory function and disease prevention, and one kind of candidate substance for enhancing NNCCS function has been confirmed.

[0009] The present inventors focused on the extract derived from skipjack tuna, and found anti-inflammatory effects, improvement of blood-brain barrier barrier properties, and activation of the non-neural non-central cardiac acetylcholine production system as new health functions of this extract derived from skipjack tuna. From the perspective of the above findings, intensive experiments and the like were conducted on its effectiveness as a functional food, and the present invention was completed.

[0010] That is, according to the present invention, the following is provided.

[0011] (1) A functional food for preventing or improving neurodegenerative diseases, characterized by containing an extract derived from skipjack tuna.

[0012] (2) The functional food according to (1) above, wherein the neuropsychiatric disorder is caused by intracerebral inflammation.

[0013] (3) The functional food according to (2) above, which has an anti-inflammatory effect.

[0014] (4) The functional food according to (3) above, wherein the anti-inflammatory effect is suppression of inflammatory cytokine production and / or suppression of microglial activation in the brain.

[0015] (5) In the functional food according to (3) above, the skipjack-derived extract reduces the production of inflammatory cytokines as compared with the amounts of DHA, EPA, and a composition having the same concentration of DHA and EPA contained in the skipjack-derived extract.

[0016] (6) In the functional food according to (3) above, the skipjack-derived extract reduces the production of inflammatory cytokines as compared with the amounts of histidine, anserine, creatine, creatinine, betaine, carnosine, inosinic acid, and a composition having the same concentration of histidine, anserine, creatine, creatinine, betaine, and carnosine contained in the skipjack-derived extract.

[0017] (7) The functional food according to (1) above, wherein the neuropsychiatric disorder is caused by disruption of the blood-brain barrier.

[0018] (8) The functional food according to (7) above, which has an effect of improving blood-brain barrier properties.

[0019] (9) In the functional food according to (8) above, the active ingredients in the effect of improving blood-brain barrier properties are histidine and inosinic acid.

[0020] (10) The functional food according to (7) above, which has an effect of activating the cardiac acetylcholine production system.

[0021] (11) In the food described in (1) above, the concentration of the extract derived from skipjack tuna is 0.1 mg / mL, a functional food.

[0022] (12) In the food described in (9) above, the concentration of the extract derived from skipjack tuna is 0.1 mg / mL, or the histidine concentration is 0.836 mg / mL, and the inosinic acid concentration is 0.0537 mg / mL, a functional food.

[0023] (13) In the food described in (10) above, the concentration of the extract derived from skipjack tuna is 10 mg / mL, a functional food.

[0024] With the above configuration, the functional food characterized by containing the extract derived from skipjack tuna according to the present invention can exhibit the effects of anti-inflammatory action, improvement of blood-brain barrier permeability, and activation of the non-neural non-central cardiac acetylcholine production system.

[0025] Regarding the features of the present invention other than those described above, they will be clarified in the description of the embodiments of the present invention described below.

Brief Description of the Drawings

[0026]

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Mode for Carrying Out the Invention

[0027] Hereinafter, an embodiment of the present invention will be described with reference to the drawings and the like.

[0028] (Embodiment of the Present Invention) First, hereinafter, the process leading to the conclusion that the extract derived from katsuobushi has the effects of anti-inflammatory action, improvement of blood-brain barrier permeability, and activation of the non-neuronal non-central cardiac acetylcholine production system will be described.

[0029] (Anti-inflammatory Action) Regarding the extracts of katsuobushi (ara-katsuobushi, hon-kare-katsuobushi, namari-katsuobushi), which are traditional fermented foods in Japan, and the extracts of various fish species (urume, saba, miyada katsuobushi, tuna), in vitro screening of the anti-inflammatory action was performed using a cultured cell line derived from mouse brain microglia. As a result, an anti-inflammatory action was recognized in most of the extracts of katsuobushi. However, since the anti-inflammatory actions of DHA and EPA, which are already known ω-3 polyunsaturated fatty acids specific to fish, were considered, it was also possible that the anti-inflammatory actions of the various extracts of katsuobushi were due to these fatty acids.

[0030] Therefore, the DHA and EPA content concentrations in various kelp extracts were calculated by GCMS analysis. With reference to these concentrations, a composition with the same concentration as each extract was prepared using DHA and EPA as reagents, and the anti-inflammatory effect was examined. As a result, it was found that the amounts of DHA and EPA contained in the skipjack kelp extract were extremely low for the anti-inflammatory effect to be recognized, and that the skipjack kelp rough extract had a higher anti-inflammatory effect than the composition with the same concentration of DHA+EPA. It was inferred that there was a component with a high anti-inflammatory effect different from DHA and EPA in the skipjack kelp extract. On the other hand, in most of the other kelp extracts, the DHA and EPA content was higher compared to the skipjack kelp extract, and the anti-inflammatory effects of each extract and the composition with the same concentration of DHA+EPA were similar. Therefore, it was considered highly likely that the responsible substances were DHA and EPA. After such a process, the skipjack kelp rough extract was used as the research material.

[0031] In addition, the anti-inflammatory effects of known components characteristic of skipjack kelp (histidine, anserine, creatine, creatinine, betaine, carnosine, inosinic acid) were examined using the same evaluation system. None of them showed an anti-inflammatory effect comparable to that of the skipjack kelp extract when used alone, and no significant anti-inflammatory effect was observed even when they were mixed (excluding inosinic acid). From this, the existence of an unknown substance showing a strong anti-inflammatory effect was considered to be present in the skipjack kelp extract.

[0032] (Improving effect on the blood-brain barrier) Using the anti-inflammatory effect of cultured cells derived from mouse brain microglia as an indicator, the active fractions obtained in the process of sequentially separating and purifying the hot water extract of dried skipjack tuna by gel filtration chromatography and reverse-phase HPLC, and the compounds (histidine, inosinic acid) estimated by LCMS analysis from the active fractions were examined for the expression of tight junction-related molecules (Claudin-5, Occludin) in rat brain vascular endothelial cells. An increase in expression was observed at the gene level and protein level. The effect was also confirmed when the hot water extract of dried skipjack tuna or the active fraction was orally administered to mice. Furthermore, in the freeze injury, which is a BBB disruption model, BBB disruption was significantly suppressed in mice orally administered the hot water extract of dried skipjack tuna.

[0033] (Activation of the non-neural non-central cardiac acetylcholine production system) As a result of orally administering the hot water extract of dried skipjack tuna to mice, the acetylcholine concentration increased in the heart, and an increase in the protein level expression of acetylcholine synthase (ChAT) was also confirmed in the heart and brain. Therefore, it was confirmed that the cardiac acetylcholine production system was activated. Furthermore, since the heart rate of mice orally administered the hot water extract of dried skipjack tuna decreased significantly, it was confirmed that the parasympathetic nerves of the whole body were also enhanced at the same time.

[0034] The following describes the experiments conducted to examine whether the skipjack-derived extract has the effects of anti-inflammatory action, improvement of blood-brain barrier properties, and activation of the non-neural non-central cardiac acetylcholine production system, and the experimental results.

[0035] [Experiment 1] MG6 cells derived from mouse brain microglia were purchased from the RIKEN BioResource Research Center (BRC), and the anti-inflammatory effects of various extracts including skipjack tuna were verified (see Figure 1). First, MG6 cells were seeded in a 96-well plate (5×10 3 cells / well·90μL), and culturing was started in a CO 2 incubator (37°C, CO 2Concentration: 5%). After 0.5 hours, various extracts were added to each well, and after another hour, lipopolysaccharide (LPS) was added to activate the MG6 cells and induce an inflammatory state. After 6 hours, 10 μL of the culture supernatant from each well was collected, and then 10 μL of a viable cell counting reagent (WST-8) was added to each well. The absorbance at 450 nm (reference wavelength: 630 nm) was measured using a plate reader after 1 and 2 hours.

[0036] The culture supernatant collected earlier was diluted 25-fold with buffer, and the amount of TNF-α, a type of inflammatory cytokine produced in the culture supernatant, was measured by ELISA (using Bio Legend's ELISA MAX (trademark) Deluxe Set Mouse TNF-α kit). The greater the reduction in the amount of TNF-α produced by the pre-addition of various extracts compared to the LPS-added test group, the greater the anti-inflammatory effect, and the less the reduction, the lower the anti-inflammatory effect.

[0037] [Experiment 2] The hot water extract and water extract (0.01mg, 0.03mg, 0.1mg, 0.3mg, 1mg / mL) of dried bonito flakes were evaluated for their anti-inflammatory effects against LPS stimulation (see Figure 2). Regardless of the part of the dried bonito flakes (surface, interior) or the extraction method, the amount of TNF-α production decreased in a concentration-dependent manner, making it clear that the extracts have an anti-inflammatory effect. In the WST-8 assay, no decrease in absorbance was observed in the test groups of the various extracts compared to the control test group (LPS ±), confirming that the extracts have an anti-inflammatory effect without affecting cell viability.

[0038] Furthermore, the concentrations of DHA and EPA in each extract were calculated by GCMS analysis, and these concentrations were used as a reference to prepare compositions of equivalent concentrations to each extract using DHA and EPA reagents to examine their anti-inflammatory effect.The amounts of DHA and EPA contained in the dried bonito extract were too low to demonstrate an anti-inflammatory effect, and the anti-inflammatory effect was greater in the dried bonito extract than in a composition of equivalent concentration DHA+EPA, suggesting the presence of a component other than DHA and EPA that has a strong anti-inflammatory effect.

[0039] [Experiment 3] The anti-inflammatory effects of the hot water extract and water extract (0.01 mg, 0.03 mg, 0.1 mg, 0.3 mg, 1 mg / mL) of dried bonito wood on LPS stimulation were evaluated (see Figure 3). Regardless of the part (surface, inside) of the dried bonito wood and the extraction method, the production amount of TNF-α decreased in a concentration-dependent manner, indicating that it has an anti-inflammatory effect. In the WST-8 assay, no decrease in absorbance value was observed in the test groups of various extracts compared with the control test group (LPS±), confirming that it shows an anti-inflammatory effect without affecting cell viability.

[0040] Furthermore, when the DHA and EPA content concentrations in each extract were calculated by GCMS analysis, since the amounts of DHA and EPA contained in the dried bonito wood extract were extremely low to recognize an anti-inflammatory effect, the presence of components showing high anti-inflammation different from DHA and EPA was inferred.

[0041] [Experiment 4] The anti-inflammatory effects of the hot water extract (0.01 mg, 0.03 mg, 0.1 mg, 0.3 mg, 1 mg / mL) of leaded dried bonito on LPS stimulation were evaluated (see Figure 4). Regardless of the part (male part, female part / surface, inside) of the leaded dried bonito, the production amount of TNF-α decreased in a concentration-dependent manner, indicating that it has an anti-inflammatory effect. In the WST-8 assay, no decrease in absorbance value was observed in the test groups of various extracts compared with the control test group (LPS±), confirming that it shows an anti-inflammatory effect without affecting cell viability.

[0042] Furthermore, when the DHA and EPA content concentrations in each leaded dried bonito extract were calculated by GCMS analysis, since the amounts of DHA and EPA contained in the leaded dried bonito extract were extremely low to recognize an anti-inflammatory effect, the presence of components showing high anti-inflammation different from DHA and EPA was inferred.

[0043] [Experiment 5] The anti-inflammatory effects of the hot water extracts and water extracts (0.01 mg, 0.03 mg, 0.1 mg, 0.3 mg, 1 mg / mL) of eel segments and mackerel segments against LPS stimulation were evaluated (see Figure 5). Regardless of the extraction method of eel segments and mackerel segments, since the production amount of TNF-α decreased in a concentration-dependent manner, it was revealed that they have anti-inflammatory effects. In the WST-8 assay, since no decrease in absorbance value was observed in the test sections of various extracts compared to the control test section (LPS±), it was confirmed that they exhibit anti-inflammatory effects without affecting cell viability.

[0044] However, when the DHA and EPA content concentrations in each extract were calculated by GCMS analysis, and compositions of the same concentrations as each extract were prepared using reagent DHA and EPA with reference to those concentrations to examine the anti-inflammatory effects, since the anti-inflammatory effects of each extract and the compositions of the same concentrations of DHA+EPA were comparable, it was considered highly likely that the responsible substances for the anti-inflammatory effects observed in each extract were DHA and EPA.

[0045] [Experiment 6] The anti-inflammatory effects of the hot water extracts and water extracts (0.01 mg, 0.03 mg, 0.1 mg, 0.3 mg, 1 mg / mL) of sardine segments and tuna segments against LPS stimulation were evaluated (see Figure 6). Regardless of the extraction method of sardine segments and tuna segments, since the production amount of TNF-α decreased in a concentration-dependent manner, it was revealed that they have anti-inflammatory effects. In the WST-8 assay, since no decrease in absorbance value was observed in the test sections of various extracts compared to the control test section (LPS±), it was confirmed that they exhibit anti-inflammatory effects without affecting cell viability.

[0046] Furthermore, when the DHA and EPA content concentrations in each extract were calculated by GCMS analysis, and compositions of the same concentrations as each extract were prepared using reagent DHA and EPA with reference to those concentrations to examine the anti-inflammatory effects, although the DHA and EPA contents were not at concentrations that showed a high anti-inflammatory effect, since the anti-inflammatory effects of each extract and the compositions of the same concentrations of DHA+EPA were comparable, it was considered highly likely that the responsible substances for the anti-inflammatory effects observed in each extract were DHA and EPA.

[0047] [Experiment 7] Since the anti-inflammatory effects of various kelp extracts were clarified by in vitro experiments using MG6 cells derived from mouse brain microglia, as the next step, an animal experiment was conducted by oral administration to mice to verify whether the effects were also shown in vivo.

[0048] The hot water extract of dried skipjack kelp was dissolved in distilled water at a concentration of 11 mg / mL and placed in a drinking bottle, which was installed in a breeding cage and allowed to drink freely for 4 days. Then, after applying restraint stress (2 hours), the gene expression of inflammatory cytokines (IL-1β, TNF-α) in the brain and the blood corticosterone concentration were measured. Furthermore, the activation of microglia in the hypothalamus was observed by immunohistological methods.

[0049] As a result, for IL-1β and TNF-α in the brain, a significant decrease was observed in the group administered with the hot water extract of dried skipjack kelp (E) (see Figure 7A). In addition, the activation of microglia (the part marked with black triangles) observed under restraint stress conditions was also significantly suppressed in the group administered with the hot water extract of dried skipjack kelp (E), and a state similar to that before the restraint stress load could be observed (see Figure 7B).

[0050] From these results, it became clear that the hot water extract of dried skipjack kelp also showed anti-inflammatory effects (inhibiting the production of inflammatory cytokines in the brain and suppressing microglia activation) in in vivo experiments.

[0051] [Experiment 8] The anti-inflammatory effect of the hot water extract of dried skipjack kelp in mice induced with inflammation by LPS was examined.

[0052] The hot water extract of skipjack katsuobushi was dissolved in water at a concentration of 10 mg / mL, placed in a drinking bottle, installed in a breeding cage, and allowed to drink freely for 3 days. Then, LPS (10 mg / kg) was intraperitoneally injected, and after 4 hours, the gene expression of inflammatory cytokines (TNF-α, IL-1β, IL-6) and blood inflammatory cytokines (TNF-α, IL-1β) in the liver were measured.

[0053] As a result, for TNF-α, IL-1β, and IL-6 in the liver, a significant decrease was observed in the administration group of the hot water extract of skipjack katsuobushi (E) (see Fig. 8A). At this time, the expression of α7 nicotinic receptor (α7 AChR) protein in the liver decreased, suggesting an inhibitory effect of the hot water extract of skipjack katsuobushi (E) on the inflammatory response (see Fig. 8B). Also, for TNF-α and IL-6 in the blood, a significant decrease was observed in the administration group of the hot water extract of skipjack katsuobushi (E) (see Fig. 8C).

[0054] From these results, it became clear that the hot water extract of skipjack katsuobushi also exhibits an anti-inflammatory effect (inhibiting the production of inflammatory cytokines in the liver and blood) in in vivo experiments.

[0055] [Experiment 9] The hot water extract of skipjack katsuobushi was separated and purified by gel filtration chromatography, and the anti-inflammatory effect of the obtained fractions was verified using MG6 cells.

[0056] First, 1851.6 mg of the hot water extract of skipjack katsuobushi (Lot: 200729) was redissolved in 12.3 mL of the mobile phase (0.1 M acetic acid) [225 mg / 1.5 mL], and this was separated and purified by gel filtration chromatography. As the conditions for gel filtration, a column XK16 / 70 (GE Health Care) was packed with Sephadex G-25 (GE Health Care, P / N: 17-0033-02 Lot: 10034186) as the gel filtration carrier, and the sample was eluted with 0.1 M acetic acid at a flow rate of 0.3 mL / min., and the absorbance (214 nm) was measured with a UV detector. The separated eluate was fractionated using a fraction collector so that each fraction was 10 minutes (3 mL / fraction). Next, each of the obtained fractions was dried under reduced pressure and redissolved in ultrapure water. Fractions with a recovered solid content weight of 5 mg or more were redissolved to 100 mg / mL, and fractions less than 5 mg were uniformly redissolved in 50 μL of ultrapure water and then filter sterilized using a 0.2 μm membrane filter. For each fraction prepared in this way, the anti-inflammatory effect was evaluated using MG6 cells.

[0057] As a result, extremely high anti-inflammatory activity was observed in fractions 26 to 28 (gel filtration active fraction I) shown in Fig. 9, and anti-inflammatory activity was also observed in fractions 34 to 36 (gel filtration active fraction II), fractions 39 to 41 (gel filtration active fraction III), and fractions 48 to 51 (gel filtration active fraction IV). However, since it was predicted that the gel filtration active fraction IV would have almost no absorbance value and would be difficult to detect, for the other anti-inflammatory active fractions, further separation and purification were carried out and an attempt was made to isolate and identify the active components.

[0058] First, for the gel filtration active fraction I obtained by separating and purifying the hot water extract of skipjack katsuobushi by gel filtration chromatography, further separation and purification were carried out using reverse phase HPLC, and the anti-inflammatory effect of the obtained fractions was verified using MG6 cells.

[0059] As separation conditions, Inertsil ODS-3, 5 μm, 10 × 250 mm (GL Science, C / N 5020-06812, S / N 0BI41240) was used for the preparative reversed-phase column, mobile phase A was 0.1% TFA, mobile phase B was 80% acetonitrile - 0.1% TFA, and a linear concentration gradient of acetonitrile was applied. The sample was eluted at a flow rate of 3 mL / min, and the absorbance (214 nm) was measured with a UV detector. The separated eluate was fractionated so that 1 fraction was collected per minute (3 mL / fraction). Next, each fraction obtained was dried to dryness under reduced pressure and redissolved in ultrapure water. Fractions with a recovered solid content weight of 5 mg or more were redissolved to 100 mg / mL, and fractions less than 5 mg were uniformly redissolved in 50 μL of ultrapure water and then filter-sterilized using a 0.2 μm membrane filter. The anti-inflammatory effects of each fraction prepared in this way were evaluated using MG6 cells.

[0060] As a result, extremely high anti-inflammatory activity was observed in fractions 5 to 7 shown in Fig. 10. Also, when compounds were estimated based on the accurate masses obtained by LCMS analysis, urea, formate, 5-hydroxyorotic acid were found in fraction 5, 5-methylcytidine, 2-methylcytidine, benserazide were found in fraction 6, and Lys-Lys, lysine anhydride, Lys-His (or His-Lys), cadralazine were suggested to be contained in fraction 7.

[0061] Next, the gel filtration active fraction II obtained by separating and purifying the hot water extract of skipjack bonito flakes by gel filtration chromatography was further separated and purified using reversed-phase HPLC, and the anti-inflammatory effects of the obtained fractions were verified using MG6 cells.

[0062] As separation conditions, Inertsil ODS-3, 5 μm, 10×250 mm (GL Science, C / N 5020-06812, S / N 0BI41240) was used for the preparative reverse-phase column, mobile phase A: 0.1% TFA, mobile phase B: 80% acetonitrile - 0.1% TFA, and a linear concentration gradient of acetonitrile was applied to elute the sample at a flow rate of 3 mL / min, and the absorbance (214 nm) was measured with a UV detector. The separated eluate was fractionated so that it was 1 fraction per minute (3 mL / fraction). Next, each of the obtained fractions was dried under reduced pressure and redissolved in ultrapure water. Fractions with a recovered solid content weight of 5 mg or more were redissolved to 100 mg / mL, and fractions less than 5 mg were uniformly redissolved in 50 μL of ultrapure water and then filter sterilized using a 0.2 μm membrane filter. The anti-inflammatory effects of each fraction prepared in this way were evaluated using MG6 cells.

[0063] As a result, anti-inflammatory activities were observed in fraction 7 and fraction 17 shown in Fig. 11. Also, when compounds were estimated based on the accurate masses obtained by LCMS analysis, it was suggested that fraction 7 might contain creatine, creatinine, glycolic acid, lactic acid, and fraction 17 might contain inosinic acid, AMP, succinic acid, ribose-5-phosphate, hypoxanthine.

[0064] In order to attempt the isolation and identification of substances showing anti-inflammatory effects, the anti-inflammatory effects of 4 components (creatine, creatinine, glycolic acid, lactic acid) detected in a large amount from fraction 7 were evaluated for each component alone.

[0065] Among the four components (creatine, creatinine, glycolic acid, lactic acid) that were highly contained in Fraction 7, anti-inflammatory activity was observed in three components (creatinine, glycolic acid, lactic acid) (see Figure 12). Among these, anti-inflammatory activity of lactic acid has already been reported (Liang et al. L-lactate inhibits lipopolysaccharide-induced inflammation of microglia in the hippocampus, International Journal of Neuroscience, 2022 Jul 26;1-8), while the other two components (creatinine, glycolic acid) were novel anti-inflammatory components.

[0066] Next, for the five components (inosinic acid, AMP, succinic acid, ribose-5-phosphate, hypoxanthine) that were detected in large amounts from Fraction 17, the anti-inflammatory effect of each component alone was evaluated.

[0067] As a result, anti-inflammatory activity was observed for each of the components of inosinic acid, AMP, succinic acid, ribose-5-phosphate, and hypoxanthine (see Figure 13).

[0068] That is, it was suggested that the seven components of creatinine, glycolic acid, inosinic acid, AMP, succinic acid, ribose-5-phosphate, and hypoxanthine might be novel anti-inflammatory components contained in the hot water extract of skipjack tuna flakes.

[0069] Finally, for the gel filtration active fraction III obtained by separating and purifying the hot water extract of skipjack tuna flakes by gel filtration chromatography, further separation and purification were performed using reverse phase HPLC, and the anti-inflammatory effect of the obtained fraction was verified using MG6 cells.

[0070] As separation conditions, an Inertsil ODS-3, 5 μm, 10 × 250 mm (GL Science, C / N 5020-06812, S / N 0BI41240) reverse-phase column for fractionation was used. Mobile phase A was 0.1% TFA, and mobile phase B was 80% acetonitrile - 0.1% TFA. A linear concentration gradient of acetonitrile was applied, and the sample was eluted at a flow rate of 3 mL / min. The absorbance (214 nm) was measured with a UV detector. The separated eluate was fractionated so that each fraction was 1 minute (3 mL / fraction). Next, each obtained fraction was dried under reduced pressure and redissolved in ultrapure water. Fractions with a recovered solid content weight of 5 mg or more were redissolved to 100 mg / mL, and fractions less than 5 mg were uniformly redissolved in 50 μL of ultrapure water and then sterilized by filtration using a 0.2 μm membrane filter. The anti-inflammatory effects of each fraction prepared in this way were evaluated using MG6 cells.

[0071] As a result, extremely high anti-inflammatory activity was observed in fraction 20 shown in Fig. 14. Also, when compounds were estimated based on the accurate masses obtained by LCMS analysis, it was suggested that fraction 20 might contain inosine and arabinosylhypoxanthine.

[0072] [Experiment 10] To examine the effect of the hot water extract of skipjack dried bonito on the BBB function, an in vitro experiment was conducted using the protein expression of claudin 5 and occludin, which are tight junction-related proteins in rat brain capillary endothelial cells, as indicators. Claudin 5 and occludin, which form the tight junction that is the barrier between vascular endothelial cells, are often used as markers for evaluating the BBB function (see Brain Circulation and Metabolism 24: 111-115, 2013).

[0073] · Protein expression of tight junction-related molecules in rat brain capillary endothelial cells (RBECs, primary cells) (see Fig. 15) Rat brain capillary endothelial cells (RBECs, primary cells) and culture medium were purchased from PharmacoCell, and the effects of the hot water extract of skipjack bonito on the protein expression of tight junction-related molecules (claudin 5, occludin) were verified.

[0074] First, RBECs were seeded in a 48-well plate (2×10 5 cells / well·440 μL), and culture was started in a CO2 incubator (37 °C, CO2 concentration 5%). Next, after 72 hours, the medium in each well was replaced with the evaluation medium prepared by adding various extracts. Then, 24 hours and 72 hours later, using the Nucleospin RNA / Protein kit (Takara Bio Inc.), RNA and protein were recovered from the cultured cells according to the protocol. The extracted protein was evaluated by Western Blot, and the DNA obtained by reverse transcription of the extracted RNA was evaluated by real-time PCR.

[0075] As a result, the protein expression of claudin 5 was enhanced at 24 hours and 72 hours compared with the serum-free medium test group (serum free), and the expression of occludin was also enhanced, especially at 24 hours, as if it interacted with claudin 5 [EXP 1]. In addition, when the same verification was carried out for RBECs (total passage number 5) that had been passaged two more times, it was confirmed that the protein expression of claudin 5 was enhanced compared with the serum-free medium test group [EXP 2].

[0076] · Acetylcholine synthase in the mouse brain (see Figure 16) Mice were orally administered with a 10 mg / mL hot water extract of skipjack bonito, and then the protein level expression of acetylcholine synthase (ChAT) was verified from the samples prepared from the whole brain (control group; 3 mice, skipjack bonito hot water extract administration group; 5 mice).

[0077] As a result, enhanced ChAT protein expression was observed in the preparations extracted from the whole brain, suggesting that the production of acetylcholine in nerve cells in the brain was enhanced.

[0078] From these results, it was revealed that the hot water extract of skipjack katsuobushi enhanced the protein expression of tight junction-related molecules (claudin 5, occludin) in brain capillary endothelial cells in in vitro experiments.

[0079] [Experiment 11] Regarding the anti-inflammatory highly active fraction obtained by sequentially separating and purifying the hot water extract of skipjack katsuobushi using gel filtration chromatography and reverse-phase HPLC, the effect on the protein expression of tight junction-related molecules (claudin 5, occludin) in rat brain capillary endothelial cells (RBECs, primary cells) was verified (see Figure 17).

[0080] Rat brain capillary endothelial cells (RBECs, primary cells) and culture media were purchased from PharMacell Co., Ltd., and the effect of the hot water extract of skipjack katsuobushi on the protein expression of tight junction-related molecules (claudin 5, occludin) was verified. First, RBECs were seeded in a 48-well plate (2×10 5 cells / well·440 μL), and culture was started in a CO2 incubator (37°C, CO2 concentration 5%). Next, after 72 hours, the medium in each well was replaced with the evaluation medium prepared by adding various extracts, and after another 24 hours and 72 hours, RNA and Protein were recovered from the cultured cells according to the protocol using the Nucleospin RNA / Protein kit (Takara Bio Inc.). The extracted Protein was evaluated by Western Blot, and the DNA obtained by reverse-transcribing the extracted RNA was evaluated by real-time PCR (real-time PCR data are not shown here).

[0081] As a result, when the addition concentration of the active fraction was 0.1 mg / mL, after 24 hours, in samples 4, 5, and 6 (corresponding to fraction 20 in Fig. 14, and fractions 5 and 6 in Fig. 10, respectively), and further after 72 hours, in samples 7 and 8 (corresponding to fraction 7 in Fig. 10 and fraction 7 in Fig. 11, respectively), the protein expression of claudin 5 was enhanced compared to the serum-free medium test group. Also, when the addition concentration of the active fraction was 1.0 mg / mL, after 24 hours, in sample 5 (corresponding to fraction 5 in Fig. 10) and sample 8 (corresponding to fraction 7 in Fig. 11), and after 72 hours, in sample 8 (corresponding to fraction 7 in Fig. 11), the protein expression of claudin 5 was enhanced compared to the serum-free medium test group.

[0082] From these results, it was clarified that the anti-inflammatory highly active fraction obtained by sequentially separating and purifying the hot water extract of dried skipjack tuna using gel filtration chromatography and reverse-phase HPLC enhanced the protein expression of tight junction-related molecules (claudin 5, occludin) in rat brain capillary endothelial cells (RBECs, primary cells).

[0083] [Experiment 12] Regarding the compounds (inosinic acid, histidine) estimated from the accurate mass numbers obtained by LCMS analysis of the components contained in the anti-inflammatory highly active fraction obtained by sequentially separating and purifying the hot water extract of dried skipjack tuna using gel filtration chromatography and reverse-phase HPLC, and Dashi-presso (a product of Katsuo Dashi manufactured by Maruhachi Muramatsu Co., Ltd.), the effects on the protein expression of tight junction-related molecules (claudin 5, occludin) in rat brain capillary endothelial cells (RBECs, primary cells) were verified (see Fig. 18).

[0084] Rat brain capillary endothelial cells (RBECs, primary cells) and culture media, etc. were purchased from Pharmakosel Co., Ltd., and the effects of the hot water extract of dried skipjack tuna on the protein expression of tight junction-related molecules (claudin 5, occludin) were verified. First, RBECs were seeded in a 48-well plate (2×10 5Cells / well (440 μL) are added and culturing is initiated in a CO₂ incubator (37 °C, 5% CO₂ concentration). Next, after 72 hours, the medium in each well is replaced with the evaluation medium prepared by adding each component and the extract. Then, after another 24 hours and 72 hours, RNA and protein are recovered from the cultured cells according to the protocol using the Nucleospin RNA / Protein kit (Takara Bio Inc.). The extracted protein is evaluated by Western Blot, and the DNA obtained by reverse transcription of the extracted RNA is evaluated by real-time PCR (real-time PCR data is not shown here).

[0085] As a result, after 24 hours, the protein expression of claudin 5 was enhanced in the serum-free medium test group compared to the inosine (added concentration 0.0537 mg / mL) and histidine (added concentration 0.836 mg / mL). Also, in the case of Dashi-presso (added concentration 0.1 mg / mL), after 72 hours, the protein expression of claudin 5 was enhanced compared to the serum-free medium test group.

[0086] From these results, it was revealed that the protein expression of tight junction-related molecules (claudin 5, occludin) in rat brain capillary endothelial cells (RBECs, primary cells) was enhanced for the components (inosine, histidine) that were predicted to be contained in the anti-inflammatory highly active fraction obtained by sequentially separating and purifying the hot water extract of dried skipjack tuna using gel filtration chromatography and reverse-phase HPLC.

[0087] [Experiment 13] The blood-brain barrier was physically damaged, and the cerebrovascular permeability was evaluated by the Evans blue method (Evans blue; EB) when the hot water extract of dried skipjack tuna was orally administered before and after the damage (see Figure 19).

[0088] Specifically, first, mice were orally administered with 10 mg / mL of hot water extract of skipjack bonito flakes for 3 days. On the 4th day, while continuing oral administration, a cooled metal rod (diameter 3 mm) was brought into contact with the right parietal skull for 5 seconds to directly damage the blood-brain barrier. 24 hours after the cryogenic injury, 3% EB was administered. 3 - 4 hours later, the right hemisphere of the brain was sliced into 3-mm thick sections and immersed in 800 μL of formaldehyde at 50 °C for 3 days. Then, the absorbance at 634 nm was measured.

[0089] As a result, in the group administered with the hot water extract of skipjack bonito flakes, the amount of EB leaked into the brain was clearly less compared to the non-administered group. Thus, it was revealed that the hot water extract of skipjack bonito flakes has a high effect of maintaining and improving the function of the blood-brain barrier.

[0090] [Experiment 14] The effects on the acetylcholine production ability in the heart and changes in hemodynamics were examined.

[0091] When mice were orally administered with 10 mg / mL of the hot water extract of skipjack bonito flakes for 2 weeks, it was found that the acetylcholine concentration in the heart was clearly increased compared to the water-administered group (see Fig. 20A), and the expression at the protein level of acetylcholine synthase (ChAT) in the brain in addition to the heart was enhanced (see Fig. 16).

[0092] The effects on blood pressure and heart rate were examined when mice were orally administered with the hot water extract of skipjack bonito flakes for 1 week and 2 weeks. It was found that at the 2nd week compared to the 1st week, the heart rate (HR) was significantly decreased compared to the water-administered group, and there was also a slightly decreasing trend in systolic blood pressure (SBP) and diastolic blood pressure (DBP) at the 2nd week. That is, it was suggested that the hot water extract of skipjack bonito flakes enhances the parasympathetic nervous system of mice and also enhances the acetylcholine production ability of the heart (see Fig. 20B).

[0093] [Experiment 15] A forced swimming test (FST) was conducted using mice orally administered with 10 mg / mL of the hot water extract of skipjack bonito flakes to examine the inhibitory effect on depressive-like behavior (see Fig. 21).

[0094] Specifically, using mice orally administered with the hot water extract of katsuobushi for 1 to 5 days, they were placed in a water-filled aquarium and forced to swim, and the length of time the mice were in a motionless state was measured during the last 4 minutes of a 10-minute observation period. It is considered that a longer immobile time indicates a stronger depressive state, and a shorter immobile time indicates an antidepressant effect.

[0095] As a result, it was revealed that in two experiments with different oral administration periods of the hot water extract of katsuobushi, it played a role related to the antidepressant effect.

[0096] [Experiment 16] A tail suspension test (TST) was conducted using mice orally administered with the hot water extract of katsuobushi at 10 mg / mL to examine the inhibitory effect on depressive-like behavior (see Fig. 22).

[0097] Specifically, using mice orally administered with the hot water extract of katsuobushi for 1 to 2 days, their tails were fixed and they were hung upside down, and the length of time the mice were in a motionless state was measured during a 10-minute observation period. It is considered that a longer immobile time indicates a stronger depressive state, and a shorter immobile time indicates an antidepressant effect.

[0098] As a result, it was revealed that in two experiments with different oral administration periods of the hot water extract of katsuobushi, it played a role related to the antidepressant effect.

[0099] [Experiment 17] Utilizing the property that mice prefer novelty, a novel object exploration test, which is a method for evaluating visual cognitive memory shown in Fig. 23, was conducted to compare the effects on the recognition memory of novel objects between two groups: a group not administered with the hot water extract of katsuobushi and a group administered with it.

[0100] First, a mouse was placed in an experimental apparatus (a cylindrical tube with a diameter of approximately 50 cm) without placing an object (the target object) and allowed to get used to the environment for 10 minutes (Habituation). Then, the mouse was allowed to freely explore for 10 minutes in the experimental apparatus with two identical objects placed in it (Training; training trial). After that, one of the objects was replaced with a novel object and the mouse was allowed to freely explore for 10 minutes (Retention; retention trial). The movement of the mouse was recorded from above by the installed camera. In the training trial and the retention trial, the exploration time for each of the two objects and the total exploration time were measured. In the training trial, the ratio (%) of the exploration time for any one object to the total exploration time was calculated, and in the retention trial, the ratio (%) of the exploration time for the novel object to the total exploration time was calculated as the exploration preference, and the latter was used as an index of visual cognitive memory.

[0101] As a result, there was almost no difference in the total moving distance on the 3rd day between the non-administered group and the administered group of the hot water extract of skipjack bonito flakes at 10 mg / mL. However, a significant increase was observed in the time the mouse invaded the area within the experimental apparatus defined as the central part, the staying time, and the moving distance in the central part in the administered group of the hot water extract of skipjack bonito flakes. Furthermore, in the non-administered group of the hot water extract of skipjack bonito flakes (control; water), the time until the mouse performed exploratory behavior (exploration latency) was longer for the novel object than for the known object, whereas there was almost no difference in the exploration latency for the novel object and the known object in the administered group of the hot water extract of skipjack bonito flakes (see Figure 24).

[0102] From these results, it became clear that by ingesting the hot water extract of skipjack bonito flakes, the fear was reduced, and thus the mouse tended to explore both novel and known objects without discrimination. That is, it was considered that the hot water extract of skipjack bonito flakes might have an effect of alleviating the fear of novel objects.

Claims

1. A functional food for preventing or improving mental and neurological disorders, comprising a bonito-derived extract.

2. 2. A functional food according to claim 1, wherein the psychiatric and neurological disorder is caused by inflammation in the brain.

3. 3. A functional food according to claim 2, having an anti-inflammatory effect.

4. 4. The functional food according to claim 3, wherein the anti-inflammatory effect is inhibition of inflammatory cytokine production and / or inhibition of microglial activation in the brain.

5. The functional food of claim 3, wherein the bonito-derived extract reduces the production of inflammatory cytokines compared to the amounts of DHA, EPA, and compositions of equivalent concentrations of DHA and EPA contained in the bonito-derived extract.

6. 4. A functional food according to claim 3, wherein the bonito-derived extract reduces the production of inflammatory cytokines compared to the amounts of histidine, anserine, creatine, creatinine, betaine, carnosine, inosinic acid contained in the bonito-derived extract, and compositions containing the same concentrations of histidine, anserine, creatine, creatinine, betaine and carnosine.

7. 2. The functional food according to claim 1, wherein the psychiatric and neurological disorder is caused by breakdown of the blood-brain barrier.

8. 8. A functional food according to claim 7, which has an effect of improving the blood-brain barrier property.

9. 9. The functional food according to claim 8, wherein the active ingredients in improving the blood-brain barrier property are histidine and inosinic acid.

10. 8. A functional food according to claim 7, which has the effect of activating the cardiac acetylcholine production system.

11. 2. The functional food according to claim 1, wherein the concentration of the bonito-derived extract is 0.1 mg / mL.

12. 10. The functional food according to claim 9, wherein the concentration of the bonito-derived extract is 0.1 mg / mL, or the histidine concentration is 0.836 mg / mL, and the inosinic acid concentration is 0.0537 mg / mL.

13. 11. The functional food according to claim 10, wherein the concentration of the bonito-derived extract is 10 mg / mL.

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