WO2019131136A1 - Brain protective agent - Google Patents

Abstract

The purpose of the present invention is to provide a medicine for effectively protecting brain tissues from inflammatory reactions induced by cerebral ischemia. The brain protective agent according to the present invention is characterized by comprising a specific carotenoid compound as an active ingredient.

Description

Brain protective agent

The present invention relates to a drug for effectively protecting brain tissue from an inflammatory reaction caused by cerebral ischemia.

Stroke (cerebrovascular disorder) is mainly classified into cerebral infarction, cerebral hemorrhage and subarachnoid hemorrhage. In cerebral infarction, cerebral blood flow is insufficient due to blockage or thinning of cerebral blood vessels due to arteriosclerosis in cerebral blood vessels and thrombus made of blood vessels other than the brain being transported to the brain. Hematological disorder is a disease that causes necrosis. In addition, intracerebral hemorrhage bleeds in the brain due to high blood pressure, aging, etc., and a blood clot compresses brain cells. Subarachnoid hemorrhage is the occurrence of hemorrhage in the subarachnoid space due to rupture of the cob in the cerebral artery. In any case, it is said that the necrotic brain tissue is not restored, and even if life is saved, not only motor paralysis, sensory disorders, speech disorders but also dementia symptoms often remain. On the other hand, in recent years, diseases called lifestyle-related diseases such as high blood pressure, heart disease, hyperlipidemia and diabetes are increasing, and the risk of stroke is increasing accordingly. In Japan, cerebrovascular disease is the third leading cause of death next to cancer and ischemic heart disease, and this is also the case in advanced Western countries. Thus, an effective means of treating stroke is needed.

However, so far no truly effective treatment for stroke has yet been found. For example, thrombolytic agents such as tissue-type plasminogen activator have been used to lyse the thrombus that caused the cerebral infarction and resume blood flow. However, disorders caused by free radicals caused by blood flow resumption are also deeply involved in the post-ischemic condition, and thrombolytic agents alone do not provide a fundamental solution for brain tissue necrosis.

Recently, it has become clear that extracellular zinc ions, which increase in the hippocampus during reperfusion following cerebral ischemia, activate zinc signals in microglia, which are central nervous system glial cells, and cause an excessive inflammatory response. Excessive inflammatory responses derived from microglia are known to impair cognitive function, and in particular the hippocampus of the limbic system involved in cognitive function is known as a brain region vulnerable to the inflammatory response. However, there are no agents that target zinc signaling, and such agents may be brain protective agents from new approaches.

In Japan, edaravone, which is a radical scavenger, is the only drug approved as a drug for improving the functional disorder associated with the acute phase of cerebral infarction. However, edaravone has side effects such as hepatic dysfunction and renal dysfunction, and there are also data that abnormal changes in clinical laboratory test values, such as abnormal liver function test values, reach as much as 21.4% of the patients who receive it. No matter how lethal the treatment is for cerebral infarction, which is a fatal disease, such high side effect rate is problematic.

By the way, the research group of the present inventors has found that peridinin, which is a natural marine carotenoid, exhibits an excellent inhibitory effect on delayed allergy, and has filed a patent application (Patent Document 1).

Patent No. 6183746

As mentioned above, despite the recent increase in stroke problems, only edaravone has been approved in Japan. On the other hand, elucidation of the mechanism leading to cerebral sequelae from cerebral ischemia is progressing, such as excessive inflammatory reaction of microglia caused by zinc ion increased during reperfusion following cerebral ischemia seriously affects cerebral sequelae. It is.
Therefore, an object of the present invention is to provide a drug for effectively protecting brain tissue from an inflammatory reaction caused by cerebral ischemia.

The present inventors have intensively studied to solve the above problems. As a result, they have found that certain carotenoid compounds can suppress the excessive release of inflammatory cytokines after reperfusion following cerebral ischemia and can reduce the excessive inflammatory response, thereby completing the present invention.
Hereinafter, the present invention is described.

［式中、
　Ｒ¹～Ｒ³は、独立して水素原子またはＣ_1-18アルカノイル基を示し、
　Ｒ⁴～Ｒ¹⁷は、独立して水素原子またはＣ_1-6アルキル基を示し、但し、ＲⁿとＲⁿ⁺²（ｎは、４以上、１５以下の整数を示す）は、結合してエステル基を形成していてもよく、
　Ｘは、－ＣＨ₂－Ｃ（＝Ｏ）－基または単結合を示す。］
　［２］　Ｒ¹が水素原子である上記［１］に記載の脳保護剤。
　［３］　Ｒ²が水素原子である上記［１］または［２］に記載の脳保護剤。
　［４］　Ｒ³がアセチル基である上記［１］～［３］のいずれかに記載の脳保護剤。
　［５］　上記式（Ｉ）で表されるカロテノイド化合物がペリジニンである上記［１］～［４］のいずれかに記載の脳保護剤。
　［６］　上記式（Ｉ）で表されるカロテノイド化合物がフコキサンチンである上記［１］～［４］のいずれかに記載の脳保護剤。
　［７］　脳虚血後の炎症反応を抑制する上記［１］～［６］のいずれかに記載の脳保護剤。
　［８］　脳虚血から脳組織を保護するための、上記式（Ｉ）で表されるカロテノイド化合物の使用。
　［９］　Ｒ¹が水素原子である上記［８］に記載の使用。
　［１０］　Ｒ²が水素原子である上記［８］または［９］に記載の使用。
　［１１］　Ｒ³がアセチル基である上記［８］～［１０］のいずれかに記載の使用。
　［１２］　上記式（Ｉ）で表されるカロテノイド化合物がペリジニンである上記［８］～［１１］のいずれかに記載の使用。
　［１３］　上記式（Ｉ）で表されるカロテノイド化合物がフコキサンチンである上記［８］～［１１］のいずれかに記載の使用。
　［１４］　脳虚血後の炎症反応を抑制するための上記［８］～［１３］のいずれかに記載の使用。
　［１５］　脳虚血から脳組織を保護するための方法であって、上記式（Ｉ）で表されるカロテノイド化合物を脳虚血患者に投与する工程を含むことを特徴とする方法。
　［１６］　Ｒ¹が水素原子である上記［１５］に記載の方法。
　［１７］　Ｒ²が水素原子である上記［１５］または［１６］に記載の方法。
　［１８］　Ｒ³がアセチル基である上記［１５］～［１７］のいずれかに記載の方法。
　［１９］　上記式（Ｉ）で表されるカロテノイド化合物がペリジニンである上記［１５］～［１８］のいずれかに記載の方法。
　［２０］　上記式（Ｉ）で表されるカロテノイド化合物がフコキサンチンである上記［１５］～［１８］のいずれかに記載の方法。
　［２１］　脳虚血後の炎症反応を抑制するための上記［１５］～［２０］のいずれかに記載の方法。 [1] A brain protective agent comprising a carotenoid compound represented by the following formula (I) as an active ingredient.

[In the formula,
R ¹ to R ³ independently represent a hydrogen atom or a C _1-18 alkanoyl group,
R ⁴ to R ¹⁷ independently represent a hydrogen atom or a C _1-6 alkyl group, provided that R ⁿ and R ^{n + 2} (n is an integer of 4 or more and 15 or less) are bonded to each other May form an ester group,
X represents a —CH ₂ —C (= O) — group or a single bond. ]
[2] The brain protective agent as described in the above [1], wherein R ¹ is a hydrogen atom.
[3] The brain protective agent according to the above-mentioned [1] or [2], wherein R ² is a hydrogen atom.
[4] The brain protective agent according to any one of the above-mentioned [1] to [3], wherein R ³ is an acetyl group.
[5] The brain protective agent according to any one of the above [1] to [4], wherein the carotenoid compound represented by the above formula (I) is peridinin.
[6] The brain protective agent according to any one of the above [1] to [4], wherein the carotenoid compound represented by the above formula (I) is fucoxanthin.
[7] The brain protective agent according to any one of the above-mentioned [1] to [6] which suppresses an inflammatory reaction after cerebral ischemia.
[8] Use of the carotenoid compound represented by the above formula (I) for protecting brain tissue from cerebral ischemia.
[9] The use according to the above-mentioned [8], wherein R ¹ is a hydrogen atom.
[10] The use according to the above-mentioned [8] or [9], wherein R ² is a hydrogen atom.
[11] The use according to any one of the above-mentioned [8] to [10], wherein R ³ is an acetyl group.
[12] The use according to any one of the above-mentioned [8] to [11], wherein the carotenoid compound represented by the above-mentioned formula (I) is peridinin.
[13] The use according to any one of the above-mentioned [8] to [11], wherein the carotenoid compound represented by the above-mentioned formula (I) is fucoxanthin.
[14] The use according to any of the above-mentioned [8] to [13] for suppressing an inflammatory reaction after cerebral ischemia.
[15] A method for protecting brain tissue from cerebral ischemia, comprising the step of administering the carotenoid compound represented by the above-mentioned formula (I) to a cerebral ischemia patient.
[16] The method according to the above-mentioned [15], wherein R ¹ is a hydrogen atom.
[17] The method according to the above-mentioned [15] or [16], wherein R ² is a hydrogen atom.
[18] The method according to any one of the above-mentioned [15] to [17], wherein R ³ is an acetyl group.
[19] The method according to any one of the above-mentioned [15]-[18], wherein the carotenoid compound represented by the above-mentioned formula (I) is peridinin.
[20] The method according to any one of the above-mentioned [15] to [18], wherein the carotenoid compound represented by the above-mentioned formula (I) is fucoxanthin.
[21] The method according to any one of the above [15] to [20] for suppressing an inflammatory reaction after cerebral ischemia.

The brain protective agent according to the present invention can protect brain tissue by suppressing excessive release of inflammatory cytokines after reperfusion following cerebral ischemia and reducing excessive inflammatory reaction that damages brain tissue. Such action mechanism is not found in conventional brain protective agents. Also, although it is an approved drug, its active ingredient is a carotenoid compound as compared with edaravone according to the present invention, as compared with edaravone, which has a short half-life due to a radical scavenger, and a severe action and a serious side effect. It can be said that there is little safety. Therefore, the brain protective agent according to the present invention is very useful as one that can reduce the sequelae after stroke.

FIG. 1 is a graph of in vitro experimental results showing the suppressive effect of peridinin according to the present invention on the excessive release of inflammatory cytokines by microglia caused by zinc ions. FIG. 2 is a graph of in vivo experimental results showing the suppressive effect of peridinin according to the present invention on the excessive release of inflammatory cytokines in the hippocampus caused by zinc ions. FIG. 3 is a photograph and a graph of in vitro experimental results showing the inhibitory effect of peridinin according to the present invention on the generation of active oxygen in microglia caused by zinc ions. FIG. 4 is a photograph and graph showing the anti-inflammatory effect of peridinin on activation of hippocampal microglia to type M1 after reperfusion following cerebral ischemia. FIG. 5 is a graph showing the results of a Y-maze test to determine whether pre-administration of peridinin has a protective effect on short-term spatial recognition memory loss.

The brain protective agent according to the present invention contains a carotenoid compound represented by the above formula (I) (hereinafter sometimes abbreviated as "carotenoid compound (I)") as an active ingredient. In the above-mentioned formula (I) and the following chemical structural formulas, “= · =” means “= C =”.

The “C _1-18 alkanoyl group” which is R ¹ to R ³ in the above formula (I) refers to a formyl or C _1-17 alkyl-carbonyl group, wherein “C _1-17 alkyl group” is carbon number 1 or more and 17 or less linear or branched monovalent saturated hydrocarbon groups. As C _1-18 alkanoyl group, in addition to formyl, for example, acetyl, ethyl carbonyl, n-propyl carbonyl, isopropyl carbonyl, n-butyl carbonyl, pivaloyl, n-hexyl carbonyl, n-octyl carbonyl, n-nonyl carbonyl, 6-methyloctyl carbonyl, 7-methyloctyl carbonyl, n-decyl carbonyl, 8-methyl nonyl carbonyl, n-undecyl carbonyl, 9-methyl decyl carbonyl, n-dodecyl carbonyl, 10-methyl undecyl carbonyl, n-tri Examples include decylcarbonyl, 11-methyldodecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl and the like. As the C _1-18 alkanoyl group, a C _1-8 alkanoyl group or a C _9-18 alkanoyl group is preferable, a C _1-6 alkanoyl group is more preferable, a C _1-4 alkanoyl group is more preferable, and a C _1-2 alkanoyl group is more preferable. Groups are more preferred, with acetyl being particularly preferred.

The “C _1-6 alkyl group” which is R ⁴ to R ¹⁷ in the above formula (I) refers to a linear or branched monovalent saturated hydrocarbon group having 1 to 6 carbon atoms. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, n-hexyl and the like can be mentioned, preferably a C _1-4 alkyl group More preferably, it is a C _1-2 alkyl group, and most preferably methyl.

In R ⁴ to R ¹⁷ of the above formula (I), an ester group which may be formed by combining R ⁿ and R ^{n + 2} (n is an integer of 4 or more and 15 or less) is —C (C) = O) -O- group or -O-C (= O)-group, preferably -C (= O) -O- group.

The following carotenoid compounds (II) can be mentioned as carotenoid compound (I).

［式中、Ｒ¹～Ｒ³は上記と同義を示す。］

Wherein R ¹ to R ³ are as defined above. ]

Among carotenoid compounds (I), peridinin is a hydrogen atom in which R ¹ and R ² are hydrogen, R ³ is acetyl, R ⁴ , R ⁵ , R ⁷ , R ⁹ and R ¹¹ to R ¹⁶ are hydrogen atoms, R ¹⁰ and R ¹⁷ Is methyl and X is a single bond.

Symbiodinium sp. Which is a dinoflagellate. Peridinin, a natural carotenoid compound isolated from OTCL2A strain and the like, has the following steric structure.

Among the carotenoid compounds (I), fucoxanthin is such that R ¹ and R ² are hydrogen atoms, R ³ is acetyl, R ⁴ , R ⁸ , R ¹³ and R ¹⁷ are methyl, R ⁵ to R ⁷ , R ⁹ to R ¹² And R ¹⁴ to R ¹⁶ is a hydrogen atom, and X is a —CH ₂ —C (= O) — group.

Fucoxanthin, which is a naturally occurring carotenoid compound mainly isolated from non-uniform algae such as brown algae, has the following three-dimensional structure.

The carotenoid compound (I) used in the present invention can be isolated and purified from algae since it is mainly a natural marine carotenoid. For example, the above peridinin is Symbiodinium sp. It can be isolated and purified from dinoflagellate such as OTCL2A strain. Hereinafter, although the isolation method of peridinin is demonstrated as an example, other carotenoid compound (I) can also be isolated by the same method from algae.

Among the dinoflagellate producing peridinin, Symbiodinium sp. Are symbiotic with symbiotic relationships with marine invertebrates. As marine animals that are hosts, there can be mentioned foraminifers, radiolarians, flatworms angut, jellyfish, corals, bivalves and the like. Thus, to isolate peridinin from nature, the dinoflagellate Symbiodinium sp. Is isolated from the marine animal and cultured, and the cultured dinoflagellate may be homogenized and then extracted. It is also possible to extract peridinin directly from marine animals that contain peridinin producing dinoflagellate. In the case of using marine animals, as in the case of using dinoflagellate, marine animals containing dinoflagellate may be homogenized and then extracted.

The extraction solvent may be selected as appropriate. For example, alcohol solvents such as methanol and ethanol; ketone solvents such as acetone and methyl ethyl ketone; halogenated hydrocarbon solvents such as dichloromethane and chloroform; aromatics such as benzene and toluene Hydrocarbon solvents; ester solvents such as methyl acetate and ethyl acetate; ether solvents such as diethyl ether and tetrahydrofuran; amide solvents such as dimethylformamide and dimethylacetamide; among these organic solvents having a concentration of 60 v / v% or more A mixed solvent of water miscible organic solvent and water can be mentioned.

The extraction conditions may be adjusted as appropriate. For example, the extraction can be performed at normal temperature, but may be heated to about 30 to 60 ° C. depending on the air temperature in order to enhance the extraction efficiency. Moreover, in order to raise extraction efficiency, it is preferable to stir at the time of extraction. Alternatively, the dinoflagellate may be ground in the extraction solvent after the extraction solvent is added to the dinoflagellate. The extraction time is not particularly limited, but can be, for example, about 1 hour or more and 10 days or less.

After the extraction operation, for example, by measuring the action of production of inflammatory cytokines in the same manner as in Examples described later or referring to physical property data of known compounds, etc., by chromatography such as thin layer chromatography, HPLC, preparative chromatography, etc. The active ingredient may be specified.

As described above, the carotenoid compound (I) may be isolated from algae, or may be produced by derivatizing the isolated compound. For example, a carotenoid compound (I) in which any one or more of R ¹ to R ³ is a C _1-18 alkanoyl group can be produced by esterifying a predetermined hydroxyl group. Such esterification can be carried out by a method known to those skilled in the art using an acid corresponding to a C _1-18 alkanoyl group, or an activated ester compound thereof or an acid chloride compound, and further, if necessary, a condensing agent. However, R ³ of peridinin and fucoxanthin which are natural compounds among the carotenoid compounds (I) is an acetyl group.

The brain protective agent according to the present invention can protect the brain by suppressing excessive inflammatory reaction caused by zinc ions after reperfusion following cerebral ischemia. Therefore, it may be administered prophylactically before cerebral ischemia, but if it is difficult, it is preferable to administer it after cerebral ischemia and further simultaneously with or after reperfusion treatment. According to the experimental findings by the present inventors, the brain protective agent according to the present invention can suppress active oxygen at the time of reperfusion following cerebral ischemia and M1 type microglia which causes excessive inflammatory reaction. it can. Examples of patients to which the brain protective agent according to the present invention should be administered include humans and animals other than humans.

There is no particular limitation on the dosage form or administration form of the brain protective agent according to the present invention, but in consideration of the emergency for cerebral ischemia, intravenous administration as an injection is preferable. In that case, as the solvent, an isotonic solution of plasma such as physiological saline or glucose aqueous solution whose pH has been adjusted can be used. Alternatively, when the carotenoid compound (I) is dried together with salts etc., pure water, distilled water, sterilized water etc. can also be used if the solution finally becomes isotonic or nearly isotonic with plasma. The concentration may also be that of a normal injection, and can be, for example, 0.05 mg / mL or more and 10 mg / mL or so.

The dosage of the brain protective agent of the present invention is not particularly limited, and may be appropriately adjusted according to the age, sex, weight, symptoms, severity, etc. of the patient. It can be about 0.5 mg / kg body weight or more and 100 mg / kg body weight or less. In addition, the administration time per one dose can be about 10 minutes or more and about 2 hours or less. The number of times of administration per day can be about 0.5 times or more and 5 times or less.

The present application claims the benefit of priority based on Japanese Patent Application No. 2017-248845 filed on Dec. 26, 2017. The entire content of the specification of Japanese Patent Application No. 2017-248845 filed on December 26, 2017 is incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be more specifically described by way of examples. However, the present invention is of course not limited by the following examples, and appropriate modifications may be made as long as the present invention can be applied to the purpose. Of course, implementation is also possible, and all of them are included in the technical scope of the present invention.

Example 1
(1) Isolation of peridinin The dinoflagellate Symbiodinium sp. From the red sea mussel Tridacna crocea. The OTCL2A strain was isolated and cultured. 100.2 g of frozen cells obtained from 156 L of culture solution are homogenized in 200 mL of 70% ethanol using a homogenizer ("Ultra-Turrax T25" made by Janke & Kunkel) and allowed to stand at 4 ° C for 3 days and then centrifuged separated. The supernatant was collected, and the above operation was repeated twice on the precipitate. All supernatants were combined and concentrated in vacuo. To the concentrate, 100 mL of water was added, and extracted three times with 150 mL of ethyl acetate. The extract was concentrated under reduced pressure to obtain 1094 mg of ethyl acetate fraction. 957.9 mg of the obtained ethyl acetate fraction was subjected to silica gel chromatography. In silica gel chromatography, 40 mL of Silica Gel 60 (manufactured by Nacalai Tesque, Inc.) is used, and dichloromethane: methanol = 99: 1 (80 mL) → 98: 2 (80 mL) → 96: 4 (120 mL) → 92: 8 (92 mL) as an eluent. 80 mL of the mixed solvent was used. 294.9 mg of the 313.5 mg fraction eluted with the eluent of dichloromethane: methanol = 96: 4, 20 mL of ODS silica gel ("Cosmosil 75C18-OPN" manufactured by Nacalai Tesque, Inc.), 60 mL of 80% methanol, subsequently 85% It was subjected to silica gel column chromatography using 140 mL of methanol. 169.1 mg of fraction eluted with 85% methanol was subjected to HPLC (column: “COSMOSIL 5C ₁₈ -AR-II” manufactured by Nacalai Tesque, 20 mmφ × 250 mm; eluent: 80% acetonitrile; flow rate: 6.0 mL / min) To give 31.7 mg of peridinin.

(2) Test for confirming the effect of peridinin on microglia Microglia were isolated from the brain of 1-day-old neonatal mouse C57BL / 6, and a medium for cell culture ("Eagle MEM" Fetal Bovine Serum produced by Nissui Pharmaceutical (Thermo) And the cells were cultured at 37.degree. C. for 2 weeks. The resulting microglia culture solution was added to a cell culture well plate at 300 μL per well (about 6.4 × 10 ⁵ cells / well). Furthermore, 50 μL of a 0.03-0.3 μg / mL aqueous solution of peridinin was added per well, and the mixture was incubated at 37 ° C. for 30 minutes. Then, 50 μL of 60 μM aqueous solution of zinc chloride was added per well, and incubated at 37 ° C. for 2 hours. After removing the culture solution and further washing the attached microglia with serum-free medium, 350 μL of fresh serum-free medium was added per well. The microglia were activated to type M1 by adding 50 μL of 1 ng / mL aqueous solution of lipopolysaccharide per well and incubating at 37 ° C. for 22 hours. The culture supernatant was collected, and the concentrations of inflammatory cytokines IL-1β, TNFα and IL-6 were measured by ELISA. In addition, for comparison, when neither zinc nor lipopolysaccharide is added (control), when adding only peridinin without adding peridinin, when adding only peridinin, when adding only lipopolysaccharide without adding peridinin, peridinin is added The same measurement was performed for the case where no addition was made. Each measurement was performed four times, and the average value was calculated. The results are shown in FIG.

(3) Discussion of Experimental Results As shown in FIG. 1, inflammatory cytokines were released from microglia activated to type M1 by lipopolysaccharide, and the concentration was obviously increased when zinc was added. However, when peridinin was added, the concentration of inflammatory cytokines was significantly reduced, and depending on the concentration of peridinin, it was reduced to almost the same level as when zinc was not added.
From these results, it has been proved that peridinin can suppress the excessive inflammatory reaction caused by the zinc signal generated in the brain of stroke patients.

Example 2
A 10 to 100 μg / mL aqueous solution of peridinin or 2 μL of a 0.1 mM aqueous solution of CaEDTA, which is a chelating agent, was administered to the intracerebroventricular zone of a male C57BL / 6N mouse weighing approximately 35 g and 12 weeks old. Within 10 minutes of administration, cerebral ischemia was achieved by occlusion of the bilateral common carotid arteries for 20 minutes, followed by reperfusion. Three days after cerebral ischemia treatment, total RNA was extracted from the hippocampus of each mouse, and gene expression levels of inflammatory cytokines TNFα, IL-6 and IL-1β were measured by real-time PCR. Each measurement was performed three times, and the average value was calculated. For comparison, measurements were performed in the same manner without cerebral ischemia treatment and without administration of peridinin or CaEDTA. The relative ratio, where the concentration of each inflammatory cytokine is 1 when no cerebral ischemia treatment is performed, is shown in FIG.
As shown in FIG. 2, administration of peridinin can suppress inflammatory cytokine production after stroke equivalent to or higher than that of the chelating agent CaEDTA, which is equivalent to the level without cerebral ischemic treatment. There was also an experimental example that could be suppressed up to
Thus, in vivo experiments also demonstrated that peridinin can suppress excessive inflammatory responses after stroke and protect brain tissue.

Example 3
It was tested whether peridinin could suppress the generation of reactive oxygen species (ROS) caused by zinc in microglia.
The microglia were precultured for 30 minutes in a medium containing or not containing 1 μM dihydroethidium (DHE) and 300 ng / mL of peridinin, and then cultured for 2 hours in the presence of 60 μM ZnCl ₂ . DHE indicates the amount of superoxide anion radical and is used to detect active oxygen. The microglial cells were observed under magnification with an all-in-one fluorescence microscope ("BZ-9000" manufactured by Keyence Corporation) to detect cells stained with DHE. The results are shown in FIGS. 3A and 3B. In addition, the result of FIG. 3B is an average value of 4 examples. In FIG. 3B, “*” indicates that there is a significant difference at p <0.05 with respect to the control, and “#” indicates that there is a significant difference at p <0.05 with respect to the ZnCl ₂ only treatment group. Indicates
As the results shown in FIGS. 3A and 3B, DHE staining was clearly increased in zinc-treated microglia, but such DHE staining was significantly reduced by peridinin.
Also, in the same manner as in Example 1 except that zinc was not used, the effect of peridinin on the active oxygen level of microglia was examined. The results are shown in FIGS. 3C and 3D. In addition, the result of FIG. 3D is an average value of 4 examples. In FIG. 3D, “*” indicates that there is a significant difference at p <0.05 with respect to the control, and “#” indicates that there is a significant difference at p <0.05 with respect to the ZnCl ₂ only treatment group. Indicates
As shown in the results shown in FIGS. 3C and 3D, in microglia treated with only peridinin, DHE-positive cells did not increase significantly relative to the control.

Example 4
In order to confirm the anti-inflammatory effect of peridinin on activation of hippocampal microglia to type M1 after reperfusion following cerebral ischemia, double immunostaining with the microglia marker Iba1 is used as a representative M1 marker The expression of certain CD16 / 32 was examined. The results are shown in FIGS. 4A and 4B. In addition, the result of FIG. 4B is an average value of six examples. In FIG. 4B, "*" indicates that there is a significant difference at p <0.05 with respect to the solvent-treated sham-operated mouse group, and "#" and "##" indicate that there is a difference with respect to the solvent-treated cerebral ischemia-operated mouse group. It shows that there is a significant difference at p <0.05.
As shown in FIG. 4, as compared with the sham-operated mice, the solvent-treated mice with cerebral ischemia have a clearly increased CD16 / 32 immune response in Iba1-positive cells in the hippocampus three days after the cerebral ischemia treatment. Was. On the other hand, CD16 / 32 immunoreactivity was reduced to low level in a dose-dependent manner in Iba1-positive cells in the hippocampus in mice treated with peridinin.
Thus, it became clear that peridinin can suppress the activation of hippocampal microglia to the M1 type after reperfusion following cerebral ischemia and reduce the excessive inflammatory response.

Example 5
To determine whether pre-administration of peridinin had a protective effect on short-term loss of spatial recognition memory, a Y-shaped maze test was performed 5 days after induction of cerebral ischemia.
Specifically, gray wood was used to form a Y-shaped maze having 40 × 2 × 3 cm identical three arms at 60 ° intervals. The mouse was placed at the end of either arm and allowed to move freely for 10 minutes and the entry to each arm was recorded with a digital video camera. If the mouse invades into an arm different from the two arms that entered the previous and previous times is defined as "alternate response", and if the mouse reenters any of the two arms that invaded the previous and previous rounds, "error" Defined as The percentage of alternation reaction was calculated by the following equation. The results are shown in FIG. In addition, the result of FIG. 5 is an average value of six examples. In FIG. 5, “*” indicates that there is a significant difference at p <0.05 with respect to the solvent-treated sham-operated mouse group.
[Number of alternation reactions / (total number of entry to arm-2)] x 100
As shown in FIG. 5, in the solvent-treated mice with cerebral ischemia, a significant reduction in spontaneous alternation was observed as compared to the sham-operated mice.
On the other hand, in mice pre-administered with peridinin, the decrease in spontaneous alternation induced by cerebral ischemia was clearly prevented.

Claims (21)

A brain protective agent comprising a carotenoid compound represented by the following formula (I) as an active ingredient.

[In the formula,
R ¹ to R ³ independently represent a hydrogen atom or a C _1-18 alkanoyl group,
R ⁴ to R ¹⁷ independently represent a hydrogen atom or a C _1-6 alkyl group, provided that R ⁿ and R ^{n + 2} (n is an integer of 4 or more and 15 or less) are bonded to each other May form an ester group,
X represents a —CH ₂ —C (= O) — group or a single bond. ] The brain protective agent according to claim ¹ , wherein R ¹ is a hydrogen atom. The brain protective agent according to claim 1 or 2, wherein R ² is a hydrogen atom. The brain protective agent according to any one of claims 1 to 3, wherein R ³ is an acetyl group. The brain protective agent according to any one of claims 1 to 4, wherein the carotenoid compound represented by the above formula (I) is peridinin. The brain protective agent according to any one of claims 1 to 4, wherein the carotenoid compound represented by the above formula (I) is fucoxanthin. The brain protective agent according to any one of claims 1 to 6, which suppresses an inflammatory response after cerebral ischemia. Use of a carotenoid compound represented by the following formula (I) for protecting brain tissue from cerebral ischemia.

[In the formula,
R ¹ to R ³ independently represent a hydrogen atom or a C _1-18 alkanoyl group,
R ⁴ to R ¹⁷ independently represent a hydrogen atom or a C _1-6 alkyl group, provided that R ⁿ and R ^{n + 2} (n is an integer of 4 or more and 15 or less) are bonded to each other May form an ester group,
X represents a —CH ₂ —C (= O) — group or a single bond. ] The use according to claim 8, wherein R ¹ is a hydrogen atom. The use according to claim 8 or 9, wherein R ² is a hydrogen atom. The use according to any one of claims 8 to 10, wherein R ³ is an acetyl group. The use according to any one of claims 8 to 11, wherein the carotenoid compound represented by the above formula (I) is peridinin. The use according to any one of claims 8 to 11, wherein the carotenoid compound represented by the above formula (I) is fucoxanthin. The use according to any of claims 8 to 13 for suppressing the inflammatory response after cerebral ischemia. A method for protecting brain tissue from cerebral ischemia, comprising the step of administering a carotenoid compound represented by the following formula (I) to a cerebral ischemia patient.

[In the formula,
R ¹ to R ³ independently represent a hydrogen atom or a C _1-18 alkanoyl group,
R ⁴ to R ¹⁷ independently represent a hydrogen atom or a C _1-6 alkyl group, provided that R ⁿ and R ^{n + 2} (n is an integer of 4 or more and 15 or less) are bonded to each other May form an ester group,
X represents a —CH ₂ —C (= O) — group or a single bond. ] The method according to claim 15, wherein R ¹ is a hydrogen atom. The method according to claim 15 or 16, wherein R ² is a hydrogen atom. The method according to any one of claims 15 to 17, wherein R ³ is an acetyl group. The method according to any one of claims 15 to 18, wherein the carotenoid compound represented by the above formula (I) is peridinin. The method according to any one of claims 15 to 18, wherein the carotenoid compound represented by the above formula (I) is fucoxanthin. The method according to any one of claims 15 to 20 for suppressing an inflammatory response after cerebral ischemia.

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