JP6966804B2 - Brain protectant - Google Patents

Description

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

Stroke (cerebrovascular accident) is mainly classified into cerebral infarction, cerebral hemorrhage and subarachnoid hemorrhage. Cerebral infarction is caused by arteriosclerosis in the cerebral blood vessels and the transport of blood clots formed in blood vessels other than the brain to the brain. A disease in which blood flow disorder tissue becomes necrotic. In addition, cerebral hemorrhage bleeds in the brain due to hypertension or aging, and a blood clot presses on brain cells. Subarachnoid hemorrhage is the rupture of a bump in the cerebral artery, causing bleeding in the subarachnoid space. In any case, it is said that the necrotic brain tissue cannot be restored, and even if the life is saved, not only motor paralysis, sensory disorder, and speech disorder but also dementia symptoms often remain. On the other hand, in recent years, diseases called lifestyle-related diseases such as hypertension, heart disease, hyperlipidemia, and diabetes have increased, and the risk of stroke has increased accordingly. Cerebrovascular accidents are the third leading cause of death in Japan after cancer and ischemic heart disease, and this is also the case in developed countries in Europe and the United States. Therefore, an effective stroke treatment method is eagerly desired.

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

In recent years, it has been revealed that extracellular zinc ions that 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 the hippocampus of the limbic system, which is particularly involved in cognitive function, is known as a brain region vulnerable to inflammatory responses. However, there are no drugs that target zinc signals, and such drugs may be brain protectants from new approaches.

In Japan, edaravone, a radical scavenger, is the only approved drug for improving dysfunction associated with the acute phase of cerebral infarction. However, edaravone has side effects such as hepatic dysfunction and renal dysfunction, and there is data that abnormal changes in clinical laboratory test values such as abnormal liver function test values amount to 21.4% of the treated patients. No matter how fatal the cerebral infarction is, there is a problem with such a high incidence of side effects.

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

Japanese Patent No. 6183746

As mentioned above, edaravone is the only drug approved in Japan, despite the increasing problem of stroke in recent years. On the other hand, the mechanism from cerebral ischemia to stroke sequelae has been elucidated, such as the excessive inflammatory reaction of microglia caused by zinc ions that increases during reperfusion following cerebral ischemia has a serious effect on stroke sequelae. I have.
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 conducted extensive research to solve the above problems. As a result, they have found that a specific carotenoid compound can suppress the excessive release of inflammatory cytokines after reperfusion following cerebral ischemia and reduce the excessive inflammatory reaction, and completed the present invention.
Hereinafter, the present invention will be shown.

［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 containing a carotenoid compound represented by the following formula (I) as an active ingredient.

[During the ceremony,
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, where R ⁿ and R ^{n + 2} (n represents an integer greater than or equal to 4 and less than or equal to 15) are bonded. It may form an ester group,
X indicates a −CH _2- C (= O) − group or a single bond. ]
[2] The brain protective agent according to the above [1], wherein ^{R 1 is a hydrogen atom.}
[3] The brain protective agent according to the above [1] or [2], wherein ^{R 2 is a hydrogen atom.}
[4] The brain protective agent according to any one of the above [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 [1] to [6], which suppresses an inflammatory reaction after cerebral ischemia.
[8] Use of a carotenoid compound represented by the above formula (I) to protect brain tissue from cerebral ischemia.
[9] The use according to the above [8], wherein ^{R 1 is a hydrogen atom.}
[10] The use according to the above [8] or [9], wherein ^{R 2 is a hydrogen atom.}
[11] The use according to any one of the above [8] to [10], wherein ^{R 3 is an acetyl group.}
[12] The use according to any one of the above [8] to [11], wherein the carotenoid compound represented by the above formula (I) is peridinin.
[13] The use according to any one of the above [8] to [11], wherein the carotenoid compound represented by the above formula (I) is fucoxanthin.
[14] The use according to any one of the above [8] to [13] for suppressing the inflammatory reaction after cerebral ischemia.
[15] A method for protecting brain tissue from cerebral ischemia, which comprises a step of administering a carotenoid compound represented by the above formula (I) to a cerebral ischemia patient.
[16] The method according to the above [15], wherein ^{R 1 is a hydrogen atom.}
[17] The method according to the above [15] or [16], wherein ^{R 2 is a hydrogen atom.}
[18] The method according to any one of the above [15] to [17], wherein ^{R 3 is an acetyl group.}
[19] The method according to any one of the above [15] to [18], wherein the carotenoid compound represented by the above formula (I) is peridinin.
[20] The method according to any one of the above [15] to [18], wherein the carotenoid compound represented by the above 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 the excessive release of inflammatory cytokines after reperfusion following cerebral ischemia and reducing the excessive inflammatory reaction that damages the brain tissue. Such a mechanism of action is not found in conventional brain protectants. In addition, the active ingredient of the brain protective agent according to the present invention is a carotenoid compound, which has side effects, as compared with edaravone, which is an approved drug but has a short half-life because it is a radical scavenger and has a strong action and has side effects. It can be said that there are few and high safety. Therefore, the cerebral protective agent according to the present invention is very useful as it can reduce the sequelae after stroke.

FIG. 1 is a graph of in vitro experimental results showing the inhibitory 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 inhibitory 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 the 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 confirm whether pre-administration of peridinin has a protective effect against the deletion of short-term spatial cognitive memory.

The brain protective agent according to the present invention contains a carotenoid compound represented by the above formula (I) (hereinafter, may be abbreviated as "carotenoid compound (I)") as an active ingredient. In addition, "= · =" in the above formula (I) and the following chemical structural formula 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, where the "C _1-17 alkyl group" has the number of carbon atoms. A linear or branched monovalent saturated hydrocarbon group of 1 or more and 17 or less. C _1-18 alkanoyl groups include, for example, acetyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, pivaloyl, n-hexylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl, in addition to formyl. 6-Methyloctylcarbonyl, 7-methyloctylcarbonyl, n-decylcarbonyl, 8-methylnonylcarbonyl, n-undecylcarbonyl, 9-methyldecylcarbonyl, n-dodecylcarbonyl, 10-methylundecylcarbonyl, n-tri Examples thereof 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 further preferable, and a C _1-2 alkanoyl group is more preferable. Groups are more preferred, and acetyls are particularly preferred.

The "C _1-6 ^{alkyl group" of R 4 to} R ¹⁷ of the above formula (I) refers to a linear or branched monovalent saturated hydrocarbon group having 1 or more carbon atoms and 6 or less 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, and a _{C 1-4 alkyl group is preferable.} , More preferably a C _1-2 alkyl group, most preferably methyl.

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

Examples of the carotenoid compound (I) include the following carotenoid compound (II).

［式中、Ｒ¹〜Ｒ³は上記と同義を示す。］

[In the formula, R ^{1 to} R ³ have the same meaning as above. ]

Of the carotenoid compounds (I), peridinins are R ¹ and R ² hydrogen atoms, R ³ acetyls, R ⁴ , R ⁵ and R ⁷ , R ⁹ and R ^{11 to} R ¹⁶ hydrogen atoms, R ¹⁰ and R ¹⁷ Has the following structure in which is a methyl and X is a single bond.

Symbiodinium sp. Peridinin, a natural carotenoid compound isolated from OTCL2A strains and the like, has the following three-dimensional structure.

Of the carotenoid compounds (I), fucoxanthin has R ¹ and R ² as hydrogen atoms, R ³ as acetyl, R ⁴ , R ⁸ and R ¹³ and R ¹⁷ as methyl, R ^{5 to} R ⁷ , R ^{9 to} R ¹² And R ^{14 to} R ¹⁶ have the following structure in which a hydrogen atom and X are −CH _2- C (= O) − groups.

Fucoxanthin, a natural carotenoid compound isolated mainly from ochrophyta dinophyceae such as brown algae, has the following three-dimensional structure.

Since the carotenoid compound (I) used in the present invention is mainly a natural marine carotenoid, it can be isolated and purified from algae. For example, the above peridinin is described in Symbiodinium sp. It can be isolated and purified from Dinophyceae such as OTCL2A strain. Hereinafter, a method for isolating peridinin will be described as an example, but other carotenoid compounds (I) can also be isolated from algae by the same method.

Among the dinophyceae that produce peridinin, Symbiodinium sp. Is a symbiotic one that has a symbiotic relationship with marine invertebrates. Examples of the marine animal as a host include foraminifera, radiolarians, acoelomorpha of flatworms, jellyfish, corals, bivalves and the like. Therefore, in order to isolate peridinin from nature, the Dinophyceae Symbiodinium sp. Is isolated from the above-mentioned marine animals, cultured, and the cultured dinoflagellates are homogenized and then extracted. It is also possible to extract peridinin directly from marine animals containing dinophyceae that produce peridinin. Even when a marine animal is used, the marine animal containing the dinophyceae may be homogenized and then extracted as in the case of using the dinophyceae.

The extraction solvent may be appropriately selected, and for example, an alcohol solvent such as methanol or ethanol; a ketone solvent such as acetone or methyl ethyl ketone; a halogenated hydrocarbon solvent such as dichloromethane or chloroform; an aromatic solvent such as benzene or toluene. Hydrocarbon-based solvent; Ester-based solvent such as methyl acetate and ethyl acetate; Ether-based solvent such as diethyl ether and tetrahydrofuran; Amido-based solvent such as dimethylformamide and dimethylacetamide; Among these organic solvents having a concentration of 60 v / v% or more. Examples thereof include a mixed solvent of a water-miscible organic solvent and water.

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

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

As described above, the carotenoid compound (I) may be produced by isolating it from algae or by derivatizing the isolated compound. For example, the 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 the C 1-18} alkanoyl group, an activated ester compound thereof, an acid chloride compound, and if necessary, a condensing agent. However, among the carotenoid compounds (I), R ³ of the natural compounds peridinin and fucoxanthin are acetyl groups.

The cerebral protective agent according to the present invention can protect the brain by suppressing an 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 at the same time as or immediately after reperfusion therapy. According to the experimental findings by the present inventors, the brain protective agent according to the present invention can suppress active oxygen during reperfusion following cerebral ischemia and M1 type microglia that cause an excessive inflammatory reaction. can. Examples of the patient to whom the brain protective agent according to the present invention should be administered include humans and animals other than humans.

The dosage form and administration form of the cerebral protective agent according to the present invention are not particularly limited, but in consideration of urgency for cerebral ischemia, intravenous administration as an injection is preferable. In that case, as the solvent, an isotonic solution of plasma such as a physiological saline solution having an adjusted pH or an aqueous glucose solution can be used. Alternatively, when the carotenoid compound (I) is dried together with salts or the like, pure water, distilled water, sterile water or the like can also be used if the solution is finally isotonic or substantially isotonic with plasma. The concentration may be that of a normal injection, and can be, for example, 0.05 mg / mL or more and about 10 mg / mL.

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

This application claims the benefit of priority under Japanese Patent Application No. 2017-248845 filed on December 26, 2017. The entire contents of the specification of Japanese Patent Application No. 2017-248845 filed on December 26, 2017 are incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can meet the purposes of the preceding and the following. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

Example 1
(1) Isolation of peridinin Symbiodinium sp. The OTCL2A strain was isolated and cultured. 100.2 g of frozen cells obtained from 156 L of culture medium was homogenized in 200 mL of 70% ethanol using a homogenizer (“Ultra-Turrax T25” manufactured by Janke & Kunkel), allowed to stand at 4 ° C. for 3 days, and then centrifuged. separated. The supernatant was collected and the above operation was repeated twice for the precipitate. All supernatants were combined and concentrated under reduced pressure. 100 mL of water was added to the concentrate, and the mixture was extracted 3 times with 150 mL of ethyl acetate. The extract was concentrated under reduced pressure to obtain 1094 mg of ethyl acetate fraction. Of the obtained ethyl acetate fraction, 957.9 mg was subjected to silica gel chromatography. In silica gel chromatography, 40 mL of Silica Gel 60 (manufactured by Nacalai Tesque) was used, and the eluent was dichloromethane: methanol = 99: 1 (80 mL) → 98: 2 (80 mL) → 96: 4 (120 mL) → 92: 8 ( 80 mL) of mixed solvent was used. 294.9 mg of the 313.5 mg fraction eluted with the eluent of dichloromethane: methanol = 96: 4 is 20 mL of ODS silica gel (“Cosmosil 75C18-OPN” manufactured by Nacalai Tesque), 60 mL of 80% methanol, followed by 85%. It was subjected to silica gel column chromatography using 140 mL of methanol. A fraction 169.1 mg eluted with 85% methanol was subjected to HPLC (column: "COSMOSIL 5C _18- AR-II" manufactured by Nacalai Tesque, 20 mmφ x 250 mm; eluent: 80% acetonitrile; flow rate: 6.0 mL / min). To obtain 31.7 mg of peridinin.

(2) Confirmation test of the effect of peridinin on microglia Microglia were isolated from the brain of 1-day-old neonatal mouse C57BL / 6 and used as a cell culture medium (“Eagle MEM” manufactured by Nissui Pharmaceutical Co., Ltd.). In addition to the product), the cells were cultured at 37 ° C. for 2 weeks. The resulting microglial cultures, 300 [mu] L was added per well to the cell culture well plates (about ^{6.4 × 10 5 cells / well)} . Further, 50 μL of a 0.03 to 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 a 60 μM aqueous solution of zinc chloride was added per well and incubated at 37 ° C. for 2 hours. After removing the culture broth and further washing the attached microglia with a serum-free medium, 350 μL of fresh serum-free medium was added per well. Microglia were activated to M1 form 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 the inflammatory cytokines IL-1β, TNFα and IL-6 were measured by ELISA. For comparison, when neither zinc nor lipopolysaccharide is added (control), when only zinc is added without adding peridinin, when only peridinin is added, when only lipopolysaccharide is added without adding peridinin, peridinin is added. The same measurement was performed when no addition was made. The measurement was performed 4 times each, and the average value was calculated. The results are shown in FIG.

(3) Consideration of experimental results As shown in the results shown in FIG. 1, inflammatory cytokines were released from microglia activated to M1 type by lipopolysaccharide, and their concentrations were clearly 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.
These results demonstrate that peridinin can suppress the excessive inflammatory response caused by zinc signals in the brains of stroke patients.

Example 2
A 10-100 μg / mL aqueous solution of peridinin or a 0.1 mM aqueous solution of CaEDTA, a chelating agent, was administered to the ventricles of a 12-week-old male C57BL / 6N mouse weighing about 35 g. Within 10 minutes of administration, bilateral common carotid arteries were occluded for 20 minutes to create a cerebral ischemic state, and then reperfusion was performed. Three days after the cerebral ischemia treatment, total RNA was extracted from the hippocampus of each mouse, and the gene expression levels of the inflammatory cytokines TNFα, IL-6 and IL-1β were measured by real-time PCR. The measurement was performed three times each, and the average value was calculated. For comparison, measurements were made in the same manner without cerebral ischemia treatment and without peridinin or CaEDTA. FIG. 2 shows a relative ratio in which the concentration of each inflammatory cytokine is 1 when cerebral ischemia treatment is not performed.
As shown in the results shown in FIG. 2, administration of peridinin can suppress inflammatory cytokine production after stroke to the same level as or higher than that of the chelating agent CaEDTA, which is at the same level as when cerebral ischemia treatment is not performed. There was also an experimental example that could be suppressed.
Thus, in vivo experiments also demonstrated that peridinin can suppress the excessive inflammatory response after stroke and protect brain tissue.

Example 3
It was tested whether peridinin could suppress the generation of active oxygen (ROS: Reactive Oxygen Species) caused by zinc in microglia.
Microglia were precultured in medium with or without 1 μM dihydroethidium (DHE) and 300 ng / mL peridinin for 30 minutes and then cultured for 2 hours in the presence of _{60 μM ZnCl 2.} DHE suggests the amount of superoxide anion radicals and is used to detect reactive oxygen species. Microglial cells were magnified and observed with an all-in-one fluorescence microscope (“BZ-9000” manufactured by KEYENCE CORPORATION), and cells stained with DHE were detected. The results are shown in FIGS. 3A and 3B. The result in FIG. 3B is an average value of 4 cases. 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 treatment group _{containing only ZnCl 2.} Is shown.
As shown in 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.
In addition, the effect of peridinin on the active oxygen level of microglia was tested in the same manner except that zinc was not used. The results are shown in FIGS. 3C and 3D. The result in FIG. 3D is an average value of 4 cases. 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 treatment group _{containing only ZnCl 2.} Is shown.
As shown in the results shown in FIGS. 3C and 3D, DHE-positive cells were not significantly increased relative to the control in microglia treated with peridinin alone.

Example 4
To confirm the anti-inflammatory effect of peridinin on the activation of hippocampal microglia to M1 type after reperfusion following cerebral ischemia, double immunostaining with the microglial marker Iba1 was performed with a representative M1 marker. The expression of certain CD16 / 32 was tested. The results are shown in FIGS. 4A and 4B. The result in FIG. 4B is an average value of 6 cases. In FIG. 4B, "*" indicates a significant difference at p <0.05 with respect to the solvent-treated pseudo-surgery mouse group, and "#" and "##" indicate that there is a significant difference with respect to the solvent-treated cerebral ischemia-surgery mouse group. It is shown that there is a significant difference when p <0.05.
As shown in the results shown in FIG. 4, the CD16 / 32 immune response in the Iba1-positive cells in the hippocampus 3 days after the cerebral ischemia treatment was clearly increased in the solvent-treated mice with cerebral ischemia as compared with the simulated surgical mice. Was. On the other hand, in mice treated with peridinin, the CD16 / 32 immune response was dose-dependently reduced to low levels in Iba1-positive cells in the hippocampus.
Thus, it was revealed that peridinin can suppress the activation of hippocampal microglia to M1 type after reperfusion following cerebral ischemia and reduce excessive inflammatory reaction.

Example 5
A Y-maze test was performed 5 days after induction of cerebral ischemia to determine if pre-administration of peridinin had a protective effect against the deletion of short-term spatial cognitive memory.
Specifically, graywood was used to form a Y-shaped maze with the same three arms of 40 x 2 x 3 cm at 60 ° intervals. The mouse was placed on the end of one of the arms and moved freely for 10 minutes, and the approach to each arm was recorded with a digital video camera. The case where the mouse invades an arm different from the two arms that entered the previous and the previous two times is defined as "alternate reaction", and the case where the mouse re-enters either of the two arms that entered the previous and the previous two times is an "error". Was defined. The percentage of alternation reaction was calculated by the following formula. The results are shown in FIG. The result in FIG. 5 is an average value of 6 cases. In FIG. 5, “*” indicates that there is a significant difference at p <0.05 with respect to the solvent-treated pseudo-surgery mouse group.
[Number of alternation reactions / (Total number of entries into the arm-2)] x 100
As shown in the results shown in FIG. 5, a significant decrease in spontaneous alternation response was observed in the solvent-treated mice with cerebral ischemia as compared with the pseudo-surgery mice.
On the other hand, in mice pre-administered with peridinin, the decrease in spontaneous alternation response induced by cerebral ischemia was clearly impeded.

Claims (6)

A brain protectant characterized by containing peridinin as an active ingredient. The brain protective agent according to claim 1, further comprising a solvent. The brain protective agent according to claim 2 , which is an injection. The brain protective agent according to any one of claims 1 to 3, which contains peridinin in an amount of 0.05 mg / mL or more and 10 mg / mL or less. The brain protective agent according to any one of claims 1 to 4, wherein peridinin is administered at a dose of 0.5 mg / kg body weight or more and 100 mg / kg body weight or less. The brain protective agent according to any one of claims 1 to 5 , which suppresses an inflammatory reaction after cerebral ischemia.

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