JP2014162768A - Cerebral protective drug - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cerebral protective drug effective against, e.g., brain cell damage caused by ischemia or ischemia reperfusion.SOLUTION: The cerebral protective drug contains a compound represented by general formula (1) as an active ingredient. (In the formula (1), Rto Reach represent hydrogen, hydroxy group, or methoxy group, and at least two of Rto Rare hydroxy group and/or methoxy group). In addition, the compound is preferably syringic acid or vanillic acid.

Description

  The present invention relates to a cerebral protective agent effective for brain cell damage caused by ischemia or ischemia / reperfusion.

  Nerve cells are particularly vulnerable to ischemia, and the brain is susceptible to damage from ischemia. For example, cerebral infarction caused by ischemia due to cerebrovascular occlusion is a serious lifestyle-related disease that is the fourth leading cause of death in Japan, and the number of patients is also increasing. In addition, even in the case of cerebral infarction, even if the life is saved, there is a high possibility that serious sequelae such as paralysis remain due to irreversible damage of nerve cells.

  A transient ischemic attack is a reversible neurological symptom that often appears prior to cerebral infarction, but it is said that brain cells may not recover from injury even after several minutes of ischemia. In addition, blood reperfusion after ischemia is said to significantly worsen damage to brain cells due to the influence of active oxygen and the like generated during reperfusion. Therefore, it is said that if brain cells can be protected from ischemia or ischemia / reperfusion injury, it is possible to avoid severe sequelae such as paralysis and speech impairment.

  For example, edaravone (trade name: “Radicut”) has been approved as a brain protective agent used to improve neurological symptoms associated with the acute phase of cerebral infarction, impairment of daily living, functional impairment, and the like. In addition, the present applicant has reported a brain protective agent comprising as an active ingredient a culture medium extract obtained by culturing mycelium of Ganoderma fungi in a medium containing plant fiber raw materials (Patent Document 1).

Japanese Patent No. 5144112

  If a new active ingredient of a brain protective agent can be provided, the range of choice of treatment and treatment is expanded. In addition, it is desirable that the active ingredient is a compound that has been isolated and identified, because it is easy to ensure its effectiveness stably regardless of the production batch.

  Accordingly, an object of the present invention is to provide a cerebral protective agent comprising an isolated / identified compound as an active ingredient and effective for brain cell damage due to ischemia, ischemia / reperfusion, and the like.

  From the culture medium extract obtained by culturing mycelium of Ganoderma fungus in a medium containing a plant fiber raw material, the present inventors provide effective components for brain cell damage due to ischemia, ischemia and reperfusion, and the like. It was isolated and identified to complete the present invention.

  That is, the brain protective agent of the present invention contains a compound represented by the following general formula (1) as an active ingredient.

(In Formula (1), R _{1 to} R ₃ each represent hydrogen, a hydroxyl group, or a methoxy group, and at least two of R _{1 to} R ₃ are a hydroxyl group and / or a methoxy group.)

  In the brain protective agent of the present invention, the compound is preferably syringic acid or vanillic acid.

  According to the brain protective agent of the present invention, brain cell damage due to ischemia or ischemia / reperfusion can be reduced, the volume of cerebral infarction can be reduced, and the occurrence of cerebral infarction or severe motor dysfunction can be suppressed. can do.

It is a graph which shows the result of the safety test (Test Example 3) with respect to PC12 cell by the concentration-dependent addition of syringic acid. 6 is a chart showing the results of evaluating the protective effect of syringic acid against cell death of PC12 cells by H ₂ O ₂ treatment in Test Example 3. 10 is a chart showing the results of evaluating the inhibitory effect of syringic acid on the production of intracellular reactive oxygen species in PC12 cells by H ₂ 0 ₂ treatment in Test Example 4. 10 is a chart showing the results of evaluating the inhibitory effect of syringic acid on the occurrence of neurological symptoms associated with hypoxic cerebral ischemia (H / I) injury in Test Example 5. It is a graph which shows the result of having evaluated the inhibitory effect of syringic acid with respect to generation | occurrence | production of the motor dysfunction accompanying hypoxic cerebral ischemia (H / I) disorder in Experimental Example 6. FIG. 6A is a chart showing the results of evaluating the inhibitory effect of syringic acid on the development of cerebral infarction lesions associated with hypoxic cerebral ischemia (H / I) injury in Test Example 7, and FIG. 6A is a photograph of an infarct lesion of a mouse brain section. FIG. 6B shows the result of comparison of cerebral infarct volume.

  The brain protective agent of the present invention contains a compound represented by the following general formula (1) as an active ingredient.

(In Formula (1), R _{1 to} R ₃ each represent hydrogen, a hydroxyl group, or a methoxy group, and at least two of R _{1 to} R ₃ are a hydroxyl group and / or a methoxy group.)

  For example, syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), vanillic acid (4-hydroxy-3-methoxybenzoic acid), 3,4,5-trimethoxybenzoic acid (3,4,5-trimethoxybenzoic acid), 3,4-dimethoxybenzoic acid, 3,5-dimethoxybenzoic acid, gallic acid (3,4,5-trihydroxybenzoic acid), protocatechuic acid (3, 4-dihydroxybenzoic acid), 3,5-dihydroxybenzoic acid (3,5-dihydroxybenzoic acid), isovanillic acid (3-hydroxy-4-methoxybenzoic acid), 3,4-dihydroxy-5-methoxybenzoic acid (3,4 -dihydroxy-5-methoxybenzoic acid), 3,5-dihydroxy-4-methoxybenzoic acid, 3-hydroxy-4,5-dimethoxybenzoic acid (3-hydroxy-4) , 5-dimethoxybenzoic acid) and the like. Since these can be synthesized by known methods and are also commercially available, such commercially available products can be used.

  Note that syringic acid has a chemical structure represented by the following chemical formula (2).

  Vanillic acid has a chemical structure represented by the following chemical formula (3).

  In the brain protective agent of the present invention, in addition to the above active ingredients, there is no particular limitation on blending other materials, and if necessary, a pharmaceutically acceptable base or carrier is added, Depending on the formulation method, it can be used in the form of tablets, granules, capsules, pills, powders, liquids, powders, jelly-forms, rod-forms, injections, inhalants, coatings, and the like.

  In the cerebral protective agent of the present invention, the administration form is not particularly limited, and for example, oral administration, intravenous administration, intracerebral administration, intraperitoneal administration, aspiration, nasal administration, transdermal administration, intracavitary administration, etc. Is mentioned. Among them, as shown in the examples described later, since the brain protective agent of the present invention can provide sufficient brain protection effect by oral ingestion, it is possible to reduce the burden on the intake and ease of taking. From the viewpoint, the oral administration form is preferable.

  The dose of the brain protective agent of the present invention is 3 mg to 300 mg per day for an adult when administered orally. If the dose is less than the range, it is difficult to obtain a sufficient effect, and if the dose is more than the range, the risk of causing some side effects increases.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

<Test Example 1>
Ganoderma mycelium culture medium extract (trade name “MAK”, Noda Shokubai Kogyo Co., Ltd.) (hereinafter referred to as “MAK”) is used as a starting material in order, followed by ultrafiltration, DIAION HP-20 column ( Adsorption), ODS column (reverse phase), and silica gel column (normal phase) MPLC fractionation means were used to isolate two compounds, Compound A and Compound B. Isolation was performed as follows using the activity of scavenging ability of DPPH free radical and superoxide anion as an index.

  MAK was adjusted to 5 g / L with water and centrifuged at 3900 × g for 15 minutes to remove insolubles. A membrane filter (ultrafiltration disk PLBC, ultracellulose 3 kDa NMWL, MILIPORE) was set in a pressure filtration device (stirring cell 8000, MILIPORE), and the MAK aqueous solution was injected. Apply pressure filtration with 0.2 to 0.3 MPa of nitrogen gas, apply the obtained elution fraction to a DIAION HP-20 column (Mitsubishi Chemical Corporation), elute with water, methanol and acetone in order, and fractions 1 to 4 Obtained. The third fraction (1.26 g) eluted with methanol was MPLC on an ODS column (ULTRA PACK ODS-SM; 26 x 300 mm; Yamazen Co., Ltd.), water: methanol (9: 1/7: 3/6: 4 / 0: 10) to give fractions 1-7. The third fraction (0.22 g) was further mixed with 9: 1/8 chloroform: [methanol (9): water (1)] on a silica gel column (Hi-Flash column; 26 × 150 mm; Yamazen Corporation) MPLC. : 2/7: 3/6: 4/10: 0, and fractions 1 to 11 were obtained. Among them, Compound A and Compound B were obtained from the second fraction.

These compounds were subjected to ¹ H-NMR, ¹³ C-NMR analysis (Agilent Technology Co., Ltd.), and LC / MS analysis (JEOL Ltd.) to identify vanillic acid having a molecular weight of 168.1 and syringic acid having a molecular weight of 198.17.

  As a result, syringic acid represented by the following chemical formula (2) and vanillic acid represented by the following chemical formula (3) were identified.

  Separately, the contents of syringic acid and vanillic acid in MAK were quantified by HPLC using pure syringic acid (Wako Pure Chemical Industries, Ltd.) and vanillic acid (Sigma-Aldrich Co., Ltd.) as standard. 1 g of MAK contained 0.511 mg of syringic acid and 0.868 mg of vanillic acid.

<Test Example 2>
In Test Example 2, a cytotoxicity test for nerve cells was performed by addition of syringic acid in a concentration-dependent manner.
Specifically, undifferentiated PC12 cells were seeded on a poly-D-lysine-coated 60 mm plate (Becton Dickinson Franklin Lakes, New Jersey, USA), and 10% inactivated horse serum, 5% inactivated fetal bovine serum ( Tissue Culture Biologicals, Tulare, CA, USA), 2 g / L NaHCO ₃ , and antibiotic complex “Antibiotic-Antimycotic” (GIBCO, Grand Island, NY, USA) (0.25 μg / ml; 100 unit / ml penicillin, 100 μg RPMI1640 medium (GIBCO) supplemented with / ml streptomycin, 100 μg / ml amphotericin B) was added and cultured in a 37 ° C., 5% CO ₂ incubator. When 80% confluence was reached, the cells were trypsinized and seeded in 96-well plates at a cell concentration of 1 × 10 ⁴ / well. After culturing for 5 days, syringic acid was replaced with a culture solution added to have a concentration of 0 mM, 0.001 mM, 0.01 mM, 0.1 mM, 1 mM, 3 mM, and further cultured for 2 days.

  Cell viability was evaluated by MTT assay according to conventional methods. After a serum-free medium containing 0.25 mg / ml MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) is allowed to act for 3 hours, DMSO (dimethyl sulfoxide) is added at a concentration of 1%. In addition, the cells were lysed with a micromixer, and the absorbance at 570 nm and 670 nm was measured. The same operation was performed on wells not seeded with cells, and the absorbance value measured was subtracted as blank.

  As a result, as shown in FIG. 1, no significant toxicity to PC12 cells was observed even when syringic acid was added to the culture solution at a final concentration of 0.001 mM to 3 mM and cultured for 2 days.

<Test Example 3>
In Test Example 3, the protective effect of syringic acid against neuronal cell death by oxidative stress treatment was evaluated.
Specifically, PC12 cells are seeded in a 96-well plate at a cell concentration of 1 × 10 ⁴ / well, and 2 days later, NGF (nerve growth factor; 50 ng / ml; Alomone Labs) is added to differentiate into neurons. It was. To the differentiated PC12 cells, syringic acid was added to a concentration of 0 mM, 0.001 mM, 0.01 mM, 0.1 mM, 1 mM, 3 mM, and H ₂ O ₂ was added to a final concentration of 250 μM after 1 hour. The culture was further continued for 6 hours. The cell viability was evaluated by MTT assay as in Test Example 2.

As a result, as shown in FIG. 2, the cell viability was significantly decreased (P <0.001) by H ₂ 0 ₂ treatment (62.24% survival rate compared to no treatment), while syringic acid culture was performed. Increased the viability of PC12 cells by H ₂ 0 ₂ treatment in a concentration-dependent manner in the final concentration range of 0.001 mM to 1 mM in the solution, especially at 0.1 to 3 mM (P <0.05 and P <0.001 ) The nerve cell protective effect was seen.

<Test Example 4>
In Test Example 4, the inhibitory effect of syringic acid on the production of reactive oxygen species in neurons by oxidative stress treatment was evaluated.
Specifically, as in Test Example 3, PC12 cells differentiated into nerve cells by addition of NGF were cultured at different syringic acid addition concentrations, and oxidative stress was induced by H ₂ 0 ₂ treatment. Production of endogenous reactive oxygen species was evaluated by DCF-DA staining. DCF-DA staining was performed as follows.

  The medium was removed, 2 ', 7',-Dichlorodihydrofluorescin diacetate (DCF-DA; Sigma) prepared in a low serum medium was added at 10 μM / well, and the mixture was incubated at 37 ° C. for 30 minutes. Subsequently, after removing the medium, 100 μL of PBS was added and mixed for 10 minutes. Fluorescence (excitation wavelength: 485 nm, fluorescence detection: 535 nm) was measured with a multi-label plate counter (Wallac 1420ARVOsx; Perkin Elmer Japan Co., Ltd.). The oxidative stress was evaluated from the fluorescence intensity with the untreated group as 100%.

As a result, as shown in FIG. 3, the intracellular reactive oxygen species increased by H ₂ 0 ₂ treatment (about 1.6 times that of no treatment), but by adding syringic acid, Production of intracellular reactive oxygen species was suppressed. In particular, production of intracellular reactive oxygen species was significantly suppressed (P <0.001) by adding 0.1 mM to 3 mM syringic acid.

<Test Example 5>
Since syringic acid was confirmed to have a neuronal cell protective effect and an active oxygen production inhibitory effect at the cellular level (in vitro), the brain protective effect at the biological level (in vivo) was next evaluated. In Test Example 5, the improvement effect of syringic acid on neurological symptoms associated with hypoxic cerebral ischemia (H / I) injury was evaluated. Specifically, the following tests were conducted.

As an experimental animal, a type 2 diabetes model mouse (male KK- ^Ay mouse, manufactured by CLEA Japan) that exhibits a cerebral infarct lesion more sensitively than normal mice against hypoxic cerebral ischemia (H / I) injury was used. The mice were divided into a control group (20 mice) and a syringic acid-administered group (6 mice). The control group was given water and the syringic acid-treated group was given syringic acid (50 mg / Kg / day). Administered.

After the breeding period, each group of mice was anesthetized with halothane, fixed in the supine position, and then the midline incision was made at the neck. The right common carotid artery was detached from the vagus nerve, double ligated, and finally the incision was sutured. Three hours after ligation of the common carotid artery, mice were placed in glass containers and loaded with hypoxic gas (8% O ₂ /92% N ₂ ) for 30 minutes. During this time, the inside of the glass container was kept at 35.5 ° C. with a water bath and an infrared lamp. The mice were then raised for 24 hours with free access to food and water in a normal atmospheric environment.

  Neurological symptoms were evaluated 24 hours after the H / I treatment. Specifically, for each item of 3 minutes of spontaneous movement, walking state, bridging, paralysis of the opposite front and rear limbs, contact reaction, reaction when touching the opposite trunk, 0: severe disability, 1: Mice were scored for neurological symptoms in 4 stages of moderate disorder, 2: mild disorder, 3: no disorder, and evaluated using the sum of these scores.

  As shown in FIG. 4, the Sham group (13 mice) that had undergone only the common carotid artery ablation was significantly decreased (P <0.001) to 8.53 in the control group compared to the score of 18.0, and neurological symptoms appeared. . This score recovered significantly (P <0.05) to 14.17 in the syringic acid-administered group, and neurological symptoms improved.

<Test Example 6>
In Test Example 6, the inhibitory effect of syringic acid on motor dysfunction associated with hypoxic cerebral ischemia (H / I) injury was evaluated. Specifically, for the mice treated with H / I in the same manner as in Test Example 5, the motor function of the mice was evaluated by the roter-rod test before and after the H / I treatment. The Rota-Rod test uses the Single Lane Rota-Rod test device MK-630B (Muromachi Kikai Co., Ltd.), puts the mouse on a rotating rod with a diameter of 4 cm, and accelerates it at 4-40 rpm to force the mouse to walk. This was done by measuring the time to fall. The exercise was performed 5 days and 2 days before the H / I treatment (5 times / day). In addition, measurement was performed three times before H / I treatment and 24 hours after treatment, and the average value was evaluated.

  As shown in FIG. 5, in the Sham operation group, the time until the mouse fell before and after the operation was similar, whereas in the control group, the time until the mouse dropped was after the operation. 72.35 seconds, which was significantly shorter than 201.0 seconds before surgery (P <0.001), clearly resulting in motor dysfunction. On the other hand, in the syringic acid-administered group, it was 135.56 seconds after the operation, which was significantly longer than that in the control group (P <0.05), and its motor dysfunction was improved.

<Test Example 7>
In Test Example 7, the inhibitory effect of syringic acid on cerebral infarction lesions associated with hypoxic cerebral ischemia (H / I) injury was evaluated.
Specifically, after the Rota-Rod test in Test Example 6, the brain of the mouse was removed and a 2 mm thick brain section was prepared. The sections were incubated in PBS (pH 7.4) containing 2% triphenyltetrazolium chloride (TTC; Wako Pure Chemical Industries, Ltd.) for 15 minutes at 37 ° C. in the dark to be stained with TTC. The stained section was immersed and fixed with formaldehyde, and a stained image was taken with a digital camera.

  As shown in FIG. 6A, in the control group, a clear infarct (white portion in the photograph) appeared by H / I treatment. In contrast, in the syringic acid administration group, the infarct size decreased. Using the image analysis software (SconImage 1.62, Scion Corporation, Fredeik, MD, USA), measure the rostral and caudal infarct areas of each piece, and calculate the infarct volume using the following formula (A). Calculated.

  Infarct volume (%) = {Left hemisphere volume− (Right hemisphere volume−Infarct volume)} / Left hemisphere volume × 100 (A)

  As shown in FIG. 6B, the infarct volume was 30.9% in the control group, but 12.51% in the syringic acid administration group, which was significantly suppressed compared to the control group (P <0.01).

Claims (2)

A brain protective agent comprising a compound represented by the following general formula (1) as an active ingredient.

(In Formula (1), R _{1 to} R ₃ each represent hydrogen, a hydroxyl group, or a methoxy group, and at least two of R _{1 to} R ₃ are a hydroxyl group and / or a methoxy group.)   The brain protective agent according to claim 1, wherein the compound is syringic acid or vanillic acid.