JP2013071911A - Brain protective agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent having brain protective action.SOLUTION: This brain protective agent includes a wheat powder extract as an active constituent. Further, in the brain protective agent, brain protection is intended for prevention of ischemic encephalopathy.

Description

  The present invention relates to an agent for preventing brain damage associated with ischemic disease.

  Ischemic neuronal cell death due to cerebral infarction, etc. shows typical pathologically disordered necrotic conditions such as organelle swelling and nuclear aggregation in the center of the ischemic foci. It has been believed to be neuronal cell death due to necrosis. However, since a pathological image completely different from necrosis is exhibited at the time of mild cerebral ischemia (Non-Patent Document 1: Brain Res. Mol., 29, pp.1-14, 1995;), in these neurons, It has come to be considered that apoptosis is induced in addition to necrosis (Non-patent Document 2: Science, 281, pp. 1302-1304, 1998).

  In apoptosis, a series of signal cascades proceed after the switch to cell death is turned on, and caspase, which is an execution molecule of cell death, is activated, and it is thought that cells eventually die. Neonatal hypoxic encephalopathy has also been implicated in apoptosis through caspase activation (Non-patent Document 3: J. Clin. Invest., 101, pp. 1992-1999, 1998). In addition, it is known that nerve cells in a remote region having nerve fiber communication with the damaged brain region are secondary to cell death, and apoptosis is also deeply involved in this.

  Apoptosis due to cerebral ischemia has been observed to appear slightly later than necrosis in the marginal part (penumbra region) of ischemic necrosis (core part). Therefore, the treatment start time (treatment time window) for suppressing brain cell death caused by apoptosis during cerebral ischemia is more tolerated than necrosis, and the drug is administered several hours after onset. May also be able to suppress cell death caused by apoptosis. This also protects cells destined to undergo apoptosis and increases the number of viable cells, and may further prevent secondary degeneration in remote regions and reduce residual neurological function. is there.

  From this point of view, Patent Document 1 (Japanese Patent Application Laid-Open No. 2000-344663) discloses a brain in which 2-phenyl-1,2-benzoisoselenazol-3 (2H) -one (generic name: Epselen) is caused by apoptosis. Since cell death is suppressed, it is described that it can be used as an agent for preventing cerebral damage associated with cerebral ischemia. Recently, natural products-derived brain protective agents and apoptosis inhibitors are attracting attention as therapeutic agents for ischemic disorders. Patent Document 2 (JP-A-6-172200) discloses lactoferrin-derived peptides, Patent Document 3 (JP-A 2002-265359) discloses tea-derived theanine, Patent Document 4 (JP-A-2008-266177). Discloses a basidiomycete or ascomycete extract, and Patent Document 5 (Japanese Unexamined Patent Application Publication No. 2009-292747) discloses a natural product-derived brain protective agent such as purple koji fermented liquid.

JP 2000-344663 A JP-A-6-172200 JP 2002-265359 A JP 2008-266177 A JP 2009-292747 A

Brain Res. Mol., 29, pp.1-14, 1995; Science, 281, pp.1302-1304, 1998 J. Clin. Invest., 101, pp. 1992-1999, 1998

  An object of the present invention is to provide an agent having a brain protective action. Moreover, let it be a subject to provide the food-drinks containing this agent.

  The inventors of the present invention propose the present invention based on the fact that, as a result of testing with a focus on wheat flour, the extract of wheat flour has an inhibitory effect on the occurrence of apoptosis caused by ischemia.

That is, the present invention
(1) A brain protective agent comprising an extract of wheat flour powder as an active ingredient.
(2) The brain protective agent according to (1), wherein the brain protection is for prevention by ischemic brain injury.
(3) The brain protective agent according to (1) or (2), wherein the brain protection is due to inhibition of apoptosis caused by ischemia.
(4) Food / beverage products comprising the brain protective agent according to any one of (1) to (3), which is an oral preparation, (5) The brain protective agent according to any one of (1) to (4)

  The present invention can provide an agent that suppresses apoptosis caused by ischemia and has a brain protective action. In addition, foods and drinks that prevent ischemic brain damage can be provided.

The graph which shows the apoptosis inhibitory effect with respect to the human neuroblastoma origin SH-SY5Y cell by the water extract of wheat flour. The image which observed the infarct state of the mouse brain which artificially generated the cerebral ischemia is shown. The graph which shows that a cerebral infarction image is image-analyzed and wheat flour flour is suppressing concentration of infarction. The brain cross-sectional image of a cerebral infarction model is shown. In this image, the infarcted area is shown in white.

  The brain protective agent of the present invention comprises an extract of wheat flour flour as an active ingredient.

There are two main methods for classifying flour. One is classified by type, and the other is classified by grade. The classification by grade is derived from the form of the raw wheat grains. The powder used in the present invention is classified by grade.
Even in the endosperm portion of the same wheat grain, the central part has less ash, white color, and the amount of protein tends to decrease. In the milling process, the powder taken mainly from this central part is classified and referred to as advanced powder. The fine powder has a low ash content and has a pale white or pale yellow color. On the contrary, the lower grade powder taken from near the epidermis has more protein, and the color becomes dull and brownish. In general, those with an ash value of 0.3 to 0.35% are special powder, those with 0.35 to 0.45% are first powder, those with 0.45 to 0.65% are second powder, those with 0.7 to 1.0% are third powder, and 1.2 ~ 2.0% is classified as powder. In addition, the wheat flour hulls, which are separately screened as bran, are usually not suitable for food, so they are used together with bran as animal feed or as an industrial paste material. The present invention uses this powder as a raw material.

In order to prepare the agent of the present invention from wheat flour powder, the powder powder is extracted with water. Hereinafter, when it is described as “powder”, it means wheat flour.
Although the powder may be commercially available for feed, the powder immediately after milling is preferable because it does not oxidize lipids.

Add 5 to 10 parts by weight of water to 1 part by weight of the powder and extract at room temperature with stirring for 0.5 to 1 hour. In order to shorten the extraction time, a physical vibration such as an ultrasonic vibration is applied to extract a water-soluble component. In the case of a small-scale extraction operation, 5 parts by weight of water is added to 1 part by weight of the powder powder, and after stirring and dispersing, the active ingredient is eluted in water by performing ultrasonic treatment for 3 to 10 minutes.
Next, solid-liquid separation is performed by centrifugation or filter filtration, and the filtrate or supernatant is collected. The collected supernatant or filtrate is dried and powdered. The drying treatment can be applied by any method such as spray drying, drum dryer or freeze drying as long as it is a method for drying a normal liquid, but it is preferable to dry at a low temperature as much as possible in order not to reduce the activity.

The brain protective agent of the present invention can be further pulverized from the dried product obtained by the above method to form ultrafine particles.
The brain protective agent of the present invention can be commercialized as a powder, granule, tablet, or capsule by a conventional method. It can also be dissolved in water or fruit juice and commercialized as a liquid or jelly-like beverage. Furthermore, it can also be used by adding to various foods and drinks.
Such a food or drink is not particularly limited.
The effective dose of the brain protective agent of the present invention is 1 to 10 g per day for an adult when taken orally.
The following examples further illustrate the present invention.

<Test Example 1>
1. Test for preventing apoptosis of nerve cells caused by oxidative stress Test materials and methods (1) Cells used and culture methods
Human neuroblastoma-derived SH-SY5Y cells (passage number X + 18) purchased from DS Pharma Biomedical Co., Ltd. were used. Normal culture medium is D-MEM / Ham's F-12 (Wako Pure Chemical Industries) inactivated fetal bovine serum (Life Technologies Japan) 15%, non-essential amino acid (Sigma) 1%, penicillin (SIGMA ) 50000 U, Streptomycin (Meiji Seika) 0.05 pot. Was used. In normal culture, cells were seeded in a 10 cm petri dish, and subcultured by detaching the cells with a 0.05% trypsin-EDTA solution at 80-90% confluence. Culturing was performed in the presence of 37 ° C, 5% carbon dioxide, and 95% air.

(2) Plate seeding Passaged SH-SY5Y cells (passage number X + 19-25) are placed on a 96-well plate (96-well micro test plate; FALCON (Nippon Becton Dickinson Co., Ltd.)) at 1.0-1.5 × 10 ⁵ cells / Inoculated with mL and cultured overnight.

(3) Reagent preparation
Preparation of 6-Hydroxydopamine 6-Hydroxydopamine (6-OHDA) was used as an oxidative stress inducer. 6-OHDA was prepared in distilled water to 150 mM and added to the medium to a final concentration of 150 μM.

Preparation of powdered powder extract Powdered powder manufactured by Nippon Flour Milling Co., Ltd. was used.
5 mL of water was added to 1 g of the powder powder, and sonication was performed for 3 minutes. After centrifugation at 1000 rpm × 10 minutes with a centrifuge, the supernatant was recovered and lyophilized to obtain a dried product. Hereinafter referred to as water extract.

  Prepare the extract with distilled water to 3, 10, 30, and 100 mg / mL, and add medium (usually added with culture medium 6-OHDA) to final concentrations of 3, 10, 30, and 100 μg / mL, respectively. Added to.

(4) MTT assay
Preparation of MTT reagent
Prepare 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) to 5 mg / mL with PBS (-) and filter with 0.45 μm filter (Adevantech Toyo Co., Ltd.) This was used as an MTT solution. The MTT solution was dispensed and stored at −20 ° C. until use. At the time of use, it was added to the normal culture medium so as to be 500 μg / mL (MTT medium).

MTT assay
MTT assay was performed 15 hours after the addition of 6-OHDA and the test substance.
The medium added with 6-OHDA and the test substance was replaced with MTT medium and incubated for 3 hours. The medium was removed, and dimethyl sulfoxide was added. After the formazan precipitated was dissolved, the absorbance at 570 nm was measured.

B. The result of measurement is shown in FIG. As a result of adding the powdered water extract simultaneously with 150 μM 6-OHDA, it was found that the powdered water extract suppressed apoptosis due to oxidative stress of 6-OHDA in a dose-dependent manner. In particular, when the concentration was 10 μg / mL or more, a remarkable apoptosis inhibitory effect could be confirmed.

<Test Example 2>
1. Confirmation test of cerebral protective effect using artificial cerebral infarction model A cerebral infarction model was prepared by artificially blocking cerebral blood flow to confirm the cerebral protective action of the present invention.
A. Test Method (1) Preparation and Administration of Brain Protective Agent The extract used in Test Example 1 was continuously orally administered to confirm the brain protective effect.
The prescribed amount of brain protective agent for the test was weighed and dispensed in advance for each day, and the administration date and group were described and stored at -20 ° C. The dosing solution was prepared at the time of use, and dissolved by inversion mixing at 10, 30, and 60 mg / mL.

(2) Test animal mice, Slc: ddY, 60 males were used.
Arrival: 4 weeks old Surgery and dissection: 7 weeks old

At the time of arrival, the species, strain, age, number of animals and sex were confirmed, the general state and appearance were observed, and the body weight was measured. The acclimatization period was 7 days.
Based on body weight, the groups were divided so that there was no difference in each group.

(3) Environmental conditions of the test system Breeding environment Set temperature: 25 ° C (allowable range 23-27 ° C), set humidity: 60% (allowable range 40-80%), lighting: 7am on, 7pm off Animals were bred in Fukuoka University School of Pharmacy, Animal Breeding Room, maintained for 12 hours. Animals were housed at 5 per cage using plastic cages.

Feed Solid feed (CE-2, Nippon Claire Co., Ltd.) was placed in a feeder and allowed to eat freely. .

Drinking water Tap water washed with a filter was freely taken from a water bottle.

(4) Administration method / administration route Forcible oral administration using a gastric tube was used. In addition, it was administered once a day for 14 days. The dose was calculated from the closest measured body weight value at 10 mL / kg.

Group composition and dosage
Dose (mg / kg) Number of animals used Control group 0 15
100 mg / kg group 100 15
300 mg / kg group 300 15
600 mg / kg group 600 15

(5) Creation of a cerebral infarction model
An 8-0 nylon monofilament (Ethilon; Ethicon, NJ, USA) was cut to a length of 11 mm, and 4 mm from the tip was coated with silicon resin (Xantopren; Bayer Dental, Osaka, Japan) as an embolus. This was inserted into a mouse by the following method to create a cerebral infarction model.

Surgery method Anesthesia was introduced into the mouse by inhalation of 2% halothane, and then the anesthesia was maintained by inhalation of 1% halothane and fixed on the operating table. An incision was made in the center of the neck of the mouse, and the left common carotid artery and external carotid artery were ligated. The common carotid artery was incised, and 9 mm was inserted through the internal carotid artery from the bifurcation of the internal carotid artery and external carotid artery so that the embolus reached the origin of the middle cerebral artery (MCA). Reperfusion was performed by pulling the embolus forward. MCA occlusion time was 4 hours, and behavioral observation (Neurological score) and 2,3,5, -Triphenyltetrazolium chloride (TTC) staining were performed 24 hours later, and changes in infarct volume were measured.

(6) Behavioral observation (Neurological score)
Reperfusion was performed 4 hours after MCA occlusion, and cerebral infarction was scored 24 hours after MCA occlusion to observe changes in the infarct. The scores are as follows.
Score (0-5)
0: No effect on motor function
1: Torso flexion, forelimb lifting
2: circling behavior in normal posture
3: circling behavior with obvious posture abnormalities
4: Rolling behavior
5: There is no spontaneous behavior and the body is tilted

(7) TTC staining and image analysis After behavioral observation, the cervical spine of the mouse was dislocated and immediately decapitated. Cool the extracted brain well with cold saline and use an acrylic slicer (Sagittal type slice thickness for mice: 2 mm; Neuroscience Inc .; Fig. 3) to prepare a pre-cut slice of the brain containing the cerebral cortex. Fabricated at intervals of 2 mm. 2% 2,3,5, -triphenyltetrazolium chloride (Sigma Aldrich Japan Co., Ltd.) was prepared with physiological saline, and the sections were incubated in the solution at 37 ° C. for 30 minutes.
Then, they were placed on a glass slide and photographed with a digital still camera (MVC-FD91, SONY). What is the infarct volume? The total area of the infarct was measured and calculated from image of the cleavage plane with image analysis software (NIH Image 1.63).

B. Results As shown in FIG. 2, the behavioral observation (Neurological score) showed that the occurrence of injury associated with cerebral infarction was suppressed in a dose-dependent manner. The behavior until the 4th hour was relatively active, but the behavior was active according to the powder extract dosage. In the powder powder extract 600 mg / Kg administration group, the neurological score was as good as 2 or less, and the effect of the brain protective agent of the present invention was also confirmed in behavior. In addition, the behavior after 24 hours deteriorated, but the behavior score of the administration group was lower than that of the non-administration group, and it was speculated that the progression of cerebral infarction was suppressed.
As shown in FIGS. 3 and 4, as a result of image analysis of the cerebral infarction, a decrease in the cerebral infarction was observed in a dose-dependent manner. In particular, in the 600 mg / Kg administration group, the cerebral infarction site was prominent compared to the control (no administration) group. This corresponded well with the results of the above behavioral observation.
From the above results, it was revealed that the brain protective agent of the present invention prevents brain damage due to ischemic cerebral infarction.

Claims (5)

  A brain protective agent containing an extract of wheat flour flour as an active ingredient.   The brain protective agent according to claim 1, wherein the brain protection is for prevention by ischemic brain injury.   The cerebral protective agent according to claim 1 or 2, wherein the cerebral protection is based on suppression of apoptosis caused by ischemia.   It is an oral agent, The brain protective agent in any one of Claims 1-3 Food / beverage products containing the brain protective agent in any one of Claims 1-4.