JPH0873360A - Suppressing agent against brain nerve cell injury - Google Patents

Abstract

PURPOSE: To obtain the subject suppressing agent composed of 2"-O- glycosylisovitexin as effective component having suppressive activity against brain nerve cell injury attributable to active oxygen atom and useful for treating ischemic cerebropathy, etc. CONSTITUTION: This is the objective suppressive agent composed of 2"-O- glycosylisovitexin as effective component. 2"-O-Glycosylisovitexin is obtained e.g. by extracting and separating it from young green barley leaves as an anti- oxidant active substance. Further, the suppressive agent is obtained e.g. by homogeneously mixing 2"-O-glycosylisovitexin, milk sugar, crystal cellulose, talc and CMC of a binding agent, granulating the mixture and pressing the granules to a tablet after adding magnesium stearate. The dose of the suppressive agent is preferably 0.2-50mg/kg-body weight/day in terms of the active component.

Description

Detailed Description of the Invention

[0001] The present invention relates to a cerebral nerve cell disorder inhibitor containing 2 "-O-glucosylisovitexin as an active ingredient.

[0002] 2 "-O-glucosylisovitexin has a strong antioxidant activity and has an oxidation-inhibiting effect on easily oxidizable substances such as lipids [Antioxidative Activi
ty of an Isoflavonoid, 2 ″ -O-Glycosylisovitexi
n Isolated from Green Barley Leaves: Kitta, K., Hag
iwara, Y., Shibamoto, T .: J. Agric. Food Chem., 4
0 , (10), 1843-1845 (1992)]. It has already been disclosed by the present inventors that 2 ″ -O-glucosylisovitexin is effective as an antioxidant for vitamins [JP-A-5-85936]. It has been reported that 2 "-O-glucosylisovitexin also shows strong antioxidant activity against the oxidation of unsaturated fatty acids such as acid, squalene and arachidonic acid [Inhibition of Malonaldehyde Formation
from Lipids by an Isoflavonoid Isolated from Young
Green Barley Leaves: Nishiyama, T., Hagiwara, Y., H
agiwara, H., Shibamoto, T .: JAOCS., 70 , 811-8
13 (1993)].

Surprisingly, the present inventors have found that 2 ″ -O-glucosylisovitexin is extremely useful as an inhibitor of cerebral nerve cell damage caused by cerebral ischemia, etc., and completed the present invention. .

[0004] Cerebral nerve cell disorders are caused by various factors, and reactive oxygen species are said to be involved in many cranial nerve disorders. Cytotoxicity due to active oxygen occurs when lipid peroxide or its secondary product generated by the involvement of active oxygen impairs the function of cell membrane, and may be due to impairment of membrane components other than lipid, It is considered that the same phenomenon occurs in the cerebral nerve cells.

Cerebral ischemic lesions are damaged by ischemia and necrosis is required, and the protection of this necrosis requires reopening of blood flow. It is said that reactive oxygen species are generated as a side reaction at the time of reopening of blood flow to damage the cranial nerve.

Excessive generation of active oxygen and accumulation of lipid peroxide in the brain tissue give cerebral nerve cells oxidative damage. 2 ″ -O-glucosylisovitexin has the action of suppressing the damage of the cerebral nerve cells due to the reactive oxygen species in this brain tissue, and thereby exhibits the effect of suppressing the damage of the cerebral nerve cells. Therefore, it is useful for treatment of ischemic brain injury and the like.

2 ″ -O-glucosylisovitexin, which is an active ingredient of the inhibitor of cerebral nerve cell damage of the present invention, can be extracted and isolated as an antioxidant active substance from, for example, green barley juice component of barley [A Novel Antioxidant Isolated from
Young Green Barley Leaves: Osawa, T., Katsuzaki, H.,
Hagiwara, Y., Hagiwara, H., Shibamoto, T .: J, Agric.
Food Chem., 40 (7), 1135-1138 (19
92)].

When 2 "-O-glucosylisovitexin is administered to humans and other animals as a cerebral nerve cell injury inhibitor, 2" -O-glucosylisovitexin is generally pharmaceutically acceptable. By blending various additives,
It is preferable to formulate into a dosage form suitable for administration.

As the additives that can be used, in addition to the additives that are usually used in the freeze-drying or spray-drying of 2 "-O-glucosylisovitexin, any of the additives that are usually used for formulating into a desired dosage form can be used. Examples of the additives for preparation include the specific examples of ascorbic acid,
Nutrients such as biotin, pantothenic acid, calcium, carotene, choline chloride, magnesium oxide, niacin, pyridoxine chloride, riboflavin, sodium pantothenate, thiamine hydrochloride, tocopherol, vitamin A, vitamin B ₁₂ , vitamin D ₂ ; metaphosphoric acid Sodium, sodium phosphate (first, second, third
Salt), sodium pyrophosphate, sodium tripolyphosphate, and other pacing agents; calcium sorbate, benzoic acid,
Preservatives such as methyl paraoxybenzoate, sodium benzoate, etc .; Arabica gum, tragacanth, sodium alginate, methylcellulose, carboxymethylcellulose,
Carriers or diluents such as calcium alginate, aluminum silicate, calcium silicate, mannite, sorbitol, lactose, fructose, soluble starch, amino acids, glucose, sugar, honey, sucrose and fatty acid esters can be mentioned.

The inhibitor of cerebral nerve cell damage of the present invention can be administered orally or parenterally, and can be formulated into any dosage form suitable for each administration route. For example, powder, granules, pellets. Or, dosage forms suitable for oral administration such as tablets, coated tablets, capsules, troches, solutions, syrups; dosage forms suitable for parenteral administration such as injections, drops, suppositories, nasal drops, sprays, etc. Can be Furthermore, it is also possible to make external preparations such as ointments, creams, tinctures and patches.

The dose of the inhibitor of cerebral nerve cell damage of the present invention can be varied within a wide range depending on the patient's symptom weight, sex, age, weight, doctor's judgment, etc., but as a general guideline, About 0.1 to about 300m as an active ingredient
g / kg body weight / day, preferably about 0.2 to about 50 mg /
It can be exemplified within the range of kg body weight / day. The above dose can be administered once or several times a day.

Hereinafter, the present invention will be described more specifically with reference to Examples.

[0013]

【Example】

Example 1: Effect of 2 "-O-glucosylisovitexin on the lethal effect of neonatal mouse cultured cerebral cortical neurons by glucose oxidase The isolated cerebral cortical neurons of the mouse are the methods of Maeda and Mikoshiba [Maeda, N., Mikoshiba, K .: The culture of cerebella
r nerve cell, In Experimental Medicine, Supplemen
t, Manual in Neuronal Biochemistry, eds. by K. Miko
shiba and H. Hatanaka, Youdosha, Tokyo ,: 136-1
42 (1990)]. Newborn baby on the first day of life d
The cerebral cortex of a dy mouse was removed and treated with a Ca 2 ^+-and Mg 2 ⁺ -free HBSS solution containing 1% trypsin and 0.05% DNase I for 13 minutes, and then 0.05%
Cells were isolated by pipetting approximately 60 times in Ca ²⁺ free HBSS solution supplemented with DNase I.
The supernatant of this cell suspension was collected and centrifuged at 1000 rpm for 5 minutes, and the precipitate was mixed with Dulbecco's modified Eagle medium (D
Resuspended in MEM medium). The number of cells was counted with a hemocytometer, diluted with DMEM medium at a ratio of 1 × 10 ⁵ cells / ml, and coated with poly-L-lysine 96.
Sprinkled on a hole microplate After culturing in a CO ₂ incubator at 37 ° C. for 2 days, 20 μM cytosine arabinoside (ara C) was added, and the cells were further cultivated for 7 days. Glucose oxidase of 10 unit / ml and 2 ″ -O-glucosylisovitexin of 1 to 100 μM were added and left for 12 hours. Stained with 0.4% trypan blue and observed under a microscope. Then, dead cells and live cells were separated to determine the cell viability.

The results are shown in FIG. The vertical axis represents the survival rate of cultured cerebral cortex cells, and the horizontal axis represents the concentration of 2 "-O-glucosylisovitexin. The control was about 88 by mouth.
% Survival rate is shown. Glucose oxidase 10mU /
The survival rate was reduced to about 8% by the addition of ml (Fig. 1: ▪). This decrease in survival rate due to glucose oxidase was confirmed by 2 μM of 2 ″ -O-glucosylisovitexin, which was about 2 μM.
0%, 10 μM recovered about 57%, and 100 μM recovered about 97%.

Example 2: Effect of 2 "-O-glucosylisovitexin on learning impairment due to cerebral ischemia Method of Kojima and Kaneto [Preparation of cerebral ischemia
-induced amnesia model in mice and ameliorative ef
fect of several compounds on the model: Kojima, M.,
Kaneto, H .: Folia pharmacol. Japon., 94 : 223
-228 (1989)], a surgical cerebral ischemia model of 6 to 7-week-old male ddy mice was prepared. The learning ability was evaluated by a passive avoidance learning experiment [Step-down type pas
sive avoidance-and escape-learning method: suitabi
lity for experimental amnesiamodels .: Kameyama, T.,
Nabeshima, T., Kozawa, T .: J.Pharmacol. Method, 1
6 , 39-52 (1986)]. ME for equipment floor grid
An electrostimulator ME 6200 manufactured by Comercial was connected. On the first day, surgery for ischemic manipulation was performed, and on the second day, training for acclimatizing the animals to the experimental apparatus was performed. That is,
Step downlatency, which is the time it takes for the mouse to rest on the platform and get off
Was measured. The mouse was left on the floor for 1 minute. 15 minutes after intraperitoneal administration of 10 mg / kg or 300 mg / kg of 2 "-O-glucosylisovitexin on the 3rd day, the mouse was placed on the platform, and immediately after all the four limbs of the mouse descended from the platform, 50V, 2
The electrical stimulation of 00 msec and 2 Hz was applied for a maximum of 30 seconds. Immediately after electrical stimulation, both carotid arteries were stopped for 10 minutes. Twenty-four hours later, the mouse was placed on the platform again, and the step-down latency (this latency is an index of memory formation), which is the time until the mouse exits the platform, was measured.

The obtained results are shown in FIG. The vertical axis shows the step-down latency, and the step-down latency in the memory retention test of the control mouse is 1
It was significantly shortened by the ischemic operation for 0 minutes. The reduction of the step-down latency by this ischemic operation is reduced by 2 "-O-
Glucosyl isovitexin 100mg / kg, 300
Administration of mg / kg before the ischemic operation showed a dose-dependent recovery tendency.

Example A: Tablet 2 ″ -O-glucosylisovitexin 17.0 mg Lactose 100.0 mg Crystalline cellulose 75.4 mg Talc 5.0 mg Binder (carboxymethylcellulose) 2.0 mg Magnesium stearate 0.6 mg 200. 0 mg 2 ″ -O-glucosylisovitexin, lactose, crystalline cellulose, talc and binder are homogenized into granules, magnesium stearate is added, and 1 tablet 200 m
g tablets.

Example B: Enteric coated tablet The tablet obtained in Example A was subjected to enteric coating having the following formulation to produce an enteric solution of 400 mg per tablet.

Methacrylic acid / ethyl acrylate copolymer 10.8% Polyethylene glycol 6000 1.6% Surfactant (Tween 80) 1.1% Talc 7.2% Purified water 79.3% 100.0% Examples C: Granules 2 ″ -O-glucosylisovitexin 100.0 mg Lactose 650.0 mg Starch 240.0 mg Gelatin 10.0 mg 100.0 mg 2 ″ -O-glucosylisovitexin, lactose and starch were uniformly mixed, After adding a small amount of water and further mixing, the mixture is granulated and dried. (Particle size 0.8 mm columnar granules) Example D: Capsule granules are made according to the following formulation, enteric coated, and filled in capsules.

Granules (particle diameter 0.8 mm, columnar granules) 2 ″ -O-glucosylisovitexin 20.0 mg lactose 130.0 mg starch 48.0 mg gelatin 2.0 mg 200.0 mg enteric coating (coating amount 400 mg /
g granules) The formulation is as in Example B above.

Capsule filling Gelatin capsule No. 2 is filled with 200 mg.

Example E: Lozenge Agent 2 ″ -O-glucosylisovitexin 100.0 mg Sucrose 820.0 mg Gum arabic 79.0 mg Purified water appropriate amount 100.0 mg 2 ″ -O-glucosylisovitexin and sucrose uniformly The mixture was mixed, gum arabic was dissolved in a small amount of purified water, and the mixture was added and kneaded to form granules, which were then dried and compressed into a troche.

Example F: Suppository 2 ″ -O-Glucosylisovitexin 15.0 g Polyoxyethylene lauryl ether (21 E.O.) 25.0 g Polyoxyethylene sorbitan monostearate (6 E.O.) 90.0 g Lipophilic glyceryl monostearate 10.0 g Polyethylene glycol 400 10.0 g Polyethylene glycol 4000 Appropriate amount of polyoxyethylene lauryl ether, polyoxyethylene sorbitan monostearate, lipophilic glyceryl monostearate, polyethylene glycol 400 and polyethylene glycol 4000 at 60 ° C. After heating to dissolve and then cooling to 45 ° C., 2 ″ -O-glucosylisovitexin was added thereto and uniformly mixed, and then molded into 2 g of suppository with a suppository molding machine.

Example G: Microcapsule Lactose 840.0 g 2 ″ -O-glucosylisovitexin 100.0 g Eudragit RS 50.0 g Magnesium stearate 10.0 g 100.0 g Lactose is put into a vacuum mixing and drying coating device. Then, while rotating and mixing, the mixture was heated to about 50 ° C., the inside of the tank was evacuated by a vacuum pump, and coating was performed with a uniform dispersion of particles prepared by using a fine powder of 2 ″ -O-glucosylisovitexin. Ivy. After coating, Eudragit R
Coating with S (Roehm Pharma Co., Ltd.) in methylene chloride solution (containing 1% magnesium stearate based on the total weight of the product) was also performed under vacuum to obtain 2 ″ -O-glucosylisovitexin microcapsules. .

[Brief description of drawings]

FIG. 1 is a graph showing the effect of 2 ″ -O-glucosylisovitexin on the lethal effect of glucose oxidase on cultured neonatal mouse cortical neurons.

FIG. 2 is a graph showing the effect of 2 ″ -O-glucosylisovitexin on learning impairment due to cerebral ischemia.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomohisa Ninomiya 4-20-17 Mihama, Urayasu City, Chiba Prefecture (72) Inventor Nobuyoshi Sanakin 686-111 Fujishin, Kashiwa City, Chiba Prefecture (72) Tsuyoshi Uruno Nagareyama, Chiba Prefecture 2-23-7 Ichinomiya (72) Inventor Yoshihide Hagiwara 4-14 Hiraisanso, Takarazuka-shi, Hyogo

Claims (1)

[Claims] 1. A cerebral nerve cell disorder inhibitor, which comprises 2 ″ -O-glucosylisovitexin as an active ingredient.