CN111182911A - Composition for protecting retinal nerve cells comprising processed product of cowberry fruit and processed product of pine bark - Google Patents

Composition for protecting retinal nerve cells comprising processed product of cowberry fruit and processed product of pine bark Download PDF

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Publication number
CN111182911A
CN111182911A CN201880065056.1A CN201880065056A CN111182911A CN 111182911 A CN111182911 A CN 111182911A CN 201880065056 A CN201880065056 A CN 201880065056A CN 111182911 A CN111182911 A CN 111182911A
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nerve
cells
test substance
processed product
cell
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CN201880065056.1A
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CN111182911B (en
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真木贤太郎
谷口孝纯
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Santen Pharmaceutical Co Ltd
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Santen Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/13—Coniferophyta (gymnosperms)
    • A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

The present invention aims to find a novel composition which inhibits nerve cell death, particularly retinal nerve cell death, and has an effect of protecting retinal nerve cells and protecting the optic nerve. A composition comprising a processed product of cowberry fruit and a processed product of pine bark remarkably inhibits nerve cell death induced by various causes. Therefore, the composition containing the processed product of the blueberry fruit and the processed product of the pine bark is expected to be useful as a composition for protecting retinal nerve cells or a composition for protecting optic nerve due to significant inhibition of nerve cell death, particularly retinal nerve cell death.

Description

Composition for protecting retinal nerve cells comprising processed product of cowberry fruit and processed product of pine bark
Technical Field
The present invention relates to a composition for protecting retinal nerve cells and/or for protecting optic nerve comprising a processed product of blueberry fruit, a processed product of pine bark or a combination thereof, and a composition for protecting nerve cells and/or for protecting nerve comprising a processed product of blueberry fruit and a processed product of pine bark.
Background
The retina is a tissue having a thickness of 0.1 to 0.5mm, and is composed of 10 layers, i.e., an inner limiting membrane, a nerve fiber layer, a ganglion cell layer, an inner plexiform layer, an inner granular layer, an outer plexiform layer, an outer granular layer, an outer limiting membrane, a visual cell layer, and a retinal pigment epithelium layer, and includes a group of retinal nerve cells such as visual cells, bipolar cells, ganglion cells, horizontal cells, amacrine cells, and muller cells.
The retinal nerve cell population plays a very important role in receiving and transmitting visual information such as converting light stimulation into an electrical signal and transmitting the electrical signal to the brain.
In detail, the transmission mechanism electrically signals visual information entering from the eye through the visual cells, and transmits the visual information to the ganglion cells via horizontal cells, bipolar cells and/or amacrine cells. This electrical signal is then conducted to the brain via the optic nerve (which is a bundle of optic nerve fibers that contain axons of ganglion cells).
However, when retinal nerve cells and the optic nerve are damaged by various causes, persistence of the retinal nerve cells and the optic nerve cannot be maintained, and conduction of visual information to the brain is hindered, resulting in visual field damage. For example, in non-patent document 1, it is known that visual field damage accompanied by functional impairment of retinal nerve cells occurs in various retinal diseases such as retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, normal tension glaucoma, macular degeneration, retinitis pigmentosa, and leber's disease.
in addition, as a drug for inhibiting retinal nerve cell death, for example, patent document 1 discloses an optic nerve protecting effect of tafluprost which is one of prostaglandin derivatives, patent document 2 discloses an optic nerve protecting effect of ebselen, patent document 3 discloses a retinal nerve cell protecting agent containing nipropranol which is one of β blockers as an active ingredient, and patent document 4 discloses an α -form such as bunazosin1As an optic ganglion cell protective agent containing a receptor blocker as an active ingredient, the neuroprotective activity of latanoprost, which is one of prostaglandin derivatives, is known in non-patent document 2, and the neuroprotective activity of ONO-AE1-259-01, which is a prostaglandin EP2 receptor agonist, is known in non-patent document 3 or non-patent document 4.
On the other hand, as for the fruit of cowberry, it has an antioxidant effect, a blood flow improving effect, etc., and contains abundant anthocyanins purportedly beneficial to the eyes, so that a supplement for the eyes containing an extract of the fruit thereof is widely known, for example, a supplement containing an extract of cowberry fruit (Mirtoselect (registered trademark)) is widely sold.
In addition, pine bark, particularly french maritime pine bark, is rich in polyphenols having an antioxidant effect, such as procyanidins, flavonoids, and catechins, and therefore, extracts of the bark are widely known as cosmetics and supplements, and for example, supplements containing french maritime pine bark extract (Pycnogenol (registered trademark)) are widely sold.
Further, it is known that a composition containing an extract of cowberry fruit (Mirtoselect (registered trademark)) and an extract of maritime pine bark (Pycnogenol (registered trademark)) which is widely sold in the form of Mirtogenol (registered trademark) shows an intraocular pressure-lowering effect on healthy persons with high intraocular pressure.
However, there have been no reports on the effect of protecting retinal nerve cells and/or protecting the optic nerve and on the effect of preventing and/or treating glaucoma without intraocular pressure increase in a composition containing a processed product of a blueberry fruit and a processed product of pine bark.
Documents of the prior art
Patent document
Patent document 1: japanese patent laid-open publication No. 2006-306862
Patent document 2: japanese laid-open patent publication No. 2009-227671
Patent document 3: japanese patent laid-open No. 2001-072591
Patent document 4: international publication No. 03/004058
Patent document 5: japanese Kohyo publication Hei 3-501025
Non-patent documents:
non-patent document 1: bull, 2004, 62(6), 447-453
Non-patent document 2: experimental Eye Res, 2001, 72, 479-486
Non-patent document 3: eur.j.pharmacol, 2007, 570, 135-141
Non-patent document 4: eur.j.pharmacol, 2009, 616, 64-67
Disclosure of Invention
Problems to be solved by the invention
The present invention addresses the problem of finding a novel composition that is useful for inhibiting nerve cell death and/or a decrease in the function of nerve cells, in particular, retinal nerve cell death and/or a decrease in the function of retinal nerve cells, has an effect of protecting retinal nerve cells and/or an optic nerve, and is useful for maintaining visual function and preventing and/or improving optic nerve damage.
Means for solving the problems
the present inventors have focused on nerve cell death and/or nerve cell function reduction (attenuation) induced by various causes (1. vesicle stress, 2. excitotoxicity, 3. trophic factor starvation stress, 4. oxidative stress, 5. inflammatory stress, 6. amyloid β 1-42, 7. amyloid β 25-35), and have studied the effects of many ingredients derived from natural substances and their combinations on nerve cell death and/or function reduction of retinal nerve cells.
Namely, the present invention provides the following embodiments.
[ means A ]
[A1] A composition for protecting retinal nerve cells and/or protecting optic nerve, which comprises a processed product of cowberry fruit and a processed product of pine bark.
[A2] The composition as described in [ A1], wherein the processed product of the blueberry fruit is a blueberry fruit extract.
[A3] The composition according to [ A1], wherein the processed product of pine bark is a pine bark extract.
[A4] The composition according to [ A3], wherein the extract of pine bark is French maritime pine bark extract.
[A5] The composition according to any one of [ A1] to [ A4] above, which is for oral or parenteral administration.
[A6] The composition according to any one of [ A1] to [ A5] above, which is a pharmaceutical composition.
[A7] The composition according to any one of [ A1] to [ A5] above, which is a food composition.
[A8] The composition according to [ A7], wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
[A9] A method for protecting retinal nerve cells and/or protecting optic nerve, characterized in that a composition comprising a therapeutically effective amount of a processed product of cowberry fruit and a processed product of pine bark is administered to a patient.
[A10] A composition comprising a processed product of cowberry fruit and a processed product of pine bark for use in protecting retinal nerve cells and/or protecting optic nerve.
[A11] Use of a composition comprising a processed product of a cowberry fruit and a processed product of pine bark in the manufacture of a medicament for protecting retinal nerve cells and/or protecting the optic nerve.
In addition, two or more of the above-described structures [ a1] to [ a11] may be arbitrarily selected and combined.
[ means B ]
[B1] A composition for protecting retinal nerve cells and/or protecting optic nerve comprises a processed product of cowberry fruit.
[B2] The composition as described in [ B1], wherein the processed product of the blueberry fruit is a blueberry fruit extract.
[B3] The composition as described in the above [ B1] or [ B2], which is for oral or parenteral administration.
[B4] The composition according to any one of [ B1] to [ B3] above, which is a pharmaceutical composition.
[B5] The composition according to any one of [ B1] to [ B3] above, which is a food composition.
[B6] The composition according to [ B5], wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
[B7] A method for protecting retinal nerve cells and/or protecting optic nerve, characterized in that a therapeutically effective amount of a processed product of cowberry fruit is administered to a patient.
[B8] Processed product of cowberry fruit for use in protecting retinal nerve cells and/or protecting the optic nerve.
[B9] Use of a processed product of a cowberry fruit for the manufacture of a medicament for protecting retinal nerve cells and/or protecting the optic nerve.
In addition, two or more of the above-described structures [ B1] to [ B9] may be arbitrarily selected and combined.
[ means C ]
[C1] A composition for protecting retinal nerve cells and/or protecting optic nerve, which comprises a processed product of pine bark.
[C2] The composition according to [ C1], wherein the processed product of pine bark is a pine bark extract.
[C3] The composition according to [ C2], wherein the extract of pine bark is French maritime pine bark extract.
[C4] The composition according to any one of the above [ C1] to [ C3], which is for oral or parenteral administration.
[C5] The composition according to any one of [ C1] to [ C4] above, which is a pharmaceutical composition.
[C6] The composition according to any one of [ C1] to [ C4] above, which is a food composition.
[C7] The composition according to [ C6], wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
[C8] A method for protecting retinal nerve cells and/or protecting the optic nerve, characterized by administering a therapeutically effective amount of a processed product of pine bark to a patient.
[C9] A processed product of pine bark for use in protecting retinal nerve cells and/or protecting the optic nerve.
[C10] Use of a processed product of pine bark in the manufacture of a medicament for protecting retinal nerve cells and/or protecting the optic nerve.
In addition, two or more of the above-described structures [ C1] to [ C10] may be optionally selected and combined.
Namely, the present invention provides the following embodiments.
[ means D ]
[D1] A composition for protecting retinal nerve cells and/or protecting the optic nerve comprising a combination of a processed product of a cowberry fruit and a processed product of pine bark.
[D2] The composition as described in [ D1], wherein the processed product of the blueberry fruit is a blueberry fruit extract.
[D3] The composition according to [ D1], wherein the processed product of pine bark is a pine bark extract.
[D4] The composition according to [ D3], wherein the pine bark extract is French maritime pine bark extract.
[D5] The composition according to any one of [ D1] to [ D4] above, which is for oral or parenteral administration.
[D6] The composition according to any one of [ D1] to [ D5] above, which is a pharmaceutical composition.
[D7] The composition according to any one of [ D1] to [ D5] above, which is a food composition.
[D8] The composition according to [ D7], wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
[D9] A method for protecting retinal nerve cells and/or protecting optic nerve, characterized by administering to a patient a therapeutically effective amount of a processed product of cowberry fruit and a processed product of pine bark in combination.
[D10] The method according to [ D9], wherein the processed product of the blueberry fruit and the processed product of the pine bark are used in combination in a therapeutically effective amount.
[D11] The method according to [ D10], wherein the therapeutically effective amounts of the processed product of cowberry fruit and the processed product of pine bark are administered simultaneously or sequentially (separately with time variation).
[D12] A combination of a processed product of cowberry fruit and a processed product of pine bark for use in protecting retinal nerve cells and/or protecting the optic nerve.
[D13] Use of a combination of a processed product of a cowberry fruit and a processed product of pine bark in the manufacture of a medicament for protecting retinal nerve cells and/or protecting the optic nerve.
In addition, two or more of the above-described structures [ D1] to [ D13] may be arbitrarily selected and combined.
[ means E ]
[E1] A composition for protecting nerve cells and/or nerves comprises a processed product of cowberry fruit and a processed product of pine bark.
[E2] The composition as described in [ E1], wherein the processed product of the blueberry fruit is a blueberry fruit extract.
[E3] The composition according to [ E1], wherein the processed product of pine bark is a pine bark extract.
[E4] The composition according to [ E3], wherein the extract of pine bark is French maritime pine bark extract.
[E5] The composition according to any one of [ E1] to [ E4] above, which is for oral or parenteral administration.
[E6] The composition according to any one of [ E1] to [ E5] above, which is a pharmaceutical composition.
[E7] The composition according to any one of [ E1] to [ E5] above, which is a food composition.
[E8] The composition according to [ E7], wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
[E9] A method for protecting nerve cells and/or protecting nerves, characterized in that a composition containing therapeutically effective amounts of a processed product of cowberry fruit and a processed product of pine bark is administered to a patient.
[E10] A composition comprising a processed product of cowberry fruit and a processed product of pine bark for use in protecting nerve cells and/or protecting nerves.
[E11] Use of a composition comprising a processed product of a cowberry fruit and a processed product of pine bark in the manufacture of a medicament for protecting nerve cells and/or protecting nerves.
In addition, two or more of the above-described structures [ E1] to [ E11] may be optionally selected and combined.
[ means F ]
[F1] A composition for protecting nerve cells and/or protecting nerves, comprising a combination of a processed product of a blueberry fruit and a processed product of pine bark.
[F2] The composition as described in [ F1], wherein the processed product of the blueberry fruit is a blueberry fruit extract.
[F3] The composition according to [ F1], wherein the processed product of pine bark is a pine bark extract.
[F4] The composition according to [ F3], wherein the extract of pine bark is French maritime pine bark extract.
[F5] The composition according to any one of the above [ F1] to [ F5], which is for oral or parenteral administration.
[F6] The composition according to any one of [ F1] to [ F5] above, which is a pharmaceutical composition.
[F7] The composition according to any one of [ F1] to [ F5] above, which is a food composition.
[F8] The composition according to [ F7], wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
[F9] A method of protecting nerve cells and/or protecting nerves, characterized by administering to a patient a therapeutically effective amount of a processed product of a blueberry fruit in combination with a processed product of pine bark.
[F10] The method according to [ F9], wherein the processed product of the cowberry fruit and the processed product of the pine bark are used in combination in a therapeutically effective amount.
[F11] The method according to [ F10], wherein the therapeutically effective amounts of the processed product of cowberry fruit and the processed product of pine bark are administered simultaneously or sequentially (separately with time variation).
[F12] A combination of a processed product of cowberry fruit and a processed product of pine bark for use in protecting nerve cells and/or protecting nerves.
[F13] Use of a combination of a processed product of a cowberry fruit and a processed product of pine bark in the manufacture of a medicament for protecting nerve cells and/or protecting nerves.
In addition, two or more of the above-described structures [ F1] to [ F13] may be arbitrarily selected and combined.
Effects of the invention
According to the present invention, since the effect of protecting retinal nerve cells and/or protecting the optic nerve can be exerted by administering the processed product of the cowberry fruit and the processed product of the pine bark, either individually or in combination or combined preparation form, maintenance of the optic function and prevention, inhibition and/or improvement of optic nerve damage can be expected, and application as a pharmaceutical composition and a food composition (particularly, use as a supplement) can be expected.
Drawings
FIG. 1 is a graph showing the cytoprotective effects of the test substance A-added group, the test substance B-added group, and the test substance A + test substance B-added group.
Detailed Description
Hereinafter, embodiments of the present invention will be described in more detail.
One embodiment of the present invention is a composition for protecting retinal nerve cells and/or protecting optic nerve, which contains a processed product of blueberry fruit and a processed product of pine bark.
In the present invention, "blueberry fruit" refers to fruits of plants of genus Vaccinium (Vaccinium) of family Ericaceae. The kind and the production place of the fruit of a plant of the genus Vaccinium (Vaccinium) of the family Ericaceae are not particularly limited. Examples of fruits such as Vaccinium myrtillus (Vaccinium myrtillus L.), Vaccinium uliginosum (Vaccinium uliginosum L), Vaccinium myrtillus (Vaccinium caespitum Michx), sweet Vaccinium vitis-idaea (Vaccinium delactiosum), Vaccinium membranaceum (Vaccinium membranaceum), and Vaccinium myrtillus (Vaccinium ovalifolium), preferably those containing abundant anthocyanins.
In the present invention, the "processed product of a blueberry fruit" is not particularly limited as long as it is a processed product prepared and produced from a blueberry fruit by a generally known processing method. For example, the processed product of the blueberry fruit can be prepared and manufactured by cutting, crushing, squeezing, extracting, centrifuging, filtering, purifying, drying, etc., the blueberry fruit (pericarp, fruit, pulp, etc.) as required. Further, the blueberry fruit and the processed product thereof can be suitably mixed to prepare and produce the blueberry fruit. In addition, in the preparation and production process, excipients and the like that can be used in pharmaceuticals and foods can be added for preparation and production.
As the processed product of the blueberry fruit in the present invention, an extract of blueberry fruit is preferable, and in addition, a commercially available blueberry fruit extract (Vaccinium myrtillus extract) can be used. In particular, Mirtoselect (registered trademark) manufactured by Indena s.p.a. company, and the like are preferably used. Mirtoselect (registered trademark) is a cowberry fruit extract containing anthocyanins.
The processed product of the blueberry fruit of the present invention can be administered to an adult in an amount of, for example, 5 to 500mg, preferably 10 to 250mg, and more preferably 90mg per day.
The composition of the present invention can be administered to an adult in an amount of, for example, 1 to 1000mg, preferably 1 to 500mg, more preferably 5 to 250mg, particularly preferably 10 to 90mg per day of at least one anthocyanin (also called anthocyanin) (or processed product of blueberry fruit containing the same).
In the present invention, "pine bark" refers to the bark of a plant of the genus Pinus (Pinus) of the family Pinaceae. The species and the place of production are not particularly limited as long as the plant bark of Pinus (Pinus) belonging to Pinaceae is used. Examples thereof include, for example, the bark of French maritime pine (Pinus Martima), New Zealand pine, Finnish pine, shortleaf pine, larch, Pinus thunbergii, Pinus densiflora, Pinus wumerica, Pinus pumila, Pinus sylvestris, slash pine, Caribbean pine, Pinus taeda, Pinus palustris, Pinus taeda, Pinus sylvestris, Pinus margarita, Pinus sylvestris, Pinus massoniana, Pinus albus, and Annedda in Quebec Canada, preferably the bark of French maritime pine, New Zealand pine, and Pinus finnishiki, and particularly preferably the bark of French maritime pine containing abundant procyanidins.
In the present invention, the "processed product of pine bark" is not particularly limited as long as it can be prepared and produced from pine bark by a generally known processing method. For example, a processed product of pine bark can be prepared and produced by cutting, crushing, squeezing, extracting, centrifuging, filtering, purifying, drying, and the like, as necessary. In addition, the composition can be prepared and produced by appropriately mixing two or more pine barks and processed products thereof. In addition, in the preparation and production process, excipients and the like that can be used in pharmaceuticals and foods can be added for preparation and production.
The processed product of pine bark of the present invention is preferably an extract of pine bark, and a commercially available extract of pine bark may be used. In particular, Pycnogenol (registered trademark) manufactured by Horphag Research is particularly preferably used. Pycnogenol (registered trademark) manufactured by Horphag Research is a pine bark extract containing proanthocyanidins.
The amount of the processed product of pine bark of the present invention to be administered to an adult is, for example, 1 to 500mg, preferably 1 to 450mg, more preferably 5 to 120mg, particularly preferably 40mg per day.
The composition of the present invention can be administered to an adult in an amount of, for example, 0.5 to 500mg, preferably 1 to 450mg, more preferably 3 to 120mg, particularly preferably 5 to 40mg per day of at least one procyanidin (or a processed product of pine bark containing the same).
In the present invention, the "retinal nerve cell" refers to a cell present in the retina that is involved in visual functions related to the conduction of visual signals to the brain. Specifically, the term "retinal nerve cell group" refers to an optic cell, a horizontal cell, a bipolar cell, a retinal ganglion cell, an amacrine cell, and a muller cell, and preferably refers to a horizontal cell, a bipolar cell, an optic ganglion cell, and an amacrine cell, which are nerve cells, and particularly preferably refers to a retinal ganglion cell.
In the present invention, "protection of retinal nerve cells" means inhibition of retinal nerve cell death and/or retinal nerve cell function reduction caused by some cause. In addition, it also means prevention of retinal nerve cell death and/or a decrease in retinal nerve cell function that may occur in the future.
In the present invention, the "optic nerve" refers to a bundle of nerve fibers formed by about 100 ten thousand axons extending from retinal ganglion cells and a cell group involved in the conduction of visual information.
In the present invention, "protection of the optic nerve" means maintenance of the optic nerve in a normal function. For example, it is intended to inhibit the optic nerve from being damaged or degenerated for some reason and then causing cell death. In addition, even a physically normal optic nerve is included in the object of optic nerve protection of the present invention if it is an optic nerve that functionally generates an abnormality.
In the present invention, the "nerve cell" refers to a cell that constitutes the nervous system and is specialized in information processing and information transmission, and examples thereof include a sensory nerve cell that responds to light or mechanical stimulation, and a motor nerve cell that outputs to a muscle fiber.
In the present invention, "protecting a nerve cell" means inhibiting death of a nerve cell and/or reduction in function of a nerve cell caused by some cause. In addition, it also means to prevent nerve cell death and/or nerve cell function reduction which may occur in the future.
In the present invention, "nerve" refers to a tissue that governs the function of the body and transmits stimulation. Examples thereof include central nerves (brain and spinal cord), peripheral nerves (brain and spinal nerves and autonomic nerves).
In the present invention, "protecting nerves" means maintaining normal functions of nerves. For example, it is intended to inhibit nerve damage or degeneration and cell death due to some cause. In addition, even a physically normal nerve is included in the neuroprotective object of the present invention if it is a nerve that functionally generates an abnormality.
The composition for protecting retinal nerve cells and/or protecting optic nerves of the present invention is useful for preventing and/or treating eye diseases involving damage to retinal nerve cells and/or damage to optic nerves. More specifically, the compound is useful for preventing and/or treating eye diseases such as visual field abnormality, retinal vessel occlusion, diabetic retinopathy, ischemic optic neuropathy, glaucoma with no increase in intraocular pressure, normal tension glaucoma, glaucomatous optic neuropathy, glaucomatous field stenosis, glaucomatous optic atrophy, PPG (pre-ocular glaucoma), age-related macular degeneration, retinal pigment degeneration, Leber's disease, retinopathy of prematurity, retinal detachment, and retinal pigment epithelium detachment.
As used herein, "patient" refers to humans and other animals, such as dogs, cats, horses, and the like. The patient is preferably a mammal, more preferably a human.
In the present invention, a "therapeutically effective amount" refers to the amount of active ingredient that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher or physician. In addition, when administered to a mammal for the treatment of a disease, "therapeutically effective amount" also includes the meaning of the amount of the effective ingredient (processed product of cowberry fruit, processed product of pine bark) sufficient to effect the treatment for the disease. The "therapeutically effective amount" may be suitably varied depending on the effective ingredient, the disease and its severity, and the age, body weight, etc. of the mammal to be treated.
Also, "effective amount" includes the meaning of the amount of the effective ingredient (processed product of blueberry fruit, processed product of pine bark) that produces acute or chronic therapeutic effect when an appropriate dose is administered. As the therapeutic effect, there can be mentioned the prevention, correction, inhibition or reversal to a detectable degree of the symptoms, signs and underlying pathology of the disease/condition and related complications.
As the effective amount, there may be mentioned an amount of the processed product of the blueberry fruit or the processed product of the pine bark of the present invention alone, an amount of the processed product of the blueberry fruit and the processed product of the pine bark of the present invention in combination, and/or an amount of the effective ingredient of the present invention in combination with other effective ingredients.
In the present invention, the combination ratio of the processed product of the blueberry fruit and the processed product of the pine bark is not particularly limited and may be appropriately selected to achieve the effect of protecting retinal nerve cells or protecting optic nerve. The combination ratio is not particularly limited, and may be appropriately selected, for example, within a range of 1: 100 to 100: 1, preferably 1: 50 to 5: 1, more preferably 1: 30 to 3: 1, further preferably 1: 2 to 3: 1, further preferably 1: 1 to 5: 2, specifically preferably 2: 1 or 9: 4, and particularly preferably 9: 4.
In the combination of the processed product of blueberry fruit and the processed product of pine bark of the present invention, the two components may be administered separately or together as a single preparation. In addition, one of the components of the combination of the invention may be administered in advance, simultaneously or subsequently with respect to the other components. These ingredients can be prepared into pharmaceutical preparations in single dosage form or in separate dosage forms.
Examples of the combination of the processed product of the blueberry fruit and the processed product of the pine bark of the present invention are not particularly limited, and the following forms (1) and (2) may be mentioned.
(1) A single preparation (a composition, a compound preparation) containing both the processed product of cowberry fruit and the processed product of pine bark; and
(2) the preparation (composition) containing the processed product of cowberry fruit and the preparation (composition) containing the processed product of pine bark have forms as separate preparations. .
In the case of the form according to (2), the preparations may be administered simultaneously or sequentially (separately at appropriate time intervals), and an appropriate administration schedule may be employed to achieve the effect of protecting retinal nerve cells or protecting optic nerves.
In the case of preparing the above components into a single formulation, there is no particular limitation, and the processed product of pine bark may be mixed in a ratio of 0.01 to 100 parts by weight, preferably 0.1 to 50 parts by weight, more preferably 0.3 to 20 parts by weight, per 1 part by weight of the processed product of blueberry fruit. The single preparation is not particularly limited, and is contained in an amount of 0.1 to 99% by weight based on the total amount of the active ingredients of the preparation, relative to the composition of the preparation.
The composition of the present invention may be appropriately compounded with other effective ingredients (e.g., substances having auxiliary effects) and additives by a method known per se as long as the effect of protecting retinal nerve cells or protecting optic nerves is not hindered.
examples of other active ingredients that can be incorporated into the composition of the present invention include carotenoids such as vitamin A, xanthophyll, zeaxanthin, racemic zeaxanthin and astaxanthin, polyphenols such as vitamin B, vitamin C, vitamin D, vitamin E, tocotrienols, glutathione and derivatives or salts thereof, proteins and derivatives and hydrolysates selected from collagen, elastin, fibronectin and keratin, α -hydroxy acids such as glycolic acid, lactic acid, malic acid, citric acid and salicylic acid, α -hydroxy acids and corresponding derivatives or salts thereof, albumin serum (proteopeptide), spleen, chicken crown, linolenic acid, yeast, xylitol, bifidobacterium, ganoderma, swertia powder (swertia), rosemary, chamomile, aloe, vitamin A, vitamin E, vitamin D, vitamin E, vitamin A, vitamin E, vitamin A, vitamin E, vitamin A, vitamin E, vitamin A, vitamin E, vitamin A, vitamin E.
The other active ingredient may be incorporated in an amount of, for example, 0.1 to 99% by weight, preferably 1 to 90% by weight, more preferably 5 to 80% by weight, still more preferably 10 to 70% by weight, and particularly preferably 20 to 60% by weight, based on the total weight of the composition. In addition, two or more other active ingredients may be used in combination.
The additive that can be incorporated into the composition of the present invention is not particularly limited as long as it is an additive acceptable as a medicine or food. Examples thereof include excipients, thickeners, and emulsifiers (beeswax and glycerin fatty acid ester).
The compositions of the invention may be administered orally or parenterally (e.g., eye drops, vitreous, subconjunctival, Tech's capsule, subcutaneously, intramuscularly, intracamerally, into the optic nerve, or intravenously). The composition of the present invention may be mixed with additives such as an excipient, a thickener, an emulsifier, etc. to prepare a desired formulation. The dosage form of the composition of the present invention is not particularly limited, and examples thereof include forms of supplements such as soft capsules, hard capsules, liquid preparations (including eye drops, vitreous injections, and the like), jellies, soft candy forms, tablets, powders, and jellies.
The composition of the present invention can be used as a pharmaceutical and a food (including a pharmaceutical for non-human mammals). In the present invention, the amount of the composition of the present invention to be incorporated into a medicine or food is not particularly limited, and can be suitably determined depending on the purpose of application (target disease, symptom type, etc.), the subject to which the composition is applied (human, non-human animal (preferably mammal, particularly preferably dog, cat, etc.)), the sex and age of the subject to which the composition is applied, the form of the medicine or food, the method of administration or ingestion thereof, the frequency, preference, and the like.
In the present invention, when the composition of the present invention is prepared into a medicine (a medicine, a quasi-medicine, etc.), the processed product of the cowberry fruit and the processed product of the pine bark may be combined with other effective components, pharmaceutically acceptable carriers and additives. For example, binders, disintegrants, lubricants, wetting agents, buffers, preservatives, flavors, and the like, as pharmaceutically acceptable carriers and additives, may be mixed to prepare a desired dosage form. The dosage form of the drug is not particularly limited, and examples thereof include injections, external preparations, inhalants, suppositories, films, troches, liquid preparations, powders, tablets, granules, capsules, syrups, eye drops, eye washes, and nasal drops. Among these dosage forms, dosage forms suitable for oral administration (i.e., pharmaceuticals for oral administration) are preferable, and, for example, troches, liquid preparations, powders, tablets, granules, capsules, soft capsules, syrups are particularly preferable. These dosage forms can be used as, for example, pharmaceuticals for protecting retinal nerve cells or protecting optic nerves.
In the present invention, when the composition of the present invention is prepared into a food composition (health food, functional food, nutritional supplement food, health functional food, food for specified health use, nutritional functional food, functional label food, etc.), the processed product of the blueberry fruit and the processed product of the pine bark may be mixed with a sweetener, a coloring agent, a preservative, a thickener, a stabilizer, a gelling agent, a paste, an antioxidant, a coupler, a bleaching agent, an antifungal agent (an antifungal agent), a yeast nutrient (yeastfood), a gum base, a perfume, an acidulant, a seasoning, an emulsifier, a pH adjuster, salt water (salt water), a leavening agent, a nutrient enhancer, or other raw materials for food and beverage to be prepared into a desired form. The form of the composition of the present invention is not particularly limited, and examples thereof include supplement-type foods such as jellified agents, granules, fine granules, capsules (hard capsules, soft capsules, etc.), tablets, powders, liquid preparations, semisolid agents, and the like; beverages such as carbonated beverages, soft drinks, milk drinks (milk drinks), alcoholic beverages, fruit juice drinks, teas, and nutritional drinks; powdered beverages such as powdered fruit juice and powdered soup; confectionery such as chewing gum (gum), tablet candy (candy), cookie (cookie), soft candy (gummi), rice cake (rice cracker), biscuit (bisguit), and jelly (jelly); bread, noodles, cereals (cereal), jam, flavoring agent, etc. The above forms can be used as foods and drinks having an effect of protecting retinal nerve cells or an effect of protecting optic nerves, for example, they can be used as nutraceuticals such as nutritional supplement foods, functional marker foods, foods for specified health uses, and foods for patients, in addition to ordinary foods and drinks.
When the composition of the present invention is a food composition, the composition may be indicated as "supporting the operation of the optic nerve", "enhancing the optic nerve and the like", "repairing the optic nerve and the like", "recovering the optic nerve and the like", "nutritionally supporting the optic nerve protection", "effective for recovering from disorder of the optic nerve system", "supporting the operation of the retinal nerve cell", "strengthening the retinal nerve cell and the like", "repairing the retinal nerve cell and the like", "recovering the retinal nerve cell and the like", "nutritionally supporting the retinal nerve cell protection", "effective for recovering from disorder of the retinal nerve cell", "strengthening the nerve and the like", "repairing the nerve and the like", "recovering the nerve and the like", "nutritionally supporting the nerve", or the like, "support neuroprotection from a nutritional aspect", "effective for recovery from a disorder of the nervous system", "strengthening nerve cells and the like", "repair nerve cells and the like", "recover nerve cells and the like", "support nerve cells from a nutritional aspect", "support nerve cell protection from a nutritional aspect", "effective for recovery from a disorder of the nerve cell system", and the like; preferably, "support of the operation of the optic nerve", "enhancement of the optic nerve and the like", "repair of the optic nerve and the like", "recovery of the optic nerve and the like", "support of the optic nerve from the trophic aspect", "support of the optic nerve protection from the trophic aspect", "effective for recovery from disorder of the optic nerve system", "support of the operation of the retinal nerve cell", "enhancement of the retinal nerve cell and the like", "repair of the retinal nerve cell and the like", "recovery of the retinal nerve cell and the like", "support of the retinal nerve cell from the trophic aspect", "support of the retinal nerve cell protection from the trophic aspect", "effective for recovery from disorder of the retinal nerve cell" and the like may be denoted by the similar contents as described above. The indication may be direct or indirect, and examples of direct indication include description of the product itself, package, container, label (1abel), label (tag), and the like on the package, and examples of indirect indication include activities such as advertisement/promotion/doctor's instruction, and the like, including transaction document, instruction manual, attached document, catalog, website, store front, exhibition, signboard, bulletin board, newspaper, magazine, television, broadcast, and e-mail.
The effects of "protecting retinal nerve cells", "protecting optic nerves", "protecting nerve cells" and "protecting nerves" according to the present invention can be confirmed by performing pharmacological tests using nerve cells or nerve-like cells such as PC-12 cells derived from rat adrenal brown cell tumor, SH-SY5Y cells derived from human neuroblastoma, Neuro2a cells derived from mouse neuroblastoma, primary cultured neurons, rat retinal cells, primary cultured retinal ganglion cells, and the like.
The effects of "protecting retinal nerve cells", "protecting optic nerves", "protecting nerve cells" and "protecting nerves" according to the present invention can be confirmed by, for example, pharmacological tests a to I and the following examples 1 to 10.
In the present invention, pharmacological test a is an "effect test against the cell death or nerve cell death induced by vesicle stress and/or the decrease (attenuation) of the function of the cell or nerve cell", and can be carried out, for example, according to the methods described in the following documents 1 to 4. According to the present invention, it is possible to inhibit, in a neuron-like cell or a nerve cell, neuron-like cell death or nerve cell death induced by vesicle stress and/or a decrease (attenuation) in the function of a neuron-like cell or a nerve cell. Also, according to the present invention, it is possible to significantly suppress vesicle-like body stress-induced nerve cell death or nerve cell death and/or a decrease (attenuation) in the function of a nerve cell or nerve cell in the nerve cell or nerve cell.
Yamauchi, M et al, Crocetin preservation differentiation induced negative and end plastic bacterium strains virus inhibition of caspase. European Journal of pharmacy.650 (2011)110-119
Wu, L et al, Exendin-4protects HUVECs from transformed apoptosis via inhibiting the IRE1a/JNK/caspase-3pathway Endocrine (2017) 55: 764-772
(2009) 144-150. J. Toxicolog.30. Miyake K and Nagai K, Inhibition of a-semiconducting acidic strain-induced neuronal cell loss. neuron will be described
Zou, C et al, The Molecular Mechanism of endo-biological Stress-Induced Apoptosis in PC-12 neural Cells: the Protective Effect of Insulin-Like Growth Factor I.Endocrinology, January 2009, 150 (1): 277-285
In the present invention, pharmacological test B is a "test of effect on neuronal-like cell death or nerve cell death induced by excitotoxicity and/or a decrease (attenuation) in the function of neuronal-like cells or nerve cells", and can be carried out, for example, according to the method described in the following document 5. According to the present invention, it is possible to inhibit excitotoxicity-induced neuronal cell death or nerve cell death and/or a decrease (attenuation) in the function of neuronal cells or nerve cells in neuronal cells or nerve cells. Further, according to the present invention, it is possible to significantly suppress excitotoxicity-induced neuronal cell death or neuronal cell death and/or a decrease (attenuation) in the function of neuronal cells or neuronal cells in neuronal cells or neuronal cells.
Penugonda, S et al, Effects of N-acetyl cysteine amide (NACA), a novel thiolantation obtained in a sodium glutamate-induced cytotoxicity in a neuronal cell line C12 brain research 1056(2005)132-138
In the present invention, pharmacological test C is an "effect test on neuronal cell death or nerve cell death induced by trophic factor starvation stress and/or a decrease (attenuation) in the function of neuronal cells or nerve cells", and can be carried out, for example, according to the method described in the following document 6. According to the present invention, it is possible to suppress, in a nerve cell or a nerve cell, nerve cell death or nerve cell death induced by trophic factor starvation stress and/or a decrease (attenuation) in function of a nerve cell or a nerve cell. Also, according to the present invention, it is possible to significantly suppress, in a nerve cell or a nerve cell, nerve cell death or nerve cell death induced by trophic factor starvation stress and/or a decrease (attenuation) in function of a nerve cell or a nerve cell.
Ji, Z et al, Ginsenoside Re attenate beta-amyloid and serum-free induced neurovirulence in PC12 cells journal of Ethnopharmacology, 107(2006)48-52
In the present invention, pharmacological test D is an "effect test on neuronal-like cell death or nerve cell death induced by oxidative stress and/or a decrease (attenuation) in the function of neuronal-like cells or nerve cells", and can be carried out, for example, according to the method described in the following document 7. According to the present invention, it is possible to inhibit, in a nerve cell or a nerve cell, nerve cell death or nerve cell death induced by oxidative stress and/or a decrease (attenuation) in function of a nerve cell or a nerve cell. Also, according to the present invention, it is possible to significantly suppress, in a nerve cell or a nerve cell, nerve cell death or nerve cell death induced by oxidative stress and/or a decrease (attenuation) in function of the nerve cell or the nerve cell.
110-119 of pharmaceutical Journal of pharmaceutical.650 (2011) from Yamauchi, M et al, Crocetin depression differentiation induced negative and end plastic stress vitamin inhibition of caspase
In the present invention, the pharmacological test E is an "effect test against neuronal cell death or nerve cell death induced by inflammatory stress and/or a decrease (attenuation) in the function of neuronal cells or nerve cells", and can be carried out, for example, according to the method described in the following documents 8 to 12. According to the present invention, it is possible to inhibit nerve cell death or nerve cell death induced by inflammatory stress and/or a decrease (attenuation) in function of a nerve cell or nerve cell in the nerve cell or nerve cell. Also, according to the present invention, it is possible to significantly inhibit, in a nerve cell or a nerve cell, nerve cell death or nerve cell death induced by inflammatory stress and/or a decrease (attenuation) in function of the nerve cell or the nerve cell.
Buntinx, M et al, Cytokine-Induced Cell Death in human Oligodroglucial cells I.Synthesis Effects of IFN-. gamma.and TNF- α on Apoptosis.journal of neuroscience Research 76: 834-845(2004)
Ray, R et al, Inhibition of Tumor Necrosis Factor (TNF- α) -media apoptosis by Hepatitis C Virus Protein The Journal of biological chemistry Vol.273, No.4, Issue of January 23, pp.2256-2259, 1998
Xia, Z et al, N-acetyl cysteine epitopes TNF- α -induced human vascular cell apoptosis and restore eNOS expression, European Journal of Pharmacology 550(2006)134-142
Haviv, R et al, New Growth Factor introduced by moving Necrosis Factor in PC12 cells journal of Neuroscience research.55: 269-277(1999)
Liang, F et al, Puerarin precursors tune reactor- α -induced apoptosis of PC12 cells via activation of the PI3K/Akt signaling pathway, ExPERIMENTAL AND THERAPEUTIC MEDICINE 14: 813-818, 2017
in the present invention, the pharmacological test F is "an effect test against the neuronal-like cell death or nerve cell death induced by amyloid β 1-42 and/or the decrease (attenuation) in the function of the neuronal-like cell or nerve cell", and can be carried out, for example, according to the method described in the following documents 13-14.
Li, X et al, Effect OF β -Amyloid Peptide 1-42 on the cytoprotective active medium by α 7 Nicotinic acid derivatives in Growth Factor-predicted Differentiated PC-12 cells. this JOURNAL OF PHARMACOLOGY AND EXPFRIMFNTAL THERAPEUCS, 307: 675-670, 2003
Sasaki, H et al, inhibition activities of biflavonoids against inhibition antibodies 42 cytoxicity in PC-12 cells bioorganic & Medicinal chemistry letters.25(2015)2831-2833
in the present invention, the pharmacological test G is "an effect test against neuronal-like cell death or nerve cell death and/or a decrease (attenuation) in the function of neuronal-like cells or nerve cells induced by amyloid β 25-35", and can be carried out, for example, according to the method described in the following documents 15-17.
Ye, J, et al, Effect of pure Sweet Potato to antibiotic on β -Amyloid-medial PC-12 cell Death by Inhibition of Oxidative stress, neurohem Res (2010) 35: 357-365. fig.
Ji, Z et al, Ginsenoside Re attenate beta-amyloid and serum-free induced neurologic in PC12 cells. journal of Ethnopharmacology, 107(2006)48-52
Peng, Q et al, Pycnogenol (registered trade Mark) protects nerves from amino- β peptide-induced apoptosis, molecular Brain research.104(2002)55-65
In the present invention, pharmacological test H is "a confirmation test of the effect of protecting retinal nerve cells (retinal ganglion cells, amacrine cells, etc.) or the effect of protecting optic nerves using an NMDA intravitreal injection retinal injury model", and can be carried out, for example, in the following documents 18 to 25. According to the present invention, an effect of protecting retinal nerve cells (retinal ganglion cells, amacrine cells, etc.) or an effect of protecting optic nerves can be obtained in vivo (in vivo).
Matsunaga N et al, Bilberry and its main control ingredients associated with a neutral neurological damage in vitro and in vivo. mol.Nutr. food Res.2009, 53, 869-877
Ohno Y et al, Oral administration of crocetin precursors in a porcine-D-aspartic-semiconductor, European Journal of Pharmacology.690, 2012, 84-89
Maekawa S et al, The neuropathic effect of heperidin in NMDA-induced transcriptional activity by lateral inhibitory activity stress and exogenous polypeptide activity, scientific Reports 7.6885(2017)
Tsutsumi T et al, Potential neuroprotectant Effects of an LSD1Inhibitor in Retinal Ganglion Cells via p38 MAPK Activity. invest OphthalmolVis Sci.2016; 57: 6461-6473
Binda NS et al, PhTx3-4, a Spider Toxin Calcium Channel packer, Reductes NMDA-Induced injection of the Retina
Vicente VG et al, neuroprotectant Effect of tauurodeoxycholic acid N-Methyl-D aliquot-Induced specific growth Cell Generation PLoS ONE10(9)2015
Sakamoto K et al, Protective effect of all-trans retinic acid on NMDA-induced neural cell death in Rat Retina. European Journal of Pharmacology, 635(2010)56-61
Siliprandi R et al, N-methyl-D-aspartic-induced neuropathy in the adolt rat retina, Vis neurosci.1992Jun; 8(6): 567-73
In the present invention, pharmacological test I is "a confirmation test of the effect of protecting retinal nerve cells or the effect of protecting optic nerves using an ischemia-reperfusion retinal injury model", and can be carried out according to the following documents such as 26 to 32. According to the present invention, an effect of protecting retinal nerve cells or an effect of protecting optic nerves in vivo can be obtained.
Yang X et al, Neuroprotective Effects of Crocin Induced by Ischemia/playback in ray Retina, Ophthalmic Res 2015; 54: 157-168
Shimouchi et al, neuroprotectant effect of water-dispersed heparin derivative in J pn J Ophthalmol.2016 January; 60(1): 51-61
Kara S et al, Protective Effect of Hespertin and Naringgen against ions in Ischemia/reproduction-Induced specific Injury in rates, the scientific World journal volume 2014
Yondeda S et al, Topiramate products exutoxic and ischemistry intrate retina, Brain research.967(2003)257-266
Chen B et al, Protective effects of catalyst on regenerative ischemia/regenerative in rates, Experimental Eye research.93(2011)599-606
Wang L et al, Curcumin inhibition neural and Vascular differentiation in regeneration after Ischemia and reproduction in, PLoS ONE. (2011)6(8)
Aydogan S et al, The effect of The clinical on vascular endovenous growth factor and tumor necrosis factor- α levels in nutritional ischemia/nutritional in J/E246: 363-368
With respect to the definitions of the various aspects of the present invention, the respective definitions of the composition containing the processed product of the blueberry fruit and the processed product of the pine bark can be applied.
Examples
The following formulation examples and pharmacological test examples are shown for better understanding of the present invention and do not limit the scope of the present invention.
[ preparation examples ]
Representative formulation examples of the present invention are shown below. In the following formulation examples, the amount of each component added is the content of each capsule when making a soft capsule.
Formulation example 1
Soft capsule
Cowberry fruit extract 80mg
Pine bark extract 40mg
Proper amount of glycerin fatty acid ester
Preparation example 2
Soft capsule
Figure BDA0002439049110000251
[ pharmacological test examples ]
Typical pharmacological tests of the present invention are shown below. The concentrations and final concentrations used in the following may be appropriately selected, or may be used in combination according to the conditions at the time of the test. The contents of the documents and patent documents described in the present specification can also be appropriately cited in the present test.
1. Test compound, reagent, instrument, and apparatus for use
In this pharmacological test, the test compound of table 1, the reagent of table 2, the instrument of table 3, and the device of table 4 were used to carry out the test and evaluation.
[ Table 1]
Figure BDA0002439049110000252
[ Table 2]
Figure BDA0002439049110000261
[ Table 3]
Figure BDA0002439049110000262
[ Table 4]
Figure BDA0002439049110000271
2. Preparation of the culture Medium
1) Preparation of multiplication Medium
To Dulbecco's modified Eagle Medium (#08456-65, Dulbecco's modified Eagle Medium, DMEM) manufactured by Nacalai Tesque, fetal bovine serum SA (#172012, fetal bovine serum, FBS) manufactured by Sigma-Aldrich was added so that the final concentration was 1, 2, 4, 6, 8, 10, 12, or 15%, horse serum (#16050-122, horse serum manufactured by Thermo Fisher scientific (# HS) was added so that the final concentration was 1, 2, 4, 6, 8, 10, 12, or 15%, and a penicillin-streptomycin mixed solution (stabilization) (#32777-44) manufactured by Nacalai Tesque was added so that the final concentration was 1%, the preparation was performed.
2) Preparation of differentiation Medium
Differentiation medium was prepared as follows: to DMEM, NGF 2.5S native mouse protein (#13257019, NGF), manufactured by Thermo Fisher Scientific, was added to a final concentration of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200ng/mL, FBS was added to a final concentration of 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0%, HS was added to a final concentration of 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0%, and a mixed penicillin-streptomycin solution (stabilized) was added to a final concentration of 1%.
3) Preparation of serum-free Medium
The preparation was carried out by adding a penicillin-streptomycin mixed solution (stabilization) to DMEM so that the final concentration was 1%.
4) Preparation of Low serum Medium
The preparation was performed by adding FBS to DMEM so that the final concentration was 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0%, adding HS so that the final concentration was 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0%, and adding a penicillin-streptomycin mixed solution (stabilization) so that the final concentration was 1%.
5) Preparation of test substance-containing Medium
A culture medium containing a test substance A, a culture medium containing a test substance B, or a culture medium containing a test substance A and a test substance B is prepared by adding a powdery test substance A or a powdery test substance B, or a test substance A solution or a test substance B solution to the above-mentioned various culture media. The test substance a solution or the test substance B solution is prepared as follows: dissolving the test substance A or B in a water solution at a final concentration of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 400000, 600000, 800000, 1000000, 2500000, 5000000, 7500000, or 10000000 μ g/mL, or at a concentration of 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3/2, 7/4, 13/4, or 15/4 to obtain a solution, Physiological saline, phosphate buffer, DPBS, acetate buffer, citrate phosphate buffer, borate buffer, tartrate buffer, Tris buffer, dimethyl sulfoxide (DMSO), DMEM, serum-free medium, proliferation medium, differentiation medium, low serum content medium, ethanol, acetone, Tetrahydrofuran (THF), pyridine, Dimethylformamide (DMF), formamide, dioxane, methanol, N-methylpyrrolidone, carboxymethyl cellulose solution, methyl cellulose solution, gum arabic solution, ethyl acetate solution, and acetonitrile. When a test substance-containing medium prepared using a test substance a solution or a test substance B solution is used for a test, a medium prepared by adding a solvent for each test substance to the test substance-containing medium so that the concentration of the solvent for each test substance is the same as the concentration of the solvent for each test substance is used as a control group (medium containing no test substance).
6) Preparation of glutamic acid-containing Medium
L-glutamic acid monosodium salt monohydrate (#49621, glutamic acid) manufactured by Sigma-Aldrich was dissolved in a medium so that the final concentration was 30, 65, 100, 135, 170, 205, 240, or 275mM, and a glutamic acid-containing medium was prepared. The glutamic acid-containing medium added to the medium was prepared for each use and used for the test.
3. Preparation of the solution
1) Preparation of tunicamycin solution
Tunicamycin (# T7765, tunicamycin) from Streptomyces species, manufactured by Sigma-Aldrich, was dissolved in DMSO, DMF, pyridine, distilled water, ethanol, methanol, dioxane, and/or THF to a final concentration of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0, 12.0, 13.0, 14.0, or 15.0 mg/mL to prepare a tunicamycin solution, which was added to the culture medium for the test.
2) preparation of tumor necrosis factor- α (TNF- α) solution
any one of recombinant rat TNF- α (#510-RT-010, TNF- α) manufactured by R & D Systems, or recombinant mouse TNF- α aa 80-235(#410-MT-010, TNF- α) manufactured by R & D Systems is dissolved in BSA/PBS solution of any one of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, or 3.0% to a final concentration of 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500. mu.g/mL, and is dispensed and cryopreserved.
3) preparation of amyloid β 1-42 oligomer solution
amyloid β 1-42, a β, ultra pure, HFIP, recombinant human (# AG968-1MG) manufactured by Merck corporation was dissolved in 1% ammonium hydroxide solution so that the final concentration was 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 mM., and then diluted in a manner of DPBS, calcium-free, magnesium-free (#14190250, DPBS), phosphate buffer or phosphate buffered saline manufactured by Thermo Fisher Scientific corporation to 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 μ M, and incubated at 4 ℃ for 24 hours, thereby preparing amyloid β 1-42 oligomer solution, amyloid β 1-42 oligomer solution prepared at each use was added to the culture medium for the test.
4) preparation of amyloid β 25-35 oligomer solution
amyloid β protein fragments 25-35(#4309-v, Beta amyloid 25-35) prepared by INC, PEPTIDE INSTITUTE, were dissolved using DPBS, phosphate buffer or phosphate buffered saline so that the final concentration was 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3.0mM, incubated at 37 ℃ for 1, 2, 3 or 4 days, thereby preparing amyloid β 25-35 oligomer solutions, and the amyloid β 25-35 oligomer solutions prepared at each use were added to the medium for the experiments.
4. Pharmacological test method and evaluation method
As the cells used in the following pharmacological tests 1 to 7 and 10, PC-12 cells derived from rat adrenal brown cell tumor, which are easily available neuroblastoma, were used, and instead of the PC-12 cells derived from rat adrenal brown cell tumor, neural cells or neuroid cells such as SH-SY5Y cells derived from human neuroblastoma, Neuro2a cells derived from mouse neuroblastoma, primary cultured neurons, rat retinal cells, and primary cultured retinal ganglion cells can be used.
In pharmacological tests 1 to 7 and 10, the differentiation medium used for (1) preparation and differentiation induction of PC-12 cells derived from rat adrenal brown cell tumor and the like may be the same as or different from the differentiation medium used for (2) evaluation of cell survival rate.
Pharmacological test 1[ test for Effect of vesicle-like cell death or nerve cell death induced by vesicular body stress and/or reduction (attenuation) of function of nerve-like cell or nerve cell ]
The test method of pharmacological test 1 is shown below, but this example is for better understanding of pharmacological test 1 and is not intended to limit pharmacological test 1. The pharmacological test can be carried out by the method described in the following documents 1 to 4.
Yamauchi, M et al, Crocetin preservation differentiation induced negative and end plastic bacterium strains virus inhibition of caspase. European Journal of pharmacy.650 (2011)110-119
Wu, L et al, Exendin-4protects HUVECs from transformed apoptosis via inhibiting the IRE1a/JNK/caspase-3pathway Endocrine (2017) 55: 764-772
(2009) 144-150. J. Toxicolog.30. Miyake K and Nagai K, Inhibition of a-semiconducting acidic strain-induced neuronal cell loss. neuron will be described
Zou, C et al, The Molecular Mechanism of endo-biological Stress-Induced Apoptosis in PC-12 neural Cells: the Protective Effect of Insulin-Like Growth Factor I.Endocrinology, January 2009, 150 (1): 277-285
(1) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
PC-12 cells were thawed in BioCoat IV T75 flash with Plugseal cap (#354523, type IV Collagen-coated T-75 Flask), manufactured by Corning Inc., using a growth medium, and subjected to direct heating CO, manufactured by Astec VNCo., Ltd2Multiple gas incubator (# SCA-165DRS, CO)2Incubator) (5% CO)237 ℃ and humidity). The cells were detached from the culture medium by replacing the culture medium with a new growth medium every 2 to 4 days, using a solution of 2.5g/l Trypsin/1 mmol/l EDTA manufactured by Nacalai Tesque corporation, phenol red-containing, filter-sterilized (#32777-44, 0.25% Trypsin-EDTA) at a time point of 60 to 90% confluence, and using the growth medium at 1.0X 103、2.0 x 103、3.0 x 103、4.0x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x 103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103The cells were seeded in a manner of 0.1 mL/well in a BioCoat (trade name) Collagen IV 96-well plate (#354429, type IV Collagen-coated 96-well culture plate) manufactured by Corning Corp. In CO2Incubator (5% CO)237 ℃ C., humidified) overnight, the medium of the plate was changed to a differentiation medium at C02Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then subjecting the cells to the evaluation of cell viability.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was replacedThe following media were used: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380. 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a differentiation medium (100 μ L) of test substance a; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)2At 37 deg.C and wet) Culturing for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and treating with the test substance. Then, equal amounts (100 μ L) of the following medium were added: a differentiation medium comprising a tunicamycin solution of any one of 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μ M; or a tunicamycin solution comprising any of 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μ M and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 100, 180, 140, 100, 360, 400, 180, 380, 320, 380, 320, 180, 380, 320, 200, 380, 200 μ M, 600. 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL; or a tunicamycin solution containing 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μ M and multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2,A differentiation medium for the test substance B at any one of concentrations of 11/4, 3, 13/4, 7/2, 15/4, or 4; or a tunicamycin solution comprising any of 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μ M and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 100, 180, 140, 100, 360, 400, 180, 380, 320, 380, 320, 180, 380, 320, 200, 380, 200 μ M, 600. 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a test substance A and a differentiation medium of a substance B at a concentration obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4, are cultured in a CO differentiation medium2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
b) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: a differentiation medium containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 2, 5, 50, 60, 65, 70, 80,400. 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL; or a differentiation medium containing a test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and treating the test substance. The medium in the plate was then changed to the following medium: contains 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μ M tunicamycin solution; or 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μ M tunicamycin solution and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 0.1, 0.2, 150, 40, 150, 70, 80, 180, 400, 180, 400, 180, 400, 180, 400, 180, 200, 700. 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a differentiation medium for test substance a; or a test substance B obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 7/4, 15/4 or 4, or a test substance B obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 7/2, 15/4 or 4; or a tunicamycin solution comprising 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μ M, and 0, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55,0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 4200, 1400, 5000, 1400, 14000, 4600, 36000, 5500, 55000, 8000, 55000, 2000000, 5500, 2000, 12000, 2000000, 55000, 2000, 12000, 3000, 12000, 7000, 3000, 2000000, 3000, 12000, 7000, 3000, 200, 300, 340, 200, 340, 200, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture of the above a) or b), the cells were cultured in a differentiation medium supplemented with 5, 10, 15 or 20% of living cell assay reagent SF (#07553-15, SF) manufactured by Nacalaitetsque, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 ℃ and humidity), and after 30, 90, 150 or 210 minutes, absorbance (absorption wavelength: 450nm, reference wavelength: 630nm) was measured using a varioska nflash assay Reader (#5250040, plate tester) manufactured by Thermo Fisher Scientific, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from both values to calculate the relative number of living cells. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results and discussion
In the test substance A or test substance B-treated nerve-like cells or nerve cells, the inhibition of vesicle-like cell death or nerve cell death induced by vesicle stress and/or the reduction (attenuation) of the function of the nerve-like cells or nerve cells is suppressed. In addition, in the nerve-like cells or nerve cells treated by combining the test substance a with the test substance B, the cell death or nerve cell death induced by vesicle stress and/or the functional decline (attenuation) of the nerve-like cells or nerve cells are significantly suppressed.
Pharmacological test 2[ test for Effect of excitotoxicity-induced neuronal cell death or neuronal cell death and/or reduction (attenuation) of neuronal cell or neuronal cell function ]
The test method of pharmacological test 2 is shown below, but this example is for better understanding of pharmacological test 2 and is not intended to limit pharmacological test 2. The pharmacological test can be carried out according to the method described in the following documents 5.
Penugonda, S et al, Effects of N-acetyl cysteine amide (NACA), a novel thiolantation obtained in sodium glutamate-induced cytotoxicity in neuronal cell line PC12 brain research 1056(2005)132-138
(1) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 1.0X 103、2.0 x103、3.0 x103、4.0 x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103Individual cells/0.1 mL/well of either type inoculated into type IV collagen coating 96And (4) culturing the plate in a hole. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then subjecting the cells to the evaluation of cell viability.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380. 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a differentiation medium (100 μ L) of test substance a; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1900, 2000, 2200, 2400, 2600, 2800, 2400, 2, 0.4, 0.5, 80, 90, 100, 180, 200,3000. 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a test substance A and a differentiation medium (100 μ L) of a test substance B at a concentration obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours, and treating the test substance. Then, equal amounts (100 μ L) of the following medium were added: a differentiation medium comprising a glutamic acid solution at any one of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, or 260 mM; or a glutamic acid solution containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, or 260mM, and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 1600, 850, 1000, 2000, 3800, 2000, 4400, 2000, 2400, 3000, 2000, 4400, 3000, 200, 1200, 2200, and combinations thereof, 4600. 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL of differentiation medium of test substance A; or glutamic acid in a concentration of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240 or 260mM, and multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1,A differentiation medium for the test substance B at any one of concentrations of 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4; or 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, or 260mM of glutamic acid and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 650, 1600, 190, 200, 4200, 1000, 4400, 200, 300, 200, 2000, 1000, 200, 2000, 4400, 200, 2000, 200, 700, 2000, 6000. 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL, and a differentiation medium of a test substance B at a concentration obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4, in CO2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
b) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: a differentiation medium containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500,1600. 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL; or a differentiation medium containing a test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours, and treating the test substance. The medium in the plate was then changed to the following medium: a differentiation medium comprising a glutamic acid solution at any one of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 mM; or comprises 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120 or 130mM of any one of the above0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360. 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL; or a differentiation medium containing glutamic acid at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130mM and test substance B at a concentration obtained by multiplying the concentration of test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or glutamic acid in an amount of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130mM, and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,/0,/5, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 1300, 1600, 850, 1000, 4200, 1000, 2000, 3000, 2400, 3000, 2200, 1, 0, 4400. 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL of test substance A, andthe differentiation medium containing the test substance B at any one of the concentrations obtained by multiplying the concentration by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4 was cultured in CO2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture of a) or b), the cells were cultured in a differentiation medium supplemented with either 5, 10, 15 or 20% SF, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 ℃ and humidity), and after 30, 90, 150 or 210 minutes, the absorbance (absorption wavelength 450nm, reference wavelength 630nm) was measured using a plate meter, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from the two values, and the relative number of living cells was calculated. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results and discussion
In the nerve-like cells or nerve cells treated with test substance A or test substance B, excitotoxicity-induced nerve-like cell death or nerve cell death and/or a decrease (attenuation) in the function of the nerve-like cells or nerve cells is suppressed. In addition, in the nerve-like cells or nerve cells treated by combining the test substance a with the test substance B, neuronal cell death or nerve cell death induced by excitotoxicity and/or functional decline (attenuation) of the neuronal cells or nerve cells is significantly suppressed.
Pharmacological test 3[ test for Effect of neuronal-like cell death or nerve cell death induced by trophic factor starvation stress and/or reduction (attenuation) of neuronal-like cell or nerve cell function ]
The test method of pharmacological test 3 is shown below, but this example is for better understanding of pharmacological test 3 and is not intended to limit pharmacological test 3. The pharmacological test can be carried out according to the method described in the following documents 6.
Ji, Z et al, Ginsenoside Re attenate beta-amyloid and serum-free induced neurovirulence in PC12 cells journal of Ethnopharmacology, 107(2006)48-52
(1) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 1.0X 103、2.0 x103、3.0 x 103、4.0 x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103Individual cells/0.1 mL/well of either were plated onto type IV collagen-coated 96-well culture plates. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then subjecting the cells to the evaluation of cell viability.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650. 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a differentiation medium (100 μ L) of test substance A; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and treating the test substance. Then, the medium in the plate was removed, washed with DMEM, and replaced with the following medium: a serum-free medium or a low-serum medium (100. mu.L) containing no test substance; or contains 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 89, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a culture medium to be tested for serum addition or for addition of a substance (without a substance in a low-serum content medium (100 μ L); or a serum-free medium or a low-serum medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, The serum-free medium or low-serum medium (100. mu.L) of the test substance B at any one of the concentrations obtained 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 was incubated in CO2Incubator (5% CO)237 ℃ and humidity) under conditions of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22. Any one of 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture in the above a), the cells were cultured in a differentiation medium supplemented with either 5, 10, 15 or 20% SF, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 ℃ and humidity), and after 30, 90, 150 or 210 minutes, the absorbance (absorption wavelength: 450nm, reference wavelength: 630nm) was measured using a plate meter, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from the two values, and the relative number of living cells was calculated. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results and discussion
In a nerve-like cell or a nerve cell treated with a test substance A or a test substance B, the death of the nerve-like cell or the death of the nerve cell induced by trophic factor starvation stress and/or the reduction (attenuation) of the function of the nerve-like cell or the nerve cell is suppressed. In addition, in the nerve-like cells or nerve cells treated by combining the test substance a with the test substance B, the death of nerve-like cells or nerve cells induced by trophic factor starvation stress and/or the reduction (attenuation) of the function of nerve-like cells or nerve cells are significantly suppressed.
Pharmacological test 4[ test for Effect of oxidative stress-induced nerve cell-like death or nerve cell death and/or reduction (attenuation) of function of nerve cell-like or nerve cell ]
The test method of pharmacological test 4 is shown below, but this example is for better understanding of pharmacological test 4 and is not intended to limit pharmacological test 4. The pharmacological test can be carried out according to the method described in the following documents 7.
110-119 of pharmaceutical Journal of pharmaceutical.650 (2011) from Yamauchi, M et al, Crocetin depression differentiation induced negative and end plastic stress vitamin inhibition of caspase
(1) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 1.0X 103、2.0 x103、3.0 x 103、4.0 x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103Individual cells/0.1 mL/well of either were plated onto type IV collagen-coated 96-well culture plates. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then subjecting the cells to the evaluation of cell viability.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 2200, 2400, 2000, 2200, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 4200, 5500, 4600, 5500, 200, 300A differentiation medium (100. mu.L) of the test substance A of any one of 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000. mu.g/mL; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and treating the test substance. Then, equal amounts (100 μ L) of the following medium were added: contains 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.44, 0.46, 0.48, 0.50, 0.52, 0.54, 0.56, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.70, 0.72, 0.74, 0.76, 0.78, 0.80, 0.82, 0.84, 0.86, 0.88, 0.90, 0.92, 0.94, 0.96, 0.98, 1.00, 1.10, 1.20, 1.30, 1.40, 1.90, 2.90, 2.50, 2.30, 2.90, 2.50, 2.30, 2.50, 2.90, 2.30, 2.0. A differentiation medium comprising hydrogen peroxide (#081-04215) produced by Wako pure chemical industries, Ltd, in a concentration of any one of 2.70, 2.80, 2.90, 3.00, 3.20, 3.40, 3.60, 4.00, 4.20, 4.40, 4.60, 5.80, 5.00, 5.20, 5.40, 5.60, 5.80, and 6.00 mM; or 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.44, 0.46, 0.48, 0.50, 0.52, 0.54, 0.56, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.70, 0.72, 0.74, 0.76, 0.78, 0.80, 0.82, 0.84, 0.86, 0.88, 0.90, 0.92, 0.94, 0.96, 0.98, 1.00, 1.10, 1.20, 1.30, 1.40, 1.80, 1.0.80, 2.5, 2.0.0.5, 2.0.0.0, 2.0.5, 2, 2.0.0.0.0.0.5, 2, 2.0.0.0.0.0.0.0.5, 2, 2.0.0.0.0.80, 2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 3, 2, 2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 4, 3, 4, 3.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 85. 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of the substance a to be detected; or 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.44, 0.46, 0.48, 0.50, 0.52, 0.54, 0.56, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.70, 0.72, 0.74, 0.76, 0.78, 0.80, 0.82, 0.84, 0.86, 0.88, 0.90, 0.92, 0.94, 0.96, 0.98, 1.00, 1.10, 1.20, 1.30, 1.40, 1.80, 1.90, 1.80, 1.90, 1.80, 1.70, 1.90, 1.80, 1.90, 1.80, 1.60A culture medium for the test substance B at any one of a concentration of 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2/4, 7/4, 2, 9/4, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 multiplied by the concentration of the test substance A, and a concentration of the test substance B at any one of a concentration of 2.00, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 3.00, 3.20, 3.40, 3.60, 3.80, 4.00 mM, 4.20, 4.40, 4.60, 4.80, 5.20.20, 5.40, 5.60, 5.80 or 6.00 mM; or 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.44, 0.46, 0.48, 0.50, 0.52, 0.54, 0.56, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.70, 0.72, 0.74, 0.76, 0.78, 0.80, 0.82, 0.84, 0.86, 0.88, 0.90, 0.92, 0.94, 0.96, 0.98, 1.00, 1.10, 1.20, 1.30, 1.40, 1.80, 1.0.80, 2.5, 2.0.0.5, 2.0.0.0, 2.0.5, 2, 2.0.0.0.0.0.5, 2, 2.0.0.0.0.0.0.0.5, 2, 2.0.0.0.0.80, 2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 3, 2, 2.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 4, 3, 4, 3.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 85. 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL of a substance A to be detected, and multiplying the concentration of the substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 3/4, 3/2, 7/4, 18000, 2/4, 3/2, 3/4, 3/2, 4, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) of the culture medium5. 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
b) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: a differentiation medium containing no test substance; or any substance tested for differentiation in culture medium comprising 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4200, 5000, 4400, 5000, 1400, 5000, 5500, 5000, 5500, 2000000, 55000, 2000000, 300, 2000, 7000, 2000, 300, 2000, 2000000, 2000; or a differentiation medium containing a test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380. 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a test substance a and a test substance a to be tested.The differentiation medium containing the test substance B at any concentration obtained by multiplying the concentration by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 is subjected to CO treatment2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and treating the test substance. The medium in the plate was then changed to the following medium: a differentiating medium comprising 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.70, 0.43, 0.44, 0.45, 0.46, 0.45, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.70, 1.90, 1.0.0.0.0.0.0.0.0.00, 1.90, 1.0.0.0.0.5, 1.0.0.0.0.0.5, 1.0.0.0.0.5, 1.0.0.0.0.0.0.0.5, 1.0.90, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.; or 0.0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.70, 0.43, 0.44, 0.45, 1.45, 1.0.0.45, 1.0.0.0.0.0.45, 1.0.0.0.0.0.0.0.5, 0.55, 0.5, 0.6, 0.65, 0.70, 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.5, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.5, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.5, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0, 85. 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360,380. 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL; or a substance having a concentration of 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.70, 0.45, 0.70, 1.5, 2.0.0.0.0.5, 1.0.0.0.0.0.5, 1/20, 1.0.0.0.0.0.0.0.5, 1/20, 1.0.0.50, 2.0.0.0.0.0.0.0.5, 1.0.0.0.0.0.0.5, 1/20, 2.0.0.0.0.10, 1.0.0.0.0.10, 1.0.0.0.0.0.0.0.0.0.5, 1.0.0.0.0.0.0.0., 3. A differentiation medium for the test substance B at any one of concentrations of 13/4, 7/2, 15/4, or 4; or 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.70, 0.43, 0.44, 0.45, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.70, 0.0.0.0.0.0.0.0.0.0.0.0.5, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.00, 1, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.00, 1, 1.0.0.0.0.0.0.0.0.0.0.0.0.0.5, 1, 1.0.0.0.0.0,40. 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2800, 3000, 2600, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL of a substance A, and multiplying the concentration of the substance A by 1/4, 1/2, 3/4, 1/4, 3/5, 2/4, 3/4, 2/5, 2/4, 3/4, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃, wet) for 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture of a) or b), the cells were cultured in a differentiation medium supplemented with either 5, 10, 15 or 20% SF, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 ℃ and humidity), and after 30, 90, 150 or 210 minutes, the absorbance (absorption wavelength 450nm, reference wavelength 630nm) was measured using a plate meter, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from the two values, and the relative number of living cells was calculated. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results
In the nerve-like cell or nerve cell treated with test substance A or test substance B, the death of the nerve-like cell or nerve cell induced by oxidative stress and/or the reduction (attenuation) of the function of the nerve-like cell or nerve cell is suppressed. In addition, in the nerve-like cells or nerve cells treated by combining the test substance a with the test substance B, the death of nerve-like cells or nerve cells induced by oxidative stress and/or the decrease (attenuation) in the functions of nerve-like cells or nerve cells are significantly suppressed.
Pharmacological test 5[ test for Effect of neuronal-like cell death or nerve cell death induced by inflammatory stress and/or reduction (attenuation) of neuronal-like cell or nerve cell function ]
The test method of pharmacological test 5 is shown below, but this example is for better understanding of pharmacological test 5 and is not intended to limit pharmacological test 5. The pharmacological test can be carried out by the method described in the following documents 8 to 12.
Buntinx, M et al, Cytokine-Induced Cell Death in human Oligodroglucial cells I.Synthesis Effects of IFN-. gamma.and TNF- α on Apoptosis.journal of neuroscience Research 76: 834-845(2004)
Ray, R et al, Inhibition of Tumor Necrosis Factor (TNF- α) -media apoptosis by Hepatitis C Virus Protein The Journal of biological chemistry Vol.273, No.4, Issue of January 23, pp.2256-2259, 1998
Xia, Z et al, N-acetyl cysteine epitopes TNF- α -induced human vascular cell apoptosis and restore eNOS expression, European Journal of Pharmacology 550(2006)134-142
Haviv, R et al, New Growth Factor introduced by moving Necrosis Factor in PC12 cells journal of Neuroscience research.55: 269-277(1999)
Liang, F et al, purifier precursors tune coefficient of PC12 cells via activation of the PI3K/Akt signaling path, ExPERIMENTAL AND THERAPEUTIC MEDICINE 14: 813-818, 2017
(1) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 1.0X 103、2.0 x103、3.0 x 103、4.0 x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103Individual cells/0.1 mL/well of either were plated onto type IV collagen-coated 96-well culture plates. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then subjecting the cells to the evaluation of cell viability.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380. 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a differentiation medium (100 μ L) of test substance a; or multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13A differentiation medium (100. mu.L) of the test substance B at any one of the concentrations obtained at/4, 7/2, 15/4 or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours, and treating the test substance. Then, equal amounts (100 μ L) of the following medium were added: contains 0, 0.0001, 0.0005, 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74. 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng/mL.0.0001, 0.0005, 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 120, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 80, 88, 90, 92, 96, 120, 600, 200, 600, 10, 600, 200, 600, 200, 600,135. 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000ng/mL of a substance A to be tested and a differentiation medium for substance B at a concentration of 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 of 1, 200, 600, 200, 210, 220, 300, 1000, 600, 200, 600, 200, 1000, 600, 900, 1000, 600, 1000, 600, 1000, 600, 900, 1000, 600, 900, 1000, 600, 1000, 900, 1000, 600, 1000, 600, 900, 1000, 600, 1000, 600, 1000, 600, 900, 1000, 600, 1000, 600, 1000, 600, 900, 600, 900, 1000, 600, 900, 1000ng/mL of a substance A to be tested substance A, 200, 1000, 200, 900, 1000, 600, 1000, 600, 900, 600, 1000, 600, 900, 600, 1000, 600, 1000, 600, 1000, 600, 1000, 600, 1000, 600, 900, 600, 1000,differentiation medium for test substance B at any of concentrations of 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
b) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: a differentiation medium containing no test substance; or any substance tested for differentiation in culture medium comprising 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4200, 5000, 4400, 5000, 1400, 5000, 5500, 5000, 5500, 2000000, 55000, 2000000, 300, 2000, 7000, 2000, 300, 2000, 2000000, 2000; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 2200, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 4200, 4400, 4200, 4,4600. 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000. mu.g/mL of a test substance A, and a differentiation medium containing the test substance B at a concentration obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4, and introducing the mixture into a CO system2Incubator (5% CO)237 ℃ and humidity), and then the test substance is treated by changing the medium in the culture plate to a medium containing 0, 0.00005, 0.00025, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 6, 8, 10, 12, 16, 7, 60, 10, 12, 14, 16, 18, 20, 28, 70, 20, 23, 20, 70, 25, 20, 23, 25, 20, 70, 25, 20, 25, 70, 25, 20, 25, 0.05, 0.1, 1, 0.1, 7, 20, 1, 25, 1, 25, 1, 25, 1, 25, 1, 25, 1, 25, 1, 25,140. 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500ng/mL of TNF- α, and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 700, 650, 180, 190, 200, 220, 240, 260, 300, 320, 600, 200, 220, 240, 200, 220, 240, 200, 300, 320, 20, 380, 20, 200, 1, 200, 1, 200, 1, 200, 1, 200, 1, 200, 1, 200, 1, 200, 1, 35, 1, 200, 1, 200, 1, 200, 1.1. 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50.25, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 200, 500, 600, 500, 600, 200, 600, 200, 600, 200, 600, 200, 600, 200, 600, 2002Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture of a) or b), the cells were cultured in a differentiation medium supplemented with either 5, 10, 15 or 20% SF, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 deg.C, moisture)After 30, 90, 150 or 210 minutes, the absorbance (absorbance wavelength 450nm, reference wavelength 630nm) was measured using a plate meter, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from both values to calculate the relative number of living cells. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results
In the nerve-like cell or nerve cell treated with test substance A or test substance B, the death of the nerve-like cell or nerve cell induced by inflammatory stress and/or the reduction (attenuation) of the function of the nerve-like cell or nerve cell is suppressed. In addition, in the nerve-like cells or nerve cells treated by combining the test substance a with the test substance B, the death of nerve-like cells or nerve cells induced by inflammatory stress and/or the decrease (attenuation) in the functions of nerve-like cells or nerve cells are significantly suppressed.
pharmacological test 6[ test for Effect of neuronal-like cell death or neuronal cell death induced by amyloid β 1-42 and/or reduction (attenuation) of neuronal-like cell or neuronal cell function ]
The test method of pharmacological test 6 is shown below, but this example is for better understanding of pharmacological test 6 and is not intended to limit pharmacological test 6. The pharmacological test can be carried out according to the methods described in the following documents 13 to 14.
Li, X et al, Effect OF β -Amyloid Peptide 1-42 on the cytoprotective active medium by α 7 Nicotinic acid Receptors in Growth Factor-predicted Differentiated PC-12 cells. this JOURNAL OF PHARMACOLOGY ANDEXPERIMENTAL THERAPEUTIC, 307: 670-675, 2003
Sasaki, H et al, inhibition activities of biflavonoids against inhibition antibodies 42 cytoxicity in PC-12 cells bioorganic & Medicinal chemistry letters.25(2015)2831-2833
(1) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 1.0X 103、2.0 x103、3.0 x 103、4.0 x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103Individual cells/0.1 mL/well of either were plated onto type IV collagen-coated 96-well culture plates. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then subjecting the cells to the evaluation of cell viability.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 4200, 5000, 5500, 5000, 5500, 55000, 12000, 55000, 20000, 7000, 8000, 7000, 8000, 7000, 2000, 7000, 200/mLA differentiation medium (100. mu.L) of the test substance A; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 20000, 55000, 12000, 20000, 55000, 2000, 20000, 2000, 12000, 55000, 3000, 2000, 12000, 3000, or 8000A/mL/1 of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and then adding an equal amount (100. mu.L) of a differentiation medium containing an amyloid β 1-42 oligomer solution of any one of 0, 5, 10, 20, 30, 40, 50, 60, 70 or 80. mu.M, or an amyloid β 1-42 oligomer solution of any one of 0, 5, 10, 20, 30, 40, 50, 60, 70 or 80. mu.M, and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 60, 180, or 80. mu.M460. 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL of a substance A to be detected in a differentiation medium containing the substance A to be detected at a concentration of either 4400, 4800, 5000, 5500, 1000, 200, 70, or 80 μ M of amyloid β 1-42 oligomer solution of 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 μ M in a medium containing the substance A to be detected substance A, 140, 1000, 500, 1000, 600, 200, 1000, 2000, 1000, 600, 1000, 600, 200, 1000, 500, 1000, 200, 1000, 200, 1000, 200, 1000, 300, 1000, 300, 1000, 1, 1000, 300, 10, 300, 10, 40, 10, 40, 50, 60, 70, 80 μ M of the substance B, 1, 200, 1, 200, 1, 1000, 200, 1000, 1, 2002Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
b) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was replaced with the following cultureBase: a differentiation medium containing no test substance; or any substance tested for differentiation in culture medium comprising 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4200, 5000, 4400, 5000, 1400, 5000, 5500, 5000, 5500, 2000000, 55000, 2000000, 300, 2000, 7000, 2000, 300, 2000, 2000000, 2000; or a differentiation medium containing a test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)237 ℃ and humidity) under conditions of 1, 2, 4, 6, 8, 10, 12, 14,16. 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours after the test substance treatment, the culture medium in the culture plate is replaced with a differentiation medium containing an amyloid β 1-42 oligomer solution of any one of 0, 2.5, 5, 10, 15, 20, 25, 30, 35 or 40 μ M amyloid β 1-42 oligomer solution, and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 42, 48, 54, 60, 66 or 72 hours after the differentiation medium containing an amyloid β 1-100, 600, 200, 600, 200, 600, 200, 600, 200, 600, 200 μ M amyloid β 1, 600, 200 μ M amyloid protein containing any one of amyloid β 1, 600,1500. 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a test substance A, and a differentiation medium for the test substance B at a concentration obtained by multiplying the concentration of the test substance A by any one of 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4, wherein the differentiation medium is subjected to CO treatment2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture of a) or b), the cells were cultured in a differentiation medium supplemented with either 5, 10, 15 or 20% SF, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 ℃ and humidity), and after 30, 90, 150 or 210 minutes, the absorbance (absorption wavelength 450nm, reference wavelength 630nm) was measured using a plate meter, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from the two values, and the relative number of living cells was calculated. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results
nerve cell death or nerve cell death induced by amyloid β 1-42 and/or a decrease (attenuation) in function of a nerve cell or nerve cell is suppressed in a nerve cell or nerve cell treated with a test substance a or a test substance B, and nerve cell death or nerve cell death induced by amyloid β 1-42 and/or a decrease (attenuation) in function of a nerve cell or nerve cell is significantly suppressed in a nerve cell or nerve cell treated with a combination of a test substance a and a test substance B.
pharmacological test 7[ test for Effect of neuronal-like cell death or neuronal cell death induced by amyloid β 25-35 and/or reduction (attenuation) of neuronal-like cell or neuronal cell function ]
The test method of pharmacological test 7 is shown below, but this example is for better understanding of pharmacological test 7 and is not intended to limit pharmacological test 7. The pharmacological test can be carried out according to the methods described in the following documents, such as 15 to 17.
Ye, J, et al, Effect of pure Sweet Potato to antibiotic on β -Amyloid-medial PC-12 cell Death by Inhibition of Oxidative stress, neurohem Res (2010) 35: 357-365. fig.
Ji, Z et al, Ginsenoside Re attenate beta-amyloid and serum-free induced neurologic in PC12 cells. journal of Ethnopharmacology, 107(2006)48-52
Peng, Q et al, Pycnogenol (registered trade Mark) protects nerves from amino- β peptide-induced apoptosis, molecular Brain research.104(2002)55-65
(1) Preparation and differentiation Induction of PC-12 cells
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 1.0X 103、2.0 x103、3.0 x 103、4.0 x 103、5.0 x 103、6.0 x 103、7.0 x 103、8.0 x 103、9.0 x 103、10.0 x103、11.0 x 103、12.0 x 103、13.0 x 103、14.0 x 103Or 15.0 x 103Individual cells/0.1 mL/well of either were plated onto type IV collagen-coated 96-well culture plates. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity), inducing differentiation, and then supplying to the cellsAnd (4) evaluating the activity rate.
(2) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380. 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a differentiation medium (100 μ L) of test substance a; or a differentiation medium (100. mu.L) containing the test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or a substance having a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4400, 5000, 1400, 5000, 5500, 55000, 5500, 5000, 55000, 200000, 55000, 20000, 55000, 2000, 20000, 12000, 3000, 7000, 8000, 1/mL of a of the substance to be detected, 1/2, 3/4, 1, 5/4,Differentiation medium (100. mu.L) of test substance B at any one of concentrations obtained at 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 in CO2Incubator (5% CO)2wetting at 37 deg.C) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, treating the test substance, adding an equal amount (100. mu.L) of a differentiation medium containing amyloid β 25-35 oligomer solution of any one of 0, 5, 10, 20, 30, 40, 50, 60, 70 or 80. mu.M amyloid β 25-35 solution, or adding a differentiation medium containing amyloid β 25-35 oligomer solution of any one of 0, 5, 10, 20, 30, 40, 50, 60, 70 or 80. mu.M amyloid β 25, 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 200, 180, 1000, 200, 600, 200, 600, 200, 600, 200, 600, 200, 10, 200, 600, 200, 600, 200, 10, 600, 200, 600, 200, 10, 200, 10, 200, 600, 10, 200, 600, 10, 200, 10, 200, 10, 200, 10, 200, 10, 200, 10, 200, 10,180. 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000, or 20000 μ g/mL of a substance A to be detected, and a differentiation medium obtained by multiplying the concentration of the substance A to be detected by any of 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3/4, 13/4, 7/4, 2, 9/4, 5/2, 11/4, 3/4, 13/4, or 15/4, in CO2Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
b) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: a differentiation medium containing no test substance; or any substance tested for differentiation in culture medium comprising 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3800, 4000, 3600, 4200, 5000, 4400, 5000, 1400, 5000, 5500, 5000, 5500, 2000000, 55000, 2000000, 300, 2000, 7000, 2000, 300, 2000, 2000000, 2000; or a differentiation medium containing a test substance B at any one of concentrations obtained by multiplying the concentration of the test substance A by 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4, or 4; or contains 0, 0.01, 0.02, 0.04,0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1500, 1600, 1800, 1900, 2000, 2200, 2400, 2600, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 5000, 4800, 6000, 14000, 6500, 55000, 8000, 9500, 8000, 16000, 8000, 1200, 12000, 20000, 1201/mL of the substance to be detected, Differentiation medium (100. mu.L) of test substance B at any of the concentrations obtained at 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 was incubated in CO2Incubator (5% CO)237 ℃ and humidity) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours, and then the test substance is treated, and then the culture medium in the culture plate is replaced with a differentiation medium containing an amyloid β 25-35 oligomer solution of any one of 0, 2.5, 5, 10, 15, 20, 25, 30, 35 or 40 μ M, or a differentiation medium containing an amyloid β 25-35 oligomer solution of any one of 0, 2.5, 5, 10, 15, 20, 25, 30, 35 or 40 μ M, and 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 1600, 30, 1000, 200, 500, 1000, 200, 1000, 200, 1000, 200, 1000, 200, 300, 200, 300, 200, 300, 200, 300, 200, 300, 200, 300, 200000. 8500, 9000, 9500, 10000, 12000, 14000, 16000, 18000 or 20000 μ g/mL of a differentiation medium for test substance A, or a differentiation medium containing 0, 2.5, 5, 10, 15, 20, 25, 30, 35 or 40 μ M of amyloid β 25-35 oligomer solution of test substance A and test substance B at a concentration of any one of 1/4, 1/2, 3/4, 1, 5/4, 3/2, 7/4, 2, 9/4, 5/2, 11/4, 3, 13/4, 7/2, 15/4 or 4 multiplied by the concentration of test substance A, or an amyloid β 25-35 oligomer solution containing 0, 2.5, 5, 10, 15, 20, 25, 30, 35 or 40 μ M of test substance B at a concentration of 0, 0.01, 0.02, 0.04, 0.06, 0.08, 1200.00, 14000, 180, 1000, 500, 1000, 500, 1000, 500, 1000, 500, 200, 500, 200, 1000, 200, 1, 200, 1, 2002Incubator (5% CO)237 ℃, wet) for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
After the completion of the culture of a) or b), the cells were cultured in a differentiation medium supplemented with either 5, 10, 15 or 20% SF, and the intracellular dehydrogenase activity was detected using WST-8 as a chromogenic substrate, thereby determining the cell survival rate. In CO2Incubator (5% CO)237 ℃, wet), measured using a plate meter after any one of 30, 90, 150, or 210 minutesThe absorbance (absorption wavelength: 450nm, reference wavelength: 630nm) was determined, and the amount of change in absorbance per 1, 2 or 3 hours was calculated from both values to calculate the relative number of living cells. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(3) Test results
the present invention relates to a method for inhibiting nerve cell death or nerve cell death and/or a decrease (attenuation) in function of a nerve cell or nerve cell induced by amyloid β 25-35 in a nerve cell or nerve cell treated with a test substance a or a test substance B, and a method for significantly inhibiting nerve cell death or nerve cell death and/or a decrease (attenuation) in function of a nerve cell or nerve cell induced by amyloid β 25-35 in a nerve cell or nerve cell treated with a test substance a in combination with a test substance B.
Pharmacological test 8[ confirmation test of the Effect of protecting retinal nerve cells (retinal ganglion cells, amacrine cells, etc.) or the Effect of protecting optic nerve Using the model of retinal injury injected intravitreally with NMDA ]
The effect of protecting retinal nerve cells (retinal ganglion cells, amacrine cells, etc.) or the effect of protecting optic nerves in vivo of the test substance A, the test substance B, or a mixture of the test substance A and the test substance B can be confirmed by intravitreal injection of a retinal injury rat model. The test method can be carried out according to the following documents 18 to 25, and various conditions such as the application amount and the compounding ratio of the test substance a, the test substance B, or the mixture of the test substance a and the test substance B can be changed as appropriate.
Matsunaga N et al, Bilberry and its main control ingredients associated with a neutral neural damage in a video and in a video. mol. Nutr. FoodRes.2009, 53, 869-877
Ohno Y et al, Oral administration of crocetin derived from fibrous amino acid by N-methyl-D-aspartic acid, European Journal of Pharmacology.690, 2012, 84-89
Maekawa S et al, The neuropathic effect of heperidin in NMDA-induced transcriptional activity by lateral inhibitory activity stress and exogenous polypeptide activity, scientific Reports 7.6885(2017)
Tsutsumi T et al, Potential Neuroprotective Effects of an LSD1Inhibitor in Retinal Ganglion Cells via p38 MAPK Activtv. invest OphthalmolVis Sci.2016; 57: 6461-6473
Binda NS et al, PhTx3-4, a Spider Toxin Calcium Channel packer, Reductes NMDA-Induced injection of the Retina
Vicente VG et al, neuroprotectant Effect of tauurodeoxycholic acid N-Methyl-D aliquot-Induced specific growth Cell Generation PLoS ONE10(9)2015
Sakamoto K et al, Protective effect of all-trans retinic acid on NMDA-induced neural cell death in Rat Retina. European Journal of Pharmacology, 635(2010)56-61
Siliprandi R et al, N-methyl-D-aspartic-induced neuropathy in the adolt rat retina, Vis neurosci.1992Jun; 8(6): 567-73
Pharmacological test 9[ confirmation test of Effect of protecting retinal nerve cells or Effect of protecting optic nerve Using ischemia-reperfusion retinal injury model ]
The effect of protecting retinal nerve cells or the effect of protecting optic nerves in vivo of the test substance a, the test substance B, or a mixture of the test substance a and the test substance B can be confirmed by an ischemia reperfusion retinal injury rat model. The test method can be carried out according to the following documents such as 26 to 32. the application amount and the compounding ratio of the test substance a, the test substance B, or the mixture of the test substance a and the test substance B can be appropriately changed.
Yang X et al, Neuroprotective Effects of Crocin Induced by Ischemia/playback in ray Retina, Ophthalmic Res 2015; 54: 157-168
Shimouchi et al, neuroprotectant effect of water-dispersed heparin derivative in J pn J Ophthalmol.2016 January; 60(1): 51-61
Kara S et al, Protective Effect of Hespertin and Naringgen against ions in Ischemia/reproduction-Induced specific Injury in rates, the scientific World journal volume 2014
Yondeda S et al, Topiramate products exutoxic and ischemistry intrate retina, Brain research.967(2003)257-266
Chen B et al, Protective effects of catalyst on regenerative ischemia/regenerative in rates, Experimental Eye research.93(2011)599-606
Wang L et al, Curcumin inhibition neural and Vascular differentiation in regeneration after Ischemia and reproduction in, PLoS ONE. (2011)6(8)
Aydogan S et al, The effect of The clinical on vascular endovenous growth factor and tumor necrosis factor- α levels in nutritional ischemia/nutritional in therapy, Graves arc Clin Exp Ophalm 0l (2008) 246: 363-368
Pharmacological test 10[ test for Effect of oxidative stress-induced nerve cell-like death or nerve cell death and/or reduction (attenuation) of function of nerve cell-like or nerve cell ]
The test method of the pharmacological test 10 (in accordance with the above pharmacological test 4) is shown below, but this example is for better understanding of the pharmacological test 10 and does not limit the pharmacological test 10. The pharmacological test can be carried out according to the method described in the above-mentioned document 7.
(1) Preparation of multiplication Medium
A mixture of Dulbecco's modified Eagle Medium (#08456-65, Dulbecco's modified Eagle Medium, DMEM) manufactured by Nacalai Tesque was added with fetal bovine serum SA (#172012, fetal bovine serum, FBS) manufactured by Sigma-Aldrich to a final concentration of 10%, horse serum (#16050-122, horse serum manufactured by Thermo Fisher Scientific, HS) manufactured by New Zealand to a final concentration of 10%, and a mixed penicillin-streptomycin solution (stabilized) (#32777-44) manufactured by Nacalai Tesque to a final concentration of 1% to prepare a mixture.
(2) Preparation of differentiation Medium
Differentiation medium was prepared as follows: the preparation was carried out by adding NGF 2.5S native mouse protein (#13257019, NGF) manufactured by Thermo Fisher Scientific Co., Ltd. to DMEM so that the final concentration was 50ng/mL, FBS so that the final concentration was 0.1%, HS so that the final concentration was 0.1%, and a penicillin-streptomycin mixed solution (stabilization) so that the final concentration was 1%.
(3) Preparation of test substance-containing Medium
The test substance A or the test substance B is prepared by adding a powdery test substance A or a powdery test substance B to the above-mentioned various culture media.
(4) Preparation and differentiation Induction of PC-12 cells derived from rat Adrena Brown cell tumor
Using proliferation medium, PC-12 cells were revived in type IV collagen-coated T-75 flasks in CO2Incubator (5% CO)237 ℃ and humidity). The cells were detached with 0.25% trypsin-EDTA at a time point of 60-90% confluence by replacing the growth medium with a new one every 2-4 days, and the growth medium was used so as to be 5.0X 103The cells were plated per 0.1 mL/well in type IV collagen-coated 96-well plates. In CO2Incubator (5% CO)237 ℃ and humidity) overnight, the medium of the plate was changed to a differentiation medium, and the plate was incubated with CO2Incubator (5% CO)237 ℃ and humidity) for 2 days, induced to differentiate, and then subjected to cell survival evaluation.
(5) Cell viability assessment
a) The differentiation medium in the plate in which the differentiation-induced PC-12 cells were cultured was changed to the following medium: differentiation medium (100. mu.L) containing no test substance; or a differentiation medium (100. mu.L) containing 10. mu.g/mL of test substance A; or a differentiation medium (100. mu.L) containing a test substance B at a concentration obtained by multiplying the concentration of the test substance A by 9/4; orDifferentiation Medium (100. mu.L) containing test substance A at 10. mu.g/mL and test substance B at a concentration obtained by multiplying the concentration of test substance A by 9/4, in CO2Incubator (5% CO)237 ℃ and wet) for 24 hours, and then the test substance was treated. Then, equal amounts (100 μ L) of the following medium were added: a differentiation medium containing 0 or 0.72mM hydrogen peroxide (#081-04215) manufactured by Wako pure chemical industries, Ltd; or a differentiation medium containing 0.72mM hydrogen peroxide and 10. mu.g/mL of test substance A; or a differentiation medium containing 0.72mM hydrogen peroxide and a test substance B at a concentration obtained by multiplying the concentration of the test substance A by 9/4; or a differentiation medium containing 0.72mM hydrogen peroxide, 10. mu.g/mL of test substance A, and test substance B having a concentration obtained by multiplying the concentration of test substance A by 9/4, in CO2Incubator (5% CO)237 ℃ and humidity) for 1 hour.
After the completion of the culture in the above a), the cells were assayed for the cell survival rate by replacing the culture medium with a differentiation medium supplemented with 10% SF and detecting the intracellular dehydrogenase activity using WST-8 as a chromogenic substrate. In CO2Incubator (5% CO)237 ℃ and humidity), and then the absorbance (absorption wavelength: 450nm, reference wavelength: 630nm) was measured using a plate meter after 30 to 150 minutes, and the amount of change in absorbance per 2 hours was calculated from the two values to calculate the relative number of viable cells. In the calculation of the relative number of living cells, other living cell detection reagents or methods or dead cell detection reagents or methods may be used instead of the WST-8 method.
(6) Test results
The ratios of the number of viable cells in the test substance non-added group, test substance a added group, test substance B added group, and test substance a + test substance B added group under the hydrogen peroxide treatment conditions, where the number of viable cells relative to the cells not subjected to the hydrogen peroxide treatment was 100%, are shown in table 5. The test substance A addition group treated cells with 10. mu.g/mL of test substance A, the test substance B addition group treated cells with 22.5. mu.g/mL of test substance B, and the test substance A + test substance B addition group treated cells with 10. mu.g/mL of test substance A and 22.5. mu.g/mL of test substance B. The difference between the ratio of the number of living cells in the group to which each test substance was added and the ratio of the number of living cells in the group to which no test substance was added was shown as the effect of protecting cells of each test substance. As shown in table 5 and fig. 1, the cell protective effect of the test substance a + test substance B-added group showed a significantly higher value than the value obtained by adding the cell protective effect of the test substance a-added group and the cell protective effect of the test substance B-added group. As can be seen from the results, test substance a and test substance B have a synergistic effect in the inhibition of neuronal cell death or neuronal cell death induced by oxidative stress and/or the reduction (attenuation) of neuronal cell or neuronal cell function. From this result, the following conclusions can be drawn.
In the nerve-like cell or nerve cell treated with test substance A or test substance B, the death of the nerve-like cell or nerve cell induced by oxidative stress and/or the reduction (attenuation) of the function of the nerve-like cell or nerve cell is suppressed. In addition, in the nerve-like cells or nerve cells treated by combining the test substance a with the test substance B, the death of nerve-like cells or nerve cells induced by oxidative stress and/or the decrease (attenuation) in the functions of nerve-like cells or nerve cells are significantly suppressed.
[ Table 5]
Figure BDA0002439049110000801
Industrial applicability
The present invention uses a processed product of a blueberry fruit derived from a natural product and a processed product of pine bark, and therefore is useful not only as a pharmaceutical composition but also as a food composition such as a beverage, a food, and a supplement, and provides a composition for protecting retinal nerve cells and/or for protecting optic nerve, which can be used in a wide range of fields and is highly safe.

Claims (8)

1. A composition for protecting retinal nerve cells and/or protecting optic nerve, which comprises a processed product of cowberry fruit and a processed product of pine bark.
2. The composition of claim 1, wherein the processed product of the blueberry fruit is a blueberry fruit extract.
3. The composition of claim 1, wherein the pine bark processed product is a pine bark extract.
4. The composition of claim 3, wherein the extract of pine bark is French maritime pine bark extract.
5. A composition as claimed in any one of claims 1 to 4 for oral or parenteral administration.
6. A composition according to any one of claims 1 to 5 which is a pharmaceutical composition.
7. The composition according to any one of claims 1 to 5, which is a food composition.
8. The composition according to claim 7, wherein the food composition is a health food, a functional food, a nutritional supplement food, a supplement, a health functional food, a food for specified health use, a nutritional functional food or a functional marker food.
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