WO2024024395A1 - Pharmaceutical or food and beverage composition for prevention or treatment of optic nerve disorder - Google Patents

Pharmaceutical or food and beverage composition for prevention or treatment of optic nerve disorder Download PDF

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WO2024024395A1
WO2024024395A1 PCT/JP2023/024440 JP2023024440W WO2024024395A1 WO 2024024395 A1 WO2024024395 A1 WO 2024024395A1 JP 2023024440 W JP2023024440 W JP 2023024440W WO 2024024395 A1 WO2024024395 A1 WO 2024024395A1
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diosgenin
optic nerve
pharmaceutical
food
treatment
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PCT/JP2023/024440
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French (fr)
Japanese (ja)
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千尋 東田
省吾 渋江
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国立大学法人富山大学
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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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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

Definitions

  • the present invention relates to a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders such as glaucoma.
  • Glaucoma has been described as ⁇ a disease characterized by functional and structural abnormalities of the eye that have characteristic changes in the optic nerve and visual field, and which can usually improve or suppress optic nerve damage by sufficiently lowering intraocular pressure.'' has been defined (see Non-Patent Document 1).
  • Lowering the intraocular pressure is the standard treatment for patients with normal-tension glaucoma, but lowering the intraocular pressure is a way to protect the optic nerve and suppress the progression of visual field damage, and it is possible to restore lost visual field. cannot be expected. Therefore, glaucoma treatment strategies other than lowering intraocular pressure are desired.
  • optic nerve damage such as glaucoma.
  • problems such as visual field defects occur due to optic nerve damage such as death of retinal ganglion cells for some reason or atrophy of the optic nerve, which is the axon of the retinal ganglion cells. If the optic nerve re-projects correctly to its projection sites, such as the lateral geniculate body and superior colliculus, and the pathway that transmits signals to the visual cortex is repaired, it is thought that visual field defects can be restored, and this is possible. Methods and drugs have not yet been established.
  • an object of the present invention is to provide a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders such as glaucoma.
  • the present invention provides a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders, which contains diosgenin or a diosgenin derivative as an active ingredient.
  • Such pharmaceutical or food/beverage compositions can contribute to an increase in retinal ganglion cells and/or elongation of optic nerves, and therefore are effective in preventing or treating optic nerve disorders, regardless of whether or not they reduce intraocular pressure.
  • the pharmaceutical or food/beverage composition of the present invention preferably contains a plant extract containing diosgenin or a diosgenin derivative. Plant extracts containing diosgenin or diosgenin derivatives are desirable because they are inexpensive and easily available.
  • the above-mentioned plant extract is preferably a plant extract with an increased content of diosgenin through at least one treatment selected from acid hydrolysis treatment, fermentation treatment, and enzyme treatment. This makes it easier to improve the medicinal efficacy of the medicine or food/beverage composition.
  • the pharmaceutical or food/beverage composition of the present invention can prevent or treat optic neuropathy, for example, by suppressing the death of retinal ganglion cells.
  • the pharmaceutical or food/beverage composition of the present invention is particularly effective when the optic nerve disorder is glaucoma. Furthermore, the pharmaceutical or food/beverage composition of the present invention is particularly effective in preventing or treating normal-tension glaucoma, since it can be prevented or treated regardless of whether or not there is a decrease in intraocular pressure.
  • a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders such as glaucoma.
  • FIG. 2 is a micrograph of a region fluorescently immunostained with pNF-H (phosphorylated NF-H) and a region fluorescently immunostained with MAP2 in Example 1.
  • FIG. 3 is a diagram showing the measurement results of axon length ( ⁇ m) per retinal ganglion cell in Example 1.
  • FIG. 3 is a diagram showing the measurement results of dendrite length per retinal ganglion cell in Example 1.
  • 3 is a diagram showing the results of intraocular pressure measurement in Example 2.
  • FIG. FIG. 2 is a diagram showing representative photographs proximal to the injury site and quantitative values of the CTB-488 positive area in Example 2.
  • FIG. 2 is a diagram showing representative photographs distal to the injury site and quantitative values of the CTB-488 positive area in Example 2.
  • FIG. 2 is a diagram showing the results of LC-MS analysis of a standard substance (diosgenin).
  • FIG. 7 is a diagram showing the analysis results for the retina in Example 3.
  • FIG. 7 is a diagram showing the analysis results for the optic nerve in Example 3.
  • FIG. 7 is a diagram showing the analysis results for the brain in Example 3.
  • FIG. 7 is a diagram showing the results of intraocular pressure measurement in Example 4. This is a representative micrograph in Example 4 when a solvent, 0.1.1 ⁇ mol/kg diosgenin olive oil solution, was administered.
  • FIG. 4 is a diagram showing retinal ganglion cell densities when the optic nerve was not crushed and when the optic nerve was crushed in Example 4.
  • FIG. 4 is a diagram showing the retinal ganglion cell density after oral administration of a solvent, 0.1, 1, and 10 ⁇ mol/kg diosgenin olive oil solution in Example 4.
  • FIG. 4 is a representative micrograph of a case where a diosgenin olive oil solution was orally administered in Example 4.
  • FIG. 7 is a diagram showing the CTB area in the lateral geniculate body in Example 4 when the optic nerve was not crushed and when the optic nerve was crushed.
  • FIG. 4 is a diagram showing the overlapping area of the CTB area and the Fluoro-Gold positive area in the lateral geniculate body when the optic nerve was not crushed and when the optic nerve was crushed in Example 4.
  • FIG. 4 is a diagram showing the CTB area in Example 4 when a solvent was administered and when a diosgenin olive oil solution was administered.
  • FIG. 4 is a diagram showing the overlap area between the CTB area and the Fluoro-Gold positive area in the case of administering a solvent and the case of administering a diosgenin olive oil solution in Example 4.
  • the pharmaceutical or food/beverage composition of this embodiment contains diosgenin or a diosgenin derivative as an active ingredient.
  • Diosgenin is a compound represented by the following chemical formula (I).
  • Diosgenin derivative refers to a compound that can be an equivalent of diosgenin.
  • Diosgenin and diosgenin derivatives are not particularly limited, and may be commercially available products, those manufactured according to known methods, methods known per se, or methods analogous thereto, and extracts from natural products. Good too.
  • diosgenin derivatives may be equivalents that can be achieved by chemical modification such as introducing or converting substituents into diosgenin, or may be diosgenin glycosides (such as dioscine) extracted from natural products. It's okay.
  • diosgenin derivatives include, for example, replacing the hydroxyl group in diosgenin with an alkoxy group, ester group (e.g. acetate ester), amino acid ester group (e.g. glycine ester), aminosulfonic acid ester group, carbamate group, halogen atom (e.g. fluorine atom). ).
  • the pharmaceutical or food/beverage composition of the present embodiment preferably contains a plant extract containing diosgenin or a diosgenin derivative.
  • Plant extracts include, for example, Dioscorea rhizome, Trigonella spp., Polygonatum spp., and Smilax, which are known to contain diosgenin (or its glycosides). Extracts of plants such as herbal medicines such as spp.) can be used. Extraction can be performed by a conventional method using an extraction solvent such as water, ethanol, 1,3-butylene glycol, or the like.
  • the plant extract is preferably a plant extract with increased diosgenin content through further processing.
  • the treatment include acid hydrolysis treatment, fermentation treatment, enzyme treatment, and the like. These treatments may be used alone or in combination of two or more. Conventionally known treatment methods can be applied to these treatments, but for example, as acid hydrolysis treatment, the method described in T Herrera et al., J Sci Food Agric 2019; 99: 3157-3167, For fermentation treatment, the methods described in Y. Chen et al., Steroids 136 (2016) 40-46 and J. Dong et al., Indian J Microbiol (Apr-June 2015) 55(2):200-206 are used.
  • the enzyme treatment the methods described in W. Huang et al., Bioresource Technology 99 (2008) 7407-7411 can be applied.
  • the form of the medicament of this embodiment is not particularly limited, and diosgenin or a diosgenin derivative may be used as it is (for example, a powder), or may be formulated with other ingredients.
  • components include components that are generally used as raw materials for pharmaceutical preparations, and are not particularly limited. Specific examples include carriers, excipients, binders, disintegrants, lubricants, coating agents, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, Examples include preservatives and antioxidants. These may be used alone or in combination of two or more.
  • the pharmaceutical or food/beverage composition of this embodiment may be administered orally or parenterally.
  • Parenteral administration forms include topical ocular administration (intravitreal administration, eye drops, intraconjunctival sac administration, subconjunctival administration, sub-Tenon's administration, etc.), intravenous administration, transdermal administration, and the like.
  • dosage forms for oral administration include solutions, suspensions, capsules, soft capsules, tablets, granules, powders, syrups, jellies, orally disintegrating tablets, and chewable tablets.
  • diosgenin or diosgenin derivative is preferably suspended or dissolved in an oil or fat.
  • oils and fats examples include soybean oil, rapeseed oil (rapeseed oil, canola oil), high oleic acid rapeseed oil, corn oil, sesame oil, sesame salad oil, Taihaku sesame oil, perilla oil, linseed oil (linseed oil), peanut oil, safflower oil ( safflower oil), high oleic safflower oil, sunflower oil, high oleic sunflower oil, cottonseed oil, grape seed oil, macadamia nut oil, hazelnut oil, peanut oil, almond oil, nut oil, walnut oil, pumpkin seed oil, walnut oil , lemon oil, camellia oil, tea seed oil, perilla oil, borage oil, olive oil (olive oil), rice oil, rice bran oil, wheat germ oil, palm oil, palm olein, palm stearin, palm kernel oil, coconut oil, cocoa butter Vegetable oils such as beef tallow, pork fat (lard), chicken fat, milk fat, animal oils such as
  • sardine oil mackerel oil, cod oil, whale oil, liver oil, etc.
  • fatty acids docosahexaenoic acid (DHA), eicosapentaene, etc.
  • examples include edible oils such as acid (EPA), fat-soluble vitamins (vitamin A, vitamin E, etc.); synthetic oils such as medium-chain fatty acid triglycerides, and iodized poppy seed fatty acid esters. These may be used alone or in combination of two or more.
  • dosage forms for parenteral administration include injections, eye drops, eye ointments, patches, gels, and inserts.
  • the food and drink composition of this embodiment can be provided in the form of drinks, foods, supplements, and the like.
  • beverages include water, soft drinks, fruit juice drinks, carbonated drinks, milk drinks, alcoholic drinks, sports drinks, nutritional drinks, and the like.
  • foods include breads, noodles, rice, tofu, dairy products, soy sauce, miso, and sweets.
  • Food and drink compositions include, for example, health foods, foods with health claims, foods with functional claims, foods for special uses, nutritional supplements, and foods for specified health uses.
  • the content of diosgenin or a diosgenin derivative as an active ingredient in the pharmaceutical or food/beverage composition of the present embodiment is not particularly limited, but is a sufficient amount to treat, improve, alleviate, or recover the symptoms associated with the disease. It is preferable to do so.
  • the dosage of the pharmaceutical or food/beverage composition of the present embodiment is appropriately selected depending on, for example, the severity of symptoms of the subject to be administered, age, sex, body weight, dosage form, type of salt, specific type of disease, etc.
  • the molar amount of diosgenin or diosgenin derivative per unit body weight of the subject to be administered may be, for example, usually about 0.001 to about 1000 ⁇ mol/kg/day, Preferably about 0.01 to about 10 ⁇ mol/kg/day, more preferably 0.01 to about 1 ⁇ mol/kg/day.
  • the subject to whom the medicine or food/beverage composition of the present embodiment is administered is not particularly limited, but is preferably a mammal including a human.
  • Mammals including humans include, but are not particularly limited to, humans, monkeys, baboon, chimpanzees, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, etc. .
  • the pharmaceutical or food/beverage composition of this embodiment is effective for preventing or treating optic nerve disorders.
  • Optic nerve disorders include optic nerve diseases such as glaucoma, optic neuropathy, and optic neuritis, and the pharmaceutical or food/beverage composition of the present embodiment may be particularly effective in preventing or treating glaucoma, particularly normal-tension glaucoma. .
  • Diosgenin (Tokyo Kasei) was dissolved in 99.5% ethanol (Fujifilm, Wako Pharmaceutical) to prepare a high concentration solution of diosgenin, and diluted with physiological saline to prepare a diosgenin ethanol solution.
  • diosgenin olive oil solution was prepared by dissolving 9.6 mg of diosgenin (Tokyo Kasei) in 790 ⁇ l of Japanese Pharmacopoeia olive oil (Maruishi Pharmaceutical).
  • mice All mice were housed in plastic cages (23 x 16 x 12 cm) in an environment with a 12-hour light/dark cycle (light period: 7:00 am to 7:00 pm) and constant temperature and humidity (22°C ⁇ 2°C, 55 ⁇ 10%). bred. Water and chow were available ad libitum.
  • mice Male 6-week-old ddY mice (Japan SLC) were used in the experiment. The animal was anesthetized by intraperitoneally administering a triple-mixed anesthesia. While pulling out the eyeball, the optic nerve in the immediate vicinity of the eyeball was compressed for one second with forceps. Optic nerve compression was performed in both eyes. Antisedan was administered intraperitoneally, and the mice were left on a hot plate at 37°C to wake up while preventing a drop in body temperature.
  • ⁇ Intraocular pressure measurement method> Immediately before exsanguination and perfusion of the mouse, intraocular pressure was measured under anesthesia. Using a Tonolab hand-held tonometer (M-E Technica for mice and rats), the average value of the values obtained by measuring 5 times per eye was used.
  • Frozen brains were wrapped in aluminum foil and stored at -30°C.
  • the washed eyeballs were placed in a PBS solution and a small hole was made in the eyeballs using a 21G injection needle under a stereomicroscope (SZ61, Olympus), and one blade of scissors was inserted through the hole to cut off the cornea. After carefully removing the lens and gel-like vitreous body, only the retina was isolated. Thereafter, the optic nerve was cut from the eyeball with microscissors, and the retina and optic nerve were each replaced with a 30% sucrose solution.
  • the secondary antibody solution [0.5% TritonX-100-PBS solution, Alexa Fluor647-labeled goat anti-rabbit IgG antibody] (1:600, Thermo Fisher Scientific)] was added at 100 ⁇ l/well, and the reaction was allowed to occur overnight at 4°C with shaking in the dark. After the reaction, the secondary antibody solution was removed and washed with PBS. Four incisions were made in the cup-shaped retina and the plate was transferred onto a slide glass. After being flat mounted, Aqua poly Mount (Polysciences, Warrington , PA, USA).
  • ⁇ Preparation of optic nerve section> The sucrose-substituted optic nerve was implanted into Tissue Tek OCT (Sakura Finetech Japan) using Cryomold No. 3 (Sakura Finetech Japan) as a mold. Sagittal sections with a thickness of 12 ⁇ m were successively prepared using a cryostat (CM3050SL, Leica). At that time, the temperature inside the chamber and on the stage was set to -22°C. The sections were attached to coated glass slides (Matsunami Glass Industries) and stored at -30°C.
  • CM3050SL a cryostat
  • Leica the brain was serially sectioned into 12- ⁇ m-thick coronal sections in a region ranging from Bregma -1.70 mm to -2.92 mm.
  • the temperature inside the warehouse and on the stage was set to -20°C.
  • the sections were attached to coated glass slides (Matsunami Glass Industries) and stored at -30C.
  • a fluorescent inverted microscope KEYENCE BZ-X800 (Keyence) and an objective lens (PlanApo10x, Nikon) were used to photograph the left and right lateral geniculate bodies.
  • Example 1 We investigated the effect of diosgenin on axonal elongation in cultured retinal ganglion cells. Details are shown below.
  • ⁇ Retinal ganglion cell primary culture> The eyeballs of 4-day-old ddY mice (Japan SLC) were removed under isoflurane anesthesia. The eyeballs were placed in Neurobasal-A medium (Life Technologies) containing B-27 [2mM L-glutamine, 1X B-27 added], and the retinas were isolated under a stereomicroscope. Thereafter, the supernatant was removed by centrifugation at 700 rpm for 3 minutes, and 2 ml of 0.05 trypsin-0.53 mM EDTA solution (Life Technologies) was added to the pellet to suspend it. It was left standing at 37°C for 7 minutes, and tapped once after 5 minutes.
  • the number of living cells was counted while separating them from dead cells by Trypanblue staining, and the cells were seeded in an 8-well chamber slide at a density of 1.0 ⁇ 10 5 cells/well/300 ⁇ l. All culture dishes were incubated with 5 ⁇ g/ml poly-D-lysin-PBS solution at 37°C for 16 hours, washed twice with PBS, and further incubated with 20 ⁇ g/ml laminin-PBS solution at 37°C for 16 hours. , and those washed twice with PBS were used. After seeding, culture was performed under 10% CO 2 at 37° C. and saturated steam, and the entire amount of the medium was replaced 24 hours after seeding. After medium exchange, culture was performed under 5% CO 2 at 37°C and saturated steam.
  • ⁇ Drug treatment on retinal ganglion cells The above-mentioned 0.1 ⁇ M and 1 ⁇ M diosgenin ethanol solutions were each dissolved in Neurobasal-A medium containing B-27 (ethanol final concentration: 9.95 ⁇ 10 ⁇ 2 %) to prepare a culture medium of each concentration. After replacing the entire volume with the medium 4 days after the start of retinal ganglion cell culture, the culture was continued for an additional 4 days.
  • Triton100-PBS solution 1% normal goat serum (Fujifilm Wako Pure Chemical Industries), rabbit anti-MAP2 antibody (1:1000, Abcam), mouse anti-phosphorylated neurofilament-H (pNF-H) monoclonal antibody ( 1:200, Convance)
  • Triton100-PBS solution 1% normal goat serum (Fujifilm Wako Pure Chemical Industries), rabbit anti-MAP2 antibody (1:1000, Abcam), mouse anti-phosphorylated neurofilament-H (pNF-H) monoclonal antibody ( 1:200, Convance)
  • pNF-H mouse anti-phosphorylated neurofilament-H
  • FIG. 1A shows micrographs of the areas fluorescently immunostained by pNF-H (corresponding to axons) and the areas fluorescently immunostained by MAP2 (corresponding to dendrites).
  • FIG. 1B shows the measurement results of the dendrite length ( ⁇ m) per retinal ganglion cell
  • FIG. 1C shows the measurement results of the dendrite length ( ⁇ m) per retinal ganglion cell.
  • Example 2 We used an optical nerve crush (ONC) model as a glaucoma mouse model to reliably evaluate optic nerve crush and reprojection, and investigated the effects of intravitreal administration of diosgenin on the optic nerve. Details are shown below.
  • OOC optical nerve crush
  • ⁇ Intravitreal drug administration> The above-mentioned 0.1 ⁇ M and 1 ⁇ M diosgenin solutions (2.5% ethanol, 97.5% physiological saline) were each administered into the vitreous body of a normal-tension glaucoma model mouse under anesthesia by intraperitoneally administering a triple-mixed anesthesia. Diosgenin concentration was expressed as the final intravitreal concentration. Under a stereomicroscope (SZ61, Olympus), the eyeball of the mouse was picked out with tweezers, and a sterilized glass syringe (Narishige, Tokyo) with an outer diameter of 0.15 mm at the tip was inserted from the ciliary body toward the vitreous body. .
  • SZ61 stereomicroscope
  • the glass syringe was connected to a 10 ⁇ l Hamilton syringe (Hamilton Company) via 0.5 mm tubing (MS Equipment) and a 21G injection needle (Terumo), and set in a syringe pump (KD Scientific). After inserting a glass syringe into the vitreous, diosgenin ethanol solution was injected at 1.0 ⁇ l/eye/min. After intravitreal injection, the same amount of antisedan as the anesthetic was administered intraperitoneally, and the mouse was left on a hot plate at 37°C to waken up while preventing a drop in body temperature. In the same manner, intravitreal administration was performed three times in total, every 5 days after the first administration.
  • ⁇ Optic nerve labeling using an antegrade tracer> Anterograde tracer injection was performed 6 days before retinal, optic nerve, and brain extraction under anesthesia using triple anesthesia administered intraperitoneally. Under a stereomicroscope (SZ61, Olympus), the mouse eyeball was picked out with tweezers, and a sterilized glass syringe (Narishige) with an outer diameter of 0.15 mm at the tip was inserted from the ciliary body toward the vitreous body.
  • the glass syringe was connected to a 10 ⁇ l Hamilton syringe (Hamilton Company) via 0.5 mm tubing (MS Equipment) and a 21G injection needle (Terumo), and set in a syringe pump (KD Scientific). After inserting a glass syringe into the vitreous body, 1.0 mg/ml Alexa Fluor 488-conjugated cholera toxin subunit B (CTB) (Molecular probes) was administered into the vitreous body of the mouse at 1.0 ⁇ l/eye/min.
  • CTB Alexa Fluor 488-conjugated cholera toxin subunit B
  • Example 3 In Example 2, intravitreal administration of diosgenin showed a tendency for optic nerve density to increase. If diosgenin acts not only on the retina but also on the optic nerve, which is the axon of retinal ganglion cells, and the lateral geniculate body neurons, which are the destinations of its projection, the neural circuits that form visual function will be strengthened as a whole. It is thought that Therefore, we investigated the migration of diosgenin into the retina, optic nerve, and brain after oral administration. Details are shown below.
  • mice Male 6-week-old ddY mice (Japan SLC) were used in the experiment. The mice were fasted 16 hours before administration, and the above-mentioned diosgenin olive oil solution was orally administered to the mice. After 1, 6, and 12 hours, plasma, retina, optic nerve, and whole brain were removed. A 10-fold volume of methanol (Fujifilm Wako Pharmaceutical) was added to the excised tissue, homogenized for 15 seconds, vortexed for 1 minute, sonicated for 5 minutes, and centrifuged at 4°C at 12,000 g for 10 minutes.
  • methanol Flujifilm Wako Pharmaceutical
  • the supernatant was transferred to a 5 ml tube and evaporated to dryness on a hot plate at 50°C. 100 ⁇ l of 100% methanol was added thereto to dissolve, and the mixture was centrifuged at 13,000 x 5 minutes at 4°C. The supernatant was passed through a 0.45 ⁇ m filter (Merck Millipore) to obtain a sample for LC-MS/MS.
  • Example 4 The effect of oral administration of diosgenin on optic nerve elongation was investigated. Details are shown below.
  • Retrograde tracer injection was performed under anesthesia by intraperitoneal administration of triple anesthesia 7 days before retinal, optic nerve, and brain extraction. The top of the head was shaved, and the scalp was incised to expose the skull. Using an electric microdrill, a hole was made at the position of Lat: 2.5 mm, Bregma: -38 mm, depth: 1.2 mm, and Fluoro-Gold (Fujifilm Wako Pharmaceutical) was injected into the left and right primary visual cortex at 1.0 ⁇ l/min. . After the surgery, antisedan was administered intraperitoneally in the same amount as the anesthetic, and the mice were left on a hot plate at 37°C to wake them up while preventing a drop in body temperature.
  • CTB Alexa Fluor 488-conjugated cholera toxin subunit B
  • FIG. 7A shows the CTB area in the lateral geniculate body when the optic nerve was not crushed and when the optic nerve was crushed
  • Fig. 7C shows the overlap area between the CTB area and the Fluoro-Gold positive area
  • 7D shows the CTB area when a diosgenin olive oil solution of 1,10 ⁇ mol/kg was administered
  • FIG. 7E shows the overlap area between the CTB area and the Fluoro-Gold positive area.

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Abstract

Provided is a pharmaceutical or food and beverage composition which is for prevention or treatment of an optic nerve disorder and which includes diosgenin or a diosgenin derivative as an active ingredient thereof.

Description

視神経障害の予防または治療のための医薬または飲食品組成物Pharmaceutical or food/beverage compositions for preventing or treating optic nerve disorders
　本発明は、緑内障等の視神経障害の予防または治療のための医薬または飲食品組成物に関する。 The present invention relates to a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders such as glaucoma.
　日本において、40歳以上の5％が緑内障であり、失明原因の第一位となる疾患である。緑内障は、「視神経と視野に特徴的変化を有し、通常、眼圧を十分に下降させることにより視神経障害を改善もしくは抑制しうる目の機能的構造的異常を特徴とする疾患」とこれまで定義されてきた（非特許文献１参照）。 In Japan, 5% of people over the age of 40 suffer from glaucoma, making it the leading cause of blindness. Glaucoma has been described as ``a disease characterized by functional and structural abnormalities of the eye that have characteristic changes in the optic nerve and visual field, and which can usually improve or suppress optic nerve damage by sufficiently lowering intraocular pressure.'' has been defined (see Non-Patent Document 1).
　しかしながら、開放隅角緑内障患者の3分の1以上は正常レベルの眼圧であること（正常眼圧緑内障）が報告されている。よって、現在の緑内障の定義は、眼圧の上昇の存在を条件とするものではなく、「眼圧およびその他の現在知られていない要因が、視神経の特徴的な後天的萎縮と網膜神経節細胞の喪失に寄与する、成人における進行性の慢性視神経障害」であるとされる（非特許文献２参照）。日本においても、緑内障患者の８割が正常眼圧緑内障患者であり、眼圧上昇による痛みを伴わずに視野狭窄が進行していく。そのため、緑内障患者の９割は無自覚のまま診断されている。 However, it has been reported that more than one-third of open-angle glaucoma patients have normal-level intraocular pressure (normal-tension glaucoma). Therefore, the current definition of glaucoma is not conditional on the presence of elevated intraocular pressure, but rather, ``intraocular pressure and other currently unknown factors are associated with characteristic acquired atrophy of the optic nerve and retinal ganglion cells.'' It is said to be a "progressive chronic optic nerve disorder in adults that contributes to the loss of vision" (see Non-Patent Document 2). Even in Japan, 80% of glaucoma patients are normal-tension glaucoma patients, and visual field narrowing progresses without pain due to increased intraocular pressure. As a result, 90% of glaucoma patients are diagnosed without knowing it.
　正常眼圧緑内障患者においても、眼圧を低下させることが標準治療となっているが、眼圧低下は視神経を保護することで視野障害の進行を抑制する方法であり、失われた視野の回復は期待できない。そのため、眼圧を下降させること以外の、緑内障治療戦略が望まれている。 Lowering the intraocular pressure is the standard treatment for patients with normal-tension glaucoma, but lowering the intraocular pressure is a way to protect the optic nerve and suppress the progression of visual field damage, and it is possible to restore lost visual field. cannot be expected. Therefore, glaucoma treatment strategies other than lowering intraocular pressure are desired.
　現在、緑内障に代表される視神経障害を予防または治療する方法は確立されていない。何らかの原因で網膜神経節細胞が死滅したり、網膜神経節細胞の軸索である視神経が萎縮する等といった視神経障害に起因して、視野欠損等の問題が生じる。視神経がその投射部位である外側膝状体や上丘に正しく再投射し、視覚野に信号を伝達する経路が修復されれば、視野欠損が回復するものと考えられるが、それを可能にする方法や薬物等は未だ確立されていない。 Currently, there is no established method to prevent or treat optic nerve damage, such as glaucoma. Problems such as visual field defects occur due to optic nerve damage such as death of retinal ganglion cells for some reason or atrophy of the optic nerve, which is the axon of the retinal ganglion cells. If the optic nerve re-projects correctly to its projection sites, such as the lateral geniculate body and superior colliculus, and the pathway that transmits signals to the visual cortex is repaired, it is thought that visual field defects can be restored, and this is possible. Methods and drugs have not yet been established.
　そこで本発明は、緑内障等の視神経障害の予防または治療のための医薬または飲食品組成物を提供することを目的とする。 Therefore, an object of the present invention is to provide a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders such as glaucoma.
　上記事情に鑑みて、本発明は、ジオスゲニンまたはジオスゲニン誘導体を有効成分として含む、視神経障害の予防または治療のための医薬または飲食品組成物を提供する。かかる医薬または飲食品組成物によれば、網膜神経節細胞の増加および／または視神経の伸長に寄与し得るので、眼圧の低下の有無にかかわらず、視神経障害の予防または治療に有効である。 In view of the above circumstances, the present invention provides a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders, which contains diosgenin or a diosgenin derivative as an active ingredient. Such pharmaceutical or food/beverage compositions can contribute to an increase in retinal ganglion cells and/or elongation of optic nerves, and therefore are effective in preventing or treating optic nerve disorders, regardless of whether or not they reduce intraocular pressure.
　本発明の医薬または飲食品組成物は、ジオスゲニンまたはジオスゲニン誘導体を含む植物エキスを含有することが好ましい。ジオスゲニンまたはジオスゲニン誘導体を含む植物エキスは、安価で容易に入手可能であることから望ましい。 The pharmaceutical or food/beverage composition of the present invention preferably contains a plant extract containing diosgenin or a diosgenin derivative. Plant extracts containing diosgenin or diosgenin derivatives are desirable because they are inexpensive and easily available.
　上記植物エキスは、酸加水分解処理、発酵処理および酵素処理から選ばれる少なくとも１種の処理によってジオスゲニンの含有量を高めた植物エキスであることが好ましい。これにより、医薬または飲食品組成物による薬効をより向上させやすい。 The above-mentioned plant extract is preferably a plant extract with an increased content of diosgenin through at least one treatment selected from acid hydrolysis treatment, fermentation treatment, and enzyme treatment. This makes it easier to improve the medicinal efficacy of the medicine or food/beverage composition.
　本発明の医薬または飲食品組成物は、例えば、網膜神経節細胞の死滅を抑制させることによって視神経障害を予防または治療することができる。 The pharmaceutical or food/beverage composition of the present invention can prevent or treat optic neuropathy, for example, by suppressing the death of retinal ganglion cells.
　本発明の医薬または飲食品組成物は、視神経障害が緑内障である場合に特に有効である。さらに、本発明の医薬または飲食品組成物は、眼圧の低下の有無にかかわらず予防または治療が可能であるため、正常眼圧緑内障の予防または治療に特に有効である。 The pharmaceutical or food/beverage composition of the present invention is particularly effective when the optic nerve disorder is glaucoma. Furthermore, the pharmaceutical or food/beverage composition of the present invention is particularly effective in preventing or treating normal-tension glaucoma, since it can be prevented or treated regardless of whether or not there is a decrease in intraocular pressure.
　本発明によれば、緑内障等の視神経障害の予防または治療のための医薬または飲食品組成物を提供することができる。 According to the present invention, it is possible to provide a pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders such as glaucoma.
実施例１における、pNF-H（リン酸化型NF-H）により蛍光免疫染色された箇所とMAP2により蛍光免疫染色された箇所の顕微鏡写真である。FIG. 2 is a micrograph of a region fluorescently immunostained with pNF-H (phosphorylated NF-H) and a region fluorescently immunostained with MAP2 in Example 1. FIG. 実施例１における、網膜神経節細胞当たりの軸索長（μｍ）の計測結果を示す図である。FIG. 3 is a diagram showing the measurement results of axon length (μm) per retinal ganglion cell in Example 1. 実施例１における、網膜神経節細胞当たりの樹状突起長の計測結果を示す図である。FIG. 3 is a diagram showing the measurement results of dendrite length per retinal ganglion cell in Example 1. 実施例２における、眼圧測定の結果を示す図である。3 is a diagram showing the results of intraocular pressure measurement in Example 2. FIG. 実施例２における、損傷部位から近位の代表的写真とCTB-488陽性面積の定量値を示す図である。FIG. 2 is a diagram showing representative photographs proximal to the injury site and quantitative values of the CTB-488 positive area in Example 2. 実施例２における、損傷部位から遠位の代表的写真とCTB-488陽性面積の定量値を示す図である。FIG. 2 is a diagram showing representative photographs distal to the injury site and quantitative values of the CTB-488 positive area in Example 2. 標準物質（ジオスゲニン）について、LC-MS分析を行った結果を示す図である。FIG. 2 is a diagram showing the results of LC-MS analysis of a standard substance (diosgenin). 実施例３における、網膜についての分析結果を示す図である。FIG. 7 is a diagram showing the analysis results for the retina in Example 3. 実施例３における、視神経についての分析結果を示す図である。FIG. 7 is a diagram showing the analysis results for the optic nerve in Example 3. 実施例３における、脳についての分析結果を示す図である。FIG. 7 is a diagram showing the analysis results for the brain in Example 3. 実施例４における、眼圧測定の結果を示す図である。FIG. 7 is a diagram showing the results of intraocular pressure measurement in Example 4. 実施例４における、溶媒、0.1,1μmol/kgのジオスゲニン　オリブ油溶液を投与した場合の代表的顕微鏡写真である。This is a representative micrograph in Example 4 when a solvent, 0.1.1 μmol/kg diosgenin olive oil solution, was administered. 実施例４における、視神経を挫滅しなかった場合と挫滅した場合の網膜神経節細胞密度を示す図である。FIG. 4 is a diagram showing retinal ganglion cell densities when the optic nerve was not crushed and when the optic nerve was crushed in Example 4. 実施例４における、溶媒、0.1,1,10μmol/kgのジオスゲニン　オリブ油溶液を経口投与した後の網膜神経節細胞密度を示す図である。FIG. 4 is a diagram showing the retinal ganglion cell density after oral administration of a solvent, 0.1, 1, and 10 μmol/kg diosgenin olive oil solution in Example 4. 実施例４における、ジオスゲニン　オリブ油溶液を経口投与した場合等の代表的顕微鏡写真である。FIG. 4 is a representative micrograph of a case where a diosgenin olive oil solution was orally administered in Example 4. 実施例４における、視神経を挫滅しなかった場合と挫滅した場合の外側膝状体におけるＣＴＢ面積を示す図である。FIG. 7 is a diagram showing the CTB area in the lateral geniculate body in Example 4 when the optic nerve was not crushed and when the optic nerve was crushed. 実施例４における、視神経を挫滅しなかった場合と挫滅した場合の外側膝状体におけるＣＴＢ面積とFluoro-Gold陽性面積との重複面積を示す図である。FIG. 4 is a diagram showing the overlapping area of the CTB area and the Fluoro-Gold positive area in the lateral geniculate body when the optic nerve was not crushed and when the optic nerve was crushed in Example 4. 実施例４における、溶媒を投与した場合とジオスゲニン　オリブ油溶液を投与した場合におけるＣＴＢ面積を示す図である。FIG. 4 is a diagram showing the CTB area in Example 4 when a solvent was administered and when a diosgenin olive oil solution was administered. 実施例４における、溶媒を投与した場合とジオスゲニン　オリブ油溶液を投与した場合におけるＣＴＢ面積とFluoro-Gold陽性面積との重複面積を示す図である。FIG. 4 is a diagram showing the overlap area between the CTB area and the Fluoro-Gold positive area in the case of administering a solvent and the case of administering a diosgenin olive oil solution in Example 4.
　以下、本発明を実施するための形態について詳細に説明する。なお、本発明は、以下の実施形態に限定されるものではない。 Hereinafter, modes for carrying out the present invention will be described in detail. Note that the present invention is not limited to the following embodiments.
　本実施形態の医薬または飲食品組成物は、ジオスゲニンまたはジオスゲニン誘導体を有効成分として含む。 The pharmaceutical or food/beverage composition of this embodiment contains diosgenin or a diosgenin derivative as an active ingredient.
　ジオスゲニンは、下記化学式（Ｉ）で表される化合物である。
 Diosgenin is a compound represented by the following chemical formula (I).
　ジオスゲニン誘導体は、ジオスゲニンの等価物であり得る化合物をいう。 Diosgenin derivative refers to a compound that can be an equivalent of diosgenin.
　ジオスゲニンおよびジオスゲニン誘導体としては、特に限定されず、市販品であってもよく、公知方法、自体公知方法又はそれらに準ずる方法に従って製造したものであってもよく、天然物からの抽出物であってもよい。 Diosgenin and diosgenin derivatives are not particularly limited, and may be commercially available products, those manufactured according to known methods, methods known per se, or methods analogous thereto, and extracts from natural products. Good too.
　例えば、ジオスゲニン誘導体は、ジオスゲニンに置換基を導入したり、置換基を変換したりする化学修飾によって達成できる等価物であってもよく、天然物から抽出したジオスゲニン配糖体（ジオシン等）であってもよい。 For example, diosgenin derivatives may be equivalents that can be achieved by chemical modification such as introducing or converting substituents into diosgenin, or may be diosgenin glycosides (such as dioscine) extracted from natural products. It's okay.
　ジオスゲニン誘導体の具体例としては、例えば、ジオスゲニンにおける水酸基を、アルコキシ基、エステル基（例えば酢酸エステル）、アミノ酸エステル基（例えばグリシンエステル）、アミノスルホン酸エステル基、カーバメート基、ハロゲン原子（例えばフッ素原子）に変換した誘導体が挙げられる。 Specific examples of diosgenin derivatives include, for example, replacing the hydroxyl group in diosgenin with an alkoxy group, ester group (e.g. acetate ester), amino acid ester group (e.g. glycine ester), aminosulfonic acid ester group, carbamate group, halogen atom (e.g. fluorine atom). ).
　本実施形態の医薬または飲食品組成物は、ジオスゲニンまたはジオスゲニン誘導体を含む植物エキスを含有することが好ましい。植物エキスは、例えば、ジオスゲニン（またはその配糖体）を含むことが知られている、サンヤク（Dioscorea rhizome）、及びトリゴネラ属（Trigonella spp.）、アマドコロ属（Polygonatum spp.）、シオデ属（Smilax spp.）のようなハーブ薬等の植物の抽出物を用いることができる。抽出は、水、エタノール、１，３－ブチレングリコール等の抽出溶媒を用いて、一般的な方法で行うことができる。 The pharmaceutical or food/beverage composition of the present embodiment preferably contains a plant extract containing diosgenin or a diosgenin derivative. Plant extracts include, for example, Dioscorea rhizome, Trigonella spp., Polygonatum spp., and Smilax, which are known to contain diosgenin (or its glycosides). Extracts of plants such as herbal medicines such as spp.) can be used. Extraction can be performed by a conventional method using an extraction solvent such as water, ethanol, 1,3-butylene glycol, or the like.
　植物エキスは、更なる処理を行うことによって、ジオスゲニンの含有量を高めた植物エキスであることが好ましい。処理としては、例えば、酸加水分解処理、発酵処理、酵素処理等が挙げられる。これらの処理は１種を単独で用いても、２種以上を組み合わせて用いてもよい。これらの処理としては、従来公知の処理方法を適用することができるが、例えば、酸加水分解処理としてはT Herrera et al., J Sci Food Agric 2019; 99: 3157-3167に記載の方法を、発酵処理としてはY. Chen et al., Steroids 136 (2018) 40-46やJ.Dong et al., Indian J Microbiol (Apr-June 2015) 55(2):200-206に記載の方法を、酵素処理としてはW. Huang et al., Bioresource Technology 99 (2008) 7407-7411に記載の方法をそれぞれ適用することができる。 The plant extract is preferably a plant extract with increased diosgenin content through further processing. Examples of the treatment include acid hydrolysis treatment, fermentation treatment, enzyme treatment, and the like. These treatments may be used alone or in combination of two or more. Conventionally known treatment methods can be applied to these treatments, but for example, as acid hydrolysis treatment, the method described in T Herrera et al., J Sci Food Agric 2019; 99: 3157-3167, For fermentation treatment, the methods described in Y. Chen et al., Steroids 136 (2018) 40-46 and J. Dong et al., Indian J Microbiol (Apr-June 2015) 55(2):200-206 are used. As the enzyme treatment, the methods described in W. Huang et al., Bioresource Technology 99 (2008) 7407-7411 can be applied.
　本実施形態の医薬の形態としては、特に限定されず、ジオスゲニンまたはジオスゲニン誘導体をそのまま剤（例えば粉剤）としてもよく、他の成分とともに製剤化してもよい。 The form of the medicament of this embodiment is not particularly limited, and diosgenin or a diosgenin derivative may be used as it is (for example, a powder), or may be formulated with other ingredients.
　他の成分としては、一般に製剤の原料として用いられる成分等が挙げられ、特に限定されない。その具体例としては、担体、賦形剤、結合剤、崩壊剤、滑沢剤、コーティング剤、着色剤、矯味矯臭剤、安定化剤、乳化剤、吸収促進剤、界面活性剤、ｐＨ調整剤、防腐剤、抗酸化剤等が挙げられる。これらは、単独で又は２種以上組み合わせて使用してもよい。 Other components include components that are generally used as raw materials for pharmaceutical preparations, and are not particularly limited. Specific examples include carriers, excipients, binders, disintegrants, lubricants, coating agents, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, Examples include preservatives and antioxidants. These may be used alone or in combination of two or more.
　本実施形態の医薬または飲食品組成物は経口で投与してもよく、非経口で投与してもよい。非経口の投与形態としては、眼局所投与（硝子体内投与、点眼投与、結膜嚢内投与、結膜下投与、テノン嚢下投与等）、静脈内投与、経皮投与等が挙げられる。 The pharmaceutical or food/beverage composition of this embodiment may be administered orally or parenterally. Parenteral administration forms include topical ocular administration (intravitreal administration, eye drops, intraconjunctival sac administration, subconjunctival administration, sub-Tenon's administration, etc.), intravenous administration, transdermal administration, and the like.
　経口投与の場合の投与剤形としては、例えば、液剤、懸濁剤、カプセル剤、ソフトカプセル剤、錠剤、顆粒剤、散剤、シロップ剤、ゼリー剤、口腔内崩壊錠、チュアブル錠等が挙げられる。投与剤形中、ジオスゲニンまたはジオスゲニン誘導体は油脂に懸濁または溶解していると好ましい。 Examples of dosage forms for oral administration include solutions, suspensions, capsules, soft capsules, tablets, granules, powders, syrups, jellies, orally disintegrating tablets, and chewable tablets. In the dosage form, diosgenin or diosgenin derivative is preferably suspended or dissolved in an oil or fat.
　油脂としては、例えば、大豆油、菜種油（ナタネ油、カノラ油）、高オレイン酸菜種油、コーン油、ゴマ油、ゴマサラダ油、太白ゴマ油、シソ油、亜麻仁油（アマニ油）、落花生油、紅花油（サフラワー油）、高オレイン酸紅花油、ひまわり油、高オレイン酸ひまわり油、綿実油、ブドウ種子油、マカデミアナッツ油、ヘーゼルナッツ油、ピーナッツ油、アーモンド油、ナッツ油、クルミ油、カボチャ種子油、クルミ油、レモン油、椿油、茶実油、エゴマ油、ボラージ油、オリーブ油（オリブ油）、米油、米糠油、小麦胚芽油、パーム油、パームオレイン、パームステアリン、パーム核油、ヤシ油、カカオ脂等の植物油；牛脂、豚脂（ラード）、鶏脂、乳脂、魚油（例えば、鰯油、鯖油、鱈油、鯨油、肝油等）等の動物油、脂肪酸類（ドコサヘキサエン酸（ＤＨＡ）、エイコサペンタエン酸（ＥＰＡ）等）、脂溶性ビタミン類（ビタミンＡ、ビタミンＥ等）等の食用油；中鎖脂肪酸トリグリセリド、ヨード化ケシ脂肪酸エステル等の合成油が挙げられる。これらは、単独で又は２種以上組み合わせて使用してもよい。 Examples of oils and fats include soybean oil, rapeseed oil (rapeseed oil, canola oil), high oleic acid rapeseed oil, corn oil, sesame oil, sesame salad oil, Taihaku sesame oil, perilla oil, linseed oil (linseed oil), peanut oil, safflower oil ( safflower oil), high oleic safflower oil, sunflower oil, high oleic sunflower oil, cottonseed oil, grape seed oil, macadamia nut oil, hazelnut oil, peanut oil, almond oil, nut oil, walnut oil, pumpkin seed oil, walnut oil , lemon oil, camellia oil, tea seed oil, perilla oil, borage oil, olive oil (olive oil), rice oil, rice bran oil, wheat germ oil, palm oil, palm olein, palm stearin, palm kernel oil, coconut oil, cocoa butter Vegetable oils such as beef tallow, pork fat (lard), chicken fat, milk fat, animal oils such as fish oil (e.g. sardine oil, mackerel oil, cod oil, whale oil, liver oil, etc.), fatty acids (docosahexaenoic acid (DHA), eicosapentaene, etc.) Examples include edible oils such as acid (EPA), fat-soluble vitamins (vitamin A, vitamin E, etc.); synthetic oils such as medium-chain fatty acid triglycerides, and iodized poppy seed fatty acid esters. These may be used alone or in combination of two or more.
　非経口投与の場合の投与剤形としては、例えば、注射剤、点眼剤、眼軟膏、貼布剤、ゲル、挿入剤等が挙げられる。 Examples of dosage forms for parenteral administration include injections, eye drops, eye ointments, patches, gels, and inserts.
　本実施形態の飲食品組成物は、飲料や食品、サプリメント等の形で提供することができる。飲料としては、例えば、水、清涼飲料水、果汁飲料、炭酸飲料、乳飲料、アルコール飲料、スポーツドリンク、栄養ドリンク等が挙げられる。食品としては、パン類、麺類、米類、豆腐、乳製品、醤油、味噌、菓子類等が挙げられる。飲食品組成物には、例えば、健康食品、保健機能食品、機能性表示食品、特別用途食品、栄養補助食品及び特定保健用食品等が含まれる。 The food and drink composition of this embodiment can be provided in the form of drinks, foods, supplements, and the like. Examples of beverages include water, soft drinks, fruit juice drinks, carbonated drinks, milk drinks, alcoholic drinks, sports drinks, nutritional drinks, and the like. Examples of foods include breads, noodles, rice, tofu, dairy products, soy sauce, miso, and sweets. Food and drink compositions include, for example, health foods, foods with health claims, foods with functional claims, foods for special uses, nutritional supplements, and foods for specified health uses.
　本実施形態の医薬または飲食品組成物における、有効成分であるジオスゲニンまたはジオスゲニン誘導体の含有量は、特に限定されないが、疾患に伴う症状を治療、改善、緩和、又は回復させるのに十分な用量とすることが好ましい。 The content of diosgenin or a diosgenin derivative as an active ingredient in the pharmaceutical or food/beverage composition of the present embodiment is not particularly limited, but is a sufficient amount to treat, improve, alleviate, or recover the symptoms associated with the disease. It is preferable to do so.
　本実施形態の医薬または飲食品組成物の投与量は、例えば投与する対象の症状の程度、年齢、性別、体重、投与形態、塩の種類、疾患の具体的な種類等に応じて、適宜選択してもよく、特に限定されないが、投与する対象の単位体重当たりのジオスゲニンまたはジオスゲニン誘導体のモル量で表わした場合、例えば、通常、約０．００１～約１０００μｍｏｌ／ｋｇ／日であってよく、好ましくは約０．０１～約１０μｍｏｌ／ｋｇ／日、より好ましくは０．０１～約１μｍｏｌ／ｋｇ／日である。 The dosage of the pharmaceutical or food/beverage composition of the present embodiment is appropriately selected depending on, for example, the severity of symptoms of the subject to be administered, age, sex, body weight, dosage form, type of salt, specific type of disease, etc. Although not particularly limited, the molar amount of diosgenin or diosgenin derivative per unit body weight of the subject to be administered may be, for example, usually about 0.001 to about 1000 μmol/kg/day, Preferably about 0.01 to about 10 μmol/kg/day, more preferably 0.01 to about 1 μmol/kg/day.
　本実施形態の医薬または飲食品組成物の投与対象は、特に限定されないが、ヒトを含む哺乳動物であることが好ましい。ヒトを含む哺乳動物としては、特に限定されないが、例えば、ヒト、サル、マントヒヒ、チンパンジー、マウス、ラット、モルモット、ハムスター、ウサギ、ネコ、イヌ、ヒツジ、ヤギ、ブタ、ウシ及びウマ等が挙げられる。 The subject to whom the medicine or food/beverage composition of the present embodiment is administered is not particularly limited, but is preferably a mammal including a human. Mammals including humans include, but are not particularly limited to, humans, monkeys, baboon, chimpanzees, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, etc. .
　本実施形態の医薬または飲食品組成物は、視神経障害の予防または治療に有効である。視神経障害としては、緑内障、視神経症、視神経炎等の視神経疾患が挙げられるが、本実施形態の医薬または飲食品組成物は、緑内障、特に正常眼圧緑内障の予防または治療に特に有効であり得る。 The pharmaceutical or food/beverage composition of this embodiment is effective for preventing or treating optic nerve disorders. Optic nerve disorders include optic nerve diseases such as glaucoma, optic neuropathy, and optic neuritis, and the pharmaceutical or food/beverage composition of the present embodiment may be particularly effective in preventing or treating glaucoma, particularly normal-tension glaucoma. .
　以下、実施例に基づいて本発明をより具体的に説明する。ただし、本発明は、以下の実施例により限定されるものではない。 Hereinafter, the present invention will be explained more specifically based on Examples. However, the present invention is not limited to the following examples.
実験材料の準備と一般的な操作
＜ジオスゲニン　エタノール溶液の調製＞
　ジオスゲニン（Diosgenin; 東京化成）を99.5％エタノール（Fujifilm 和光製薬）に溶解させ、ジオスゲニン高濃度溶液を調製し、生理食塩水で希釈してジオスゲニン　エタノール溶液を調製した。ジオスゲニン　エタノール溶液としては、0.1μM、１μMの濃度のものをそれぞれ準備した。 Preparation of experimental materials and general operations <Preparation of diosgenin ethanol solution>
Diosgenin (Tokyo Kasei) was dissolved in 99.5% ethanol (Fujifilm, Wako Pharmaceutical) to prepare a high concentration solution of diosgenin, and diluted with physiological saline to prepare a diosgenin ethanol solution. Diosgenin ethanol solutions with concentrations of 0.1 μM and 1 μM were prepared, respectively.
＜ジオスゲニン　オリブ油溶液の調製＞
　ジオスゲニン（東京化成）9.6mgを日本薬局方オリブ油（丸石製薬）790μlに溶解させジオスゲニン　オリブ油溶液を調製した。 <Preparation of diosgenin olive oil solution>
A diosgenin olive oil solution was prepared by dissolving 9.6 mg of diosgenin (Tokyo Kasei) in 790 μl of Japanese Pharmacopoeia olive oil (Maruishi Pharmaceutical).
＜マウス＞
　すべてのマウスはプラスチックケージ（23×16×12cm）に入れ、12時間の明暗周期（明期：７時～19時）、恒温恒湿（22℃±2℃,55±10％）の環境で飼育した。水および固形飼料は自由に摂取させた。 <Mouse>
All mice were housed in plastic cages (23 x 16 x 12 cm) in an environment with a 12-hour light/dark cycle (light period: 7:00 am to 7:00 pm) and constant temperature and humidity (22°C ± 2°C, 55 ± 10%). bred. Water and chow were available ad libitum.
＜正常眼圧緑内障モデルマウスの作製＞
　実験には雄性6週齢ddYマウス（日本SLC）を用いた。三種混合麻酔を腹腔内に投与して麻酔した。眼球を引き出しながら、眼球直近の視神経をピンセットで一秒間圧迫した。両目とも視神経の圧迫を行った。アンチセダンを腹腔内投与し、マウスを37℃のホットプレート上に静置し、体温の低下を防ぎながら覚醒させた。 <Preparation of normal tension glaucoma model mouse>
Male 6-week-old ddY mice (Japan SLC) were used in the experiment. The animal was anesthetized by intraperitoneally administering a triple-mixed anesthesia. While pulling out the eyeball, the optic nerve in the immediate vicinity of the eyeball was compressed for one second with forceps. Optic nerve compression was performed in both eyes. Antisedan was administered intraperitoneally, and the mice were left on a hot plate at 37°C to wake up while preventing a drop in body temperature.
＜眼圧測定法＞
　マウスを脱血灌流する直前、麻酔下で眼圧計測を行った。トノラボ手持眼圧計(マウス・ラット専用エムイーテクニカ)を用い、片目につき5回測定して得られた値の平均値を採用した。 <Intraocular pressure measurement method>
Immediately before exsanguination and perfusion of the mouse, intraocular pressure was measured under anesthesia. Using a Tonolab hand-held tonometer (M-E Technica for mice and rats), the average value of the values obtained by measuring 5 times per eye was used.
＜網膜、視神経および脳の摘出＞
　マウスに深麻酔をかけ、胸部を切開し、右心耳に切れ込みを入れて翼状針を左心室に刺入し、氷冷した生理食塩水を25-35ml灌流脱血した。灌流後、ピンセットを用いて眼球をつまみ出し、視神経ごとゆっくりと眼球を引き出した。取り出した眼球および視神経をPBSで洗浄した後、4％パラホルムアルデヒド-PBS溶液に1時間浸して固定し、PBS溶液で洗浄した。マウスを断頭して全脳を摘出した後、脳をドライアイス上で15分間急速凍結させた。凍結した脳はアルミホイルで包み、-30℃で保存した。洗浄した眼球はPBS溶液に入れ実体顕微鏡(SZ61、Olympus)下で21G注射針を用いて眼球に小さな穴を開けた後、穴からハサミの片方の刃を入れ込み、角膜を切り離した。水晶体およびゲル状の硝子体を丁寧に取り除いた後、網膜だけを単離した。その後、視神経を眼球からマイクロシザーズで切断し、網膜と視神経それぞれを30%スクロース溶液で置換した。 <Extraction of retina, optic nerve and brain>
The mouse was deeply anesthetized, an incision was made in the chest, an incision was made in the right atrial appendage, a winged needle was inserted into the left ventricle, and blood was removed by perfusion with 25-35 ml of ice-cold physiological saline. After perfusion, the eyeball was pinched out using tweezers and the eyeball was slowly pulled out along with the optic nerve. The removed eyeballs and optic nerves were washed with PBS, fixed by immersion in 4% paraformaldehyde-PBS solution for 1 hour, and washed with PBS solution. After decapitating the mouse and removing the whole brain, the brain was quickly frozen on dry ice for 15 minutes. Frozen brains were wrapped in aluminum foil and stored at -30°C. The washed eyeballs were placed in a PBS solution and a small hole was made in the eyeballs using a 21G injection needle under a stereomicroscope (SZ61, Olympus), and one blade of scissors was inserted through the hole to cut off the cornea. After carefully removing the lens and gel-like vitreous body, only the retina was isolated. Thereafter, the optic nerve was cut from the eyeball with microscissors, and the retina and optic nerve were each replaced with a 30% sucrose solution.
＜網膜の蛍光免疫染色＞
　網膜は、あらかじめ0.5%TritonX-100(和光純薬)-PBS溶液を150μl/well入れておいた96穴ウェルプレートに1網膜ごと入れた。ウェル中で5分間静置させ、溶液を交換しさらに5分間静置させたのち溶液をすべて丁寧に除去した。その後、一次抗体溶液[0.5%TritonX-100-PBS、1% normal goat serum(和光純薬)、rabbit抗RNA-binding protein with multiplesplicing (RBPMS) polyclonal抗体(1:200,GeneTex)]を100μl/well加え、4Cで2日間揺らしながら反応させた。一次抗体液を除去して0.5%TritonX-100-PBS溶液で15分間の洗浄を2回行った後、二次抗体液[0.5%TritonX-100-PBS溶液、Alexa Fluor647標識 goat anti-rabbit IgG抗体(1:600,Thermo Fisher Scientific)]を100μl/well加え、遮光下、4℃で一晩揺らしながら反応させた。反応後、二次抗体溶液を除去してPBSで洗浄した後、カップ状になっている網膜に4ヵ所切れ込みをいれてスライドグラス上に移し、フラットマウントとした後、Aqua poly Mount(Polysciences, Warrington, PA, USA)で封入した。 <Fluorescent immunostaining of the retina>
Each retina was placed into a 96-well plate in which 150 μl/well of 0.5% TritonX-100 (Wako Pure Chemical Industries, Ltd.)-PBS solution had been added in advance. The well was allowed to stand for 5 minutes, the solution was exchanged, and the solution was left to stand for another 5 minutes, after which all the solution was carefully removed. Then, add 100 μl/well of primary antibody solution [0.5% TritonX-100-PBS, 1% normal goat serum (Wako Pure Chemical Industries), rabbit anti-RNA-binding protein with multiple splicing (RBPMS) polyclonal antibody (1:200, GeneTex)]. In addition, the reaction was performed at 4C for 2 days with shaking. After removing the primary antibody solution and washing twice for 15 minutes with 0.5% TritonX-100-PBS solution, the secondary antibody solution [0.5% TritonX-100-PBS solution, Alexa Fluor647-labeled goat anti-rabbit IgG antibody] (1:600, Thermo Fisher Scientific)] was added at 100 μl/well, and the reaction was allowed to occur overnight at 4°C with shaking in the dark. After the reaction, the secondary antibody solution was removed and washed with PBS. Four incisions were made in the cup-shaped retina and the plate was transferred onto a slide glass. After being flat mounted, Aqua poly Mount (Polysciences, Warrington , PA, USA).
＜網膜神経節細胞数の定量＞
　蛍光免疫染色後の網膜組織視察には、蛍光倒立顕微鏡KEYENCE BZ-X800 (Keyence)および対物レンズ(PlanApo10x,Nikon)を用い画像を取得した。画像解析には、ハイブリットセルカウント(Keyence)ソフトウェアを用い、網膜フラットマウント上における視神経乳頭と外周の中間地点にあたる部分で、かつ染まりムラや破れが生じていない箇所のRBPMS陽性細胞数を定量した。
(n = 3-6 optic nerves, 8-12 retinas, **P < 0.01, vs Non-crush,unpaired t-test,  *P < 0.05 , **P < 0.01, vs Vehicle, One-way ANOVA,Dunnett's test) <Quantification of retinal ganglion cell number>
For observation of retinal tissue after fluorescent immunostaining, images were acquired using a fluorescent inverted microscope KEYENCE BZ-X800 (Keyence) and an objective lens (PlanApo10x, Nikon). For image analysis, hybrid cell counting (Keyence) software was used to quantify the number of RBPMS-positive cells on the retinal flat mount at the midpoint between the optic disc and the periphery, where there were no uneven staining or tears.
(n = 3-6 optic nerves, 8-12 retinas, **P < 0.01, vs Non-crush,unpaired t-test, *P < 0.05 , **P < 0.01, vs Vehicle, One-way ANOVA,Dunnett's test)
＜視神経切片の作製＞
　スクロース置換を行った視神経は、クリオモルド3号(サクラファインテックジャパン)を型にしてTissue Tek OCT(サクラファインテックジャバン)中に埋め込んだ。クリオスタット(CM3050SL, Leica)を用いて連続的に12μm厚矢状断切片を作製した。その際、庫内およびステージの温度は-22℃に設定した。切片はコーティング済みスライドグラス(松浪硝子工業)に貼り付け、-30℃に保存した。 <Preparation of optic nerve section>
The sucrose-substituted optic nerve was implanted into Tissue Tek OCT (Sakura Finetech Japan) using Cryomold No. 3 (Sakura Finetech Japan) as a mold. Sagittal sections with a thickness of 12 μm were successively prepared using a cryostat (CM3050SL, Leica). At that time, the temperature inside the chamber and on the stage was set to -22°C. The sections were attached to coated glass slides (Matsunami Glass Industries) and stored at -30°C.
＜視神経切片の画像解析＞
　視神経切片観察には蛍光倒立顕微鏡KEYENCE BZ-X800(Keyence)および対物レンズ(PlanApo10x, Nikon)を用い、眼球近位の視神経圧迫部位を含む場所から遠位にわたり画像を取得した。画像解析にはImageJ(National Institutes of Health)を用い、各蛍光画像を損傷部位から近位（～500μm）と遠位（500～1000μm）の領域に分けて、それぞれの面積中におけるCTB陽性面積割合を算出した。
（スケール100μm．n=3-4 mice、One-way ANOVA、post hoc Dunnett's test） <Image analysis of optic nerve sections>
For observation of optic nerve sections, an inverted fluorescent microscope KEYENCE BZ-X800 (Keyence) and an objective lens (PlanApo10x, Nikon) were used to acquire images from a location including the optic nerve compression site proximal to the eyeball to a distal location. ImageJ (National Institutes of Health) was used for image analysis, and each fluorescence image was divided into regions proximal (~500 μm) and distal (500 to 1000 μm) from the injury site, and the percentage of CTB-positive area in each area was calculated. was calculated.
(Scale 100μm, n=3-4 mice, One-way ANOVA, post hoc Dunnett's test)
＜脳切片の作製＞
　脳は、クリオスタット(CM3050SL, Leica)を用いて、Bregma -1.70mm～-2.92mmにわたる領域で、連続的に12μm厚冠状断切片を作製した。その際庫内およびステージの温度は-20℃に設定した。切片はコーティング済みスライドグラス(松浪硝子工業)に貼り付け、-30Cに保存した。脳切片観察には蛍光倒立顕微鏡KEYENCE BZ-X800(Keyence)および対物レンズ(PlanApo10x, Nikon)を用い、左右外側膝状体の範囲を撮影した。画像解析には、MetaMorphソフトウェアを用い、CTB陽性面積を算出した。
（n = 2-5 , *P < 0.01, vs Non-crush, unpaired t-test.） <Preparation of brain sections>
Using a cryostat (CM3050SL, Leica), the brain was serially sectioned into 12-μm-thick coronal sections in a region ranging from Bregma -1.70 mm to -2.92 mm. At that time, the temperature inside the warehouse and on the stage was set to -20°C. The sections were attached to coated glass slides (Matsunami Glass Industries) and stored at -30C. For brain section observation, a fluorescent inverted microscope KEYENCE BZ-X800 (Keyence) and an objective lens (PlanApo10x, Nikon) were used to photograph the left and right lateral geniculate bodies. For image analysis, MetaMorph software was used to calculate the CTB positive area.
(n = 2-5, *P < 0.01, vs Non-crush, unpaired t-test.)
（実施例１）
　ジオスゲニンの作用による培養網膜神経節細胞の軸索伸長作用の検討を行った。詳細を以下に示す。 (Example 1)
We investigated the effect of diosgenin on axonal elongation in cultured retinal ganglion cells. Details are shown below.
＜網膜神経節細胞初代培養＞
　生後４日齢のddYマウス(日本SLC)の眼球をイソフルラン麻酔下で取り出した。眼球はB-27入りNeurobasal-A培地（Neurobasal-A medium ;Life Technologies)[2mM L-glutamine, 1X B-27付加]に入れ、実体顕微鏡下で網膜を単離した。その後700rpmで3分間遠心して上清を除去し、沈査に0.05 trypsin-0.53mM EDTAsolution(Life Technologies)を2ml加えて懸濁した。37℃で7分間静置し、5分経過後に一度タッピングを行った。インキュペーション後、培地を2ml加え700rpmで3分間遠心して上清を除去し、沈査に600U/ml DNase I (Life Technologies)-0.03% trypsin inhibitor (LifeTechnologies)-PBS溶液を1ml加えて懸濁した。37℃で5分間静置したのち、懸濁して培地を2ml加え、700mmで3分間遠心した。上清を除去し、沈査に培地を1ml加え、先端を炙りなめしたバスツールピペットで細胞塊が見えなくなるまで穏やかに懸濁した後、70μm nylon cell strainer (Becton Diskinsonand Company)で濾過した。Trypanblue染色で死細胞と分別しながら生細胞数をカウントし、1.0×105cells/well/300μlとなるように8 wellチャンバースライドに播種した。すべての培養皿は5μg/ml poly-D-lysin-PBS溶液を入れ37℃で16時間インキュベートし、PBSで2回洗浄した後、さらに20μg/ml laminin-PBS溶液で37℃、16時間インキュベートし、PBSで2回洗浄したものを用いた。播種後は10% CO2 37℃、飽和蒸気下で培養を行い、播種から24時間後に培地の全量を交換した。培地交換後は、5% CO2 37℃、飽和蒸気下で培養を行った。 <Retinal ganglion cell primary culture>
The eyeballs of 4-day-old ddY mice (Japan SLC) were removed under isoflurane anesthesia. The eyeballs were placed in Neurobasal-A medium (Life Technologies) containing B-27 [2mM L-glutamine, 1X B-27 added], and the retinas were isolated under a stereomicroscope. Thereafter, the supernatant was removed by centrifugation at 700 rpm for 3 minutes, and 2 ml of 0.05 trypsin-0.53 mM EDTA solution (Life Technologies) was added to the pellet to suspend it. It was left standing at 37°C for 7 minutes, and tapped once after 5 minutes. After incubation, 2 ml of medium was added and centrifuged at 700 rpm for 3 minutes to remove the supernatant, and 1 ml of 600 U/ml DNase I (Life Technologies)-0.03% trypsin inhibitor (Life Technologies)-PBS solution was added to the pellet to suspend it. After standing at 37°C for 5 minutes, the suspension was suspended, 2 ml of culture medium was added, and centrifuged at 700 mm for 3 minutes. The supernatant was removed, 1 ml of culture medium was added to the pellet, and the suspension was gently suspended using a Barstool pipette with a burnt tip until no cell mass was visible, followed by filtration using a 70 μm nylon cell strainer (Becton Diskinson and Company). The number of living cells was counted while separating them from dead cells by Trypanblue staining, and the cells were seeded in an 8-well chamber slide at a density of 1.0×10 5 cells/well/300 μl. All culture dishes were incubated with 5μg/ml poly-D-lysin-PBS solution at 37℃ for 16 hours, washed twice with PBS, and further incubated with 20μg/ml laminin-PBS solution at 37℃ for 16 hours. , and those washed twice with PBS were used. After seeding, culture was performed under 10% CO 2 at 37° C. and saturated steam, and the entire amount of the medium was replaced 24 hours after seeding. After medium exchange, culture was performed under 5% CO 2 at 37°C and saturated steam.
＜網膜神経節細胞への薬物処置＞
　上述の0.1μM、１μMジオスゲニン　エタノール溶液を、それぞれB-27入りNeurobasal-A培地に溶解(エタノール 終濃度:9.95×10-2%)して各濃度の培地を作製した。これを網膜神経節細胞培養開始から４日後の培地と全量交換した後に、更に４日間培養を行った。 <Drug treatment on retinal ganglion cells>
The above-mentioned 0.1 μM and 1 μM diosgenin ethanol solutions were each dissolved in Neurobasal-A medium containing B-27 (ethanol final concentration: 9.95×10 −2 %) to prepare a culture medium of each concentration. After replacing the entire volume with the medium 4 days after the start of retinal ganglion cell culture, the culture was continued for an additional 4 days.
＜網膜神経節細胞の蛍光免疫染色＞
　薬物処置後の網膜神経節細胞の培養終了後、培地を除去してPBSで洗浄を行った。4%パラホルムアルデヒド-PBS溶液を加え、30分間常温で静置し、細胞の固定を行った。固定液を除去した後、0.3% TritonX-100(和光純薬)-PBS溶液で5分間の洗浄を2回行った。一次抗体溶液[0.3%Triton100-PBS溶波、1% normal goat serum(Fujifilm和光純薬)、rabbit抗MAP2抗体(1:1000,Abcam)、mouse 抗phosphorylated neurofilament-H (pNF-H)monoclonal抗体(1:200,Convance)]を100μl/well加え、4℃で一晩反応させた。翌日、一次抗体液を除去して0.3%TritonX-100-PBS溶液で5分間の洗浄を2回行った後二次抗体液[0.3%TritonX-100-PBS溶液AlexFluoro594標識goat anti-rabbit IgG抗体(1:600,Thermo Fisher Scientific), AlexaFluor 488標識 goat anti-mouse IgG 抗体(1:600,Thermo FisherScientific)]を100μl/well加え、遮光下、室温で2時間反応させた。反応後、二次抗体溶液を除去してPBSで洗浄した後、4,6-diamidino-2-phenylindole(DAPI)(Enzo Life Science, Farmingdale)をPBSで1μg/mlとし100μl/well加え、遮光下、室温で5分間反応させた。反応後、DAPI溶液を除去してPBSで5分間の洗浄を1回行った後、AquaPolyount(Polyscience)で封入した。 <Fluorescent immunostaining of retinal ganglion cells>
After culturing the retinal ganglion cells after drug treatment, the medium was removed and the cells were washed with PBS. A 4% paraformaldehyde-PBS solution was added, and the cells were fixed by standing at room temperature for 30 minutes. After removing the fixative, washing was performed twice for 5 minutes with 0.3% TritonX-100 (Wako Pure Chemical Industries)-PBS solution. Primary antibody solution [0.3% Triton100-PBS solution, 1% normal goat serum (Fujifilm Wako Pure Chemical Industries), rabbit anti-MAP2 antibody (1:1000, Abcam), mouse anti-phosphorylated neurofilament-H (pNF-H) monoclonal antibody ( 1:200, Convance)] was added at 100 μl/well, and the reaction was allowed to occur overnight at 4°C. The next day, remove the primary antibody solution and wash twice for 5 minutes with 0.3% TritonX-100-PBS solution. 1:600, Thermo Fisher Scientific), AlexaFluor 488-labeled goat anti-mouse IgG antibody (1:600, Thermo Fisher Scientific)] was added at 100 μl/well, and the mixture was allowed to react at room temperature for 2 hours in the dark. After the reaction, remove the secondary antibody solution and wash with PBS, then add 100 μl/well of 4,6-diamidino-2-phenylindole (DAPI) (Enzo Life Science, Farmingdale) at 1 μg/ml in PBS, and add it under light protection. , and reacted for 5 min at room temperature. After the reaction, the DAPI solution was removed and washed once with PBS for 5 minutes, followed by encapsulation with AquaPolyount (Polyscience).
＜培養網膜神経節細胞の画像解析＞
　蛍光免疫染色後のスライド観察には、蛍光倒立顕微鏡Cell Observer (Carl Zeiss)、Axio Vision4.8ソフトウェア(CarlZeiss)を用い、一枚当たり432.49×322.81μmの大きさの画像を取得した。網膜神経節細胞当たりの軸索長、樹状突起長、および細胞数の計測にはMetaMorph version 7.8(Molecular Devices)を用いた。 <Image analysis of cultured retinal ganglion cells>
For observation of slides after fluorescent immunostaining, an inverted fluorescence microscope Cell Observer (Carl Zeiss) and Axio Vision 4.8 software (Carl Zeiss) were used to obtain images of 432.49 x 322.81 μm per slide. MetaMorph version 7.8 (Molecular Devices) was used to measure axon length, dendrite length, and cell number per retinal ganglion cell.
　pNF-Hにより蛍光免疫染色された箇所（軸索に相当）とMAP2により蛍光免疫染色された箇所（樹状突起に相当）の顕微鏡写真を図１Ａに、網膜神経節細胞当たりの軸索長（μｍ）の計測結果を図１Ｂに、網膜神経節細胞当たりの樹状突起長（μｍ）の計測結果を図１Ｃに、それぞれ示す。 Figure 1A shows micrographs of the areas fluorescently immunostained by pNF-H (corresponding to axons) and the areas fluorescently immunostained by MAP2 (corresponding to dendrites). FIG. 1B shows the measurement results of the dendrite length (μm) per retinal ganglion cell, and FIG. 1C shows the measurement results of the dendrite length (μm) per retinal ganglion cell.
　これらの結果から明らかであるように、0.1μM、1μMジオスゲニン溶液（エタノール０．１％、培地９９．９％）で処置することにより、網膜神経節細胞における軸索及び樹状突起の有意な伸長が見られた。 As is clear from these results, treatment with 0.1 μM and 1 μM diosgenin solutions (ethanol 0.1%, medium 99.9%) resulted in significant elongation of axons and dendrites in retinal ganglion cells. It was observed.
（実施例２）
　緑内障モデルマウスとして、視神経の挫滅と再投射を確実に評価するためにONC（Optic nerve crush）モデルを用い、ジオスゲニンの硝子体投与による視神経への作用を検討した。詳細を以下に示す。 (Example 2)
We used an optical nerve crush (ONC) model as a glaucoma mouse model to reliably evaluate optic nerve crush and reprojection, and investigated the effects of intravitreal administration of diosgenin on the optic nerve. Details are shown below.
＜薬物硝子体内投与＞
　上述の0.1μM、１μMジオスゲニン溶液（エタノール2.5%、生理食塩水97.5%）を、それぞれ三種混合麻酔を腹腔内に投与して麻酔下で、正常眼圧緑内障モデルマウスの硝子体に投与した。ジオスゲニン濃度は硝子体内での最終濃度として表した。実体顕微鏡（SZ61, Olympus）下でマウスの眼球をピンセットでつまみ出し、先端の外径を0.15mmに引いた滅菌済ガラスシリンジ（Narishige, 東京）を毛様体部分から硝子体内に向けて差し込んだ。ガラスシリンジを0.5mmチュービング（エムエス機器）と21G注射針（テルモ）を介して10μlHamilton注射器（Hamilton Company）と連結させ、シリンジポンプ（KD Scientific）にセットした。硝子体内へのガラスシリンジ挿入後にジオスゲニン　エタノール溶液を1.0μl/eye/minで注入した。硝子体内注射後、アンチセダンを麻酔薬と同量、腹腔内投与し、マウスを37℃のホットプレート上に静置し、体温の低下を防ぎながら覚醒させた。同様の方法で、最初の投与から５日おきに計３回硝子体投与を行った。 <Intravitreal drug administration>
The above-mentioned 0.1 μM and 1 μM diosgenin solutions (2.5% ethanol, 97.5% physiological saline) were each administered into the vitreous body of a normal-tension glaucoma model mouse under anesthesia by intraperitoneally administering a triple-mixed anesthesia. Diosgenin concentration was expressed as the final intravitreal concentration. Under a stereomicroscope (SZ61, Olympus), the eyeball of the mouse was picked out with tweezers, and a sterilized glass syringe (Narishige, Tokyo) with an outer diameter of 0.15 mm at the tip was inserted from the ciliary body toward the vitreous body. . The glass syringe was connected to a 10 μl Hamilton syringe (Hamilton Company) via 0.5 mm tubing (MS Equipment) and a 21G injection needle (Terumo), and set in a syringe pump (KD Scientific). After inserting a glass syringe into the vitreous, diosgenin ethanol solution was injected at 1.0 μl/eye/min. After intravitreal injection, the same amount of antisedan as the anesthetic was administered intraperitoneally, and the mouse was left on a hot plate at 37°C to waken up while preventing a drop in body temperature. In the same manner, intravitreal administration was performed three times in total, every 5 days after the first administration.
＜順行性トレーサーを用いた視神経の標識＞
　順行性トレーサーの注入は、三種混合麻酔を腹腔内に投与して麻酔下で、網膜、視神経および脳の摘出の６日前に行った。実体顕微鏡（SZ61, Olympus）下でマウスの眼球をピンセットでつまみ出し、先端の外径を0.15mmに引いた滅菌済ガラスシリンジ（Narishige）を毛様体部分から硝子体内に向けて差し込んだ。ガラスシリンジを0.5mmチュービング（エムエス機器）と21G注射針（テルモ）を介して10μlHamilton注射器（Hamilton Company）と連結させ、シリンジポンプ（KD Scientific）にセットした。硝子体内へのガラスシリンジ挿入後に1.0mg/mlAlexa Fluor 488-conjugated cholera toxin subunit B (CTB)(Molecular probes)を1.0μl/eye/minでマウスの硝子体内に投与した。 <Optic nerve labeling using an antegrade tracer>
Anterograde tracer injection was performed 6 days before retinal, optic nerve, and brain extraction under anesthesia using triple anesthesia administered intraperitoneally. Under a stereomicroscope (SZ61, Olympus), the mouse eyeball was picked out with tweezers, and a sterilized glass syringe (Narishige) with an outer diameter of 0.15 mm at the tip was inserted from the ciliary body toward the vitreous body. The glass syringe was connected to a 10 μl Hamilton syringe (Hamilton Company) via 0.5 mm tubing (MS Equipment) and a 21G injection needle (Terumo), and set in a syringe pump (KD Scientific). After inserting a glass syringe into the vitreous body, 1.0 mg/ml Alexa Fluor 488-conjugated cholera toxin subunit B (CTB) (Molecular probes) was administered into the vitreous body of the mouse at 1.0 μl/eye/min.
＜眼圧測定＞
　上述の眼圧測定法に従って、３回目の硝子体投与の５日後に麻酔下にて眼圧を測定した。その結果を図２に示す。この結果から明らかであるように、視神経挫滅によっても、ジオスゲニン投与によっても眼圧は殆ど変化しなかった。なお、図中、対照のため、視神経挫滅を行わなかったものを「コントロール」として、ジオスゲニン溶液を投与せずに、溶媒を投与したものを「溶媒」として、それぞれ解析した結果を併せて示す（実施例２について以下同様）。
（n=8 eyes、One-way ANOVA、post hoc Dunnett's test） <Intraocular pressure measurement>
Five days after the third vitreous administration, intraocular pressure was measured under anesthesia according to the intraocular pressure measurement method described above. The results are shown in FIG. As is clear from these results, there was almost no change in intraocular pressure either by optic nerve crush or by diosgenin administration. In addition, in the figure, for comparison purposes, those in which optic nerve crush was not performed are referred to as "controls", and those in which diosgenin solution was not administered but a solvent was administered as "vehicle", and the results of the respective analyzes are also shown ( The same applies below for Example 2).
(n=8 eyes, One-way ANOVA, post hoc Dunnett's test)
＜硝子体投与による視神経への作用の評価＞
　眼圧測定後に、上述の方法で、網膜、視神経および脳の摘出、視神経切片の作製、および視神経切片の画像解析を行った。その結果について、損傷部位から近位の代表的写真とCTB-488陽性面積の定量値を図３Ａ（ａ）、（ｂ）に、損傷部位から遠位の代表的写真とCTB-488陽性面積の定量値を図３Ｂ（ａ）、（ｂ）に、それぞれ示す。 <Evaluation of the effect of intravitreal administration on the optic nerve>
After measuring the intraocular pressure, the retina, optic nerve, and brain were extracted, optic nerve sections were prepared, and image analysis of the optic nerve sections was performed using the method described above. Regarding the results, representative photographs proximal to the injury site and quantitative values of the CTB-488 positive area are shown in Figure 3A (a) and (b). The quantitative values are shown in FIGS. 3B (a) and (b), respectively.
　これらの結果中、「コントロール」と「溶媒」との対比から明らかであるように、挫滅しなかった群と比べて、挫滅後溶媒を投与した群は、視神経密度が著しく減少した。また、0.1μM、１μMジオスゲニン溶液で処置した場合に、近位では視神経密度が増加する傾向が見られ（図３Ａ）、遠位でもわずかに視神経密度が増加する傾向が見られた（図３Ｂ）。 Among these results, as is clear from the comparison between "control" and "vehicle," the optic nerve density was significantly reduced in the group to which the vehicle was administered after crushing compared to the group that was not crushed. Furthermore, when treated with 0.1 μM and 1 μM diosgenin solutions, there was a tendency for optic nerve density to increase proximally (Figure 3A), and a tendency for optic nerve density to slightly increase distally (Figure 3B). .
（実施例３）
　実施例２において、ジオスゲニン硝子体投与により視神経密度が増加する傾向が示された。もしジオスゲニンが網膜だけでなく、網膜神経節細胞の軸索である視神経や、その投射先である外側膝状体神経細胞にも作用すれば、視機能を形成する神経回路が全体的に強化されると考えられる。そこで、ジオスゲニン経口投与後の網膜、視神経、脳への移行性を検討した。詳細を以下に示す。 (Example 3)
In Example 2, intravitreal administration of diosgenin showed a tendency for optic nerve density to increase. If diosgenin acts not only on the retina but also on the optic nerve, which is the axon of retinal ganglion cells, and the lateral geniculate body neurons, which are the destinations of its projection, the neural circuits that form visual function will be strengthened as a whole. It is thought that Therefore, we investigated the migration of diosgenin into the retina, optic nerve, and brain after oral administration. Details are shown below.
＜ジオスゲニンの網膜移行性の検討＞
　実験には雄性6週齢ddYマウス（日本SLC）を用いた。投与16時間前に絶食を行い、上述のジオスゲニン　オリブ油溶液をマウスに経口投与した。1時間後、6時間後、12時間後に血漿、網膜、視神経、全脳を摘出した。摘出した組織に10倍量のメタノール（Fujifilm和光製薬）を加え15秒間ホモジネート、1分間ボルテックス、5分間超音波処理し、4℃、12000g×10分間遠心した。上清を5mlチューブに移し、50℃のホットプレート上で蒸発乾固させた。そこに100%メタノール100μlを加えて溶解し、4℃、13000×5分間遠心した。上清を0.45μmフィルター(Merck Millipore)に通し、LC-MS/MS用のサンプルを得た。
(n = 3 mice, *p < 0.05, **p < 0.01, ****p < 0.0001, vsVehicle, Two-way ANOVA, post hoc, Bonferroni multiple comparisons)
　調製したサンプルを、Accela HPLCシステム(Thermo Fisher scientific)、LTQ-Orbitrap XL質量分析計(Thermo Fisherscientific)およびCLQ Xcaliburソフトウェア(ThemoFisherscientific)を用いて解析した。HPLCの条件は以下のとおりである。
溶媒A:0.1%ギ酸水溶液
溶媒B:100%メタノール
カラム:Cat.No.92620 type:MGIII(内径20mm、高さ150mm)
グラジエント設定：溶媒A-溶媒B、35%-65%(5分)、5%-95%(9分)、35%-65%(2分)の順に線形溶離
カラム温度:40℃
　MS解析の条件は以下のとおりである。
イオン化法:エレクトロスプレーイオン化法(positive mode)
キャピラリー温度:330℃
キャピラリー電圧:19V
スプレー電圧:4.5kV
チュープレンズ電圧:150V
シースガス流速:50(arbitrary units)
AUXガス流速:10(arbitrary units) <Study of retinal migration of diosgenin>
Male 6-week-old ddY mice (Japan SLC) were used in the experiment. The mice were fasted 16 hours before administration, and the above-mentioned diosgenin olive oil solution was orally administered to the mice. After 1, 6, and 12 hours, plasma, retina, optic nerve, and whole brain were removed. A 10-fold volume of methanol (Fujifilm Wako Pharmaceutical) was added to the excised tissue, homogenized for 15 seconds, vortexed for 1 minute, sonicated for 5 minutes, and centrifuged at 4°C at 12,000 g for 10 minutes. The supernatant was transferred to a 5 ml tube and evaporated to dryness on a hot plate at 50°C. 100 μl of 100% methanol was added thereto to dissolve, and the mixture was centrifuged at 13,000 x 5 minutes at 4°C. The supernatant was passed through a 0.45 μm filter (Merck Millipore) to obtain a sample for LC-MS/MS.
(n = 3 mice, *p < 0.05, **p < 0.01, ****p < 0.0001, vsVehicle, Two-way ANOVA, post hoc, Bonferroni multiple comparisons)
The prepared samples were analyzed using an Accela HPLC system (Thermo Fisher scientific), LTQ-Orbitrap XL mass spectrometer (Thermo Fisherscientific) and CLQ Xcalibur software (ThemoFisherscientific). The conditions for HPLC are as follows.
Solvent A: 0.1% formic acid aqueous solution Solvent B: 100% methanol Column: Cat. No. 92620 type: MGIII (inner diameter 20 mm, height 150 mm)
Gradient settings: Solvent A - Solvent B, linear elution in the order of 35%-65% (5 minutes), 5%-95% (9 minutes), 35%-65% (2 minutes) Column temperature: 40 °C
The conditions for MS analysis are as follows.
Ionization method: Electrospray ionization method (positive mode)
Capillary temperature: 330℃
Capillary voltage: 19V
Spray voltage: 4.5kV
Tuple lens voltage: 150V
Sheath gas flow rate: 50 (arbitrary units)
AUX gas flow rate: 10(arbitrary units)
　標準物質(ジオスゲニン:10μg/ml)(m/z=415.3171)について、LC-MS分析を行った結果を図４Ａに示す。図４Ａに示されるように、保持時間13-14分でピークが確認された。 The results of LC-MS analysis of the standard substance (diosgenin: 10 μg/ml) (m/z=415.3171) are shown in FIG. 4A. As shown in FIG. 4A, a peak was observed at a retention time of 13-14 minutes.
　溶媒投与したマウスの組織からジオスゲニンのピークは確認されなかったが、ジオスゲニン投与、6、24時間後の網膜、視神経および全脳からジオスゲニンのピークが検出された。網膜についての分析結果を図４Ｂ（ａ），（ｂ）に、視神経についての分析結果を図４Ｃ（ａ），（ｂ）に、脳についての分析結果を図４Ｄ（ａ），（ｂ）に、それぞれ示す。これらの結果から、少なくともジオスゲニンの経口投与後6時間後には、ジオスゲニンが網膜、視神経、全脳内に移行することが示された。 Although no diosgenin peak was observed in the tissues of mice administered the vehicle, diosgenin peaks were detected in the retina, optic nerve, and whole brain 6 and 24 hours after diosgenin administration. The analysis results for the retina are shown in Figure 4B (a), (b), the analysis results for the optic nerve are shown in Figure 4C (a), (b), and the analysis results for the brain are shown in Figure 4D (a), (b). , respectively. These results showed that diosgenin migrates into the retina, optic nerve, and whole brain at least 6 hours after oral administration of diosgenin.
（実施例４）
　ジオスゲニン経口投与による視神経伸長への作用を検討した。詳細を以下に示す。 (Example 4)
The effect of oral administration of diosgenin on optic nerve elongation was investigated. Details are shown below.
＜ジオスゲニンの経口投与＞
　緑内障モデルマウスの視神経挫滅直後に、上述のジオスゲニン　オリブ油溶液を、１回当たりそれぞれ0.1,1または10μmol/kgとなるように、３週間の間毎日１回ずつ経口投与した。 <Oral administration of diosgenin>
Immediately after optic nerve crush of glaucoma model mice, the above-mentioned diosgenin olive oil solution was orally administered once daily for 3 weeks at a dose of 0.1, 1, or 10 μmol/kg, respectively.
＜逆行性トレーサーを用いた一次視覚野に投射する外側膝状体神経節細胞の標識＞
　逆行性トレーサーの注入は、三種混合麻酔を腹腔内に投与して麻酔下で、網膜、視神経および脳の摘出の７日前に行った。頭頂部を剃毛し、頭皮を切開し頭蓋骨を露出させた。電動マイクロドリルを用いて、Lat:2.5mm, Bregma:-38mm, depth:1.2mmの位置に穴をあけ、Fluoro-Gold（Fujifilm和光製薬）を1.0μl/minで左右の一次視覚野に注入した。術後、アンチセダンを麻酔薬と同量腹腔内投与し、マウスを37℃のホットプレート上に静置し、体温の低下を防ぎながら覚醒させた。 <Labeling of lateral geniculate ganglion cells projecting to the primary visual cortex using a retrograde tracer>
Retrograde tracer injection was performed under anesthesia by intraperitoneal administration of triple anesthesia 7 days before retinal, optic nerve, and brain extraction. The top of the head was shaved, and the scalp was incised to expose the skull. Using an electric microdrill, a hole was made at the position of Lat: 2.5 mm, Bregma: -38 mm, depth: 1.2 mm, and Fluoro-Gold (Fujifilm Wako Pharmaceutical) was injected into the left and right primary visual cortex at 1.0 μl/min. . After the surgery, antisedan was administered intraperitoneally in the same amount as the anesthetic, and the mice were left on a hot plate at 37°C to wake them up while preventing a drop in body temperature.
＜順行性トレーサーを用いた視神経の標識＞
　順行性トレーサーの注入は、三種混合麻酔を腹腔内に投与して麻酔下で、網膜、視神経および脳の摘出の４日前に行った。実体顕微鏡（SZ61, Olympus）下でマウスの眼球をピンセットでつまみ出し、先端の外径を0.15mmに引いた滅菌済ガラスシリンジ（Narishige）を毛様体部分から硝子体内に向けて差し込んだ。ガラスシリンジを0.5mmチュービング（エムエス機器）と21G注射針（テルモ）を介して10μlHamilton注射器（Hamilton Company）と連結させ、シリンジポンプ（KD Scientific）にセットした。硝子体内へのガラスシリンジ挿入後に1.0mg/mlAlexa Fluor 488-conjugated cholera toxin subunit B (CTB)(Molecular probes)を1.0μl/eye/minでマウスの硝子体内に投与した。 <Optic nerve labeling using an antegrade tracer>
Anterograde tracer injection was performed 4 days before retina, optic nerve, and brain extraction under anesthesia using triple anesthesia administered intraperitoneally. Under a stereomicroscope (SZ61, Olympus), the mouse eyeball was picked out with tweezers, and a sterilized glass syringe (Narishige) with an outer diameter of 0.15 mm at the tip was inserted from the ciliary body toward the vitreous body. The glass syringe was connected to a 10 μl Hamilton syringe (Hamilton Company) via 0.5 mm tubing (MS Equipment) and a 21G injection needle (Terumo), and set in a syringe pump (KD Scientific). After inserting a glass syringe into the vitreous body, 1.0 mg/ml Alexa Fluor 488-conjugated cholera toxin subunit B (CTB) (Molecular probes) was administered into the vitreous body of the mouse at 1.0 μl/eye/min.
＜眼圧測定＞
　上述の眼圧測定法に従って、経口投与開始後３週間後に麻酔下にて眼圧を測定した。その結果を図５に示す。この結果から明らかであるように、視神経挫滅によっても、ジオスゲニン投与によっても眼圧は殆ど変化しなかった。なお、図中、対照のため、視神経挫滅を行わなかったものを「コントロール」として、ジオスゲニン　オリブ油溶液を投与せずに、溶媒を投与したものを「溶媒」として、それぞれ解析した結果を併せて示す（実施例４について以下同様）。
（n =8-12 eyes, One-way ANOVA, post hoc Dunnett's test） <Intraocular pressure measurement>
Three weeks after the start of oral administration, intraocular pressure was measured under anesthesia according to the intraocular pressure measurement method described above. The results are shown in FIG. As is clear from these results, there was almost no change in intraocular pressure either by optic nerve crush or by diosgenin administration. In addition, in the figure, for the purpose of comparison, the results of the analysis are shown as "control" in which the optic nerve crush was not performed, and "vehicle" in which the vehicle was administered without administering the diosgenin olive oil solution. (The same applies to Example 4 below).
(n =8-12 eyes, One-way ANOVA, post hoc Dunnett's test)
＜経口投与による視神経への作用の評価＞
　眼圧測定後に、上述の方法で、網膜、視神経および脳の摘出、網膜の蛍光免疫染色、網膜神経節細胞数の定量、視神経切片の作製、および視神経切片の画像解析、脳切片の作製、および脳切片の画像解析を行った。 <Evaluation of effects on optic nerve by oral administration>
After intraocular pressure measurement, the retina, optic nerve, and brain were extracted using the methods described above, fluorescent immunostaining of the retina, quantification of the number of retinal ganglion cells, preparation of optic nerve sections, image analysis of optic nerve sections, preparation of brain sections, and Image analysis of brain sections was performed.
　網膜神経節細胞数の定量について、コントロール（視神経を挫滅しなかったもの）、溶媒（ジオスゲニン　オリブ油溶液に代えて溶媒を投与したもの）、0.1または1μmol/kgのジオスゲニン　オリブ油溶液を投与した場合の代表的顕微鏡写真を図６Ａに示す。さらに、視神経を挫滅しなかった場合と挫滅した場合の網膜神経節細胞密度を図６Ｂに、溶媒、0.1,1,10μmol/kgのジオスゲニン　オリブ油溶液を投与した後の網膜神経節細胞密度を図６Ｃに、それぞれ示す。 Regarding quantification of retinal ganglion cell numbers, control (in which the optic nerve was not crushed), vehicle (in which vehicle was administered instead of diosgenin olive oil solution), and cases in which 0.1 or 1 μmol/kg diosgenin olive oil solution was administered. A representative micrograph is shown in Figure 6A. Furthermore, Figure 6B shows the retinal ganglion cell density when the optic nerve was not crushed and when the optic nerve was crushed, and the retinal ganglion cell density after administration of the solvent and 0.1, 1, and 10 μmol/kg diosgenin olive oil solution is shown in Figure 6B. 6C, respectively.
　図６Ａおよび図６Ｂから明らかであるように、視神経挫滅により網膜神経節細胞は有意に減少した。これに対して、図６Ｃから明らかであるように、ジオスゲニンの経口投与により有意に網膜神経節細胞の死滅が抑制された。 As is clear from FIGS. 6A and 6B, the number of retinal ganglion cells decreased significantly due to optic nerve crush. On the other hand, as is clear from FIG. 6C, oral administration of diosgenin significantly suppressed the death of retinal ganglion cells.
　また、脳切片の作製について、コントロール（視神経を挫滅しなかったもの）、溶媒（ジオスゲニン　オリブ油溶液に代えて溶媒を投与したもの）、0.1,1,10μmol/kgのジオスゲニン　オリブ油溶液を投与した場合の代表的顕微鏡写真を図７Ａに示す。さらに、視神経を挫滅しなかった場合と挫滅した場合の外側膝状体におけるＣＴＢ面積を図７Ｂに、ＣＴＢ面積とFluoro-Gold陽性面積との重複面積を図７Ｃに、溶媒を投与した場合と0.1,1,10μmol/kgのジオスゲニン　オリブ油溶液を投与した場合におけるＣＴＢ面積を図７Ｄに、ＣＴＢ面積とFluoro-Gold陽性面積との重複面積を図７Ｅに、それぞれ示す。 In addition, for the preparation of brain slices, a control (in which the optic nerve was not crushed), a solvent (in which a solvent was administered in place of the diosgenin olive oil solution), and a 0.1, 1, and 10 μmol/kg diosgenin olive oil solution were administered. A representative micrograph of the case is shown in FIG. 7A. Furthermore, Fig. 7B shows the CTB area in the lateral geniculate body when the optic nerve was not crushed and when the optic nerve was crushed, and Fig. 7C shows the overlap area between the CTB area and the Fluoro-Gold positive area. 7D shows the CTB area when a diosgenin olive oil solution of 1,10 μmol/kg was administered, and FIG. 7E shows the overlap area between the CTB area and the Fluoro-Gold positive area.
　図７Ａおよび図７Ｂから明らかであるように、視神経挫滅により、外側膝状体に投射する軸索密度が有意に減少した。一方、図７Ｄから明らかであるように、ジオスゲニンの経口投与により用量依存的に軸索密度が増加した。また、図７Ｃから明らかであるように、外側膝状体に投射する視神経と、一次視覚野に投射する外側膝状体神経細胞の重なりが有意に減少した。一方、図７Ｅから明らかであるように、ジオスゲニンの経口投与により用量依存的に増加した。これらの結果は、ジオスゲニン経口投与により、網膜神経節細胞から一次視覚野までのつながりが増加したことを示す。 As is clear from FIGS. 7A and 7B, optic nerve crush significantly reduced the density of axons projecting to the lateral geniculate body. On the other hand, as is clear from FIG. 7D, oral administration of diosgenin increased axon density in a dose-dependent manner. Furthermore, as is clear from Figure 7C, the overlap between the optic nerves that project to the lateral geniculate body and the lateral geniculate neurons that project to the primary visual cortex was significantly reduced. On the other hand, as is clear from FIG. 7E, oral administration of diosgenin resulted in a dose-dependent increase. These results indicate that oral administration of diosgenin increased connectivity from retinal ganglion cells to the primary visual cortex.

Claims (5)

  1. 　ジオスゲニンまたはジオスゲニン誘導体を有効成分として含む、視神経障害の予防または治療のための医薬または飲食品組成物。 A pharmaceutical or food/beverage composition for the prevention or treatment of optic nerve disorders, containing diosgenin or a diosgenin derivative as an active ingredient.
  2. 　ジオスゲニンまたはジオスゲニン誘導体を含む植物エキスを含有する、請求項１に記載の医薬または飲食品組成物。 The pharmaceutical or food/drink composition according to claim 1, which contains a plant extract containing diosgenin or a diosgenin derivative.
  3. 　前記植物エキスが、酸加水分解処理、発酵処理および酵素処理から選ばれる少なくとも１種の処理によってジオスゲニンの含有量を高めた植物エキスである、請求項２に記載の医薬または飲食品組成物。 The pharmaceutical or food/beverage composition according to claim 2, wherein the plant extract is a plant extract whose diosgenin content has been increased by at least one treatment selected from acid hydrolysis treatment, fermentation treatment, and enzyme treatment.
  4. 　網膜神経節細胞の死滅を抑制させることによって視神経障害を予防または治療する、請求項１～３のいずれか一項に記載の医薬または飲食品組成物。 The pharmaceutical or food/beverage composition according to any one of claims 1 to 3, which prevents or treats optic nerve damage by suppressing death of retinal ganglion cells.
  5. 　視神経障害が緑内障である、請求項１～３のいずれか一項に記載の医薬または飲食品組成物。 The pharmaceutical or food/beverage composition according to any one of claims 1 to 3, wherein the optic nerve disorder is glaucoma.
PCT/JP2023/024440 2022-07-29 2023-06-30 Pharmaceutical or food and beverage composition for prevention or treatment of optic nerve disorder WO2024024395A1 (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008516972A (en) * 2004-10-14 2008-05-22 ジョージタウン ユニバーシティー Pharmaceutical composition of spirostenol having neuroprotective action
JP2010500287A (en) * 2006-08-03 2010-01-07 オンコロジー リサーチ インターナショナル リミテッド Methods and compositions for inhibiting angiogenesis
WO2014112145A1 (en) * 2013-01-21 2014-07-24 レジリオ株式会社 Therapeutic agent and therapeutic method relating to 1,25d3-marrs for neurological disease such as alzheimer's disease
CN104324038A (en) * 2013-07-24 2015-02-04 四川京华创生物科技有限公司 Application of diosgenin-3-site derivative
WO2015163318A1 (en) * 2014-04-25 2015-10-29 レジリオ株式会社 Therapeutic agent for diseases associated with nerve axon dysfunction, including therapeutic agent for alzheimer's disease
JP2015218134A (en) * 2014-05-16 2015-12-07 タカラバイオ株式会社 Composition for prevention and/or treatment of memory impairment

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008516972A (en) * 2004-10-14 2008-05-22 ジョージタウン ユニバーシティー Pharmaceutical composition of spirostenol having neuroprotective action
JP2010500287A (en) * 2006-08-03 2010-01-07 オンコロジー リサーチ インターナショナル リミテッド Methods and compositions for inhibiting angiogenesis
WO2014112145A1 (en) * 2013-01-21 2014-07-24 レジリオ株式会社 Therapeutic agent and therapeutic method relating to 1,25d3-marrs for neurological disease such as alzheimer's disease
CN104324038A (en) * 2013-07-24 2015-02-04 四川京华创生物科技有限公司 Application of diosgenin-3-site derivative
WO2015163318A1 (en) * 2014-04-25 2015-10-29 レジリオ株式会社 Therapeutic agent for diseases associated with nerve axon dysfunction, including therapeutic agent for alzheimer's disease
JP2015218134A (en) * 2014-05-16 2015-12-07 タカラバイオ株式会社 Composition for prevention and/or treatment of memory impairment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHOGO SHIBUE, CHIHIRO HIGASHIDA: "O-10: Examination of drugs that promote optic nerve elongation in normal-tension glaucoma model mice", 39TH ACADEMIC CONFERENCE OF JAPANESE AND CHINESE MEDICINE SOCIETY; AUGUST 27-28, 2022 WEB EVENT, JAPANESE AND CHINESE MEDICINE SOCIETY, JP, 10 August 2022 (2022-08-10) - 28 August 2022 (2022-08-28), JP, pages 73, XP009553650, ISSN: 2435-4953 *

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