Oral composition for intraocular pressure reduction, rock inhibitor, and autophagy enhancer

Abstract

To provide a novel agent for reducing intraocular pressure.SOLUTION: (A) Theobromine and / or (B) hesperidins selected from the group consisting of hesperidin, glucosyl hesperidin, and hesperetin are effective as active ingredients in an oral composition for intraocular pressure reduction, in a ROCK inhibitor, and in an autophagy enhancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oral composition for reducing intraocular pressure, a ROCK inhibitor, and an autophagy enhancer. [Background technology]

[0002] Aqueous humor produced from blood vessels in the ciliary body or iris fills the anterior chamber via the pupil, then passes through the trabecular meshwork tissue and is discharged from the Schlemm's canal. The intraocular pressure is kept normal by the circulation of the aqueous humor, but if the drainage of the aqueous humor is impaired due to clogging of the trabecular meshwork tissue, the intraocular pressure rises. It is believed that the increase in intraocular pressure damages the optic nerve and leads to glaucoma, and the main treatment for glaucoma is to control the intraocular pressure with intraocular pressure-reducing eye drops.

[0003] As intraocular pressure-reducing eye drops, drugs classified according to their mechanism of action as parasympathomimetics, sympathomimetics, sympatholytics, prostaglandin drugs, or carbonic anhydrase inhibitors are used, and when multiple drugs are used in combination, they are used in combination in such a way that their respective pharmacological actions do not cancel each other out (Non-Patent Document 1). β-blockers have little effect on reducing intraocular pressure at night, and prostaglandin drugs and carbonic anhydrase inhibitors, although inferior to their daytime intraocular pressure-reducing effects, still show a significant effect on reducing intraocular pressure at night, and the more drugs that have a weaker effect on reducing intraocular pressure at night are used in combination, the more likely intraocular pressure after treatment will be higher at night than during the day (Non-Patent Document 2).

[0004] Currently, there are more than 10 types of eye drops used to treat glaucoma, and when it is determined that one type of eye drop alone is not effective, a combination of multiple eye drops is often prescribed. Treatment with eye drops is chosen as a treatment method that can be effective with as few side effects as possible. There are also oral medications that can lower intraocular pressure, but these can cause severe systemic side effects and cannot be taken orally in some cases (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Folia Pharmacol. Jpn. 128, 250-254 (2006) [Non-Patent Document 2] Journal of the Japan Medical University Medical Association 2023;19(2),156-163 [Non-Patent Document 3] “Eye Diseases - Glaucoma Treatment - (1) Drug Therapy”, [online], Japan Ophthalmological Society [searched on July 25, 2022], Internet<URL:https: / / www.nichigan.or.jp / public / disease / treatment / item05.html> Summary of the Invention [Problem to be solved by the invention]

[0006] Once the optic nerve is damaged by an increase in intraocular pressure, even if the intraocular pressure can be reduced reversibly by treatment, the optic nerve damage is basically irreversible. Therefore, it is ideal to suppress the condition in which intraocular pressure is likely to increase before the optic nerve damage becomes serious. It is desirable for the agent to be orally ingested as a form of application for disease prevention, but considering the severe side effects of oral medications that lower intraocular pressure, it is not realistic to use it for prevention. Therefore, a new option for intraocular pressure-reducing agents is desired.

[0007] Therefore, an object of the present invention is to provide a new intraocular pressure-reducing agent. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above problems and found that theobromine and / or hesperidins, which are also contained in foods, have an effect of reducing intraocular pressure and can be used as new active ingredients for intraocular pressure-reducing agents. Furthermore, the inventors found that these ingredients have a ROCK inhibitory effect and an autophagy enhancing effect, and that these effects are thought to be responsible for at least a part of the mechanism of action for reducing intraocular pressure, and that they can be used not only for reducing intraocular pressure but also for other applications that utilize the ROCK inhibitory effect or the autophagy enhancing effect. The present invention was completed based on these findings and through further research.

[0009] That is, the present invention provides the following aspects. Item 1. An oral composition for reducing intraocular pressure, comprising (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin. Item 2. The oral composition for reducing intraocular pressure according to Item 1, comprising a cacao extract as a source of the component (A). Item 3. An oral composition for reducing intraocular pressure according to Item 1 or 2, which is used to suppress nocturnal increase in intraocular pressure. Item 4. A ROCK inhibitor comprising (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin. Item 5. An autophagy enhancer comprising (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin. Item 6. The autophagy enhancer according to Item 5, which is an autophagy enhancer for trabecular meshwork cells. Item 7. A method for reducing intraocular pressure, comprising administering to a subject in need of reduction in intraocular pressure an effective amount of (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin. Item 8. Use of (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin, for the manufacture of an oral composition for reducing intraocular pressure. Item 9. (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin for use in the treatment of a disease associated with high intraocular pressure. Item 10. Non-therapeutic use of (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin for reducing intraocular pressure. Item 11. A method for reducing intraocular pressure, comprising administering to a subject in need of ROCK inhibition or autophagy enhancement an effective amount of (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin. Item 12. Use of (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin for the manufacture of a ROCK inhibitor or an autophagy enhancer. Item 13. (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin for use in the treatment of a disease associated with increased ROCK activity or decreased autophagy activity. Item 14. Non-therapeutic use of (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin, for inhibiting ROCK or enhancing autophagy. Effect of the Invention

[0010] According to the present invention, theobromine and / or hesperidins, which are also contained in foods, can be used as active ingredients of an intraocular pressure-reducing agent, providing a new oral composition for intraocular pressure reduction. Furthermore, according to the present invention, theobromine and / or hesperidins can be used as active ingredients with ROCK inhibitory effect and autophagy enhancing effect, and therefore the composition can be used not only for reducing intraocular pressure, but also for other applications utilizing ROCK inhibitory effect or autophagy enhancing effect. [Brief description of the drawings]

[0011] [Figure 1A] 1 shows the intraocular pressure lowering effect of an oral composition for lowering intraocular pressure containing a cacao extract. [Figure 1B] 1 shows the intraocular pressure-reducing effect of an oral composition for reducing intraocular pressure containing glucosyl hesperidin. [Figure 2A] 1 shows the intraocular pressure lowering effect (reduction in maximum intraocular pressure increase value) of an oral composition for lowering intraocular pressure containing a cacao extract. [Figure 2B] 1 shows the intraocular pressure-reducing effect (reduction in maximum intraocular pressure increase value) of an oral composition for reducing intraocular pressure that contains glucosyl hesperidin. [Figure 3A] 1 shows the effect of lowering intraocular pressure (reduction in the accumulated change in the dark phase) by an oral composition for lowering intraocular pressure containing a cacao extract. [Figure 3B] 1 shows the effect of reducing intraocular pressure (reducing the amount of accumulated change during the dark period) by an oral composition for reducing intraocular pressure containing glucosyl hesperidin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Oral composition for reducing intraocular pressure The oral composition for reducing intraocular pressure of the present invention is characterized by containing, as active ingredients, (A) theobromine (hereinafter also referred to as component (A)) and / or (B) hesperidins (hereinafter also referred to as component (B)). The oral composition for reducing intraocular pressure of the present invention will be described in detail below.

[0013] (A) Theobromine Theobromine (3,7-dimethylxanthine) is a type of alkaloid and is a known component that is known to have vasodilatory, cerebral vasoconstrictor, tracheal dilator, central nervous system stimulant, diuretic, and fat accumulation inhibitory effects.

[0014] In the present invention, natural materials containing theobromine can be used as theobromine sources. Examples of such natural materials include plants containing theobromine or their extracts. Examples of such plants include cacao (Theobroma cacao Linn.), mate tea (Ilex paraguariensis), tea plant (Camellia sinensis), cola (Cola acuminata), guarana (Paullinia cupana), coffee plant (Coffea arabica), etc.

[0015] Among the theobromine and natural materials containing theobromine, cacao extract is preferably used from the viewpoint of further enhancing the intraocular pressure lowering effect. The cacao extract is an extract obtained by extracting cacao seeds (cacao beans). The raw cacao beans may be fermented, roasted, and then crushed or powdered, and then subjected to extraction. The extraction process for obtaining the cacao extract may be a general extraction method used in the production of plant extracts, such as solvent extraction, supercritical extraction, and steam distillation. Among these, solvent extraction is preferably used. The extraction solvent used in the solvent extraction process may be water, monohydric lower alcohols having 1 to 4 carbon atoms, and mixtures thereof, more preferably water, ethanol, and aqueous ethanol, and even more preferably aqueous ethanol. Furthermore, the cacao extract may be processed to increase the concentration of theobromine by removing a portion of components other than theobromine from the extract obtained by the extraction process. When cacao extract is used to formulate component (A), the amount of theobromine contained in 100 parts by weight of cacao extract is, for example, 1 to 30 parts by weight, preferably 3 to 20 parts by weight, more preferably 5 to 15 parts by weight, and even more preferably 7 to 12 parts by weight.

[0016] The amount of component (A) in the oral composition for reducing intraocular pressure of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but may be, for example, 1 to 4% by weight, preferably 2 to 4% by weight. When the theobromine source includes a theobromine-containing plant or an extract thereof, the amount of component (A) refers to the amount of the plant or extract thereof converted into the amount of theobromine contained therein.

[0017] When cacao extract is contained as the theobromine source, the amount of cacao extract in the oral composition for reducing intraocular pressure of the present invention is not particularly limited as long as the effects of the present invention are obtained, but can be, for example, 10 to 40% by weight, preferably 20 to 40% by weight, calculated as the dry weight.

[0018] (B) Glucosyl hesperidins The glucosyl hesperidins are selected from the group consisting of hesperidin, glucosyl hesperidin (a glycosylated form of hesperidin), and hesperetin (an aglycone of hesperidin). Glucosyl hesperidins are known components that are known to have capillary strengthening effects, blood flow improving effects, blood neutral fat lowering effects, blood cholesterol level improving effects, antiallergic effects, rheumatic symptom improving effects, etc.

[0019] As component (B), one of hesperidin, glucosyl hesperidin (a sugar transfer product of hesperidin), and hesperetin (an aglycone of hesperidin) may be used alone or in combination of two or more. Among these hesperidins, glucosyl hesperidin is preferred.

[0020] The amount of component (B) in the oral composition for reducing intraocular pressure of the present invention is not particularly limited as long as the effects of the present invention are obtained, but may be, for example, 10 to 95% by weight, preferably 40 to 95% by weight, and more preferably 80 to 95% by weight.

[0021] Other Ingredients The oral composition for reducing intraocular pressure of the present invention may or may not contain additives and / or bases as other ingredients in addition to the above-mentioned active ingredient depending on the formulation form.

[0022] The additives and bases, which may or may not be contained, are not particularly limited as long as they are pharma- ceutically acceptable, and examples thereof include excipients, binders, disintegrants, lubricants, isotonicity agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, coating agents, glossing agents, water, oils and fats (medium-chain fatty acid triglycerides, vegetable oils, animal oils, etc.), hydrocarbon oils, higher (carbon number 8-22) fatty acids, higher (carbon number 8-22) alcohols, ester oils, water-soluble polymers, surfactants, monohydric lower (carbon number 1-5) alcohols, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV protection agents, preservatives, flavorings, fragrances, powders, thickeners, dyes, and chelating agents. These additives and bases may be used alone or in combination of two or more. The content of these additives and bases is appropriately set according to the formulation form and the type of ingredients used.

[0023] Furthermore, the oral composition for reducing intraocular pressure of the present invention may or may not contain other nutritional components and / or pharmacological components as other components, as necessary.

[0024] Nutritional components and pharmacological components, which may or may not be contained, are not particularly limited as long as they are pharmacologic acceptable, and examples thereof include antacids, stomachics, digestive aids, intestinal regulators, antispasmodics, mucosal repair agents, nonsteroidal or steroidal anti-inflammatory agents, astringents, antiemetics, antitussives, expectorants, anti-inflammatory enzymes, sedatives, hypnotics, antihistamines, caffeine, cardiac diuretics, antibacterial agents, vasoconstrictors, vasodilators, local anesthetics, herbal extracts, and other vitamins. These nutritional components and pharmacological components may be used alone or in combination of two or more. The content of these components is appropriately set according to the purpose of the oral composition for reducing intraocular pressure and the type of components used.

[0025] Formulation and product classification The formulation of the oral composition for reducing intraocular pressure of the present invention is not particularly limited, and examples thereof include liquid, semi-solid (e.g., gel, paste, etc.), and solid (e.g., granules, fine granules, powder, tablets, capsules, etc.) The formulation may be either a non-emulsified composition or an emulsified composition.

[0026] The product classification of the oral composition for reducing intraocular pressure of the present invention is not particularly limited as long as it is in a form for internal use (including oral ingestion and oral administration), and may be any of foods and beverages [including special purpose foods and health functional foods (nutrient functional foods, foods with functional claims, foods for specified health uses, etc.), as well as general foods (nutritional supplements, health supplements, nutritionally fortified foods, nutritionally regulated foods, supplements, etc.)] and oral pharmaceuticals [including oral quasi-drugs].

[0027] Use / Dosage The oral composition for reducing intraocular pressure of the present invention is used for reducing intraocular pressure in subjects for whom reduction in intraocular pressure is required. In the present invention, reducing intraocular pressure includes both lowering the current intraocular pressure (including both abnormal intraocular pressure and normal intraocular pressure) to a lower level and suppressing an increase in intraocular pressure in the presence of a cause of intraocular pressure increase (including both slowing down the rate of increase and preventing an increase).

[0028] Specific applications include treatment of glaucoma with abnormal intraocular pressure, treatment of glaucoma with normal intraocular pressure (e.g., lowering intraocular pressure to reduce the burden on the optic nerve), lowering intraocular pressure in subjects who do not have glaucoma (e.g., people with relatives who have glaucoma, people who have lifestyle habits that increase intraocular pressure, etc.), suppression of transient increases in intraocular pressure after ophthalmic surgery (e.g., laser surgery, etc.), reduction of diurnal fluctuations in intraocular pressure, suppression of increases in intraocular pressure at night, and lowering intraocular pressure during the day.

[0029] The subject of the oral composition for reducing intraocular pressure of the present invention may be any mammalian animal including human, and preferably human.

[0030] Examples of the intake / administration amount of the oral composition for reducing intraocular pressure of the present invention include a daily dose of theobromine for a human (60 kg) of 20 to 300 mg, preferably 50 to 300 mg, and more preferably 100 to 300 mg, a daily dose of cacao extract (converted into dry weight) for a human of 100 to 3,000 mg, preferably 500 to 3,000 mg, and more preferably 1,000 to 3,000 mg, and a daily dose of hesperidins (equivalent to hesperetin) for a human of 100 to 10,000 mg, preferably 1,000 to 10,000 mg, and more preferably 5,000 to 10,000 mg.

[0031] 2.ROCK inhibitors The ROCK (Rho-associated coiled-coil forming protein kinase: Rho kinase) inhibitor of the present invention is characterized by containing (A) theobromine and / or (B) hesperidins as active ingredients.

[0032] Details of the active ingredients (A) and (B), other ingredients, and dosage are as described above in "1. Oral composition for reducing intraocular pressure."

[0033] The formulation of the ROCK inhibitor of the present invention is not particularly limited, and examples thereof include liquid, semi-solid (e.g., gel, paste, etc.), and solid (e.g., granules, fine granules, powder, tablets, capsules, etc.) The formulation may be either a non-emulsified composition or an emulsified composition.

[0034] The product classification of the ROCK inhibitor of the present invention includes any product classification that can be taken in any intake or administration form, including internal and external use, and may be, for example, any of foods and beverages [including special purpose foods and health functional foods (nutritional functional foods, functional foods, foods with functional claims, foods for specified health uses, etc.), as well as general foods (nutritional supplements, health supplements, nutritionally fortified foods, nutritionally regulated foods, supplements, etc.)], oral medicines [including oral quasi-drugs], and external medicines [including external quasi-drugs].

[0035] The ROCK inhibitor of the present invention can be used for any purpose utilizing ROCK inhibitory activity. Such purpose may be any purpose that improves a condition or disease associated with enhanced ROCK activity, such as lowering intraocular pressure; treating or preventing diabetes, Parkinson's disease, amyotrophic lateral sclerosis, pulmonary hypertension, cancer, or Ewing's sarcoma; and protecting the kidney in nephropathy. The lowering of intraocular pressure is as described above in "1. Oral composition for lowering intraocular pressure."

[0036] 3. Autophagy enhancers The autophagy enhancer of the present invention is characterized by containing (A) theobromine and / or (B) hesperidins as active ingredients.

[0037] Details of the active ingredients (A) and (B), other ingredients, and dosage are as described above in "1. Oral composition for reducing intraocular pressure."

[0038] The formulation forms and product classifications of the autophagy enhancer of the present invention are as described above in "2. ROCK inhibitors."

[0039] The autophagy enhancer of the present invention can be used for any application that utilizes the autophagy enhancing effect. Such applications may be any application that improves conditions or diseases associated with decreased autophagy activity, such as lowering intraocular pressure; treatment or prevention of neurodegenerative diseases such as Huntington's disease, type 2 diabetes, etc.; inhibition of aging; treatment or prevention of osteoporosis, treatment or prevention of age-related macular degeneration, and treatment or prevention of arteriosclerosis. The reduction of intraocular pressure is as described above in "1. Oral composition for reducing intraocular pressure."

[0040] The target site of the autophagy enhancer of the present invention is not particularly limited, but preferably includes trabecular meshwork cells. EXAMPLES

[0041] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0042] Test Example 1: Evaluation of intraocular pressure lowering effect (1) Cacao extract (obtained by extracting cacao (Theobroma cacao LINN) seeds with aqueous ethanol) or glucosyl hesperidin was dissolved in water for injection to prepare a test sample. Water for injection was used as a control sample.

[0043] (2) Purchased female rabbits (Kbs:JW) were quarantined and acclimated to the breeding environment (temperature: 23.0±3.0°C, humidity: 50±20%), and then acclimated to a 12-hour dark (14:00-2:00) and 12-hour light (2:00-14:00) rhythm.

[0044] (3) Intraocular pressure was measured every two hours using a pneumatometer (Model 30 Classic, RE Medical Co., Ltd.), and the diurnal variation of intraocular pressure over a 24-hour period was examined. (4) Using the two parameters of intraocular pressure value at time 0 (10:00) during diurnal variation measurement and intraocular pressure difference as indicators, stratified randomization was performed using SAS Ver. 9.3 (SAS Institute Japan, Inc.) so that each group was approximately uniform.

[0045] (5) Seven female rabbits were used in each group, and the test sample or control sample was orally administered at a dose of 10 mL / kg [cocoa extract dose: 125 mg / kg (equivalent theobromine dose of 12.5 mg / kg), i.e., a human equivalent dose of cocoa extract of 2,400 mg / 60 kg (human equivalent dose of theobromine of 240 mg / 60 kg); glucosyl hesperidin dose: 500 mg / kg, i.e., a human equivalent dose of glucosyl hesperidin of 9,678 mg / 60 kg] repeatedly once daily for 21 days. (6) On days 1, 7, 14, and 21 after the start of oral administration, intraocular pressure in the left eye was measured before administration and 2 hours, 4 hours, 6 hours, and 8 hours after administration, and the average value was calculated from 3 measurements per eye.

[0046] The changes in intraocular pressure due to administration of glucosyl hesperidin or cocoa extract are shown in Figures 1A and 1B. The maximum increase in intraocular pressure, i.e., the maximum amount of change in intraocular pressure on each measurement day, is shown in Figures 2A and 2B. The accumulated change in the dark phase, i.e., the sum of the intraocular pressure increase values ​​during the dark phase, is shown in Figures 3A and 3B.

[0047] As shown in these figures, oral ingestion of the test sample was confirmed to have an effect of lowering intraocular pressure, such as a decrease in the maximum intraocular pressure increase value and a decrease in the accumulated amount of change during the dark period. When the accumulated amount of change during the light period for glucosyl hesperidin and the cacao extract, i.e., the sum of intraocular pressure increases during the light period, was compared with the accumulated amount of change during the dark period shown in Figures 3A and 3B, it was found that the inhibition of intraocular pressure increase in the accumulated amount of change during the dark period was significantly more remarkable.

[0048] Test Example 2: Evaluation of ROCK activity inhibitory effect (1) The same cacao extract or glucosyl hesperidin used in Test Example 1 was dissolved in water for injection to prepare a test sample of 300 μg / ml. Water for injection was used as a control sample.

[0049] (2) A 96-Well ROCK Activity Assay Kit (CELL BIOLABS, INC., product number: STA-416) was prepared, and active ROCK-II (0.00125 ng / ml) diluted with 80 μl of 1× kinase reaction buffer and 10 μl of a test sample or control sample were added to the wells of a substrate plate. (3) 10 μl of 10× kinase reaction buffer containing DTT (final concentration 10 mM) and ATP (final concentration 2 mM) was added and thoroughly mixed to initiate the kinase reaction. (4) The plate was covered with a plate cover and reacted at 30°C for 60 minutes with gentle shaking. (5) After the reaction, the solution in the wells was discarded into a tray by inverting the plate to stop the kinase reaction, and the wells were washed three times with 250 μl of 1× wash buffer. (6) After washing, turn the plate upside down and tap it gently against a Kimtowel to completely remove excess wash buffer. (7) Anti-phospho-MYPT1 antibody diluted 1,000-fold with 100 μl of Assay Diluent was added to each well. (8) Cover the plate with a plate cover and react at room temperature for 1 hour. (9) After the reaction, the solution was discarded into a tray and the wells were washed in the same manner as in (6). (10) After washing, 100 μl of HRP-conjugated secondary antibody diluted 1,000-fold with Assay Diluent was added to each well. (11) Cover the plate with a plate cover and react with shaking at room temperature for 1 hour. (12) After the reaction, the solution was poured into a tray and washed in the same manner as in (6). (13) After washing, 100 μl of Substrate Solution that had been brought to room temperature was added to each well, including the blank (control) well, and reacted at room temperature for 20 minutes. (14) 100 μl of STOP Solution was added to each well, including the blank (control) well, to stop the reaction. The solution in each well was then transferred to a new 96-well plate, and the absorbance at 450 nm was measured using a microplate reader. (15) The ROCK inhibitory effect of each material was calculated from the absorbance. The ROCK inhibitory effect is the ROCK activity inhibition rate (%) calculated by the following formula.

number

[0050] The results obtained are shown in the table below. [Table 1]

[0051] As shown in Table 1, the ROCK inhibitory effect of the test sample was confirmed.

[0052] Test Example 3: Evaluation of autophagy enhancement effect (1) 75cm in advance 2 Human trabecular meshwork cells (HTMC, #6590, ScienCell Research Laboratories) cultured in a flask were seeded into a 6-well plate containing Humedia KG-2 (Kurabo Industries, Ltd.) at 400,000 cells / well and cultured overnight at 37°C. (2) After the incubation, the test ingredient (cacao extract or glucosyl hesperidin) was added to each petri dish to a concentration of 100 μg / ml, and the dishes were incubated for 24 hours at 37° C. After the incubation in step (1), the dishes were incubated for 24 hours at 37° C. without adding the test ingredient, which served as a control. (3) Each test compound was divided into two groups: one treated with chloroquine diphosphate (CQ) and the other untreated with CQ. The control was also divided into two groups. (4) The CQ-treated group was treated with 10 μM CQ 4 hours before protein extraction. CQ is known to be an autophagy pathway inhibitor and inhibits the fusion of autophagosomes and lysosomes. The untreated group was cultured for 4 hours in the same manner as the CQ-treated group, except that CQ was not added. (5) Proteins were extracted from the treated cells using standard techniques, and the expression levels of the autophagy marker LC3-II were examined by Western blotting. (6) The bands obtained by Western blotting were quantified using ImageJ, and the autophagy activity (%) was calculated for the control and each component added using the following formula.

number

[0053] The results are shown in the table below. [Table 2]

[0054] As shown in Table 2, the autophagy-enhancing effect of the test ingredients was confirmed.

Claims

1. An oral composition for reducing intraocular pressure, comprising (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin.

2. The oral composition for reducing intraocular pressure according to claim 1 , comprising a cacao extract as a source of the component (A).

3. The oral composition for reducing intraocular pressure according to claim 1, which is used to suppress nocturnal increase in intraocular pressure.

4. A ROCK inhibitor comprising (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosylhesperidin, and hesperetin.

5. An autophagy enhancer comprising (A) theobromine and / or (B) a hesperidin selected from the group consisting of hesperidin, glucosyl hesperidin, and hesperetin.

6. The autophagy enhancer according to claim 5 , which is an autophagy enhancer for trabecular meshwork cells.

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