KR102138860B1 - Composition for lowering intraocular pressure regulating comprising extract of maple leaves - Google Patents

Abstract

The present invention relates to a composition for lowering intraocular pressure, comprising a maple leaf extract, a method for lowering intraocular pressure, comprising administering the composition to the eyes of an individual other than a human, a pharmaceutical composition for treating ocular disease induced by increased intraocular pressure , It relates to a method for treating ocular diseases induced by an increase in intraocular pressure, a quasi-drug composition and a health functional food composition comprising administering the composition to the eyes of an individual other than a human.

Description

Composition for lowering intraocular pressure regulating comprising extract of maple leaves}

The present invention relates to a composition for lowering intraocular pressure, comprising a maple leaf extract, a method for lowering intraocular pressure, comprising administering the composition to the eyes of an individual other than a human, a pharmaceutical composition for treating ocular disease induced by increased intraocular pressure , It relates to a method for treating ocular diseases induced by an increase in intraocular pressure, a quasi-drug composition and a health functional food composition comprising administering the composition to the eyes of an individual other than a human.

The maple tree is 100m high, the small branches are hairless, and are reddish brown trees. In addition, the leaves of the maple tree face apart and are deeply divided into 5-7 pieces in the shape of a palm. The cracked pieces are broad, lanceolate, pointed at the ends, have double teeth at the edges, and are 5-6 cm long. The petiole has a red color and is 3-5cm long. Flowers bloom in male and bisexual flowers. It blooms in dark red in May and runs at the end of the branch in a mountain flower turn. There are 5 calyx pieces with soft hairs and 5 petals. There are 8 stamens.

These maple trees use root bark and branches as a medicinal material for gradation axis in oriental medicine, and when they are severely affected by knee arthritis, they are taken by boiling in water, and when they are injured, they use a combination of agapi and have anti-inflammatory and detoxifying effects. Is known.

Intraocular pressure (IOP) or intraocular pressure is the fluid pressure inside the eye. Because it is an area without blood vessels, such as the cornea, lens (lens), and vitreous of the eye, there is water called waterproof, which supplies nutrients and transports waste products. The intraocular pressure is formed by this waterproofing, but the waterproofing comes out as normal, but when the part where the waterproofing escapes is damaged, the pressure of the eye increases. As such, when the pressure of the eye is increased, blood cannot be transported to the eye that needs to receive a lot of blood, and continuous pressure is applied to the optic nerve, thereby causing fatal damage to the optic nerve, resulting in a loss of vision and vision. Glaucoma (glaucoma) is a representative disease that causes an abnormality in the functioning of the optic nerve due to atrophy of the optic nerve or impaired blood supply due to such an increase in intraocular pressure. Glaucoma occurs at a frequency of 0.5 to 2% among adults over 40 years of age, while the normal value of intraocular pressure is 15 to 20 mmHg, while intraocular pressure increases when glaucoma progresses, the inside of the pupil has a characteristic of green (KR 10 -1185106 B1).

Currently, treatment for glaucoma cannot be cured because most of it prevents an increase in intraocular pressure using a drug or a surgical method. Therefore, lifelong medication, laser treatment, and surgery should be used to control intraocular pressure to minimize optic nerve disorders. .

Accordingly, the present inventors tried to develop a composition comprising a natural extract having no side effects and preventing or treating ocular diseases induced by an increase in intraocular pressure by lowering intraocular pressure, resulting in lower intraocular pressure in maple leaf extracts and through this It has been confirmed that there is a therapeutic effect on eye diseases induced by an increase in intraocular pressure, and the present invention has been completed.

The main object of the present invention is to provide a composition for reducing intraocular pressure, which comprises a maple leaf extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a method for lowering intraocular pressure, comprising administering the composition for lowering intraocular pressure to the eyes of an individual other than a human.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ocular diseases induced by an increase in intraocular pressure, which includes a maple leaf extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating ocular diseases induced by increased intraocular pressure, comprising administering the pharmaceutical composition to the eyes of an individual other than a human.

Another object of the present invention is to provide a quasi-drug composition for lowering intraocular pressure, comprising a maple leaf extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition for lowering intraocular pressure, comprising maple leaf extract or a fraction thereof as an active ingredient.

The present inventors discovered that the maple leaf extract shows an effect of treating ocular diseases caused by increased intraocular pressure by lowering intraocular pressure during various studies in order to develop a formulation derived from natural products that can safely and effectively lower intraocular pressure. Did. In general, maple trees are known to exhibit various pharmacological activities. However, there has been no report on the use of the maple tree for the therapeutic effect of ocular diseases induced by increased intraocular pressure. In addition, only invasive treatments exist, and no technology has been reported so far for pharmaceutical compositions and drug treatments that have a therapeutic effect on eye diseases induced by increased intraocular pressure.

The present inventors predicted that a therapeutic effect would be exhibited for ocular diseases induced by an intraocular pressure drop and an intraocular pressure increase from the maple leaf extract, and conducted various studies to identify the effective component showing an intraocular pressure drop, and as a result, the foliage It was confirmed that the tree leaf extract has an effect of treating ocular diseases induced by an increase in intraocular pressure.

One aspect of the present invention for achieving the above object, to provide a composition for reducing intraocular pressure comprising a maple leaf extract or a fraction thereof as an active ingredient.

Hereinafter, a composition for lowering intraocular pressure, comprising a maple leaf extract or a fraction thereof in the present invention will be described in detail.

In the present invention, the term "maple tree" is 100m in height, small branches have no hairs, and are reddish brown trees, which means that the leaves of the maple tree face each other and are deeply divided into five palms. Meanwhile, the maple tree can be largely divided into roots, stems, and leaves, and the composition for reducing intraocular pressure of the present invention may include the maple leaf extract or a fraction thereof. At this time, the extract for each part of the maple tree or a fraction thereof can be achieved for the effect of lowering the intraocular pressure without particular limitation.

In the present invention, the term “intraocular pressure (IOP)” or intraocular pressure is the fluid pressure inside the eye. Measurement of intraocular pressure is a method used to measure intraocular pressure, and intraocular pressure is an important factor in assessing the risk of glaucoma. In the present invention, the decrease in intraocular pressure may mean lowering the intraocular pressure elevated outside the normal range into the normal range, and the normal range of the intraocular pressure may generally mean 10 to 21 mmHg.

In the present invention, the term, "extract" is a product obtained by immersing the desired substance in various solvents, and then extracting it for a period of time at a normal temperature or a warm state, resulting in solids obtained by removing the solvent from the liquid component. Can mean In addition, in addition to the above results, it can be comprehensively interpreted as including all of the dilutions of the above results, their concentrates, their modulators, and tablets. The method for obtaining the extract is not particularly limited as long as the extract for lowering the eye can be obtained, but specifically, the maple leaf, its dried product, and the like are immersed in the solvent, and at 10 to 25°C. Methods such as cold immersion extraction at room temperature, heating extraction at 40 to 100° C., ultrasonic extraction at extraction with ultrasonic waves, and reflux extraction using a reflux cooler can be used. For example, the extract may be included in an amount of 0.01 to 100% by weight, more specifically 1 to 80% by weight, based on the total weight of each composition.

For example, the maple leaf extract of the present invention may be a hot water extract.

In addition, the maple leaf extract of the present invention may be an ethanol extract.

In the present invention, the maple leaf extract may be prepared and used as a fraction thereof.

In the present invention, the term "fraction" means a result obtained by performing a fraction to separate a specific component or a specific component group from a mixture containing various various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Solvent fractionation performed by treating various solvents, ultrafiltration fractionation performed by passing through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity) ), and combinations thereof. In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. As a non-limiting example of the fractional solvent, water, an organic solvent, or a mixed solvent thereof may be used, and the organic solvent is an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dicro A non-polar solvent of methane or a mixed solvent of these can be used. In addition, specifically, water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof may be used, and more specifically, ethanol may be used. The fraction may be included in 0.001 to 100% by weight, more preferably 0.1 to 80% by weight, based on the total weight of each composition.

In one embodiment of the present invention, as a result of observing intraocular pressure in a high-pressure animal model, intraocular pressure was decreased (FIGS. 1, 2, 7 and 8 ).

In another embodiment of the present invention, the inhibitory efficacy of histological changes of the retina was confirmed in a high pressure animal model (FIGS. 3 and 4 ).

In another embodiment of the present invention, the inhibitory effect of glial cell and astrocyte activity in the high pressure animal model and the cell death inhibition effect of retinal tissue and optic nerve cell were confirmed (FIGS. 5 and 6).

Therefore, it was found from the above results that the maple leaf extract has a very good effect on the decrease in intraocular pressure.

In another aspect, the present invention provides a method for lowering intraocular pressure, comprising administering a composition for lowering intraocular pressure comprising a maple leaf extract or a fraction thereof to the eyes of an individual other than a human.

At this time, the maple, extract, fraction and intraocular pressure are the same as described above.

In the present invention, the term "individual" refers to all animals that do not include humans who have or are likely to develop ocular diseases due to increased intraocular pressure, and the composition of the present invention is administered to an individual who needs to lower intraocular pressure. , Can effectively treat the individual.

The route of administration of the composition for reducing intraocular pressure of the present invention can be administered through any general route as long as it can reach the target tissue.

The composition for lowering intraocular pressure of the present invention is not particularly limited thereto, but may be administered by eye drop, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, as desired. It may be administered through a route such as administration or intrarectal administration, and specifically, may be administered through a route of eye drop administration.

As used in the present invention, the term "administration" means to introduce the composition of the present invention to an individual by any suitable method, and the nature of the maple leaf extract or a fraction thereof of the present invention has an effect of reducing intraocular pressure. The route of administration may be administered through the eye.

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of ocular diseases induced due to an increase in intraocular pressure including a maple leaf extract or a fraction thereof.

At this time, the maple, extract, fraction and intraocular pressure are the same as described above.

In the present invention, the term "prevention" refers to all actions to suppress or delay the onset of ocular disease induced by an increase in intraocular pressure through administration of the pharmaceutical composition of the present invention, and "treatment" refers to the pharmaceutical composition of the present invention It means all actions that improve symptoms or change beneficially through a decrease in intraocular pressure. Specific examples of ocular diseases induced by an increase in intraocular pressure according to the present invention include anterior uveitis, retinal depression, glaucoma or hypertension due to high pressure.

In the present invention, the term, "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and can be used by being formulated in various forms according to a conventional method. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be used in the form of external preparations and sterile injectable solutions.

For example, the pharmaceutical composition may be in the form of an external preparation for the eye, and may be specifically formulated as any one selected from the group containing eye ointment, eye drop, and spray, but if it is a formulation used for administration to the eye in the art It is not limited.

In the present invention, the pharmaceutical composition comprising the maple leaf extract may further include a pharmaceutically acceptable carrier.

In the present invention, the term, "pharmaceutically acceptable carrier" can mean a carrier or diluent that does not inhibit the biological activity and properties of the extract being injected, without stimulating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier that is commonly used in the art and is pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more. The carrier may include a non-naturally occurring carrier. In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostatic agents can be added and used, and additionally added diluents, dispersants, surfactants, binders, lubricants, etc., such as aqueous solutions, suspensions, emulsions, etc. It can be formulated and used in dosage forms, pills, capsules, granules or tablets.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the upper leaf extract and its fractions, for example, starch, calcium carbonate , Sucrose (lactose), gelatin, etc. are prepared by mixing. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use may include various excipients, such as wetting agents, humectants, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are simple diluents commonly used for suspending agents, intravenous solutions, emulsions, syrups, etc. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

In addition, the pharmaceutical composition of the present invention may include a pharmaceutically effective amount of maple leaf extract or a fraction thereof. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and generally in an amount of 0.001 to 1000 mg/kg, specifically 0.05 It may be administered in an amount of 200 to 200 mg/kg, more specifically, 0.1 to 100 mg/kg once to several times a day. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular subject may include specific composition, patient age, weight, general health status, including the type and extent of reaction desired to be achieved and, in some cases, other agents being used, It is desirable to apply differently depending on various factors, including sex and diet, time of administration, route of administration and composition, secretion rate of treatment, duration of treatment, drugs used with or concurrently with the specific composition, and similar factors well known in the pharmaceutical arts.

The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount capable of obtaining a maximum effect in a minimum amount without causing side effects, and can be easily determined by those skilled in the art.

As another aspect of the present invention, there is provided a method of treating ocular diseases induced by increased intraocular pressure, comprising administering a pharmaceutical composition comprising a maple leaf extract or a fraction thereof to the eyes of an individual other than a human.

At this time, the maple, extract, fraction, individual, administration and intraocular pressure are the same as described above.

The route of administration of the pharmaceutical composition of the present invention is the same as the route of administration of the composition for reducing intraocular pressure, and can be administered through any general route as long as it can reach the target tissue.

Another aspect of the present invention provides a quasi-drug composition for reducing intraocular pressure comprising a maple leaf extract or a fraction thereof.

At this time, the maple, extract, fraction and intraocular pressure are the same as described above.

In the present invention, the term "quasi-drug" refers to articles that are used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases of humans or animals, which have less action than pharmaceuticals, for example, according to the pharmacist law. Quasi-drugs are products that are used for the purpose of pharmaceuticals, and are fiber or rubber products used for the treatment or prevention of disease in humans and animals, and have little or no direct action on the human body and are similar to non-equipment or machinery. This includes sterilization and pesticides to prevent infectious diseases. The type or formulation of the quasi-drug composition of the present invention is not particularly limited, but may preferably be an eye wash or an eye drop.

Another aspect of the present invention provides a health functional food composition for lowering intraocular pressure comprising a maple leaf extract or a fraction thereof.

At this time, the maple, extract, fraction, intraocular pressure and improvement are the same as described above.

Health functional foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, dairy products, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, health There are supplementary foods, etc., and include all foods in the ordinary sense.

Since the health functional food composition of the present invention can be ingested on a daily basis, an effect of lowering the elevated intraocular pressure can be expected, and thus can be very useful for health promotion purposes.

The functional food (functional) food (food for special health use) (food for special health use, FoSHU) in the same terms, in addition to nutrition, processed foods, so that the bio-control function to appear efficiently, high medical effect food Means Herein, the term'function (sex)' means obtaining a useful effect for health use such as adjusting nutrients or physiological effects on the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and in the case of the preparation, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the food can be prepared without limitation as long as the formulation is recognized as food. The composition for food of the present invention can be manufactured in various types of formulations, and unlike general medicines, it has the advantage that there is no side effect that can occur when taking medicines for a long time using natural substances as raw materials, and it is excellent in portability. The food of the invention can be consumed as an adjuvant to enhance the effect of lowering intraocular pressure.

The health food refers to food having an active health maintenance or enhancement effect compared to general food, and health supplement food means food for health supplement purposes. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.

Specifically, the health functional food composition is a food prepared by adding maple leaf extract or fractions thereof of the present invention to food materials such as beverages, teas, spices, gums, confectionery, or encapsulating, powdering, suspension, etc. This means that it has a certain effect on health when ingested, but unlike general medicines, it has the advantage that there is no side effect that can occur when taking medicines for a long time using food as a raw material.

The health functional food composition may further include a food-acceptable carrier, and the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the health functional foods are commonly used in food compositions, and may include additional ingredients capable of improving smell, taste, and vision. For example, vitamin A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantotenic acid, and the like. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr); And amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the health functional food composition is a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleached and highly bleached, sodium hypochlorite, etc.), antioxidants (butyl hydroxyanisole (BHA), Butylhydroxytoluene (BHT, etc.), colorants (such as tar pigment), colorants (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (such as MSG sodium glutamate), sweeteners (dulcine, cyclate) , Saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen sulphate, etc.), strengthening agents, emulsifiers, thickeners (foams), coating agents, gum starting agents, foam inhibitors, solvents, improvers, etc. May include food additives. The additive may be selected according to the type of health functional food and used in an appropriate amount.

As an example of the health functional food composition of the present invention, it can be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates, etc., as additional components, such as a normal beverage. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame. The ratio of the natural carbohydrate may be generally about 0.0001 to 0.4 g, specifically about 0.0002 to 0.3 g per 100 mL of the health drink composition of the present invention.

In addition to the above, the health drink composition includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohol or carbonic acid. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected from 0.0001 to 0.10 parts by weight per 100 parts by weight of the health drink composition of the present invention.

The pharmaceutical composition comprising the maple leaf extract or fractions thereof of the present invention as an active ingredient is effective in preventing, improving or treating ocular diseases induced by intraocular pressure through lowering of intraocular pressure.

1 is a final intraocular pressure measured as a result of injecting dexamethasone eye drops for 4 weeks, and then injecting maple leaf hydrothermal extract (KIOM-2015EW) for 5 weeks. It was confirmed that intraocular pressure was lowered when the maple leaf hydrothermal extract was administered in eye drops compared to the negative control (IOP). Nor is normal control, IOP is negative control exposed to dexamethasone, IOP+EW 1mg is exposed to dexamethasone and then treated with maple leaf hydrothermal extract 1mg/mL, IOP+EW 2mg is exposed to dexamethasone, then maple leaf Experimental group treated with hot water extract 2mg/mL, Nor+EW 2mg is an experimental group treated with maple leaf hot water extract 2mg/mL in normal mice. At this time, ###P<0.001 is compared with normal, *P<0.01 is statistical significance when compared with IOP.
FIG. 2 is the final intraocular pressure measured as a result of oral administration of dexamethasone eye drops for 4 weeks, followed by oral administration of maple leaf hot water extract (KIOM-2015EW) for 5 weeks. It was confirmed that intraocular pressure was lowered when the maple leaf hydrothermal extract was administered orally compared to the negative control (IOP). Nor is the normal control group, IOP is the negative control exposed to dexamethasone, IOP+EW 100mg is exposed to dexamethasone, and the experimental group is treated with 100mg/kg of maple leaf hydrothermal extract, IOP+EW 200mg is exposed to dexamethasone, then maple leaf The experimental group treated with hot water extract 200mg/kg, Nor+EW 200mg is the experimental group treated with maple leaf hot water extract 200mg/kg in normal mice. At this time, ###P<0.001 is compared with normal, and ***P<0.001 is statistical significance when compared with IOP.
3 is a result of observation by the immunohistostaining method after instillation of maple leaf extracts (KIOM-2015EW and KIOM-2015EE), it was confirmed that a significant protective effect appears when the maple leaf extract is administered in eye drops. IOP is negative control exposed to dexamethasone, IOP+EW 1mg/mL is exposed to dexamethasone, and then treated with 1mg/mL of maple leaf hydrothermal extract, IOP+EW 2mg/mL is exposed to dexamethasone, and then leaves maple leaves Experimental group treated with extract 2mg/mL, Nor+EW 2mg/mL treated with maple leaf hydrothermal extract 2mg/mL in normal mice, IOP+EE 1mg/mL exposed to dexamethasone, maple leaf ethanol extract 1mg /mL treated experimental group, IOP+EE 2mg/mL was exposed to dexamethasone, and then treated with maple leaf ethanol extract 2mg/mL, Nor+EE 2mg/mL was normal mouse, maple leaf ethanol extract 2mg/mL It is the experimental group treated.
4 is a result of observation by the immunohistostaining method after oral administration of maple leaf extracts (KIOM-2015EW and KIOM-2015EE), confirming that a significant protective effect appears when the maple leaf extract is administered in eye drops. Nor is normal control, IOP is negative control exposed to dexamethasone, IOP+EW 100mg/kg is exposed to dexamethasone and then treated with maple leaf hot water extract 100mg/kg, IOP+EW 200mg/kg is exposed to dexamethasone After the maple leaf hydrothermal extract 200mg/kg treatment group, Nor+EW 200mg/kg is normal mice treated with maple leaf hydrothermal extract 200mg/kg, IOP+EE 100mg/kg leaves after exposure to dexamethasone Experimental group treated with 100 mg/kg of ethanol extract from tree leaves, IOP+EE 200 mg/kg treated with maple leaves after exposure to dexamethasone, experimental group treated with ethanol extract 200 mg/kg, Nor+EE 200 mg/kg with maple leaves in normal mice It is an experimental group treated with 200 mg/kg of ethanol extract.
5 is a result of observing by immunohistostaining after instillation of maple leaf extracts (KIOM-2015EW and KIOM-2015EE), the maple leaf extract when stained with GFAP, a Muller cell indicator protein It was confirmed that GFAP expression was reduced in the treated group and that it was protected by maple extract when OPN1SW (photoreceptor cell antibody) was used. IOP is negative control exposed to dexamethasone, IOP+EW 1mg/mL is exposed to dexamethasone, and then treated with 1mg/mL of maple leaf hydrothermal extract, IOP+EW 2mg/mL is exposed to dexamethasone, and then leaves maple leaves Experimental group treated with extract 2mg/mL, Nor+EW 2mg/mL treated with maple leaf hydrothermal extract 2mg/mL in normal mice, IOP+EE 1mg/mL exposed to dexamethasone, maple leaf ethanol extract 1mg /mL treated experimental group, IOP+EE 2mg/mL was exposed to dexamethasone, and then treated with maple leaf ethanol extract 2mg/mL, Nor+EE 2mg/mL was normal mouse, maple leaf ethanol extract 2mg/mL It is the experimental group treated.
6 is a result of observation by the immunohistostaining method after oral administration of maple leaf extracts (KIOM-2015EW and KIOM-2015EE), the maple leaf extract when stained with GFAP, a Muller cell indicator protein It was confirmed that GFAP expression was reduced in the treated group and that it was protected by maple extract when OPN1SW (photoreceptor cell antibody) was used. Nor is normal control, IOP is negative control exposed to dexamethasone, IOP+EW 100mg/kg is exposed to dexamethasone and then treated with maple leaf hot water extract 100mg/kg, IOP+EW 200mg/kg is exposed to dexamethasone After the maple leaf hydrothermal extract 200mg/kg treatment group, Nor+EW 200mg/kg is normal mice treated with maple leaf hydrothermal extract 200mg/kg, IOP+EE 100mg/kg leaves after exposure to dexamethasone Experimental group treated with 100 mg/kg of ethanol extract from tree leaves, IOP+EE 200 mg/kg treated with maple leaves after exposure to dexamethasone, experimental group treated with ethanol extract 200 mg/kg, Nor+EE 200 mg/kg with maple leaves in normal mice It is an experimental group treated with 200 mg/kg of ethanol extract.
7 is the final intraocular pressure measured as a result of injecting dexamethasone eye drops for 4 weeks, and then injecting maple leaf ethanol extract (KIOM-2015EE) in drops for 5 weeks. It was confirmed that intraocular pressure was lowered when the maple leaf ethanol extract was administered in eye drops compared to the negative control group (IOP). Nor is normal control, IOP is negative control exposed to dexamethasone, IOP+EE 1mg is exposed to dexamethasone, and then treated with maple leaf ethanol extract 1mg/mL, IOP+EE 2mg is exposed to dexamethasone, maple leaves Experimental group treated with ethanol extract 2mg/mL, Nor+ EE 2mg is an experimental group treated with maple leaf ethanol extract 2mg/mL in normal mice. At this time, ###P<0.001 is compared with normal, *P<0.01 is statistical significance when compared with IOP.
8 is the final intraocular pressure measured as a result of oral administration of ethanol extract of maple leaf (KIOM-2015EE) for 5 weeks after administration of dexamethasone eye drop for 4 weeks. It was confirmed that intraocular pressure was lowered when the maple leaf ethanol extract was administered orally compared to the negative control (IOP). Nor is the normal control group, IOP is the negative control exposed to dexamethasone, IOP+EE 100mg is exposed to dexamethasone and then treated with maple leaf ethanol extract 100mg/kg, IOP+EE 200mg is exposed to dexamethasone, then maple leaves Experimental group treated with ethanol extract 200mg/kg, Nor+EE 200mg is an experimental group treated with maple leaf ethanol extract 200mg/kg in normal mice. At this time, ###P<0.001 is compared with normal, *P<0.01 is statistical significance when compared with IOP.

Hereinafter, the configuration and effects of the present invention will be described in more detail through manufacturing examples and examples. These production examples and examples are only intended to illustrate the present invention, and the scope of the present invention is not limited thereby.

Preparation Example 1: Preparation of maple leaf extract

Preparation Example 1-1: Maple leaf hot water extract (KIOM-2015EW) Preparation

After 1700 g of maple leaf crushed product was put in 17 ℓ of water and immersed for 1 hour, hot water was extracted for 3 hours using a Gyeongseo Super-High-Speed Vacuum Extractor (Gyeongseo Extractor, COSMOS 660, Incheon, Korea). Then, the extract was lyophilized to obtain 98.77 g of maple leaves.

The extracted maple leaf extract was filtered using a standard test sieve (sieve) (150 μm, Retsch, Han, Germany) and concentrated to dryness in a freeze dryer. The freeze-dried maple leaf extract powder (50 mg) was dissolved in 1 ml of distilled water, filtered through a 0.22 μm disk filter, and stored at -20° C. until use.

Preparation Example 1-2: Maple Leaf Ethanol Extract (KIOM-2015EE) Preparation

1700 g of the maple leaf crushed product was added to 17 L of 25% ethanol, and was extracted by depositing at room temperature for 24 hours. Thereafter, the extract was concentrated under reduced pressure and lyophilized to obtain 78.57 g of maple leaf 25% ethanol extract.

Preparation Example 2: Preparation of fractions of maple leaf extract

Preparation Example 2-1: Preparation of fraction of maple leaf hot water extract (KIOM-2015EW)

Solvent fractionation using an organic solvent was performed to separate the components in the extract from the maple leaf hot water extract (KIOM-2015EW) prepared in Preparation Example 1-1 according to polarity. That is, 4 g of KIOM-2015EW extract was suspended in 80 mL of water, and then 80 mL of hexane was added to obtain the components eluted with the hexane layer by repeating three times (hexane fraction; Hexane Fr.). In addition, dichloromethane (DCM), ethyl acetate fraction (EtOAc), n-butanol fraction (n-) by applying the same method in the order of dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (n-BuOH) BuOH) and water fractions (H ₂ O Fr.) were obtained (FIG. 1), and the yields for each organic solvent were confirmed and are shown in Table 1.

Preparation Example 2-2: Preparation of fraction of maple leaf ethanol extract (KIOM-2015EE)

Solvent fractionation using an organic solvent was performed to separate the components in the extract from the maple leaf ethanol extract (KIOM-2015EE) prepared in Preparation Example 1-2 according to polarity. That is, 4 g of KIOM-2015EW extract was suspended in 80 mL of water, and then 80 mL of hexane was added to obtain the components eluted with the hexane layer by repeating three times (hexane fraction). In addition, dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (n-BuOH) were applied in the same order, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and water (H ₂ O) fraction Obtained (FIG. 1), it was shown in Table 1 to confirm the yield by organic solvent.

Fraction yield by organic solvent Fraction Yield by organic solvent (%) KIOM-2015EW
Hot water extract KIOM-2015EE
25% Ethanol extract Hexane 0.16 0.10 Dichloromethane (DCM) 1.44 1.43 Ethyl Acetate (EtOAc) 9.78 6.41 n-butanol (n-BuOH) 17.51 17.02 Water (H ₂ O) 58.70 74.98

Example 1: Confirmation of the effect of KIOM-2015EW in a high pressure animal model

Example 1-1: High pressure reduction effect of KIOM-2015EW in high pressure animal model

High-pressure animal models were prepared using C57BL/6 mice. Mice were dosed with dexamethasone eye drops (Maxidex®, Alcon Korea, Korea) three times a day for 4 weeks (9 am, 1 pm, and 5 pm). Subsequently, KIOM-2015EW was administered to the steroid-induced high pressure animal model three times a day for 5 weeks (in the morning at 9:00 a.m., 9:00 a.m.) through eye drops (1 mg/mL or 2 mg/mL) or orally (100 mg/kg or 200 mg/kg). 1 hour, 5 pm). The intraocular pressure of the mice was measured once weekly. In other words, the measurement of intraocular pressure used a tool that can accurately measure the intraocular pressure of rodent animals such as mice and rats. The principle of rebound measurement using Icare's famous products (Tonometer, Icare® TonoLab, Vantaa, Finland) Based on the intraocular pressure was measured in such a way that the probe contacts the cornea in an instant.

The mice were classified into 8 groups, and each group was set as follows.

As a control group (Normal), mice raised without treatment were used; As a negative control (IOP), mice receiving only dexamethasone eye drops were used; Experimental group 1 (IOP+EW 1mg/mL) was treated with dexamethasone eye drops for 4 weeks, and the mice fed with the maple leaf hydrothermal extract obtained in Preparation Example 1-1 were administered eye drops at a dose of 1mg/mL for 5 weeks. Was used; Experimental group 2 (IOP+EW 2mg/mL) was administered with dexamethasone eye drops for 4 weeks, and the mice fed with the maple leaf hydrothermal extract obtained in Preparation Example 1-1 were administered eye drops at a dose of 2mg/mL for 5 weeks. Was used; Experimental group 3 (Nor + EW 2mg / mL) was used as a mouse mice raised while administering eye drops at a dose of 2mg/mL for 5 weeks to the maple leaf hot water extract obtained in Preparation Example 1-1; Experimental group 4 (IOP+EW 100mg/kg) was administered with dexamethasone eye drops for 4 weeks, and the mice fed with the maple leaf hydrothermal extract obtained in Preparation Example 1-1 were administered orally at a dose of 100mg/kg for 5 weeks. Was used; Experimental group 5 (IOP+EW 200mg/kg) was administered with dexamethasone eye drop for 4 weeks, and the mice fed with the maple leaf hydrothermal extract obtained in Preparation Example 1-1 were administered orally at a dose of 200mg/kg for 5 weeks. Was used; Experimental group 6 (Nor + EW 200mg / kg) was used as a mouse mice raised while administering eye drops at a dose of 200mg/kg for 5 weeks with the maple leaf hot water extract obtained in Preparation Example 1-1.

As a result, as can be seen in FIGS. 1 and 2, on the 64th day, the intraocular pressure of the negative control group was significantly increased compared to the control group, but it was confirmed that intraocular pressure decreases when KIOM-2015EW is administered to the high pressure mouse or instilled or orally. Did.

Example 1-2: Confirmation of retinal histological changes by KIOM-2015EW in high pressure animal model

To confirm histological changes of the retina, the harvested eyeballs were fixed in a 4% PFA solution, and finally, paraffin was infiltrated and the tissue was embedded, and then cut to a thickness of 3 μm using a microtome. After removing and hydrating paraffin for 1 hour in a drying oven, 5 minutes with Hematoxylin solution, staining with eosin solution, washing, mounting, and morphology of the retina using microscopy Histological changes were acquired.

3 and 4, it was observed that the number of retinal ganglion cells (RGCs) of the ganglion cell layer (GCL) was significantly reduced in the negative control (IOP) exposed to dexamethasone compared to the normal control. . However, it was observed that this change was inhibited in all groups including ophthalmic and oral administration of KIOM-2015EW.

Example 1-3: Inhibition of activity of glial cells and astrocytes by KIOM-2015EW in a high pressure animal model

After fixing the tissues in 10% neutral formalin, the tissues embedded in paraffin were continuously sliced by 4 μm, and one sheet was subjected to Hematoxylin-Eosin (H&E) staining, and the rest was put on slides for immunohistochemical staining. Attached. To remove the endogenous peroxidase, H ₂ O ₂ was treated for 30 minutes. Anti-DAPI (anti-DAPI), anti-GFAP (anti-GFAP), and anti-GFAP antibody (anti-GFAP antibody) to be used for immunohistochemical staining 1:50, anti-OPN1SW (anti-OPN1SW) 1: After diluting each with 50, it was incubated overnight at 4° C. (overnight incubation) and then washed (washed), and then labeled with a secondary antibody and observed under a microscope.

5 and 6 show the results of immunofluorescence staining using glial fibrillary acidic protein (GFAP) antibodies, which are glioma and astrocyte markers. Astrocytes and glial cells that are activated around the damage include astrocytes and glial cells, which are frequently activated when tissues are damaged, and confirmed the increase or decrease of GFAP, a marker of astrocytes. In the case of high-pressure mice (IOP) exposed to dexamethasone, it was confirmed that the expression of GFAP was significantly increased compared to the control group, and it was confirmed that KIOM-2015EW significantly decreased the expression in all groups including instillation and oral administration. .

Example 1-4: Confirmation of cell death inhibition of retinal tissue and optic ganglion cells by KIOM-2015EW in high pressure animal model

As a result of observation by the immunohistostaining method of Example 1-3, when staining using OPN1SW (photoreceptor cell antibody), it was confirmed through FIG. 5 and FIG. 6 that the OPN1SW was rarely expressed by steroids in the IOP group. . However, it was confirmed that OPN1SW was protected in all groups including instillation and oral administration of KIOM-2015EW.

Example 2: Confirmation of the effect of lowering intraocular pressure of KIOM-2015EE in a high pressure animal model

Example 2-1: High pressure reduction effect of KIOM-2015EE in high pressure animal model

High-pressure animal models were prepared using C57BL/6 mice. Mice were dosed with dexamethasone eye drops (Maxidex®, Alcon Korea, Korea) three times a day for 4 weeks (9 am, 1 pm, and 5 pm). Thereafter, KIOM-2015EE was administered to the steroid-induced high pressure animal model three times a day for 5 weeks (in the morning at 9:00 a.m. and 9:00 a.m.) through instillation (1 mg/mL or 2 mg/mL) or orally (100 mg/kg or 200 mg/kg). 1 hour, 5 pm). The intraocular pressure of the mice was measured once weekly.

The mice were classified into 8 groups, and each group was set as follows.

As a control group (Normal), mice raised without treatment were used; As a negative control (IOP), mice receiving only dexamethasone eye drops were used; Experimental group 7 (IOP+EE 1mg/mL) was administered with dexamethasone eye drops for 4 weeks, and mice raised with eye drops of maple leaf ethanol extract obtained in Preparation Example 1-2 at a dose of 1mg/mL for 5 weeks. Was used; Experimental group 8 (IOP+EE 2mg/mL) was treated with dexamethasone eye drops for 4 weeks, and the mice fed with maple leaf ethanol extract obtained in Preparation Example 1-2 were administered eye drops at a dose of 2mg/mL for 5 weeks. Was used; Experimental group 9 (Nor + EE 2mg/mL) was used in normal mice, the mice raised with the maple leaf ethanol extract obtained in Preparation Example 1-2 were administered in drops at a dose of 2mg/mL for 5 weeks; Experimental group 10 (IOP+EE 100mg/kg) was treated with dexamethasone eye drops for 4 weeks, and mice raised with oral administration of maple leaf ethanol extract obtained in Preparation Example 1-2 at a dose of 100mg/kg for 5 weeks Was used; Experimental group 11 (IOP+EE 200mg/kg) was administered with dexamethasone eye drops for 4 weeks, and mice raised with oral administration of maple leaf ethanol extract obtained in Preparation Example 1-2 at a dose of 200mg/kg for 5 weeks Was used; Experimental group 12 (Nor + EE 200mg / kg) was used in normal mice, mice raised and raised while orally administering the maple leaf ethanol extract obtained in Preparation Example 1-2 at a dose of 200mg/kg for 5 weeks.

As a result, as can be seen in FIGS. 7 and 8, on the 64th day, the intraocular pressure of the negative control group was significantly increased compared to the control group, but it was confirmed that intraocular pressure was decreased when KIOM-2015EE was administered to the high-pressure mouse instilled or orally. Did.

Example 2-2: Confirmation of retinal histological changes by KIOM-2015EE in high pressure animal model

Histological changes of the retina were obtained in the same manner as in Example 1-2.

3 and 4, it was observed that the number of retinal ganglion cells (RGCs) of the ganglion cell layer (GCL) was significantly reduced in the negative control (IOP) exposed to dexamethasone compared to the normal control. . However, it was observed that this change was inhibited in all groups including ophthalmic and oral administration of KIOM-2015EE.

Example 2-3: Neurofiber protection effect by KIOM-2015EE in high pressure animal model

Immunohistostaining was performed in the same manner as in Example 1-3.

5 and 6 show the results of immunofluorescence staining using glial fibrillary acidic protein (GFAP) antibodies, which are glioma and astrocyte markers. Astrocytes and glial cells that are activated around the damage include astrocytes and glial cells, which are frequently activated when tissues are damaged, and confirmed the increase or decrease of GFAP, a marker of astrocytes. In the case of high-pressure mice (IOP) exposed to dexamethasone, it was confirmed that the expression of GFAP was significantly increased compared to the control group, and it was confirmed that KIOM-2015EE significantly decreased its expression in all groups including instillation and oral administration. .

Example 2-4: Confirmation of cell death inhibition of retinal tissue and optic ganglion cells by KIOM-2015EW in high pressure animal model

As a result of observation by the immunohistostaining method of Example 1-3, when staining using OPN1SW (photoreceptor cell antibody), it was confirmed through FIG. 5 and FIG. 6 that the OPN1SW was rarely expressed by steroids in the IOP group. . However, it was confirmed that OPN1SW was protected in all groups including instillation and oral administration of KIOM-2015EW.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the claims, which will be described later, rather than the above detailed description, and all modified or modified forms derived from the equivalent concepts thereof.

Claims (10)

A composition for reducing intraocular pressure, comprising a maple leaf extract or a fraction thereof as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is obtained by extracting maple leaves with water, C1 to C4 alcohol, or a mixed solvent thereof. A method of lowering intraocular pressure, comprising administering the composition of any one of claims 1 or 2 to the eye of an individual other than a human. A pharmaceutical composition for the prevention or treatment of ocular diseases induced by an increase in intraocular pressure, which includes a maple leaf extract or a fraction thereof as an active ingredient. The pharmaceutical composition of claim 4, wherein the ocular disease is selected from anterior uveitis, retinal depression, glaucoma due to high pressure, or hypertension. The pharmaceutical composition according to claim 4, wherein the composition is in the form of a topical external preparation. The pharmaceutical composition of claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier. A method for preventing or treating ocular diseases induced by increased intraocular pressure, comprising administering the composition of any one of claims 4 to 7 to the eyes of an individual other than a human. A quasi-drug composition for reducing intraocular pressure, comprising a maple leaf extract or a fraction thereof. A health functional food composition for reducing intraocular pressure, comprising maple leaf extract or a fraction thereof.

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