WO2018106009A1

WO2018106009A1 - Composition containing maple tree leaf extract or fractions thereof for preventing or treating inflammatory ocular diseases

Info

Publication number: WO2018106009A1
Application number: PCT/KR2017/014224
Authority: WO
Inventors: 마진열; 김연희; 오태우; 박은희; 조원경; 임남희; 오유창; 구민정
Original assignee: 한국한의학연구원
Priority date: 2016-12-07
Filing date: 2017-12-06
Publication date: 2018-06-14
Also published as: KR20180065933A

Abstract

The present invention relates to: a pharmaceutical composition containing maple tree leaf extract or fractions thereof for preventing or treating inflammatory ocular diseases; a method for treating the diseases comprising a step of administering the composition into the eye of an individual; a quasi-drug composition; a composition for an eye makeup additive; a functional health food composition; and an animal feed composition.

Description

Inflammatory eye disease preventing or treating composition comprising maple leaf extract or a fraction thereof

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory eye disease, including maple leaf extract or a fraction thereof; A method of treating said disease comprising administering said composition to an eye of a subject; Quasi-drug compositions; Eye cosmetic additive compositions; Health functional food composition; And feed compositions.

Inflammatory disease is a generic term that refers to inflammation as the primary lesion, and is known to be involved in numerous human diseases. In fact, the list of specific diseases of inflammatory diseases shows that there are many types of inflammatory diseases such as acne, asthma, autoimmune diseases, periodic fever syndrome, colitis, pelvic inflammatory disease, rheumatoid arthritis and vasculitis. Can be. Dry eye syndrome is not simply a lack of tears, but causes eye discomfort, decreased vision, and instability of the tear layer due to tears and inflammation of the eye surface (cornea and conjunctiva), causing damage to the eye surface. There is a high risk of developing irregular corneal surfaces, blurred vision and increased corneal ulcers. However, the pathogenesis has not yet been fully understood, but studies have shown that inflammation plays an important role, such as invasion of inflammatory cells, increased immune activating molecule and adhesion molecule expression, Th1 and Th17 responses, apoptosis markers and abnormal changes in chemokines. have.

Inflammatory eye disease is a disease that occurs in the eye due to inflammation of the eye or its surrounding tissues among numerous types of inflammatory diseases. Until now, studies on inflammatory eye disease have mainly been carried out to activate autologous T cells and B cells through them. It focuses on the immune response that occurs through the disease. Specifically, the disease includes dry eye, conjunctivitis, hyperemia, keratitis, and the like, all of which are diseases directly occurring in the eye due to inflammation in the eye or its surrounding tissues.

Dry eye syndrome is a common disease that occurs in 5.5 to 15% of adults worldwide. It is not just a lack of tears, but an eye discomfort due to tears and inflammation of the eye surface (cornea and conjunctiva). It is known to cause instability and damage the ocular surface. The disease is characterized by eye pain, irregular corneal surfaces, corneal ulcers, and decreased vision. The altered corneal permeability in chronic dry eye and dry keratitis has been very well known to cause inflammation, which is known to be induced by increased cytokine mediating inflammation in tears. In general, it is recommended to eat foods rich in potassium and anthocyanin for the treatment of dry eye. In addition, it is also highly recommended to eat fruits such as kale, kiwi, and apples. Some modalities may be used.

Conjunctivitis is an inflammatory condition of the conjunctiva caused by microorganisms such as bacteria, viruses, and fungi, and environmental factors such as pollen and chemical stimulation, and is the most common eye disease because the conjunctiva is exposed to the outside. It usually heals well, but in some cases it can be fatal, resulting in blindness due to tissue damage. Conjunctivitis is largely classified into infectious conjunctivitis and non-infectious conjunctivitis depending on the cause.

Hyperemia is a condition in which the conjunctiva's blood vessels expand and the whites of the eyes appear red. Inflammation of the eyes such as all types of conjunctivitis, keratitis, and iris phloemitis can cause redness, and if the eyelids do not provide enough protection for the eyes due to dry eye syndrome, the redness develops well, and the cornea can be worn even when contact lenses are worn for a long time. Many new blood vessels are created around the redness.

Keratitis accounts for more than 90% of corneal diseases. Defects in the outermost part of the eye, such as corneal epithelium, and cloudiness caused by inflammatory reactions to the corneal parenchyma can lead to decreased vision. With more than 50,000 keratitis occurring each year in the United States, keratitis is the most important disease that can cause blindness in the United States. If you have keratitis, you may have redness, foreign body, and pain, and you may be sensitive to light, tears, or blurry symptoms. Keratitis is also classified into infectious keratitis and non-infectious keratitis depending on the cause.

Patients with inflammatory eye disease make up more than half of patients with eye disease, and therefore, drugs with ocular anti-inflammatory effects play an important role in medicine. Specific examples include steroidal drugs or nonsteroidal drugs. In particular, recently, as an agent for treating inflammatory eye disease, an unpreserved fluorometholone (FML), a steroidal drug, or cyclosporin A, a nonsteroidal drug, is used. Cyclosporine A, CsA) eye drops are widely used.

However, the steroidal drug for the treatment of inflammatory eye disease is known to have side effects such as increased intraocular pressure or the occurrence of cataracts or glaucoma when used for a long period of time. It is known that this leads to a decrease in white blood cells, thereby weakening its immunity. In addition, as the use of computers or smartphones is rapidly increasing, inflammatory eye diseases are also rapidly increasing. Therefore, there is a demand for the development of safer and inflammatory eye disease treatments that can be used by various age groups.

Meanwhile, maple ( Acer palmatum ) is a deciduous broad-leaved arboreous tree native to Korea and widely distributed in Japan and China. It often grows in the mountains of sub-central Jeju and North Jeolla provinces, and has strong cold resistance, disease resistance, and pollution resistance. In autumn, leaves with leaves contain a compound called vitexin, which has been reported to be an antioxidant against cells that have been exposed to anti-aging or excessive exposure to UV light.

Accordingly, the present inventors, as a result of intensive efforts to develop a composition comprising a natural extract or a fraction thereof having a prophylactic or therapeutic effect of inflammatory eye disease, specifically, dry eye syndrome, conjunctivitis, hyperemia or keratitis and without side effects, maple leaf extract Or the fraction thereof has been confirmed to increase the viability of corneal epithelial cells and conjunctival epithelial cells in the osmotic stress environment, and to treat inflammatory eye disease through the inhibitory effect of the expression of inflammation-induced cytokines, to complete the present invention.

It is a main object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory eye diseases, including maple leaf extract or fractions thereof.

Another object of the present invention is to provide a method for preventing or treating inflammatory eye disease, comprising administering the composition to an eye of an individual.

Another object of the present invention is to provide a quasi-drug composition for the prevention or treatment of inflammatory eye diseases, including maple leaf extract or fractions thereof.

Still another object of the present invention is to provide an eye cosmetic additive composition for preventing or improving inflammatory eye disease including maple leaf extract or a fraction thereof.

Still another object of the present invention is to provide a nutraceutical composition for the prevention or improvement of inflammatory eye disease, including maple leaf extract or a fraction thereof.

Still another object of the present invention is to provide a feed composition for preventing or improving inflammatory eye disease, including a maple leaf extract or a fraction thereof.

Maple leaf extract or a fraction thereof according to the present invention is effective in the prevention, improvement or treatment of inflammatory eye diseases, specifically dry eye, conjunctivitis, hyperemia and keratitis.

Figure 1 shows the manufacturing process of the fraction according to an embodiment of the present invention.

Figure 2 is a graph showing the cell viability of human corneal epithelial cells according to the fraction of the maple leaf hydrothermal extract (KIOM-2015EW) and the maple leaf ethanol extract (KIOM-2015EE) according to an embodiment of the present invention (KIOM-2015EW Frac. BuOH ; N-butanol fraction of maple leaf hydrothermal extract, KIOM-2015EW Frac.H ₂ O; water fraction of maple leaf hydrothermal extract, KIOM-2015EE Frac.EtOA; ethyl acetate fraction of maple leaf ethanol extract, KIOM-2015EE Frac.BuOH; maple leaf ethanol N-butanol fraction of the extract, KIOM-2015EE Frac. H ₂ O; water fraction of the maple leaf ethanol extract, ** p <0.01, *** p <0.001 vs. control, the same below).

Figure 3 is a graph showing the cell viability of human conjunctival epithelial cells according to the fraction of the maple leaf hydrothermal extract (KIOM-2015EW) and the maple leaf ethanol extract (KIOM-2015EE) according to an embodiment of the present invention (KIOM-2015EW Frac. DCM) Dichloromethane fraction of maple leaf hydrothermal extract, KIOM-2015EW Frac.EtOA; ethyl acetate fraction of maple leaf hydrothermal extract, KIOM-2015EE Frac.Hexane; hexane fraction of maple leaf ethanol extract).

Figure 4 is a graph showing the inflammation-related cytokine expression inhibitory activity of n-butanol fraction (KIOM-2015EW Frac. BuOH) of maple leaf hydrothermal extract on human corneal epithelial cells according to an embodiment of the present invention (** p <0.01 , *** p <0.001 vs. control; #p <0.05, ## p <0.01, ### p <0.001 vs. HOS.

5 is a graph showing the inhibitory activity of inflammation-related cytokine expression of the water fraction (KIOM-2015EW Frac. H ₂ O) of maple leaf hydrothermal extract on human corneal epithelial cells according to an embodiment of the present invention.

Figure 6 is a graph showing the inflammation-related cytokine expression of the n-butanol fraction (KIOM-2015EE Frac. BuOH) of the maple leaf ethanol extract on human corneal epithelial cells according to an embodiment of the present invention (** p <0.01 , *** p <0.001 vs. control; #p <0.05, ## p <0.01, ### p <0.001 vs. HOS.

Figure 7 is a graph showing the inhibitory activity of inflammation-related cytokine expression of maple leaf ethanol extract water fraction (KIOM-2015EE Frac. H ₂ O) for human corneal epithelial cells according to an embodiment of the present invention.

8 is a diagram showing the antioxidant protein expression ability of the maple leaf extract and fractions for human corneal epithelial cells according to an embodiment of the present invention (1; control, 2; high osmotic stress-induced human corneal epithelial cells (HOS)) 3, n-butanol fraction treatment of maple leaf hydrothermal extract (50 μg / ml), 4; water fraction treatment of maple leaf hydrothermal extract (50 μg / ml), 5; butanol fraction treatment of maple leaf ethanol extract (50 μg / ml) 6; water fraction treatment of maple leaf ethanol extract (50 μg / ml), 7; positive control dry eye treatment FML treatment, 8; positive control dry eye treatment CsA treatment, 9; maple leaf hydrothermal extract treatment (100 μg / ml), 10; maple leaf ethanol extract treatment (100 μg / ml).

9 is a graph showing the inflammation-related cytokine expression inhibitory activity of the n-butanol fraction of maple leaf hydrothermal extract on human conjunctival epithelial cells according to an embodiment of the present invention.

10 is a graph showing the inflammation-related cytokine expression inhibiting ability of the water fraction of the maple leaf hydrothermal extract for human conjunctival epithelial cells according to an embodiment of the present invention.

11 is a graph showing the inflammation-related cytokine expression inhibitory activity of the n-butanol fraction of the maple leaf ethanol extract on human conjunctival epithelial cells according to an embodiment of the present invention.

12 is a graph showing the inflammation-related cytokine expression inhibitory ability of the water fraction of the maple ethanol extract for human conjunctival epithelial cells according to an embodiment of the present invention.

The present invention as one aspect to achieve the above object, provides a pharmaceutical composition for the prevention or treatment of inflammatory eye disease comprising a maple leaf extract or a fraction thereof.

The present invention also provides a prophylactic or therapeutic use of an inflammatory eye disease of maple leaf extract or a fraction thereof.

As used herein, the term “inflammatory eye disease” may have various forms of eye diseases involving various pains depending on the location of inflammation, and may include itching, redness, edema, ulcers, and the like. More than half of patients with ocular disease account for inflammatory ocular disease, so drugs with ocular anti-inflammatory efficacy play an important role in the medical field. Specific examples of inflammatory eye diseases according to the present invention may be dry eye, conjunctivitis, hyperemia and keratitis.

In the present invention, the term "foliage leaves" is maple ( Acer palmatum ) leaves. The maple tree is a deciduous lumbered arbor tree, about 10 to 20m high, and refers to a plant having a circular shape.

In the present invention, the term "extract" refers to a liquid component obtained by immersing a desired substance in various solvents, and then extracted for a predetermined time at room temperature or warm state, and a resultant such as a solid component obtained by removing the solvent from the liquid component. Can mean. In addition, in addition to the result, it can be comprehensively interpreted to include all of the dilution of the resultant, their concentrates, their modifiers, purified products and the like. The method for obtaining the extract is not particularly limited as long as an extract having a prophylactic or therapeutic effect of inflammatory eye disease can be obtained, but specifically, the maple leaf, its dried product, processed products, etc. are immersed in the solvent, Cold extraction method extracted at room temperature of 10 to 25 ℃, heating extraction method to be extracted by heating to 40 to 100 ℃, ultrasonic extraction method by applying ultrasonic waves, reflux extraction method using a reflux cooler and the like 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 the pharmaceutical 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.

As used herein, the term "fraction" refers to the result obtained by performing the fractionation to separate a specific component or a specific component group from a mixture comprising various various components.

In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. Solvent fractionation by treatment of various solvents, ultrafiltration fractionation through passage of ultrafiltration membranes with constant molecular weight cut-off values, chromatographic chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) Chromatography), and combinations thereof. The kind of solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. As a non-limiting example of the fractionation solvent, water, an organic solvent or a mixed solvent thereof may be used. The organic solvent may be a C 1-4 alcohol, a polar solvent such as ethyl acetate or acetone, hexane or dichloromethane. A nonpolar solvent or a mixed solvent thereof can be used. In addition, specifically, water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof may be used, and more specifically, ethanol may be used. The fractions may each comprise 0.001 to 100% by weight, more specifically 0.1 to 80% by weight relative to the total weight of the pharmaceutical composition.

For example, the fraction of the maple leaf extract may be a fraction of maple leaf hydrothermal extract or ethanol extract.

As used herein, the term "dry eye syndrome" refers to abnormal tear evaporation due to tear secretion or meibomian gland dysfunction or eyelid dysfunction due to inflammation of the tear glands and denervation of the cornea. It may be a disease caused by the dry eye, and it may be a corneal epithelial disorder caused by excessive dryness of the tear due to the dry eye, which may damage the tear gland, blockage of the tear tube, and tear formation. Increased tear evaporation due to ocular surface disorders such as decreased tear secretion, meibomian gland dysfunction, eyelid openness, low eyelid disease, vitamin A deficiency, topical medications and preservatives, contact lens wear and allergy And keratoconjunctival epithelial disorders.

As used herein, the term "conjunctival" refers to a tissue that forms the surface of the eye and protects the eye from foreign substances, and is a tissue involved in the formation of the mucus layer of the tear and immune function. In the present invention, the term "conjunctivitis" refers to an inflammatory disease occurring in the conjunctiva, which is a tissue surrounding the eye, and has typical epidemic conjunctivitis.

For example, the conjunctivitis may be non-infectious conjunctivitis. In the case of non-infectious conjunctivitis, it is caused by an allergic reaction to foreign substances.

As another example, the conjunctivitis may be infectious conjunctivitis. The infectious conjunctivitis is caused by infection of various pathogens such as bacteria, viruses, and fungi, and may be, for example, epidemic conjunctivitis, acute hemorrhagic conjunctivitis (Apollo conjunctivitis), and bacterial conjunctivitis.

As used herein, the term "cornea" refers to a structure that allows the eye to be protected from the outside, through the light, and refracted as a transparent film on the ocular surface at the center of the eye. In the present invention, the term "keratitis" refers to a disease in which the inflammation of the cornea causes pain, decreased visual acuity, corneal clouding, and the like, and ocular hyperemia may occur as a representative symptom.

For example, the keratitis may be non-infectious keratitis. For example, it may mean exposed keratitis caused by continuous exposure to external air, toxic keratitis caused by drugs, neurotrophic keratitis caused by corneal nerve damage, disorders caused by contact lenses, trauma, or allergic keratitis. Non-infectious keratitis is difficult to treat with general treatment for infectious keratitis.

As another example, the keratitis may be infectious keratitis caused by bacteria, viruses, fungi (fungus).

As used herein, the term "hyperemia" refers to a symptom in which the conjunctival blood vessels are expanded to open the whites of the eyes. Congestion due to conjunctivitis is most severe in the far side of the eye, and congestion due to inflammation of the cornea or iris is most severe in the eye conjunctiva. Inflammation of the eye, such as conjunctivitis and keratitis of all kinds, can cause hyperemia, and congestion can occur if tears do not provide enough protection for the eyes due to dry eye.

In one embodiment of the present invention, in order to confirm the dry eye improvement effect of the maple leaf extract or fractions thereof, as a result of observing the cell protective effect on human corneal epithelial cells and human conjunctival epithelial cells, dry eye syndrome induced by osmotic stress It was confirmed that the treatment of maple leaf hot water and ethanol extract in the environment inhibits the toxicity of the cells and increases the viability. Thus, the maple leaf extract or fractions thereof may be used for the prevention or treatment of inflammatory eye diseases including dry eye, conjunctivitis, hyperemia or keratitis (FIGS. 2 and 3).

As used herein, the term "prevention" refers to any action that inhibits or delays the symptoms of inflammatory eye disease by administration of a composition comprising a maple leaf extract or a fraction thereof.

In the present invention, the term "treatment" may mean any action that improves or advantageously changes the symptoms of the disease by administration of a composition comprising a maple leaf extract or a fraction thereof.

As used herein, the term "improvement" may mean any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.

In the present invention, the composition may be to inhibit inflammation-related cytokines. When inflammatory eye disease is induced, cytokines related to inflammation such as IL-6, TNF-α, IL-1β, and the like are expressed, and the composition of the present invention may be to suppress the amount of expression thereof.

In one embodiment of the present invention as a result of confirming the inhibitory effect of the expression of inflammation-related cytokine protein in order to confirm the anti-inflammatory effect on human corneal epithelial cells and human conjunctival epithelial cells, the fraction of the maple leaf hot water and ethanol extract is IL-6 The concentration of TNF-α and IL-1β was confirmed to decrease in a concentration-dependent manner (FIGS. 4 to 7 and 9 to 12). Therefore, it was confirmed that the fraction of the extract can be usefully used for the improvement or treatment of inflammatory eye disease.

In the present invention, the term "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and may be used in various forms, each formulated in accordance with conventional methods. For example, they may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like, and may be used in the form of external preparations and sterile injectable solutions.

For example, the pharmaceutical composition may be an external topical formulation, and may be specifically formulated into any one selected from the group including eye ointments, eye drops, and sprays, but formulations used for administration to the eye in the art. Is not limited.

In the present invention, the pharmaceutical composition including the maple leaf extract or a fraction thereof may further include a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be injected without stimulating the organism. The kind of the carrier usable in the present invention is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art. 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 thereof. The carrier may include a non-naturally occuring carrier. In addition, if necessary, other conventional additives such as antioxidants, buffers and / or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, lubricants, and the like may be additionally added to provide a solution such as an aqueous solution, a suspension, an emulsion, or the like. It may be formulated into a use formulation, pills, capsules, granules or tablets.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, and the like in the above extracts and fractions thereof. , Sucrose or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can 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. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, generally in an amount of 0.001 to 1000 mg / kg, specifically 0.05 To 200 mg / kg, more specifically, the amount of 0.1 to 100 mg / kg can be administered once to several times a day. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health of the patient, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or co-specific with the specific composition.

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

As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, wherein the maple leaf extract of the present invention or a fraction thereof is effective in the prevention or treatment of inflammatory eye disease. By nature, the route of administration of the composition can be administered through the eye.

In another aspect of the present invention, there is provided a method of treating inflammatory eye disease, comprising administering the composition to an eye of an individual.

At this time, the inflammatory eye disease, administration and treatment is as described above.

As used herein, the term "individual" may refer to an animal other than a human who has or is likely to develop an inflammatory eye disease. The animal may be a mammal such as, but not limited to, a human, a cow, a horse, a sheep, a pig, a goat, a camel, a antelope, a dog, a cat, and the like, which require treatment of similar symptoms.

The composition may be administered in single or multiple amounts in a pharmaceutically effective amount.

The route of administration of the pharmaceutical composition for preventing or treating inflammatory eye disease of the present invention may be administered via any general route as long as it can reach the target tissue.

The pharmaceutical composition of the present invention is not particularly limited, but as desired, instillation, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, pulmonary administration, It may be administered through a route such as rectal administration, and specifically, may be administered through a route of eye drop administration.

As another aspect of the present invention, there is provided a quasi-drug composition for the prevention or treatment of inflammatory eye disease comprising a maple leaf extract or a fraction thereof.

At this time, the maple leaf, extract, fraction, inflammatory eye disease, prevention and treatment are as described above.

In the present invention, the term "quasi drug" refers to articles that are less active than drugs among those used for the purpose of diagnosing, treating, ameliorating, alleviating, treating, or preventing a disease of a human or animal. Quasi-drugs, except those used for the purpose of medicines, are textiles and rubber products used in the treatment or prevention of diseases of humans and animals, and have a slight or no direct action on the human body; This includes sterilizing and insecticides for preventing infectious diseases. The kind or formulation of the quasi-drug composition of the present invention is not particularly limited, but may preferably be a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler.

In another aspect of the present invention, there is provided an eye cosmetic additive composition for the prevention or improvement of inflammatory eye disease comprising a maple leaf extract or a fraction thereof. The additive may be included in eye cosmetic products to exhibit an effect on the prevention or improvement of the disease.

At this time, the maple leaf, extract, fraction, inflammatory eye disease, prevention and improvement are as described above.

In the present invention, the composition may include eye cosmetic products, and the eye cosmetic products may be any one selected from the group comprising eyebrow pencil, eyeliner, eye shadow, mascara, and eye makeup remover. have.

In another aspect of the present invention, there is provided a dietary supplement composition for preventing or improving inflammatory eye disease, including a maple leaf extract or a fraction thereof.

When the composition of the present invention is used as a health functional food additive, the maple leaf extract or a fraction thereof may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), and the composition of the present invention is environmentally friendly and there is no problem in terms of stability, so there is no big limitation on the blending amount.

Since the health functional food composition of the present invention can be ingested on a daily basis, an improvement effect on inflammatory eye disease can be expected, and thus can be very useful for health promotion purposes.

The term "health functional food" of the present invention is the same term as a food for special health use (FOSHU), and foods having high medical effects and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. Means. Here, the term 'function' refers to obtaining a useful effect for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a food. The health functional food composition of the present invention can be prepared in various forms of formulations, unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material, and excellent portability In addition, the health functional food of the present invention can be ingested as an adjuvant to enhance the improvement effect of inflammatory eye disease.

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

Specifically, the health functional food is a food prepared by adding the compound of the present invention to food materials such as beverages, teas, spices, gums, confections, or the like, encapsulated, powdered, suspensions, etc. Means to bring effect, but unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material.

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

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

In addition, the health functional food composition is a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), Butylhydroxytoluene (BHT), etc.), colorant (such as tar pigment), coloring agent (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (such as MSG glutamate), sweetener (ducin, cyclate , Saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen titanate, etc.), reinforcing agents, emulsifiers, thickeners (pigments), coatings, gum herbicides, foam inhibitors, solvents, modifiers, etc. May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

An example of the health functional food composition of the present invention can be used as a health beverage composition, in which case it may contain various flavors or natural carbohydrates as additional ingredients, such as conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin, cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumartin, stevia extract; Synthetic sweeteners such as saccharin and aspartame; The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohol or carbonation agent and the like. Others may contain fruit flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The present invention also provides a feed composition for the prevention or improvement of inflammatory eye disease, including the maple leaf extract or a fraction thereof.

At this time, the definition of the maple leaf, extract, fraction, inflammatory eye disease, prevention and improvement are as described above.

The content of the maple leaf extract in the feed composition for the prevention or improvement of inflammatory eye disease may be appropriately selected according to the species, age, weight, and breeding conditions of the feed livestock, and 0.01 to 95% by weight based on the total weight of the feed composition. May be a ratio of 0.1 to 80% by weight.

The feed composition may include a feed additive. The feed additive of the present invention corresponds to a feed supplement in the Feed Control Act.

As used herein, the term "feed" may refer to any natural or artificial diet, one meal, or the like or a component of the one meal for the animal to eat, ingest, and digest.

The kind of the feed is not particularly limited, and may be used a feed commonly used in the art. Non-limiting examples of the feed may include plant feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

In addition, the feed additive may additionally contain a carrier that is acceptable to the unit animal. In the present invention, the feed additive may be added as it is, or a known carrier, stabilizer, or the like. If necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants, and other additives may be added. Powders, granules, pellets, suspensions and the like may be in a suitable state. In the case of supplying the feed additive of the present invention, the unit animal may be supplied alone or mixed with feed.

Hereinafter, the configuration and effects of the present invention through the production examples and examples will be described in more detail. These preparation examples and examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Preparation Example 1 Maple Leaf Extract Preparation

Preparation Example 1-1: Preparation of Maple Leaf Hot Water Extract (KIOM-2015EW)

1700 g of the maple leaf crushed into 17 L of water and immersed for 1 hour, followed by hydrothermal extraction for 3 hours using a Gyeongseo ultrafast vacuum extractor (Gyeongseo Extractor, COSMOS 660, Incheon, Korea). Thereafter, the extract was lyophilized to obtain 98.77 g of maple leaf 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 maple leaf pulverized product was put in 17 L of 25% ethanol and extracted by immersion 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 Extracts

Preparation Example 2-1: Preparation of Fraction of Maple Leaf Hydrothermal Extract (KIOM-2015EW)

From the maple leaf hydrothermal extract (KIOM-2015EW) prepared in Preparation Example 1-1, a solvent fraction using an organic solvent was performed to separate components in the extract according to polarity. That is, 4 g of KIOM-2015EW extract was suspended in 80 mL of water, and then 80 mL of hexane (Hexane) was added to obtain a component eluted three times with hexane (hexane fraction; Hexane Fr.). In addition, dichloromethane fraction (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 organic solvent yields are shown in Table 1 below.

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

From the maple leaf ethanol extract (KIOM-2015EE) prepared in Preparation Example 1-2, a solvent fraction using an organic solvent was performed to separate the components in the extract according to polarity. In other words, 4 g of KIOM-2015EW extract was suspended in 80 mL of water, and then 80 mL of hexane was added to obtain hexane eluted three times. Dichloromethane fractions, ethyl acetate fractions, n-butanol fractions and water (H ₂ O) fractions were applied in the same manner in the order of dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (n-BuOH). Acquired (Fig. 1), the yield by organic solvents are shown in Table 1.

Fraction yield by organic solvent

분획물Fraction	유기용매 별 수율(%)Yield per organic solvent (%)
분획물Fraction	KIOM-2015EW 열수 추출물KIOM-2015EW Hot Water Extract	KIOM-2015EE 25% 에탄올 추출물KIOM-2015EE 25% Ethanol extract
헥산(Hexane) Hexane	0.160.16	0.100.10
디클로로메탄(DCM) Dichloromethane (DCM)	1.441.44	1.431.43
에틸아세테이트(EtOAc) Ethyl Acetate (EtOAc)	9.789.78	6.416.41
n-부탄올(n-BuOH) n-butanol (n-BuOH)	17.5117.51	17.0217.02
물(H₂O) Water (H ₂ O)	58.7058.70	74.9874.98

Example 1 Cytotoxicity Evaluation of Fractions of Maple Leaf Extracts

In order to evaluate the toxicity of the fraction of the maple leaf hydrothermal extract (KIOM-2015EW) or the ethanol extract (KIOM-2015EE), the hexane, dichloromethane, ethyl acetate, prepared in Preparation Example 2-1 and Preparation Example 2-2, The n-butanol and water fractions were treated with human corneal epithelial cells (HCEC) and human conjunctival epithelial cells, respectively, to observe the viability of the cells.

First, in order to produce a dry eye invitro model, the cells were grown in a growth medium and stabilized, and then the cells were treated with a fraction of maple leaf hydrothermal extract and maple leaf ethanol extract and cultured for 24 hours. Then, add 100 μl of MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide) at a concentration of 5 mg / ml for 2 hours, remove supernatant, and then remove DMSO ( 100 ul of dimethyl sulfoxide) was added to dissolve the precipitate, and the viability of the cells was measured at an absorbance of 570 nm.

As a result, as shown in Figure 2, in the case of human corneal epithelial cells, n-butanol fraction and water fraction of maple leaf hydrothermal extract did not show cytotoxicity at the concentration of 50 μg / mL and 150 μg / mL, respectively (Fig. 2 (A) and (b)). In addition, the ethyl acetate fraction, n-butanol fraction and water fraction of maple leaf ethanol extract did not show significant cytotoxicity at concentrations of 50 μg / mL, 50 μg / mL and 150 μg / mL, respectively (FIG. 2C). To (e)).

In addition, as shown in FIG. 3, in the case of human conjunctival epithelial cells, as a result of confirming the cytotoxicity of each fraction of the maple leaf hydrothermal extract, the n-butanol fraction did not show cytotoxicity even at a concentration of 500 μg / mL, and the water fraction was There was no significant cytotoxicity at the concentration of 100 μg / mL (FIGS. 3C and 3D). Among the fractions of the maple leaf ethanol extract, the hexane fraction and the ethyl acetate fraction did not show cytotoxicity even at the concentration of 100 μg / mL, respectively (FIG. 3 (e) and (f)).

Through the above results, the fraction of the maple leaf hot water or ethanol extract of the present invention was able to confirm the cell viability of human corneal epithelial cells and human conjunctival epithelial cells in the same environment as dry eye syndrome induced by osmotic stress.

Example 2: Maple Leaf Extract on Human Corneal Epithelial Cells Fraction Inhibitory Effect of Inflammation-related Cytokines

Example 2-1: Confirmation of the Inhibitory Effect of Inflammation-Induced Cytokines of Fractions of Maple Leaf Hot Water Extracts

When dry eye syndrome is triggered, inflammation-related cytokines are overexpressed. Therefore, in order to confirm the anti-inflammatory effects of the maple leaf extract fractions on human corneal epithelial cells in the same environment as dry eye induced by osmotic stress, the representative inflammation-inducing cytokines IL-6, TNF-α and IL-1β Protein expression levels were analyzed.

Specifically, human corneal epithelial cells are grown in a growth medium and stabilized, followed by adding 450 mOsM of DMEM / F-12 medium to osmotic stress (HOS) to the same environment as dry eye caused by osmotic stress. Human corneal epithelial cells were exposed. At this time, the n-butanol (n-BuOH) fraction of KIOM-2015EW of Preparation Example 2-1 in consideration of the cytotoxicity of each fraction to the cells at a concentration of 25, 50 and 100 μg / ml, water (H ₂ O) Fractions were treated at concentrations of 50, 100 and 150 μg / ml and incubated for 24 hours, and the supernatant of the cell culture was recovered. Thereafter, Human IL-6 ELISA kit II (eBioscience catalog No. 88-7066), Human TNF ELISA kit II (eBioscience catalog No. 88-7346), and Human IL-1β ELISA kit II (eBioscience catalog No. 88-7261) The amount of expression of IL-6, TNF-α, and IL-1β included in the cell culture was measured using.

As a result, as shown in Figure 4, when treated with n-butanol fraction of the maple leaf hydrothermal extract, IL-6 expression level is reduced depending on the concentration, the expression amount of TNF-α and IL-1β is 25 μg / ml concentration In addition, it was confirmed that the marked decrease (Fig. 4 (a) to (c)).

In addition, as shown in Figure 5, when treated with the water fraction of the maple leaf hydrothermal extract, IL-6 and TNF-α expression decreases in a concentration-dependent manner, respectively, the amount of IL-1β expression is significantly even at a concentration of 50 μg / ml It was confirmed that the decrease (Fig. 5 (a) to (c)).

Example 2-2: Maple Leaf Ethanol Extract Fraction Confirmation of Inhibitory Effect of Inflammatory Cytokines

IL-6, TNF-α and IL-1β after the KIOM-2015EE fraction prepared in Preparation Example 2-2 was treated in the same manner as in Example 2-1. Expression level was measured.

As a result, as shown in Figure 6, when treated with n-butanol fraction of the maple leaf ethanol extract, it was confirmed that the expression level of IL-6 and TNF-α increased by osmotic stress decreased in a concentration-dependent manner (Fig. (A) and (b) of 6). In addition, it was confirmed that the expression level of IL-1β significantly reduced to the level of control cells not subjected to osmotic stress even at a concentration of 25 μg / ml (Fig. 6 (c)).

In addition, as shown in Figure 7, when treating the water fraction of the maple leaf ethanol extract, it was confirmed that the expression level of IL-6, TNF-α and IL-1β increased by osmotic stress all decreased (Fig. 7 ( a) to (c))

Through the results of the above examples, n-butanol and water fractions fractionated from KIOM-2015EW and KION-2015EE of the present invention are TNF-α, IL-1β and IL in human corneal epithelial cells induced with the same environment as dry eye syndrome. -6 of protein Since inflammation is improved by reducing expression, it has been confirmed that it can be usefully used for the improvement and treatment of dry eye syndrome.

Example 3: Confirmation of Antioxidant Related Protein Expression in Human Corneal Epithelial Cells

To evaluate the effects of KIOM-2015EW on the inhibition of apoptosis in human corneal epithelial cells by osmotic stress, the expression levels of antioxidant-related proteins were investigated.

Specifically, the cells were treated with osmotic stress at the same time concentrations of 50 μg / ml and n-butanol fractions of KIOM-2015EW and KIOM-2015EE at 100 μg / ml, KIOM-2015EW 100 μg / ml and KIOM-2015EE 100 μg / ml was treated. At this time, it was incubated for 24 hours using CsA 5 μg / ml and FML 10 μg / ml used as a treatment for dry eye as a positive control group.

Then, the expression level of the protein related to antioxidant in the cells was confirmed by Western blot analysis. After incubation of the cells three times with 1 × PBS, using lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 μg / ml aprotinin) Dissolved. Then, the supernatant was recovered to quantify the protein present in each sample, and 30 μg of protein was mixed with 4 × sample buffer to mix 12% SDS-polyacrylamide gel (1.5 M Trisma base, 10% SDS, 30% acrylamide, 10%). ammonium persulfate, TEMED). The electrophoresed gel-like proteins were transferred to NC membranes, and each membrane was blocked by reaction for 1 hour at room temperature with 5% skim milk to prevent nonspecific binding with antibodies. Antioxidant primary antibodies were reacted overnight at 4 ° C. (GPX-1; Anti-Glutathione Peroxidase 1 antibody (Abcam, ab26604), SOD-1; Cu / Zn SOD polyclonal antibody (Enzo, ADI-SOD-100) , After washing three times with TBS containing 0.05% Tween, and reacted with the secondary antibody anti-IgG conjugated HRP for 1 hour at room temperature, washed three times with TBS containing 0.05% Tween and then ECL solution The amount of protein expression was detected using.

As shown in Figure 8, when the n-butanol fraction or water fraction of the maple leaf hydrothermal extract and ethanol extract, the antioxidant proteins reduced by HOS GPX-1 and SOD-1 It was confirmed that the expression is increased. In particular, the expression of GPX-1 was confirmed that the expression is increased at a concentration of 50% lower than the maple leaf extract concentration (100 μg / ml).

Through the above results, it was confirmed that the fractions of KIOM-2015EW and KIOM-2015EE inhibit the human corneal oxidative stress induced by HOS by increasing the expression of antioxidant inducing proteins.

Example 4: human On conjunctival epithelial cells For maple leaf extract Fraction Inhibitory Effect of Inflammation-related Cytokines

Example 4-1: Confirmation of Inhibitory Effect of Inflammatory Cytokines on Fractions of Maple Leaf Hydrothermal Extracts

Inflammation-related cytokines are overexpressed when dry eye syndrome is induced, and the representative inflammation-induced cytokines are investigated to confirm the anti-inflammatory effects of the hot water extract fraction of maple leaf on human conjunctival epithelial cells in the same environment as ocular dryness induced by osmotic stress. Protein expression levels of IL-6, TNF-α and IL-1β were analyzed. As a positive control, 5 μg / mL of CsA and 10 μg / mL of FML used as a dry eye treatment were used.

The specific experimental method is the same as that of the human corneal epithelial cell of Example 2-1.

As a result, as shown in Figure 9, when the n-butanol fraction of the maple leaf hydrothermal extract was treated, the expression levels of IL-6, TNF-α and IL-1β were significantly decreased, respectively, in particular, IL- It was confirmed that the expression amount of 6 and IL-1β is significantly reduced to or above the positive control level (Fig. 9 (a) to (c)).

In addition, as shown in Figure 10, even when treated with water fraction of the maple leaf hydrothermal extract, it was confirmed that the expression level of IL-6, TNF-α and IL-1β decreased similarly to the positive control (Fig. 10 ( a) to (c)).

Example 4-2: Maple Leaf Ethanol Extract Fraction Confirmation of Inhibitory Effect of Inflammatory Cytokines

After treating the KIOM-2015EE fraction prepared in Preparation Example 2-2 in the same manner as in Example 2-1, the IL-6, TNF-α and IL-1β Expression level was measured.

As a result, as shown in Figure 11, when treated with n-butanol fraction of maple leaf ethanol extract, it was confirmed that the expression level of IL-6, TNF-α and IL-1β increased by osmotic stress significantly decreased. ((A) to (c) of FIG. 11).

In addition, as shown in Figure 12, even when treated with the water fraction of the maple leaf ethanol extract, it was confirmed that the expression level of IL-6, TNF-α and IL-1β increased by osmotic stress significantly decreased (Fig. (A) to (c) of 12).

Through the results of the above examples, the fractions of KIOM-2015EW and KIOM-2015EE of the present invention are characterized in that the IL-6, TNF-α and IL-1β proteins in human conjunctival epithelial cells Since inflammation is improved by reducing expression, it has been confirmed that it can be usefully used for the improvement and treatment of dry eye syndrome.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims

Pharmaceutical composition for the prevention or treatment of inflammatory eye disease comprising maple leaf extract or a fraction thereof as an active ingredient.
The pharmaceutical composition of claim 1, wherein the inflammatory eye disease is dry eye, conjunctivitis, hyperemia or keratitis.
The pharmaceutical composition of claim 1, wherein the fraction of the maple leaf extract is a fraction of the maple leaf hydrothermal extract.
The pharmaceutical composition of claim 1, wherein the fraction of the maple leaf extract is a fraction of the maple leaf ethanol extract.
The pharmaceutical composition of claim 2, wherein the conjunctivitis and keratitis are infectious or non-infectious.
The pharmaceutical composition of claim 1, wherein the composition is in an ocular topical formulation.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
A method of preventing or treating inflammatory eye disease, comprising administering the composition of any one of claims 1 to 7 to an eye of an individual except a human.
The method of claim 8, wherein the inflammatory eye disease is dry eye, conjunctivitis, hyperemia or keratitis.
The method of claim 9, wherein the conjunctivitis and keratitis are infectious or non-infectious.
A quasi-drug composition for the prevention or treatment of inflammatory eye disease, including maple leaf extract or a fraction thereof.
Eye cosmetic additive composition for the prevention or improvement of inflammatory eye disease comprising a maple leaf extract or a fraction thereof as an active ingredient.
Health functional food composition for the prevention or improvement of inflammatory eye disease comprising maple leaf extract or a fraction thereof as an active ingredient.
Feed composition for the prevention or improvement of inflammatory eye disease comprising a maple leaf extract or a fraction thereof as an active ingredient.