KR20240154479A - Composition for preventing, ameliorating or treating eye diseases - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating eye diseases, comprising extracts of Usul, licorice or a mixture thereof.
According to the present invention, a composition comprising extracts of Usul, licorice or a mixture thereof as an effective ingredient has an excellent antioxidant effect and an excellent effect of restoring oxygen consumption and ATP production in mitochondria, and further has an effect of protecting mitochondria and inhibiting retinal cell death caused by blue light stress, and thus can be applied as a pharmaceutical, health functional food or quasi-drug composition for preventing, improving or treating various eye diseases.

Description

Composition for preventing, ameliorating or treating eye diseases

The present invention relates to a composition for preventing or treating eye diseases, and more particularly, to a composition for preventing, improving or treating eye diseases comprising extracts of Usul, licorice or a mixture thereof.

Age-related macular degeneration (AMD), one of the world's three major eye diseases, is divided into dry and wet. The total number of AMD patients reached 196 million in 2020. 20-30% of dry patients develop wet AMD, which causes rapid vision loss and blindness. Among them, dry AMD is rapidly increasing in all age groups due to environmental changes such as the aging population and frequent smartphone use. It is a degenerative disease in which drusen, a waste product, accumulates in the macula due to deterioration of retinal cell function, causing photoreceptors to die and progress from vision loss to blindness. It is reported to account for 90% of macular degeneration. It is reported that complex and diverse mechanisms such as oxidative stress, inflammation, decreased mitochondrial function, and abnormal neovascularization are involved in the development of dry AMD.

In particular, mitochondria play an important role in energy production, reactive oxygen species, apoptosis, and retrograde signaling information, and mitochondrial retrograde signaling regulates complement, inflammation, and innate immune pathways involved in the progression of macular degeneration. Therefore, the severity of AMD depends on the degree of mitochondrial DNA damage and fragmentation in retinal pigment epithelial cells. In addition, the onset or progression of AMD can be delayed or controlled by utilizing substrates or regulatory factors that prevent apoptosis by regulating mitochondrial permeability and suppressing oxidative stress (ROS) damage that affects mitochondrial function.

It has been reported that protecting retinal mitochondria, which are responsible for various functions, may prevent, delay, or treat the onset of macular degeneration (J Ophthalmic Vis Res 2017; 12　4): 424-8).

Until now, there has been no treatment to restore vision in most patients with dry AMD, but in February 2023, the US FDA approved Syfovre (Apellis Pharmaceuticals, USA) as the first new drug for patients with geographic atrophy (GA) due to late-stage dry macular degeneration. The new drug inhibits excessive activity of complement factor 3 involved in the immune cascade reaction, is injected at intervals of 25 to 60 days, and is scheduled to be launched from March 2023 at $2,190 (about 2.8 million won) per vial (PHARM EDAILY; 2023. 02.21). However, the new drug is expected to have significant limitations in being applied to most patients due to its very high treatment cost.

In addition, ‘Zimura (Iveric Bio; USA)’ is a drug currently in phase 3 clinical trials, and its mechanism is to prevent the decomposition of complement factor C5 into C5a and C5b, thereby causing chronic inflammation or cell death. In addition, Allegro and Hanmi Pharmaceuticals in the USA are preparing phase 3 trials for ‘Luminate’, a new drug candidate for dry AMD patients, and unlike Sipobre, which targets late-stage dry AMD, Luminate is reported as an early treatment strategy.

However, most of the currently approved or under development treatments are oral injections into the vitreous body, and there is a problem that there are no products that can be administered in combination with the currently used treatments.

Meanwhile, licorice ( Glycyrrhiza uralensis; Glycyrrhizae glabra; Glycyrrhizae Radix et Rhizoma ) is a perennial herb belonging to the legume family, and its dried root is used as a medicinal herb. In general, licorice is used to treat inflammation, gastritis, stomatitis, pharyngitis, hepatitis, eczema, peptic ulcers, and alcohol detoxification.

Usul ( Achyranthes japonica; Achyranthes bidentata; Achyranthes Radix ) belongs to the Amaranthaceae family and protects the liver and kidneys and relieves blood stasis caused by poor circulation. It is also used to strengthen muscles and bones, improve arthritis, and treat irregular menstruation, bruises, and back pain. However, it is reported that pregnant women should be careful when taking it because it has a uterine contraction effect.

Against this backdrop, the inventors of the present invention have completed the present invention by confirming that extracts of Usul, licorice or a mixture thereof have excellent antioxidant effects and excellent effects of restoring oxygen consumption and ATP production in mitochondria, and further have effects of protecting mitochondria and inhibiting retinal cell death caused by blue light oxidative stress.

One object of the present invention is to provide a food composition for preventing or improving eye diseases, which contains extracts of Usul, licorice or a mixture thereof as an effective ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating eye diseases, comprising extracts of Usul, licorice or a mixture thereof as an effective ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or improving eye diseases, which comprises extracts of Usul, licorice or a mixture thereof as an effective ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving eye diseases, which contains extracts of Usul, licorice or a mixture thereof as an effective ingredient.

Another object of the present invention is to provide a method for preventing or treating eye diseases, comprising a step of administering the pharmaceutical extract to a subject.

Another object of the present invention is to provide a composition containing extracts of Usul, licorice or a mixture thereof as an effective ingredient for preventing or treating eye diseases.

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention cannot be considered limited by the specific description described below.

Furthermore, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention is based on the novel demonstration that extracts of Usul, licorice or a mixture thereof have excellent antioxidant effects and excellent effects of restoring oxygen consumption and ATP production in mitochondria, and further have the effect of protecting mitochondria and inhibiting retinal cell death caused by blue light oxidative stress.

In particular, the present invention is based on the newly confirmed that when the solvent for extracting Usul, licorice or a mixed extract thereof is 30% to 50% ethanol, the antioxidant effect in retinal epithelial cells is significantly excellent at a specific mixing ratio, the effect of restoring oxygen consumption and ATP production in mitochondria is excellent, and further, the effect of protecting mitochondria and inhibiting retinal cell death caused by blue light oxidative stress is observed.

One aspect of the present invention to achieve the above object provides a food composition for preventing or improving eye disease, comprising extracts of Usul, licorice or a mixture thereof as an effective ingredient.

In this specification, the term '우슬(膝)' refers to Achyranthes japonica; Achyranthes bidentata; Achyranthis Radix , which belongs to the Amaranthaceae family and has the scientific name of Achyranthes japonica; Achyranthes bidentata; and Achyranthis Radix. It is reported to protect the liver and kidneys, relieve blood stasis caused by blood circulation disorders, strengthen muscles and bones, improve arthritis, and treat irregular menstruation, bruises, and lumbago. It is also reported to have a uterine contraction effect, so pregnant women should be careful when taking it.

The herbal medicine material used in the extract in this specification may be at least one selected from the group consisting of roots, rhizomes, leaves, flowers, seeds, and whole plants.

In this specification, the term 'licorice' refers to a perennial herb belonging to the legume family with scientific names Glycyrrhizae uralensis; Glycyrrhizae glabra; Glycyrrhizae Radix et Rhizoma , and the dried root is used as a medicinal herb. It is generally reported to be used in the treatment of inflammation, gastritis, stomatitis, pharyngitis, hepatitis, eczema, peptic ulcer, alcohol detoxification, etc.

In this specification, the licorice used as a medicinal herb for the extract may be at least one selected from the group consisting of aerial parts such as flowers, leaves, stems, roots, seeds, and whole plants.

The above-mentioned licorice and/or licorice may be purchased commercially or may be used without limitation as those collected or grown in nature.

In this specification, the term 'extract' means a resultant such as a liquid component obtained by immersing a target substance in various solvents and then extracting it at room temperature or in a heated state for a certain period of time, or a solid component obtained by removing the solvent from the liquid component. In addition, it can be comprehensively interpreted to include, in addition to the resultant, a dilution of the resultant, a concentrate thereof, a conditioning agent, a purified product thereof, etc. Accordingly, the Usul, Glycyrrhiza uralensis or mixed extracts thereof provided in the present invention can be interpreted to include an extract itself and all formulations that can be formed using the extract, such as an extracted extract, a mixed solution thereof, a mixed solution extracted by mixing, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a conditioning agent or purified product of the extract, or a mixture thereof.

In addition, the above-mentioned extracts of Usul, licorice or a mixture thereof can be extracted from various organs of natural, hybrid or mutant plants, and specifically, they can be extracted from roots, aerial parts, stems, leaves, flowers, body of fruits, peel of fruits, as well as plant tissue cultures.

In addition, the above extract is a concept that includes all active ingredients separated from natural products (herbal medicines) with a suitable solvent, regardless of the method of separation or type of ingredients. That is, it is a broad concept that includes all things such as extracting a solvent-soluble ingredient from a natural product using water or an organic solvent, extracting only a specific ingredient of a natural product, such as oil, etc., and can be used to encompass all things such as extracts and/or purified products obtained at each stage of extraction and/or purification, dilutions and/or concentrates of the extracts and/or purified products, and dried products of the extracts, purified products, dilutions, and/or concentrates.

The mixed extract of Usul and licorice, which is an effective ingredient of the composition provided herein, refers to a mixture of Usul extract and licorice extract (a mixture of herbal extracts), or may be a mixed extract obtained by mixing Usul and licorice and then extracting them.

The above-mentioned Usul extract, licorice extract, and/or extracts thereof may be a crude extract, a solvent (polar solvent or nonpolar solvent) soluble extract, a fraction, or a combination thereof obtained by extracting, separating, and/or fractionating from nature using extraction, separation, and/or fractionation methods known in the art. In one specific embodiment, the Usul extract, licorice extract, and/or extract of the mixture may be, but is not limited to, a crude extract or a solvent soluble extract (e.g., a polar solvent soluble extract).

The above-mentioned Usul, Glycyrrhiza glabra or mixed extracts thereof may be extracted using Usul, Glycyrrhiza glabra and/or a mixture thereof using at least one selected from water, an organic solvent and a combination thereof as an extraction solvent. The organic solvent may be at least one selected from a linear or branched alcohol having 1 to 4 carbon atoms (specifically, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, etc.), specifically, it may be water-soluble ethanol, and specifically, it may be an ethanol aqueous solution of 25 to 55%, more specifically, 30 to 50% (v/v).

In one specific embodiment, the extraction solvent may be water, ethanol, methanol, or a combination thereof (specifically, an aqueous ethanol solution and/or an aqueous methanol solution, etc.), and specifically, 10 to 80% (v/v), 15 to 80% (v/v), 20 to 80% (v/v), 25 to 80% (v/v), 28 to 80% (v/v), 30 to 80% (v/v), 10 to 70% (v/v), 15 to 70% (v/v), 20 to 70% (v/v), 25 to 70% (v/v), 28 to 70% (v/v), 30 to 70% (v/v), 10 to 60% (v/v), 15 to 60% (v/v), 20 to 60% (v/v), 25 It may be ethanol or methanol of 10 to 60% (v/v), 28 to 60% (v/v), 30 to 60% (v/v), 10 to 50% (v/v), 15 to 50% (v/v), 20 to 50% (v/v), 25 to 50% (v/v), 28 to 50% (v/v), or 30 to 50% (v/v), and specifically, it may be an ethanol aqueous solution of 30% to 50% (v/v).

In one example, the extract of Usul, licorice or a mixture thereof,

(1) (i) Usul (e.g., root) 10 to 80% (v/v), 15 to 80% (v/v), 20 to 80% (v/v), 25 to 80% (v/v), 28 to 80% (v/v), 30 to 80% (v/v), 10 to 70% (v/v), 15 to 70% (v/v), 20 to 70% (v/v), 25 to 70% (v/v), 28 to 70% (v/v), 30 to 70% (v/v), 10 to 60% (v/v), 15 to 60% (v/v), 20 to 60% (v/v), 25 to 60% (v/v), 28 to 60% (v/v), 30 to An extract of Usul extracted with ethanol or methanol at 60% (v/v), 10 to 50% (v/v), 15 to 50% (v/v), 20 to 50% (v/v), 25 to 50% (v/v), 28 to 50% (v/v), or 30 to 50% (v/v), and (ii) licorice (e.g., aerial part) at 10 to 80% (v/v), 15 to 80% (v/v), 20 to 80% (v/v), 25 to 80% (v/v), 28 to 80% (v/v), 30 to 80% (v/v), 10 to 70% (v/v), 15 to 70% (v/v), 20 to 70% (v/v), 25 to 70% (v/v), A mixture of licorice extracts extracted with ethanol or methanol at 28 to 70% (v/v), 30 to 70% (v/v), 10 to 60% (v/v), 15 to 60% (v/v), 20 to 60% (v/v), 25 to 60% (v/v), 28 to 60% (v/v), 30 to 60% (v/v), 10 to 50% (v/v), 15 to 50% (v/v), 20 to 50% (v/v), 25 to 50% (v/v), 28 to 50% (v/v), or 30 to 50% (v/v);

(2) A mixture of Usul (e.g., root) and licorice (e.g., aerial part) at 10 to 80% (v/v), 15 to 80% (v/v), 20 to 80% (v/v), 25 to 80% (v/v), 28 to 80% (v/v), 30 to 80% (v/v), 10 to 70% (v/v), 15 to 70% (v/v), 20 to 70% (v/v), 25 to 70% (v/v), 28 to 70% (v/v), 30 to 70% (v/v), 10 to 60% (v/v), 15 to 60% (v/v), 20 to 60% (v/v), 25 to 60% (v/v), 28 to An extract of a mixture extracted with ethanol or methanol at 60% (v/v), 30 to 60% (v/v), 10 to 50% (v/v), 15 to 50% (v/v), 20 to 50% (v/v), 25 to 50% (v/v), 28 to 50% (v/v), or 30 to 50% (v/v);

Or (3) it may be a combination thereof, and more specifically, it may be an extract of Usul, a licorice extract, a mixture thereof, or an extract extracted after mixing them, extracted with an ethanol aqueous solution of 30 to 50% (v/v).

The above extraction can be performed at an extraction temperature of, but is not limited to, 30 to 100°C, 40 to 100°C, 50 to 100°C, 60 to 100°C, 30 to 95°C, 40 to 95°C, 50 to 95°C, 30 to 90°C, 40 to 90°C, 50 to 90°C, 30 to 85°C, 40 to 85°C, 50 to 85°C, 40 to 70°C, or 50 to 70°C.

The above extraction may be performed under the above extraction solvent and/or extraction temperature conditions for 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or 3 hours or more, for example, 10 minutes to 12 hours, 10 minutes to 6 hours, 10 minutes to 5 hours, 10 minutes to 4 hours, 10 minutes to 3 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 5 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 1 to 12 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 2 to 12 hours, 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 12 hours, 3 to 6 hours, 3 to 5 hours, or 3 to 4 hours. However, it is not limited to these.

The above extraction can be performed by any extraction method commonly used, and specifically, it can be performed by at least one method selected from the group consisting of hot water extraction, ultrasonic extraction, reflux extraction, cold infusion extraction, reflux cooling extraction, solvent extraction, steam distillation, elution, pressing, etc., and specifically, it can be reflux extraction, but is not limited thereto.

After obtaining the extract using the extraction solvent as described above, a liquid can be obtained by cooling, heating and/or filtering at room temperature using conventional methods known in the art, and optionally, the solvent can be additionally evaporated, spray-dried or freeze-dried. In addition, the extract extracted as described above can be manufactured into a powder state by an additional process such as vacuum concentration, vacuum distillation, freeze-drying or spray drying, etc.

The above-mentioned Usul extract, licorice extract, and/or mixed extract can also be obtained as further purified fractions using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc.

The mixing ratio of the Usul extract and the licorice extract in the above mixed extract or the mixing ratio of Usul and licorice in the mixture of Usul and licorice may be, by weight, 9:1 to 1:9, 3:1 to 1:9, 2:1 to 1:9, 1:1 to 1:9, 1:3 to 1:9, 1:6 to 1:9 (or more, Usul weight or Usul extract solid weight: licorice weight or licorice extract solid weight), and the licorice extract or the content of the licorice is 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more (specifically, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:7, 1:1 to 1:6, 1:1 to It can be 1:5 1:1 to 1:4, 1:1 to 1:3, 1:1 to 1:2 (weight of Usul or solid weight of Usul extract: weight of licorice or solid weight of licorice extract)), and more specifically, Usul:licorice can be a mixing ratio of 1:3 to 1:9, more specifically, a mixing ratio (weight ratio) of 1:6 to 1:9.

The above solid weight refers to the weight of the solid remaining after removing the solvent component of the extract. This is a term used to indicate that, when the above mixture is a mixture of a Usul extract and a licorice extract, the mixing ratio refers to the ratio between the weights of the effective ingredients from which the extraction solvent has been removed so that it is not affected by the properties and/or concentration of the extract.

The extracts of Usul, licorice or a mixture thereof included as effective ingredients in the above food composition may be in the form of a dried product, a concentrate or a concentrated dried product.

In addition, the extract of the present invention may optionally include an extract of a known natural product or a known ingredient that enhances the effect of preventing or improving eye diseases.

In this specification, the term 'eye disease' means a disease occurring in the eye, and specifically, may be, but is not limited to, macular degeneration, glaucoma, diabetic retinopathy, retinal edema, epiretinal membrane, retinal tear, cataract, dry eye, superficial keratitis, or retinal and corneal abnormalities of unknown cause.

The term "macular degeneration" used in the present invention refers to age-related macular degeneration (AMD), which is one of the irreversible diseases that occurs in many elderly people and is a representative ophthalmic disease that leads to blindness. The incidence rate is gradually increasing worldwide due to the aging population. In the United States, macular degeneration is the main cause of blindness, and environmental and genetic factors also affect the onset, and in particular, smoking has been reported to be the most fatal cause. In addition, obesity, excessive intake of antioxidants and dietary fat also induce macular degeneration and affect its further progression. Therefore, a healthy diet, weight control, appropriate exercise, and smoking cessation reduce the onset of macular degeneration.

The term 'dry eye syndrome' used in the present invention may be a disease caused by inhibition of tear secretion due to inflammation of the lacrimal gland and denervation of the cornea, abnormal tear evaporation due to Meibomian gland dysfunction or eyelid dysfunction, and may be a keratoconjunctival epithelial disorder in which tears dry excessively and appear as corneal wounds due to the dry eye syndrome.

The term 'glaucoma' used in the present invention refers to a disease that causes abnormalities in the function of the optic nerve due to a progressive optic neuropathy and induces defects in the corresponding visual field. Since the optic nerve is a nerve that transmits light received by the eye to the brain and 'allows us to see', if there is a problem here, visual field defects appear and, in the final stage, vision is lost. Open-angle glaucoma refers to glaucoma that occurs while the anterior chamber angle does not close and maintains its normal shape, and closed-angle glaucoma refers to glaucoma that occurs when the iris moves toward the cornea due to suddenly increased posterior pressure, closing the anterior chamber angle. The angle formed by the back surface of the cornea and the front surface of the iris is called the anterior chamber angle, and when this is pressed, the passage through which the aqueous humor drains is blocked, causing the intraocular pressure to rapidly increase.

The term 'retinopathy' used in the present invention is called retinopathy and refers to persistent or severe damage to the retina of the eye.

The term 'retinal edema' used in the present invention is also called macular edema and refers to edema that occurs in the macula, the central part of the retina. We can see objects because images are formed on the retina and transmitted to the brain through the optic nerve. The macula is located in the center of the retina and is responsible for central vision. Therefore, if macular edema is accompanied and there is a problem with the structure of the macula, objects appear distorted or vision deteriorates, resulting in visual abnormalities.

In this specification, the term food composition means a health functional food, and unless otherwise specified, the terms food composition and health functional food may be interchangeably used with the same meaning.

'Health functional food' refers to any food manufactured (processed) using nutrients that are easily deficient in daily meals or raw materials or ingredients (hereinafter, 'functional raw materials') that have functions useful to the human body, and helps maintain health or prevent and/or improve certain diseases or symptoms. There are no special restrictions on the final product form. For example, the health functional food may be selected from the group consisting of various foods, beverages, food additives, etc., but is not limited thereto.

The above food composition may include acceptable food additives and may further include suitable carriers, excipients and diluents commonly used in the manufacture of health functional foods.

When the above food composition is used as a food additive, the composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

The content of the effective ingredient (usul, licorice or mixed extracts thereof) contained in the food composition can be appropriately determined without special limitation according to the form of the food, the desired effect, the purpose, etc., and specifically, 0.001 to 99 wt%, 0.001 to 90 wt%, 0.01 to 99 wt%, 0.01 to 95 wt%, 0.01 to 90 wt%, 0.01 to 80 wt%, 0.01 to 50 wt%, 0.1 to 99 wt%, 0.1 to 95 wt%, 0.1 to 90 wt%, 0.1 to 80 wt%, 0.1 to 70 wt%, 0.1 to 60 wt%, 0.1 to 50 wt%, 0.1 to 30 wt%, 0.1 to 10 wt%, 1 to 99 wt% of the total food weight. % by weight, 1 to 95 wt%, 1 to 90 wt%, 1 to 80 wt%, 1 to 70 wt%, 1 to 60 wt%, 1 to 50 wt%, 1 to 30 wt%, 1 to 10 wt%, 10 to 99 wt%, 10 to 95 wt%, 10 to 90 wt%, 10 to 80 wt%, 10 to 70 wt%, 10 to 60 wt%, 10 to 50 wt%, 10 to 30 wt%, 25 to 99 wt%, 25 to 95 wt%, 25 to 90 wt%, 25 to 80 wt%, 25 to 70 wt%, 25 to 60 wt%, 25 to 50 wt%, 25 to 30 wt%, 40 to 99 wt%, It can be, but is not limited to, 40 to 95 wt%, 40 to 90 wt%, 40 to 80 wt%, 40 to 70 wt%, 40 to 60 wt%, 40 to 50 wt%, 50 to 99 wt%, 50 to 95 wt%, 50 to 90 wt%, 50 to 80 wt%, 60 to 99 wt%, 60 to 95 wt%, 60 to 90 wt%, or 60 to 80 wt%.

There is no particular limitation on the type of the food composition above. Examples of foods to which the food composition above can be added include dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and can be manufactured into powder, granules, tablets, capsules, syrups, or beverages, and can be manufactured without limitation in a known form, and can include all foods designed to sufficiently express the body's regulatory function related to regulation of the biological defense rhythm, disease prevention, and recovery, etc. of food groups or food compositions that have added value so that the food function in the conventional sense can be performed and expressed for a specific purpose.

The above food composition may additionally contain at least one additive selected from the group consisting of various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents or natural flavoring agents, coloring agents, pectic acid or its salts, alginic acid or its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks. The proportion of such additives is generally selected in a range of about 0.0001 to about 20 parts by weight per 100 parts by weight of the total health functional food, but is not limited thereto.

In this specification, the term 'prevention' means preventing in advance the deterioration of ocular ability by applying the extract of Usul, licorice or a mixture thereof of the present invention to a food composition, and 'improvement' means that the composition of the present invention promotes, enhances or strengthens ocular ability.

Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating eye diseases, comprising an extract of Usul, licorice or a mixture thereof as an active ingredient.

In addition, the present invention provides a method for preventing or treating an eye disease, comprising a step of administering to a subject a pharmaceutical composition comprising extracts of Usul, licorice or a mixture thereof.

In this specification, the terms 'usul', 'licorice', 'mixed extract', 'eye disease' and 'prevention' are as described above.

In this specification, 'treatment' is used to mean alleviation or improvement of symptoms, reduction of the extent of a disease, delay or palliation of disease progression, improvement, alleviation or stabilization of a disease state or symptoms, partial or complete recovery, prolongation of survival, and other beneficial treatment results. 'Prevention' is used to mean all mechanisms and/or effects that act on a subject who does not have a specific symptom or disease to prevent the occurrence of said specific symptom or disease, or to delay the onset thereof.

As used herein, 'administering' may mean providing a pharmaceutically effective amount of a pharmaceutical composition provided herein to a subject by any suitable method.

The pharmaceutical composition above can be administered to mammals including humans, dogs, cats, horses, cows, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures thereof isolated therefrom, via various routes. The administration method can be any method commonly used, and for example, can be oral administration, or parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, rectal administration, or local administration to a lesion site. The pharmaceutical composition can be formulated and used as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, or aerosols, or parenteral formulations in the form of transdermal agents, suppositories, or sterile injectable solutions, according to a common method.

The above pharmaceutical composition may further contain, in addition to the mixed extract of Usul and Glycyrrhiza uralensis as an effective ingredient, an auxiliary agent such as a pharmaceutically and/or physiologically acceptable carrier, excipient, and/or diluent. Examples of the carrier, excipient, or diluent include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, one or more diluents or excipients selected from the group consisting of commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants can be used. Solid preparations for oral administration include one or more selected from the group consisting of tablets, pills, powders, granules, capsules, syrups, powders, and suspensions, and these solid preparations can be prepared by mixing the extract with at least one excipient, for example, one or more selected from the group consisting of starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include at least one selected from the group consisting of suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, the preparations may include at least one selected from the group consisting of various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include at least one selected from the group consisting of sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and transdermal agents. Non-aqueous solvents and suspending agents may include at least one selected from the group consisting of propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The above pharmaceutical composition can be administered in a pharmaceutically effective amount. The term 'pharmaceutically effective amount' means an administration dosage of an effective ingredient that can exert a beneficial effect (e.g., prevention, alleviation, improvement, relief, inhibition, elimination, and/or treatment) on a symptom and/or disease, and can be prescribed in various ways depending on various factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and reaction sensitivity, and can be administered once a day or several times in divided doses at regular intervals depending on the judgment of a doctor or pharmacist.

The above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art. In addition, the pharmaceutically effective amount may vary depending on the subject being administered.

In one specific example, the dosage (pharmaceutically effective amount) of the pharmaceutical composition is based on the weight of the mixed extract of the effective ingredients, Usul and licorice.

1 mg/kg/day or more, 2 mg/kg/day or more, 3 mg/kg/day or more, 4 mg/kg/day or more, 5 mg/kg/day or more, 6 mg/kg/day or more, 7 mg/kg/day or more, 8 mg/kg/day or more, 9 mg/kg/day or more, or 10 mg/kg/day or more; and/or 15 mg/kg/day or less, 30 mg/kg/day or less, 50 mg/kg/day or less, 90 mg/kg/day or less, 120 mg/kg/day or less, or 150 mg/kg/day or less.

In one embodiment, the pharmaceutical composition may be administered at a dosage of, but is not limited to, 50-150 mg/kg/day, 60-140 mg/kg/day, 70-130 mg/kg/day, 80-120 mg/kg/day, 90-110 mg/kg/day, or about 100 mg/kg/day, based on the weight of the mixed extract of Usul and Glycyrrhiza uralensis.

The composition containing the above-mentioned Usul, licorice or mixed extracts thereof as an effective ingredient can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral, rectal or intravenous, intramuscular or subcutaneous administration.

In this specification, the term 'subject' means all animals, including rats, mice, and livestock, including humans, that have developed or can develop an eye disease, and specific examples thereof include, but are not limited to, mammals, including humans.

In addition, the pharmaceutical composition may be used alone or in combination with methods using dietary therapy, physical therapy, and/or biological response modifiers for the prevention, improvement, and/or treatment of eye diseases.

Another embodiment of the present invention provides a pharmaceutical composition for preventing or improving eye diseases, comprising extracts of Usul, licorice or a mixture thereof as an effective ingredient.

In this specification, the terms 'usul', 'licorice', 'mixed extract', 'eye disease', 'prevention' and 'improvement' are as described above.

In the present invention, the term 'quasi-drug composition' includes products for diagnosing, treating, or improving diseases of humans or animals, oil and rubber products, products that have a mild or no direct effect on the human body, and things similar thereto that are not instruments or machines, sterilizing or insecticidal agents for preventing infectious diseases, etc. The type or formulation of the quasi-drug composition of the present invention is not particularly limited, but specifically, it may be an eye cleansing agent or eye drops.

Another embodiment of the present invention provides a feed composition for preventing or improving eye diseases, comprising extracts of Usul, licorice or a mixture thereof as an effective ingredient.

In this specification, the terms 'usul', 'licorice', 'mixed extract', 'eye disease', 'prevention' and 'improvement' are as described above.

The term "feed" of the present invention means any natural or artificial diet, meal, etc. or a component of said meal for eating, ingesting, and digesting by an animal or suitable therefor.

The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant feed such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; and animal feed such as proteins, inorganic substances, fats, mineral substances, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

The amount of the extract of Usul and licorice or fractions thereof included in the feed composition of the present invention may vary depending on the intended use and conditions of use of the feed, and for example, may be included in an amount of 0.01 to 100 wt%, more specifically, 1 to 80 wt%, based on the total weight of the livestock feed composition, but is not limited thereto.

In one aspect, the composition of the present invention comprising Usul, licorice or a mixed extract thereof as an effective ingredient has an excellent antioxidant effect in retinal epithelial cells, an excellent effect of restoring oxygen consumption and ATP production in mitochondria, and further has an effect of protecting mitochondria and significantly inhibiting retinal cell death caused by blue light oxidative stress, and thus can be utilized as a pharmaceutical, health functional food, and quasi-drug composition for preventing, improving or treating eye diseases. In addition, since the number of herbal medicines essentially included is small and drug interactions are low, there are advantages of excellent commerciality and low concern about side effects.

Figure 1 is a graph showing the cell viability of each extract in ARPE-19 cells, a retinal epithelial cell line, of Usul, licorice or a mixed extract thereof according to one embodiment.
Figure 2 is a graph showing the cytoprotective efficacy of an extract (200 μg/ml) in ARPE-19 cells treated with tBHP (300 μM) of Usul, licorice or a mixed extract thereof according to one embodiment.
Figure 3 is a graph showing the cytoprotective efficacy of extracts (1 to 200 μg/㎖) in ARPE-19 cells treated with tBHP (300 μM) of Usul, licorice or a mixed extract thereof according to one embodiment.
FIG. 4 is a graph showing the efficacy of JK-502, 504, 505, 508, 509, 514 and 520 on the mitochondrial respiratory system in ARPE-19 treated with tBHP of Usul, Glycyrrhiza or a mixed extract thereof according to one embodiment (Oligo (oligomycin, ATP synthesis inhibitor), FCCP (fluoro-carbonyl cyanide phenylhydrazone (FCCP), hydrogen ions transporter), ROT/AA (rotenone/antimycin A; complex I and III inhibitor).
FIG. 5 is a graph showing the mitochondrial protective effect of JK-504, 505, 508, 509, 514 and 520 in ARPE-19 treated with tBHP of Usul, licorice or a mixed extract thereof according to one embodiment.
Figures 6 to 9 are graphs showing the inhibitory effect of extracts of Usul, licorice or a mixture thereof on retinal cell death caused by blue light oxidative stress according to one embodiment.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to describe the present invention more specifically, but the following examples, comparative examples, experimental examples, and manufacturing examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples, comparative examples, experimental examples, and manufacturing examples. The examples, comparative examples, experimental examples, and manufacturing examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

Experimental Example 1. Preparation of extracts of Usul, licorice or their mixture

Usul and licorice were mixed in the ratios shown in Table 1 and refluxed for 2 hours at 50℃ with 30% aqueous ethanol and 50% aqueous ethanol. At this time, the amount of aqueous ethanol is about 10 times the amount of the herb. Approximately 300 g of the herb was extracted, and approximately 300㎖ of aqueous ethanol was used. After refluxing twice, the first and second extracts were combined, concentrated under reduced pressure at 45-50℃, and then dried in a freeze dryer (Table 1).

number Experimental conditions JK-502 30% 1:6 (Usul:Licorice = 1:6) JK-503 30% 6:1 (Usul:Licorice = 6:1) JK-504 30% 1:9 (Usul:Licorice = 1:9) JK-505 50% 1:6 (Usul:Licorice = 1:6) JK-506 50% 6:1 (Usul:Licorice = 6:1) JK-507 50% 1:1 (Usul:Licorice = 1:1) JK-508 50% 1:3 (Usul:Licorice = 1:3) JK-509 50% 1:9 (Usul:Licorice = 1:9) JK-510 50% 3:1 (Usul:Licorice = 3:1) JK-511 30% 1:1 (Usul:Licorice = 1:1) JK-512 30% 1:3 (Usul:Licorice = 1:3) JK-514 50% licorice JK-515 50% 9:1 (Usul:Licorice = 9:1) JK-516 30% 3:1 (Usul:Licorice = 3:1) JK-517 30% 9:1 (Usul:Licorice = 9:1) JK-519 30% off JK-520 50% off

Experimental Example 2. Cell Culture

Human retinal pigment epithelial cell line (ARPE-19 cells) were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in a humidified atmosphere of 37°C in DMEM/F12 (Gibco, Carlsdad, CA, USA), a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12, supplemented with 10% fetal _bovine serum (Gibco, Burlington, ON, Canada), and 1% penicillin-streptomycin (Gibco, Life Technologies Limited, Paisley, UK). The cells were subcultured when they reached a confluent density of 70% or more and used in the experiment.

Experimental Example 3. Confirmation of cell viability in human retinal epithelial cell line (ARPE-19)

To evaluate the cytotoxicity of the candidate substance, a cell counting kit-8 (CCK-8; Dojindo, Japan) assay was performed. First, to confirm the cytotoxicity of the candidate substance itself, ARPE-19 cells were seeded on a 96-well plate ( ^1Х104 cells/well). When 70% confluence was reached, the candidate substance was treated at various concentrations and cultured for 24 hours. 20 ㎕ of CCK-8 solution was added to each well, and the cells were cultured for 2 hours in a 37°C, 5% _CO2 incubator. Next, the absorbance value was measured at 450 nm using a microplate reader (Molecular Devi-ces, Sunnyvale, CA, USA). Meanwhile, to confirm the cytoprotective effect of candidate substances against oxidative stress by tert-Butyl Hydroperoxide (tBHP, Sigma-Aldrich Chemical Co., USA) treatment, tBHP (300 μM) and 17 extracts (200 μg/ml) were simultaneously treated for 24 hours, and then 20 μl of CCK-8 was treated to each well, and the absorbance value was measured at 450 nm using a microplate reader.

Experimental Example 4. Confirmation of antioxidant effect in retinal epithelial cell line

To determine whether the above extracts of Usul, Glycyrrhiza glabra or their mixture protect ARPE-19 cells from oxidative stress induced by tert-Butyl hydroperoxide (tBHP), 300 μM tBHP and 17 kinds of extracts (200 μg/ml) were simultaneously treated, and cell viability was determined after 24 hours. The efficacy was proven at a concentration of 200 μg/ml, and the efficacy was reconfirmed at lower concentrations of 1, 10, 100, and 200 μg/ml.

Experimental Example 5. Mitochondrial oxygen consumption rate (OCR) and Measuring ATP production

To measure the intracellular oxygen consumption rate, ARPE-19 cells ( ^1Х104 cells/well) were cultured in DMEM/F-12 medium containing 10% fetal bovine serum (GIBCO, USA) on plates for XF analyzer (Seahorse Bioscience, USA). To measure the oxygen consumption rate (OCR) of the cells, the medium was replaced with bicarbonate-free medium, and the cells were cultured for 1 hour in a CO2-free cell incubator. The basal level OCR value of the cells was measured for 20 minutes, and then the cells were co-treated with 300 μM tBHP or each candidate substance for 4 hours, and the OCR change was measured for 60 minutes. Afterwards, the OCR change was measured again while sequentially treating with four respiratory chain inhibitors, including 0.5 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), 1 μM rotenone, and 1 μg/ml antimycin A.

Experimental Example 6. Measurement of intracellular mitochondrial shape and active oxygen

To measure mitochondrial morphology and intracellular ROS (reactive oxygen species) due to intracellular oxidative stress, ^1.5Х104 cells/well of ARPE-19 cells were cultured in μ-slide well plates using DMEM/F-12 medium containing 10% fetal bovine serum (GIBCO, USA). Then, the cells were simultaneously treated with 300 μM tBHP or each candidate substance for 4 h. To measure the change in ROS production, 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen) staining was used, and to confirm the mitochondrial morphology, MitoTracker™ Red CMXRos (Thermo fisher scientific, USA) staining was performed for 40 minutes. After that, the cells were washed twice with PBS and replaced with medium containing DAPI. After washing twice with PBS for 30 min, replacing the medium, images were obtained using a confocal laser scanning microscope (LSM 710; Carl Zeiss, Inc.). ROS intensity was measured using a ROS detection cell-based assay kit (Cayman chemical, USA), and mitochondria intensity was measured using a fluorescence microplate reader in the range of λex 556 and λem 575 nm after cell lysis.

Experimental Example 7. Statistical Processing

Statistical analysis was performed using one-way ANOVA in GraphPad Prism ^® version 7.04 (Graphpad Inc., San Diego, CA, USA), and a post hoc T-test was used. A value of p<0.05 was considered significant and statistically processed.

Example 1. Preparation of ethanol aqueous solution reflux extract

The results of extraction, concentration, and drying according to the above Experimental Example 1 were summarized in Table 2 as follows (Table 2).

number Experimental conditions Yield (g)
(B)-(A) HM tube self weight (g)
(A) HM tube + dry sample (g)
(B) JK-502 30% 1:6 (Usul:Licorice = 1:6) 5.64 9.50 15.13 JK-503 30% 6:1 (Usul:Licorice = 6:1) 6.83 9.37 16.21 JK-504 30% 1:9 (Usul:Licorice = 1:9) 6.72 9.47 16.18 JK-505 50% 1:6 (Usul:Licorice = 1:6) 7.20 9.41 16.61 JK-506 50% 6:1 (Usul:Licorice = 6:1) 8.33 9.42 17.75 JK-507 50% 1:1 (Usul:Licorice = 1:1) 7.10 9.37 16.47 JK-508 50% 1:3 (Usul:Licorice = 1:3) 8.42 9.50 17.92 JK-509 50% 1:9 (Usul:Licorice = 1:9) 2.22 9.38 11.60 JK-510 50% 3:1 (Usul:Licorice = 3:1) 7.97 9.43 17.41 JK-511 30% 1:1 (Usul:Licorice = 1:1) 7.39 9.38 16.78 JK-512 30% 1:3 (Usul:Licorice = 1:3) 7.76 9.36 17.11 JK-514 50% licorice 7.55 9.43 16.99 JK-515 50% 9:1 (Usul:Licorice = 9:1) 9.84 9.45 19.30 JK-516 30% 3:1 (Usul:Licorice = 3:1) 4.15 9.50 13.65 JK-517 30% 9:1 (Usul:Licorice = 9:1) 8.39 9.32 17.71 JK-519 30% off 5.53 9.43 14.97 JK-520 50% off 9.21 9.37 18.58

Example 2. Confirmation of cytotoxicity in human retinal epithelial cell line (ARPE-19)

As a result of confirming cell viability in a human retinal epithelial cell line (ARPE-19) cell line, as can be seen in Fig. 1, it was confirmed that Usul, licorice, or a mixed extract thereof (200 μg/㎖) had no cytotoxicity in the ARPE-19 cell line (Fig. 1).

Example 3. Confirmation of antioxidant effect in retinal epithelial cell line

As a result of confirming the antioxidant effect of the present invention's Usul, licorice or mixed extracts thereof on retinal epithelial cell lines, as can be seen in Fig. 2, it was confirmed that the mixed extracts JK-502, 504, 508, 509, licorice extract 514, and Usul extract 520 (200 μg/㎖) had antioxidant efficacy that protected ARPE-19 cells from oxidative stress caused by tBHP (300 uM) (Fig. 2).

As a result of confirming the efficacy of extracts whose antioxidant efficacy was verified at 200㎍/㎖ at lower concentrations (1, 10, 100, 200㎍/㎖), it was confirmed that extracts 504, 508, 514, and 520 showed antioxidant effects at 100 and 200㎍/㎖, and extracts 502, 505, and 509 showed antioxidant effects at 200㎍/㎖, respectively (Fig. 3).

In particular, in the case of the Usul extract and the licorice extract, when extracted with a 30% ethanol aqueous solution, it was confirmed that there was no antioxidant effect in the retinal epithelial cell line, whereas only when extracted with a 50% ethanol aqueous solution, the antioxidant effect was very excellent. In the case of the mixed extract of Usul and licorice, when Usul and licorice were mixed in an equal ratio, there was no effect, and even when Ausul was mixed in a high weight ratio to licorice, there was no antioxidant effect. In contrast, it was confirmed that when licorice was mixed in a high weight ratio to Ausul, it had an antioxidant effect in the retinal epithelial cell line. In particular, it was confirmed that there was a very excellent antioxidant effect at a mixing weight ratio of 1:3 to 1:9.

Through this, it was confirmed that the effect differs depending on the content of ethanol in the aqueous solution, and among them, the effect was excellent in a 50% ethanol aqueous solution for individual extracts, and in the case of mixed extracts, it was confirmed that the effect was observed when extracted with a 30 to 50% ethanol aqueous solution, but the mixing ratio of Usul and licorice was effective within a specific range.

Example 4. Effect of restoring oxygen consumption and ATP production in mitochondria

The effects of extracts JK-502, 504, 505, 508, 509, 514, and 520, which were confirmed to have antioxidant effects in the above Example 3, on mitochondrial stress response due to oxidative stress were confirmed.

As a result, as can be confirmed in FIG. 4, the reduced oxygen consumption rate (OCR) in the mitochondria was significantly recovered in the Achyranthes extract and licorice extract extracted with a 50% ethanol aqueous solution of the present invention and the mixed extracts of Achyranthes and licorice extracted with a 30 to 50% ethanol aqueous solution at a ratio of 1:3 to 1:9, and at the same time, the reduced ATP productivity was recovered to almost normal levels in JK-504, 505, 508, 509, 514, and 520. In addition, it was confirmed that the proton leak increased by oxidative stress was significantly reduced by each extract (FIG. 4).

This suggests that the extracts of Usul, licorice, or a mixture thereof of the present invention have the effect of restoring oxygen consumption and ATP production in mitochondria, thereby having the effect of preventing, improving, and treating eye diseases.

Example 5. Mitochondrial protection effect

In order to qualitatively and quantitatively confirm the effect of each extract on mitochondrial damage caused by oxidative stress in extracts that previously showed a cell protective effect due to oxidative stress, mitotracker and DCFDA reagents were used.

Specifically, as a result of confirming the intensity of mitochondria with mitotracker, as can be confirmed in FIG. 5, it was qualitatively and quantitatively confirmed that the intensity of mitotracker reduced by tBHP was recovered in the 50% ethanol aqueous solution of the present invention extracted from the Usul extract and the licorice extract, and the 1:3 to 1:9 mixed extract of Usul and licorice extracted from the 30 to 50% ethanol aqueous solution.

At the same time, when DCFDA was oxidized to DCF by active oxygen and turned green by a fluorescence analysis method to measure intracellular active oxygen, it was confirmed that, unlike the strong intensity of DCFDA due to the increased oxidative stress within the cell by tBHP, the intensity was significantly reduced in the treatment groups of the Achyranthes root extract and licorice root extract extracted with a 50% ethanol aqueous solution of the present invention and the Achyranthes root and licorice mixed extract treatment group of 1:3 to 1:9 extracted with a 30 to 50% ethanol aqueous solution (Fig. 5).

This also suggests that the extracts of Usul, licorice, or a mixture thereof of the present invention have the effect of restoring oxygen consumption and ATP production in mitochondria, thereby having the effect of preventing, improving, and treating eye diseases.

Example 6. Inhibitory effect on retinal cell death caused by blue light oxidative stress

Using zebrafish as an animal model, the inhibitory effect of the extracts of Usul, licorice or their mixture on retinal cell death by blue light oxidative stress was confirmed.

Oxidative stress caused by blue light damages retinal pigment epithelium (RPE) cells and photoreceptor cells, and the photoreceptor layer is the outer nuclear layer, which includes rod cell nuclei and cone cell nuclei.

The specific experimental method is as follows.

Zebrafish were maintained using Westerfield's husbandry technique, maintained at a temperature of 28.5℃ and under a light control of 200 Lux lighting with a light cycle of 14 hours and dark cycle of 10 hours (14/10 light/dark cycle), and fed a fixed amount of commercial feed mixed with fish meal, shrimp meal, krill meal, and grains 1-2 times a day.

Next, the samples of the extract of Usul and licorice extracted with a 50% ethanol aqueous solution of the present invention and the mixed extracts of Usul and licorice extracted with a 30 to 50% ethanol aqueous solution at a ratio of 1:3 to 1:9 were dissolved in PBS solvent and used, and N-acetylcysteine was selected as a positive control and confirmed together.

Zebrafish were used as adults 6 months after spawning, and 30 fish were transferred to a 3L tank equipped with a water warmer (28.5 ^o C) and moved to a dark room for 48 hours of dark adaptation. Feed was provided once during dark adaptation.

Zebrafish that had been dark-adapted were divided into three each of the Light treatment group, N-acetylcysteine, and the JK502, JK 504, JK505, JK508, JK509, JK514, and JK520 groups of the present invention, and divided into 200 mL system water using a 500 mL beaker, and blue light stress was applied for 24 hours using a blue light emitting diode lamp (Blue light, 440 nm, 12,000 Lux). Considering the amount of solvent used for each group, the system water was adjusted to make the 200 mL volume the same, and at this time, the extract of the present invention and each concentration were used at the same concentration of 100 μg per 200 mL system tank water for three zebrafish.

After the end of light exposure, zebrafish were euthanized using ice-cold water mixed with ice, fixed in a 70% aqueous ethanol solution for 24 hours, and then placed again in a 4% paraformaldehyde solution and fixed for more than 24 hours at room temperature. Afterwards, to prepare tissue samples for examination of the zebrafish eye tissue, the eyes were removed using fine forceps and paraffin blocks were made. For each group, retinal tissues including the optic disc and close to the optic nerve were selected to prepare 4 μm tissue sections, and H&E staining (Hematoxylin and Eosin Staining) was performed.

Next, changes in the tissue structure of the H&E-stained retinas of each test group were observed using a light source microscope (KB-600 Biological Microscope, KCS3-63SS Sony CMOS color camera).

At this time, tissues containing the optic nerve were selected from the H&E-stained ocular tissue sections, and the thickness of the outer nuclear layer (OUTL), inner nuclear layer (INL), inner plexiform layer (IPL), and the entire retinal thickness (NFL-RPE) at a distance of 100 μm from the optic nerve were measured.

Statistical analysis was performed using Excel functions, and the statistical significance of ONL thickness and ONL cell nucleus counts for each group of zebrafish included in the experiment was analyzed. One-way ANOVA was performed to compare the results between each group, and a p-value less than 0.05 in the analysis results was considered statistically significant.

The experimental results are shown in Figs. 6 to 9.

As a result, it was confirmed that the thickness of the outer nuclear layer, inner nuclear layer, inner plexiform layer, and the entire retinal tissue of zebrafish were all significantly reduced under blue light stress compared to the retinal tissue after dark adaptation in zebrafish compared to the normal state and dark-adapted state.

In contrast, in the case of the outer core layer (ONL) thickness, as can be confirmed in FIG. 6, in the extracts of Acerola and Glycyrrhizae Radix extracted with a 50% ethanol aqueous solution of the present invention and the mixed extracts of Acerola and Glycyrrhizae Radix extracted with a 30 to 50% ethanol aqueous solution of 1:3 to 1:9, JK 502, JK 504, JK 505, JK 508, and JK 509, the ONL thickness under blue light stress was significantly increased from 6.91±0.23 μm to 8.56±021 μm (19.3%), 9.49±0.33 μm (27.2%), 9.41±033 μm (26.6%), 9.41±030 μm (26.6%), and 8.66±0.18 μm, respectively. In the case of the single extracts, the ONL thickness of the licorice and Acerola extracts, respectively, It was confirmed that this also increased to 8.72 μm and 9.20 μm, and it was confirmed that this effect was similar to or better than the positive control group, NAC (Figure 6).

In particular, the mixed extracts of JK 504, JK 505, and JK 508, which mixed Usul and Glycyrrhiza uralensis in a mixing ratio of 1:3 to 1:9, significantly increased the outer nuclear layer thickness compared to the single extracts of JK 514 and JK 520, confirming that the mixed extracts had a greater effect on inhibiting retinal cell death than the respective single extracts.

Next, as can be confirmed in Fig. 7, the inner nuclear layer (INL) thickness under blue light stress was 7.51±0.73 μm, and in the groups treated with JK 502, JK 504, JK 505, JK 508, and JK 509, which are mixed extracts of Achyranthes root and Glycyrrhiza glabra extracted with a 50% ethanol aqueous solution of the present invention and Achyranthes root and Glycyrrhiza glabra mixed extracts of 1:3 to 1:9 extracted with a 30 to 50% ethanol aqueous solution, it was confirmed that the INL thickness increased to 9.77±1.04 μm, 10.85±0.32 μm, 9.07±0.21 μm, 8.89±0.56 μm, and 7.92±0.27 μm, respectively (Fig. 7).

It was also confirmed that the effect was better in the mixed extract of Usul and Glycoris in a ratio of 1:3 to 1:9 than in the single extract of Usul and Glycoris.

Next, as can be confirmed in Fig. 8, the thickness of the inner plexiform layer (IPL) under blue light stress was 8.69±1.40 μm, and in the extracts of Usul and licorice extracted with 50% ethanol aqueous solution of the present invention and the mixed extracts of Usul and licorice extracted with 30 to 50% ethanol aqueous solution of 1:3 to 1:9, JK 502, JK 504, JK 505, JK 508, and JK 509, it was confirmed that it increased to 10.46±0.64 μm (16.9%), 11.69±0.33 μm (25.75%), 10.72±0.61 μm (18.9%), 10.59±0.29 μm (17.9%), and 9.69±0.40 μm (10.3%), respectively, which was also higher than that of the positive control group. It was confirmed that it had similar or better effects.

In particular, in terms of the thickness of the inner reticular layer, it was confirmed that the mixed extract of Usul and Glycyrrhiza glabra in a ratio of 1:3 to 1:9 was more effective than the single extract of Usul and Glycyrrhiza glabra.

Finally, as can be confirmed in Fig. 9, the thickness of the entire retina from the retinal neural layer (NFL) to the retinal pigment epithelium (RPE) was measured to be 47.32±1.10 μm under blue light stress after dark adaptation, but in the 50% ethanol aqueous solution of the present invention extracted with the Achyranthes root extract and the licorice extract, and the 1:3 to 1:9 mixed extracts of Achyranthes root and licorice extracted with the 30 to 50% ethanol aqueous solution, JK 502, JK 504, JK 505, JK 508, JK 509, JK514, and JK520, it was confirmed that it significantly increased compared to the value measured under blue light stress, and among them, the mixed extract JK 504 treatment group increased to 63.03±1.58 μm, showing a very excellent increasing effect of 25.5%, and it can be confirmed that this value is an excellent effect compared to the positive control group.

In summary, through the above Examples 1 to 6, it can be confirmed that the Achyranthes root extract and the licorice root extract extracted with a 50% ethanol aqueous solution as effective ingredients and the Achyranthes root and licorice mixed extract of 1:3 to 1:9 extracted with a 30 to 50% ethanol aqueous solution, Achyranthes root, licorice or a mixed extract thereof, have excellent antioxidant effects in retinal epithelial cells and excellent effects on restoring oxygen consumption and ATP production in mitochondria, and further have the effect of protecting mitochondria and effectively inhibiting retinal cell death caused by blue light oxidative stress, and thus these extracts can be utilized as drugs, health functional foods, etc. for preventing, improving or treating eye diseases. In addition, it can be confirmed that there are advantages such as excellent commerciality and low concern about side effects because the number of herbal medicines included is small and there are few drug interactions.

An example of a formulation of a composition according to one embodiment of the present invention is described below, but it can be applied to various other formulations, and this is not intended to limit the present invention but to specifically explain the present invention.

Preparation Example 1. Preparation of pharmaceutical preparations

1-1. Manufacturing of mountain products

100 mg of the mixed extract of licorice and ginseng prepared in any one of Examples 1.3 to 1.8, 100 mg of lactose, and 10 mg of talc were mixed and filled into an airtight pouch to prepare a powder.

1-2. Purification manufacturing

100 mg of the mixed extract of Usul and Glycyrrhiza prepared in any one of Examples 1.3 to 1.8, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and then compressed into tablets according to a conventional tablet manufacturing method.

1-3. Capsule manufacturing

According to a conventional capsule manufacturing method, 100 mg of the mixed extract of Usul and Glycyrrhiza prepared in any one of Examples 1.3 to 1.8, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and filled into a gelatin capsule to manufacture a capsule.

1-4. Manufacturing of injections

According to the manufacturing method of a conventional injection, 100 mg of the mixed extract of Usul and Glycyrrhiza prepared in any one of Examples 1.3 to 1.8, an appropriate amount of sterilized distilled water for injection, and an appropriate amount of a pH regulator were mixed per 1 ampoule (2 mL) according to the manufacturing method of a conventional injection, and the above ingredient contents were manufactured per 1 ampoule (2 mL).

1-5. Liquid preparation

According to the conventional method for preparing a liquid, 20 mg of the mixed extract of Usul and Glycyrrhiza prepared in any one of Examples 1.3 to 1.8, 10 g of isomerized sugar, and 5 g of mannitol were dissolved in purified water, an appropriate amount of lemon flavor was added, and the above ingredients were mixed. Then, purified water was added to adjust the total volume to 100 mL, and the mixture was filled into a brown bottle and sterilized to prepare a liquid.

Preparation Example 2. Preparation of food composition

2-1. Manufacturing of health functional foods

100 mg of a mixed extract of Usul and Glycyrrhiza prepared in any one of Examples 1.3 to 1.8, an appropriate amount of a vitamin mixture, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of vitamin C, 10 g of biotin, 1.7 mg of nicotinamide, 50 g of folic acid, 0.5 mg of calcium pantothenate, an appropriate amount of a mineral mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of monobasic potassium phosphate, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate and 24.8 mg of magnesium chloride were mixed, and then granules were prepared and a health functional food was prepared according to a conventional method. At this time, the composition ratio of the vitamin and mineral mixture is a preferred example of mixing ingredients relatively suitable for health functional foods, but the mixing ratio may be arbitrarily modified.

2-2. Manufacturing of health functional beverages

According to a conventional health beverage manufacturing method, 100 mg of the mixed extract of Usul and Glycyrrhiza uralensis prepared in any one of Examples 1.3 to 1.8, 15 g of vitamin C, 100 g of vitamin E (powder), 19.75 g of ferrous lactate, 3.5 g of zinc oxide, 3.5 g of nicotinamide, 0.2 g of vitamin A, 0.25 g of vitamin B1, 0.3 g of vitamin B2, and an appropriate amount of water were mixed, stirred and heated at 85°C for about 1 hour, and the resulting solution was filtered, placed in a sterilized 2 L container, sealed and sterilized, and then stored in the refrigerator to manufacture a health beverage. At this time, the composition ratio is a preferred example of mixing ingredients relatively suitable for a favorite beverage, but the mixing ratio may be arbitrarily modified according to regional or ethnic preferences such as the demand class, demand country, and intended use.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the claims described below rather than the above detailed description and their equivalent concepts within the scope of the present invention.

Claims (11)

A food composition for preventing or improving eye disease comprising extracts of licorice, licorice or a mixture thereof.
In the first paragraph,
A food composition, wherein the extract is extracted with a solvent selected from the group consisting of water, straight-chain or branched-chain alcohols having 1 to 6 carbon atoms, organic solvents, and mixed solvents thereof.
In the first paragraph,
A food composition wherein the above extract is an aqueous ethanol solution extract.
In the third paragraph,
A food composition, wherein the above ethanol aqueous solution is a 25 to 55% ethanol aqueous solution.
In the first paragraph,
A food composition wherein the above mixed extract is a mixture of Usul and licorice in a weight ratio of 1:1 to 1:9.
In the first paragraph,
A food composition, wherein the extract is contained in an amount of 0.001 to 90 wt% based on the total weight of the food composition.
In the first paragraph,
A food composition, wherein the food composition is manufactured in the form of a powder, granules, tablets, capsules, syrup or beverage.
A food composition in claim 1, wherein the eye disease is macular degeneration, glaucoma, diabetic retinopathy, retinal edema, epiretinal membrane, retinal tear, cataract, dry eye, superficial keratitis, or retinal and corneal abnormalities.
A pharmaceutical composition for preventing or treating eye diseases comprising extracts of Usul, licorice or a mixture thereof.
A pharmaceutical composition for preventing or improving eye diseases, comprising extracts of Usul, licorice or a mixture thereof.
A feed composition for preventing or improving eye diseases, comprising extracts of licorice, licorice or a mixture thereof.