A composition for preventing eye damage containing extract of *Leptochloa chinensis*

Abstract

The compositions containing *Pyracantha fortuneana* extract of the present invention exhibit excellent activity in inhibiting A2E oxidation, inhibiting A2E-induced retinal pigment epithelial cell death, and inhibiting the accumulation of drusen, and therefore can be used to prevent eye damage. The compositions containing *Pyracantha fortuneana* extract of the present invention can even be developed into therapeutic agents and health foods for various eye diseases.

Description

Technical Field

[0001] This invention relates to a composition containing Dendropanax morbifera extract as an active ingredient and its use. Background Technology

[0002] Since the eyes are the most important sensory organs, damage or loss of eye function is one of the biggest factors reducing quality of life. Modern people spend most of their time in front of various digital screens such as computers and smartphones, and because LEDs are frequently used on smart device screens, blue light deteriorates eye health.

[0003] Blue light is blue light in the visible light spectrum, ranging from 400 nm to 500 nm. Prolonged exposure to blue light can stimulate the optic nerve, cause eye fatigue, and contribute to various eye diseases. Due to its high energy and penetrating power, blue light causes the focal point on the retina to become opaque and reduces sharpness when it enters the eye as is. Long-term exposure to blue light can also cause retinal aging and degeneration. Known effects of blue light on the human body include dry eye and eye fatigue, decreased visual acuity and various eye diseases, accelerated retinal aging, macular degeneration, and insomnia caused by the inhibition of melatonin production (Ganka Gakkai Zasshi. 2001 Oct; 105(10):687–95; Archives of Ophthalmology 1992; 110:99–104; Review of Ophthalmology Oct 15 2003; 10(10)).

[0004] Metabolic byproducts are known to accumulate in retinal cells throughout life due to the effects of blue light, with the most active accumulation occurring in the subcentral retinal pigment epithelium (SNPE), where rod cells, photoreceptors, are highly concentrated. One of the most important of these metabolic waste byproducts is the lipofuscin component N-retinyl-N-retinyl-ethanolamine (A2E), and high concentrations of A2E are a major contributor to SNPE cell death. Furthermore, even at low concentrations, A2E is photooxidized by blue light, transforming into peroxide A2E and furan A2E in the form of epoxides. The highly reactive epoxide forms of A2E induce oxidative stress and inflammation, and alter mitochondrial protein activity, thereby inducing apoptosis. As a result, the loss of normal SNPE cell function leads to secondary photoreceptor cell death. Particularly in the case of the macula, where photoreceptors are densely concentrated, it is severely damaged, leading to macular degeneration. In other words, the difference between macular degeneration and diabetic retinopathy is that macular degeneration is caused by A2E oxidation due to oxidative stress in retinal cells caused by blue light, while diabetic retinopathy is caused by the effects of glycation products and angiogenic factors such as VEGF caused by high blood sugar levels.

[0005] Age-related macular degeneration (AMD) can be classified into two types: dry (non-exudative) AMD and wet (exudative) AMD. Dry AMD accounts for approximately 90% of all AMD cases and is characterized by the accumulation of waste products called drusen between the retinal pigment epithelium and Bruch's membrane, leading to atrophy or thinning of the macular tissue. Conversely, wet AMD accounts for approximately 10% of all AMD cases and refers to a condition where normal blood and nutrients are not supplied to the macula, leading to the formation and rupture of abnormal new blood vessels, resulting in leakage of blood or mucus into the macula and causing severe vision loss. Early symptoms of dry AMD are often asymptomatic or easily overlooked, as they are mistaken for presbyopia. However, if the progression of dry AMD is not inhibited or treated, vision loss can occur due to continuous retinal damage, and it may progress to wet AMD and lead to blindness; therefore, early prevention of dry AMD is crucial.

[0006] Previous eye health-related products have been synthetic treatments, which have caused side effects due to long-term use of synthetic compounds. Therefore, there is a need to develop materials for improving eye health derived from natural products containing specific compounds that are typically consumed as food rather than artificially synthesized. Summary of the Invention

[0007] Technical issues

[0008] The object of this invention is to provide a composition for preventing eye damage caused by blue light.

[0009] Another objective of this invention is to provide a health functional food for preventing or improving eye damage caused by blue light.

[0010] Another object of the present invention is to provide a composition for the prevention or treatment of eye diseases caused by blue light.

[0011] Technical solution

[0012] To achieve the above objectives, one aspect of the present invention provides a composition for preventing eye damage, which contains extract of *Leptochloa chinensis* as an active ingredient.

[0013] Another aspect of the present invention provides a health functional food for preventing or improving eye damage, which contains extract of *Leptochloa chinensis* as an active ingredient.

[0014] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of eye diseases, which contains extract of *Leptochloa chinensis* as an active ingredient.

[0015] Beneficial effects

[0016] The composition containing *Leptochloa chinensis* extract of the present invention exhibits excellent activity in inhibiting A2E oxidation induced by blue light, inhibiting A2E-induced retinal pigment epithelial cell death, and inhibiting the accumulation of drusen. Therefore, it can be used to prevent eye damage caused by blue light. Furthermore, the composition containing *Leptochloa chinensis* extract of the present invention can be developed into a therapeutic agent and a health food for various eye diseases caused by blue light.

[0017] The effects of the present invention are not limited to those described above, and those skilled in the art will clearly understand from the following description other effects not mentioned. Attached Figure Description

[0018] Figure 1 This is a graph showing the photooxidation of A2E in human retinal pigment epithelial cells ARPE-19, which was increased by blue light treatment and significantly inhibited by treatment with lacquerwood extract.

[0019] Figure 2 This figure shows the effect of treating human retinal pigment epithelial cells ARPE-19 with extract of *Lysimachia christinae* on inhibiting the accumulation of drusen.

[0020] Figure 3 This figure shows that in human retinal pigment epithelial cells ARPE-19, cell activity decreased by blue light treatment and significantly increased by treatment with lacquerwood extract.

[0021] Figure 4The image shows the thickness of the outer nuclear layer (ONL) of the retina as observed under a microscope after administration of the test drug to an animal model, which induced optic nerve cell loss similar to dry macular degeneration.

[0022] Figure 5 It is a graph showing the thickness of the outer layer of the retina, which is reduced in animal models by excessive exposure to light that induces optic nerve cell loss similar to dry macular degeneration, and increased by treatment with extracts of *Lysimachia christinae*.

[0023] Figure 6 This is a graph showing the activity of photoreceptor cells, which is reduced in an animal model by excessive exposure to light that induces optic nerve cell loss similar to dry macular degeneration, and increased by treatment with extracts of *Lysimachia christinae*.

[0024] Figure 7 The diagram shows the thickness of the outer nuclear layer (ONL) of the retina after administration of the test drug to an animal model, where optic nerve cell loss similar to that in dry macular degeneration was observed under a microscope after administration of N-methyl-N-nitrosourea (MNU).

[0025] Figure 8 This is a graph showing the thickness of the outer nuclear layer of the retina, which was reduced in an animal model by administration of N-methyl-N-nitrosourea to induce optic nerve cell loss similar to dry macular degeneration, and increased by treatment with extracts of *Lysimachia christinae*.

[0026] Figure 9 The diagram shows the location observed under a microscope, where folding deformation of retinal tissue occurs by administering the test drug to an animal model, and where morphological deformation is induced in the retinal tissue located above it by administering sodium iodate (NaIO3).

[0027] Figure 10 This is a diagram showing damage to retinal pigment epithelial cells, which increased in animal models by morphological deformation of the retinal tissue located above it induced by administration of sodium iodate (NaIO3), and decreased by treatment with extracts of *Leptochloa chinensis*.

[0028] Figure 11 The image shows cells observed under a microscope, where a TUNEL-positive response was observed in the nuclear layer of optic nerve cells after administration of the test drug to an animal model, which induced optic nerve cell loss similar to dry macular degeneration.

[0029] Figure 12 This is a graph showing an increased apoptotic response in an animal model, inducing optic nerve cell loss similar to dry macular degeneration, which was reduced by treatment with extracts of *Lysimachia christinae*.

[0030] Figure 13The figure shows that in an animal model induced with optic nerve cell loss similar to dry macular degeneration, treatment with *Lysimachia christinae* extract resulted in decreased expression of Bax (a pro-apoptotic factor that induces apoptosis), increased expression of Bcl-2 (an anti-apoptotic factor), and increased / decreased expression of the cleaved form of asparagine 3.

[0031] Figure 14 The image shows a microscopic observation of ZO-1 after administration of the test drug to an animal model. This protein forms tight junctions, and the retinal tissue in the upper part of the model induced morphological deformation.

[0032] Figure 15 This diagram illustrates the mechanism of action of lacquerwood extract in improving macular degeneration. Detailed Implementation

[0033] The present invention will now be described in detail.

[0034] One aspect of the present invention provides a composition for preventing eye damage, which contains extract of *Leptochloa chinensis* as an active ingredient.

[0035] "Yellow Lacquer Wood" belongs to the Araliaceae family, and naturally includes its subspecies and varieties. Furthermore, the Yellow Lacquer Wood of this invention includes herbs of the same genus that are readily apparent in the art, and can be used for the same or similar purposes of prevention, improvement, and treatment as described in this invention.

[0036] "Extract" includes the extract itself, as well as extracts of all formulations that can be formed using liquid extracts, such as extracts obtained by extraction treatment of *Leptochloa chinensis*, diluted or concentrated solutions of extracts, dried products obtained by drying extracts, crude or purified products of extracts, and mixtures thereof. For example, the extract of the present invention can be prepared and used in dry powder form after extraction.

[0037] This extract of *Leptochloa chinensis* can be extracted from natural, hybrid, or variant *Leptochloa chinensis* plants, as well as from plant tissue cultures. Furthermore, this extract can be obtained from the leaves, stems, roots, flowers, and seeds of *Leptochloa chinensis*.

[0038] There are no particular limitations on the extraction method for the above-mentioned extract of *Leptochloa chinensis*, and extraction can be carried out according to methods commonly used in the art. Non-limiting examples of extraction methods may include: hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., which may be used alone or in combination of two or more.

[0039] There is no particular limitation on the type of extraction solvent used to extract the above-mentioned *Pyracantha fortuneana* extract; any solvent known in the art may be used. Non-limiting examples of extraction solvents may include water; C1 to C4 lower alcohols, such as methanol, ethanol, propanol, and butanol; polyols, such as glycerol, butanediol, and propylene glycol; and hydrocarbon-based solvents, such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; or mixtures thereof. For example, the *Pyracantha fortuneana* extract of the present invention can be extracted from the leaves and stems of *Pyracantha fortuneana* using water, C1 to C4 lower alcohols, or mixtures thereof.

[0040] "Eye damage" can be caused by blue light, and can specifically refer to eye damage caused by the photo-oxidation of A2E caused by blue light and its accumulation. More specifically, eye damage can refer to eye damage caused by oxidative damage to the eye, eye aging, eye fatigue, degeneration of eye function, or eye diseases, but is not limited to these.

[0041] Eye diseases can be caused by blue light, and can specifically refer to retinal diseases associated with A2E photooxidation and drusen accumulation caused by blue light. Specifically, the eye diseases can refer to one or more types selected from the group consisting of retinal degeneration, macular degeneration, glaucoma, retinitis pigmentosa, Stagger's disease, choroidal atrophy, retinal detachment, optic neuropathy, and dry eye syndrome, but are not limited thereto.

[0042] In one specific embodiment of the present invention, in order to confirm the effect of the yellow lacquer wood extract on preventing eye damage caused by blue light, the photo-oxidation level of retinal pigment epithelial cells was measured by treating retinal pigment epithelial cells with the yellow lacquer wood extract and blue light. As a result, it was confirmed that the increased photo-oxidation of A2E caused by blue light was significantly inhibited by treatment with the yellow lacquer wood extract.

[0043] In addition, to confirm the effect of preventing cell damage caused by A2E oxidation, retinal pigment epithelial cells were treated with lacquer wood extract along with blue light to confirm cell activity. The results showed that the reduced cell activity caused by blue light was significantly increased by treatment with lacquer wood extract.

[0044] In addition, the accumulation of drusen was confirmed by irradiating with blue light, and the accumulation of drusen was confirmed to be reduced by treatment with extract of lacquerwood.

[0045] Furthermore, it was confirmed that the thickness of the outer nuclear layer of the retina, which is thinned due to light exposure, was increased by treatment with extracts of *Leptospermum jasminoides*, and that the decreased activity of photoreceptor cells was improved by treatment with extracts of *Leptospermum jasminoides*.

[0046] Furthermore, it was confirmed that damage or deformation of retinal pigment epithelial cells in a drug-induced macular degeneration animal model was improved by treatment with extracts of *Lysimachia christinae* to inhibit retinal tissue deformation.

[0047] Furthermore, it was confirmed that treatment with *Pyracantha fortuneana* extract inhibited light-induced retinal cell death, thereby improving optic nerve cell damage and maintaining tight junctions of retinal pigment epithelial cells, thus improving retinal pigment epithelial cell damage.

[0048] Therefore, it is evident that the extract of *Leptochloa chinensis* prevents damage to retinal pigment epithelial cells by inhibiting the oxidation of A2E, and has the activity of inhibiting retinal tissue atrophy or morphological changes by inhibiting the accumulation of drusen. Thus, the extract of *Leptochloa chinensis* can be effectively used as an active ingredient in compositions for preventing eye damage.

[0049] Another aspect of the present invention provides a health functional food for preventing or improving eye damage, which contains extract of *Leptochloa chinensis* as an active ingredient.

[0050] The yellow lacquer wood, extract, and eye damage are described above.

[0051] Because yellow lacquer wood has long been used as a natural material and has been proven safe for human use, it can be prepared and consumed as food, which can prevent or improve eye damage when consumed normally.

[0052] "Prevention" refers to all actions that inhibit or delay eye damage through the intake of healthy foods.

[0053] "Improvement" refers to all behaviors that reduce parameters associated with a condition to be treated, such as the severity of symptoms, by consuming healthy foods.

[0054] In addition to extracts from *Leptochloa chinensis*, the aforementioned health functional foods may also contain nutritionally acceptable food additives.

[0055] Food additives are components that can be added to food and can be appropriately selected and used by those skilled in the art as additives to prepare various formulations of health-functional foods. Examples of food additives include, but are not limited to, various nutrients, vitamins, minerals (electrolytes), flavorings (such as synthetic and natural flavorings), colorants and fillers, pectic acids and their salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerol, alcohols, and carbonating agents used in carbonated beverages.

[0056] Health functional foods refer to foods prepared and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients that have beneficial functions for the human body. In particular, "function" refers to achieving beneficial effects for health purposes in terms of the structure and function of the human body, such as regulating nutritional or physiological functions. The health functional foods of this invention can be prepared using methods commonly used in the art, or by adding raw materials and ingredients typically added in the art during the preparation process. Furthermore, the formulation of health functional foods can be prepared without limitation, as long as the formulation is considered a health functional food.

[0057] This functional health food can be made into various types of preparations. Unlike ordinary drugs, its advantage is that, since food is used as a raw material, long-term use will not produce side effects, and it has excellent portability. Therefore, the health benefits of the functional food of this invention can be used as a supplement to enhance and improve the effect of eye damage.

[0058] There are no limitations on the form of health functional foods; they can include all foods in the conventional sense and can be used interchangeably with terms known in the art, such as functional foods. Furthermore, the health functional foods of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in the food according to the selection of those skilled in the art. Examples of foods to which they can be added include meat, sausages, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, chewing gum, dairy products containing ice cream, various soups, beverages, tea, alcoholic beverages, vitamin complexes, etc., and can be manufactured by adding the extracts of the present invention as a main ingredient to fruit juices, teas, jellies, etc. It also includes foods used as animal feed.

[0059] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of eye diseases, which contains extract of *Leptochloa chinensis* as an active ingredient.

[0060] Yellow lacquer wood extract is as described above.

[0061] "Prevention" refers to all effects of inhibiting or delaying the occurrence of eye diseases by administering the compositions of the present invention.

[0062] "Treatment" refers to all effects of improving or beneficially altering the symptoms of an eye disease by administering the composition of the present invention.

[0063] "Eye disease" can refer to eye diseases caused by blue light, specifically retinal diseases related to A2E photooxidation and its accumulation caused by blue light, or drusen accumulation and photooxidation. More specifically, the eye disease can refer to one or more types selected from the group consisting of retinal degeneration, macular degeneration, glaucoma, retinitis pigmentosa, Stagger's disease, choroidal atrophy, retinal detachment, optic neuropathy, and dry eye syndrome, but is not limited to these.

[0064] Macular degeneration (MD) refers to the phenomenon where central vision is impaired by damage to the macula. Macular degeneration diseases can include dry macular degeneration, wet macular degeneration, age-related macular degeneration, myopic macular degeneration, idiopathic macular degeneration, etc., and specifically, it can be age-related macular degeneration (AMD).

[0065] Although the exact etiology of age-related macular degeneration has not been determined, it is generally known that the accumulation of excess pigment in retinal pigment epithelial cells due to aging occurs in the early stages of age-related macular degeneration; and A2E, produced by the synthesis of all-trans retina and ethanolamine, is a substance that usually accumulates in retinal epithelial cells. It generates singlet oxygen through blue light and may cause damage to retinal pigment epithelial cells while oxidizing carbon-carbon double bonds.

[0066] By inhibiting apoptosis induced by A2E oxidized by blue light or suppressing the accumulation of drusen, the prevention or treatment of eye diseases can be achieved. In one specific embodiment of the present invention, it was confirmed that apoptosis occurs when retinal pigment epithelial cells are irradiated with blue light after A2E accumulation, but treatment with *Leptochloa chinensis* extract has an inhibitory effect on apoptosis. Furthermore, it was confirmed that the accumulation of drusen was suppressed when retinal pigment epithelial cells were treated with *Leptochloa chinensis* extract. That is, the *Leptochloa chinensis* extract of the present invention can be effectively used for the prevention or treatment of eye diseases by inhibiting the death of retinal pigment epithelial cells caused by exposure to blue light or suppressing the accumulation of drusen.

[0067] The pharmaceutical composition comprises a therapeutically effective amount of *Pyracantha fortuneana* extract and a pharmaceutically acceptable carrier.

[0068] "Therapeutic effective amount" refers to an amount sufficient to enable the composition of the present invention to treat or prevent eye diseases caused by blue light.

[0069] Pharmaceutically acceptable carriers are those commonly used in the preparation of formulations and may contain, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc.

[0070] The pharmaceutical composition can be administered orally or parenterally, and in the case of parenterally administration, it can be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, or transdermally.

[0071] The appropriate dosage of a pharmaceutical composition varies depending on factors such as preparation method, administration method, patient age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and responsiveness. Skilled practitioners are usually able to easily determine and prescribe an effective dosage for the desired treatment or prevention.

[0072] The daily dose of the pharmaceutical composition is from 0.0001 mg / kg to 100 mg / kg, and preferably from 0.001 mg / kg to 30 mg / kg, administered once or several times daily. Furthermore, the dosing period can be from 1 day to 2 months, but can be administered indefinitely until the preventive or therapeutic effect on the disease is achieved.

[0073] The pharmaceutical composition is prepared in unit dose form by using pharmaceutically acceptable carriers and / or excipients, according to methods readily available to those skilled in the art, or it can be prepared by placing it in a multi-dose container. In particular, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or it may be in the form of an extract, powder, granules, tablet, or capsule, and may further contain dispersants or stabilizers.

[0074] Pharmaceutical compositions can also be formulated as ophthalmic compositions, such as eye drops and eye ointments. This form can encompass all ophthalmic preparations intended for topical application to the eyes in the ophthalmic field. Eye drops are prepared by dissolving the active ingredient in a sterile aqueous solution (e.g., saline and buffer). Eye drops can be provided as a powder composition dissolved before use, or in combination with a powder composition dissolved before use. Eye ointments are prepared by mixing the active ingredient into an ointment base.

[0075] The present invention will now be described in detail through examples and experimental cases. However, the examples and experimental cases below are merely illustrative of the present invention, and the content of the present invention is not limited to the examples and experimental cases below. Specific Implementation

[0077] [Manufacturing Example]

[0078] Preparation of Yellow Lacquer Wood Extract

[0079] Add 15 times its weight of 30% (v / v) ethanol aqueous solution to 2 kg of *Pyracantha fortuneana* leaves and branches, and extract 1–3 times at 80°C for 8 hours each time, mixing the extracts obtained from each extraction. Filter the extract using a 5 μm filter. Concentrate the filtrate at 55°C using a rotary vacuum concentrator (BUCHI, R-220). Add sterile distilled water to the concentrate to dilute to a Brix level of 20, and freeze-dry to obtain 210 g of *Pyracantha fortuneana* extract powder.

[0080] [Experimental Example 1]

[0081] The inhibitory effect of yellow lacquer wood extract on the photo-oxidation of A2E

[0082] A2E, which accumulates in retinal pigment epithelial cells and is a cause of macular degeneration, is known to undergo photo-oxidation upon exposure to blue light, inducing cytotoxicity. To confirm whether *Lysimachia christinae* extract has an inhibitory effect on A2E photo-oxidation, human retinal pigment epithelial cell line ARPE-19 cells and A2E were used.

[0083] Specifically, ARPE-19 cells (CRL-2302, ATCC, USA) were treated with 90 μM A2E (Gene and Cell Technologies), then with the *Pyracantha fortuneana* extract prepared in the above manufacturing example at concentrations of 0 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL, and then treated with blue light (430 nm, intensity: 2.0 mW / cm²). 2 Up to 2.5mW / cm 2 Irradiation was performed for 10 minutes. To determine the degree of A2E photo-oxidation, absorbance at 440 nm was measured using a microplate reader before and after blue light irradiation.

[0084] As a result, Figure 1 As shown, in the group treated with *Rubus tectorum* extract, the accumulation of oxidized A2E was inhibited by 40% to 75% compared with the negative control group not treated with *Rubus tectorum* extract.

[0085] As can be seen from the above, the extract of *Leptochloa chinensis* has the effect of inhibiting the accumulation of intracellular A2E and inhibiting the photo-oxidation of A2E.

[0086] [Experimental Example 2]

[0087] The effect of extract from *Pyracantha fortuneana* on inhibiting photo-oxidation and the accumulation of drusen.

[0088] It is known that when drusen accumulate in retinal epithelial cells, the macular tissue thins or atrophies, leading to impaired visual function and visual acuity, thereby inducing macular degeneration. Following photooxidation, drusen produces reactive dicarbonyl substances, such as glyoxal and methylglyoxal (MGO) (which are cytotoxic), in retinal cells via A2E peroxide. These substances then non-specifically bind to intracellular proteins to form MGO-modified adducts that accumulate in retinal cells. Since these MGO-modified adducts are also known to exacerbate retinal disease by being a component of drusen, this study investigated whether *Lysimachia christinae* extract inhibited photooxidation of drusen and the accumulation of MGO-modified adducts.

[0089] Specifically, 0.7 mL of bovine serum albumin (Sigma, St. Louis, Missouri, USA) dissolved at 10 mg / mL in 50 mM phosphate buffer (pH 7.4) and 0.1 mL of 0.22 M methylglyoxal were added, and the extract of *Lysimachia christinae* prepared in the manufacturing example was added at concentrations of 0 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL to a total volume of 1 mL. The product was incubated at 37°C for 7 days to induce the production of glycation products. After one week, the absorbance at 450 nm was measured using a microplate reader.

[0090] As a result, Figure 2 As shown, the accumulation of MGO-modified adducts was inhibited in the group treated with *Rubus idaeus* extract compared to the negative control group that was not treated with *Rubus idaeus* extract.

[0091] As can be seen from the above, the extract of *Lysimachia christinae* has the effects of inhibiting the photo-oxidation of drusen and the accumulation of MGO-modified adducts, preventing eye damage, and inhibiting the occurrence and deterioration of macular degeneration.

[0092] [Experiment Example 3]

[0093] The protective effect of *Lysimachia christinae* extract against blue light-induced photooxidation in retinal pigment epithelial cells.

[0094] Considering that A2E accumulation in retinal pigment epithelial cells during blue light irradiation causes photo-oxidation, which induces cytotoxicity leading to apoptosis, this study investigated whether extracts of *Lysimachia christinae* could inhibit blue light-induced cytotoxicity and apoptosis.

[0095] Specifically, ARPE-19 cells were treated for 24 hours with 0 μg / mL and 50 μg / mL of *Lysimachia christinae* extract, 50 μg / mL of chlorogenic acid, 50 μg / mL of neochlorogenic acid, 50 μg / mL of stigmasterol, or 50 μg / mL of lutein prepared in the above manufacturing example, followed by treatment with 20 μM A2E for 24 hours, and then irradiated with blue light for 10 minutes. Cell viability of ARPE-19 cells was then measured using a cell counting kit (Dojin Chemical Research Institute, Japan), and the cell viability of each treatment group relative to the control group with accumulated A2E and no blue light irradiation was expressed as a percentage (%).

[0096] As a result, the cell viability of the negative control group, which accumulated A2E and was irradiated with blue light, was significantly reduced compared to the control group, and apoptosis was induced. Conversely, cell viability was confirmed to be increased in the group treated with *Leptochloa chinensis* extract compared to the negative control group treated with A2E alone, and even increased compared to the positive control group treated with lutein.

[0097] As can be seen from the above, the extract of *Leptochloa chinensis* can prevent or treat macular degeneration by inhibiting apoptosis in retinal pigment epithelial cells caused by the accumulation and oxidation of A2E.

[0098] [Experiment Example 4]

[0099] The protective effect of *Lysimachia christinae* extract against blue light-induced photooxidation in retinal pigment epithelial cells.

[0100] Considering that A2E accumulation in retinal pigment epithelial cells during blue light irradiation causes photo-oxidation, which induces cytotoxicity leading to apoptosis, this study investigated whether extracts of *Lysimachia christinae* could inhibit blue light-induced cytotoxicity and apoptosis.

[0101] Specifically, ARPE-19 cells were treated for 24 hours with 0 μg / mL and 50 μg / mL of *Lysimachia christinae* extract, 50 μg / mL of chlorogenic acid, 50 μg / mL of neochlorogenic acid, 50 μg / mL of stigmasterol, or 50 μg / mL of lutein prepared in the above manufacturing example, followed by treatment with 20 μM A2E for 24 hours, and then irradiated with blue light for 10 minutes. Cell viability of ARPE-19 cells was then measured using a cell counting kit (Dojin Chemical Research Institute, Japan), and the cell viability of each treatment group relative to the control group with accumulated A2E and no blue light irradiation was expressed as a percentage (%).

[0102] As a result, Figure 3 As shown, the cell viability of the negative control group, which accumulated A2E and was irradiated with blue light, was significantly reduced compared to the control group, and apoptosis was induced. Conversely, cell viability was confirmed to be increased in the group treated with *Leptochloa chinensis* extract compared to the negative control group treated with A2E alone, and even increased compared to the positive control group treated with lutein.

[0103] As can be seen from the above, the extract of *Leptochloa chinensis* can prevent or treat macular degeneration by inhibiting apoptosis in retinal pigment epithelial cells caused by the accumulation and oxidation of A2E.

[0104] [Experiment Example 5]

[0105] Effects of Yellow Lacquer Wood Extract on Improving Photoreceptor Cell Damage or Deformation in Animal Models of Macular Degeneration

[0106] When drusen accumulate in the macular region due to dry macular degeneration, the macular region is photo-oxidized, causing damage to surrounding tissues and optic nerve cells, leading to decreased visual acuity. In severe cases, geographic atrophy occurs, a condition characterized by optic nerve cell loss and retinal apoptosis. To confirm whether *Ilex cornuta* extract has an effect in improving photoreceptor damage or deformation in an animal model of optic nerve cell damage and optic nerve cell loss similar to dry macular degeneration caused by excessive exposure to blue light sources, changes in the thickness of the outer nuclear layer (ONL) of the retina were measured.

[0107] Specifically, 6-week-old male BALB / c mice (Orient Bio) were acclimatized for one week, and then subjected to 14 hours of dark acclimatization in the dark from the evening before exposure to a white LED containing blue light. The pupils were then dilated with 1% tropicamide eye drops the following morning. Subsequently, the mice were exposed to a specially manufactured cooled white LED light panel with a brightness of 5,000 lux for 0.5 hours, followed by 24 hours in a dark room. Following light exposure, the mice were orally administered the test drug once daily for 7 days. The drug concentrations administered are shown in Table 1 below. Neochlorogenic acid, known to help prevent macular degeneration by activating aging retinal nerve cells, and lutein, a component widely known as an eye nutrient, were set as positive controls, and *Lysimachia christinae* extract, manufactured according to the above-described example, was administered at various concentrations. After administration, necropsy was performed, and eyeballs were extracted, fixed in 10% formalin for one day, and then embedded in paraffin to prepare slide sections. Slides were stained with hematoxylin and eosin (H&E) and observed under an optical microscope. The changes in the thickness of the outer nuclear layer of the retinal tissue were measured to evaluate the damage or deformation of photoreceptor cells.

[0108] [Table 1]

[0109] Test group Group Name Administer drugs Dosage concentration (mg / kg / day) Head count normal control group normal – – 8 negative control group LRD – – 8 Groups given extracts of *Leptochloa chinensis* DM-25 Yellow lacquer wood extract 25 8 Groups given extracts of *Leptochloa chinensis* DM-50 Yellow lacquer wood extract 50 8 Groups given extracts of *Leptochloa chinensis* DM-100 Yellow lacquer wood extract 100 8 Groups given extracts of *Leptochloa chinensis* DM-200 Yellow lacquer wood extract 200 8 Positive control group NCA-6.5 Neochlorogenic acid 6.5 8 Positive control group Lutein-20 Lutein 20 8

[0110] As a result, Figure 4 and 5 As shown, in the negative control group, the thickness of the outer nuclear layer (ONL), containing the nuclei of optic nerve cells, decreased. Furthermore, when *Lysimachia christinae* extract was administered, the thickness of the ONL increased in a concentration-dependent manner, with a better effect than lutein. However, the effect of increasing the thickness of the ONL was slightly less in the group administered 200 mg / kg of *Lysimachia christinae* extract compared to the group administered 100 mg / kg. This is attributed to the competitive inhibition of the absorption of active substances by various plant compounds in the extract as the concentration increases. The thickness of the ONL also increased in the group administered neochlorogenic acid (another positive control group), but the results were not statistically significant.

[0111] As can be seen from the above, the extract of *Leptochloa chinensis* has the effect of improving photoreceptor cell damage or deformation by increasing the thickness of the outer nuclear layer (ONL) of the retina.

[0112] [Experiment Example 6]

[0113] Confirming the effect of lacquerwood extract in improving decreased photoreceptor cell activity in an animal model of macular degeneration.

[0114] Electroretinography (ERG) measures the electrical potential of the optic nerve in the retina, representing the electrical signal generated by the optic nerve's response to light, and is used as an indicator to confirm the presence or absence of retinal damage or disease. ERG consists of wave A and wave B, where wave A is generated by photoreceptors, and wave B is generated by the depolarization of bipolar cells in the inner nuclear layer of the retina due to light stimulation. To confirm whether extracts from *Lysimachia christinae* (a type of retinal flamingowood) have an effect on improving reduced optic cell activity in an animal model of optic nerve cell damage induced by excessive light exposure, resulting in optic nerve cell loss similar to that in dry macular degeneration, electroretinography was measured.

[0115] Specifically, the animal model used in Experiment 5 was acclimatized to darkness for 16 hours or longer the day before the autopsy. It was then anesthetized by inhalation with isoflurane (Poran, JW Pharmaceutical), and its pupils were dilated with 0.5% tropicamide. While maintaining body temperature under general anesthesia, an appropriate amount of 1% methylcellulose was added to the eyeball. The gold wire loop of the recording electrode was grounded to the cornea, the reference electrode was placed in the cheek region, and the grounded ELEC solder electrode was grounded to the tail. Electroretinography (ERG) was then measured using a Retipot (Roland Conference, Germany). After illumination with white LED light, waves a and b were obtained from the damascene flash response. Waves a and b were measured by administering the test drugs described in Table 1 above, thereby determining the activity level of the photoreceptor cells.

[0116] As a result, Figure 6 As shown, administration of the above-mentioned *Pyracantha fortuneana* extract confirmed that it improved nerve conduction of waves a and b, and mitigated the reduction in photoreceptor cell activity, with effects superior to lutein. However, the effect of improving nerve conduction was slightly lower in the group administered 200 mg / kg of *Pyracantha fortuneana* extract compared to the group administered 100 mg / kg, but the difference was not significant. The group administered neochlorogenic acid served as another positive control group, also showing improvement in retinal nerve conduction, but this was not statistically significant.

[0117] As can be seen from the above, the extract of *Leptochloa chinensis* has the effect of improving the reduced activity of photoreceptor cells.

[0118] [Experiment Example 7]

[0119] Effects of Yellow Lacquer Wood Extract on Improving Photoreceptor Cell Damage or Deformation in Animal Models of Macular Degeneration

[0120] To confirm the effect of the *Pyracantha fortuneana* extract on improving damage or deformation of photoreceptor cells, N-methyl-N-nitrosourea (MNU), which is toxic to photoreceptor cells, was administered, and changes in the thickness of the outer nuclear layer (ONL) of the retina were measured in an animal model, inducing the loss of optic nerve cells similar to dry macular degeneration in the animal model.

[0121] Specifically, 6-week-old male BALB / c mice (Orient Bio) were acclimatized for one week. A 1% concentration of N-methyl-N-nitrosourea (MNU) (Sigma, USA) was prepared in a 0.05% acetic acid solution and administered intraperitoneally at a concentration of 60 mg / kg per mouse. In the negative control, the same amount of 0.05% acetic acid was administered intraperitoneally only. Starting on the same day after N-methyl-N-nitrosourea administration, the test drug was administered orally once daily for 7 days. The drug concentrations administered are shown in Table 2 below. Neochlorogenic acid, known to help prevent macular degeneration by activating aging retinal nerve cells, and lutein, a component widely known as an ocular nutrient, were set as positive controls, and extracts of *Lysimachia christinae* from the above-described manufacturing example were administered at various concentrations. After administration, necropsy was performed, and eyeballs were extracted, fixed in 10% formalin for one day, and then embedded in paraffin to prepare slide sections. Slides were stained with hematoxylin and eosin (H&E) and observed under an optical microscope. The changes in the thickness of the outer nuclear layer of the retinal tissue were measured to evaluate the damage or deformation of photoreceptor cells.

[0122] [Table 2]

[0123] Test group Group Name Administer drugs Dosage concentration (mg / kg / day) Head count normal control group normal – – 5 negative control group MNU – – 5 Groups given extracts of *Leptochloa chinensis* DM-10 Yellow lacquer wood extract 10 5 Groups given extracts of *Leptochloa chinensis* DM-25 Yellow lacquer wood extract 25 5 Groups given extracts of *Leptochloa chinensis* DM-50 Yellow lacquer wood extract 50 5 Groups given extracts of *Leptochloa chinensis* DM-100 Yellow lacquer wood extract 100 5 Positive control group NCA-6.5 Neochlorogenic acid 6.5 5 Positive control group NCA-13 Neochlorogenic acid 13 5 Positive control group Lutein Lutein 20 5

[0124] As a result, Figure 7 and 8 As shown, in the negative control group, the thickness of the outer nuclear layer (ONL), containing the nuclei of retinal nerve cells, decreased, and when *Pyracantha fortuneana* extract was administered, the thickness of the ONL increased in a concentration-dependent manner, with a better effect than lutein. The thickness of the ONL also increased in the group administered neochlorogenic acid (another positive control group), but the result was not statistically significant. Therefore, *Pyracantha fortuneana* extract can improve photoreceptor damage or deformation by increasing the thickness of the ONL.

[0125] [Experiment Example 8]

[0126] The effect of extract of *Leptochloa chinensis* on the improvement of retinal pigment epithelial cell damage in an animal model of macular degeneration.

[0127] To confirm whether the extract of *Leptochloa chinensis* has the effect of improving retinal pigment epithelial cell damage, the locations of retinal tissue folding deformation were counted and evaluated in an animal model, in which sodium iodate (NaIO3), which is toxic to retinal pigment epithelial cells, was administered to the retinal pigment epithelial cells, thereby inducing morphological deformation in the retinal tissue located above them.

[0128] Specifically, 6-week-old male SD rats (Orient Bio) were acclimatized for one week. A 3.5% concentration of sodium iodate (NaIO3) (Sigma-Aldrich, USA) was prepared in sterile saline to induce retinal pigment epithelial cell damage and degeneration. Each rat was administered intraperitoneally at a dose of 60 mg / kg. In the negative control group, an equal volume of saline was administered intraperitoneally. Starting on the same day as sodium iodate administration, the test drug was administered orally once daily for 14 days. The drug concentrations administered are shown in Table 3 below. Neochlorogenic acid, known to help prevent macular degeneration by activating aging retinal nerve cells, and lutein, a component widely known as an ocular nutrient, were designated as positive controls. Yellow lacquer wood extract, according to the above-described manufacturing example, was administered at various concentrations. After administration, necropsy was performed, and eyeballs were extracted, fixed in 10% formalin for one day, and then embedded in paraffin to prepare glass slides. Slides were stained with hematoxylin and eosin (H&E) and observed under an optical microscope. Damage or deformation of retinal pigment epithelial cells was evaluated by counting the locations where retinal tissue folding and deformation occurred.

[0129] [Table 3]

[0130] Test group Group Name Administer drugs Dosage concentration (mg / kg / day) Head count normal control group normal – – 5 negative control group <![CDATA[NaIO3]]> – – 5 Groups given yellow lacquer wood extract DM-25 Yellow lacquer wood extract 25 5 Groups given extracts of *Leptochloa chinensis* DM-50 Yellow lacquer wood extract 50 5 Groups given extracts of *Leptochloa chinensis* DM-100 Yellow lacquer wood extract 100 5 Positive control group NCA Neochlorogenic acid 6.5 5 Positive control group Lutein Lutein 50 5

[0131] As a result, Figure 9 and 10 As shown, in the negative control group, retinal tissue deformation was observed due to damage to retinal pigment epithelial cells, and numerous folding sites were observed (red arrows). However, when *Lysimachia christinae* extract was administered, the folding sites were inhibited due to the concentration-dependent tissue deformation, and its effect was superior to that of lutein. Therefore, *Lysimachia christinae* extract has the effect of inhibiting retinal tissue deformation, thereby improving damage or deformation of retinal pigment epithelial cells.

[0132] [Experiment Example 9]

[0133] Mechanism of action of yellow lacquer wood extract in improving macular degeneration

[0134] [9-1] Confirming the mechanism of action of *Pyracantha fortuneana* extract in improving light-induced optic nerve cell damage.

[0135] To confirm the mechanism of action of the effect of the extract of *Lysimachia christinae* in improving optic nerve cell damage caused by light irradiation as confirmed in Experiment 5, it was determined whether the damage and loss of optic nerve cells caused by excessive exposure to light sources was due to apoptosis, and whether the effect of *Lysimachia christinae* extract in improving damage or deformation of photoreceptor cells in the macular degeneration animal model was due to its anti-apoptotic effect.

[0136] Specifically, in the animal model of Experiment 5, retinal tissue slides prepared for analyzing optic nerve cell damage and loss were used to analyze the degree of optic nerve cell apoptosis. After administration of the test drug, an autopsy was performed, and the eyeballs were extracted. The eyeballs were fixed in 10% formalin for one day and then embedded in paraffin to prepare slide sections. The tissue sections were dewaxed and hydrated, washed with PBS, treated with a 20 μg / ml proteinase K solution at 37°C for 15 minutes, and then washed with PBS. After reacting in TUNEL reaction mixture solution (In Situ Apoptosis Detection Kit AP, Roche, Germany) at 37°C for 1 hour, the products were observed under a microscope.

[0137] As a result, Figure 11 and 12 As shown, in the retina with macular degeneration induced by light irradiation, apoptosis was induced in all cells of the three layers (i.e., the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL)). In particular, a strong TUNEL-positive reaction was confirmed in the nuclei of optic nerve cells in the outer nuclear layer, confirming that apoptosis mainly occurs in the outer nuclear layer. However, treatment with *Lysimachia christinae* extract confirmed that the reduction in TUNEL positivity in the optic nerve cell nuclear layer inhibited apoptosis in a concentration-dependent manner, and its effect was superior to that of lutein.

[0138] As shown above, the extract of *Leptochloa chinensis* improves the damage to optic nerve cells by inhibiting light-induced retinal cell apoptosis.

[0139] [9-2] Confirming the mechanism of action of *Pyracantha fortunei* extract in inhibiting apoptosis

[0140] To confirm the results of Experiment 9-1, the expression of apoptosis-related proteins in retinal tissue was confirmed.

[0141] Specifically, the retinal tissue used in Example 9-1 was rapidly frozen in liquid nitrogen and then stored at -70°C. After protein quantification of the sample homogenized with homogenization buffer (pH 7.6), 30 μg of each sample was subjected to SDS-PAGE electrophoresis. The proteins were then transferred to a PVDF membrane (Bio-Rad Laboratories, California, USA) using transfer buffer (0.25 M Tris, 1.92 M glycine, pH 8.3–8.4). After dissolving 5% skim milk in TBST (200 mM Tris, 1.37 M NaCl, 0.05% Tween 20) and blocking for 30 minutes, the primary antibody was incubated at 4°C, followed by washing three times with TBST for 10 minutes each time, then reacting with HRP-bound secondary antibody, and washing three more times with TBST for 10 minutes each time. The resulting product was then reacted with an enhanced chemiluminescence (ECL) solution, and the expression levels of Bax, Bcl-2, and hemiasparaginase 3, which are apoptosis-related signaling substances, were confirmed using a LAS-3000 (Fujifilm, Japan).

[0142] As a result, Figure 13 As shown, treatment with *Pyracantha fortuneana* extract significantly decreased the expression of Bax, a pro-apoptotic factor that induces apoptosis, while treatment with *Pyracantha fortuneana* extract increased the expression of Bcl-2, an anti-apoptotic factor. Furthermore, it was confirmed that the expression of a truncated form of hemispartase 3, which is involved in apoptosis, was significantly increased during macular degeneration induction, but this expression was decreased by treatment with *Pyracantha fortuneana* extract.

[0143] As can be seen from the above, the extract of *Leptochloa chinensis* inhibits apoptosis factors while activating anti-apoptotic factors, thereby improving damage to optic nerve cells.

[0144] [9-3] Confirming the mechanism of action of *Pyracantha fortuneana* extract in improving retinal pigment epithelial cell damage.

[0145] When macular degeneration occurs, issues arise regarding damage and permeability of retinal pigment epithelial cells (RPE). To verify the inhibition of this damage and permeability changes in RPE, alterations in the cell-cell tight junction protein ZO-1 were identified.

[0146] Specifically, to separate the retinal pigment epithelial (RPE)-choroid-sclera complex containing the retinal pigment epithelium layer after removing the eyeball during autopsy, the lens, anterior segment (anterior chamber), and retina were removed from the eyeball for observation under a dissecting microscope. The posterior eye cup was then immersed in 4% paraformaldehyde (PFA) and fixed at 4°C for one day. After one day, the posterior eye cup was washed several times with PBS to remove the fixative, and the four corners of the eye cup were cut off and spread flat on a glass slide. The product was treated with blocking solution (10% normal goat serum) for 10 minutes, treated with anti-ZO-1 antibody (1:200, Cat#61-7300, Invitrogen, Carlsbad, California) for 1 hour, and then washed several times. Then, the product was treated with Alexa Fluor 488 goat anti-rabbit (1:500; Cat#A-11008, Invitrogen) as a second antibody for 1 hour, and the flat retinal pigment epithelial cells-choroid-sclera complex was examined under a fluorescence microscope (Olympus Tokyo, Japan).

[0147] As a result, Figure 14 As shown, no changes were observed in the retinal pigment epithelial cell membrane in the normal group, while in the group where sodium iodate (NaIO3) administration caused morphological deformation of the retinal tissue (dAMD), the size of the retinal pigment epithelial cells changed and the cell membrane ruptured. However, it was confirmed that the changes in retinal pigment epithelial cells were inhibited in a concentration-dependent manner in the group treated with *Leptochloa chinensis* extract.

[0148] As shown above, the extract of *Leptochloa chinensis* improves the damage to retinal pigment epithelial cells by maintaining the tight junction proteins that make up retinal pigment epithelial cells.

[0149] In summary, it can be seen that *Rubus idaeus* extract not only inhibits optic nerve cell damage by suppressing the photo-oxidation of drusen, a pathogenic factor in macular degeneration, and inhibits optic nerve cell apoptosis by suppressing the expression of apoptosis signaling substances, but also maintains tight junction proteins in retinal pigment epithelial cells, thereby exhibiting an effect of improving macular degeneration. Figure 15 ).

Claims

1. Use of *Pyracantha fortuneana* extract as an active ingredient in the preparation of a medicament for the prevention or treatment of eye injuries, wherein the eye injury is caused by… N -ATV Huang Ji- N The accumulation of retinyl-ethanolamine oxidation products or drusen is caused by the drusen extract, wherein the extract is obtained by extraction with a 30% v / v aqueous ethanol solution.

2. The use as claimed in claim 1, wherein the eye injury is caused by blue light.

3. The use as described in claim 1, wherein the eye injury is selected from one or more types selected from the group consisting of retinal degeneration, macular degeneration, glaucoma, and dry eye syndrome.

4. The use as described in claim 1, wherein the drug inhibits the effects of blue light. N -ATV Huang Ji- N -Oxidation of retinyl-ethanolamine, inhibition of oxidation N -ATV Huang Ji- N -Retinyl-ethanolamine induces cell death, inhibits the accumulation of drusen, increases the thickness of the outer nuclear layer of the retina, improves the reduction of photoreceptor cell activity, and inhibits retinal tissue deformation.

5. The use as described in claim 1, wherein the eye injury is dry macular degeneration.

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