Composition for preventing or treating glaucoma comprising the extracts of gac pulp

Abstract

The present invention relates to a composition for the prevention or treatment of glaucoma comprising a gac pulp extract. A composition comprising the gac pulp extract of the present invention as an active ingredient can effectively protect optic nerve cells against oxidative stress, and thus can be utilized in various ways, such as pharmaceutical compositions and food compositions, for the prevention, improvement, or treatment of glaucoma. Furthermore, the gac pulp extract of the present invention is derived from natural products and has almost no side effects, allowing for safe and continuous use.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention or treatment of glaucoma comprising a sac pulp extract. Background Technology

[0002] Glaucoma is defined as a disease in which the visual field narrows as the optic nerve inside the eye gradually weakens, and the exact cause of glaucoma has not yet been definitively identified. Glaucoma is an optic neuropathy in which various risk factors, including intraocular pressure, act to induce apoptosis in retinal ganglion cells, and it is one of the leading causes of blindness. Currently, the ultimate goal of glaucoma treatment is to prevent the progression of optic nerve damage; however, since intraocular pressure is a risk factor for progression rather than the cause of the disease, it is impossible to completely block the progression of optic nerve damage solely through the classic treatment method of lowering intraocular pressure.

[0003] Glaucoma can occur due to the accumulation of oxidative stress on the optic nerve caused by factors such as elevated intraocular pressure, which ultimately leads to the death of the optic nerve. In the early stages, the visual field narrows starting from the margins. Currently, no specific treatment for glaucoma has been developed. Although xalatan is used as a symptomatic treatment to lower intraocular pressure, its use is restricted due to side effects associated with long-term use or in pregnant women. Furthermore, while xalatan is effective in lowering intraocular pressure, it does not provide optic nerve protection. Given that the majority of glaucoma patients in Korea develop the condition at normal intraocular pressure, xalatan is largely ineffective in treating the disease; therefore, there is a need for a medication specifically designed for patients with normal-pressure glaucoma. Prior art literature

[0004] Republic of Korea Published Patent No. 10-2016-0104217 The problem to be solved

[0005] The inventors completed the present invention by researching to develop a natural product capable of effectively preventing or treating glaucoma, and confirming that glaucoma pulp extract has optic nerve protective efficacy against oxidative stress.

[0006] The objective of the present invention is to provide a composition for preventing, improving, or treating glaucoma comprising sac pulp extract as an active ingredient.

[0007] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0008] The present invention provides a pharmaceutical composition for the prevention or treatment of glaucoma comprising sac pulp extract as an active ingredient.

[0009] In addition, the present invention provides a food composition for preventing or improving glaucoma or a health functional food composition comprising sac pulp extract as an active ingredient.

[0010] The above glaucoma can occur due to the accumulation of oxidative stress in the optic nerve.

[0011] The above composition may have a protective effect on optic nerve cells.

[0012] The above composition can reduce MAPK (Mitogen-activated protein kinase) activity. Effects of the invention

[0013] A composition containing the gac pulp extract of the present invention as an active ingredient can effectively protect optic nerve cells against oxidative stress, and can be utilized in various ways, such as pharmaceutical compositions and food compositions for the prevention, improvement, or treatment of glaucoma. In addition, the gac pulp extract of the present invention is derived from natural products and has almost no side effects, so it can be safely used continuously. Brief explanation of the drawing

[0014] Figure 1 shows the results of analyzing the protective efficacy of optic nerve cells against oxidative stress of ginkgo pulp extract. Figure 2 shows the results of confirming the viability of optic nerve cells through flow cytometry analysis. Figure 3 shows the results of analyzing the effect of ginkgo pulp extract on MAPK (Mitogen-activated protein kinase) signaling. Figures 4 and 5 show the results of confirming the neuroprotective efficacy of ginkgo pulp extract using a rodent ONC model, which is an animal model of glaucoma. (5 mg / kg, p = 0.0156; 50 mg / kg, p = 0.0034; xalatan, p = 0.006). Specific details for implementing the invention

[0015] The inventors completed the present invention by experimentally confirming that an extract of gac pulp, a natural product with little to no side effects, has the effect of protecting scleroderma cells against oxidative stress.

[0016] The present invention will be described in detail below.

[0017] The present invention provides a composition for preventing, improving, or treating glaucoma comprising a gac pulp extract as an active ingredient.

[0018] The above glaucoma may have been caused by the accumulation of oxidative stress in the optic nerve.

[0019] The above composition may have a protective effect on optic nerve cells.

[0020] The above composition can reduce MAPK (Mitogen-activated protein kinase) activity.

[0021] In this specification, "prevention" refers to any act of delaying the onset of glaucoma by administering the composition of the present invention, and "treatment" and "improvement" refer to any act of improving or beneficially altering the symptoms of glaucoma by administering the composition of the present invention.

[0022] Glaucoma is a disease in which the visual field narrows as the optic nerve inside the eye gradually weakens. Although the cause of glaucoma has not yet been definitively identified, it is known that damage to the optic nerve leads to visual impairment as a result.

[0023] Gac is a fruit widely cultivated in Vietnam within Southeast Asia and also grows wild in southern China and northeastern Australia; its scientific name is M omordica cochinchinensis Gac is a traditional Vietnamese medicinal herb known for its efficacy against inflammation, cancer, and skin diseases. Gac is edible and is listed in the Korean Food Code. Additionally, gac pulp is a byproduct that is discarded as it is not utilized as part of the shell.

[0024] The above-mentioned prickly ash pulp extract can be extracted using a polar solvent or / and a non-polar solvent.

[0025] The above polar solvent may include one or more selected from the group consisting of (i) water, (ii) alcohol (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMFO (dimethyl-formamide), and (v) DMSO (dimethyl sulfoxide). The above non-polar solvent may include one or more selected from the group consisting of acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkanes, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

[0026] The above ginkgo pulp extract can preferably be extracted with alcohol, and more preferably with ethanol. Most preferably, it can be extracted with 100% ethanol. 100% ethanol may be the most advantageous in terms of extracting the expected active ingredients.

[0027] As used in this specification, the term 'extract' has the meaning commonly used in the art as a crude extract, as described above, but in a broader sense, it also includes fractions obtained by further fractionating the extract. That is, the above-mentioned peat extract includes not only that which is obtained using the extraction solvent described above, but also that which is obtained by additionally applying a purification process thereto. For example, fractions obtained through various additional purification methods, such as a fraction obtained by passing the above-mentioned extract through an ultrafiltration membrane having a certain molecular weight cut-off value, or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity), are also included in the natural product extract of the present invention.

[0028] The ginkgo pulp extract used in the present invention can be produced in a powder state by additional processes such as vacuum distillation and freeze-drying or spray-drying.

[0029] Specifically, the above gac pulp extract is preferably extracted by adding a solvent to the gac pulp in an amount of 2 to 50 times the weight of the gac pulp, and more preferably by adding 10 to 30 times the weight of the gac pulp, but is not limited thereto. The extraction temperature is preferably 30°C to 150°C, and more preferably 35°C to 45°C, but is not limited thereto. The extraction time is preferably 10 minutes to 20 hours, and more preferably 10 minutes to 30 minutes, but is not limited thereto. As an extraction method, accelerated solvent extraction, cold maceration, ultrasonic extraction, or reflux cooling extraction methods may be used, but accelerated solvent extraction and cold maceration extraction are preferred. Accelerated solvent extraction and cold maceration extraction may be the most advantageous in terms of extracting the expected active ingredients.

[0030] It is preferable that the number of extractions be 1 to 5 times, and it is more preferable to extract 2 to 3 times, but is not limited thereto. Additionally, the extract may be used after diluting, concentrating, or purifying and drying it after dilution or concentration.

[0031] In this specification, the term "containing as an active ingredient" means containing an amount sufficient to achieve the efficacy or activity of the gac pulp extract. The present invention is a composition extracted from gac pulp, which is a natural plant material, and the upper limit of the quantity of said gac pulp extract included in the composition of the present invention may be selected and implemented by a person skilled in the art within an appropriate range. For example, said gac pulp extract may be included in an amount of 0.00001 to 100 weight%, 0.01 to 80 weight%, or 0.1 to 30 weight% relative to the total weight of the composition, but is not limited thereto.

[0032] Pharmaceutical composition for the prevention or treatment of glaucoma comprising gac pulp extract as an active ingredient

[0033] The composition of the present invention can be prepared as a pharmaceutical composition.

[0034] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0035] According to a preferred embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the gac pulp extract of the present invention described above; and (b) a pharmaceutically acceptable carrier. As specified herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the gac pulp extract described above.

[0036] Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0037] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0038] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The general dosage of the pharmaceutical composition of the present invention is within the range of 0.001 to 100 mg / kg for adults. Administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosages.

[0039] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0040] Food composition for preventing or improving glaucoma containing ginkgo pulp extract as an active ingredient

[0041] The composition of the present invention may be provided as a food composition or a health functional food composition. When a composition for the prevention, improvement, or treatment of glaucoma containing the gac pulp extract of the present invention as an active ingredient is prepared as a food composition, it includes not only the gac pulp extract as an active ingredient but also ingredients that are typically added during food preparation, such as, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of the carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as conventional sugars like dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavorings, natural flavorings [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavorings (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to the natural product extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0042] The formulation of the above food composition or health functional food composition can be in the form of powder, granule, pill, tablet, or capsule, as well as any form of general food or beverage.

[0043] There are no specific restrictions on the types of food mentioned above, and examples of food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all food in the conventional sense.

[0044] Generally, when manufacturing food or beverages, the above-mentioned ginkgo pulp extract may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or for the purpose of health control, the above amount may be less than the above range, and furthermore, since the present invention uses natural substances, there is no problem in terms of safety, so it may be used in an amount greater than the above range.

[0045] The present invention will be explained in more detail below through examples. The purpose, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the examples described herein and may be embodied in other forms. The examples introduced herein are provided to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

[0046] Example 1. Preparation of Gac Pulp Extract 1

[0047] 15g of gac pulp powder was dried and then extracted using accelerated solvent extraction to obtain 1.413g of gac pulp extract (pressure 1500psi, temperature 40℃, heating for 15 minutes, 3 cycles). 75ml of 100% ethanol was used as the extraction solvent.

[0048] Example 2. Preparation of Gac Pulp Extract 2

[0049] 1 g of gac pulp powder was dried and extracted using the cold maceration method to obtain 147.2 mg of gac pulp extract. 100 ml of 100% ethanol was used as the extraction solvent. The extraction conditions were as follows: 100 ml of 100% ethanol was added to 1 g of gac pulp powder and stirred at room temperature for 48 hours. After removing the solid by filtering it through filter paper at room temperature, the ethanol solution was evaporated under reduced pressure to obtain 147.2 mg of extract.

[0050] Experimental Example 1. Analysis of the viability of RGC5 optic neurons under oxidative stress

[0051] The protective efficacy of the gac pulp extract of Example 1 against optic neuronal apoptosis was analyzed. After inducing oxidative stress in RGC5 cells by treating with Glutamate 10 mM + BSO (butionine sulfoximine) 0.5 mM, The viability of optic nerve cells was confirmed after 2 hours of incubation following treatment with Gac pulp extract (Fig. 1). In Fig. 1a, NAC is the positive control, Nontreat ASE is the Gac pulp extract of Example 1, and Residue ASE is a sample obtained by extracting the residue after the first extraction according to the method of Example 1 using the same method. It was confirmed that treatment with Gac pulp extract exhibited a protective efficacy of over 80% on optic nerve cells even at low concentrations.

[0052] Figure 2 shows the results of verifying cell viability using flow cytometry. It was confirmed that about 70% of the cells died in the group treated with Glutamate 10 mM + BSO 0.5 mM, and it was confirmed that cell death was significantly inhibited in the group treated with ginkgo pulp extract.

[0053] Experimental Example 2. MAPK Signal Analysis

[0054] The degree of MAPK activity was confirmed after inducing oxidative stress for 10 hours by treating RGC5 cells with 10 mM Glutamate + 0.5 mM BSO (buthionine sulfoximine). In addition, after treating with the ginkgo pulp extract of Example 1 and culturing for 2 hours, the degree of MAPK activity was confirmed after inducing oxidative stress for 10 hours in RGC5 cells by treating with 10 mM Glutamate + 0.5 mM BSO (buthionine sulfoximine).

[0055] When oxidative stress was induced in RGC5 cells by treating them with 10 mM Glutamate + 0.5 mM BSO (buthionine sulfoximine), MAPK signaling increased, but it was confirmed that MAPK activity decreased upon treatment with ginkgo pulp extract (Fig. 3).

[0056] Experimental Example 3. Confirmation of the inhibitory effect of gac pulp extract on the death of retinal photoreceptors in C57BL / 6J rats induced by a partial optic nerve crush (PONC) model.

[0057] The inhibitory effect of the Gac pulp extract of Example 1 on the death of retinal photoreceptor cells in C57BL / 6J rats induced by a partial optic nerve compression model was confirmed. Starting one week before the experiment, C57BL / 6J rats were orally administered Gac pulp extract and the control group, 0.01 M PBS, according to concentration. After anesthesia by intramuscularly injecting 1 ml / kg of a mixture of 2% Rumpoon injection solution and Zolletil, the posterior conjunctiva was carefully incised to expose the optic nerve without damaging blood vessels, and a compression injury was applied for 5 seconds to the optic nerve located 2 mm posterior to the eyeball using cross-action forceps. To prevent infection after surgery, one drop of 0.5% Vigamox eye drops (ALcon Laboratories, Inc.) was instilled. The experimental group was orally administered 5 mg / kg and 50 mg / kg of Gac pulp extract once daily, while the control group was orally administered the same dose of 0.01 M PBS.

[0058] Six days after optic nerve injury, the mice were anesthetized again, and the scalp was incised. To stain optic nerve cells, a surgical drill was used to perforate the superior colliculus of the brain, which is connected to the optic nerve, and 3% FluoroGold was injected. The incision was sutured, and antibiotic ointment was applied to prevent infection. Twelve days after optic nerve injury, fundus photography and retinal tomography were performed using Phoenix's micron4 to assess the extent of damage to the retinal ganglion cell layer. Additionally, the eyeballs were enucleated and fixed in 4% parafromaldehyde for four hours. The fixed eyeballs were separated from the choroid, four radial incisions were made, and the eyeballs were mounted on slides in a clover leaf shape. For each experimental group, fluorescent optic nerve cells were identified using a fluorescence microscope, and images were taken at magnifications of 40x and 200x.

[0059] The experimental results are as follows. Optic nerve compression not only reduced the thickness of the retinal ganglion cell layer but also induced apoptosis, thereby reducing cell viability. When gac pulp extract was administered, the reduction in the thickness of the retinal ganglion cell layer was prevented, and cell viability was increased by significantly protecting against retinal cell apoptosis, confirming that gac pulp extract has a protective effect against external damage to the retina (Figs. 4 and 5).

[0060] Hereinafter, examples of the preparation of a pharmaceutical or food containing the above-mentioned ginkgo pulp extract as an active ingredient according to the present invention are described; however, the present invention is not intended to be limited but merely to be described in detail. Using the above-mentioned active ingredient, the pharmaceutical or food composition of Preparation Example 1 or 2 was prepared according to a conventional method according to the following compositional ingredients and compositional ratios.

[0061] [Preparation Example 1] Preparation of a pharmaceutical composition

[0062] <1-1> Preparation of Powders

[0063] Ginkgo pulp extract 20 mg

[0064] Lactose monohydrate 100 mg

[0065] Talc 10 mg

[0066] The above ingredients were mixed and filled into an airtight bag to produce a powder.

[0067] <1-2> Preparation of Tablets

[0068] Ginkgo pulp extract 10 mg

[0069] 100 mg corn starch

[0070] Lactose monohydrate 100 mg

[0071] Magnesium stearate 2 mg

[0072] After mixing the above ingredients, tablets were manufactured by compressing them according to the conventional method of manufacturing tablets.

[0073] <1-3> Manufacture of Capsules

[0074] Ginkgo pulp extract 10 mg

[0075] Microcrystalline cellulose 3 mg

[0076] Lactose monohydrate 14.8 mg

[0077] Magnesium stearate 0.2 mg

[0078] After mixing the above ingredients, a capsule was manufactured by filling it into a gelatin capsule according to a conventional method for manufacturing capsules.

[0079] <1-4> Preparation of Injectables

[0080] Ginkgo pulp extract 10 mg

[0081] Mannitol 180 mg

[0082] 2974 mg sterile distilled water for injection

[0083] Sodium monohydrogen phosphate 26 mg

[0084] After mixing the above ingredients, the above ingredients were prepared in an amount of 2 mL per ampoule according to the conventional method of preparing injectables.

[0085] <1-5> Preparation of Liquid Formulations

[0086] Ginkgo pulp extract 10 mg

[0087] 10 mg isomerized sugar

[0088] Mannitol 5 mg

[0089] Appropriate amount of purified water

[0090] Appropriate amount of lemon flavor

[0091] The above ingredients are dissolved in purified water according to a conventional manufacturing method, an appropriate amount of lemon scent is added, then purified water is added to adjust the total volume to 100 mL, sterilized, and filled into a brown bottle to prepare a liquid preparation.

[0092] [Preparation Example 2] Preparation of a health functional food

[0093] <2-1> Manufacturing of Health Supplements

[0094] Ginkgo pulp extract 10 mg

[0095] Appropriate amount of vitamin mixture

[0096] Vitamin A acetate 70 µg

[0097] Vitamin E 1.0 mg

[0098] Vitamin B1 0.13 mg

[0099] Vitamin B2 0.15 mg

[0100] Vitamin B6 0.5 mg

[0101] Vitamin B 12 0.2 µg

[0102] Vitamin C 10 mg

[0103] Biotin 10 µg

[0104] Nicotinamide 1.7 mg

[0105] Folic acid 50 µg

[0106] Calcium pantothenate 0.5 mg

[0107] Appropriate amount of mineral mixture

[0108] Ferrous sulfate 1.75 mg

[0109] Zinc oxide 0.82 mg

[0110] Magnesium carbonate 25.3 mg

[0111] Potassium dihydrogen phosphate 15 mg

[0112] 55 mg dicalcium phosphate

[0113] Potassium citrate 30 mg

[0114] Calcium carbonate 100 mg

[0115] Magnesium chloride 24.8 mg

[0116] The composition ratio of the above vitamin and mineral mixture is composed of ingredients that are relatively suitable for health food as a preferred example, but the mixing ratio may be modified at will. The above ingredients may be mixed according to a conventional method for manufacturing health food, then granules may be prepared and used to manufacture a health food composition according to a conventional method.

[0117] <2-2> Preparation of Health Drinks

[0118] Ginkgo pulp extract 10 mg

[0119] 15 g of Vitamin C

[0120] Vitamin E (powder) 100 g

[0121] Iron lactate 19.75 g

[0122] 3.5 g zinc oxide

[0123] Nicotinamide 3.5 g

[0124] Vitamin A 0.2 g

[0125] Vitamin B1 0.25 g

[0126] Vitamin B2 0.3 g

[0127] Quantification of purified water

[0128] The above ingredients are mixed according to a conventional method for manufacturing health drinks, then stirred and heated at 85°C for about 1 hour, the resulting solution is filtered, obtained in a sterilized 2-liter container, sealed and sterilized, and then refrigerated and used to manufacture the health drink composition of the present invention.

[0129] Although the above composition ratio was formulated by mixing ingredients relatively suitable for beverages as a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences, such as consumer groups, countries of demand, and intended uses.

[0130] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of glaucoma comprising gac pulp extract as an active ingredient, wherein the composition is characterized by reducing MAPK (Mitogen-activated protein kinase) activity. Claim 2 A pharmaceutical composition according to claim 1, wherein the glaucoma is caused by the accumulation of oxidative stress in the optic nerve. Claim 3 A pharmaceutical composition according to claim 1, characterized in that the composition has an optic nerve cell protective effect. Claim 4 delete Claim 5 A food composition for preventing or improving glaucoma comprising gac pulp extract as an active ingredient, wherein the composition is characterized by reducing MAPK (Mitogen-activated protein kinase) activity.

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