Patent Application
20220047664
The present invention relates to a novel use of a Centella asiatica extract for protecting retinal nerve cells from oxidative stress, improving the survival rate of retinal nerve cells or retinal pigment epithelial cells through increased sugar availability, inhibiting the apoptosis of retinal nerve cells, or preventing, ameliorating, or treating glaucoma or macular degeneration.
Glaucoma is a disease caused by damage to the optic nerve due to an increase in intraocular pressure or abnormal blood circulation of the optic nerve, is characterized by temporary or permanent visual field defects and decreased vision, and may lead to permanent blindness if not treated. The general cause of the onset thereof is increased intraocular pressure due to circulatory disorders of the aqueous humor, and at the age group of 40s or older, an aqueous humor outlet becomes narrower and the amount of aqueous humor produced increases, resulting in increased intraocular pressure, which causes pressure on and damage to the optic nerve. In addition, although intraocular pressure is normal, glaucoma develops in some cases, and especially in Korea and Japan, there are more patients with glaucoma with normal intraocular pressure (about 70-80% of glaucoma patients), and the cause is known as an optic nerve blood circulation disorder. Practically, it is known that there are many cases of peripheral blood circulation disorders caused by metabolic diseases in patients within the range of normal intraocular pressure. Ultimately, glaucoma leads to the death of optic nerve cells such as retinal node cells (Almasieh et al., 2012), and substances capable of preventing and treating the death of optic nerve cells can be used as foods and therapeutic agents for inhibiting the onset and progression of glaucoma.
The macula is the part of the eye's retina that receives light most clearly and accurately because optic cells are concentrated, and plays a very important role in vision, and the macular is degenerated by various causes, causing vision disorders, which is referred to as macular degeneration. Macular degeneration is a disease caused by gradual degeneration of retinal epithelial cells, the retina, and the capillary layer of the choroid, and in early stages, macular degeneration is characterized by observation of abnormal retinal epithelial cells in which extracellular deposits called drusen are formed between the Bruch's membrane and retinal epithelial cells. In addition, in later stages, macular degeneration is broadly divided into exudative (wet) macular degeneration and atrophic (dry) macular degeneration, and in both types of macular degeneration, optic cells in the macula are gradually destroyed, and thus the function of the macula deteriorates over time and vision at the central portion decreases. The main etiology of macular degeneration is known to be oxidative stress and inflammation, and particularly, when A2E molecules, which are by-products after intracellular metabolism, are not excreted in retinal pigment epithelial cells and gradually accumulate, they undergo an oxidative stress process and eventually leads to cell death (Sparrow and Cai, 2001). Thus, it is known that, since blocking the accumulation of the causative agent A2E molecule itself can prevent oxidation in retinal cells, the risk itself of macular degeneration can be reduced.
Glaucoma and macular degeneration are one of the three major causes of blindness, along with diabetic retinopathy, and the death of optic nerve cells or retinal nerve cells can be the main cause, and it is known that these two diseases are rapidly increasing in patients in their 30s to 50s as well as the elderly due to the recent increase in the prevalence of metabolic diseases and the like.
Recently, medicines and health functional foods for maintaining eye health such as the prevention of oxidative damage to the eyes have been actively developed, and particularly, energy improvement agents using wild blueberry-derived anthocyanoside as a main ingredient have been developed, and herbal medicines such as cassia seeds and Lycium have long been used for vision maintenance and vision improvement. However, there are no studies on a method of preventing or treating glaucoma and macular degeneration of the retina, which are related to eye health, using Centella asiatica.
Therefore, the inventors of the present invention have recognized the importance of prevention and treatment of glaucoma, macular degeneration, and the like related to eye health, and used various natural substances to select natural resources exhibiting proliferative efficacy on retinal nerve cells and retinal pigment epithelial cells and capable of protecting oxidative damage to retinal cells. As a result, a Centella asiatica extract was found to exhibit excellent proliferative efficacy on optic nerve cells and retinal cells, and particularly, to inhibit oxidative damage caused by A2E in retinal cells. In addition, the Centella asiatica extract has greatly increased sugar availability in retinal cells, which indicates excellent effects of recovery and protection from cell damage, and thus the Centella asiatica extract of the present invention has a very high possibility of commercialization as a composition for preventing, ameliorating, or treating glaucoma and macular degeneration.
Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a pharmaceutical composition for preventing, ameliorating, or treating glaucoma or macular degeneration including a Centella asiatica extract.
It is another object of the present invention to provide a health functional food composition for preventing and ameliorating glaucoma and macular degeneration including a Centella asiatica extract.
The objects of the present invention are not limited to the aforementioned objects. Other objects of the present invention will become more apparent from the following description, and will be realized by the means described in the claims and combinations thereof.
In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a food composition for protecting retinal nerve cells from oxidative stress, including a Centella asiatica extract as an active ingredient.
In one embodiment of the present invention, the Centella asiatica extract improves the survival rate of retinal nerve cells or retinal pigment epithelial cells through increased sugar availability.
In one embodiment of the present invention, the Centella asiatica extract inhibits the apoptosis of retinal nerve cells.
In one embodiment of the present invention, the Centella asiatica extract prevents the death of retinal nerve cells from oxidative stress caused by A2E.
In one embodiment of the present invention, the food composition prevents or ameliorates a retinal nerve cell damage syndrome caused by oxidative stress.
In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for preventing, ameliorating, or treating a retinal nerve cell damage syndrome caused by oxidative stress, including a Centella asiatica extract as an active ingredient.
In one embodiment of the present invention, the Centella asiatica extract improves the survival rate of retinal nerve cells or retinal pigment epithelial cells through increased sugar availability.
In another embodiment of the present invention, the Centella asiatica extract inhibits the apoptosis of retinal nerve cells.
In another embodiment of the present invention, the Centella asiatica extract prevents the death of retinal nerve cells from oxidative stress caused by A2E.
In another embodiment of the present invention, the disease is macular degeneration or glaucoma.
In one or more embodiments of the present invention, the Centella asiatica extract is an extract obtained through extraction with water, a C1-6 lower alcohol, or a mixed solvent thereof.
In one or more embodiments of the present invention, the Centella asiatica extract is a 30%-90% aqueous ethanol extract.
In one or more embodiments of the present invention, the Centella asiatica extract is administered at a dose of 0.004 mg/kg/day to 40 mg/kg/day.
In another embodiment of the present invention, the composition is a composition for topical administration.
In another embodiment of the present invention, the composition is an ophthalmic formulation.
In one or more embodiments of the present invention, the Centella asiatica extract is included in the composition at a concentration of 5 μg/ml to 500 μg/ml with respect to the total weight of the composition.
The Centella asiatica extract of the present invention effectively promotes the proliferation of retinal nerve cells and retinal pigment epithelial cells by increasing glucose uptake, and protects retinal cells from oxidative damage caused by A2E. Thus, the Centella asiatica extract can ameliorate, prevent, or treat eye diseases caused by damage to retinal nerve cells and retinal pigment epithelial cells, i.e., glaucoma and macular degeneration.
The effects of the present invention are not limited to the aforementioned effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Unless otherwise specified, all numbers, figures, and/or expressions that represent ingredients, reaction conditions, and contents of ingredients used in the specification are approximations that reflect various uncertainties of measurement occurring inherently in obtaining these figures, among other things. For this reason, it should be understood that, in all cases, the term “about” should be considered to modify all numbers, figures, and/or expressions. In addition, when numeric ranges are disclosed in the description, these ranges are continuous and include all numbers from the minimum to the maximum including the maximum within the range, unless otherwise defined. Furthermore, when the range refers to an integer, it includes all integers from the minimum to the maximum including the maximum within the range, unless otherwise defined.
It should be understood that, in the specification, when the range refers to a parameter, the parameter encompasses all figures including end points disclosed within the range. For example, the range of “5 to 10” includes figures of 5, 6, 7, 8, 9, and 10, as well as arbitrary sub-ranges such as ranges of 6 to 10, 7 to 10, 6 to 9, and 7 to 9, and any figures, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9, between appropriate integers that fall within the range. In addition, for example, the range of “10% to 30%” encompasses all integers that include figures such as 10%, 11%, 12%, and 13%, as well as 30%, and any sub-ranges of 10% to 15%, 12% to 18%, or 20% to 30%, as well as any figures, such as 10.5%, 15.5%, and 25.5%, between appropriate integers that fall within the range.
Hereinafter, the present invention will be described in detail.
The present invention relates to a composition for preventing, ameliorating, or treating glaucoma and macular degeneration, including a Centella asiatica extract. Specifically, the composition of the present invention induces the proliferation of retinal nerve cells and retinal pigment epithelial cells by increasing glucose uptake and protects cells from oxidative stress caused by A2E, and may be effectively used as a composition for preventing, ameliorating, or treating glaucoma and macular degeneration for eye health.
Centella asiatica is a perennial creeping herb belonging to the family Umbelliferae, and grows naturally in hot and humid areas, and in oriental medicine has been used as a herbal medicine for skin diseases, antipyretic action, hemoptysis, diuresis, tonicity, agenesis, leukorrhea, arthritis, and the like (Youngno LEE, 2006). As known main ingredients of Centella asiatica, triterpenic acid sugar esters such as asiaticoside, madecassoside, brahmoside, and brahminoside are reported, and it is known that, when they are hydrolyzed, asiatic acid, madecassic acid, thankunic acid, isothankunic acid, and the like are produced. Asiaticoside and madecassoside, which are major ingredients of Centella asiatica and are pentacyclic triterpene glycosides belonging to the α-amyrin-ursolic acid group, have long been used for the treatment of skin wounds, chronic ulcers, or the like, and are reported to have leprosy treatment efficacy, antimicrobial efficacy, and therapeutic effects on wounds, gastric ulcers, various skin diseases, mental diseases, tuberculosis, venous diseases, dementia, and the like. Due to these effects, Centella asiatica is used in various applications such as a food product, a skin therapeutic agent, a wound therapeutic agent, a memory enhancement agent, and a tonic, and even in Korea, is used in fields such as pharmaceuticals, cosmetics, and functional foods, but no literature published to date has revealed that a Centella asiatica extract and main ingredients thereof are effective in preventing, ameliorating, or treating glaucoma and macular degeneration.
The present invention provides a pharmaceutical composition for preventing or treating glaucoma and macular degeneration, including a Centella asiatica extract. The composition may be a liquid extract or a lyophilized powder form of the liquid extract.
The present invention also provides a health functional food composition for preventing or ameliorating glaucoma and macular degeneration including a Centella asiatica extract. The composition may be a liquid extract or a lyophilized powder form of the extract.
Hereinafter, various embodiments of the present invention will be described.
An embodiment of the present invention provides a food composition for protecting retinal nerve cells from oxidative stress including a Centella asiatica extract as an active ingredient.
The health functional food composition according to the present invention includes a Centella asiatica extract as an active ingredient, and may be ingested to improve vision through the inhibition of glaucoma and macular degeneration. The active ingredient may be ingested as a food prepared as a tablet, capsule, powder, granule, pill, liquid, suspension, or the like, or may be added to general foods and ingested. Since the health functional food uses food as a raw material, unlike general drugs, there are no side effects that may occur when administered for a long time. The content of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or amelioration). Generally, 0.1 wt % to 90 wt % of the active ingredient may be included in the health functional food composition. However, in the case of long-term ingestion for health and hygienic purposes or for health control purposes, the amount may fall within the above range or less, and since there is no problem in terms of safety, the active ingredient may also be used in an amount within or exceeding the above range.
The type of the food is not particularly limited. Examples of foods to which the active ingredient may be added include drinks, meats, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionaries, pizzas, instant noodles, other kinds of noodles, gum, dairy products including ice creams, various kinds of soup, beverages, alcoholic beverages, vitamin complexes, dairy products, processed dairy products, and the like, and all health functional foods in the ordinary sense are included. The form of the food is not particularly limited, and may be any form such as a solid form, a semi-solid form, a gel form, a liquid form, or a powder form.
The health functional food composition of the present invention may be prepared as a beverage. Ingredients included in the beverage other than the active ingredient may be selected without any particular limitation, and may include additional ingredients such as various flavoring agents or natural carbohydrates, as in general beverages. Examples of the above-described natural carbohydrates include general sugars such as monosaccharides, e.g., glucose and fructose, disaccharides, e.g., maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent other than the above-described flavoring agents, a natural flavoring agent (thaumatin and stevia extracts (e.g., rebaudioside A and glycyrrhizin) and a synthetic flavoring agent (saccharin and aspartame) are preferably used. The proportion of the natural carbohydrates generally ranges from about 1 g to about 20 g, preferably about 5 g to about 12 g, with respect to 100 ml of the composition of the present invention.
In addition, the health functional food composition of the present invention may include, as additives, in addition to the active ingredient, various nutritional supplements, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, and enhancers (cheese, chocolate, and the like), pectic acid and salts thereof, alginic acid and salts thereof, an organic acid, a protective colloid thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohols, a carbonating agent used in carbonated beverages, and the like. In addition, the health functional food composition may include flesh for the preparation of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients may be used alone, or a combination thereof may be used. The proportion of these additives is not very important, but the amounts of the additives in the health functional food composition of the present invention generally range from about 0.1 wt % to 20 wt %.
In one embodiment of the present invention, the Centella asiatica extract improves the survival rate of retinal nerve cells or retinal pigment epithelial cells through increased sugar availability.
In one embodiment of the present invention, the Centella asiatica extract inhibits the apoptosis of retinal nerve cells.
In one embodiment of the present invention, the Centella asiatica extract prevents the death of retinal nerve cells from oxidative stress caused by A2E.
In one embodiment of the present invention, the food composition prevents or ameliorates a retinal nerve cell damage syndrome caused by oxidative stress.
Another embodiment of the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating a retinal nerve cell damage syndrome caused by oxidative stress, including a Centella asiatica extract as an active ingredient.
In one embodiment of the present invention, the Centella asiatica extract improves the survival rate of retinal nerve cells or retinal pigment epithelial cells through increased sugar availability.
In another embodiment of the present invention, the Centella asiatica extract inhibits the apoptosis of retinal nerve cells.
In another embodiment of the present invention, the Centella asiatica extract prevents the death of retinal nerve cells due to oxidative stress caused by A2E.
In another embodiment of the present invention, the disease is macular degeneration or glaucoma.
When the pharmaceutical composition is used clinically, the pharmaceutical composition may be mixed with general carriers in the pharmaceutical field and formulated as general preparations in the pharmaceutical field, for example, various preparations such as preparations for oral administration such as tablets, capsules, powders, granules, pills, liquids, and suspensions, injectable preparations in the form of a ready-to-use-type injectable dry powder and the like that can be prepared and used as an injectable solution or suspension or distilled water injectable at the time of injection, or ointments. Pharmaceutical preparations prepared using general carriers may be administered orally, or may be applied parenterally, for example, intravenously, subcutaneously, intraperitoneally, or topically. Thus, the pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of drugs.
A carrier, an excipient, and a diluent that may be included in the pharmaceutical composition of the present invention may include one or more selected from lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, hydroxymethyl cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, povidone, crospovidone, croscarmellose sodium, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, Neusilin, colloidal silicon dioxide, lactose, talc, magnesium stearate, colloidal stearyl magnesium, and mineral oil.
The pharmaceutical composition is formulated using generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, troches, lozenges, capsules, and the like, and such solid preparations are formulated by mixing the composition with at least one excipient, e.g., lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, calcium carbonate, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, elixirs, syrups, and the like, and these liquid preparations may include, in addition to simple commonly used diluents such as water and liquid paraffin, various excipients, for example, a wetting agent, a sweetener, a flavoring agent, a preservative, and the like. Preparations for parenteral administration include an aqueous sterile solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, and a suppository. Non-limiting examples of the non-aqueous solvent and the suspension include propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, and an injectable ester such as ethyl oleate. Examples of suppository bases include Witepsol, Macrogol, Tween 61, cacao butter, laurin, glycerogelatin, and the like. Parenteral administration may be generally performed via subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
A suitable dose of the pharmaceutical composition of the present invention varies depending on the age and body weight of the patient, the severity of the disease, the drug form, and the administration route and period, but may be appropriately selected by those of ordinary skill in the art. To obtain desired effects, the pharmaceutical composition of the present invention may be administered in an amount of 0.01 mg/kg/day to 10 g/kg/day, preferably 1 mg/kg/day to 1 g/kg/day. The pharmaceutical composition may be administered multiple times a day, preferably one to six times a day, at certain time intervals in accordance with the determination of a doctor or a pharmacist.
In one embodiment, the composition may be safely used for preventing, ameliorating, or treating glaucoma and macular degeneration as a natural product capable of being ingested and taken on a daily basis.
The Centella asiatica extract may be obtained by a conventional extraction method, and the extract may be an extract having a powder form through drying under reduced pressure and freeze drying.
In one or more embodiments of the present invention, the Centella asiatica extract is an extract obtained through extraction with water, a C1-6 lower alcohol, or a mixed solvent thereof.
In the composition of the present invention for preventing, ameliorating, or treating glaucoma and macular degeneration, which has the above-described composition, the extraction process may be repeated as needed, and an extract obtained by performing extraction 2 to 5 times may be used. In addition, a dry powder-type extract may be prepared by freeze-drying the extract, and may be used in a composition for preventing, ameliorating, or treating glaucoma and macular degeneration.
It is desirable from the aspect of extraction efficiency that the content of alcohol in the extraction solvent be maintained at a concentration of 0 wt % to 95 wt %, preferably 30 wt % to 95 wt %, and more preferably 50 wt %. Any alcohol may be applied, as long as it is generally used in the art, preferably a C1-5 alcohol. The alcohol may be one or more selected from methanol, ethanol, isopropanol, propanol, and butanol. More preferably, ethanol or the like may be used as the alcohol.
In one or more embodiments of the present invention, the Centella asiatica extract is a 30%-90% aqueous ethanol extract.
The extraction method is a method commonly known in the art, for example, a method using an extraction device, such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, or ultrasonic extraction, a method using an adsorption resin including XAD and HP-20, or the like.
In addition, the extract is concentrated under reduced pressure using a vacuum rotary evaporator or the like to obtain an extract. In addition, the obtained extract may also be subjected to drying under reduced pressure, vacuum drying, boiling drying, spray drying, room-temperature drying, freeze drying, or the like as needed. In particular, the freeze-drying method is advantageous in that the loss of volatile organic substances in the extract can be reduced.
In one embodiment of the present invention, the Centella asiatica extract is a 40% to 60% aqueous ethanol solution extract.
In one embodiment of the present invention, the composition promotes the proliferation of optic nerve cells and retinal cells or protects cells from oxidative stress, for eye health.
In one or more embodiments of the present invention, the Centella asiatica extract is administered at a dose of 0.01 mg/kg/day to 10 g/kg/day.
In another embodiment of the present invention, the composition is a composition for topical administration.
In another embodiment of the present invention, the composition is an ophthalmic formulation.
In one or more embodiments of the present invention, the Centella asiatica extract is included in the composition at a concentration of 5 μg/ml to 500 μg/ml with respect to the total weight of the composition.
The composition has an effect of effectively proliferating retinal nerve cells and retinal pigment epithelial cells and protecting the cells from oxidative stress. In addition, the composition may be safely used for preventing, ameliorating, or treating glaucoma and macular degeneration as a natural product capable of being ingested and taken on a daily basis.
The Centella asiatica extract may be obtained using a conventional extraction method, and the extract may be an extract in a powder form, obtained through drying under reduced pressure and freeze drying.
In one embodiment of the present invention, the extract is a water extract, a C1-C5 alcohol extract, or a C1-C5 aqueous alcohol solution extract.
In the composition of the present invention for preventing, ameliorating, or treating glaucoma and macular degeneration, which has the above-described composition, the extraction process may be repeated as needed, and an extract obtained by performing extraction 2 to 5 times may be used. In addition, a dry powder-type extract may be prepared by freeze-drying the extract, and may then be used in a composition for preventing, ameliorating, or treating glaucoma and macular degeneration.
It is desirable from the aspect of extraction efficiency that the content of alcohol in the extraction solvent be maintained at a concentration of 0 wt % to 95 wt %, preferably 30 wt % to 95 wt %, and more preferably 50 wt %. Any alcohol may be applied as long as it is one generally used in the art, preferably a C1-5 alcohol. The alcohol may be one or more selected from methanol, ethanol, isopropanol, propanol, and butanol. More preferably, ethanol or the like may be used as the alcohol.
The extraction method is a method commonly known in the art, for example, a method using an extraction device, such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, or ultrasonic extraction, a method using an adsorption resin including XAD and HP-20, or the like.
In addition, the extract is concentrated under reduced pressure using a vacuum rotary evaporator or the like to obtain an extract. In addition, the obtained extract may also be subjected to drying under reduced pressure, vacuum drying, boiling drying, spray drying, room-temperature drying, freeze drying, or the like as needed. In particular, the freeze-drying method is advantageous in that the loss of volatile organic substances from the extract can be reduced.
The Centella asiatica extract obtained through the above method effectively increases the proliferation of optic nerve cells and retinal cells by increasing the glucose uptake of cells, and effectively inhibits cell death from oxidative stress caused by A2E.
In one embodiment of the present invention, the Centella asiatica extract is a 40% to 60% aqueous ethanol solution extract.
Hereinafter, the present invention will be described in more detail with reference to specific examples. These examples are provided for illustrative purposes only to aid understanding of the present invention and are not intended to limit the scope of the present invention.
Centella asiatica was purchased from a farmhouse in Hapcheon, Gyeongnam and dried in the shade, the whole plant was pulverized, and then 1 kg of pulverized Centella asiatica was extracted with 50% alcohol for 5 hours at 80° C., concentrated, and freeze-dried to thereby obtain 135 g (yield: 13.5%) of a Centella asiatica extract.
As comparative examples, a hot-water extract of Centella asiatica (Comparative Example 1), a 30% alcohol extract of Centella asiatica (Comparative Example 2), a 70% alcohol extract of Centella asiatica (Comparative Example 3) and a 95% alcohol extract of Centella asiatica (Comparative Example 4) were used.
In order to select the extract having the strongest efficacy from among the Centella asiatica extract obtained in Preparation Example and the extracts of Comparative Examples, the effect of proliferating retinal nerve cells was measured.
RGC-5 retinal nerve cells were incubated in a DMEM medium containing 10% serum and an antibiotic in an incubator supplied with 5 vol % of carbon dioxide and maintained at 37° C. GH3 cells were dispensed into a 96-well plate at a concentration of 5×103 cells/well and cultured for 24 hours. In order to measure the effect of each Centella asiatica extract on the proliferation of retinal nerve cells, the medium was replaced with a DMEM medium containing 10% charcoaled FBS, and then the cells were treated with 100 μg/mL of each of the Centella asiatica extracts prepared according to Preparation Example and Comparative Examples 1-4. After 48 hours, the growth rate of RGC-5 cells was measured at an absorbance of 550 nm using an MTT reagent, and the absorbance of each test solution was measured based on the control to represent a cell proliferation rate (%).
The proliferation rate of retinal nerve cells by each Centella asiatica extract is illustrated in
As illustrated in
Meanwhile, in order to measure the effect of the 50% alcohol extract of Centella asiatica, which exhibited the strongest proliferative efficacy, on the protection of retinal nerve cells and retinal pigment epithelial cells, the effect on the proliferation of the corresponding retinal cells was measured.
RGC-5 retinal nerve cells were incubated in the same manner as described above, and ARPE-19 retinal pigment epithelial cells were incubated in a DMEM/F12 medium containing 10% serum and an antibiotic in an incubator supplied with 5 vol % of carbon dioxide and maintained at 37° C. The RGC-5 cells and the ARPE-19 cells were dispensed into a 96-well plate at a concentration of 5×103 cells/well and cultured for 24 hours. To measure the effect of the Centella asiatica extract on the proliferation of retinal nerve cells and retinal pigment epithelial cells, the medium was replaced with a DMEM medium containing 10% charcoaled FBS and a DMEM/F12 medium, and then the cells were treated with 50 μg/mL, 100 μg/mL, and 200 μg/mL of the 50% alcohol extract of Centella asiatica. After 48 hours, the growth rates of the respective cells were measured at an absorbance of 550 nm using an MTT reagent, and the absorbance of each test solution was measured and compared with the control to represent a cell proliferation rate (%).
The proliferation rates of retinal nerve cells and retinal pigment epithelial cells due to the 50% alcohol extract of Centella asiatica are illustrated in
As illustrated in
A 2-NBDG uptake experiment was conducted to study the glucose uptake efficacy of the 50% alcohol extract of Centella asiatica of Preparation Example in retinal pigment epithelial cells.
As test cells, ARPE-19 cells, which are retinal pigment epithelial cells, were used, and the medium, medium composition, and experimental conditions were the same as in Experimental Example 1.
ARPE-19 cells were dispensed into a 96-well plate at a concentration of 1×104 cells/well and cultured for 24 hours. To measure the glucose uptake efficacy, the cells were treated with 25 μg/mL, 50 μg/mL, and 100 μg/mL of the 50% alcohol extract of Centella asiatica and cultured for 24 hours, followed by treatment with a serum-free DMEM/F12 medium and 100 μM of 2-NBDG for 30 minutes. The cells were washed twice with cold phosphate-buffered saline (PBS), and then the glucose uptake amount was measured using a fluorescence photometer (excitation 485 nm; emission 525 nm).
The glucose uptake efficacy of the Centella asiatica extract is shown in
As illustrated in
To determine the effect of inhibiting macular degeneration, the cell death inhibitory effect of A2E was measured.
As test cells, ARPE-19 cells, which are retinal pigment epithelial cells, were used, and the medium, medium composition, and experimental conditions were the same as in Experimental Example 1.
ARPE-19 cells were dispensed into a 24-well plate at a concentration of 3×104 cells/well and cultured for 24 hours, and then A2E was accumulated at a concentration of 3 μM for 3 days. In order to measure the effect of protecting cells from oxidative stress caused by A2E, the cells were treated with 25 μg/mL, 50 μg/mL, and 100 μg/mL of the 50% alcohol extract of Centella asiatica, and as a positive control, the cells were treated with 30 μM of lutein, which is known to have an inhibitory effect on macular degeneration, and cultured for 48 hours, and after 48 hours, the cells were irradiated with ultraviolet rays using a UV lamp for 5 minutes. The cell growth rate was measured at an absorbance of 570 nm using an MTT reagent, and the absorbance of each test solution was measured and compared with a control not treated with A2E.
The effect of the Centella asiatica extract on the protection of cells from oxidative stress caused by A2E is shown in Table 1.
As shown in Table 1, the 50% alcohol extract of Centella asiatica exhibited a cell survival rate of about 16% at 100 μg/mL as compared to the control, and protected cells from oxidative damage caused by A2E at the same level as that in lutein as a positive control. Therefore, the present invention may provide a composition for preventing, ameliorating, or treating glaucoma and macular degeneration for eye health including a Centella asiatica extract.
The foregoing description of the present invention is provided for illustrative purposes only, and it will be understood by those of ordinary skill in the art to which the present invention pertains that the present invention may be easily modified in other particular forms without changing the technical spirit or essential characteristics of the present invention. Thus, the above-described embodiments should be construed as being provided for illustrative purposes only and not for purposes of limitation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0122947 | Oct 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/011310 | 9/3/2019 | WO | 00 |
