KR20130037110A - Extract of crepidiastrum denticulatum for prevention or treatment of retinal diseases - Google Patents

Abstract

PURPOSE: A Crepidiastrum denticulatum extract is provided to protect degeneration of retinal neuronal cells induced by oxidative stress and to be used as a therapeutic agent, a functional food, and a pharmaceutical material for retinal diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating retinal diseases contains a Crepidiastrum denticulatum extract. The retinal diseases include age-related macular degeneration. The extract is prepared using water, C1-C4 alcohol, acetone, or a mixture solvent thereof.

Description

The present invention relates to a method for the prevention and treatment of retinopathy,

The present invention relates to the use of an extract of Crepidiastrum denticulatum as a pharmaceutical composition or a health food for the prevention and treatment of retinal diseases.

Apoptosis is a spontaneous and programmed cell death within the cell that is distinct from necrosis, and various mechanisms have been suggested to induce apoptosis. Typical inducers are oxidative stress, nitric oxide synthase, and mitochondria-mediated caspase-dependent or caspase-independent mechanisms. . In particular, many eye diseases are known to occur due to cell apoptosis of certain cells, and typical eye diseases include age-related macular degeneration and glaucoma. In order to inhibit and treat these eye diseases, researches on drugs that can inhibit or slow down the progression of apoptosis have been continuing.

Since the eye cornea, lens (lens), and vitreous body are areas free of blood vessels, there is water called waterproofing that supplies nutrients and transports waste materials. However, the waterproofing is normal, but when the escaping part is broken, the water pressure keeps coming out, so the pressure of the eyes will rise. As the pressure of the eye increases, the blood does not enter the eye, which is supposed to be supplied with a lot of blood. Continuous pressure is applied to the weak part of the optic nerve, and the sustained pressure of the optic nerve causes severe damage. . Glaucoma is a serious disease that causes loss of sight at the end of the glaucoma due to increased pressure of the optic nerve and impaired optic nerve function or blood supply. These glaucoma will occur in 0.5 to 2% of adults over 40 years of age. While the normal intraocular pressure is 15 to 20 mmHg, the intraocular pressure becomes green when the glaucoma progresses.

Currently, the treatment of glaucoma is the most important risk factor of glaucoma, the use of drugs or surgical methods to prevent the rise of glaucoma, glaucoma can not be cured, lifetime drugs, laser therapy, There is no other way to minimize the disability of the optic nerve.

In addition, glaucoma is caused by damage of the optic nerve caused by apoptotic cell apoptosis of the retinal nerve cell. Such optic nerve damage is accompanied by characteristic glaucomatous visual field damage. The apoptosis of retinal nerve cells in glaucoma is known to be apoptosis and cell apoptosis of retinal nerve cells was observed in glaucoma patients through post-mortem examination of glaucoma patients as well as animal models with elevated IOP [Quigley HA et et al., Invest Ophthalmol Vis Sci., 36, 774-786, 1995; Garcia-Valenzuela E et al., Exp Eye Res., 61, 33-44, 1995]. The fate of the cell is determined by the balance between the mechanisms that induce apoptosis in cells and the mechanism of inhibition. In glaucoma, glutamate, cytochrome C, tumor necrosis factor-alpha It is known that the increase or expression of Bad (BCL2-antagonist of cell death) protein causes retinal nerve cells to lead to apoptosis, and oxidative stress in cells is also known to be an important cause of glaucoma. In glaucoma, retinal nerve cells undergo oxidative stress to directly damage the optic nerve. In addition, the trabecular meshwork cell, which constitutes a waterproof drainage pathway, undergoes oxidative stress, , And ultimately cell apoptosis occurs, leading to elevated intraocular pressure [Sacca 'SC et al, Arch Ophthalmol., 123, 458-463, 2005; Zhou L et al., J Cell Physiol., 180, 182-189, 1999]. The decrease in the function of the trabecular cells causes the increase of the intraocular pressure by affecting the pinocytosis which absorbs water inherently possessed by the cell. Especially, the decrease of the trabecular cell function causes the primary occlusion It is known that trabecular cells of patients with primary open angle glaucoma are more vulnerable to oxidative stress (Izzotti A et al., Am. J. Med., 114, 638-646, 2003]. The total antioxidant potential of the patient was lower than that of the healthy subjects [Ferreira SM et al., Am J Ophthalmol., 137, 62-69, 2004] It has been observed that the glutathione-S-transferase, which regulates oxidative stress, is increased [Yang J et al., Invest Ophthalmol Vis Sci., 42, 1273-1276, 2001]. Recently, it has been known that the damage of trabecular cells in primary glaucoma patients is inhibited by antioxidants [Yuan He et al., Invest Ophthalmol Vis Sci., 49, 1447-1458, 2008]. In this way, inhibition of cell apoptosis of retinal nerve cells and trabecular meshwork cells and suppression of oxidative stress of the cells is emerging as a new approach.

On the other hand, Crepidiastrum denticulatum is a 1-2 year old plant that grows in mountains and fields throughout the country. The growth environment grows in semi-shade or sunny places. The height is 30 to 70 cm, the leaves are 6 to 11 cm in length, 3 to 7 cm in width, are dull and have irregular sawtooth. The flower is yellow and spreads at the tip of the branch and at the end of the main stem. The flower stalk is straight when the flower is bloomed, but after the pin it stays. Fruit is caught around October to November, length is 0.3 to 0.4 cm, brown or black, feathers are white, and length is about 0.3 cm.

In any of the documents reported so far, it has not been reported to be used for the purpose of preventing or treating retinal diseases, since the extract and extract have the effect of protecting retinal nerve cells.

The inventors of the present invention have found that when extracting a retinal nerve cell cytotoxic agent against Korean native plants, the extracts of the extracts inhibit oxidative stress-induced retinal nerve cells to inhibit apoptosis and exhibit an inhibitory effect on apoptosis, The present invention has been accomplished by confirming that it can be used for prevention and treatment of retinal diseases caused by stress-induced retinal neuronal cell degeneration.

It is an object of the present invention to provide an extract of Crepidiastrum denticulatum which is excellent in activity of inhibiting apoptosis and oxidative stress of retinal nerve cells and trabecular meshwork cells.

In order to solve the above-mentioned problems, the present invention is characterized by a composition for preventing and treating retinopathy diseases containing an extract of Crepidiastrum denticulatum as an active ingredient.

In addition, the present invention is characterized by a health food for preventing and ameliorating a retinal disease which contains an extract of Crepidiastrum denticulatum as an active ingredient.

The extract of Crepidiastrum denticulatum of the present invention has an effect of protecting the degradation of retinal nerve cells induced from oxidative stress.

Therefore, the extract of Crepidiastrum denticulatum is useful as a preventive and therapeutic agent for retinal diseases such as senile macular degeneration and glaucoma or as a functional food and pharmaceutical material.

FIG. 1 shows the results of MTT assay to determine the cell survival rate after pretreatment of the retinal nerve cell RGC-5 and extracting oxidative stress, This is a graph showing the protective effect of cells.
FIG. 2 is a photograph of a retinal nerve cell RGC-5 pretreated with a minus extract to induce oxidative stress, followed by double staining using Hoechst 33342 and propidium iodide, and then observing it with a fluorescence microscope.
A: Control,
B: group treated with 0.5 mM BSO and 10 mM glutamate only,
C: group treated with 0.5 mM BSO and 10 mM glutamate after pretreatment with 1 mM N-acetyl-L-cysteine as a positive control,
D, E, and F: 10, 1, and 0.1 μg / mL, respectively. Pretreatment of extracts and treatment with 0.5 mM BSO and 10 mM glutamate.
FIG. 3 is a graph showing changes in the production of reactive oxygen species (ROS) by oxidative radicals in the retinal nerve cell RGC-5 after pretreatment with a minus extract.
FIG. 4 is a photograph of the retinoic neuron RGC-5 pretreated with a minus extract, inducing oxidative stress, staining the production of reactive oxygen species in the cell with DHE (Dihydroethidium), and confirming it with a Confocal microscope and its quantitative graph .
FIG. 5 is a table showing the inhibitory effect of SNP (sodium nitroprusside) -mediated lipid peroxidation by pretreatment of the extract of RGC-5 and extracts.
FIG. 6 is a graph showing the expression of apoptosis-related protein by inducing oxidative stress by pretreating extracts of RGC-5 and extracts.
FIG. 7 is a graph showing the expression of antioxidative-related proteins by inducing oxidative stress by pretreating extracts of RGC-5 and extracts.
FIG. 8 shows the effect of inhibiting retinal cell death in rats pretreated with N -methyl-D-aspartate (NMDA) by pretreatment with extracts of hematoxylin and eosin Fig.
FIG. 9 is a cross-sectional view of a retina obtained by pretreating extracts with and without N -methyl-D-aspartate (NMDA) to inhibit retinal cell death in TUNEL.

The present invention relates to a composition for the prevention and treatment of retinal diseases, which contains an extract of Crepidiastrum denticulatum as an active ingredient.

The extract of the extract of the present invention is produced by a conventional natural material extraction method, and the present invention does not have any particular limitation on the extraction method.

A representative preparation method of the extract of the present invention includes the following steps:

1) extracting the dried and extracted Crepidiastrum denticulatum with an extraction solvent;

2) filtering the extract of step 1); And

3) preparing an extract by concentrating the filtered extract of step 2) under reduced pressure; And 4) further extracting the extract of step 3) with an organic solvent to prepare a fraction.

Crepidiastrum denticulatum used in the above extraction can be used without limitation, such as farmed, picked in mountains or commercially available. The above and the subtraction may utilize the entire plant, and the leaf, the stem or the root may be partially used, but the present invention is not limited thereto.

The extraction solvent may be water, alcohol, acetone or a mixture thereof, preferably a lower alcohol having 1 to 4 carbon atoms, or a mixture of these alcohols or an aqueous solution of these alcohols, more preferably methanol, An aqueous solution thereof is used, but the present invention is not limited thereto. The amount of the extraction solvent is preferably in the range of 1 to 20 times, more preferably in the range of 5 to 10 times, but is not limited thereto. The extraction method may be an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. Preferably, the extraction method may be performed once to five times by an ultrasonic extraction method. The extraction temperature is preferably from 10 to 100 ° C, more preferably room temperature, but is not limited thereto. The extraction time is preferably 1 day to 7 days, more preferably 3 days to 7 days, but is not limited thereto. In the above extraction, a conventional extraction method such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, ultrasonic extraction or the like or adsorption resin including XAD and HP-20 is used And it is preferably warmed and refluxed or extracted at room temperature, but the present invention is not limited thereto. The extraction number is preferably 1 to 5 times, more preferably 3 times, but is not limited thereto.

The vacuum concentration after the extraction or fractionation is preferably performed using a vacuum rotary evaporator, but is not limited thereto. The drying may be carried out under reduced pressure, vacuum drying, boiling, spray drying, room temperature drying or freeze drying, but is not limited thereto.

The organic solvent used for the fraction is preferably n-hexane, methylene chloride, ethyl acetate or n-butanol, but is not limited thereto. The fractions were prepared by suspending the extracts in water and then extracting the fraction of normal-hexane, methylene chloride, ethyl acetate, and n-hexane obtained by sequential fractionation with normal-hexane, methylene chloride, ethyl acetate, n-butanol and water, Butanol fraction or water fraction, and it is more preferable that it is an ethyl acetate fraction. However, the present invention is not limited thereto. The fraction may be obtained by repeating the fractionation process from 1 to 5 times, preferably 3 times, from the extract and the extract, and the fraction is preferably concentrated under reduced pressure, but is not limited thereto.

In a specific embodiment of the present invention, the dried extracts are pulverized to an appropriate size, placed in an extraction vessel, and an aqueous ethanol solution is added thereto. After standing at room temperature, the filtrate and filtrate are filtered and dried to give an extract , And the above extracts were subjected to sequential phylogenetic analysis using a fractionation funnel with normal-hexane, methylene chloride, ethyl acetate, n-butanol and water fractions, and the subtracted fractions were prepared.

In order to measure the protective effect of the retinal nerve cell (RGC-5) of the extract prepared by extracting and extracting the cells, the cells were cultured, and then the extracts were pretreated by concentration and 0.5 mM BSO And 10 mM glutamate were treated at 37 ° C for 24 hours, and MTT method was performed to confirm cell viability. As a result, when the oxidative stress was induced by the hydrogen peroxide in the retinal nerve cell RGC-5, the cell survival rate was higher than that of the group treated with only 0.5 mM BSO and 10 mM glutamate when the above extract and the extract obtained were pretreated 1).

As a result, it was confirmed that the extract obtained by extracting the extract of the present invention has a cell death protection effect on oxidative stress causing apoptosis of retinal nerve cells.

In addition, Hoechst 33342, which has membrane permeability and dyes living cells or dead cells in blue, is not transmissive in order to determine whether the extracts deprived of retinal nerve cells (RGC-5) Cell death by oxidative stress was analyzed using propidium iodide (PI), which is stained only in dead cells. As a result, in the cell death induced by 0.5 mM BSO and 10 mM glutamate treatment on retinal neuron RGC-5, the pretreatment of the extract with the extracts showed a significant decrease compared with the group treated with only 0.5 mM BSO and 10 mM glutamate It was confirmed that fluorescent red labeling by propidium iodide stained by cell death was decreased in a concentration-dependent manner (see FIG. 2).

In addition, to determine if it inhibits the production of reactive oxygen species (RGC-5) in retinal nerve cell extracts, the H ₂ O ₂ radical corresponding to active oxygen, OH ^-. Radical and O ₂ ^-. The radicals were induced, and the resultant extracts significantly inhibited the production of active oxygen (see FIG. 3).

In order to determine whether the extracts deprived of retinal nerve cells (RGC-5) cell death, DHE (Dihydroethidium), which is membrane-permeable and stains red cells with active oxygen, And the extent of active oxygen production in the cells was analyzed. As a result, in the cell death induced by RGC-5 with 0.5 mM BSO and 10 mM glutamate, the pretreatment of the extract with the extracts was compared with the treatment with 0.5 mM BSO and 10 mM glutamate alone , It was confirmed that fluorescence red labeling by DHE (dihydroethidium) stained by active oxygen generation decreased in a concentration-dependent manner (see FIG. 4).

Therefore, it was confirmed that the extract obtained by extracting and extracting had the effect of inhibiting the apoptosis of retinal nerve cells by oxidative stress.

In addition, the amount of lipid peroxidation in rat brain induced by SNP (sodium nitropurrusid) was measured by the TBARS method (thiobarbituric acid reactive substances assay method). And when pretreated with the extract to extract, the lipid peroxidation was decreased in a concentration-dependent manner (see FIG. 5).

Therefore, it was confirmed that the extracts deprived of lipids suppress lipid peroxidation.

Western blotting assays were also performed to confirm the expression of oxidative stress induced apoptosis and antioxidant related proteins. And pretreatment of the minus extract inhibited the expression of apoptosis-related protein and increased the expression of antioxidant protein (see FIGS. 6 and 7).

 In addition, to determine the changes in retinal thickness of the NMDA-induced rats, histological sections were stained with hematoxylin and eosin to observe the retina. And inhibited thinning of the ganglion cell layer (GL) and inner nuclear layer (INL) of the retina when pretreated with the extract obtained by extraction (see FIG. 8).

In addition, NMDA - induced apoptosis - induced retinal cell death was measured by TUNEL staining. And when the extract was subjected to pretreatment, it was confirmed to inhibit apoptosis-induced apoptosis (see FIG. 9).

On the other hand, in the present invention, the extract of cynthia extract protects oxidative stress-induced retinal neurons, inhibits apoptosis, exhibits an inhibitory effect of apoptosis, and prevents and treats retinal diseases due to degeneration of retinal neurons. Can be used as a drug Therefore, the present invention provides a pharmaceutical composition for the prevention and treatment of retinal diseases, which contains the extract of cynaceae as an active ingredient.

As the active ingredient contained in the composition of the present invention, it is preferable to contain 0.1 to 50% by weight based on the total weight of the composition, but the present invention is not limited thereto.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.

The compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the composition of the present invention, for example, starch, calcium carbonate, sucrose (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the patient's condition, weight, age, sex, The range varies depending on the diet, the rate of excretion, the severity of the disease, the form of the drug, the time of administration, the method of administration, the route of administration and the duration of administration. The daily dose is 0.0001 to 500 mg / kg, preferably 0.001 to 100 mg / kg, when the extract obtained by lyophilizing the extract according to the present invention is lyophilized, can do.

In applying the composition of the present invention to food, there is no particular limitation on the kind of food. Examples of food to which the composition of the present invention can be added include food products such as drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen noodles, gums, ice cream, Soups, beverages, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The extract of the present invention can be directly added to the food or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the extract in the health functional food may be 0.1 to 90% by weight of the total food weight. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition, the extract of the extract of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies such as cheese, chocolate, , Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, the extract of the extract of the present invention can contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. Although the proportion of such additives is not so important, it is common that the extract of the present invention is selected from the range of 0.1 to 20% by weight based on the total weight of the extract as an additive.

The present invention as described above is explained in detail by the following examples, experimental examples and preparation examples. EXAMPLES The following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Experimental Examples and Preparation Examples.

[Example]

Example. ≪ / RTI > and subtracted extract

( Crepidiastrum denticulatum ) were collected in the middle of April (young) and mid August (adult) in Pyeongchang, Gangwon province. After drying, the filtrate was diluted with 2 L of ethanol to 180 g, refluxed for 3 hours, and the extract was filtered. After filtration of the extract, 2 L of ethanol was added again to the remaining residue, and the resulting mixture was heated and refluxed for 3 hours. This procedure was further repeated twice to obtain 6 L of the total extract. 6 L of the extract was concentrated under reduced pressure at 35 DEG C to obtain 8.5 g of an ethanol extract. Sangha. The extract was dissolved in dimethylsulfoxide (DMSO) and used as a sample (D043) in the following Experimental Example.

Experimental Example

Experimental Example 1. Measurement of protective effect of retinal nerve cell (RGC-5) on extract of extract and extract

Retinal ganglion cells (RGC-5) were supplied free from Alcon Research, Ltd. and were cultured in DMEM medium (Dulbecco's modified Eagle's medium, Hyclone, USA) containing 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin ) Was used. At 37 ℃ to 75-T tissue culture flasks and cultured in 5% CO _2, 95% air and saturated humidity under thermostat. Cells grown to saturation after 2 to 3 days were removed from the flask using trypsin-EDTA solution and then subcultured. Cells were placed in a 96-well culture dish and placed in a thermostat for 24 hours to attach the cells. The medium was removed and replaced with DMEM medium containing 1% low-density fetal bovine serum (FBS). The samples were pretreated at final concentrations of 10, 5, 1, and 0.1 μg / mL 1 hour before the addition of oxidative stress. After treatment with 0.5 mM BSO and 10 mM glutamate at 37 ° C. for 24 hours, MTT, a tetrazolium salt, was insoluble formazan by the mitochondrial dehydrogenase of cells. The survival rate of retinal ganglion cells was measured by MTT method using the change to (formazan crystal).

 Specifically, in order to perform the MTT method, the treated medium of the RGC-5 cells was removed, MTT solution was added to a final concentration of 0.5 mg / mL per well, and the mixture was reacted for 1 hour in a thermostat. Then, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, shaken for 15 minutes, and then eluted with a synergy HT microplate reader (Bio-Tek instruments, Winooski, VT, USA) at 570 nm Absorbance was measured.

As a result, when oxidative stress was induced by 0.5 mM BSO and 10 mM glutamate treatment in retinal nerve cell RGC-5, the subtracted extract (sample D043) was significantly reduced compared with the group treated with 0.5 mM BSO and 10 mM glutamate alone, And showed significant cell viability (Fig. 1).

Therefore, it has been confirmed that the extract obtained by extracting and extracting has an effect of protecting the retinal nerve cells from oxidative stress.

Experimental Example 2. Measurement of cytotoxicity of retinal nerve cell (RGC-5)

(RGC-5). To determine whether the extracts inhibited the cell death of retinal nerve cells (RGC-5), Hoechst 33342, which stains live or dead cells, ie whole cells in blue, Cells were stained with propidium iodide (PI) and stained.

 Specifically, the cells were placed in a 96-well culture dish and adhered. Then, the medium was exchanged with a low concentration of 1% fetal bovine serum (FBS), and the extract (sample D043) mL for 1 hour. After treatment with 0.5 mM BSO and 10 mM glutamate for 24 hours at 37 ° C, the cells were washed with cold D-PBS, and Hoechst 33342 and 1.5 μM PI at a final concentration of 8 μM were added to the cells Stained at 37 캜 for 15 minutes, and then visualized using a fluorescence microscope (Fig. 2).

As a result, in the cell death by oxidative stress induced by 0.5 mM BSO and 10 mM glutamate in retinal neuron RGC-5, pretreatment with extract minerals resulted in the case of treatment with only 0.5 mM BSO and 10 mM glutamate In comparison, it was confirmed that fluorescence red labeling by propidium iodide, which shows apoptosis, is decreased (Fig. 2).

Therefore, it was confirmed that the extract obtained by extracting and extracting had the effect of inhibiting the apoptosis of retinal nerve cells by oxidative stress.

Experimental Example 3. Measurement of Inhibitory Effect of Reactive Oxygen Species on Retinal Nerve Cells (RGC-5) of Extracted Extracts

(RGC-5) in the extracts of rats. The production of reactive oxygen species is caused by the oxidation of 2 ', 7'-dichlorofluorescein diacetate (DCFH-DA, Sigma, USA) Were analyzed using the DCFH-DA method.

Specifically, the cells were cultured in a 96-well culture dish at a concentration of 5.0 × 10 ³ cells / well at 37 ° C. for 24 hours. The cells were treated with DMEM supplemented with 1% fetal bovine serum (FBS), treated with 1 μM DCFH-DA for 15 min. The medium was removed and diluted with 1 mM H ₂ O ₂ (H ₂ O ₂ ) to induce hydrogen peroxide (H ₂ O ₂ ), hydroxyl radical (OH ^- ) and superoxide radical (O ₂ ^- The cells were exposed to 1 mM H ₂ O ₂ , 100 μM Fe (Ⅱ) perchlorate hexahydrate (OH ^- ) and 1 mM KO ₂ (O ₂ ^- Olecine (DCF) was measured for fluorescence at excitation wavelength 485 nm and emission wavelength 535 nm. As a result, when pre-extracted extract (sample name D043), H ₂ O ₂ radical, OH- ^. Radical and O ₂ ^-. H ₂ O ₂ radicals in the retinal nerve cell RGC-5, OH ^-. Radical and O ₂ ^-. And significantly inhibited the increase of active oxygen by the radicals induced by radicals (Fig. 3).

Experimental Example 4. Measurement of Inhibitory Effect of Extracted Extract of Retinal Nerve Cell (RGC-5) on Intracellular Active Oxygen

(RGC-5) was inhibited by the addition of DHE (Dihydroethidium), which is membrane-permeable and red dyeing cells containing reactive oxygen species And the extent of active oxygen production in the cells due to stress was analyzed.

 Specifically, the cells were placed in a 96-well culture dish and adhered. Then, the medium was exchanged with a low concentration of 1% fetal bovine serum (FBS) and the extract (sample D043) was diluted to a final concentration of 10, 5, mL for 1 hour. Then, cells were treated with 0.5 mM BSO and 10 mM glutamate for 24 hours at 37 ° C, washed with cold D-PBS, added to DHE (dihydroethidium) at a final concentration of 1 μg / And visualized using a Confocal fluorescence microscope (Fig. 4).

As a result, in the cell death by oxidative stress induced by 0.5 mM BSO and 10 mM glutamate in the retinal nerve cell RGC-5, in the pretreatment of extract minerals and in the case of treatment with 0.5 mM BSO and 10 mM glutamate only , It was confirmed that fluorescence red labeling by dihydroethidium (DHE), which confirms the production of active oxygen in the cells, is decreased [Fig. 4].

Therefore, it was confirmed that the extract obtained by extracting and extracting had the effect of inhibiting the production of active oxygen in the cell by oxidative stress.

Experimental Example 5: Inhibition of lipid peroxidation of rat brain by SNP-induced extract of extract

And thiobarbituric acid reactive substance (TBARS), which is derived from MDA (malondialdehyde), was measured in the SNP - induced lipid peroxidation inhibition of the brain.

Specifically, the extracted brain was crushed into 10 times as much brain volume as cold saline (pH 7.0), and centrifuged at 4 ° C (1000 g ). The supernatant was transferred to a clean test tube and analyzed by Bio-Rad Protein assay Was used to quantify the protein concentration. The supernatant was transferred to a test tube in an amount of 0.5 mL, and incubated at 37 ° C in a thermostatic chamber for 5 minutes. Then, 20 μM sodium nitroprusside (SNP) Was added to a final volume of 1 mL and incubated in a 37 ° C thermostatic chamber for 30 minutes.

8.1% w / vol SDS, 0.8% TBA and 20% v / v acetic acid (pH 3.5) were added to the above oxidation reaction mixture, followed by heating in a water bath at 90 to 95 ° C for 30 minutes and then cooling to room temperature. To reduce turbidity, n-butanol: pyridine (15: 1 v / v) was added, mixed and centrifuged, and the absorbance of the supernatant was measured at 532 nm.

As a result, it was confirmed that the SNP-induced lipid peroxidation was decreased in a concentration-dependent manner when pretreatment of the extract and extract (sample D043) was performed [Fig. 5].

Experimental Example 6. Measurement of cell death and antioxidant-related protein expression activity of extracts

(RGC-5) were used for Western blotting assay to determine the effect of the extract on the apoptosis and protein expression of antioxidant-related proteins.

Specifically, the DMEM / 10% FBS solution retinal nerve cell number of the culture medium and mixed with cells ⁴ × 10 4 cells / after mL prepared as by treatment by 5 mL to 60 mm plate 5% CO ₂ / under the conditions of 37 ℃ Lt; / RTI > After the cells were stabilized, the extracts (Sample D043) were treated at 0, 1, 5, 10 μg / mL and cultured for 24 hours at 5% CO ₂ and 37 ° C. After the completion of the culture, proteins were extracted from the cell lysate and analyzed for apoptosis and antioxidant antigens (FIGS. 6 and 7).

As shown in FIG. 6 and FIG. 7, when the extract and extract were treated, the expression of the apoptotic protein was inhibited and the expression of the antioxidant protein was increased.

As a result, it has been found that the extracts with and without depressants have an effective cytotoxic effect of inhibiting apoptotic proteins and increasing the activity of antioxidant proteins.

EXPERIMENTAL EXAMPLE 7: Protective effect of NMDA-induced retinal detoxification

And NMDA - induced retinal detoxification of rat extracts.

Specifically, 2% rumon injections and Zoletil were mixed and anesthetized with 1 ml / kg intramuscular injection. A 33-G needle was used to inject the Hamilton syringe with NMDA (5 nmol / eye) Were injected into the vitreous body in an amount of 2 μL into the eyeball. After injection, one drop of 0.5% non-Gavocin eye drops (ALcon Laboratoriss, Inc.) was applied to prevent infection. In the control group, the same volume of 0.01 M PBS was injected. The left eye was injected with NMDA and the right eye as control. Histopathologic examination of retinal thickness was performed at 7 days after injection of NMDA. The extracted eyeballs were placed in 10% formalin, which was 10 times the volume of the eyeball, and fixed at room temperature for one day. Retinal sections were prepared and hematoxylin and eosin staining were performed. The tissue on the slide was fixed with acetic acid, the nuclei were stained with hematoxylin, and the cytoplasm was stained with eosin. The thickness of the retina was observed 100 times using an optical microscope. To measure the response to TUNEL, eyeballs were removed after 24 hours. After extraction, the lens and vitreous were removed and the retina was isolated. Detached retinal tissue was sectioned and fixed in 4% paraformaldehyde (pH 7.4) for 20 minutes and washed with PBS. The cells were stained with labeled TdTmediated dUTP nick end labeling (TUNEL) by binding to the exposed 3'-OH group of intracellular internucleosomally cleaved DNA using an in situ cell death detection kit, POD (Roche, Mannheim, Germany) And the location and degree of expression of the cells where the cells were generated were observed.

As a result, it was confirmed that when the retinal degeneration was induced by NMDA, the ganglion cell layer (GL) and inner nuclear layer (INL) of the retina became thinner, and the extracts (sample D043) It was confirmed that the retinal layer was prevented from being thinned (Fig. 8). In addition, in order to confirm apoptosis-induced apoptosis, NMDA-induced cells were increased in TUNEL-positive cells, and in the pretreatment of the extract and extracts, the TUNEL-positive cells were significantly decreased , And inhibited apoptosis-induced apoptosis (Fig. 9).

From the results of the above Experimental Example, it is clear that the extract obtained by extracting the roots of the present invention exhibits the protective effect of retinal nerve cells and the therapeutic effect of retinal diseases, so that it can be used as a medicament for the prevention and treatment of retinal diseases, It is useful as an active substance.

[Example]

The following pharmaceutical preparations are exemplified by pharmaceutical preparations containing the extract as an active ingredient or a preparation for the manufacture of a health functional food, but the present invention is not limited thereto.

Formulation Example 1. Preparation of pharmaceutical preparations

1-1. Manufacture of powder

And subtract Extract 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

And subtract 100 mg of extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of capsules

And 100 mg of the subtracted extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

1-4. Injection preparation

And 100 mg of the subtracted extract

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

2974 mg of distilled water

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

Formulation Example 2. Preparation of Health Functional Foods

2-1. Manufacturing of beverages

Honey 522 mg

5 mg of chitosanic acid ami

Nicotinic acid amide 10 mg

3 mg of sodium riboflavin hydrochloride

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Wild Perilla Extract 0.48 ～ 1.28 mg

200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

2-2. Preparation of Chewing Gum

Gum base 20 wt%

Sugar 76.36∼76.76 wt%

Wild Perilla Extract 0.24 ～ 0.64 wt%

Fruit flavor 1 wt%

Water 2 wt%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

2-3. Manufacture of candy

50-60 wt% sugar

Starch syrup 39.26∼49.66 wt%

Wild Perilla Extract 0.24 ～ 10.64 wt%

0.1% by weight of orange flavor

The composition and the content of the candy were prepared using a conventional method.

2-4. Manufacture of flour food products

5 parts by weight of the extract obtained by adding the extract according to the present invention to 100 parts by weight of wheat flour and preparing a bread, a cake, a cookie, a cracker and a noodle by using the mixture.

2-5. Manufacture of dairy products

5 to 10 parts by weight of the extract of the extract according to the present invention was added to 100 parts by weight of milk and various dairy products such as butter and ice cream were prepared using the milk.

2-6. Manufacture of wires

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then powdered with a grinder to prepare 60 mesh powder. Black beans, black sesame, and perilla were steamed and dried by a known method, and then powdered with a powder of 60 mesh size by a grinder. The grains and seeds prepared above and the grains and seeds according to the invention The extracts were prepared in the following proportions.

Brown rice 30 wt%

Yulmu 20 wt%

Barley 25 wt%

Perilla 7%

Black bean 7%

Black sesame 7%

≪ / RTI > and 3%

0.5%

0.5 wt%

Claims (8)

A pharmaceutical composition for preventing and treating retinal diseases, which contains an extract of Crepidiastrum denticulatum as an active ingredient.
2. The pharmaceutical composition for preventing and treating retinal disease according to claim 1, wherein the retinal disease is age-related macular degeneration or glaucoma.
The pharmaceutical composition for preventing and treating retinal disease according to claim 1, wherein the extract is extracted from Crepidiastrum denticulatum with water, alcohol having 1 to 4 carbon atoms, acetone, or a mixed solvent thereof.
The pharmaceutical composition for preventing and treating retinal diseases according to claim 3, wherein the alcohol is methanol or ethanol.
Health food for preventing and improving retinal diseases, containing extract of Crepidiastrum denticulatum as an active ingredient
The method of claim 5, wherein the retinal disease is age-related macular degeneration or glaucoma characterized in that the retina disease prevention and improvement health food.
The method of claim 5, wherein the extract is Crepidiastrum denticulatum ( Crepidiastrum denticulatum ) of water, alcohol having 1 to 4 carbon atoms, acetone or a mixed solvent thereof, characterized in that retinal disease prevention and improvement health foods.
The method of claim 7, wherein the alcohol is methanol or ethanol, retinal disease prevention and improvement health food, characterized in that.

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