WO2014116013A1 - Composition for treating, preventing, or alleviating macular degeneration, containing phenolic compound seperated from vaccinium uliginosum extract as active ingredient - Google Patents

Abstract

The present invention provides a pharmaceutical composition for treating, preventing, or alleviating macular degeneration containing, as an active ingredient, a phenolic compound which is separated from a vaccinium uliginosum extract and a pharmaceutically acceptable salt thereof. The phenolic compound of the present invention effectively protects ARPE-19 cell from blue light, thereby treating, preventing, or alleviating macular degeneration.

Description

Composition for the treatment, prevention or improvement of macular degeneration containing phenolic compounds isolated from blueberry extract as an active ingredient

The present invention relates to a composition for treating, preventing or ameliorating macular degeneration containing a compound isolated from natural products.

In general, the nerve tissue located in the central part of the inner retina of the eye is called the macular. Most of the cells are gathered here, and the place where the image of the object is also the center of the macular and plays a very important role in vision. Ocular disease that causes degeneration of the macula due to various causes and causes vision disorders is called macular degeneration (macular degeneration), and macular degeneration is known as one of the three major blindness diseases along with glaucoma and diabetic retinal disease.

This macular degeneration affects central vision, causing blurred vision in the center of vision, making it difficult to perform sophisticated activities such as reading and driving due to central dark spots, schizophrenia (a symptom of being distorted) or loss of vision in the local area. It is a very serious disease that, in severe cases, can lead to blindness.

Age-related Macular Degeneration (AMD) is the most common cause of vision loss in people 55 and older in developed countries. It is estimated that 10 million people in the United States suffer from various forms of macular degeneration. Life expectancy and current demographic growth are expected to triple this by 2020. However, not all age-related macular degenerations cause sudden, severe vision loss.

There are two types of senile macular degeneration, dry and wet senile macular degeneration. Wet senile macular degeneration results in a sudden loss of vision due to causing choroidal neovascularization in the central retina. However, only about 10% of senile macular degeneration is wet senile macular degeneration. Dry senile macular degeneration characterized by the presence of drusen (yellow precipitate between the retina and choroid) does not cause any leakage of blood or plasma. Dryness therefore does not result in a sudden loss of vision.

The underlying mechanism of the disease is still unknown, but senile macular degeneration is characterized by a loss of central vision following death of photoreceptor cells and is thought to have a multifactorial etiology. Recently, various factors (eg smoking, obesity, eating habits, increased cholesterol in the blood, irradiation with blue light, etc.) have been reported to cause the promotion of senile macular degeneration. Damage to photoreceptor cells and subsequent cell death leads to degeneration (degeneration) of retinal pigment epithelium.

In addition, the accumulation of fat brown pigment (lipofuscin) in the center of the eye retina is known to be the largest in the retinal pigment epithelial cells below the center of the retina. This reflects the fact that there are many dense rod photoreceptors.

N-retinyl-N-retinylidene ethanolamine (A2E) (produced as a by-product of all-trans-retinal in the outer disk) is the main chromophore of lipofucin and causes the formation of reactive oxygen species when exposed to blue light .

Blue light belongs to light having a relatively high amount of energy. When exposed to blue light of about 440 nm, photooxidation is induced to damage cells. Since A2E is sensitive to these blue light, it is thought that senile macular degeneration is related to lipofucin in retinal pigment epithelial cells. That is, continuous light exposure further promotes the death of retinal pigment epithelial cells of senile macular degeneration, which is one of the main causes of degeneration of photoreceptor cells.

Age-related macular degeneration is most common in older people, causing blindness and blindness. However, the exact mechanism of development of the disease has not yet been identified, and some researchers report that AMD is responsible for the death of RPE cells due to excessive accumulation of A2E. Korean Patent Laid-Open Publication No. 10-2011-0102246 reports that the blueberry extract has an antioxidant effect on various types of cells. Blueberry extract treatment on ARPE-19 cells reduced the concentration of blue light induced apoptosis, and among the blueberry extract fractions, the polyphenol fraction had the greatest effect on cell survival. However, among the blueberry extract components, the compound showing the protective effect has not been specifically identified.

Meanwhile, lutein is a representative example of a health functional food for preventing macular degeneration. Lutein protects the macula of the retina and maintains macular pigment density to prevent macular degeneration, but studies show that prolonged use of supplements containing carotenoids, such as lutein, increases the risk of developing lung cancer, especially for smokers Appeared. Therefore, it can be dangerous for smokers to take lutein-related products.

The present invention is to provide a composition for the treatment, prevention or improvement of macular degeneration comprising a phenolic compound isolated from the blueberry extract.

Polyphenols and anthocyanins are the most abundant components of blueberry extracts and the most active. Previous studies have shown that polyphenols and anthocyanins protect various cells under oxidative stress, but the effects of polyphenols and anthocyanins on cell damage following blue light induction are unknown. The present inventors have completed the present invention by confirming that the phenolic compound separated from the blueberry extract can inhibit the death of blue light-induced retinal pigment epithelial cells by inhibiting intracellular accumulation of A2E.

The present invention provides a pharmaceutical composition for treating, preventing or ameliorating macular degeneration, which contains a phenolic compound isolated from the blueberry extract and a pharmaceutically acceptable salt thereof as an active ingredient.

In a more preferred aspect of the present invention, the present invention is fractionated using chloroform, ethyl acetate, butanol, and then subjected to reverse phase HPLC to the ethyl acetate fraction separated phenol compounds and pharmaceutically acceptable salts thereof as an active ingredient. It provides a pharmaceutical composition for the treatment, prevention or improvement of macular degeneration comprising.

In a more preferred aspect of the invention, the invention is a phenolic compound isolated from blueberry extract, mycetin-3-O-galactoside and / or quercetin-3-O-arabinofuranoside, and its pharmaceutically It provides a pharmaceutical composition for the treatment, prevention or improvement of macular degeneration comprising an acceptable salt as an active ingredient.

The present invention also provides a method for treating, preventing or ameliorating macular degeneration by administering a phenolic compound isolated from the blueberry extract and a pharmaceutically acceptable salt thereof to a mammal including human.

In a more preferred aspect of the invention, the invention is a phenolic compound isolated from blueberry extract, mycetin-3-O-galactoside and / or quercetin-3-O-arabinofuranoside, and its pharmaceutically Provided are methods of treating, preventing or ameliorating macular degeneration in which an acceptable salt is administered to a mammal, including a human.

The present invention also provides the use of a phenolic compound and its pharmaceutically acceptable salts isolated from blueberry extract for the preparation of a pharmaceutical composition for treating, preventing or improving macular degeneration.

More preferably, the present invention provides mycetin-3-O-galactoside and / or quercetin-3-O-arabinofurano isolated from blueberry extract for the preparation of a pharmaceutical composition for treating, preventing or improving macular degeneration. Seed, and pharmaceutically acceptable salts thereof.

The present invention also provides a dietary supplement for preventing or improving macular degeneration, which contains a phenolic compound isolated from the blueberry extract and a pharmaceutically acceptable salt thereof as an active ingredient.

More preferably, the present invention provides a macula, wherein the phenolic compound isolated from the blueberry extract is mycetin-3-O-galactoside and / or quercetin-3-O-arabinofuranoside, and a pharmaceutically acceptable salt thereof. Provide dietary supplements to prevent or improve degeneration

In the present invention, the phenolic compound isolated from the blueberry extract is obtained by fractionating the blueberry extract using chloroform, ethyl acetate, butanol and the like, and then separating and purifying the ethyl acetate fraction using reverse phase HPLC. The phenolic compounds thus obtained include mycetin, mycetin-3-O-galactosid, quercetin, quercetin-3-O-galactosid, quercetin-3-O-arabinofuranoside, and the like. The structure is shown below.

Myricetin

Myricetin-3-O-galactoside

<Quercetin>

<Quercetin-3-O-galactosid>

<Quercetin-3-O-arabinofuranoside>

In particular, the phenolic compounds isolated from the preferred blueberry extract in the present invention are mycetin-3-O-galactosid, quercetin-3-O-arabinofuranoside, and the most preferred phenolic compounds are quercetin-3-O-ara Vinofuranoside.

Phenolic compounds isolated from the blueberry extract, which are contained as an active ingredient in the present invention, may include pharmaceutically acceptable salt forms that exist in nature or may be conventionally prepared by those skilled in the art.

The blueberry extract may be extracted from the flowers, fruits, leaves or bark of the blueberry (Vaccinium uliginosum), preferably from the fruit of the blueberry. The blueberry extract includes water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and extracted with a solvent, preferably water or ethanol, 1 to 96 hours at an extraction temperature of 5 ℃ to 100 ℃ It is obtained by an extraction method comprising the step of extracting repeatedly from five to five times.

From the blueberry extract thus obtained, phenolic compounds can be separated according to a compound separation method commonly used in the art. As described in this example, blueberry extract is fractionated using a solvent such as chloroform, ethyl acetate, butanol, and the like. Specifically, the blueberry extract is dissolved in distilled water and continuously fractionated using chloroform, ethyl acetate and butanol, and the ethyl acetate fraction in the solvent fraction may be separated and purified by reverse phase HPLC to separate phenolic compounds.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the phenolic compound isolated from the above-mentioned blueberry extract. Such pharmaceutically acceptable carriers are those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition, the pharmaceutical composition of the present invention may include diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and other pharmaceutically acceptable additives. For formulation, reference may be made to Remington's Pharmaceutical Science (Recent), Mack Publishing Company, Easton PA, and others.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on such factors as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. For example, it can be administered in a dose of 0.001 to 1000 mg / kg per day, the administration can be administered once or several times a day.

The pharmaceutical composition of the present invention can be administered orally or parenterally. Examples of the dosage form include eye drops, eye ointments, injections, tablets, capsules, granules, powders, and the like, and eye drops or eye ointments are particularly preferable. These can be formulated using techniques that are widely used.

For example, eye drops include isotonic agents such as sodium chloride and concentrated glycerin, buffering agents such as sodium phosphate and sodium acetate, surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl stearic acid 40 and polyoxyethylene hydrogenated castor oil, and citric acid Stabilizers, such as sodium and sodium edetate, and preservatives, such as benzalkonium chloride and paraben, etc. can be used as needed. Although pH should just be in the range permissible for ophthalmic preparations, the range of 4-8 is preferable.

As another embodiment of the invention, the composition of the invention may be provided in the form of a food composition, in particular a functional food composition, ie as a nutraceutical. The functional food composition of the present invention includes ingredients that are commonly added during food production and include proteins, carbohydrates, fats, nutrients and seasonings. Natural carbohydrates include monosaccharides including glucose, fructose and the like; Disaccharides including maltose, sucrose and the like; oligosaccharide; Polysaccharides including dextrin, cyclodextrin and the like; And sugar alcohols including xylitol, sorbitol, erythritol, and the like. As the flavoring agent, natural flavoring agents including tautin, stevia extract and the like, and synthetic flavoring agents including saccharin, aspartame and the like can be used.

In another embodiment of the present invention, the present invention provides a method for treating macular degeneration comprising administering the above-mentioned pharmaceutical composition or food composition to a patient with macular degeneration or a subject at risk for macular degeneration. Provide preventive or preventive measures. The subject is a mammal, including a human. Preferably, macular degeneration is senile macular degeneration, in particular macular degeneration due to blue light-induced retinal pigment epithelial cell death.

The matters mentioned in the uses, compositions and methods of the present invention apply equally unless they contradict each other.

Phenol compounds isolated from the blueberry extract of the present invention inhibits the intracellular accumulation of A2E, inhibits the death of blue light-induced retinal pigment epithelial cells, and exhibits the effect of treating, preventing or improving macular degeneration.

1 is a diagram illustrating a process of fractionating blueberry extract through a column.

Figure 2 is a schematic diagram showing the process of separating the phenolic compounds from the blueberry extract.

FIG. 3 shows the UV absorbance of compounds separated by reverse phase HPLC from ethyl acetate fraction of blueberry extract.

Figure 4 shows the results confirming that the compound of EA-1 is mycetin-3-O-galactoside in the phenol compounds.

FIG. 5 shows the results of confirming that the compound of EA-3 in the phenolic compound is quercetin-3-O-galactoside. FIG.

FIG. 6 shows the results of confirming that the compound of EA-5 in the phenolic compound is quercetin-3-O-arabinofuranoside. FIG.

7 is a view showing the results confirming that the compound of EA-6 is mycetin in the phenol compounds.

8 is a view showing the results confirming that the compound of EA-10 of the phenolic compound quercetin.

9 shows the prophylactic effect of phenolic compounds on ARPE-19 cell death by blue light.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Test Examples are merely to illustrate the present invention, the contents of the present invention is not limited to the following Examples and Experimental Examples.

EXAMPLE

Example 1 Preparation of Blueberry Extract

Dried blueberries (100 g of blueberry) were ground with a grinder and extracted twice with hot water (90 ° C., 2000 mL of water) for 16 hours to obtain 10% of blueberry extracts. The obtained extract was lyophilized and stored at -80 ° C until use.

Example 2 Isolation and Purification of Phenolic Compounds from Blueberry Extracts

25 g of the blueberry extract (freeze dried) obtained in Example 1 was dissolved in distilled water as shown in FIG. 2 and fractionated using chloroform, ethyl acetate, and n-butanol continuously. Specifically, the phenolic compounds of the ethyl acetate fraction were separated by the following method. The blueberry extract lyophilized powder was dissolved in distilled water. Separately, two C18 Sep-Pak cartridges were connected, preconditioned with 10 ml ethyl acetate, anhydrous methanol, and 0.01 N aqueous HCl was flowed into the cartridge. Blueberry extract dissolved in distilled water was loaded into the cartridge and carbohydrates / acids and other water-soluble compounds were removed with 6 ml of 0.01 N aqueous HCl. The layer removed was the carbohydrate / acid fraction. Nitrogen gas was passed through a connected C18 Sep-Pak cartridge for 10 minutes to dry the cartridge. Phenolic compounds were collected in a test tube by pouring 20 ml of ethyl acetate into the dried cartridge. The pigment fraction was collected in another test tube using 10 ml of anhydrous methanol containing 0.1% (v / v) HCl. The solvent in the fractions was removed at 40 ° C. using a concentrator. For reference, the pigment fraction and the carbohydrate / acid fraction were dissolved in distilled water, and the polyphenol fraction was dissolved in 50% aqueous ethanol and stored at -70 ° C Deer Freezer for use in the experiment.

Fractions of the phenolic compounds were dissolved in methanol and separated and purified using reversed phase HPLC. The structure of a single material was analyzed by NMR and mass spectrometry.

All single active ingredients showed absorption wavelengths at 255 and 365 nm where flavonoids were found and showed similar UV spectra (FIG. 3). Isolated flavonoids and flavonoid glycosides were divided into four groups by aglycones.

VU-EA-H1 (16.6 mg), VU-EA-H3 (19.8 mg), VU-EA-H5 (6.5 mg) and VU-EA-6 (extracted with HPLC using methanol: water 35:65). 23.5 mg) were isolated and the compounds were mycetin-3-O-galactoside (VU-EA-H1) and quercetin-3-O-galactoside (quercetin-3- O-galactoside: VU-EA-H3), quercetin-3-O-arabinofuranoside (quercetin-3-O-arabinofuranoside: VU-EA-H5) and myricetin (EA-6) (See FIGS. 4-7).

Meanwhile, the fractions obtained using methanol-water (50: 50 vol / vol) were concentrated and purified using reverse phase HPLC in the same manner as above to separate EA-10 (12.4 mg), and the compound was quercetin: VU-EA-10) (see FIG. 8).

Example 3. A2E (N-retinyl-N-retinylidene ethanolamine) Synthesis

Add all-trans retinal (30 mg, 105.6 μmol) and ethanolamine (2.85 mg, 46.5 μmol) in ethanol (3.0 mL), add acetic acid (2.79 μL, 46.5 μmol), cover with a light barrier cover and remove 3 Synthesized daily. After concentration in vacuo, the mixture was subjected to cation exchange resin (Ambelite  Purification using IRC-50 ion exchange). The mixture attached to the resin was washed with 80% methanol (pH 12), and then A2E and iso-A2E were eluted with 100% methanol (pH 2) solvent added with 0.1% trifluoroacetic acid (TFA). Purified A2E and iso-A2E were concentrated using a rotary distillation and then dissolved in methylene chloride (CH ₂ Cl ₂ ). Distilled water was added to the separatory funnel to remove TFA and shaken. The methylene chloride fractions were concentrated in a rotary distiller. The mixture was stored at -20 ° C until next use.

Experimental Example

Experimental Example 1. Confirmation of inhibitory effect of phenolic compounds on ARPE-19 cell death by blue light

Inhibition of blue light-induced ARPE-19 cell death of the phenol compounds (myricetin-3-O-galactoside, quercetin-3-O-galatoside, quercetin-3-O-arabinofuranosie, myricetin, quercetin) isolated and purified in Example 2 The experiment on the effect was carried out as follows.

Adult human RPE cells (ARPE-19, ATCC), which were cells without any A2E, were used, and the cells were made with DMEM containing 10% FBS, 100 μ / ml penicillin, and 100 mg / mL streptomycin to prepare a medium at 37 ° C. Incubated in humid conditions with 5% CO ₂ .

In the experimental group, first, phenolic compounds (EA-1, 3, 5, 6, 10) were pretreated with APRE-19 cells at 20 μM each. The cells were then treated with A2E dissolved in 20 μM in PBS for 3 days to accumulate A2E in APRE-19 cells. Lutein 30 μM instead of the phenolic compound was treated as a positive control before A2E accumulation, and the negative control accumulated only A2E without prior drug treatment. After accumulating A2E for 3 days, blue light (430 nm, 4.02 J / cm ² ) was irradiated for 7 minutes. Cells were then incubated for 24 hours in complete medium and scraped on ice to obtain cells. Cell survival was measured using protein quantitation, protein quantification was performed using the Lowry method, and bovine serum albumin (BSA) was used as a standard. Cell viability was calculated relative to the blue light unirradiated A2E treated group.

The results are shown in FIG.

As shown in FIG. 9, when blue light was irradiated to APE-treated ARPE-19 cells without prior drug treatment, it was confirmed that the total protein amount was reduced by about 55%. That is, when A2E-accumulated APRE cells were irradiated with blue light, the survival rate of the cells was reduced to about 45% or less, from which the cytotoxicity of blue light-induced A2E accumulation could be confirmed (A2E accumulation / blue light irradiation group: A2E / BL group). .

In the quercetin-3-O-galactoside (EA-3) treated group, the cell survival rate was increased by only 7.7% compared to the A2E / BL group, indicating a slight protective effect of the retinal cells. While mycetin-3-O-galactoside (EA-1), mycetin (EA-6), quercetin (EA-10) and quercetin-3-O-arabinofuranoside (EA-5) In one experimental group, the cell survival rate was increased by 25%, 26.1%, 27.7%, and 39.7%, respectively, compared to the A2E / BL group, which showed a significant protective effect of the retinal cells.

Through the above results, it was confirmed that myricetin and quercetin effectively inhibit the death of retinal cells by the action of blue light and A2E. In particular, even in the case of quercetin glycosides, quercetin-3-O-arabinofuranoside was found to exhibit about 12% higher retinal cell protective effect than quercetin, whereas quercetin-3-O-galactosidate was compared to quercetin. It was found to show a significantly inferior effect. On the other hand, even in the same 3-O-galactosid derivatives, myricetin-3-O-galactoside did not show a worse effect than myricetin, and it was confirmed that the same excellent retinal cell protective effect as mycetin.

Phenolic compounds isolated from the blueberry extract of the present invention can inhibit the accumulation of A2E by inhibiting intracellular accumulation of blue light-induced retinal pigment epithelial cells. Therefore, the phenolic compounds of the present invention can be usefully used for the treatment, prevention or improvement of macular degeneration.

Claims (6)

Pharmaceutical composition for the treatment, prevention or improvement of macular degeneration containing phenolic compounds and pharmaceutically acceptable salts thereof isolated from the blueberry extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the phenolic compound is fractionated using chloroform, ethyl acetate, butanol, and then separated by reverse phase HPLC. The pharmaceutical composition according to claim 2, wherein the phenolic compound is mycetin-3-O-galactoside or quercetin-3-O-arabinofuranoside. The pharmaceutical composition according to claim 3, wherein the phenolic compound is quercetin-3-O-arabinofuranoside. The pharmaceutical composition according to any one of claims 1 to 4, wherein the macular degeneration is senile macular degeneration. A health functional food for preventing or improving macular degeneration, which contains a phenolic compound isolated from the blueberry extract and a pharmaceutically acceptable salt thereof as an active ingredient.

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