KR20190133978A - Pharmaceutical composition for preventing or treating of macular degeneration comprising Prunella vulgaris var. lilacina extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, treating or alleviating macular degeneration containing an extract of Prunella vulgaris as an active component. Specifically, the extract of Prunella vulgaris of the present invention inhibits A2E accumulation in retinal pigment epithelial cells, inhibits the death of retinal pigment epithelial cells induced by blue light, and inhibits damage of a visual cell outer nuclear layer in a blue light-induced macular degeneration animal model, thereby being able to be usefully used as the composition for preventing, treating or alleviating macular degeneration.

Description

Pharmaceutical composition for preventing or treating of macular degeneration comprising Prunella vulgaris var. lilacina extract as an active ingredient}

The present invention relates to a composition for the prevention, treatment or improvement of macular degeneration containing an extract of Hagocho as an active ingredient.

The macula is a neural tissue located in the center of the retina, where the cells of the eye are concentrated, and the central image is the place where the image of the object is formed. Macular degeneration may occur due to various causes such as aging, genetic factors, toxicity and inflammation, which may cause visual disturbances. Macular degeneration is one of the three major causes of blindness along with glaucoma and diabetic retinopathy. Degeneration of the macula leads to decreased vision, central dark spots, and opacity.

Macular degeneration can be divided into dry (non-exudative) macular degeneration and wet (exudative) macular degeneration. About 90% of macular degeneration is equivalent to dry macular degeneration. Dry macular degeneration is a condition in which macular tissues become thin or atrophy due to the accumulation of drusen waste in the retinal pigment epithelium. On the other hand, wet macular degeneration is an abnormal growth of new blood vessels in the macula rupture blood and mucus leaking into the macula causes severe vision loss. Dry macular degeneration is not subjective at first, and even if the disease progresses, symptoms may be mistaken for simple presbyopia. However, if dry macular degeneration is left unattended, severe macular degeneration may occur, and it is important to prevent dry macular degeneration because it may be converted into wet macular degeneration with high blindness.

The underlying mechanism of macular degeneration is not yet known, but multifactorial etiology affects, and retinal pigment epithelial deformity is expected to be a major factor. As the eyes age, liposomes accumulate in the lysosomes of the retinal pigment epithelium, consisting of a mixture of lipids and proteins, as well as a fluorescent pigment called A2E ( N- retinyl - N - retinylidene ethanolamine). A2E is a major factor that kills retinal pigment epithelial cells and accumulates in lysosomes and inhibits their function, thereby killing retinal pigment epithelial cells. In particular, when A2E is irradiated with ultraviolet light or blue light, it becomes a highly reactive photooxidized form and induces apoptosis by changing the protein activity of mitochondria. In addition, A2E killing of retinal pigment epithelial cells leads to secondary killing of photoreceptor cells, which are supplied with major substances from retinal pigment epithelial cells. The most damaging is the cause of macular degeneration.

Prunella Spike, on the other hand, is a flowering plant of the perennial herb, Prunella vulgaris Linne var. Lilacina Nakai, or P. vulgaris Linne, belonging to Labiatae (Korean Pharmacopoeia, 11 Amendment, 2017). ). Higocho has been widely used as a folk medicine in China, Japan, Korea, and medicine in Europe. In Asia, it has been used for cirrhosis and hypertension. In Europe, it has been used to reduce throat inflammation, fever and wound treatment. In recent years, physiological activity studies of haejucho were reported to be anti-allergic, anti-inflammatory, antioxidant, antimicrobial and antiviral (Psotov et al., 2003).

Therefore, the present inventors have been using for a long time, while developing a treatment for macular degeneration using natural products that are safe for human body, the extract of Gojicho inhibits A2E accumulation in retinal pigment epithelial cells, and inhibits the death of blue light induced retinal pigment epithelial cells. The present invention was completed by confirming the inhibition of outer nuclear layer (ONL) damage in an animal model of blue light induced macular degeneration.

It is an object of the present invention to provide a composition for the prevention, treatment or improvement of macular degeneration, which contains an extract of Hagocho as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of macular degeneration, containing the extract of Hagocho as an active ingredient.

The present invention also provides a health functional food for the prevention or improvement of macular degeneration containing the extract of Hagocho as an active ingredient.

Higocho extract of the present invention inhibits A2E accumulation in retinal pigment epithelial cells, inhibits the death of retinal pigment epithelial cells induced by blue light, and inhibits damage of visual cell outer nucleus layer in blue light induced macular degeneration animal model. It can be usefully used as a composition for preventing, treating or improving sex.

1 is a diagram showing the inhibitory effect of A2E accumulation in retinal pigment epithelial cells of PV extract and Lutein (A2E-BDP: fluorescently labeled A2E).
2 is a quantitative diagram showing the inhibitory effect of A2E accumulation in retinal pigment epithelial cells of PV extract and Lutein (A2E-BDP: fluorescently labeled A2E).
Figure 3 is a diagram showing the effect of inhibiting the retinal pigment epithelial cell death of the extract (PV extract) and lutein (Lutein) (BL: blue light).
Figure 4 is a diagram showing the animal experiment schedule for confirming the macular degeneration treatment effect of Hachocho extract.
FIG. 5 is a diagram showing the effect of inhibiting damage to the cytoplasmic outer nuclear layer (ONL) of Prunella vulgaris (PV) and lutein (Lu) in a blue light (BL) induced macular degeneration animal model.
FIG. 6 is a quantitative diagram showing the effect of inhibiting the damage to the cytocellular extranuclear layer of PV extract and lutein in the blue light induced macular degeneration animal model.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of macular degeneration, which contains an extract of Hagocho as an active ingredient.

The hachoweed extract may be prepared by a manufacturing method comprising the following steps:

1) preparing an extract by adding an extraction solvent to hacho sec;

2) filtering the extract of step 1); And

3) drying the filtered filtrate of step 2) under reduced pressure.

The extractant may be water, alcohol or a mixture thereof. The alcohol may be C ₁ to C ₄ lower alcohol, specifically, the lower alcohol may be ethanol or methanol. The extraction solvent may be added in an amount of 1 to 50 mL per 1 g of the weight of the vinegar used for extraction.

The extraction method may be shaking extraction, Soxhlet extraction or reflux extraction. At this time, the extraction time may be 1 to 50 hours, 10 to 40 hours or 15 to 30 hours. The extraction may be repeated 3 to 5 times.

On the other hand, the reduced pressure concentration of step 3) may be used a vacuum pressure reducer or vacuum rotary evaporator. In addition, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, specifically freeze drying.

The macular degeneration may be wet macular degeneration or dry macular degeneration.

The inventors of the present invention, after preparing a hyacinth extract, confirmed that the hyacinth extract inhibits A2E accumulation in retinal pigment epithelial cells (see FIGS. 1 and 2) and inhibits the death of retinal pigment epithelial cells induced by blue light. (See Figure 3). In addition, the Hagocho extract alleviated damage of the extracellular nucleus layer in the macular degeneration animal model induced by blue light (see FIGS. 4 and 5). Therefore, the Hacho herb extract may be usefully used for preventing or treating macular degeneration.

The pharmaceutical composition according to the present invention may include 10 to 95% by weight of the hachocho extract as an active ingredient based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the active ingredient.

The pharmaceutical compositions of the present invention may comprise carriers, diluents, excipients or mixtures thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers can be used as long as they are suitable for delivery of the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added as necessary.

When formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used may be added.

The composition of the present invention may be formulated as an oral or parenteral preparation. Oral formulations may include solid and liquid formulations. The solid preparation may be tablets, pills, powders, granules, capsules or troches, and such solid preparations may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or mixtures thereof. In addition, the solid preparation may include a lubricant, for example magnesium styrate, talc and the like. On the other hand, the liquid formulation may be a suspension, a liquid solution, an emulsion or a syrup. At this time, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, preservatives and the like.

The parenteral preparations may include injections, suppositories, respiratory inhalation powders, spray aerosols, powders and creams, and the like. The injection may include a sterile aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. In this case, as the non-aqueous solvent or suspending solvent, vegetable oil such as propylene glycol, polyethylene glycol, olive oil, or injectable ester such as ethyl oleate may be used.

The composition of the present invention can be administered orally or parenterally according to the desired method. Parenteral administration can include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection modes.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, the drug being used simultaneously, and the like. However, for the desired effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration can be from one to several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration may be sequential or simultaneous.

The present invention also provides a health functional food for the prevention or improvement of macular degeneration containing the extract of Hagocho as an active ingredient.

The extract of Hagocho may be prepared by adding an extraction solvent to Hagocho to prepare an extract, and filtering the extract, followed by concentration under reduced pressure and drying.

The extractant may be water, alcohol or a mixture thereof. The alcohol may be C ₁ to C ₄ lower alcohol, specifically, the lower alcohol may be ethanol or methanol. The extraction solvent may be added in an amount of 1 to 50 mL per 1 g of the weight of the vinegar used for extraction.

The macular degeneration may be wet macular degeneration or dry macular degeneration.

The inventors of the present invention, after preparing a hyacinth extract, confirmed that the hyacinth extract inhibits A2E accumulation in retinal pigment epithelial cells (see FIGS. 1 and 2) and inhibits the death of retinal pigment epithelial cells induced by blue light. (See Figure 3). In addition, the Hagocho extract alleviated damage of the extracellular nucleus layer in the macular degeneration animal model induced by blue light (see FIGS. 4 and 5). Therefore, the Hacho herb extract may be usefully used for preventing or improving macular degeneration.

The composition of the present invention may be added as it is to food, or used with other foods or food ingredients. In this case, the amount of the active ingredient added may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total food weight.

The form and type of dietary supplement are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The dietary supplement may include various flavors, sweeteners or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include taumartin, stevia extract, and the like. On the other hand, examples of the synthetic sweeteners include saccharin, aspartame, and the like. In addition, the natural carbohydrate may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.

In addition to the above-mentioned additional ingredients, the health functional food of the present invention is a nutrient, vitamin, electrolyte, flavoring agent, coloring agent, pectane and salt thereof, alginic acid and salt thereof, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, and preservative. , Glycerin, alcohol, and the like may be further included. These components can be used independently or in combination. The proportion of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following Examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1 Preparation of Seaweed Extract

After crushing the stalk portion (Lot No. JH123-140203, Harmonic Pharmaceuticals) of dried nectar ( Prunella vulgaris var. Lilacina), distilled water was added at a rate of 100 g / L and extracted at 100 ° C for 2 hours. The extract was filtered, concentrated using a rotary concentrator, and freeze-dried.

Experimental Example 1. Inhibitory effect of A2E accumulation on retinal pigment epithelial cells

Since A2E accumulation of retinal pigment epithelial cells plays a major role in the development of macular degeneration, in order to determine whether the extract of Higocho has an effect of inhibiting A2E accumulation in retinal pigment epithelial cells, human retinal pigment epithelial cell line ARPE-19 cells and fluorescence Labeled A2E was used.

Specifically, ARPE-19 cells (American Type Culture Collection, USA) were treated with the ultrahigh extract 0, 25, 50, 100 or 200 μg / mL or lutein 17.04 μg / mL (30 μM) obtained in Example 1 above. Then, 20 μM of fluorescently labeled A2E (A2E-BDP) was treated with ARPE-19 cells, and after 24 hours, the supernatant was removed and washed with phosphate buffered saline (PBS). The ARPE-19 cells were observed with a fluorescence microscope (Nikon Eclipse Ti-U Fluorescence, Japan), and fluorescence was measured using a fluorescence photometer (FLUOstar Omega, BMG Labtech, USA) to quantify A2E accumulated in cells.

As a result, the lutein-treated group inhibited A2E accumulation in cells by 25% compared to the negative control group that induced A2E accumulation, whereas the Higocho extract inhibited A2E accumulation in 25, 50, 100 or 200 ㎍ / mL treated groups by 13, 20, 37 or 49% inhibition (FIGS. 1 and 2). This suggests that the treatment group of 100 μg / mL or more of the Hagocho extract has a superior inhibitory effect on A2E accumulation in cells than the lutein 17.04 μg / mL (30 μM) treatment group.

Experimental Example 2. Protective effect of retinal pigment epithelial cells on blue light induced photooxidation

It is known that A2E accumulated in retinal pigment epithelial cells causes cytotoxicity when blue light is irradiated, and it is cytotoxic caused by irradiation of blue light after A2E accumulation in ARPE-19 cells. Confirmed.

Specifically, ARPE-19 cells were treated with the haecho extract 0, 25, 50, 100 or 200 μg / mL or lutein 17.04 μg / mL (30 μM) obtained in Example 1 for 24 hours, followed by A2E 20 μM was treated for 24 hours and blue light (4,000 lux) was irradiated for 10 minutes. Afterwards, the cell viability of ARPE-19 cells was measured by a cell counting kit (Dojindo Labs, Japan), and A2E was accumulated and the percentage of cell viability of each treated group was compared to the control group not irradiated with blue light. ) (FIG. 3).

As a result, apoptosis was induced as A2E was accumulated and the cell viability of the negative control group irradiated with blue light was significantly reduced compared to the control group. The cell protection effect of each drug was calculated using the difference in cell viability of the control group and the negative control group as 100%, and the cell protection effect of lutein was 53%, and the cell protection effect of the haejucho extract was 25, 50, 100 or 200 ㎍ /. 34.8, 48.6, 62.1 or 69.5%, respectively, in the mL treatment group (FIG. 3). This suggests that the treatment group of 100 μg / mL or more of the Hagocho extract has a superior cell protective effect than the lutein 17.04 μg / mL (30 μM) treatment group.

Experimental Example 3 Inhibitory Effect of Extracts from Hagocho on Blue Light Induced Macular Degeneration

In order to verify the protective effect of retinal pigment epithelial cells of the haejucho extract confirmed in Experimental Examples 1 and 2 in an animal model, the following experiment was performed in the macular degeneration animal model induced by blue light.

3-1. Preparation and Experimental Schedule of Macular Degeneration Animal Models

After 5 weeks old BALB / C mice were incubated for 2 days, the haecho extract (50, 100 or 200 mg / kg) or lutein (100 mg / kg) obtained in Example 1 was administered once daily for 5 days. . Thereafter, after 24 hours of dark acclimatization, the haegocho extract (50, 100 or 200 mg / kg) or lutein (100 mg / kg) was applied every 10 minutes before the blue light irradiation while irradiating blue light at 10,000 lux for 1 hour every day for 14 days. Administered. Seven days after the end of blue light irradiation and drug administration, mice were sacrificed to extract eyeballs, and the retinal progenitor protective effect was analyzed.

3-2. Protective Effect of Rhizome Extracts on Retinal Cells

The extracted ocular tissues were fixed in neutral buffered formalin, washed, dehydrated with alcohol, transparent with xylene, and embedded in paraffin. . Tissue sections were prepared by trimming the embedded tissue block to 4 μm thickness, followed by hematoxylin & eosin (H & E) staining. Specifically, the tissue sections were treated with hematoxylin for 30 seconds, washed with water for 10 minutes, and then treated with eosin for 1 minute. Photographs of ocular tissue sections stained with a microscope (Olympus Optical, Japan), followed by image J software (Image J software, National Institute of Health, USA) thickness of the extracellular nucleus layer at 600-900 μm from the optic nerve Was measured and the number of nuclei was counted.

As a result of the measurement of the thickness of the cell outer core layer, it was significantly decreased in the negative control group irradiated with blue light compared to the control group not irradiated with blue light, and significantly increased in the negative control group in the Pleurotus chinensis extract or lutein administration group. As a result of calculating the difference in the thickness of the extracellular nuclear layer in the negative control group of the control group and the negative control group as 100%, 49.1% in the lutein group and the 50, 100, or 200 mg in the plethyrum extract group 35.8, 50.5 or 71.2% in the / kg administration group, respectively (Figs. 5 and 6).

As a result of measuring the number of nuclei of the cell nucleus layer, it was significantly decreased in the negative control group irradiated with blue light compared to the control group not irradiated with blue light, and significantly increased in the negative control group in the pleated vinegar extract or lutein administration group. As a result of calculating the difference in cell number in the nuclear cell layer of the negative control group of the control group and the negative control group as 100%, the lutein-treated group was 29.3%, the pleated extract group was 50, 100 or In the 200 mg / kg administration group was 31.6, 33.9 or 50.8%, respectively (Figs. 5 and 7).

This suggests that, in the blue light induced macular degeneration model, the hydrangea extract has an effect of significantly repairing retinal cell damage at levels similar to lutein.

In conclusion, it was confirmed from the above examples and experimental examples that the Hagocho extract showed excellent inhibition of A2E accumulation in retinal pigment epithelial cells, protection against blue light-induced retinal pigment epithelial cell toxicity, and blue light induced macular degeneration. . Therefore, Hachocho extract can be usefully used for preventing, treating or improving macular degeneration.

Claims (8)

A pharmaceutical composition for the prevention or treatment of macular degeneration, containing the extract of Hagocho as an active ingredient.
The pharmaceutical composition of claim 1, wherein the extract is extracted with water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof.
The pharmaceutical composition of claim 2, wherein the lower alcohol is ethanol or methanol.
The pharmaceutical composition of claim 1, wherein the macular degeneration is wet macular degeneration or dry macular degeneration.
Health functional food for the prevention or improvement of macular degeneration containing the extract of Hagocho as an active ingredient.
The health functional food according to claim 5, wherein the extract is extracted with water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof.
The dietary supplement of claim 6, wherein the lower alcohol is ethanol or methanol.
The dietary supplement according to claim 5, wherein the macular degeneration is wet macular degeneration or dry macular degeneration.

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