KR102399209B1 - Compositions for prevention or treatment of macular degeneration comprising ginseng berry extract and lutein - Google Patents

Abstract

The present invention provides a composition for preventing or treating macular degeneration containing a ginseng fruit extract and lutein. The composition for preventing or treating macular degeneration containing a ginseng fruit extract and lutein according to an embodiment of the present invention reduces migration of NF-κB of retinal cells from cytoplasm to a nucleus, decomposition of IκB, expression of IL-1β, expression of IL-6, expression of TNFα, and expression of VEGFα; and reduces abnormal inflammatory reactions, apoptosis, and angiogenesis. The present invention induces prevention, improvement, or treatment of the macular degeneration. In addition, since the composition inhibits death of retinal pigment epithelial cells induced by light, A2E photooxidation within the retinal pigment epithelial cells, and damage of a retinal cell layer, the composition is usefully used as a pharmaceutical or food composition for preventing, improving, and treating the macular degeneration.

Description

Composition for preventing or treating macular degeneration comprising ginseng fruit extract and lutein {COMPOSITIONS FOR PREVENTION OR TREATMENT OF MACULAR DEGENERATION COMPRISING GINSENG BERRY EXTRACT AND LUTEIN}

The present invention relates to a composition for preventing or treating macular degeneration comprising a ginseng fruit extract and lutein, and more particularly, to a ginseng fruit extract obtained by extracting ginseng fruit with water as a solvent; And it relates to a pharmaceutical and food composition for preventing or treating macular degeneration, including lutein.

The nerve tissue located in the center of the inner retina of the eye is called the macula. Most of the photoreceptor cells are gathered here, and the place where the image of an object is formed is also the center of the macula, so the macula plays a very important role in vision. Visual acuity refers to the ability to recognize the existence and shape of an object, and it is most sensitive when the image of an object is formed in the fovea of the macula (central vision), and decreases toward the periphery of the retina (peripheral vision). When you see an object, you see through the macula, the central part of the retina. In general, central vision is called vision. The macula can cause visual impairment due to various causes such as aging, genetic factors, toxicity, and inflammation, which is called macular degeneration.

Macular degeneration affects central vision, making it appear blurry in the center of the field of vision, and can make it difficult to perform sophisticated activities such as reading and driving through central scotoma, metaplasia, or focal loss of vision. In severe cases, it can lead to blindness. It is a very serious disease.

There are two types of macular degeneration: non-exudative (dry) macular degeneration and exudative (wet) macular degeneration, and 90% of people with macular degeneration have the dry form. In dry macular degeneration, waste products can build up in the tissue under the macula, forming yellow deposits called drusen. The presence of drusen interferes with blood flow to the retina, especially the macula, and when blood flow is reduced, it reduces the supply of nutrients to the macula, stopping or atrophying the efficient action of photosensitive cells, and can cause a chronic inflammatory response. is being revealed In wet macular degeneration, new weak blood vessels can grow in or under the retina, causing fluid and blood to leak into the space under the macula. Wet macular degeneration is sometimes also described as choroidal neovascularization, where the choroid is the subretinal vascular region and angiogenesis refers to the growth of new blood vessels within the tissue. This causes new blood vessels to grow.

The underlying mechanism of this macular degeneration is still unknown. Age-related macular degeneration, a type of macular degeneration, is characterized by loss of central vision due to the death of photoreceptor cells, and is believed to have a multifactorial etiological mechanism. Recently, it has been reported that various factors (eg, smoking, obesity, eating habits, increase in blood cholesterol, blue light irradiation, etc.) cause the promotion of age-related macular degeneration, which causes damage to photoreceptor cells and consequent apoptosis in the retina. It causes degeneration (degeneration) of the pigment epithelium. On the other hand, it is known that the accumulation of fat brown pigment (lipofuscin) in the center of the retina of the eye is greatest in the retinal pigment epithelial cells below the center of the retina. This reflects the fact that this area has a high concentration of rod photoreceptors. N-retinyl-Nretinylidene ethanolamine (A2E) is a major chromophore of lipofuscin and causes the generation of reactive oxygen species when exposed to blue light. Blue light is light with a relatively high energy with a wavelength of about 440 nm, and when exposed to it for a short time, damage to cells increases, which increases as the wavelength of light increases. That is, continuous light exposure further promotes the death of retinal pigment epithelial cells in age-related macular degeneration, which in turn becomes one of the main causes of photoreceptor cell degeneration. In addition, the deterioration of visual acuity due to retinal cell dysfunction is closely related to the oxidation of A2E and thus may be the main cause of age-related macular degeneration.

To date, there are very few treatments available for such macular degeneration. Moreover, there are some treatments such as anti-VEGF for wet macular degeneration, but there is no known treatment for dry macular degeneration.

On the other hand, lutein (lutein) is a naturally occurring carotenoid (carotenoid) that exists in a natural state without vitamin A activity, it is contained in a large amount in green and yellow vegetables. Lutein, one of the most abundant carotenoids in food and human blood, is known to perform antioxidant and plant protection functions from UV rays in the plant body. It is known as an ingredient that plays an important role in improving eyesight.

However, recently, a research team at Moran Eye Hospital affiliated with Utah State University in the U.S. published a 'JAMA Ophthalmology', a journal of the American Medical Association (JAMA Ophthalmology), inside the central retinal fossa of a woman who took too much lutein for a long time. reported that the substance was found. In the case of the above patient, it was found that he took a lutein supplement by 20 mg daily for 8 years, and consumed lutein-rich spinach, broccoli, kale, and avocado steadily. Therefore, the research team assumed that lutein precipitated in the eyeball, crystals were formed, and macular degeneration occurred.

Therefore, although lutein has an excellent therapeutic effect in the treatment of macular degeneration, side effects that cause macular degeneration rather appear when consumed for a long period of time or in excess, so it is necessary to develop a method for preventing, alleviating or suppressing it.

The inventors of the present invention have previously revealed that the ginseng fruit extract having a composition different from that of the ginseng root extract can have the effect of preventing, improving or treating macular degeneration (Korean Application No. 10-2020-0187943). The inventors have completed the present invention by confirming that the previously known lutein and ginseng fruit extract can exhibit a more excellent eye protection effect even at a lower concentration than when each substance is used individually.

The technical problem to be achieved by the present invention is to provide a composition capable of inducing prevention, improvement or treatment of macular degeneration.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above technical object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating macular degeneration comprising a ginseng fruit extract and lutein.

In one embodiment, the ginseng fruit extract is Korean ginseng ( Panax ginseng ), ginseng ( P. quiquefolius ), whole ginseng ( P. notoginseng ), bamboo japonicus ( P. japonicus ), trifolium ( P. trifolium ), himalayan ginseng ( P. pseudoginseng ), Vietnamese ginseng ( P. vietnamensis ) and American ginseng ( Panax quinquefolium ) It may be characterized as an extract extracted from the fruit of one or more types of ginseng selected from the group consisting of.

In one embodiment, the ginseng fruit extract may be characterized in that it is extracted using water as a solvent.

In one embodiment, the lutein may be characterized in that it is extracted from a natural product by a supercritical extraction method.

In one embodiment, the composition may include the ginseng fruit extract and the lutein in a weight ratio of 5:1 to 1:5.

In one embodiment, the macular degeneration may be characterized by light-induced death of retinal pigment epithelial cells, A2E photooxidation in retinal pigment epithelial cells, or retinal cell layer damage.

Another aspect of the present invention provides a food composition for preventing or treating macular degeneration comprising a ginseng fruit extract and lutein.

The composition for preventing or treating macular degeneration, comprising the ginseng fruit extract and lutein according to an embodiment of the present invention, is a retinal cell NF-κB movement from the cytoplasm to the nucleus, IκB degradation, IL-1β expression , IL-6 expression, TNFα expression and VEGFα expression can be reduced, thereby reducing abnormal inflammatory response, apoptosis and angiogenesis, thereby inducing prevention, improvement or treatment of macular degeneration. In addition, the composition can prevent, improve and treat macular degeneration by inhibiting light-induced death of retinal pigment epithelial cells, A2E photooxidation in retinal pigment epithelial cells, and retinal cell layer damage.

In addition, the composition is known to have a preventive, ameliorating and therapeutic effect on macular degeneration, but has been reported to have toxic effects such as causing macular degeneration when consumed excessively and preventing, improving and treating lutein and macular degeneration. It is possible to have a higher prevention, improvement and treatment effect of macular degeneration while including the ginseng fruit extract at a lower concentration, which the inventors have revealed.

The effects of the present invention are not limited to the above effects, but it should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

1 is a result of analyzing the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on apoptosis of retinal pigment epithelial cells (ARPE-19) by blue light.
2 is a result of analyzing the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the photooxidation of A2E by blue light in retinal pigment epithelial cells (ARPE-19).
3 is a representative image confirming the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on retinal photoreceptor cell layer damage in an animal model of blue light-induced macular degeneration (ONL, Outer nuclear layer); Inner nuclear layer (INL), photoreceptor layer (PL, Photoreceptor segment layer), the whole retina layer (Whole retina)).
4 is a result of analyzing the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on damage to the retinal photoreceptor cell layer in an animal model of blue light-induced macular degeneration.
5 is an image and statistical results analyzed by Western blot analysis of the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the amount of NF-κB in the cytoplasm and nucleus in retinal pigment epithelial cells (ARPE-19); FIG. .
6 is an image and statistical results of Western blot analysis of the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the amount of intracytoplasmic IκB in retinal pigment epithelial cells (ARPE-19).
7 is a PCR analysis result of the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the IL-1β mRNA expression level of retinal pigment epithelial cells (ARPE-19).
8 is a PCR analysis result of the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the IL-6 mRNA expression level of retinal pigment epithelial cells (ARPE-19).
9 is a PCR analysis result of the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the TNFα mRNA expression level of retinal pigment epithelial cells (ARPE-19).
10 is a PCR analysis result of the effect of a composition comprising a ginseng fruit extract and lutein according to Example 1 on the VEGFα mRNA expression level in retinal pigment epithelial cells (ARPE-19).

The pharmaceutical composition for preventing or treating macular degeneration according to one aspect of the present invention includes a ginseng fruit extract and lutein.

Ginseng ( Panax ginseng ) is a plant belonging to the genus Panax of the Araliaceae family . The ginseng fruit extract is Korean ginseng ( Panax ginseng ), Hoegi ginseng ( P. quiquefolius ), Jeonchilsam ( P. notoginseng ), Bamboo shoot ginseng ( P. japonicus ), Trifolium ginseng ( P. trifolium ), Himalayan ginseng ( P. pseudoginseng ), Vietnamese ginseng ( P. vietnamensis ) and American ginseng ( Panax quinquefolium ) It may be characterized as an extract extracted from the fruits of one or more species selected from the group consisting of ginseng. Preferably, the ginseng fruit used in the present invention may be the fruit of Korean ginseng (Panax ginseng).

Ginseng fruit is different from ginseng root (root), which is commonly used as a part of ginseng, in the type and content composition of the components it contains. Specifically, ginseng fruit contains more vitamins and minerals than ginseng root. In addition, the ginseng fruit contains more ginsenoside than the ginseng root, and the composition of the ginsenoside is different. Accordingly, in one embodiment of the present invention, the ginseng fruit extract contains ginsenoside PT (Protopanaxatriol) system containing ginsenoside Re, Rg1, Rg2, etc. Ginsenoside containing ginsenoside Rb1, Rb2, Rc, Rd, etc. It may contain more than the PD (Protopanaxadiol) system, and thus may exhibit different effects. For example, it has been reported that ginseng fruit exhibits superior antidiabetic efficacy than ginseng root (Dey et al., Phytomedicine , 2003, 10; 600-605). In addition, as the ginseng fruit matures from green like leaves, the flesh changes to red or yellow depending on the variety. As the color of the flesh changes, anthocyanins are formed in the ginseng fruit. Anthocyanins include, for example, cyanine chloride, delphinidin chloride, malvidin chloride, pelargonin chloride, cyanin chloride, idea chloride ), keracyanin chloride, kuromanin chloride, pelagonidin chloride, petunidin chloride, and the like (Crozier et al., Nat. Prod. REP. , 2009, 26, 1001-1043). And anthocyanins are effective in antioxidant, anti-allergy, anti-inflammatory, anti-viral, anti-proliferative, anti-mutagenic, anti-bacterial, anti-carcinogenic, cardiovascular damage and allergy protection, microcirculation protection, peripheral capillary resistance protection, diabetes improvement, etc. It has been reported that there is (Ghosh & Konishi, Asia Pac J Clin Nutr. , 2007, 16(2):200-8).

As used herein, the term "extract" refers to a material extracted from a raw material by any method, and is used in the meaning of including, without limitation, the extracted extract, the concentrate obtainable therefrom, the dried product and the powder of the concentrate.

The extract may be obtained by extraction from a raw material or a dried product thereof, and the raw material of the extract may be used without limitation, such as cultivated or commercially available ones.

When the extract is obtained by extracting it from the raw material, all conventional known extraction methods such as solvent extraction, ultrasonic extraction, high-pressure extraction, ultra-high pressure extraction, filtration, and reflux extraction may be used as the extraction method, preferably solvent extraction or It can be manufactured by using the reflux extraction method. The extraction process may be repeated several times, and thereafter, a step such as concentration or freeze-drying may be additionally performed. Specifically, the obtained extract is concentrated under reduced pressure to obtain a concentrate, and after freeze-drying the concentrate, a high concentration extract powder can be prepared using a grinder. The extract also includes a fraction obtained by further fractionating the extract.

The ginseng fruit extract may preferably be extracted by a water-soluble fractionation method, and may be prepared by a manufacturing method comprising the following steps: 1) preparing an extract by adding an extraction solvent to the ginseng fruit; 2) filtering the extract of step 1); and 3) drying the filtered filtrate of step 2) under reduced pressure.

In one embodiment, the ginseng fruit may be one selected from the group consisting of ginseng seeds, fruits, immature fruits, mature fruits, pericarp and combinations thereof, and the rinds of ginseng fruits (all except the seeds of the fruits) Or it may contain seeds, preferably the peel of a ginseng fruit.

In one embodiment, the ginseng fruit extract may be characterized in that it is extracted using an aqueous solvent, preferably water, alcohol, or a mixture thereof as a solvent, and more preferably, it may be extracted using water as a solvent. The solvent may be added in an amount of 1 to 10 ml per 1 g of the ginseng fruit used for extraction, preferably in an amount of 1 to 5 ml.

The ginseng fruit extract may contain ginsenoside Re. According to the inventor of the present invention, the ginseng fruit extract obtained by extracting the ginseng fruit by the water-soluble fractionation method has a higher ginsenoside content including ginsenoside Re than the ginseng root extract, and polysenol (anthocyanin, etc.) not present in the root. ), etc., were found to contain various active ingredients (Korean Patent Application No. 10-2020-0187943). The inventor has confirmed that the ginsenoside Re is the most contained among the active ingredients in the ginseng fruit water extract extracted by the water-soluble fractionation method as described above.

The extraction method of step 1) may be shaking extraction, Soxhlet extraction, reflux extraction, or ultrasonic extraction. In an embodiment of the present invention, the extraction method may be ultrasonic extraction. At this time, the extraction temperature may be 15 to 45 ℃, specifically 15 to 35 ℃, more specifically 20 to 30 ℃. In addition, the extraction pressure may be 200 to 1,000 MPa, specifically 300 to 900 MPa, more specifically 400 to 800 MPa. The extraction time may be 10 seconds to 1 hour, specifically 20 seconds to 30 minutes, and more specifically 30 seconds to 5 minutes. In addition, the number of times of extraction may be 1 to 5 times, specifically, may be 1 to 3 times. In a range outside the conditions of the extraction temperature, extraction pressure, extraction time, or number of times of extraction, extraction is not sufficiently performed, and the content of active ingredients in the ginseng fruit extract may be insignificant. Alternatively, the extraction yield may no longer increase and the efficiency of the extraction operation may decrease.

The vacuum concentration in step 3) may use a vacuum vacuum concentrator or a vacuum rotary evaporator. In addition, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying.

Lutein, along with chlorophyll, is present in green leaves and as esters in various flowers. In addition, it is contained together with zeaxanthin in the egg yolk and is the main pigment component of the alfalfa extract pigment. Lutein is a type of xanthophylls among carotenoids, and is a component concentrated in the macula, lens, brain, skin, heart, and spinal tissues of the eye. Protects the eyes and reduces free radicals. It is not synthesized in the body, so it must be supplied through food.

Foods that contain a large amount of lutein include spinach, kale, broccoli, lettuce, lettuce, sprouted cabbage, pumpkin, peas, yellow carrots, and some seaweeds, and especially green and yellow vegetables are known for their high content. In addition, it is known to contain a large amount in flowers such as marigold.

In one embodiment, the lutein may be extracted from natural products including the aforementioned plants, and may be commercially available lutein for food. The lutein may be a powder or oil suspension, and may be selected from lutein (alone), lutein-zeaxanthin complex extract, lutein ester, and combinations thereof.

When the lutein is extracted from a natural product, hexane, carbon dioxide, and alcohol (fermented alcohol) may be used alone or mixed as the solvent, and when used in combination, they may be used sequentially. At this time, the extraction temperature may be, for example, 10 to 80 ℃, for example, may be extracted for 1 to 10 hours. The lutein may preferably be characterized in that it is extracted by a supercritical extraction method.

In one embodiment, the composition contains the ginseng fruit extract and the lutein in an amount of 50:1 to 1:1, preferably 10:1 to 1:1, more preferably 5:1 to 1:5, most preferably 2.5: It may be characterized in that it is included in a weight ratio of 1 to 1:2.5.

The 'macular degeneration' of the present invention means that degeneration occurs in the macula, which is a nervous tissue located in the center of the inner retina of the eye, resulting in visual impairment such as reduced visual acuity, central scotoma, and metamorphosis (symptoms in which objects appear distorted). Macular degeneration is largely divided into non-exudative (dry) and exudative (wet). There is no specific treatment for non-exudative cases, and most of them do not significantly affect visual acuity, whereas exudative cases have a very poor visual prognosis. Among the structures that make up the outside of the eyeball that exist in the order of sclera, choroid, and retina from the outside, the middle membrane is called the choroid. causes Age-related macular degeneration is considered the leading cause of blindness in the elderly. In one embodiment, the macular degeneration may be characterized by light-induced death of retinal pigment epithelial cells, A2E photooxidation in retinal pigment epithelial cells, or retinal cell layer damage.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent, and each of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, It may be formulated in the form of suppositories and sterile injection solutions. The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and the like. It also includes diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral use include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose, or lactose. ), gelatin, and the like, and lubricants such as magnesium stearate and talc. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and may include water and diluents such as liquid paraffin, wetting agents, sweetening agents, fragrances, and preservatives. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, creams, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, and vegetable oils such as olive oil. oil, injectable esters such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The dosage of the active ingredient contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of disease, the form of the active ingredient, the route and period of administration, and may be appropriately adjusted according to the patient. For example, the ginseng fruit extract and the lutein may be administered in a dose of 0.0001 to 1000 mg/kg per day, preferably 0.01 to 100 mg/kg, respectively, and the administration may be performed per day. It may be administered once or in several divided doses. In addition, the pharmaceutical composition of the present invention may include the ginseng fruit extract and the lutein in an amount of 0.001 to 90% by weight based on the total weight of the composition.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, oral, skin, abdominal cavity, rectal or intravenous, muscle, subcutaneous, intrauterine dura mater or intracerebroventricular (intracerebroventricular). It may be administered by injection, preferably orally.

Another aspect of the present invention provides a food composition for preventing or treating macular degeneration comprising a ginseng fruit extract and lutein.

The weight ratio of the ginseng fruit extract, the lutein, the ginseng fruit extract, and lutein, macular degeneration, etc. will be omitted since it has been previously described in the pharmaceutical composition for preventing or treating macular degeneration containing the ginseng fruit extract and lutein.

In the food composition according to an embodiment of the present invention, there is no particular limitation on the type of food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, dairy products, various soups, It may be beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and may include all foods in a conventional sense.

In addition, it may be added to food or beverage for the purpose of preventing, improving or treating macular degeneration. At this time, the amount of the ginseng fruit extract and lutein in the food or beverage may be added in an amount of 0.01 to 15% by weight of the total food weight, respectively, and the health drink composition is 0.02 to 5 g, preferably 0.3 to 1, respectively, based on 100 ml g can be added.

The food composition of the present invention is not particularly limited in other ingredients other than containing the ginseng fruit extract and lutein as essential ingredients in the indicated proportions, and may contain additional ingredients such as various flavoring agents or natural carbohydrates like a conventional beverage. can 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 polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents such as taumatin, stevia extract, such as rebaudioside A, glycyrrhizin, and the like; and synthetic flavoring agents such as saccharin, aspartame and the like may be advantageously used. The proportion 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 to the above, the food composition comprising the ginseng fruit extract and lutein of the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavorants, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition comprising the ginseng fruit extract and lutein extract of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. In this case, the proportion of the additive is not very important, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition comprising the ginseng fruit extract and lutein of the present invention.

The food composition according to an embodiment of the present invention can be applied to animals as well as humans.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example 1. Preparation of composition for preventing or treating macular degeneration containing ginseng fruit extract and lutein>

100 g of ginseng fruits harvested from ginsengs aged 4 years or older were washed, and the washed ginseng fruits were placed in a seed separator with about three times purified water and subjected to a separation process to obtain the rinds of ginseng fruits excluding seeds. The obtained product including the peel is put in water and extracted under reflux for 2 to 5 hours at room temperature (within 25° C.), then filtered and the moisture is evaporated, and the powdery ginseng fruit extract obtained through spray drying is obtained, which is used until use. Stored at -20 °C. The production yield of the ginseng fruit extract obtained through the above process was about 2.5%, and the ginseng fruit extract was standardized to contain 6% of ginsenoside Re. The ginseng fruit extract and lutein (LU) were mixed in a 2:1 weight ratio, and this was dissolved in 0.5% carboxymethyl cellulose (CMC) at 20% (w/w), so that ginseng fruit extract and macular degeneration containing lutein A composition for sexual prophylaxis or treatment was obtained.

A2E accumulated in human retinal pigment epithelial cells (ARPE-19 cells) is known to induce cytotoxicity when irradiated with blue light (BL). > and <Example 3>.

At this time, ARPE-19 cells, which are cells without A2E in the cells, were used as the starting material, and these cells were used as a starting material in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. of the culture medium and incubated at 37 °C in humid conditions of 5% CO2.

<Example 2. Effect of a composition for preventing or treating macular degeneration containing pre-treated ginseng fruit extract and lutein on ARPE-19 cell death by blue light>

Human retinal pigment epithelial cells (ARPE-19 cells) were seeded in a 96-well tissue culture plate at a density of 1 x 10 ⁴ cells/well and cultured for 24 hours. Afterwards, as an experimental group, each test substance was treated prior to A2E accumulation in cultured ARPE-19 cells, and A2E dissolved in DMEM at 20 μM was treated 24 hours later to accumulate A2E in ARPE-19 cells. After culturing for 24 hours after A2E treatment, blue light (BL, 430 nm, 6000 lux) was irradiated for 20 minutes, and cell viability was measured after 24 hours using a cell count kit. At this time, the group in which A2E accumulation only (normal group, Normal), and A2E accumulation and blue light treatment (BL) were negative and positive controls, respectively, and the group treated with A2E accumulation, test substance and blue light was used as the experimental group. Test substances were ginseng fruit extract (GBE 25, 50, or 100 μg/ml) alone, lutein (LU 17) alone, or ginseng fruit extract (25 μg/ml) and lutein (10 μM) mixture (GBE 25+ LU 10), and the experiment was carried out.

As a result, as shown in Figure 1, cell death according to blue light irradiation was reduced by the pretreatment of the test substance. In more detail, in the case of the control group that only accumulated A2E without pretreatment with the test substance, the cell viability was reduced to 68.9% in the blue light treatment group (BL) compared to the group not treated with blue light (normal group, Normal). In contrast, in the group treated with 25, 50 and 100 μg/ml of ginseng fruit extract, 78.9, 82.6 and 84.1%, respectively, compared to the normal group, 1.15, 1.20, and 1.22 times higher than that of the blue light treatment group (BL), respectively. It was confirmed that the fruit extract could inhibit apoptosis caused by blue light in a concentration-dependent manner. In the group treated with lutein 17 μM, the cell viability was 76.7% compared to the normal group, and 1.16 times higher than that of the blue light treatment group. It was confirmed that apoptosis induced by A2E photooxidation according to this blue light irradiation could be inhibited. In addition, in the group treated with 25 μg/ml of ginseng fruit extract at the lowest concentration among the tested ginseng fruit extract concentrations and 10 μM lutein at a lower concentration than the tested lutein (GBE 25+ LU 10), the cell viability was higher than that of the normal group. 85%, which is 1.23 times higher than that of the blue light treatment group, which was higher than the four groups treated with a single substance, and even though each substance was mixed at a lower concentration than when treated with a single substance, the cell viability was higher appeared to be high. Therefore, when the composition of the present invention containing the ginseng fruit extract and lutein is pretreated, apoptosis induced by A2E photooxidation by blue light can be inhibited, and synergistic effect compared to treatment with the ginseng fruit extract or lutein alone was able to confirm that it has

<Example 3. Effect of a composition for preventing or treating macular degeneration comprising post-treated ginseng fruit extract and lutein on ARPE-19 cell death by blue light>

Human retinal pigment epithelial cells (ARPE-19 cells) were seeded in a 96-well tissue culture plate at a density of 1 x 10 ⁴ cells/well and cultured for 24 hours. A2E was treated at a concentration of 20 μM and cultured for 24 hours, the experimental group was post-treated, and after 24 hours, blue light (6000 lux, 20 min) was irradiated, and then the cell viability was measured using a cell count kit 24 hours later. . At this time, the group in which A2E accumulation only (normal group, Normal), and A2E accumulation and blue light treatment (BL) were negative and positive controls, respectively, and the group treated with A2E accumulation, test substance and blue light was used as the experimental group. Test substances were ginseng fruit extract (GBE 25, 50, or 100 μg/ml) alone, lutein (LU 17) alone, or ginseng fruit extract (25 μg/ml) and lutein (10 μM) mixture (GBE 25+ LU 10), and the experiment was carried out.

As a result, as shown in Figure 2, apoptosis according to blue light irradiation was reduced by post-treatment of the test material. In more detail, in the case of the control group that only accumulated A2E without pre-treatment with the test substance, the cell viability decreased to 69.94% in the blue light treatment group (BL) compared to the group not treated with blue light (normal group, Normal). On the other hand, the group treated with 25, 50 and 100 μg/ml of ginseng fruit extract showed 80.17, 83.70 and 88.18%, respectively, 1.15, 1.20, and 1.27 times higher than that of the blue light treatment group (BL), respectively. It was confirmed that the fruit extract could inhibit apoptosis caused by blue light in a concentration-dependent manner. In the group treated with lutein 17 μM, the cell viability was 84.08% compared to the normal group, and 1.21 times higher than that of the blue light treatment group. It was confirmed that apoptosis induced by A2E photooxidation according to this blue light irradiation could be inhibited. In addition, in the group treated with 25 μg/ml of ginseng fruit extract at the lowest concentration among the tested ginseng fruit extract concentrations and 10 μM lutein at a lower concentration than the tested lutein (GBE 25+ LU 10), the cell viability was higher than that of the normal group. 90%, which is 1.23 times higher than that of the blue light treatment group, which was higher than the four groups treated with a single substance, and even though each substance was mixed at a lower concentration than when treated with a single substance, the cell viability was higher appeared to be high. Therefore, when the composition of the present invention containing the ginseng fruit extract and lutein is post-treated, apoptosis induced by A2E photooxidation by blue light can be inhibited, and it is increased compared to when the ginseng fruit extract or lutein is treated alone. It was confirmed that it had an effect.

<Example 4. Confirmation of therapeutic effect of a composition for preventing or treating macular degeneration containing ginseng fruit extract and lutein in an animal model of blue light-induced macular degeneration>

After acclimatization of Balb/C mice (place of purchase: DBL) for one week, the test substance was administered to the test group once a day for 5 days, and after 24 hours dark adaptation, blue light was applied at 10000 lux for 2 weeks (14 days). After administration of irradiation and test substances once a day for 1 hour a day, an autopsy was performed 24 hours later, and the eyeballs including the optic nerve were removed. The extracted eye tissue was fixed in Davidson's solution for 10 days, fixed with formalin for 1-2 days, and then paraffin blocks were fabricated and H&E staining was performed. At this time, the group not treated with blue light and the test substance (normal group, Normal) and the group treated with blue light without treatment with the test substance (BL) were used as negative and positive controls, respectively, and the group treated with the test substance and blue light was used as the experimental group. . The test substance was ginseng fruit extract (GBE100, 100 mg per kg of test animal weight) alone, lutein (LU50, 50 mg per kg of test animal weight) alone, or ginseng fruit extract (50 mg per kg of test animal weight), respectively, depending on the group ) and lutein (25 mg per kg of experimental animal weight) mixture (GBE50+LU25) was used for the experiment.

By observing the eye tissue that had been stained, the thickness of the photoreceptor layer (PL) and the whole retina were confirmed (Fig. 3). As shown in Figure 4 and Table 1, the retinal layer thickness reduction according to the blue light irradiation was suppressed by the treatment of the test material.

Normal BL BL+
GBE100 BL+
LU50 BL+
GBE50+LU25 outer core layer
(ONL) Thickness (μm) 36.6 18.1 23 25.5 29.5 Normal % - 49.5 62.8 69.7 80.6 BL contrast % - - 127.1 140.9 163.0 inner core layer
(INL) Thickness (μm) 26.4 18.3 21 20 24.2 Normal % - 69.3 79.5 75.8 91.7 BL contrast % - - 114.8 109.3 132.2 photoreceptor layer
(PL) Thickness (μm) 23.1 14.4 16 16 16.5 Normal % - 62.3 69.3 69.3 71.4 BL contrast % - - 111.1 111.1 114.6 entire retinal layer
(Whole retina) Thickness (μm) 139.9 87.7 95 100 114.1 Normal % - 62.7 67.9 71.5 81.6 BL contrast % - - 108.3 114.0 130.1

In more detail, in the case of the control group not treated with the test substance, the photoreceptor outer nuclear layer (ONL), inner nuclear layer (INL), and photoreceptors in the blue light treatment group (BL) compared to the group not treated with blue light (normal group, Normal) Layer (PL) and whole retina thicknesses were reduced to 45.5, 69.3, 62.3 and 62.7%, respectively. In contrast, the group treated with ginseng fruit extract at 100 mg/kg (GBE100) had ONL, INL, PL, and whole retina thicknesses of 62.8, 79.5, 69.3, and 67.9%, respectively, compared to the normal group, and lutein at 50 mg/kg In group (LU50), ONL, INL, PL and whole retina thickness were 69.7, 75.8, 69.3, and 71.5%, respectively, higher than that of the normal group, which was about 8 to 40% higher than that of the blue light treatment group. In addition, in the group treated with 50 mg/kg of ginseng fruit extract, which is half the concentration of the single-treated ginseng fruit extract, and 25 mg/kg of lutein, which is half the concentration of lutein treated alone (GBE50+LU25), ONL, INL, The thickness of PL and whole retina was 80.6, 91.7, 71.4 and 81.6% compared to the normal group, which was about 9 to 60% higher than that of the blue light treatment group, which was higher than that of each group treated with a single substance as well as treated with a single substance. It was found that the effect of inhibiting the retinal layer thickness reduction was higher even though each material was mixed at a lower concentration compared to Si. Therefore, it was confirmed that the composition of the present invention comprising the ginseng fruit extract and lutein can suppress damage to the retinal layer due to blue light, and has a higher effect than when the ginseng fruit extract or lutein was treated alone.

<Example 5. Effect of a composition for preventing or treating macular degeneration containing ginseng fruit extract and lutein on NF-κB signaling in retinal pigment epithelial cells (ARPE-19)>

NF-κB is a protein complex, and NF-κB/Rel heterodimer is present in the cytoplasm in a state of being bound to IκB. When IκB is degraded, it enters the nucleus in the heterodimer state to induce gene transcription. Genes regulated by NF-κB are known to be related to inflammation/immune response, angiogenesis, cell survival or cell proliferation. In order to investigate how the composition containing the ginseng fruit extract and lutein of the present invention affects NF-κB signaling, the amounts of NF-κB and IκB in retinal pigment epithelial cells (ARPE-19) were checked.

Human retinal pigment epithelial cells (ARPE-19 cells) were seeded in a 96-well tissue culture plate at a density of 1 x 10 ⁴ cells/well and cultured for 24 hours. A2E was treated at a concentration of 20 μM and cultured for 24 hours, the experimental group was post-treated, and after 24 hours, blue light (6000 lux, 20 min) was irradiated, and then the cell viability was measured using a cell count kit 24 hours later. . At this time, the group in which A2E accumulation only (normal group, Normal), and A2E accumulation and blue light treatment (BL) were negative and positive controls, respectively, and the group treated with A2E accumulation, test substance and blue light was used as the experimental group. The test substance was ginseng fruit extract (GBE 50 μg/ml) alone, lutein (LU 30 μM) alone, or a mixture of ginseng fruit extract (25 μg/ml) and lutein (15 μM) at different concentrations depending on the group (GBE 25+ LU 15) was used for the experiment.

Cells were subjected to cell lysis and nuclear/cytoplasmic fractionation according to Rosner & Hengstschlaeger ( Hum. Mol. Genet. , 2008) to extract proteins from each fraction. In this regard, Western blot was performed to confirm the amount of each NF-κB in the nucleus and cytoplasm and the expression of IκB-α in the cytoplasm, respectively. At this time, the group not treated with blue light and the test substance (normal group, Normal) and the group treated with blue light without treatment with the test substance (control) were respectively negative and positive controls, and the group treated with the test substance and blue light was the experimental group. .

As a result, the amount of NF-κB in the nucleus increased by 3.48 times in the blue light treatment group (control) compared to the normal control group ( FIG. 5B ), and the expression in the cytoplasm was decreased to match it ( FIG. 5A ). In the case of the blue light treatment group to which the test substance was administered, NF-κB in the nucleus increased compared to the normal group, but compared to the untreated blue light treatment group (control), 50 μg/ml ginseng fruit extract and 30 μM lutein were administered. In each group, the levels were as low as 59% and 67%, respectively, and in the group administered with the composition of the present invention containing 25 μg/ml ginseng fruit extract and 15 μM lutein, which is half the concentration of each single test substance, nuclear It was confirmed that my NF-κB was lower at a level of 50% compared to the blue light treatment group (control) (FIG. 5B).

As a result of confirming the amount of IκB in the cytoplasm (FIG. 6), the amount of IκB was reduced to 58% in the blue light treatment group (control) compared to the normal control group, but the blue light treatment group administered with the test substance was higher than the control. . That is, in the group administered with 50 μg/ml ginseng fruit extract and 30 μM lutein, the amount of IκB in the cytoplasm was higher at 150% and 129%, respectively, compared to the blue light treatment group (control). In the group administered with the composition of the present invention containing the extract and 15 μM lutein, the amount of IκB in the cytoplasm was higher than that of the blue light treatment group (control) at 160% level.

Taken together, the composition comprising the ginseng fruit extract and lutein of the present invention inhibits the degradation of IκB, thereby further enhancing the effect of the ginseng fruit extract and lutein alone, which allows less NF-κB to migrate from the cytoplasm to the nucleus. It was confirmed that it could be raised.

<Example 6. Effect of a composition for preventing or treating macular degeneration containing ginseng fruit extract and lutein on NF-κB target gene expression in retinal pigment epithelial cells (ARPE-19)>

As described above, it has been reported that NF-κB can induce the expression of a target gene when it moves from the cytoplasm to the nucleus. In order to check whether the inhibition of NF-κB intranuclear migration by the composition of the present invention actually led to an effect on the expression of the target gene, IL-1β, IL-6 among the target genes in retinal pigment epithelial cells (ARPE-19) , the mRNA expression levels of TNFα and VEGFα were confirmed.

Human retinal pigment epithelial cells (ARPE-19 cells) were seeded in a 96-well tissue culture plate at a density of 1 x 10 ⁴ cells/well and cultured for 24 hours. A2E was treated at a concentration of 20 μM and cultured for 24 hours, the experimental group was post-treated, and after 24 hours, blue light (6000 lux, 20 min) was irradiated, and then the cell viability was measured using a cell count kit 24 hours later. . At this time, the group in which only A2E accumulation (normal group, Normal), and the group treated with A2E accumulation and blue light (control) were negative and positive controls, respectively, and the group treated with A2E accumulation, test substance and blue light was used as the experimental group. The test substance was ginseng fruit extract (GBE 50 μg/ml) alone, lutein (LU 30 μM) alone, or a mixture of ginseng fruit extract (25 μg/ml) and lutein (15 μM) at different concentrations depending on the group (GBE 25+ LU 15) was used for the experiment. For each group, RNA was extracted from the cells using TRIzol agent (Invitrogen) and quantified 1 μg/μl RNA and cDNA were synthesized using the reverse transcription system (Promega). The expression pattern of each gene was measured using the synthesized cDNA and pre-designed primers and probes for each gene (Applied biosystems). PCR reaction and analysis were performed using a Rotor-Gene 3000 system (Corbett Research, Sydney, Australia).

As a result, in the case of IL-1β, which is known to be induced through NF-κB by tissue/cell damage, the mRNA expression level in the blue light-treated group (control) was nearly 4 times higher than that of the normal control group. However, in the blue light treatment group to which the test substance was administered, it was decreased compared to the control group (FIG. 7). That is, compared to the blue light treatment group (control), the IL-1β mRNA expression level was lower in the group administered with 50 μg/ml ginseng fruit extract and 30 μM lutein, respectively, at 44% and 60% levels, respectively, and at 25 μg/ml In the group administered with the composition of the present invention containing ginseng fruit extract and 15 μM lutein, the IL-1β mRNA expression level was as low as 44% of that of the blue light treatment group (control).

IL-6 is secreted by T cells and macrophages to stimulate immune responses, and is known to mediate heat and rapid immune responses. In RPE cells, IκB degradation is induced by oxidative stress and it is known that NF-κB moves into the nucleus to induce IL-6 expression. 6 It was found that the mRNA expression level was nearly three times higher (FIG. 8). Compared to the blue light treatment group (control), the IL-1β mRNA expression levels were lower in the group administered with 50 μg/ml ginseng fruit extract and 30 μM lutein, respectively, at 41% and 79%, and in particular, 25 μg/ml ginseng In the group administered with the composition of the present invention containing the fruit extract and 15 μM lutein, the IL-1β mRNA expression level was 34% of that of the blue light treatment group (control), which was a normal control group not treated with blue light (normal) is at a similar level to

TNFα is mainly secreted by macrophages, but is also secreted by other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons, and activates NF-κB, MAPK signaling, and death signaling. It is known to act as an inducer. TNFα was found to be nearly 4 times higher in mRNA expression in the blue light treatment group than in the normal control group, but it was lower in the blue light treatment group to which the test substance was administered than in the control group (FIG. 9). That is, in the group administered with 50 μg/ml ginseng fruit extract and 30 μM lutein compared to the blue light treatment group (control), IL-1β mRNA expression levels were lower at 36% and 48%, respectively, and at 25 μg/ml In the group administered with the composition of the present invention containing ginseng fruit extract and 15 μM lutein, the IL-1β mRNA expression level was as low as 31% of that of the blue light treatment group (control), which was a normal control group not treated with blue light ( normal) was found to be at a similar level.

VEGFα, which mediates angiogenesis, showed that the mRNA expression level was nearly four times higher in the blue light treatment group than in the normal control group, whereas in the blue light treatment group treated with the test substance, both were lower than the control group. level (Fig. 10). That is, compared to the blue light treatment group (control), the VEGFα mRNA expression levels were lower in the group administered with 50 μg/ml ginseng fruit extract and 30 μM lutein, respectively, at 36% and 41% levels, and in particular, 25 μg/ml ginseng In the group administered with the composition of the present invention containing the fruit extract and 15 μM of lutein, the expression level of VEGFα mRNA was low at 31% of that of the blue light treatment group (control).

In summary, the composition for preventing or treating macular degeneration comprising the ginseng fruit extract and lutein according to an embodiment of the present invention, the movement of NF-κB from the cytoplasm to the nucleus of retinal cells, degradation of IκB, and IL-1β expression , IL-6 expression, TNFα expression and VEGFα expression can be reduced, thereby reducing abnormal inflammatory response, apoptosis and angiogenesis, thereby inducing prevention, improvement or treatment of macular degeneration. In addition, it can be expected that the composition can prevent, improve and treat macular degeneration by inhibiting light-induced apoptosis of retinal pigment epithelial cells, A2E photooxidation in retinal pigment epithelial cells, and retinal cell layer damage.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (7)

As a pharmaceutical composition for preventing or treating macular degeneration comprising ginseng fruit extract and lutein,
The ginseng fruit extract was extracted using water as a solvent,
The macular degeneration is a pharmaceutical composition for preventing or treating macular degeneration, characterized in that due to light-induced death of retinal pigment epithelial cells, A2E photooxidation in retinal pigment epithelial cells, or retinal cell layer damage.
The method of claim 1,
The ginseng fruit extract is Korean ginseng ( Panax ginseng ), Hoegi ginseng ( P. quiquefolius ), Jeonchilsam ( P. notoginseng ), Bamboo shoot ginseng ( P. japonicus ), Trifolium ginseng ( P. trifolium ), Himalayan ginseng ( P. pseudoginseng ), Vietnamese ginseng ( P. vietnamensis ) and American ginseng ( Panax quinquefolium ) A pharmaceutical composition for preventing or treating macular degeneration, characterized in that it is an extract extracted from the fruit of one or more types of ginseng selected from the group consisting of.
delete The method of claim 1,
The lutein is a pharmaceutical composition for preventing or treating macular degeneration, characterized in that it is extracted from a natural product by a supercritical extraction method.
The method of claim 1,
The composition is a pharmaceutical composition for preventing or treating macular degeneration, comprising the ginseng fruit extract and the lutein in a weight ratio of 5:1 to 1:5.
delete As a food composition for preventing or improving macular degeneration comprising ginseng fruit extract and lutein,
The ginseng fruit extract was extracted using water as a solvent,
The macular degeneration is a food composition for preventing or improving macular degeneration, characterized in that due to light-induced death of retinal pigment epithelial cells, A2E photooxidation in retinal pigment epithelial cells, or retinal cell layer damage.

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