KR20210142445A - Retinal disease prevention and treatment composition comprising turmeric extract - Google Patents

Abstract

The present invention relates to a composition for preventing and treating retinal diseases containing a Curcuma longa extract, wherein by using an effect of regulating the activity of caspase and ATPase of the Curcuma longa extract to induce apoptosis prevention and regeneration of human retinal pigment epithelial cells, the composition can be usefully used for preventing and treating retinal diseases. Furthermore, it is possible to provide a pharmaceutical composition and a functional food composition for preventing and treating retinal diseases containing the Curcuma longa extract as an active ingredient.

Description

A composition for preventing and treating retinal diseases comprising turmeric extract {RETINAL DISEASE PREVENTION AND TREATMENT COMPOSITION COMPRISING TURMERIC EXTRACT}

The present invention relates to a composition for preventing and treating retinal diseases comprising a turmeric extract. More specifically, it has the effect of preventing apoptosis of human retinal pigment epithelial cells by regulating the activity of caspase and ATPase and further inducing the regeneration of retinal pigment epithelial cells, so it is useful for the prevention and treatment of retinal diseases. It relates to a composition that can be used for

Normal vision occurs when light enters the front of the eye, focusing it onto very sensitive and weak cells called photoreceptors that line the inside of the back of the eye with lenses. Photoreceptors together with other neurons make up the retina of the eye. The retina is a transparent and thin film that is located in the innermost part of the eyeball wall and is in contact with the vitreous body in the eyeball. It serves as the primary visual information agency.

The retina is made up of over 100 million light-sensing cells (light-sensitive photoreceptor cells), over 1 million optic nerve cells called ganglion cells, and numerous nerve cells that act as wires connecting them. , is therefore the most sophisticated tissue in our body. The macula lutea, the central part of the retina that distinguishes colors and objects and shows vision, consists of a photoreceptor cell layer and a ganglion cell layer composed of cone cells, so the thickness of the retina is thin. It is converted into a signal and transmitted to the brain via the optic nerve, which is the axon of the ganglion cell.

About 30% of our brain cells are used to process the visual information sent by the retina, but as an abnormality occurs in the retina due to aging or external factors, it gradually leads to blindness along with visual impairment that causes problems with vision and vision. Retinal diseases cause abnormalities in the tissues surrounding the retina and when the neural retina is separated from the pigment epithelial cell layer, the retina is separated to the back of the eyeball and causes visual impairment, retinal detachment, and peripheral retinal degeneration, which causes abnormalities in the tissues surrounding the retina degeneration) and macular degeneration, which causes abnormalities in the macula, are broadly classified into three categories: once the retina is separated from the pigment epithelium, optical information about the image cannot be transmitted, and nutrition from the choroid is not provided. Therefore, nerve cells fail to function, and if this condition is left untreated, permanent retinal atrophy occurs, leading to blindness.

Although this retinal disease is not a fatal disease that leads to death, the number of patients is rapidly increasing in recent years due to industrialization, changes in diet, and an increase in the elderly population. It is important, and if discovered late, there is a problem that it is difficult to cure.

Accordingly, the present applicant has found that turmeric extract, such as curcumin, inhibits apoptosis of human retinal pigment epithelial cells and induces its regeneration, and has completed the present invention.

KR 10-2045044 B1 KR 10-1330850 B1

An object of the present invention is to provide a composition for preventing and treating retinal diseases comprising a turmeric extract as an active ingredient, and using the effect of regulating the activity of caspase and ATPase of the turmeric extract, human retinal pigment epithelial cells It is to provide a composition that can be usefully used for preventing and treating retinal diseases that can induce apoptosis and regeneration.

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating retinal diseases comprising a turmeric extract as an active ingredient.

Another object of the present invention is to provide a functional food composition for preventing retinal disease comprising a turmeric extract as an active ingredient.

In order to achieve the above object, the composition for preventing and treating retinal diseases according to an embodiment of the present invention contains turmeric extract as an active ingredient, and has a protective and regenerative effect on human retinal pigmented epithelial cells. it is excellent

The composition for preventing and treating retinal diseases is excellent in regulating the activity of caspase and ATPase.

In the present invention, the turmeric extract is curcumin.

In the present invention, the extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof.

The retinal disease is retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt's disease, Usher's syndrome, choroidal insufficiency, Rod-Con or Con-Rod's dystrophy, ciliopathy, mitochondrial disorder, progressive retinal atrophy , degenerative retinal disease, age-related macular degeneration (AMD), wet macular degeneration, dry macular degeneration, map atrophy, familial or acquired maculopathy, choroidal melanoma, sickle cell retinopathy, diabetic retinopathy, macular edema, cystic macular edema, Uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vessel occlusion, choroidal angiopathy nodosa, It is selected from the group consisting of retinal nerve cell degeneration and ischemic optic neuropathy due to retinal hypoxia, retinal vascular disease, ophthalmic vascular disease, and glaucoma.

A pharmaceutical composition for preventing and treating retinal disease according to another embodiment of the present invention includes the composition.

A functional food composition for preventing retinal disease according to another embodiment of the present invention includes the composition.

Hereinafter, the present invention will be described in more detail.

As used herein, the term 'extract' has the meaning commonly used as a crude extract in the art, but broadly includes a fraction obtained by additionally fractionating the extract. That is, the plant extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the extract of the present invention.

The extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

As used herein, the term “prevention” refers to blocking the appearance of a disease or condition associated with it, in an asymptomatic subject at risk of developing the disease, for example, by exposure to a cause of the disease.

As used herein, the term “treatment” refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it has been developed. Treatment of retinal disease may be demonstrated by, for example, improvement of visual acuity, reduction of clinical symptoms due to retinal disease, or other parameters well known in the art as being specific to, for example, retinal disease, the degree of dryness of the eye. can

As used herein, the term "comprising as an active ingredient" means an amount sufficient to affect a desired biological effect, such as, but not limited to, beneficial results, including, but not limited to, prevention, reduction, alleviation or elimination of signs or symptoms of a disease or disorder. is meant to include, and this amount is referred to as an “effective amount”. Accordingly, the total amount of each active ingredient of a composition or method is sufficient to exhibit a significant individual benefit. Accordingly, the effective amount will vary depending on the circumstances in which it is administered. An effective amount may be administered in one or more prophylactic or therapeutic administrations.

The composition for preventing and treating retinal diseases according to an embodiment of the present invention contains turmeric extract as an active ingredient, and has excellent protective and regenerative effects on human retinal pigmented epithelial cells.

In addition, the composition for preventing and treating retinal diseases is excellent in regulating the activity of caspase (Caspase) and ATPase.

The turmeric (Curcuma long L.) is a perennial plant belonging to the ginger family, and dried and powdered bulbs are called turmeric. is being used as It is native to India, China, and Japan, and grows wild in hot and humid southern Asia, Africa, and Central and South America, and it is known that cultivation began in the East India region (Ryuat al., Applied chemistry, 9: 57-60 (2005)).

Turmeric has been used for a long time in South and Southeast Asia in the tropics as herbal medicine, spice and food. Acceleration, diuretic, detoxification, anticancer, anti-inflammatory and antioxidant effects are known (Kim Chang-ryeol. Ministry of Food and Drug Safety, pp 1-98 (2006)). In addition, the components of the root and stem of turmeric perform antioxidant and cytoprotective roles, and the main components are known as curcumin, curcumin derivatives, and aromatic turmerone (Chatterjee, S. et al., Food Research). International. 32.487-490. 1999.). Also, turmeric affects lipid metabolism. It is known to affect the increase of blood cholesterol level when taken as a decoction or powder, and when administered to rabbits or rats, forms a mass in the main and coronary arteries and reduces the deposition of lipids, and curcuma amada (curcuma amada). ) is known to reduce blood cholesterol levels when administered.

Human retinal pigmented epithelial cells exist outside the retinal sensory nerve part, and refer to cells with pigment. It exists as a single layer in the outermost part of the retina, and it is involved in the development of the retina, plays a role in protecting rods and cones, and plays an important role in maintaining the functions of photoreceptors and retina.

Specifically, human retinal pigment epithelial cells not only function to recycle photopigment, transport, metabolize, and store vitamin A, but also engulf rod photoreceptor outer segments, and between the retina and choroid, iron and small molecules transports and maintains the Bruch membrane. Furthermore, it maintains photoreceptor function by absorbing stray light for better image resolution.

Such degeneration of human retinal pigment epithelial cells may cause photoreceptor damage, such as panchoroidal atrophy, diabetic retinopathy, macular degeneration, retinitis pigmentosa, and Stargardt's disease, and further retinal detachment, retinal It can cause dysplasia and retinal atrophy.

In the present invention, the human retinal pigment epithelial cells may be ARPE-19 cells, but are not limited thereto, and may be used without limitation if they correspond to the types of human retinal pigment epithelial cells.

Apoptosis is a type of programmed cell death seen in multicellular organisms, in which cells die due to changes in cell shape and internal biochemical. This process proceeds with cell expansion and cracking, cell membrane change, nuclear fragmentation, chromatin condensation and chromosome cleavage, and the cell is engulfed by other cells and eventually annihilated. .

The caspase is a type of cysteine protease and is an enzyme that plays an important role in apoptosis, necrosis and inflammatory reactions. Specifically, in relation to apoptosis, the caspase is first synthesized as an inactive precursor.

These precursors have a two-unit precursor region, with the initiator caspases (caspase-1,2,4,5,8,10) being more complex than the executive caspases (caspase-3,6,7,9). It has a long bulb. Caspase becomes active through cleavage of the precursor region (specifically cleaving after aspartic acid), which is mostly composed of two units. Initiator caspases are activated by other proteins or molecules that transmit apoptosis, and executive caspases are activated by upstream initiator caspases. This chain-activated caspase is

In apoptosis initiates the step of degrading cells in response to substances that induce cell death.

The ATPase is an enzyme that hydrolyzes ATP, and is a class of enzymes that catalyze the production of adenosine diphosphate (ADP) and free phosphate by decomposing adenosine triphosphate (ATP).

When the turmeric extract is used as a composition for preventing and treating retinal diseases, it inhibits the death of human retinal pigment epithelial cells by regulating the activity of the caspase and ATPase in relation to the death of human retinal pigment epithelial cells. Furthermore, it has an effect of protecting human retinal pigment epithelial cells by inducing regeneration.

Specifically, when apoptosis of human retinal pigment epithelial cells is induced, caspase is activated, and further, ATP production is reduced. When the turmeric extract is used as a composition for preventing and treating retinal diseases, it is possible to prevent apoptosis of human retinal pigment epithelial cells by inhibiting the activity of caspase, as well as inhibit the activity of ATPase to produce ATP It has the effect of restoring abilities.

Preferably, the turmeric extract may further include a natural extract selected from the group consisting of a ginseng leaf extract, a genus oleifera leaf extract, a S. asiatica extract, an extract of Albania tree bark, and mixtures thereof.

The gulpi tree (Cornus kousa) is a deciduous small tree of the Astragalus family, and mainly grows in the Yangji or coastal waters at the foot of the mountains in Korea (South Chungcheong Province), Japan, Taiwan, and China. The leaves are odd pinnate leaves, and consist of 7 to 19 small leaves without petioles. The small leaves are oval lancet-shaped or egg-shaped lanceolate with a sharp tip and deep serrations on the edge. There are white hairs on both sides of the leaves, but they gradually disappear. There is also hair on the peduncle, but it gradually disappears. The flowers are yellowish green flowers, 5-8cm in male ear and 2-4cm in female ear, and mature female ear of female flower is in the shape of a pine cone. Ears of fruit are long oval, blackish-brown, and hairless. The bract does not fall off and is lanceolate, and the fruit is a winged nut and ripens in September to October.

The fruit bark (peel) of gulpi tree contains eugloon and the fruit contains tannins, so it is used in folk medicine as a phlegm, insecticide, and pregnancy emetic.

The Heotgae tree is also called a bolge tree, a horike tree, or a zigzag tree and belongs to the angiosperm phylum. It is a broad-leaved, tall tree with straight stems and slightly curved stems, growing to a height of 10 to 17 m. Usually about 10m. The branches extend upwards and the top is rounded. The stem bark is cracked like the bottom of a rice field, and the fruits are bulging. It is equivalent to a large tree that can grow up to one rhododendron in diameter.

In particular, hemp tree has excellent liver protection and hangover relief due to ingredients such as ampheropsin and hobenitins, and contains a lot of calcium that helps bone and muscle formation, so you can drink water and extract from the plant. It has an improvement effect on diseases such as arthritis and osteoporosis.

The Smilax chine L. is distributed in mountainous areas nationwide, and is widely distributed in Korea, China, and Japan. According to the Korean Pharmacopoeia, it is also called Tobokryeong (土茯笭) or oxen according to the Korean Pharmacopoeia, and is a stem-rooted plant belonging to the family Liliaceae. was used as a natural food preservative to preserve In addition, Smilax china root is used to relieve symptoms such as fever, detoxification, and diuresis, to improve physical strength, and as a treatment for inflammation such as dermatitis, nephritis, and cystitis.

The Albizzia julibrissin DURAZZ is also called cassia, wild ash, hyacinth, and hyacinth by other names. It grows in mountains and fields, and the stems are curved or slightly lying down. The height of the tree is 3 to 5 m, and the large branches are sparsely spread, and the small branches have ridges, and the leaves are alternate phyllotaxis and have pinnate compound leaves. The small leaves are curved like a sickle and have a long oval shape with not equal left and right edges. The length of the small leaves is 6 to 15 mm and the width is about 2.5 to 4.0 mm. There are no hairs on both sides or hairs on the veins on the back side. The flowers are pale pink, bloom from June to July, and hang in an umbrella shape by 15 to 20 at the tip of small branches. The calyx and corolla are shallowly divided into 5 and turn green, and the stamens are about 25, and they come out long and the upper part is red. The reason the flowers look red is because of the color of the stamens. The fruit ripens in September-October and is a flat pod, about 15 cm long, and contains 5-6 seeds.

Algae bark is included in the Korean herbal medicine standard book other than the Korean Pharmacopoeia, and the bark collected in early summer is dried and used. It is cylindrical or semi-cylindrical, and the outer surface is grayish brown or brown, and the inner surface is light yellowish brown or yellowish white. It is odorless, has a mild taste, is astringent, and slightly irritates the tongue. In previous studies, it is known that phytochemicals such as tannins, saponins, and sitosterols are abundantly contained. It is known to have liver, excitatory, diuretic, anthelmintic, antitussive, and analgesic effects. The bark of Albania tree has been studied in various fields. Recently, it has been reported that the ethanol extract of hyaluronic acid is effective in treating kidney cancer and brain cancer.

When the turmeric extract further comprises a natural extract selected from the group consisting of a turmeric leaf extract, a serratus leaf extract, a Smilax china L. extract, an extract of Albania tree bark, and a mixture thereof, the activity of caspase and ATPase is increased. By further maximizing the regulatory effect, it may be more effective in inhibiting the death of human retinal pigment epithelial cells and inducing regeneration.

More preferably, the turmeric extract, gulberry leaf extract, sycamore leaf extract, Smilax china L. extract and Albania bark extract are in a weight ratio of 1:0.4:0.4:0.5:0.5 to 1:1:1:1:1 by weight. By using a mixture, it can exhibit excellent regulating effects of caspase and ATPase activity within the above weight range.

In the present invention, the turmeric extract is curcumin.

The curcumin (curcumin) is a natural polyphenol as a major active ingredient contained in turmeric (Curcuma long L.).

Curcumin has been used for the treatment of diseases related to inflammation and oxidative stress, and has been studied for cancer chemoprevention and cancer growth inhibition because of its ability to inhibit inflammation and scavenge free radicals. In addition, curcumin is a naturally occurring antioxidant and exhibits antioxidant properties by influencing low histone acetylation under certain conditions. Curcumin exerts an anticancer effect on human leukemia cells through elimination of reactive oxygen species generation at low concentrations, which exhibits various pharmacological effects including apoptosis, anti-inflammatory and anticancer, and also tumor-derived T cells. It protects against apoptosis, and in vitro experimental evidence and clinical trials have shown it to be useful for disease prevention of human colorectal cancer. In addition, it has been reported that curcumin can reduce cognitive decline and oxidative damage associated with aging, and as a result of animal experiments, curcumin is effective in neurodegenerative conditions such as Alzheimer's and focal brain ischemia. It was reported that As such, curcumin has received commercial interest as a useful pharmaceutical agent.

However, research on how curcumin affects the protection and regeneration of damaged human retinal pigment epithelial cells has not been reported. No support was provided either.

The curcumin is represented by the following formula (1):

[Formula 1]

In the present invention, the extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof. The 'turmeric extract' means an extract obtained by extracting from turmeric.

The turmeric extract is a pulverized product of dry turmeric, water, ethanol, alcohol having 1 to 4 carbon atoms, such as methanol, a polar solvent such as dimethyl sulfoxide, or alcohol and water having a mixing ratio of 1:0.1 to 1:10. Elution may be performed with a mixed solvent, and more specifically, ethanol may be used as an extraction solvent. At this time, the extraction temperature may be 10 ℃ to 100 ℃, preferably the extract extracted at 25 ℃.

The extraction solvent may be used in an amount of 2 to 50 times, preferably 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 72 hours for leaching in the extraction solvent, and in particular, from 1 to 24 hours.

It may be a result obtained by concentrating the filtered extract under reduced pressure with a vacuum rotary concentrator, but as long as it is a turmeric extract capable of exhibiting the retinal disease prevention and treatment effect of the present invention, it is not limited thereto. All of the dried product obtained by drying, or a crude product or purified product thereof.

The leaf extract of Gyulpi tree, the leaf extract of H. oleifera, the extract of Smilax china L. asiatica, and the extract of Albania tree bark can also be prepared using the same method as the turmeric extract.

The retinal disease is retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt's disease, Usher's syndrome, choroidal insufficiency, Rod-Con or Con-Rod's dystrophy, ciliopathy, mitochondrial disorder, progressive retinal atrophy , degenerative retinal disease, age-related macular degeneration (AMD), wet macular degeneration, dry macular degeneration, map atrophy, familial or acquired maculopathy, choroidal melanoma, sickle cell retinopathy, diabetic retinopathy, macular edema, cystic macular edema, Uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vessel occlusion, choroidal angiopathy nodosa, Retinal hypoxia, retinal vascular disease, ophthalmic vascular disease and retinal nerve cell degeneration due to glaucoma and ischemic optic neuropathy, but is not limited thereto.

A pharmaceutical composition for preventing and treating retinal disease according to another embodiment of the present invention includes the composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration may include tablet pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In addition, the pharmaceutical composition of the present invention is from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories It may have any one formulation selected.

The turmeric extract of the present invention has been used for food and medicine since ancient times, and there is no particular restriction on its dosage, body absorption, body weight, age, sex, health status, diet, administration time, administration method, excretion rate. , and the severity of the disease. In general, the turmeric extract may be administered in an amount of about 10 to 1000 mg per 1 kg of body weight, and more specifically, about 50 to 500 mg per kg of body weight.

The pharmaceutical composition comprising the turmeric extract of the present invention as an active ingredient is prepared in consideration of the effective amount range, and the unit dosage form formulated in this way is determined by the judgment of experts and individuals who monitor or observe the administration of the drug as needed. Depending on the patient, a specialized dosing regimen may be used, or several doses may be administered at regular time intervals.

A functional food composition for preventing retinal disease according to another embodiment of the present invention includes the composition.

There is no particular limitation on the type of the functional food of the present invention. Examples of health functional foods to which the extract mixture can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in a conventional sense, and may include foods used as feed for animals. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin , alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage.

According to the composition for preventing and treating retinal diseases comprising the turmeric extract of the present invention, the apoptosis prevention and regeneration of human retinal pigment epithelial cells is induced by using the effect of regulating the activity of caspase and ATPase of the turmeric extract. By doing so, it can be usefully used for prevention and treatment of retinal diseases.

Furthermore, it is possible to provide a pharmaceutical composition and a functional food composition for preventing and treating retinal diseases, including turmeric extract as an active ingredient.

1 is an experimental result for determining the optimal treatment concentration of curcumin for the study of protection and regeneration of human retinal pigment epithelial cells (ARPE-19) according to an embodiment of the present invention.
2 is an experimental result for determining the optimal treatment time of curcumin for the study of protection and regeneration of human retinal pigment epithelial cells (ARPE-19) according to an embodiment of the present invention.
3 is an experimental result for constructing a cell death model under optimized conditions for the study of protection and regeneration of human retinal pigment epithelial cells (ARPE-19) according to an embodiment of the present invention.
4 is a result of testing the protective effect of curcumin (curcumin) against apoptosis of human retinal pigment epithelial cells (ARPE-19) according to an embodiment of the present invention.
5 is a human retinal pigment epithelial cell (ARPE-19) through the inhibition of the activity of caspase-3 and caspase-9 of curcumin according to an embodiment of the present invention. This is the result of testing the protective effect of
6 is a result of testing the protective effect of human retinal pigment epithelial cells (ARPE-19) through the improvement of ATP production of curcumin (curcumin) according to an embodiment of the present invention.
7 is a result of testing the regenerative effect of curcumin on the death of human retinal pigment epithelial cells (ARPE-19) according to an embodiment of the present invention.
8 is a human retinal pigment epithelial cell (ARPE-19) through the inhibition of the activity of caspase-3 and caspase-9 of curcumin according to an embodiment of the present invention. It is the result of an experiment on the regenerative effect of
9 is a result of testing the regenerative effect of human retinal pigment epithelial cells (ARPE-19) through the improvement of ATP production of curcumin according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

[Preparation Example 1: Preparation of turmeric extract (curcumin)]

Curcumin (Sigma Chemical Co., St Louis, MO) was dissolved in DMSO (dimethyl sulfoxide) as a solvent, and then stored at -20 °C to prepare a turmeric extract (curcumin). Thereafter, the stock solution was diluted to the treatment concentration applied in each of the following experiments before use.

[Preparation Example 2: Manufacture of Gulpe biloba leaf extract, Hengaea leaf extract, Smilax china L. extract and Albania bark extract]

Gyulpi tree leaves were pulverized with a blender and then prepared into powder. 50% ethanol as an extraction solvent was added to the powder sample at a ratio of 1;10 (w; v), and then completely immersed, and extracted three times for 3 hours while refluxing at 80°C. The extract is Whatman No. 2 filtered with filter paper. The filtrate was concentrated under reduced pressure at 60° C. to prepare an extract of Gyulpi tree leaf.

Heotgae leaf extract, Smilax china L. extract and Albania bark extract were prepared in the same manner as in the preparation method of the Gulpi leaf extract.

[Preparation Example 3: Preparation of mixed extract]

The weight ranges of the turmeric extract, curcumin (CE), Gyulphus leaf extract (GE), H. asiatica leaf extract (HE), Smilax china L. extract (JE) and Albania bark extract (KE) are shown in Table 1 below. mixed extracts were prepared.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 CE 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 GE - 70 70 70 70 70 30 40 70 100 120 HE - 70 70 70 70 70 30 40 70 100 120 JE - 75 75 75 75 75 40 50 75 100 130 KE - 75 75 75 75 75 40 50 75 100 130

(Unit: parts by weight)

[Experimental Example 1: Protective and regenerative effect experiment on human retinal pigment epithelial cells (ARPE-19)]

1. Experimental method

cell culture

ARPE-19 cells, which are human retinal pigmented epithelial cells, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). ARPE-19 cells, which are human retinal pigment epithelial cells, were cultured in DMEM/F12 medium (1:1, Thermo Fisher Scientific, Carlsbad, CA, USA) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). % fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific) were added and cultured overnight at _{37° C., 5% CO 2 in an incubator.} The medium was changed after the first 2 days and every 4 days thereafter. After culturing for 2-3 days, when the primary cultured cells reached a density of 70 to 80%, they were subcultured using a 0.025% trypsin solution.

Cell viability assay

Cell viability was assessed by visual cell count through a Trypan blue exclusion experiment. In all viability assays, triplicate wells were used for each condition, and each experiment was repeated at least 3 times.

In order to confirm the effect of the concentration of curcumin prepared with the turmeric extract on cell viability, cell viability was measured using trypan blue exclusion. Specifically, in order to determine whether curcumin is cytotoxic or not, the cultured human retinal pigment epithelial cells (ARPE-19) ^{were inoculated in a 10 cm dish at a density of 5 x 10 5} , and containing 2% FBS. Stabilized in DMEM/F12 for 8 hours, 37° C., 5% CO ₂ incubator. After stabilization, the cells were treated with curcumin at concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, and 50 μM, respectively, and cultured in serum-free DMEM/F12 medium (0% FBS) for 3 days. Cell viability was measured by counting the number of visualized cells in combination with trypan blue exclusion, and in all viability assays, triplicate wells were used for each condition. and each experiment was repeated at least 3 times.

In addition, in order to optimize the treatment time of curcumin for human retinal pigment epithelial cell protection and regeneration studies, cultured human retinal pigment epithelial cells (ARPE-19) were inoculated into a 10 cm dish at a density of ^{5 x 10 5} and incubated in DMEM/F12 medium at 37° C. in a _{CO 2 incubator.} Cells were then treated with 0.05 μM curcumin for 3 days. Cell viability was assessed by visual cell counting with trypan blue exclusion. In all viability assays, triplicate wells were used for each condition, and each experiment was repeated at least three times.

Experimental on the protective effect of curcumin against human retinal pigment epithelial cells (ARPE-19) death

In order to investigate the protective effect of curcumin on human retinal pigment epithelial cell death, human retinal pigment epithelial cells (ARPE-19) cultured for pretreatment with curcumin were placed in a 10 cm dish at a density ^{of 5 x 10 5} , and incubated in DMEM/F12 containing 2% FBS for 8 hours at 37° C., CO _{2 in an} incubator. Cells were pretreated with 0.05 μM of curcumin for 24 hours and then incubated for 72 hours after UV irradiation with _{0.3 mM H 2} O _{2 or 30 seconds.} After H ₂ O ₂ or UV irradiation, the relative viability of untreated human retinal pigment epithelial cells and human retinal pigment epithelial cells pretreated with 0.05 μM curcumin for 24 hours was measured by trypan blue exclusion. evaluated.

Experiment on the regenerative effect of curcumin on the death of human retinal pigment epithelial cells (ARPE-19)

To investigate the regenerative effect of curcumin on human retinal pigment epithelial cell death, 80% density human retinal pigment epithelial cells (ARPE-19) were _{irradiated with 0.5 mM H 2} O ₂ or UV light for 50 seconds at a concentration of 0.05 μM. of curcumin was treated for 48 hours. After UV irradiation with 0.5 mM H ₂ O ₂ or 50 seconds, the relative viability of untreated human retinal pigment epithelial cells and human retinal pigment epithelial cells treated with 0.05 μM curcumin for 2 days was evaluated by trypan blue exclusion (Trypan blue). exclusion).

Caspase activity analysis and quantitative experiment

For the assay of caspase-3 and caspase-9 activity, 10 μg of protein in 50 μl total volume was mixed with 50 μl of equilibrated Caspase-Glo 3, or 9 It was mixed with reagent (Promega). The blank value was subtracted and the fold increase in activity was calculated based on the activity measured from untreated cells. Each sample was measured in triplicate.

Measurement of ATPase activity

The amount of protein was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. 150 mM/l KCl, 25 mM/l Tris-HCl; Cells at a concentration of 1 mg protein/ml buffer in buffer containing pH 7.6, 2 mM/l EDTA pH 7.4, 10 mM/l KPO ₄ pH 7.4, 0.1 mM/l MgCl _{2 and 0.1% (w/v) BSA} was resuspended. ATP synthesis was carried out in 250 ml of cell suspension in 750 µl of substrate buffer (10 mM/l malate, 10 mM/l pyruvate, 1 mM/l ADP, 40 µg/ml digitonin and 0.15 mM/l adenosine pentaphosphate). was started by adding Cells were incubated at 37° C. for 10 min. At 0 and 10 min, 50 μl aliquots of the reaction mixture were taken, quenched in 450 μl boiling 100 mM/l Tris-HCl, 4 mM/l EDTA pH 7.75 for 2 min and further 1/ in quench buffer. 10 diluted. The amount of ATP was measured in a luminometer (Berthold, Detection Systems, Pforzheim, Germany) using an ATP Bioluminescence Assay Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Raw data from each experiment was analyzed using analysis of variance using Fisher or t-test.

statistical analysis

All data are presented as mean ± S.E.M. from at least 5 independent experiments. The statistical significance of differences between groups was calculated using Student's two tailed t-test (*, P < 0.05; **, P < 0.01).

2. Experimental results

Determination of optimal treatment conditions (concentration and time) of curcumin

An experiment was conducted to determine the optimal treatment concentration and optimal treatment time of curcumin for the study of protection and regeneration of human retinal pigment epithelial cells (ARPE-19).

As a result of the experiment, cytotoxicity was observed at a treatment concentration of 50 μM of curcumin, whereas no cytotoxicity was observed at a treatment concentration of 1 μM or less. In particular, when curcumin at a concentration of 0.05 μM was treated, the survival rate of human retinal pigment epithelial cells (ARPE-19) was the best, and this was determined as the optimal concentration of curcumin for cell protection and regeneration studies. (FIG. 1).

In addition, it was confirmed that curcumin administered for 24 hours significantly increased the survival rate of human retinal pigment epithelial cells (ARPE-19) ( FIG. 2 ).

Based on the above results, the optimal treatment method (concentration and time) of curcumin for protection and regeneration of human retinal pigment epithelial cells (ARPE-19) can be determined at a concentration of 0.05 μM and treatment for 24 hours.

Experimental results for establishing a cell death model under optimized conditions for the study of protection and regeneration of human retinal pigment epithelial cells (ARPE-19)

For the study of protection and regeneration of human retinal pigment epithelial cells (ARPE-19), which is the object of the present invention, an experiment was conducted to construct a cell death model under optimized conditions.

When the results see the basis, for the protection studies, a wide range of concentrations (0.1 to 1 mM) to determine the optimal concentration of H ₂ O ₂ for human RPE cell death induction (50% survival based) H ₂ After treatment with O ₂ , the cytotoxicity and viability effects were measured. As a result, it was determined that the 0.3 mM concentration was optimal for the present invention. In addition, in order to determine the optimal irradiation time of ultraviolet (UV-B) for inducing apoptosis of human retinal pigment epithelial cells, cytotoxicity and survival effects were measured after treatment with ultraviolet rays for various times (10 to 60 seconds). was determined to be optimal for the present invention when irradiated for 30 seconds (FIG. 3).

In addition, in the case of regeneration studies, in order to determine the optimal concentration of _{H 2} O ₂ for inducing human retinal pigment epithelial cell death (based on 10% survival rate), after treatment with _{H 2} O _{2 at various concentrations (0.1 to 1 mM)} As a result of measuring the cytotoxicity and survival effect, it could be determined that the 0.5 mM concentration was optimal for the present invention. In addition, in order to determine the optimal irradiation time of ultraviolet (UV-B) for inducing apoptosis of human retinal pigment epithelial cells, after UV treatment for various times (10 to 60 seconds), cytotoxicity and survival effects were measured. When irradiated for 50 seconds, it could be determined to be optimal for the present invention (FIG. 3).

The protective effect of curcumin against the death of human retinal pigment epithelial cells (ARPE-19)

An experiment was conducted to investigate the protective effect of curcumin, a turmeric extract, against the death of human retinal pigment epithelial cells (ARPE-19).

Cell death after H ₂ O ₂ or UV irradiation was clearly observed in the 24-hour culture of human retinal pigment epithelial cells, but 0.05 μM curcumin-pretreated culture prevented apoptosis significantly. In particular, when incubated for 72 hours in a 0.05 μM concentration of curcumin-pretreatment conditions, it showed a high cytoprotective effect compared to the control ( FIG. 4 ).

The above results can be considered to indicate that curcumin, a turmeric extract, has an effect of inhibiting apoptosis of human retinal pigment epithelial cells (ARPE-19).

Mechanism of action of curcumin to protect human retinal pigment epithelial cells (ARPE-19)

Curcumin inhibits caspase-3 and caspase-9 in human retinal pigment epithelial cells treated with _{H 2} O _{2 or UV irradiation, and regulates ATPase activity in the human retina} It was tested whether it can protect pigment epithelial cells.

As a result, the activity of caspase-3 and caspase-9 was remarkably increased for 24 hours and 48 hours, respectively, after treatment with _{0.3 mM H 2} O _{2 or UV irradiation for 30 seconds, but} , 0.05 μM concentration of curcumin pretreatment showed significant inhibition of caspase activation (FIG. 5).

Furthermore, H ₂ O ₂ or UV irradiation treatment after a result of the experiment the effect of curcumin (curcumin) for ATP production, 0.3 mM H ₂ O ₂ or 30 seconds after the ultraviolet irradiation processing ATP production for 12 hours and 24 hours, respectively, is significantly Although decreased, pretreatment with curcumin at a concentration of 0.05 μM significantly restored the ATP-producing ability of the cells ( FIG. 6 ).

H ₂ O ₂ or UV irradiation effectively induces oxidative stress and apoptosis of cells, causing the activation of caspase and reducing ATP production.

The results show that curcumin inhibits caspase-3 and caspase-9, and regulates the activity of ATPase to restore ATP production capacity through the mechanism of action, It indicates that it may play a role in protecting human retinal pigment epithelial cells by preventing apoptosis.

Regeneration effect of curcumin on the death of human retinal pigment epithelial cells (ARPE-19)

An experiment was conducted to determine whether curcumin, a turmeric extract, had the effect of regenerating apoptotic human retinal pigment epithelial cells (ARPE-19).

As shown in FIG. 7 , _{cell death was clearly observed in culture for 24 hours after irradiation with H 2} O ₂ or UV light, but apoptosis was prevented in a 0.05 μM concentration of curcumin-treated culture. In particular, it can be seen that the 0.05 μM concentration curcumin-treated condition in 48-hour culture exhibits an excellent cell regeneration effect compared to 24-hour culture ( FIG. 7 ).

Mechanism of action of curcumin acting on the regeneration of human retinal pigment epithelial cells (ARPE-19)

Curcumin inhibits caspase-3 and caspase-9 in human retinal pigment epithelial cells treated with _{H 2} O _{2 or UV irradiation, and regenerative ability through regulation of ATPase activity} It was tested whether it can be improved.

As a result, the activity of caspase-3 (caspase-3) and caspase-9 (caspase-9) increased remarkably for 24 hours and 48 hours, respectively, after treatment with _{0.5 mM H 2} O _{2 or UV irradiation for 50 seconds.} However, after treatment with curcumin at a concentration of 0.05 μM, caspase activation was significantly inhibited ( FIG. 8 ).

Furthermore, as a result of testing the effect of curcumin on ATP production after _{H 2} O _{2 or UV irradiation treatment, ATP production for 12 hours and 24 hours after 0.5 mM H 2} O ₂ or UV irradiation treatment for 50 seconds, respectively Although significantly decreased, after treatment with curcumin at a concentration of 0.05 μM, the ATP production capacity of the cells was significantly restored ( FIG. 9 ).

The results show that curcumin inhibits caspase-3 and caspase-9, and regulates the activity of ATPase to restore ATP production capacity through the mechanism of action, It indicates that it may play a role in regenerating dead human retinal pigment epithelial cells.

Synergistic effect by the combined treatment of turmeric extract (curcumin), ginseng leaf extract, sycamore leaf extract, Smilax china L. extract and Albania bark extract

In order to test whether the protective and regenerative effect of human retinal pigment epithelial cells (ARPE-19) is increased by the combined treatment of the extracts,

The caspase ( caspase) inhibitory effect, the effect of restoring the ATP production capacity through regulation of ATPase activity, and the protective and regenerative effects of human retinal pigment epithelial cells were tested using a Trypan blue exclusion experiment.

For relative comparison, the caspase inhibitory effect of the case where the turmeric extract, curcumin, was treated alone (T1) was set to index 5, and the caspase inhibitory effect by the mixed extract was evaluated as an index. .

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 caspase 5 4.8 4.9 5.1 5.5 4.5 3.9 6.5 5.5 4.8 3.8 6.2 8.8 9 8 4.3

According to Table 2, the turmeric extracts curcumin, ginseng leaf extract, hemp leaf extract, Smilax china L. extract and Albania bark extract were 1:0.4:0.4:0.5:0.5 to 1:1:1:1. It was proved that there is a more excellent caspase inhibitory effect than when mixed in a weight ratio of :1.

On the other hand, when the mixing ratio of the turmeric extract, curcumin, ginseng leaf extract, hemp leaf extract, Smilax china L. extract and Albania bark extract is less than or greater than the above range, the caspase inhibitory effect is low. it has been proven

Furthermore, the effect of restoring the ATP production capacity by controlling the activity of ATPase was confirmed through the experiment, and for a relative comparison, the recovery effect of the ATP production capacity in the case where curcumin, a turmeric extract, was treated alone (T1). was set as the index 5, and the effect of recovering ATP production capacity by the mixed extract was evaluated as an index.

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 ATP production capacity recovery effect 5 4.8 3.8 5.2 5 4.7 5.5 6 3.9 5.1 3.9 4 8 8.9 8.6 4.3

According to Table 3 above, the turmeric extract curcumin, Gyulpi leaf extract, Hengae leaf extract, Smilax china L. extract and Albania bark extract were 1:0.4:0.4:0.5:0.5 to 1:1:1:1. It has been proven that there is a better recovery effect of ATP production capacity than when mixed with a weight ratio of :1.

On the other hand, when the mixing ratio of the turmeric extract curcumin, ginseng leaf extract, hemp leaf extract, Smilax china L. extract and Albania bark extract is less than or greater than the above range, the recovery effect of ATP production capacity is lowered. proved

Furthermore, the protective effect of the mixed extract against the death of human retinal pigment epithelial cells (ARPE-19) was confirmed through an experiment, and for relative comparison, the human retina in the case of treatment with curcumin, a turmeric extract, alone (T1) The protective effect on apoptosis of pigmented epithelial cells (ARPE-19) was set to an index of 5, and the protective effect on apoptosis of human retinal pigmented epithelial cells (ARPE-19) by the mixed extract was evaluated as an index.

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 protective effect 5 4.8 5.2 3.2 4.7 5 6 5.5 5.1 3.9 4 3.9 8.8 9.2 8.7 4.8

According to Table 4, the turmeric extracts curcumin, ginseng leaf extract, sycamore leaf extract, Smilax china L. extract and Albania bark extract were mixed with 1:0.4:0.4:0.5:0.5 to 1:1:1:1. It has been demonstrated that there is a more superior protective effect against the death of human retinal pigment epithelial cells (ARPE-19) than when mixed in a weight ratio of :1.

On the other hand, when the mixing ratio of the turmeric extract, curcumin, ginseng leaf extract, hemp leaf extract, Smilax china L. extract and Albania bark extract is less than or greater than the above range value, human retinal pigment epithelial cells (ARPE-19) It has been proven to be less effective in protecting against death.

Furthermore, an experiment was conducted on the effect of the mixed extract to regenerate apoptotic human retinal pigment epithelial cells (ARPE-19). For relative comparison, the effect of regenerating apoptotic human retinal pigment epithelial cells (ARPE-19) when treated with turmeric extract curcumin alone (T1) was set to index 5, and cell death by the mixed extract The effect of regenerating human retinal pigment epithelial cells (ARPE-19) was evaluated as an index.

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 play effect 5 5.1 4.8 4.9 3.9 4.5 5.5 4.8 6.5 4.8 6.2 4.5 8.5 9 8.6 4.8

According to Table 5, the turmeric extracts curcumin, ginseng leaf extract, hemp leaf extract, Smilax china L. extract and Albania bark extract were mixed with 1:0.4:0.4:0.5:0.5 to 1:1:1:1. It has been demonstrated that the effect of regenerating apoptotic human retinal pigment epithelial cells (ARPE-19) is more excellent when mixed and used in a weight ratio of :1.

On the other hand, when the mixing ratio of the turmeric extract, curcumin, ginseng leaf extract, cypress leaf extract, Smilax china L. extract, and Albania bark extract is less than or greater than the above range, apoptosis of human retinal pigment epithelial cells (ARPE) -19) has been proven to be less effective.

Looking at the results of the above experiments, compared to the case where the turmeric extract, curcumin, was treated alone, the turmeric extract curcumin, gulberry leaf extract, hemp leaf extract, Smilax china L. extract, and Albania bark When the extract is mixed in a weight ratio of 1:0.4:0.4:0.5:0.5 to 1:1:1:1:1, caspase inhibitory effect and ATP production capacity are restored by controlling ATPase activity It can be seen that the protective and regenerative effect of human retinal pigment epithelial cells (ARPE-19) is further enhanced by making the effect to be more excellent.

Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (7)

Contains turmeric extract as an active ingredient,
A composition for preventing and treating retinal diseases with excellent protective and regenerative effects of human retinal pigmented epithelial cells. According to claim 1,
The composition for preventing and treating retinal diseases is
Excellent regulating effect of caspase and ATPase
A composition for preventing and treating retinal diseases. According to claim 1,
The turmeric extract is curcumin
A composition for preventing and treating retinal diseases. According to claim 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof.
A composition for preventing and treating retinal diseases. According to claim 1,
The retinal disease is retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt's disease, Usher's syndrome, choroidal insufficiency, Rod-Con or Con-Rod dystrophy, ciliopathy, mitochondrial disorder, progressive retinal atrophy , degenerative retinal disease, age-related macular degeneration (AMD), wet macular degeneration, dry macular degeneration, map atrophy, familial or acquired maculopathy, choroidal melanoma, sickle cell retinopathy, diabetic retinopathy, macular edema, cystic macular edema, Uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vessel occlusion, choroidal ganglionopathy nodosa, Retinal hypoxia, retinal vascular disease, ophthalmic vascular disease, and retinal nerve cell degeneration due to glaucoma and ischemic optic neuropathy.
A composition for preventing and treating retinal diseases. A composition comprising the composition according to any one of claims 1 to 5
A pharmaceutical composition for preventing and treating retinal diseases. A composition comprising the composition according to any one of claims 1 to 5
Functional food composition for preventing retinal disease.

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