KR20130028261A - Composition for preventing or treating parkinson's disease using extract of ecklonia cava or phloroglucinol - Google Patents

Abstract

PURPOSE: A composition containing an Ecklonia cava extract or phloroglucinol isolated from the extract is provided to suppress DNA damages and nerve cell injuries and to prevent or treat Parkinson's disease. CONSTITUTION: A composition for preventing or treating Parkinson's disease contains an Ecklonia cava extract as an active ingredient. The extract is prepared using water or an organic solvent. The extract contains phloroglucinol. The composition for preventing or treating Parkinson's disease contains phloroglucinol as an active ingredient. The Ecklonia cava extract or phloroglucinol suppresses or prevents nerve cell injury caused by 6 -hydroxydopamine(6-OHDA).

Description

Composition for preventing or treating parkinson's disease using extract of Ecklonia cava or phloroglucinol}

The present invention relates to a novel use of Ecklonia cava , which exhibits a prophylactic or therapeutic effect of Parkinson's disease. Specifically, the present invention relates to a composition for the prevention or treatment of Parkinson's disease, comprising an Ecklonia cava extract or a phloroglucinol compound isolated therefrom as an active ingredient.

Parkinson's disease is a chronic progressive neurological disease that is the second most common degenerative neurological disease.It is one of the representative intractable diseases that are becoming a social and economic problem due to the increasing incidence of the elderly population. . Parkinson's disease is currently known to have about 4 million people worldwide, and the number of patients in the United States is increasing by 50,000 every year. The incidence rate is one in 1,000, and the higher the age, the more frequent the occurrence. This disease is known to be the most associated with aging, and environmental factors, free radicals and genetic factors (5-10%) caused by neurotoxins such as pesticides are known to affect the onset. The cause of the disease is unknown. Genetic factors are known to be associated with gene mutations such as alpha-synuclein, Parkin, PINK-1, UCH-L1, DJ-1, and the like.

Parkinson's disease is caused by extensive degeneration of dopaminergic neurons (substantia Nigra) located in the basal ganglia of the brain, such as sudden degeneration or a significant decrease in the number of cells that produce the neurotransmitter dopamine in the substantia nigra area of the midbrain. do. When about 60-80% of the dopamine produced by the neurons is damaged, the extraramidal tract line can no longer effectively promote movement, resulting in Parkinson's disease symptoms. The cause is not yet known in detail, but it is known that metabolism due to arteriosclerosis of the brain, carbon monoxide poisoning, drugs, hypoparathyroidism, trauma encephalitis, etc. are involved. Decreased dopamine in neurotransmitters disrupts the balance of the entire neurotransmitter system, resulting in tremor, stiffness, bradykinesia, and postural instability. Back symptoms appear.

Parkinson's disease is not known for its exact etiology, so treatment is mainly used to improve symptoms rather than radical treatment. Drugs for the treatment of Parkinson's disease include l-dopa preparations, dopamine receptor agonists, anticholinergic drugs, and eldepryl (delpyl). Most of these drugs control symptoms rather than cause treatment. It requires a constant and continuous medication. However, long-term administration of these drugs causes problems with drug side effects. For example, anticholinergic drugs may show autonomic nervous system abnormalities or mental dysfunctions, and thus are limited to continuous administration to older patients. In addition, in the case of the L-dopa formulation, the effect gradually decreases according to the long-term administration, and the side effects such as twisting the body and abnormal movements of the hands or feet are generated. Anti-muscarinics and NMDA antagonists have been developed and used as neurotransmitters to improve neurotransmitters other than dopamine. Cerebral nerve cell protective agents, antioxidants, inhibitors of brain cell death, and anti-brain function are used as treatments. Efforts are ongoing.

In particular, there is an active development of a therapeutic agent using an active ingredient extract with fewer side effects as an active ingredient. Specifically, a pharmaceutical composition for the prevention or treatment of Parkinson's disease, including ginger extract or shogaol (Korean Patent Publication No. 2010-060723), a composition for the prevention and treatment of Parkinson's disease containing the cheonma extract as an active ingredient (Korea Patent Publication No. 2011-0080544), such as the extract of Parkinson's disease of extracts from various plants are currently being identified.

The present inventors have Ecklonia cava (Ecklonia chloroglucinol , a bioactive substance in cava ) extract, showed cell-protective activity against neurotoxicity and cell damage caused by 6-hydroxydopamine (6-OHDA). Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for the prevention or treatment of Parkinson's disease containing the Ecklonia cava extract as an active ingredient.

Another object of the present invention is to provide a composition for preventing or treating Parkinson's disease, which contains a phloroglucinol compound as an active ingredient.

It is also an object of the present invention to provide a functional food for preventing Parkinson's disease containing an Ecklonia cava extract or a phloroglucinol compound as an active ingredient.

In order to achieve the above object, the present invention is Ecklonia cava ) provides a composition for the prevention or treatment of Parkinson's disease containing the extract as an active ingredient.

In one embodiment of the present invention, the Ecklonia cava extract may be a solvent extract solubilized by adding water or an organic solvent.

In one embodiment of the present invention, the Ecklonia cava extract may include a phloroglucinol compound.

The present invention also provides a composition for the prevention or treatment of Parkinson's disease comprising a phloroglucinol compound as an active ingredient.

In one embodiment of the present invention, the phloroglucinol may be extracted from Ecklonia cava .

In one embodiment of the present invention, the Ecklonia cava extract or phloroglucinol may exhibit an effect of inhibiting or preventing nerve cell damage caused by 6-hydroxydopamine.

Furthermore, the present invention provides a functional food for preventing Parkinson's disease containing the Ecklonia cava extract or phloroglucinol compound as an active ingredient.

Ecklonia cava extract according to the present invention or phloroglucinol isolated therefrom inhibits the production of reactive oxygen species (ROS) induced by 6-hydroxydopamine (6-OHDA, 6-hydroxydopamine) and antioxidant It restores the activity of enzymes (CAT, GSH-Px, etc.) and has the effect of inhibiting intracellular DNA damage, lipid peroxidation and protein modification. Therefore, by inhibiting neuronal damage caused by 6-hydroxydopamine, it is possible to effectively prevent or treat Parkinson's disease induced by 6-hydroxydopamine, known as neurotoxin. In addition, it is useful in the manufacture of therapeutics and functional foods that can prevent or treat Parkinson's disease because it does not cause various side effects due to toxicity compared to conventional Parkinson's disease treatment agents because of its excellent biological stability using extracted from natural substances. Can be used.

1 is Ecklonia according to an embodiment of the present invention ( Ecklonia) cava ) and phloroglucinol are shown to have an effect on cell viability attenuated by 6-OHDA. * Indicates significant difference from control group (P <0.05), ** indicates significant difference from 6-OHDA treated cells (P <0.05).
Figure 2 shows the effect of radical scavenging activity of phloroglucinol according to an embodiment of the present invention. (A) Intracellular ROS was measured using a spectrophotometer. (B) Intracellular ROS was measured using a flow cytometer. (C) Intracellular ROS was measured using a confocal microscope after DCF-DA staining. The measurement result is shown. * Indicates significant difference from control group (P <0.05), ** indicates significant difference from 6-OHDA treated cells (P <0.05). In addition, FI shows the fluorescence intensity of DCF-DA.
Figure 3 shows the effect of phloroglucinol on the damage of cellular components caused by 6-OHDA according to one embodiment of the present invention. (A) analysis of the amount of 8-isoprostane to measure lipid peroxidation, (B) analysis of carbonyl formation to analyze protein oxidation, and (C) determination of DNA damage. The result of analyzing the amount of 8-OHdG in DNA is shown. * Indicates significant difference from control group (P <0.05), ** indicates significant difference from 6-OHDA treated cells (P <0.05).
Figure 4 shows the effect of phloroglucinol on the activity of CAT and GSH-Px according to an embodiment of the present invention. (A) CAT activity, (B) GSH-Px activity measured using GPx assay kit.

The present invention relates to a novel use of Ecklonia cava extract, specifically, to provide a composition for the prevention or treatment of Parkinson's disease comprising Ecklonia cava extract or phloroglucinol isolated therefrom as an active ingredient. have.

In the present invention, " Ecklonia cava ) is a perennial brown algae and belongs to the Laminariales Alariaceae family. In Korea, they inhabit about 10m of water in Jeju coast. The length is 1 ~ 2m, the stem is cylindrical, and the base is root shaped. It is a food for abalone and turban shells, and is used as a raw material for making alginic acid. In the previous studies, there have been many useful physiological activities such as anticancer activity, anticoagulant activity, thrombogenicity inhibitory activity, cardiovascular protective effect and antiviral activity.

In addition, phloroglucinol is a basic unit constituting phlorotannin, which is a type of polyphenolic compound, and is one of aromatic compounds, benzenetriol (trihydroxybenzenes), which is substituted in the benzene ring. It is a polyphenol compound with three hydroxyl groups. In nature, it is found in marine plants, especially brown algae. The chemical name is benzene-1,3,5-triol, and the chemical formula is as follows.

<Formula>

Parkinson's disease, on the other hand, is a chronic and degenerative neurological disorder caused by neurological damage that occurs next to Alzheimer's disease. Significantly reduced dopamine in the striatum results in the selective loss of dopamine-activated neurons in the substantia nigra. The cause of Parkinson's disease is not exactly known yet, but recent studies have reported that oxidative stress affects neuronal damage processes and causes Parkinson's disease. Oxidative stress causes damage to proteins, DNA and lipids, causes structural and functional disruption of cell membranes, inactivation of enzymes and consequently apoptosis. Oxidative stress occurs as the production of active free radicals such as reactive oxygen species increases. Brain tissue is particularly sensitive to oxidative stress because of its high polyunsaturated acid content.

Accordingly, the present inventors have found that Ecklonia cava extract and phloroglucinol compound isolated therefrom are converted to 6-hydroxydopamine (6-hydroxydopamine, 6-OHDA) in the SH-SY5Y cell model, which is a human dopaminergic neuroblastoma. It has been shown for the first time that it has an effect of preventing neurotoxicity caused by it, and thus it can be useful for treating Parkinson's disease, which is known to be caused by the loss of dopaminergic neurons in the brain.

6-hydroxydopamine (6-OHDA) is a common neurotoxin that causes oxidative apoptosis of dopaminergic neurons and is generally known to induce Parkinson's disease. It is widely used in experiments to confirm the effect of preventing or treating Parkinson's disease.

Therefore, the present inventors conducted a cell experiment pretreated with 6-OHDA to confirm the effects of Ecklonia cava extract and phloroglucinol on Parkinson's disease, and the target cells were SH-, a human dopaminergic neuroblastoma associated with Parkinson's disease. SY5Y was used.

According to one embodiment of the present invention, in the SH-SY5Y, a neuroblastoma cell pretreated with Ecklonia cava extract or phloroglucinol, cellular damage caused by 6-OHDA is attenuated (see Example 2), and 6-OHDA stress It was confirmed to reduce reactive oxygen species (ROS) in the cells generated by (see Example 3). In addition, phloroglucinol was found to inhibit lipid peroxidation, protein carbonyl formation and DNA damage caused by 6-OHDA (see Example 4). Furthermore, it was confirmed that phloroglucinol improves catalytic activity (CAT) and glutathione peroxidase (GSH-Px), which are weakened by 6-OHDA (see Example 5).

Therefore, the Ecklonia cava extract according to the present invention and the phloroglucinol compound isolated therefrom can be effectively used for the treatment of neurodegenerative diseases caused by oxidative stress, particularly Parkinson's disease.

The present invention is Ecklonia cava ) It is characterized by providing a composition for the prevention or treatment of Parkinson's disease comprising an extract or phloroglucinol represented by the following formula separated from it as an active ingredient.

<Formula>

The "extract" as defined in the present invention is extracted from the Ecklonia cava using a suitable solvent, and includes, for example, crude extract of Ecklonia cava, polar solvent soluble extract or nonpolar solvent soluble extract. In addition, the desired extract can be purified using purification methods known to those of ordinary skill in the art. There is no limitation on the preparation method of the Ecklonia cava extract, and any known method may be used.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the preparation method of the Ecklonia cava extract, and any known method may be used.

As a suitable solvent for extracting the extract from the Ecklonia cava, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto. For example, purified water, carbon number 1 Lower alcohols (methanol, ethanol, propanol, isopropanol, butanol, etc.) to 4, acetone, diethyl ether, ether, benzene, chloroform, ethyl acetate Various solvents such as acetate), methylene chloride, hexane and cyclohexane may be used alone or in combination. More preferably, methanol can be used.

Extracting and purifying phloroglucinol from the Ecklonia cava comprises the steps of extracting once or twice or more by adding an organic solvent to the Ecklonia dry powder; Solvent fractionating the extracted material once or twice; And purifying the solvent fractionated component by chromatography.

The Ecklonia cava extract according to the present invention is pulverized and then pulverized Ecklonia cava in natural shade, and then, about 3 to 20 times the dry weight of Ecklonia cava, preferably about 3 to 5 times the volume of water, methanol, ethanol and butanol Lower alcohols such as these or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably 1 to 36 hours at an extraction temperature of 20 to 100 ° C., preferably 20 to 70 ° C., with methanol. Preferably it is obtained by extracting from 1 to 5 times, preferably 3 times in succession by extraction methods such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 12 to 24 hours, and then filtered under reduced pressure and vacuum filtrate Concentration under reduced pressure at 20 to 100 ℃, preferably 40 to 70 ℃ to obtain the crude Ecklonia cava extract soluble in water, lower alcohol or a mixed solvent thereof.

The crude Ecklonia cava extract is suspended in water and extracted with a solvent in the order of n-hexane, dichloromethane, ethyl acetate, n-butanol, and the soluble extract of Ecklonia cava polar solvent of the present invention, preferably dichloromethane or ethyl acetate soluble. Fractions can be obtained.

Silica gel column chromatography may be performed on the nonpolar solvent soluble fraction to obtain a diethyl ether fraction, which may be purified by Sephadex to separate and identify the compounds.

Ecklonia cava extract in the present invention can be used in a concentration of 20 ~ 100㎍ / ㎖, preferably 40 ~ 70㎍ / ㎖, phloroglucinol is 1 ~ 40㎍ / ㎖, preferably 5 ~ 20㎍ / ㎖ Can be used at the concentration of.

Ecklonia cava extract or phloroglucinol according to the present invention may inhibit the active oxygen species (ROS) in nerve tissues or cells, and may promote the antioxidant activity of tissues or cells in vivo through the active action of antioxidant enzymes. For example, the antioxidant enzyme may include CAT, glutathione peroxidase (GSH-Px) and the like.

In addition, the Ecklonia cava extract or phloroglucinol according to the present invention exhibits the effect of inhibiting intracellular DNA damage, lipid peroxidation or protein modification. Examples of protein modification include, but are not limited to, the formation of carbonyl groups on the side of the amino acid.

In addition, the Ecklonia cava extract or phloroglucinol compound according to the present invention exhibits the effect of inhibiting apoptosis, and the apoptosis inhibitory effect is to suppress the expression of apoptosis factor or increase the expression of apoptosis inhibitory factor in vivo. It may appear through, but is not limited to such.

Furthermore, the Ecklonia cava extract or phloroglucinol compound according to the present invention exhibits the effect of inhibiting or preventing nerve cell damage caused by 6-hydroxydopamine (6-OHDA).

Therefore, the composition containing the Ecklonia cava extract of the present invention or phloroglucinol isolated therefrom can be used as a pharmaceutical composition for the prevention or treatment of Parkinson's disease, and the pharmaceutically effective amount of the Ecklonia cava extract or its Soluble fraction extract, or phloroglucinol isolated from Ecklonia cava extract, may be included alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.

The "pharmaceutically effective amount" as used herein refers to an amount sufficient for the physiologically active component to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the age, weight, health condition, sex, route of administration and duration of treatment.

The pharmaceutically effective amount of the Ecklonia cava extract or phloroglucinol according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein means physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness or the like when administered to a human. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, and can be formulated for various oral or parenteral administration. It may be formulated in a form. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

Representative of formulations for parenteral administration are isotonic aqueous solutions or suspensions which are suitable for injectable use. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups and wafers, etc. These formulations may contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, , Sucrose, mannitol, sorbitol, cellulose and / or glycine) and lubricants (e.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols). The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, A disintegrating agent such as sodium salt, an absorbent, a coloring agent, a flavoring agent and / or a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.

In addition, the composition of the present invention may further include auxiliaries such as preservatives, hydrating agents, emulsifiers, salts or buffers for controlling osmotic pressure and other therapeutically useful substances, and may be formulated according to conventional methods.

The composition according to the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of the active ingredient being determined by several factors such as the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on. In addition, the composition of the present invention can be administered in parallel with known compounds that can enhance the desired effect.

Routes of administration of the compositions according to the invention can be administered to humans and animals orally or parenterally, such as intravenously, subcutaneously, intranasally or intraperitoneally. Oral administration also includes sublingual application. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

Therefore, the present invention can provide a medicament capable of preventing and treating the symptoms of Parkinson's disease, including a composition comprising an Ecklonia cava extract or a phloroglucinol compound extracted therefrom as an active ingredient. Furthermore, the composition according to the present invention is a composition derived from natural products, there is no toxicity and side effects for the drug and can be used with confidence even during long-term use.

In addition, the composition for the prevention or treatment of Parkinson's disease of the present invention can be added to food for the purpose of preventing and improving the disease caused by Parkinson's disease, the composition of the present invention for the prevention and improvement of Parkinson's disease When used as a food composition, a functional food for preventing Parkinson's disease can be provided.

Thus, the present invention can provide a food composition for preventing or treating Parkinson's disease, including the Ecklonia cava extract or phloroglucinol isolated therefrom as an active ingredient.

The food composition according to the present invention can be easily used as a food, such as a main ingredient, an auxiliary ingredient, a food additive, a functional food or a beverage, which is effective in preventing and improving Parkinson's disease symptoms.

As used herein, the term “food” refers to a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step, It includes all foods, food additives, functional foods and drinks.

Examples of the food to which the composition for food containing the Ecklonia cava extract according to the present invention or phloroglucinol separated therefrom as an active ingredient may be added, for example, various foods, beverages, gums, teas, vitamin complexes, and functional foods. Etc. In addition, in the present invention, the food may include special nutritive foods (e.g., crude oil, spirits, baby food, etc.), meat products, fish meat products, tofu, mackerel, noodles (Such as soy sauce, soybean paste, kochujang, mixed potatoes), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, milk products (eg, fermented milk, cheese, But are not limited to, pickled foods (various kinds of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages and the like) and natural seasonings (e.g. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the term "functional food" refers to the control of biological defense rhythms and disease prevention of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body's regulatory function regarding recovery and the like, and specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In addition, in the present invention, the "beverage" refers to a generic term for drinking to quench thirst or to enjoy a taste and includes a functional drink. The beverage contains, as essential ingredients, a composition for preventing and ameliorating the symptoms of Parkinson's disease as an essential ingredient, and there are no special limitations on the other ingredients. It may contain as.

Furthermore, in addition to the above, food containing a composition for preventing and treating Parkinson's disease according to the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents such as flavoring agents, colorants and fillers ( Cheeses, chocolates, and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The components can be used independently or in combination.

Therefore, the present invention can provide a dietary supplement comprising Ecklonia cava extract or phloroglucinol separated therefrom and a food acceptable additive.

The type of dietary supplement is not limited thereto, but may be in the form of powder, granules, tablets, capsules, or beverages.

In addition, in the food containing the composition for food of the present invention, the amount of the composition according to the present invention may comprise from 0.001% to 90% by weight of the total food weight, preferably 0.1% to 40% by weight In the case of a beverage, it may be included in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g based on 100ml, for a long time for the purpose of health and hygiene or for health control In the case of ingestion may be less than the above range, the active ingredient is not limited to the above range because it may be used in an amount above the above range because there is no problem in terms of safety.

Hereinafter, the present invention will be described in detail with reference to embodiments and drawings. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

Ecklonia  Extracts and separated therefrom Of phloroglucinol  Produce

The Ecklonia cava used in the experiments were collected from the coast of Jeju Island, and dried in the room and made into a powder state using a grinder, and then stored in a refrigerator.

2 kg of Ecklonia cava powder was added to 40 L of 80% methanol, stirred at room temperature for 24 hours to leach (repeated three times), and the filtrate was concentrated under reduced pressure to obtain 260 g of an extract. The obtained extract was suspended in distilled water and treated with ethyl acetate to obtain an organic layer. The ethyl acetate fraction (30 g) was loaded on a celite column and eluted with each solvent to obtain a diethyl ether fraction (18 g). This fraction was purified using Sephadex column chromatography (developing solvent: chloroform-methanol) to purely obtain phloroglucinol (0.5 g).

< Example  2>

According to the invention Ecklonia  Extracts and isolates therefrom Of phloroglucinol  Neuronal cell death inhibitory effect

Ecklonia ( Ecklonia) obtained in Example 1 In order to confirm the protective effect of 6-OHDA toxicity of cava ) extract and phloroglucinol isolated therefrom, the present inventors first simulated human dopaminergic neuroblastoma SH-SY5Y cells at different concentrations. After pre-incubated with extract (25-100 μg / ml) or phloroglucinol (5-40 μg / ml) for 1 hour, 6-OHDA 90 μM was treated for 48 hours.

Cell viability was measured via MTT assay.

As a result, Ecklonia cava extract, an oligomeric compound of phloroglucinol, was found to increase neuronal cell viability attenuated by 6-OHDA when treated with 25, 50 and 100 μg / ml, and treated with 50 μg / ml. It was found that the survival rate of neurons was increased the most (see FIG. 1A).

In addition, when 5, 10 and 20 µg / ml of the phloroglucinol compound was treated to SH-SY5Y cells, it was shown to have a significant cell protective effect and a survival increase effect on 6-OHDA induced apoptosis. In particular, it was found that the most excellent effect when treated with 10 ㎍ / ㎖ (see Figure 1B).

Through the above results, it was confirmed that the Ecklonia cava extract and the phloroglucinol compound included therein have an effect of inhibiting the death of neurons induced by 6-OHDA.

< Example  3>

According to the invention Ecklonia  origin Of phloroglucinol  Neuronal Radical  Scavenging activity

In order to confirm the radical scavenging activity of phloroglucinol against reactive oxygen species (ROS) generated by 6-OHDA, SH-SY5Y cells were first treated with 10 μg / ml phloroglucinol for 1 hour, and then 90 μM of 6-OHDA was treated for 48 hours. Next, the ROS level was measured using a spectrofluorometer.

As a result, ROS was decreased in cells treated with 6-OHDA and phloroglucinol as compared to ROS levels in cells treated with 6-OHDA alone (see FIG. 2A).

In addition, as a result of staining ROS with a DCF-DA fluorescent dye and measuring the fluorescence intensity using a flow cytometer, 6-OHDA and phloroglucinol in cells treated with 6-OHDA alone compared to 142 In the cells treated with the fluorescence intensity was 114 (see Fig. 2B).

Furthermore, as a result of the confocal microscope, 6-OHDA-treated cells showed strong red fluorescence intensity of ROS, whereas 6-OHDA-treated cells were treated with phloroglucinol. Red fluorescence intensity was found to decrease (see FIG. 2C).

From the above results, it was confirmed that phloroglucinol had ROS scavenging activity caused by 6-OHDA.

< Example  4>

According to the invention Ecklonia  origin Of phloroglucinol  In nerve cells DNA  Inhibitory effects of damage, lipid peroxidation and protein modification

To determine the effect of phloroglucinol on the damage of cellular components caused by 6-OHDA, we measured lipid peroxidation, carbonyl formation, DNA damage. SH-SY5Y cells were treated with 10 μg / ml phloroglucinol for 1 hour followed by 90 μM of 6-OHDA for 48 hours.

<4-1> lipid peroxidation ( Lipid peroxidation ) Measure

In order to measure lipid peroxidation, the amount of 8-isoprostane was measured and analyzed. 8-Isoprostane is a product of lipid peroxidation and is detected in most body fluids in the body, and has a chemically stable structure, which has recently attracted attention as a specific and reliable index for quantifying oxidative stress in the body. .

As a result, the concentration of 8-isoprostane was significantly increased when 6-OHDA alone was treated, but the concentration of 8-isoprostane was decreased when floroglucinol was treated (see FIG. 3A). These results confirmed that phloroglucinol significantly inhibited lipid peroxidation.

<4-2> protein Carbonyl  Formation measurement

Oxidative stress introduces carbonyl groups into the amino acid side chains of proteins. Accordingly, protein carbonyl formation was analyzed to analyze protein oxidation.

As a result, 6-OHDA increased the intracellular protein carbonyl content in SH-SY5Y cells, whereas phloroglucinol inhibited the protein carbonyl formation induced by 6-OHDA (see FIG. 3B).

<4-3> DNA  Damage measurement

DNA damage caused by 6-OHDA is a major factor in reducing cell viability. Intracellular DNA damage following 6-OHDA exposure can be detected by the amount of 8-OHdG. To determine the extent of oxidative DNA damage, the amount of 8-OHdG in the DNA was analyzed.

As a result, treatment with 6-OHDA increased the concentration of 8-OHdG to 204 µg / ml (50 µg / ml for control cells), whereas the concentration of 8-OHdG was 63 µg / ml when treated with phloroglucinol. It was shown to decrease up to (see Figure 3C).

These results confirm that phloroglucinol has an activity of protecting cellular component damage caused by 6-OHDA.

< Example  5>

According to the invention Ecklonia  origin Of phloroglucinol  Antioxidant Enzyme Activation Effects in Neurons

In order to determine how the radical scavenging activity of phloroglucinol affects the activity of antioxidant enzymes, the activity of CAT and GSH-Px was measured in SH-SY5Y cells treated with phloroglucinol.

As a result, the CAT activity was reduced to 26U / mg protein when treated with 6-OHDA, but the reduced CAT activity was recovered to 47U / mg protein when treated with phloroglucinol (see Figure 4A).

In addition, GSH-Px activity was reduced to 66mU / ml protein in cells exposed to 6-OHDA, but when treated with phloroglucinol, GSH-Px activity was restored to 115mU / ml protein (Fig. 4B). Reference).

Through the above results, it was confirmed that phloroglucinol increased the activity of antioxidant enzymes such as CAT and GSH-Px that were reduced in 6-OHDA treated cells.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

Ecklonia cava ) A composition for preventing or treating Parkinson's disease containing the extract as an active ingredient. The method of claim 1,
The Ecklonia cava extract is a composition for the prevention or treatment of Parkinson's disease, characterized in that the solvent extract extracted by adding water or organic solvent to the Ecklonia soluble. The method of claim 1,
The Ecklonia cava extract is a composition for preventing or treating Parkinson's disease, characterized in that it comprises a phloroglucinol compound. A composition for preventing or treating Parkinson's disease comprising a phloroglucinol compound represented by the following formula as an active ingredient:
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. 5. The method of claim 4,
The phloroglucinol compound is a composition for preventing or treating Parkinson's disease, characterized in that extracted from Ecklonia cava . The method according to claim 1 or 4,
The Ecklonia cava extract or phloroglucinol compound is a composition for the prevention or treatment of Parkinson's disease, characterized in that it exhibits the effect of inhibiting or preventing neuronal damage caused by 6-hydroxydopamine (6-OHDA). Functional food for preventing Parkinson's disease containing Ecklonia cava extract or phloroglucinol compound represented by the following formula as an active ingredient:
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