KR20190081753A - Pharmaceutical composition comprising ishige okamurae extracts for prevention and treatment of neurodegenerative disorders as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating neurodegenerative diseases and improving cognitive functions, comprising an extract of Ishige okamurae as an active ingredient. The extract of Ishige okamurae has been shown to inhibit cell death by reducing the ROS concentration in cells due to an increase in glutamate through Akt signaling regulation in glutamate-induced HT22 cells. In addition, when Aβ peptide-induced PC12 cells are treated with the extract of Ishige okamurae, the expression of pro-inflammatory mediators, such as COX2 and iNOS, is inhibited, and the inhibitory effect on inflammation is shown through the inactivation of MAPKs signaling pathway. The extract of Ishige okamurae has been shown to attenuate cytotoxicity by decreasing the ROS concentration in cells due to an increase in Aβpeptide, thereby suggesting a potential as a therapeutic agent for neurodegenerative disease.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing and treating degenerative brain diseases, which comprises, as an active ingredient, a compound of formula (I)

The present invention relates to a pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising an extract of Ishige okamurae as an active ingredient, and a composition for improving cognitive function.

Ishige okamurae is a brown algae belonging to the lacunae. It is distributed in China, Japan, and Thailand coast, and it is attached to the rocks in the west coast, southern coast and Jeju island coast in Korea. Phlorotannin, which is known as a polyphenol component of plaquettes, is an excellent source of alginate, anti-inflammation, and antioxidants in a wide range of products containing xanthopophyll and fucoxanthin. Phlorotannin, a physiologically active substance, .

When the amount of reactive oxygen species (ROS) in the body is greatly increased in the process of oxygen metabolism in the body or by various external factors such as ultraviolet rays, drugs, environmental pollutants and various other reasons, Death, disease outbreaks and cell aging. That is, the balance between the production of ROS and the antioxidant capacity of cells determines oxidative stress, and oxidative stress damages intracellular proteins, lipids and DNA. Oxidative damage of cellular or intracellular substances by these reactive oxygen species is known to be an important factor in degenerative brain diseases of the central nervous system such as Alzheimer's syndrome, Parkinson's syndrome, and Huntington's syndrome.

Apoptosis is a programmed death mechanism that occurs in cells and is regulated by a number of apoptosis-related factors such as hormones or chemicals. It is distinct from necrosis, a cell necrosis, Because of its close relationship, studies on the induction of apoptosis in various cancer cells have been carried out. Characteristically, there are contraction of nucleus and chromatin due to condensation, DNA fragmentation, protein cleavage, apoptotic body The same phenomenon appears. This apoptosis is caused by the activation of Bcl2 family, caspase activation and poly (ADP-ribose) polymerase (PARP) in the cells. Recently, apoptosis has been reported to be associated with reactive oxygen species (ROS). ROS is produced in cell metabolism and is inactivated immediately, but when the balance of redox is broken, it causes cell damage. These ROSs play a major role in intracellular signaling in stress-free situations, but ROS causes oxidative stress, resulting in DNA damage, senescence and apoptosis.

Korean Patent No. 10-0981184

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing Ishige okamurae extract as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing and treating degenerative brain diseases comprising an extract of Ishige okamurae as an active ingredient.

The extracts of glutamate-induced HT22 cells inhibited cell death by decreasing the intracellular ROS concentration by increasing glutamate through the control of Akt signaling. When the extract of PC12 cells treated with Aβ peptide, And iNOS, inhibited the inflammation by inactivating the MAPKs signaling pathway, and decreased the cytotoxicity by decreasing intracellular ROS concentration by increasing the Aβ peptide.

Figure 1 is a diagram showing the apoptosis signal pathway, showing the protein kinase signaling pathway and the apoptotic cascade.
Figure 2 depicts possible inhibition mechanisms of cardiac extracts on glutamate mediated neurotoxicity in HT-22 cells.
Figure 3 shows the possible inhibition mechanism of plaque extract on glutamate mediated neurotoxicity in PC-12 cells.
FIG. 4 is a graph showing the inhibitory effect of the extract of Lactobacillus on the viability of HT22 cells. (a) Cells were treated with L. extract at a concentration of 0.01 to 0.7 mg / mL for 24 hours. (b) Cells were treated with different concentrations of glutamate ranging from 1 mM to 10 mM for 24 hours. Cell viability for L. extract was evaluated using WST-1. Statistical analysis was performed using at least three experiments using one-way ANOVA (***: P <0.001).
FIG. 5 is a graph showing the inhibitory effect of the extract of Lactobacillus on HT22 cell viability. Lactobacillus extracts were treated at 0.05, 0.1, and 0.2 mg / mL, respectively. Absorbance was measured with a microplate reader at 450 nm (## p <0.01, ### p <0.001 versus glutamate alone; ** p <0.01, *** p <0.001 versus control).
FIG. 6 is a photograph of HT22 cell treated with L-eluate (0.05, 0.1 mg / mL) for 2 hours 30 minutes and cultured for 4 hours with 5 mM glutamate, followed by optical microscopy. (a) control group; (b) 5 mM glutamate; (c) 0.05 mg / mL Linalool extract and 5 mM glutamate treatment; (d) Treatment with Lactobacillus extract 0.1 mg / mL and glutamate 5 mM.
FIG. 7 is a graph showing the protective effect of Lactobacillus root extract on glutamate cell death in HT22 cells. (a) control group; (b) 5 mM glutamate; (c) Lactobacillus extract 0.5 mg / mL and glutamate 5 mM; (d) Lactose extract 0.1 mg / mL and glutamate 5 mM.
FIG. 8 is a graph showing the inhibitory effect of Lactobacillus root extract on intracellular ROS formation in glutamate-treated HT22 cells. The fluorescence intensity of DCF was measured by irradiating with excitation at 485 nm at 530 nm (*** p < 0.001 versus control).
9 is a graph showing (a) Western blot photograph and (b) p-Akt quantification results showing the effect of a roe extract on p-Akt and Akt protein expression in glutamate-treated HT22 cells p < 0.001 versus glutamate alone; *** p < 0.001 versus control).
10 is a graph showing the ratio of (a) western blot photograph and (b) Bax / Bcl-2 showing the effect of rosacea extract on the expression of Bcl-2 and Bax in glutamate-treated HT22 cells (*** p <0.001 compared to control group; *** p <0.001 compared to glutamate group).
Figure 11 is a western blot photograph showing the effect of Lactobacillus extract on the expression of active caspase-3 in glutamate-treated HT22 cells.
FIG. 12 is a graph showing mRNA levels of HO-1 and Nrf2 when glutamate-induced HT22 cells were treated with L. extract. MRNA levels of HO-1 and Nrf2 were measured using q-PCR (### p <0.001 versus glutamate alone; *** p <0.001 versus control).
FIG. 13 is a graph showing the inhibitory effect of Lactobacillus root extract on the viability of PC12 cells. Statistical analysis was performed using at least three experiments using one-way ANOVA (***: P <0.001).
FIG. 14 is a graph showing the toxic protective effect of the Lactobacillus extract on PC12 cells induced by Aβ peptide. FIG. Absorbance was measured with a microplate reader at a wavelength of 450 nm (## p <0.01, ### p <0.001 versus glutamate alone; *** p <0.001 versus control).
FIG. 15 is an optical microscope photograph showing the cytotoxic effect and cytotoxic effect of A? Peptide-induced PC extract on the cell extract. FIG. (a) control group; (b) 70 [mu] M a [beta] peptide treatment; (c) 70 [mu] M a [beta] peptide and L. extract 0.125 mg / ml.
FIG. 16 is a graph showing the ROS inhibitory effect when A? Peptide-induced PC12 cell was treated with L. extract. The fluorescence intensity of DCF was measured by irradiating with excitation at 485 nm at 530 nm (*** p < 0.001 versus control).
FIG. 17 shows Western blot results showing the effects of Aβ peptide-induced PC12 cells on the MAPKs pathway when treated with L. extract.
FIG. 18 is a graph showing the effect of the extract of Aβ peptide-induced PC12 cells on the iNOS and COX2 levels at the mRNA level. mRNA levels of iNOS and COX2 were measured using q-PCR (### p <0.001 versus glutamate alone; *** p <0.001 versus control).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing an extract of Ishige okamurae as an active ingredient.

The extract may be extracted with a solvent which is a lower alcohol of C1 to C2 or a mixture thereof, and then dried to be dissolved in 1,3-butylene glycol, but not limited thereto

The lower alcohol may be, but is not limited to, methanol or ethanol.

As the method of extracting the Linalool extract, a conventional method in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction may be used, and extraction may be performed once to five times by hot water extraction method. Specifically, it may be repeated three times, but is not limited thereto.

The extraction solvent may be added in an amount of 0.1 to 10 times, preferably 0.3 to 5 times.

The extraction temperature may be 20 to 40, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.

The drying may be carried out under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, preferably freeze drying.

The Lactobacillus root extract may be, but is not limited to, phosphorylating AKT to reduce intracellular ROS concentration with increasing glutamate.

The Lactobacillus root extract may inhibit the expression of pro-imflammatory mediator to reduce the intracellular ROS concentration upon increase of A [beta] peptide, but is not limited thereto.

The compositions of the present invention may be of various oral or parenteral formulations. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances or preservatives are included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations or suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select the intraperitoneal, rectal, rectal, intravenous, intramuscular, subcutaneous, intrauterine or intracerebral injection methods for parenteral administration, It is used for external skin.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, The dose is 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and can be administered 1 to 6 times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention provides a health functional food for prevention and improvement of degenerative brain diseases containing Ishige okamurae extract as an active ingredient.

There is no particular limitation on the kind of the food. Examples of the foods to which the extract can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The Lactobacillus extract or fraction thereof of the present invention can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the Lard extract of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the Lard extract of the present invention can contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of these additives is not so important, it is general that the roots extract of the present invention is selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

reagent And devices

(FBS, corning, USA) and penicillin / streptomycin were purchased from Hyclone, FBS was purchased from Corning, HS, PBS, 0.25% Trypsin-EDTA was purchased from Gibco. WST-1 was purchased from cayman chemical. N-Acrtyl-L-cysteine, glutamic acid, 2 ', 7'-Dichlorofluorescin purchased from Sigma-Aldrich, amyloid β peptide (25-35) purchased from abcam and TRIzol ^® Reagent purchased ambion. SYBR ^® Green Realtime PCR Master Mix, reverTra Ace ^® qPCR RT Master Mix with gDNA Remover purchased from TOYOBO. Acrylamide solution was purchased from Tech & Innovation, and ECL solution was purchased from BIO-RAD.

All antibiotics and inhibitors used in the experiments are summarized in Table 1 below. The instrument used in the present invention was an ELISA microplate reader (iMark Microplate Reader, Bio-Rad Laboratories, Inc.), Real-Time PCR Detection Systems (iMark Microplate Reader, Bio- Rad Laboratories, Inc.), Flow cytometry (Beckman Coulter) , Nicon Eclipse Ti-S mieroscope (Nikon), and Automatic X-ray film processor (JPI).

Name Company clonal Isotype p44 / 42 MARK (ERK1 / 2) Cell Signaing Polyclonal Rabbit IgG phospho-p44 / 42 Cell Signaing Polyclonal Rabbit IgG MARK (Erk1 / 2) Cell Signaing Polyclonal Rabbit IgG phospho-Akt Cell Signaing Polyclonal Rabbit IgG Akt Cell Signaing Polyclonal Rabbit IgG phospho-p38 Cell Signaing Polyclonal Rabbit IgG p38 Cell Signaing Polyclonal Rabbit IgG Cleaved Caspase-3 Cell Signaing Polyclonal Rabbit IgG Caspase-3 Cell Signaing Polyclonal Rabbit IgG Cleaved Caspase-9 Cell Signaing Polyclonal Rabbit IgG Caspase-9 Cell Signaing Polyclonal Rabbit IgG Cleaved PARP Cell Signaing Polyclonal Rabbit IgG PARP Cell Signaing Polyclonal Mouse IgG Anti-mouse IgG Santa Cruz Polyclonal Rabbit IgG Anti-rabbit IgG Santa Cruz Polyclonal Rabbit IgG

Experimental Method

<Cell culture>

mouse hippocampus-derived cell line, HT22 cells were used to received pre-sale in Korea Cell Line Bank, a 100 unit / ml penicillin, 100μg / ml streptomycin and 10% fatal bovine serum was added in DMEM medium, 5% CO ₂ in a 37 ℃ contain And cultured in an incubator. Rat chromium affinity cell line, PC12 cells were used to received pre-sale in Korea Cell Line Bank, it was added to 100 unit / ml penicillin, 100μg / ml streptomycin and 5% fatal bovine serum, 10% Horse serum in RPMI medium, 5% CO ₂ Lt; RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt;

<Cell viability>

HT22 cells were plated at a density of 5 × 10 ³ cells / well in a 96-well plate. After culturing for 24 hours, the cell extract-free extracts were treated for 2 hours before culturing for 18 hours. 10 μl of WST-1 (Tetrazolium Salts) was added and incubated for 2 hours. PC12 cells were plated at a density of 1 × 10 ⁴ cells / well in a 96-well plate, cultured for 24 hours, and cultured for 2 hours before the treatment for 18 hours. 10 μl of WST-1 was added and incubated for 4 hours.

< Apoptosis  assay>

HT22 and PC12 cells were plated at 5 × 10 ⁶ cells in a 10 cm cell culture dish and stabilized for 24 hours. O / N was performed with serum-free medium, and then L-lysine extract was treated for 24 hours. 1 × 10 ⁶ cells were collected, washed twice with PBS, stained with annexin V / PI reagent and 1 × binding buffer for 20 min in the dark room, and the stained cells were analyzed by flow cytometry Respectively.

<Measurement of ROS >

HT22 and PC12 cells were seeded at 5 × 10 ⁵ cells / well in 6 wells and stabilized for 24 hours. Then, the cells were treated with O / N with serum free medium and cultured for 24 hours. Cells were separated, washed with PBS, and treated with 10 μM DCFH-DA (dichlorodihydrofluorescein diacetate), stained at 37 ° C for 30 min, and then measured using flow cytometry.

<Quantitative real-time PCR >

HT22 and PC12 cells were plated in 6 wells at 5 × 10 ⁵ cells and stabilized for 24 hours. Then, O / N was added to serum-free medium, followed by culturing for 24 hours. RNA was extracted using Trizol, cDNA was prepared, and RT-PCR was performed with SYBR kit. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.

<Western blotting>

HT22 and PC12 cells were seeded at 6 × 10 ⁵ cells / well in 6 wells and stabilized for 24 hours. Then, O / N was added to serum-free medium, Cells were separated and washed with PBS. The cells were then washed with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% (w / v) IGEPAL ^® CA- 630, 5 mM sodium pyrophoahate, glycerophosphate, 1 mM phenylmehylsulfonyl fluoride and a protease inhibitor mixture. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 10% and transferred to a polyvinylidene difluoride membrane. The transferred membrane (PVDF) was blocked with TBS-T buffer (20 mM Tris-HCl buffer, pH 7.4, 136 mM NaCl, 0.1% Tween 20) containing 5% skim milk for 1 hour. The primary antibody was then incubated at 4 ° C, washed three times with TBS-T buffer, and the secondary antibody was treated at room temperature for 40 minutes. After washing three times with TBS-T buuffer, ECL reagent was treated and examined using an Autometic X-ray film processor. Identified protein bands were measured using Image J.

Example : Preparation of Lard Extracts

The plaques used in this experiment were collected from the shore of Jeju Island, washed, cut into three pieces with 80% methanol, concentrated under reduced pressure, and lyophilized, powdered and stored at -80 ℃ deep freezer. The powdered medicinal material was dissolved in 85% 1,3-butylene glycol, and the solution was centrifuged and only the supernatant was transferred.

1. Glutamate-mediated cell of Lactobacillus sp. Extract using HT22 cells Neuroprotection against toxicity

Experimental Example  1-1. Cells treated with L. extract Survival rate (Cell Viability) change

To determine the cell viability rate of HT22 cells, WST-1 (Tetrazolium Salts) assay was performed at different concentrations of Lactobacillus extract. Cell viability was decreased depending on the cell extract concentration, and cell toxicity was observed at a concentration of 0.5 mg / mL or more in HT22 cells compared with the control group, and cell viability was decreased in a concentration-dependent manner even when glutamate was treated And cytotoxicity was observed from 1 mM when compared to the control (Fig. 4).

Experimental Example  1-2. Toxicity of glutamate-induced HT22 cell line extract

WST-1 was performed to determine the effect of Lactobacillus extract on glutamate-induced apoptotic cell death. HT22 cells were pretreated for 2 hours 30 minutes and treated with 5 mM glutamate for 14 hours. The cell viability of the glutamate - treated group was decreased by about 80%. However, the viability of cell extracts treated with 0.05-0.2 mg / mL L was increased at a similar rate as the control. Lactobacillus extract inhibits glutamate-induced toxicity in HT22 cells and seems to have the effect of protecting glutamate-induced cell death (Fig. 5).

Experimental Example  1-3. Cell survival rate of Glutamate-induced HT22 cells treated with L. extract

HT22 cells were pretreated with 0.05% (0.1 mg / mL) for 2 hours 30 minutes and cultured with 5 mM glutamate for 14 hours. The cells were observed to be more glutamate-induced It seems to have a protective effect on the cells (Fig. 6).

Experimental Example  1-4. Cell survival rate of Glutamate-induced HT22 cells treated with L. extract

When glutamate is induced in HT22 cells, apoptosis is induced. When glutamate-induced HT22 cells are treated with LAH extract, apoptosis cells are reduced. Annexin V and PI staining. HT22 cells were pretreated for 2 hours 30 minutes and cultured with 5 mM glutamate for 14 hours. Then, 10 μl of annexin V-FITC and 5 μl of PI were incubated for 15 minutes. And then analyzed by flow cytometer.

The results showed that the amount of annexin V staining decreased with cell extract concentration, cell death was 9.82%, glutamate increased to 34.43%, cell extract was increased to 24.96%, 18.55% %. Experimental results show that glutamate-induced cells are protected from toxicity due to Lactobacillus extract (Fig. 7).

Experimental Example  1-5. When glutamate-induced HT22 cells were treated with Lactobacillus root extract ROS  Inhibitory effect

The inhibitory activity against the production of oxidative stress in HT22 cells was measured by DCF-DA assay. The results are as follows. Oxidative stress plays an important role in glutamate - induced cell damage. The amount of oxidative stress produced in the HT22 cells was 168% of the oxidative stress in the glutamate-treated group compared to the control group, and the positive control glutamate and L-eluate (0.1 mg / mL) In the treated group, the amount of oxidative stress was decreased to 120%, which was similar to that of the control group (FIG. 8).

Experimental Example  1-6. When glutamate-induced HT22 cells were treated with L. extract P13K / Akt  Effect on pathway

Glutamate - induced HT22 cell death was confirmed by P13K / Akt activation when the extract was treated. The phosphorylation of Akt was reduced in the part treated with 5 mM glutamate. The Akt phosphorylation was increased in the pretreated portion of the extract (Fig. 9).

Experimental Example  1-7. When glutamate-induced HT22 cells were treated with L. extract Bcl -2/ Bax  ratio

Bax and Bcl-2 protein levels. When HT23 cells were treated with 5 mM glutamate for 14 h, they increased in pro-apoptotic Bax and decreased in anti-apoptotic Bcl-2. However, when Lactobacillus extract and glutamate were treated together, they were similar to the control group in a concentration-dependent manner. The ratio of Bax / Bcl-2 was further reduced in the group treated with glutamate and L-glutamate than in glutamate alone (Fig. 10).

Experimental Example  1-8. When glutamate-induced HT22 cells were treated with L. extract caspase -3 activation effect

Caspase-3 activation plays an important role in apoptosis. Western blotting revealed caspase-3 activity. glutamate-induced cells are reduced when cleaved caspase-3 protein expression is reduced (Fig. 11).

Experimental Example  1-9. When glutamate-induced HT22 cells were treated with L. extract, HO-1, Nrf2 mRNA  Effect at level

HT22 cells were pretreated for 2 hours 30 minutes and cultured in 5 mM glutamate for 14 hours. The levels of HO-1 and Nrf2 mRNA were increased in the group treated with Lactobacillus lutein extract (FIG. 12).

2. Cell extracts of PC12 cells Aβ  peptide-mediated cell Neuroprotection against toxicity

Experimental Example  2-1. Changes in Cell viability by Treatment with Lard Extracts

First, to determine the cell viability rate of PC12 cells, WST-1 was performed at different concentrations of Lactobacillus extract. The cell viability of PC12 cells was found to be more than 0.15 mg / mL and the cell viability was decreased in Aβ peptide depending on the concentration as compared with the control group. And cytotoxicity was observed from 20 [mu] M (Fig. 13).

Experimental Example  2-2. Aβ  Toxicity of Peptide-Derived PC12 Cells

WST-1 was performed to confirm the effect of extracts on apoptotic cell death induced by Aβ peptide. PC12 cells were pretreated with L. extract (0.05-0.125 mg / mL) for 2 hours 30 minutes and treated with 70 μM Aβ peptide for 14 hours. The cell viability of Aβ peptide treated group was decreased by 80%. However, the viability of cells treated with L. extract (0.05-0.125 mg / mL) increased at a similar rate to the control group. Lattice extracts are shown to have the effect of preventing Aβ peptide induced to toxicity in PC12 cells and protecting Aβ peptide from being killed (FIG. 14).

Experimental Example  2-3. Aβ  Toxicity and cell morphology of peptide extract induced PC12 cell

PC12 cells were treated with L. extract (0.125 mg / mL) for 2 hours 30 minutes and cultured for 70 hours with 70 μM Aβ peptide for 14 hours. As a result, the cell extract was treated with Lβ Lt; / RTI &gt; cells (FIG. 15).

Experimental Example  2-4. Aβ  peptide-induced PC12 cells were treated with Lactobacillus casei extract ROS  Inhibitory effect

The inhibitory activity against oxidative stress production in PC12 cells was measured by DCF-DA assay. The results were as follows. Oxidative stress plays an important role among Aβ peptide - induced damage factors. The amount of oxidative stress produced in the PC12 cells was 250% of the oxidative stress produced in the Aβ peptide-treated group compared with the control group (100%), and the positive control glutamate and L-eluate (0.125 mg / mL) In the treated group, the amount of oxidative stress was decreased to 117%, similar to that of the control group (FIG. 16).

Experimental Example  2-5. Aβ  When peptide extracts were treated with peptide-induced PC12 cells MAPKs  Effect on pathway

Aβ peptide - induced PC12 cell death was found to be a protective effect and a decrease in MAPK phosphorylation when the extract was treated. The phosphorylation of MAPKs was increased in the area treated with 70 μM Aβ peptide. MAPKs phosphorylation was reduced in the group treated with Lactobacillus root extract (Fig. 17).

Experimental Example  2-6. Aβ  When peptide extracts were treated with peptide-induced PC12 cells mRNA  at level iNOS and On COX2  Effect

PC12 cells were treated with L. extract (0.075, 0.125 mg / mL) for 2 hours 30 minutes and cultured for 14 hours with 70 μM Aβ peptide. The levels of iNOS and COX2 mRNA were increased in the group treated with the extract of Aβ peptide (FIG. 18).

<Results and Discussion>

Nerve excitotoxicity is known to be one of the main mechanism of brain damage caused by oxidative stress, which is caused by neuronal cell death due to excessive stimulation of receptors by excitatory amino acids such as excess glutamate. Neuronal damage induced by glutamate may cause damage by inducing oxidative stress without receptor mediation as well as toxicity by excessive excitations of glutamate receptors. Thus, the present experiment demonstrated the neurotoxic protective effect of glutamate-induced HT22 cells in the parasites of marine natural products.

HT22 cells were pretreated with 0.05% fetal bovine serum (0.05 mg / mL) and 0.1 mg / mL for 2 hours 30 minutes and treated with 5 mM glutamate for 14 hours. The neuroprotective effect of glutamate-induced HT22 cells was determined by WST- Of the total. It was confirmed that cell death was decreased in a concentration-dependent manner by AnnexinV and PI staining, and it was confirmed that cytotoxic protection effect was induced by Western blotting through reduction of Bax / Bcl2 ratio and Cleaved Caspase-3 . DCFH-DA staining showed that ROS decreased in a concentration-dependent manner. Activation of the Akt / P13K pathway reduces glutamate-induced cell death. Treatment of glutamate-induced cells with L. extract phosphorylates AKT. When the drug is treated and the Akt pathway is activated, Nrf2 activity increases and HO-1 increases, thereby inhibiting ROS, thereby reducing cascade activity and reducing cell death. When glutamate-induced cells were treated with L. extract, Nrf2 and HO-1 were increased by q-PCR. Lt; RTI ID = 0.0 &gt; ROS &lt; / RTI &gt; concentration with increasing glutamate.

The present study demonstrates the neuroprotective effect of glutamate - induced HT22 cells through the regulation of Akt signaling, and suggests great potential for the development of AD medicines through oxidative stress.

Aβ peptide-induced neuroinflammation is an important mechanism of Alzheimer's. PC12 cells were pretreated with 0.075 and 0.125 mg / mL of protein extract for 2 hours and 30 minutes and treated with Aβ peptide for 14 hours to determine the neuroprotective effect of Aβ peptide-induced PC12 cells on the concentration by WST-1. Of the total. Thus, it is shown that the L. extract against Aβ peptide-induced has a neurotoxic protective effect. The expression of pro-imflammatory mediators such as COX2 and iNOS is inhibited when PC12 cells induced by Aβ peptide are treated with L. extract. In addition, the extract of Lappa shows inhibitory effect on inflammation through inactivation of MAPKs signaling pathway. Aβ peptide, it is found that it decreases cytotoxicity by decreasing intracellular ROS concentration.

This study suggests that the anti - inflammatory effect of L - extract on PC12 cells induced Aβ peptide has great potential for development of AD medicines in the future.

Claims (11)

A pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising an extract of Ishige okamurae as an active ingredient.
The method according to claim 1,
Wherein the extract is extracted with a C1 to C2 lower alcohol or a mixture thereof as a solvent, and then dried to be dissolved in 1,3-butylene glycol.
3. The method of claim 2,
Wherein the lower alcohol is methanol. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
The method according to claim 1,
Wherein the degenerative brain disease is Alzheimer's disease, and is a pharmaceutical composition for preventing and treating degenerative brain diseases.
The method according to claim 1,
Wherein said extract of Rhodophyll is phosphorylated with AKT to reduce intracellular ROS concentration upon increase of glutamate.
The method according to claim 1,
The pharmaceutical composition for the prevention and treatment of degenerative brain diseases according to claim 1, wherein the extract of Raloxifene inhibits expression of a pro-imflammatory mediator to reduce intracellular ROS concentration upon increase of A? Peptide.
(Ishige okamurae) extract as an active ingredient for the prevention and improvement of degenerative brain diseases.
8. The method of claim 7,
Wherein the extract is obtained by extracting C1 to C2 lower alcohols or a mixture thereof as a solvent and then drying and dissolving the 1,3-butylene glycol in a 1,3-butylene glycol.
8. The method of claim 7,
Wherein the degenerative brain disease is Alzheimer's disease, and is a health functional food for preventing and improving degenerative brain diseases.
8. The method of claim 7,
Wherein said extract is phosphorylated with AKT to reduce intracellular ROS concentration upon increase of glutamate.
8. The method of claim 7,
Wherein the extract of Lahm is a compound for inhibiting the expression of pro-imflammatory mediator and reducing intracellular ROS concentration upon increase of A? Peptide.

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