KR102366476B1 - Composition comprising Erigeron annuus (L.) Pers. for preventing or treating of Degenerative Brain Diseases - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of degenerative brain disease, comprising an extract of Amanita fenugreek as an active ingredient. More specifically, the extract of the present invention increases the expression of antioxidant enzymes SOD2 (superoxide dismutase-2), CAT (catalase), and GPx (glutathione peroxidase), and promotes apoptosis, protein Bax (Bcl-2 Antagonist) X), inhibits the expression of caspase 3 and caspase 8 and promotes the expression of Bcl-XL (B-cell lymphoma-extra large) protein, which inhibits apoptosis Because it is excellent, it can be widely used as a pharmaceutical composition for preventing or treating degenerative brain disease or a food composition for preventing or improving degenerative brain disease.

Description

A pharmaceutical composition for the prevention or treatment of degenerative brain disease, comprising an extract of aloe vera as an active ingredient, comprising: Erigeron annuus (L.) Pers. for preventing or treating of Degenerative Brain Diseases}

The present invention relates to a composition for the prevention or treatment of degenerative brain disease, which includes an extract of cyanobacteria as an active ingredient, and more specifically, to a composition for preventing or treating degenerative brain disease, comprising, as an active ingredient, an algae extract having excellent neuroprotective effect by inhibiting apoptosis of nerve cells due to oxidative stress. It relates to a pharmaceutical composition for preventing or treating degenerative brain disease.

It is known that degenerative brain diseases are caused by the death of cranial nerve cells or loss of function of cranial nerve cells due to neurodegeneration according to aging, genetic factors, and the like. The exact cause of the onset of degenerative brain disease is not known, and research is being actively conducted to identify the cause.

It is known that the ultimate cause of degenerative neurological brain diseases, such as Parkinson's disease, Alzheimer's disease, and stroke, is cell death leading to nerve cell damage and loss. Free radicals have been pointed out as the main cause of neuronal cell death in degenerative nervous system brain diseases. Active oxygen refers to oxygen with strong oxidizing power that is generated in various metabolic processes and attacks living tissues and damages cells as oxygen that enters the body during respiration is used for oxidation. These reactive oxygen species irreversibly damage DNA, cell constituent proteins, lipids, etc. in living cells (Valko et al., Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 39(2007),44) -84). In particular, when a large amount of reactive oxygen species is generated in brain cells, the resulting oxidative stress causes changes in the structure and function of mitochondria in cells, leading to neurological brain diseases such as Parkinson's disease and Alzheimer's (Knott et al., Mitochondrial fragmentation in neurodegeneration). Nat Rev Neurosci., 9 (2008), 505-18).

With the recent increase in the elderly population, the problems of cognitive decline and degenerative brain disease are emerging as serious social problems. The development of therapeutic agents is in progress, and the development of new materials with fewer side effects is necessary.

Erigeron annuus (L.) Pers. ) is a biennial plant of the genus Asteraceae, native to North America and distributed worldwide. It is the most diverse plant genus, with 173 species of plants of the genus Albania growing wild in North America alone. Alfalfa root is beta-sitosterol (-sitosterol), daidzein (daidzein), apigenin (apigenin), quercetin (quercetin), protocatechuic acid, pyromeconic acid (pyromeconic acid), breviscapine Since it contains physiologically active substances such as, fenugreek herb has been used in folklore for gastroenteritis, indigestion, infectious hepatitis, hematuria, haemophilia and malaria.

Currently, for the treatment or prevention of degenerative brain disease, the development of a therapeutic agent derived from a natural substance that does not show side effects is required. This is newly recognized.

Therefore, while the present inventors were researching to develop a treatment for preventing and treating degenerative brain disease using a natural substance-derived substance, the A. serrata extract inhibited nerve cell death due to oxidative stress and confirmed that it has excellent neuroprotective effect. The present invention was completed by revealing that it can be usefully used as an active ingredient in a composition for preventing or treating degenerative brain disease.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative brain diseases, comprising, as an active ingredient, an extract of Alfalfa root, which has excellent neuroprotective effect by inhibiting neuronal cell death due to oxidative stress.

In addition, it is an object of the present invention to provide a food composition for preventing or improving degenerative brain disease, comprising the extract according to the present invention as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain diseases, comprising, as an active ingredient, an extract of Astragalus which has excellent neuroprotective effect by inhibiting neuronal cell death due to oxidative stress.

In addition, the present invention provides a food composition for preventing or improving degenerative brain disease, comprising the extract according to the present invention as an active ingredient.

The extract according to the present invention increases the expression of antioxidant enzymes SOD2 (superoxide dismutase-2), CAT (catalase), GPx (glutathione peroxidase), and promotes cell death, protein Bax (Bcl-2 Antagonist X), It suppresses the expression of caspase 3 and caspase 8 and promotes the expression of Bcl-XL (B-cell lymphoma-extra large) protein, which inhibits apoptosis, and suppresses neuronal cell death due to oxidative stress, thereby providing excellent neuroprotective effect. . Therefore, it can be widely used as a pharmaceutical composition for preventing or treating degenerative brain disease or a food composition for preventing or improving degenerative brain disease.

1 is a diagram showing the results of HPLC analysis of phenolic compounds of standard solution PAs (A), an extract of the above-ground part of the Aertifolium planta (B), and an extract of the Aertifolia flower (C).
(The chromatography of PAs, EAA and EAF using by HPLC analysis. Peak identification; 1: Homogenistisic acid, 2: Gallic acid, 3: Protocatechnic acid, 4: Chlorogenic acid, 5: (+)-catechin, 6: caffeic acid, 7: Phloretic acid, 8: p-Coumaric acid, 9: Ferulic acid, 10: Veratric acid, 11: Salicylic acid, 12: Naringin, 13: Hesperidin, 14: Quercetin, 15: Cinnamic acid, 16: Naringenin, 17: Kaempferol, 18: Hesperitin. (A): Chromatogram of standard solutions for PAs analysis (100 μg/mL each). (B) Chromatogram of EAA (10 mg/mL). (C) Chromatogram of EAF (10 mg/mL) PAs: phenolic acids, EAA: aerial parts of Erigeron annuus, EAF: flowers of Erigeron annuus)
2 is a diagram showing the results of measurement of toxicity in the nerve cells (pc-12) of the above-ground part extract and the flower extract of A. serrata.
3 is a view showing the inhibitory effect of reactive oxygen species production in nerve cells (pc-12) (A and B) and oxidized protein expression analysis results (C, D and E) of the above-ground part extract and the flower extract of Alfalfa root.
Figure 4 is a diagram showing the expression analysis of apoptosis-related protein of the above-ground part extract and the flower extract of A. serrata.
Figure 5 is a diagram showing the process (A) of the flower fractionation of A. serrata and the inhibitory effect (B) of the active oxygen production in the nerve cells (pc-12) of the flower fraction of A. serrata.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising, as an active ingredient, an extract of A. serrata, which is excellent in protecting nerve cells by inhibiting apoptosis of nerve cells due to oxidative stress.

The term “composition” refers to a mixture of the extract of the present invention with other chemical components such as diluents or carriers.

The term “carrier” is defined as a compound that facilitates the addition of the compound into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into cells or tissues of living organisms.

The term “diluent” is defined as a compound that not only stabilizes the biologically active form of the subject compound, but is diluted in water which will dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

Unless otherwise defined, all technical terms used in the present invention have the meanings commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention.

The present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain disease, comprising an extract of Amanita fenugreek as an active ingredient.

In the present invention, the algae extract may be extracted using any one part selected from above or flowers of Erigeron annuus (L.) Pers .

The extract of the present invention can prevent or treat degenerative brain diseases because it suppresses neuronal cell death due to oxidative stress and has excellent neuroprotective effect.

The degenerative brain disease may be any one selected from Alzheimer's disease, Pick disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease.

The extract is extracted with a solvent selected from the group consisting of water, alcohol, organic solvents, and mixtures thereof, and is not limited thereto for the purpose of effectively extracting the extract.

The extract may be obtained in the form of a powder by removing the solvent after extracting the sagebrush extract with the solvent, and the method of removing the solvent is a solvent removal method commonly used in the art, for example, vacuum filtration can be used. However, the present invention is not limited thereto.

On the other hand, the pharmaceutical composition for preventing or treating degenerative brain disease according to the present invention is a powder, granules, tablets, capsules, suspensions, emulsions, syrups, and oral dosage forms such as aerosols, external preparations, suppositories, and sterile injections, respectively, according to conventional methods. It can be used by formulating in the form of a solution.

In addition, the pharmaceutical composition is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container.

The pharmaceutical composition for preventing or treating degenerative brain disease of the present invention can be used by being included in the pharmaceutical, and the pharmaceutically acceptable carrier included in the pharmaceutical is commonly used in the formulation, and includes lactose, dextrose, water Cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydro hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto.

In addition, when the pharmaceutical composition for preventing or treating degenerative brain disease of the present invention is included in a pharmaceutical product, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. may be additionally included in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceuticals may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. can Since the protein or peptide is digested upon oral administration, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. The active agent may also be administered by any device capable of transporting it to a target cell.

A suitable dosage of the drug varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, and usually skilled A physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The drug may be administered as an individual prophylactic or therapeutic agent, or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

In addition, the present invention provides a food composition for the prevention or improvement of degenerative brain disease, comprising the extract of aloe vera as an active ingredient.

The type of the food composition is not particularly limited as long as it is commonly manufactured and/or sold. For example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc. It can be used in the form of pills, powders, granules, needles, tablets, capsules or beverages, and includes all health functional foods in the ordinary sense.

The health drink composition is not particularly limited in the liquid component other than containing the extract according to the present invention, and may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage.

In general, the amount of the algae extract contained in the health functional food may be included in an amount of 0.1 to 50% by weight, preferably 1 to 40% by weight of the total food weight.

In addition, in the case of long-term intake for the purpose of health and hygiene or health control, it 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 greater than the above range.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. In addition, various modifications and changes within the scope of the technical spirit of the present invention based on these examples can be easily determined by those skilled in the art.

Example 1. Preparation of extract

Ginseng plant was cultivated and used by the Ginseng Specialty Department of the Academy of Horticultural and Herbal Science, and was extracted with 70% ethanol and used in the experiment.

Example 2. Neuronal cell line (pc-12) culture

Neuronal cell line (pc-12) was purchased from American Type Culture Collection (ATCC) and used. The cell line was cultured in DMEM (Dulbecco's modified Eagles Medium) medium supplemented with 5% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 units/ml streptomycin using an incubator at 37° C. and 5% CO ₂ . .

Experimental Example 1. Analysis of total phenol content, total flavonoid content, ABTS radical scavenging activity, and DPPH radical scavenging activity by part of Astragalus

In order to measure the antioxidant activity of the A. serrata extract, the scavenging ability of 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was measured.

DPPH scavenging activity was measured using the method of a known paper (Blois ML, Nature, 181 (1958) 1199-1224).

The degree of reduction of DPPH radicals due to the electron donating effect with the sample solution for DPPH (Sigma, MO, USA) was measured with a spectrophotometer (Biotek instrument, WT, USA). After taking 0.5 ml of the algae extract, 1 ml of 0.1mM DPPH solution was added, and the mixture was mixed using a vortex and reacted at room temperature for 30 minutes to measure the absorbance at 520 nm.

Next, to measure ABTS scavenging activity, first, 7 mM of ABTS and 2.45 mM of potassium persulfate were dissolved in distilled water, and left in the dark for 12 hours to generate ABTS cation radicals. After adding 3 ml of the ABTS solution in which the radicals were formed and 3 ml of the ABTS solution, the mixture was reacted for 6 minutes, and the absorbance value was measured at 734 nm. The absorbance measurement results are shown in Table 1 below.

As a result, it was confirmed that phenols were 9.8±0.1 and 20.8±0.1 in the above Aerial Aerial Extract (EAA) and Aeriale Flower Extract (EAF), respectively. In addition, it was confirmed that the flavonoids were 8.9±0.1 and 10.1±0.1, respectively. It was confirmed that the above-ground part extract (EAA) and the flower extract (EAF) of Alyssus serrata were excellent in ABTS radical scavenging activity and DPPH radical scavenging activity.

Experimental Example 2. Analysis of phenolic compounds for each part of Apifolius

Phenol content analysis of aloe vera was quantitatively and qualitatively analyzed using PDA by Waters 2996 of Waters HPLC 2790/5 system. INNO was used for the column, and the temperature was separated at 35 °C.

In the case of the above-ground part extract of Albaniasis, as shown in FIG. 1(B), protocatechnic acid, chlorogenic acid, (+)-catechin ((+)-catechin), caffeic acid, p -Coumaric acid (p-Coumaric acid), veratric acid (Veratric acid), salicylic acid (Salicylic acid), naringin (Naringin), hesperidin (Hesperidin), quercetin (Quercetin), naringenin (Naringenin), and camphor Kaempferol was detected.

In addition, in the case of Astragalus flower extract, as shown in FIG. 1(C), protocatechnic acid, chlorogenic acid, (+)-catechin ((+)-catechin), caffeic acid, p-Coumaric acid, Veratric acid, Salicylic acid, Naringin, Hesperidin, Quercetin, Cinnamic acid, Narin Naringenin, and Kaempferol were detected.

Experimental Example 3. Evaluation of neuronal (pc-12) toxicity for each part of A.

In order to evaluate the neuronal toxicity of A. serrata, the neuronal toxicity was evaluated as follows using the nerve cells cultured in Example 2 above.

After treatment with the algae extract at a concentration of 50 to 400 μg/ml, the absorbance was measured using a microplate reader at a wavelength of 490 nm after incubation for 24 hours and MTS treatment.

As a result, as shown in FIG. 2 , neither the Alystifolia extract and Aertifolia flower extract did not show cytotoxicity, and even in the case of the H ₂ 0 ₂ (50 μM) experimental group, both the above Aerial of A. serrata extract and Aeriales oleracea flower extract up to a concentration of 200 μg/mL. It was confirmed that there was no toxicity.

Experimental Example 4. Confirmation of active oxygen inhibitory effect and oxidized protein expression analysis on nerve cells (pc12 cell) by region

Neurotoxicity was evaluated as follows using the neurons cultured in Example 2 in order to evaluate the effect of inhibiting reactive oxygen species on nerve cells and oxidized protein expression analysis of A. serrata.

Rat cranial nerve cells (Rat PC-12) were treated with a pharmaceutical composition containing the extract prepared in Example 1 at a concentration of 50, 100, and 200 μg/ml, and after 12 hours, 50 μM of H ₂ O ₂ processed.

The amount of reactive oxygen production was measured at excitation 485 nm and emission 535 nm by fluorescently staining cells using DCFDA assay.

As a result, as shown in FIG. 3 , it was confirmed that, in the case of the flower extract of Albaniasis, the ability to inhibit active oxygen production was excellent at a concentration of 200 μg/mL.

In addition, it was confirmed that the expression of the antioxidant enzymes SOD2 (superoxide dismutase-2), CAT (catalase), and GPx (glutathione peroxidase) proteins was significantly increased in both the Apifolius flower extract and Aerophyllum Aeriiformes extract. It was confirmed that it can protect nerve cells by increasing gene expression.

Experimental Example 5. Neuronal (pc-12) apoptosis protein expression analysis for each part of A.

Bax (Bcl-2 Antagonist X) protein is involved in apoptosis due to DNA damage, and a representative protein that inhibits the action of Bax protein is Bcl-XL (B-cell lymphoma-extra large), which is By inhibiting the formation of oligomers of proteins, it interferes with the action of the Bax protein and ultimately inhibits apoptosis to promote cell survival. In addition, caspase 8 activates caspase 3 and at the same time serves to amplify the apoptosis signal through Bax, so in order to check the inhibitory ability of nerve cell apoptosis for each part of Apifolius extract, Bax, Bcl-XL, caspase 3 and caspase 8 The expression level of the protein was measured.

Rat cranial nerve cells (Rat PC-12) were treated with a pharmaceutical composition containing the extract prepared in Example 1 at a concentration of 50, 100, and 200 μg/ml, and after 12 hours, 50 μM of H ₂ O ₂ processed.

As a result, with respect to the effects of inhibiting Bax protein expression and promoting Bcl-XL protein expression, as shown in FIG. 4(A), in the case of the above-ground part of the Aertifolium extract, it showed a significant result at 200 µg/mL, and in the case of the Aertifolia extract, the cell concentration-dependently It was confirmed that the effect of inhibiting apoptosis appears.

In addition, in the case of Caspase 3 and caspase 8 proteins, as shown in Fig. 4(B), both the above extracts of Alysinus serrata and Apifolius flower extracts were shown to inhibit caspase 3 and caspase 8 protein expression in a concentration-dependent manner.

Experimental Example 6. Confirmation of inhibitory effect on neuronal (pc-12) active oxygen production of the flower fraction

In order to confirm the inhibitory effect of the active oxygen production of the flower fractions of the flower fractions, the flowers of the flowers of A. serrata were fractionated as shown in FIG. 5(A).

First, the solvent fraction for each polarity is by dissolving the flower powder of alfalfa in water, mixing it with the same amount of n-hexane and fractionation, then the water layer is again fractionated with chloroform, and the water layer is again ethyl After fractionation with acetate (Ethyl acetate), the remaining water layer was finally fractionated with n-butanol (n-butanol) to obtain five fractions together with the final water layer. The fractions were concentrated under reflux, and then freeze-dried. The extract was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and used in each experiment.

As a result of measuring the active oxygen inhibitory ability of the flower fraction obtained above, it was confirmed that, as shown in FIG.

Claims (8)

delete A method of increasing the expression of CAT (catalase) by treating nerve cells with an ethanol extract of Erigeron annuus (L.) Pers. flowers in vitro. delete delete delete delete delete delete

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