Pharmaceutical composition for preventing or treating neurodegenerative diseases comprising aconogonon alpinum extract, fraction thereof, or compound isolated therefrom

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising an Aconogonon alpinum extract, a fraction thereof, or a compound of Chemical Formula 1 isolated therefrom and, more specifically, to a pharmaceutical composition comprising an Aconogonon alpinum extract, a fraction thereof, or a compound of Chemical Formula 1 isolated therefrom to activate autophagy and inhibit the production of amyloid beta, thereby exhibiting a prophylactic or therapeutic effect against neurodegenerative diseases.

Description

Pharmaceutical composition for preventing or treating degenerative brain diseases comprising a Singa extract, a fraction thereof, or a compound isolated therefrom

[0001] The present invention relates to the use of a singa extract or the like for the prevention, improvement or treatment of degenerative brain diseases.

[0002]

[0003] Degenerative brain disease refers to a brain disease that occurs due to aging, and is known to occur due to damage to brain cell groups in specific areas of the brain, or damage to the function of neurons or synapses, which prevents neural transmission from occurring properly.

[0004] While the precise causes of neurodegenerative diseases remain unknown, one common characteristic is protein toxicity. In Alzheimer's disease, amyloid beta fibrils form, and in Parkinson's disease, Lewy bodies form. The aggregation of these proteins can cause toxicity and lead to brain disease, and is understood to be a key pathological mechanism of neurodegenerative diseases.

[0005] Representative degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

[0006] Parkinson's disease is an insidiously progressive, irreversible neurodegenerative disease characterized primarily by motor dysfunction. One cause of Parkinson's disease is dopamine depletion due to the destruction or damage of dopaminergic neurons. This can also be caused by impaired proteolytic function in mitochondria, which are damaged by various factors, preventing the proper breakdown of abnormal proteins and organelles.

[0007] Autophagy is the process by which cells degrade proteins and organelles via lysosomes. Large proteins and damaged organelles are primarily degraded through autophagy via lysosomes. Various protein complexes, formed by the expression of autophagy-related genes (ATGs), are involved in autophagy induction, the formation, expansion, and removal of autophagosomes, and the recognition of substrate endocytosis.

[0008] The process of forming autophagosomes and autolysosomes for autophagy is as follows. When LC3-Ⅰ is converted to LC3-Ⅱ by E3 ligase and binds to the forming autophagosome, substrates can bind to the autophagosome through the LC3-interacting region (LIR), such as p62. The mature autophagosome fuses with the lysosome to form the autolysosome, thereby initiating autophagy.

[0009] Currently, Parkinson's disease is treated by directly increasing dopamine levels through injections of Levodopa, a dopamine precursor. However, long-term use can result in various side effects. Therefore, there is a pressing need to develop more fundamental and safer drugs for the prevention and treatment of Parkinson's disease, leveraging the aforementioned autophagy mechanism.

[0010] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by cognitive impairment, psychosis, and brain damage. While the exact cause of AD remains unknown, it can be triggered by a variety of factors, including environmental and genetic factors, mitochondrial haplotypes, age, and gender. Key features of AD include senile plaques, a result of the accumulation of amyloid beta (Aβ), and neurofibrillary tangles, a result of hyperphosphorylation of the tau protein.

[0011] Amyloid beta is created by the cleavage of APP (Amyloid β precursor protein) in the cell membrane, and the enzymes involved in the cleavage of APP include alpha-, beta-, and gamma-cleavage enzymes.

[0012] Alpha-cleaving enzymes include ADAM 9, 10, and 17 (A Disintegrin and Metalloproteinase 9, 10, and 17), and APP is cleaved into sAPPα (Soluble APP ectomain fragment α) and α-CTF (α C-terminal fragment) by alpha-cleaving enzymes. Among these, α-CTF is further cleaved into P3 and amyloid precursor protein intracellular domain (AICD) by gamma-cleaving enzymes. This is a pathway that does not produce amyloid beta and is called the non-amyloid pathway.

[0013] The beta-cleaving enzyme is BACE 1 (β-site APP cleaving enzyme 1), and APP is cleaved by the beta-cleaving enzyme into sAPPβ (Soluble APP ectomain fragment β) and β-CTF (β C-terminal fragment). β-CTF is then cleaved by the gamma-cleaving enzyme into amyloid beta and AICD. This is the pathway that produces amyloid beta, and is called the amyloidogenic pathway.

[0014] Among the amyloid beta produced by beta- and gamma-cleavage enzymes, the primary types that accumulate early and form senile plaques are Aβ42 and Aβ40, with Aβ42 exhibiting the highest aggregation response. Therefore, these two types of amyloid beta represent potential targets for the development of Alzheimer's disease treatments. Aducanumab, a treatment targeting amyloid beta production, was recently approved by the US Food and Drug Administration (FDA). Thus, inhibiting amyloid beta production could have therapeutic and preventative effects on Alzheimer's disease.

[0015] Meanwhile, Aconogonon alpinum (All.) Schkuhr is a vascular plant belonging to the Aconogonaceae family in the Aconogonales order, and is a perennial herb that mainly grows in forest edges, riverbanks, and meadows. It has been revealed that Aconogonon alpinum (All.) Schkuhr is used as an astringent and cough remedy in oriental medicine, and Aconogonon root extract is known to have hepatoprotective and antioxidant effects, and Aconogonon flower extract is known to contain substances with antioxidant and anti-inflammatory effects. However, it has not been confirmed that Aconogonon extract, its fractions, and compounds isolated from it have a preventive or therapeutic effect on degenerative brain diseases by activating autophagy and inhibiting the production of amyloid beta.

[0016] Accordingly, the inventors of the present invention completed the present invention by searching for a natural material that can be used as a drug for the prevention and treatment of degenerative brain diseases and that has no side effects even when taken for a long period of time, and confirmed that the extract of Singa has the effect of activating autophagy and reducing amyloid beta secretion, and thus can be used for the prevention and treatment of degenerative brain diseases.

[0017]

[0018] The present invention aims to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising a singa extract, a fraction thereof, or a compound isolated therefrom.

[0019] The present invention aims to provide a food composition for preventing or improving degenerative brain diseases, comprising a singa extract, a fraction thereof, or a compound isolated therefrom.

[0020]

[0021] 1. A pharmaceutical composition for preventing or treating degenerative brain diseases, comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom or a pharmaceutically acceptable salt thereof:

[0022] [Chemical Formula 1]

[0023]

[0024] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

[0025] 2. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the singa extract in the above 1 is an extract of a first solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof.

[0026] 3. A pharmaceutical composition for preventing or treating degenerative brain disease, wherein the fraction of the extract of the singa plant in the above 1 is a fraction of a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof.

[0027] 4. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the degenerative brain disease in the above 1 is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, cerebral apoplexy, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

[0028] 5. A food composition for preventing or improving degenerative brain disease, comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom or a food-related acceptable salt thereof:

[0029] [Chemical Formula 1]

[0030]

[0031] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

[0032] 6. A food composition for preventing or improving degenerative brain disease, wherein the singa extract in the above 5 is an extract of a first solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof.

[0033] 7. A food composition for preventing or improving degenerative brain disease, wherein the fraction of the singa extract in the above 5 is a fraction of a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof.

[0034] 8. A food composition for preventing or improving a degenerative brain disease, wherein the degenerative brain disease in the above 5 is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, cerebral apoplexy, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

[0035] 9. A functional food composition for improving cognitive function or memory, comprising a singa extract or a fraction thereof.

[0036] 10. In the above 9, the singa extract is an extract of a first solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof, a functional food composition for improving cognitive function or memory.

[0037] 11. A functional food composition for improving cognitive function or memory, wherein the fraction of the singa extract in the above 9 is a fraction of a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof.

[0038] 12. In the above 9, the formulation of the food composition is any one selected from the group consisting of powder, granules, pills, tablets, capsules, candy, syrup, and beverage, a functional food composition for improving cognitive function or memory.

[0039] 13. In the above 9, the food composition is a functional food composition for improving cognitive function or memory, which is any one selected from the group consisting of drinks, meat, sausage, bread, candy, snacks, noodles, ice cream, dairy products, soups, sports drinks, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

[0040]

[0041] The pharmaceutical composition of the present invention exhibits a preventive and therapeutic effect on degenerative brain diseases by activating autophagy and reducing amyloid beta secretion.

[0042] The food composition of the present invention has the effect of preventing and improving degenerative brain diseases by activating autophagy and reducing amyloid beta secretion.

[0043]

[0044] Figure 1 is a schematic diagram showing the process of obtaining a singa extract from a singa dried material, suspending the singa extract in distilled water, and then sequentially adding hexane, ethyl acetate, and butanol to obtain singa fractions of each layer.

[0045] Figure 2 is a schematic diagram showing the process of obtaining six fractions (Bu1 to 6) from the singa butanol fraction, obtaining four fractions (Bu4.1 to 4) from the Bu4 fraction, and obtaining nine fractions (Bu4.1.1 to 9) from the Bu4.1 fraction.

[0046] Figure 3 is a schematic diagram showing the process of obtaining seven fractions (EA1 to 7) from the Singa ethyl acetate fraction, obtaining eight fractions (EA3.1 to 8) from the EA3 fraction, and isolating Compound 1 from the EA3.8 fraction.

[0047] Figure 4 shows the LC-MS results of compound 1.

[0048] Figure 5 is a diagram of compound 1. 1 H NMR shows the results.

[0049] Figure 6 is a diagram of compound 1. 13 C NMR results are shown.

[0050] Figure 7 is a diagram of compound 1. 1 H- 1 H COSY NMR results are shown.

[0051] Figure 8 shows the HMBC NMR results of compound 1.

[0052] Figure 9 shows the HSQC NMR results of compound 1.

[0053] Figure 10 shows the LC-MS results of compound 2.

[0054] Figure 11 is a diagram of compound 2. 1 H NMR shows the results.

[0055] Figure 12 is a diagram of compound 2.13 C NMR results are shown.

[0056] Figure 13 is a diagram of compound 2. 1 H- 1 H COSY NMR results are shown.

[0057] Figure 14 shows the HMBC NMR results of compound 2.

[0058] Figure 15 shows the HSQC NMR results of compound 2.

[0059] Figure 16 shows the LC-MS results of compound 3.

[0060] Figure 17 is a diagram of compound 3. 1 H NMR shows the results.

[0061] Figure 18 is a diagram of compound 3. 13 C NMR results are shown.

[0062] Figure 19 is a diagram of compound 3. 1 H- 1 H COSY NMR results are shown.

[0063] Figure 20 shows the HMBC NMR results of compound 3.

[0064] Figure 21 shows the HSQC NMR results of compound 3.

[0065] Figure 22 is a diagram of compound 4. 1 H NMR results are shown.

[0066] Figure 23 shows the UPLC-Oribtrap-MS spectrum of compound 4.

[0067] Figure 24 is a diagram of compound 5. 1 H NMR results are shown.

[0068] Figure 25 shows the UPLC-Oribtrap-MS spectrum of compound 5.

[0069] Figure 26 is a diagram of compound 6. 1 H NMR results are shown.

[0070] Figure 27 shows the UPLC-Oribtrap-MS spectrum of compound 6.

[0071] Figure 28 shows the results of Western blot analysis confirming the cell viability measurement results and LC3-Ⅱ increasing efficacy of the Singa extract in SH-SY5Y cells. A shows the cell viability measurement results of the Singa extract, and B shows the results confirming the LC3-Ⅱ increasing efficacy of the Singa extract. ** indicates P<0.01 versus the control group.

[0072] Figure 29 shows the results of Western blot analysis confirming the cell viability measurement results and LC3-Ⅱ increasing efficacy of the Singa butanol fraction in SH-SY5Y cells. A shows the cell viability measurement results of the Singa butanol fraction, and B shows the results confirming the LC3-Ⅱ increasing efficacy of the Singa butanol fraction. * indicates P<0.05 versus the control, and ** indicates P<0.01 versus the control.

[0073] Figure 30 shows the results of cell viability measurement and Western blot confirming the LC3-Ⅱ increasing efficacy of Bu1 to 6 fractions and Bu4 fraction among the singa-butanol fractions in SH-SY5Y cells. A shows the results of cell viability measurement of Bu1 to 6 fractions, and B shows the results confirming the LC3-Ⅱ increasing efficacy of Bu1 to 6 fractions. C shows the results of cell viability measurement at each concentration of Bu4 fraction, and D shows the results confirming the LC3-Ⅱ increasing efficacy at each concentration of Bu4 fraction. * indicates P<0.05 versus the control group, *** indicates P<0.001 versus the control group, and **** indicates P<0.0001 versus the control group.

[0074] Figure 31 shows the results of Western blot analysis confirming the cell viability measurement result and LC3-Ⅱ increasing effect of Bu4.1 fraction among the singa butanol fractions in SH-SY5Y cells. A shows the cell viability measurement result of Bu4.1 fraction, and B shows the result confirming the LC3-Ⅱ increasing effect of 4.1 fraction. ** indicates P<0.01 versus the control, *** indicates P<0.001 versus the control.

[0075] Figure 32 shows the results of a Western blot confirming the LC3-Ⅱ increasing effect of the Bu4.1.8 fraction among the butanol fractions of safflower in SH-SY5Y cells. * indicates P<0.05 compared to the control group.

[0076] Figure 33 shows the results of measuring the motor function of MPTP-induced Parkinson's disease mice administered with Singa extract. A shows the results of measuring motor ability using a rota-rod, and B shows the results of measuring motor ability using a pole test. *** indicates P<0.001 versus the control group, and **** indicates P<0.0001 versus the control group. # indicates P<0.05 versus the MPTP group, ## indicates P<0.01 versus the MPTP group, ### indicates P<0.001 versus the MPTP group, and #### indicates P<0.0001 versus the MPTP group.

[0077] Figure 34 shows the results of the enzyme-linked immunosorbent assay for measuring cell viability and confirming the amyloid beta 42 secretion inhibitory effect of the Singa extract in APPsw HeLa cells. A shows the results of measuring the cell viability of the Singa extract, and B shows the results of confirming the amyloid beta 42 secretion inhibitory effect of the Singa extract. ** indicates P<0.01 versus the control group, *** indicates P<0.001 versus the control group, and **** indicates P<0.0001 versus the control group.

[0078] Figure 35 shows the results of measuring the cell viability of the Singa fraction in APPsw HeLa cells. A shows the results of measuring the cell viability of the Singa hexane fraction, B shows the results of measuring the cell viability of the Singa ethyl acetate fraction, C shows the results of measuring the cell viability of the Singa butanol fraction, and D shows the results of measuring the cell viability of the Singa water fraction. * indicates P<0.05 versus the control, and ** indicates P<0.01 versus the control.

[0079] Figure 36 shows the results of enzyme-linked immunosorbent assay for confirming the amyloid beta 42 secretion inhibitory effect of the Singa fraction in APPsw HeLa cells. A shows the result confirming the amyloid beta 42 secretion inhibitory effect of the Singa hexane fraction, B shows the result confirming the amyloid beta 42 secretion inhibitory effect of the Singa ethyl acetate fraction, C shows the result confirming the amyloid beta 42 secretion inhibitory effect of the Singa butanol fraction, and D shows the result confirming the amyloid beta 42 secretion inhibitory effect of the Singa water fraction. * indicates P<0.05 versus the control group, ** indicates P<0.01 versus the control group, *** indicates P<0.001 versus the control group, and **** indicates P<0.0001 versus the control group.

[0080] Figure 37 shows the results of the enzyme-linked immunosorbent assay for measuring cell viability and confirming the amyloid beta 42 secretion inhibitory effect of the EA1-7 fractions among the ethyl acetate fractions of Singa in APPsw HeLa cells. A shows the results of measuring cell viability of the EA1-7 fractions, and B shows the results of confirming the amyloid beta 42 secretion inhibitory effect of the EA1-7 fractions. *** indicates P<0.001 versus the control group, and **** indicates P<0.0001 versus the control group.

[0081] Figure 38 shows the results of the enzyme-linked immunosorbent assay for measuring cell viability and confirming the amyloid beta 42 secretion inhibitory effect of the EA3.1-8 fraction among the ethyl acetate fractions of Singa in APPsw HeLa cells. A shows the results of measuring cell viability of the EA3.1-8 fraction, and B shows the results of confirming the amyloid beta 42 secretion inhibitory effect of the EA3.1-8 fraction. * indicates P<0.05 versus the control group, ** indicates P<0.01 versus the control group, and **** indicates P<0.0001 versus the control group.

[0082] Figure 39a shows the results of cell viability measurements of compounds 1 to 6 (12.5, 25, 50, 75, and 100 μM) in APPsw HeLa cells. ** indicates P<0.01 versus the control, and **** indicates P<0.0001 versus the control.

[0083] Figure 39b shows the results of enzyme-linked immunosorbent assay (ELISA) confirming the inhibitory efficacy of compounds 1 to 6 (12.5, 25, 50, 75, and 100 μM) on amyloid beta 42 secretion in APPsw HeLa cells. ** indicates P<0.01 versus the control group, and **** indicates P<0.0001 versus the control group.

[0084] Figure 40 shows the results of enzyme-linked immunosorbent assay confirming the inhibitory efficacy of compounds 1 (A) and 2 (B) on amyloid beta 40 secretion in APPsw HeLa cells. ** indicates P<0.01 versus the control group, *** indicates P<0.001 versus the control group, and **** indicates P<0.0001 versus the control group.

[0085] Figure 41 shows the results of enzyme-linked immunosorbent assay that confirmed the change in sAPPα secretion by compound 1 (A) and compound 2 (B) in APPsw HeLa cells. * indicates P<0.05 versus the control group, ** indicates P<0.01 versus the control group, and *** indicates P<0.001 versus the control group.

[0086] Figure 42 shows the results of enzyme-linked immunosorbent assay that confirmed the change in sAPPβ secretion by compound 1 in APPsw HeLa cells. ** indicates P<0.01 versus the control group, and **** indicates P<0.0001 versus the control group.

[0087] Figure 43 shows the results of a Western blot to confirm the change in the expression level of alpha-cleavage enzyme by compound 1 in APPsw HeLa cells. A and B represent the results confirming the change in the expression level of ADAM 9, C and D represent the results confirming the change in the expression level of ADAM 10, and E and F represent the results confirming the change in the expression level of ADAM 17. * indicates P<0.05 versus the control, ** indicates P<0.01 versus the control.

[0088] Figure 44 shows the results of confirming the change in activity of alpha-cleavage enzyme by compound 1 in APPsw HeLa cells.

[0089] Figure 45 shows the results of Western blot to confirm the change in the expression level of BACE 1, a beta-cleaving enzyme, by compound 1 (A) and compound 2 (B) in APPsw HeLa cells. * indicates P<0.05 versus the control, ** indicates P<0.01 versus the control, and **** indicates P<0.0001 versus the control.

[0090] Figure 46 shows the results of confirming the change in the activity of beta-cleavage enzyme by compound 1 in APPsw HeLa cells. * indicates P<0.05 compared to the control group, *** indicates P<0.001 compared to the control group, and **** indicates P<0.0001 compared to the control group.

[0091] Figure 47 shows the results of a Western blot to confirm the change in the expression level of PS1 (presenilin 1), which acts as an active site among the four proteins that form gamma-cleavage enzyme, by compound 1 (A) and compound 2 (B) in APPsw HeLa cells. * indicates P<0.05 compared to the control group (Control).

[0092]

[0093] The present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising a Singa extract, a fraction thereof, or a compound of chemical formula 1 isolated therefrom.

[0094] Singa can be purchased commercially, cultivated, or harvested from nature.

[0095] The term "extract" refers to a substance separated by a solvent from a liquid mixture or a solid mixture.

[0096] The extract of Singa may be in the form of a solution, concentrate or powder.

[0097] The Singa extract can be extracted using a conventional method known in the art for extracting extracts from natural products, i.e., using a conventional solvent under conditions of conventional temperature and pressure.

[0098] For example, the extraction method of the singa extract may be, but is not limited to, hot water extraction, alcohol extraction, ultrasonic extraction, cold extraction, distillation extraction, or reflux heating extraction.

[0099] For example, the extract of Singa can be extracted using a first solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof, but is not limited thereto.

[0100] The term "fraction" refers to the result obtained through fractionation to separate a specific desired component from a mixture containing various components.

[0101] The method for obtaining a fraction of the Singa extract can be performed according to a conventional method known in the art, i.e., fractionation can be performed using a conventional solvent under conditions of conventional temperature and pressure.

[0102] For example, the fraction of the Singa extract can be fractionated using a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof, but is not limited thereto.

[0103] The fraction of the Singa extract may be at least one of a hexane fraction, an ethyl acetate fraction and a butanol fraction obtained by suspending the Singa extract in water, adding hexane, shaking and leaving to separate into a hexane layer and an aqueous layer, adding ethyl acetate to the remaining aqueous layer, shaking and leaving to separate into an ethyl acetate layer and an aqueous layer, and then adding butanol to the remaining aqueous layer, shaking and leaving to separate into a butanol layer and a final aqueous layer.

[0104] In one embodiment, the fraction of the singa extract may be a hexane fraction, an ethyl acetate fraction, or a butanol fraction.

[0105] The present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom or a pharmaceutically acceptable salt thereof:

[0106] [Chemical Formula 1]

[0107]

[0108] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

[0109] The compound of formula 1 may be a compound isolated from a Singa extract or a fraction of a Singa extract. In one embodiment, the compound of formula 1 may be isolated from an ethyl acetate fraction of a Singa extract.

[0110] In one embodiment, R1 in formula 1 may be H and R2 may be CH2OH.

[0111] In one embodiment, R1 in formula 1 may be OH and R2 may be CH3.

[0112] In one embodiment, the compound of formula 1 can be any one of the following compounds:

[0113] ,

[0114] .

[0115] The extract of the present invention, its fraction, or the compound of formula 1 isolated therefrom, or a pharmaceutically acceptable salt thereof, can have blood-brain barrier (BBB) ​​penetration ability by binding to a substance having blood-brain barrier (BBB) ​​penetration activity.

[0116] For example, by combining the compound of chemical formula 1 with a peptide, ligand or functional group having blood-brain barrier penetration activity, the compound can be delivered to the brain by penetrating the blood-brain barrier.

[0117] For example, by loading the compound of chemical formula 1 into a nanocarrier, nanostructure, exosome, or carrier having blood-brain barrier penetration activity, it can be delivered to the brain by penetrating the blood-brain barrier.

[0118] The term "pharmaceutically acceptable" means that the compound or composition exhibits the property of not causing serious irritation to the subject, cell, tissue, etc. to which it is administered and does not impair the biological activity and physical properties of the compound.

[0119] The term "pharmaceutically acceptable salt" refers to a salt prepared using a specific compound according to the invention and a relatively non-toxic acid or base. The pharmaceutically acceptable salt may be, for example, an acid addition salt or a metal salt.

[0120] Acid addition salts can be formed from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous or phosphorous acids and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propylates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexanoate-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, It may include terephthalate, benzenesulfonate, tert-butyl sulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate. The metal salt may be a sodium, potassium or calcium salt. The metal salt may be prepared using a base, for example, an alkali metal or alkaline earth metal salt may be obtained by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering off the undissolved compound salt and evaporating and / or drying the filtrate.

[0121] In the present invention, degenerative brain disease is a brain disease caused by aging, and includes Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

[0122] In one embodiment, the degenerative brain disease may be Alzheimer's disease or Parkinson's disease.

[0123] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The carriers may be used without limitation as long as they are known in the art, such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, and lubricants.

[0124] Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0125] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., topical preparations, suppositories, and sterile injection solutions according to conventional methods. Furthermore, it can be used in the form of topical preparations for skin in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels. When formulating, it can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0126] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0127] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0128] The pharmaceutical composition of the present invention can be appropriately adjusted according to the form and purpose of use, patient condition, type and severity of symptoms, etc.

[0129] The present invention can provide a method for treating a degenerative brain disease, comprising administering to a subject in need of treatment a composition comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom.

[0130] [Chemical Formula 1]

[0131]

[0132] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

[0133] In the present invention, the “subject” may be a mammal including a human, but is not limited thereto.

[0134] The present invention can provide a use of a composition comprising a Singa extract, a fraction thereof, or a compound of formula 1 isolated therefrom for the treatment of degenerative brain diseases.

[0135] The present invention provides a food composition for preventing or improving degenerative brain diseases, comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom or a food-related acceptable salt thereof:

[0136] [Chemical Formula 1]

[0137]

[0138] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

[0139] The term “food-acceptable” means that the compound or composition exhibits the property of not causing serious irritation to the subject, cell, tissue, etc. to which it is administered and does not impair the biological activity and physical properties of the compound.

[0140] The term "pharmaceutically acceptable salt" refers to a salt prepared using a specific compound according to the invention and a relatively non-toxic acid or base. A pharmaceutically acceptable salt may be, for example, an acid addition salt or a metal salt.

[0141] The present invention provides a functional food composition for improving cognitive function or memory, comprising a singa extract or a fraction thereof.

[0142] In the food composition and functional food composition of the present invention, the same applies as long as it does not contradict the matters mentioned above with respect to the pharmaceutical composition of the present invention.

[0143] The extract of Singa or its fractions may be added to foods for the purpose of preventing or improving degenerative brain diseases. When using the extract of Singa or its fractions as a food additive, the extract of Singa or its fractions may be added directly or in combination with other food ingredients, and may be used appropriately according to conventional methods.

[0144] The food composition of the present invention may contain conventional food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the Food Additives Codex include, but are not limited to, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sucrose pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations. In addition, the food composition of the present invention may contain an appropriate carrier commonly used in the manufacture of food compositions.

[0145] The formulation of the food composition of the present invention is manufactured according to a conventional method, and can be dried together with a carrier and then encapsulated or formulated in the form of powder, granules, pills, tablets, capsules, candy, syrup, and beverages.

[0146] The food composition of the present invention can be manufactured into any food form. For example, the food may be any one selected from the group consisting of drinks, meat, sausage, bread, candy, snacks, noodles, ice cream, dairy products, soups, sports drinks, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

[0147]

[0148] Hereinafter, in order to specifically explain the present invention, manufacturing examples and examples will be described in detail.

[0149]

[0150] Manufacturing example

[0151] Manufacturing Example 1. Manufacturing of Singa Extract

[0152] To prepare the singa extract, 14 kg of singa purchased fresh from Cheonga Farm was washed and freeze-dried to obtain 3.9 kg of dried singa.

[0153] 500 g of dried Singa was extracted twice with 50% ethanol under reflux cooling for 3 hours. The extract was then concentrated using a rotary evaporator and freeze-dried to obtain 69 g of the final yellow powder extract.

[0154]

[0155] Manufacturing Example 2. Manufacturing of Singa Fraction

[0156] 69 g of the Singa extract prepared in the above Manufacturing Example 1 was suspended in 1 L of triple-distilled water, and an equal amount of hexane was added. After shaking and leaving, 4.33 g of a hexane fraction was obtained from the hexane layer. 1 L of ethyl acetate was added to the remaining aqueous layer, shaking and leaving, and 20.649 g of an ethyl acetate fraction was obtained from the ethyl acetate layer. Next, 1 L of saturated butanol was added to the remaining aqueous layer, shaking and leaving, and 17.452 g of a butanol fraction was obtained from the butanol layer. 26.566 g of a final water fraction was obtained from the remaining aqueous layer after separation (Fig. 1).

[0157] Six fractions (Bu1 to 6) were obtained by HP-20 column chromatography using 17.452 g of the above butanol fraction as a mobile phase in a mixed solvent of water and methanol (100:0, 80:20, 60:40, 40:60, 20:80, 0:100). Among these, Bu4 fraction (3.921 g) was obtained by C using a mixed solvent of water and methanol (30:70, 50:50, 70:30, 100:0) as a mobile phase. 18 -Four fractions (Bu4.1 to 4) were obtained using RP column chromatography. Among these, nine fractions (Bu4.1.1 to 9) were obtained by preparative-HPLC using the Bu4.1 fraction (848 mg) as a mobile phase in a mixed solvent of water and acetonitrile (90:10, 80:20, 20:80, 5:95) on a YMC-Triart C18 column (Fig. 2).

[0158] Seven fractions (EA1 to 7) were obtained from 14.06 g of the above ethyl acetate fraction using silica gel column chromatography with a mixed solvent of ethyl acetate and methanol (100:0, 95:5, 90:10, 80:20, 70:30, 0:100) as the mobile phase. Of these, eight fractions (EA3.1 to 8) were obtained from 1.32 g of the EA3 fraction using Sephadex LH column chromatography with methanol as the mobile phase (Fig. 3).

[0159]

[0160] Manufacturing Example 3. Isolation and structural determination of compounds from ethyl acetate fractions

[0161] 3-1. Method for separating compounds and determining their structure from ethyl acetate fractions

[0162] Among the eight fractions (EA3.1 to 8) obtained from the ethyl acetate fraction of the above Preparation Example 2, 657.4 mg of the EA3.8 fraction was separated into seven single substances by preparative-HPLC through a YMC 250×4.6 ID, 5 m column using a mixed solvent of acetonitrile and water (25:75) as the mobile phase.

[0163] Among the seven single substances above, six compounds were purified and isolated in sufficient quantities for use in the experiment by LC-MS. 1 H NMR and 13 The structure was elucidated based on the results of C NMR.

[0164] The mass of a single compound was measured by LC-MS using a Phenomenex C18(2) column (Luna, 100 mm × 4.6 mm, 5 μm) and solvent (flow rate, 0.7 ml / min; 10–100% aqueous CH3CN gradient solvent system with 0.1% formic acid over 20 min). In addition, UPLC-Orbitrap-MS mass spectrometry was performed using an ACQUITY UPLC BEH® C18 column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA) and solvent (flow rate 0.4 mL / min; 10–95% aqueous CH3CN gradient solvent system with 0.1% formic acid over 20 min).

[0165] The structure of a single substance was determined by NMR spectrum (Bruker AVANCE Ⅲ 600 NMR spectrometer) 1 H-NMR (600 MHz), 13 It was revealed under C-NMR (150 MHz) conditions. The NMR solvents used were (CD3)2SO (deuterated DMSO) and CD3OD (deuterated methanol).

[0166]

[0167] 3-2. Structure determination of compound 1

[0168] The molecular weight of compound 1 is m / z639.1 [M+Na] + Based on the results, it was estimated (Fig. 4), and the structure and molecular formula are 1 H NMR and 13 It was estimated based on the results of C NMR (Figs. 5 and 6). 1 δ in H NMR H7.54 (d,J= 2.0 Hz), 7.51 (dd,J= 8.5, 2.0 Hz) and 6.80 (d,J= 8.5 Hz) show that compound 1 has a 1, 3, 4-substituted benzene ring, and δ H 6.31 (d,J= 2.0 Hz) and 6.14 (d,J= 2.0 Hz) show that it has a meta-coupled benzene ring. Therefore, based on these results, it can be seen that compound 1 has a quercetin structure. In addition, δ H 7.10 (2H, s) and 13 δ of C NMR c 165.9 shows that compound 1 has a gallic acid structure. δ H 5.73 (d,J= 8.0 Hz) and δ H Compound 1 was confirmed to have β-glucose by 3.77 ~ 3.42. Finally, with reference to 2D NMR (COSY, HMBC and HSQC) (Figs. 7 to 9) and previous research results (Xue, YL, T. Miyakawa, Y. Hayashi, K. Okamoto, F. Hu, N. Mitani, K. Furihata, Y. Sawano, and M. Tanokura. 2011. 'Isolation and tyrosinase inhibitory effects of polyphenols from the leaves of persimmon, Diospyros kaki', J Agric Food Chem, 59: 6011-7.), the structure of compound 1 was confirmed as quercetin-3-O-(2''-O-galloyl-β-D-glucopyranoside).

[0169]

[0170]

[0171] 3-3. Structure determination of compound 2

[0172] The molecular weight of compound 2 is m / z639.1 [M+Na]+ Based on the results, the structure and molecular formula were estimated. 1 H NMR, 13 The structure of compound 2 was estimated based on C NMR and 2D NMR (COSY, HMBC, and HSQC) (Figs. 10 to 15). As a result, the structure of compound 2 was confirmed as myricetin 3-O-(2''-O-galloyl)-α-L-rhamnopyranoside.

[0173]

[0174]

[0175] 3-4. Structure determination of compound 3

[0176] The molecular weight of compound 3 is m / z623.1 [M+Na] + Based on the results, the structure and molecular formula were estimated. 1 H NMR, 13 The structure of compound 3 was estimated based on C NMR and 2D NMR (COSY, HMBC, and HSQC) (Figs. 16 to 21). As a result, the structure of compound 3 was confirmed as quercetin 3-O-(2''-O-galloyl)-α-L-rhamnopyranoside.

[0177]

[0178]

[0179] 3-5. Structure determination of compound 4

[0180] The molecular weight of compound 4 is m / z447.1 [M+H] + Based on the results, the structure and molecular formula were estimated. 1 It was estimated based on the results of H NMR and UPLC-Orbitrap-MS mass spectrometry (Figs. 22 and 23). As a result, the MS2 values ​​and similar to those of compound 1 were 1 H NMR was observed, but gallic acid was absent instead of δ. H 5.34 (d, J = 8.0 Hz) and δ HAccording to 4.21 ~ 3.33, compound 4 was confirmed to contain α-rhamnose instead of β-glucose. Referring to the results of previous studies, the structure of compound 4 was confirmed to be quercetin-3-O-α-L-rhamnopyranoside as follows.

[0181]

[0182]

[0183] 3-6. Structure determination of compound 5

[0184] The molecular weight of compound 5 is m / z585.1 [MH] - Based on the results, the structure and molecular formula were estimated. 1 It was estimated based on the results of H NMR and UPLC-Orbitrap-MS mass spectrometry (Figs. 24 and 25). Similar MS2 values ​​to compound 1 and 1 H NMR was performed, and it was confirmed that the compound contained α-arabinose instead of β-glucose. Referring to the results of previous studies, the structure of compound 5 was confirmed as quercetin-3-O-(2''-O-galloyl-α-L-arabinopyranoside).

[0185]

[0186]

[0187] 3-7. Structure determination of compound 6

[0188] The molecular weight of compound 6 is m / z599.1 [MH] - Based on the results, the structure and molecular formula were estimated. 1 It was estimated based on the results of H NMR and UPLC-Orbitrap-MS mass spectrometry (Figs. 26 and 27). Similar MS2 values ​​to compound 1 and 1 H NMR was shown. With reference to previous research results, the structure of compound 6 was identified as quercetin-3-O-(3''-O-galloyl-α-L-rhamnopyranoside).

[0189]

[0190]

[0191] Manufacturing Example 4. Cell Culture

[0192] 4-1. SH-SY5Y cell line culture

[0193] SH-SY5Y is a dopaminergic cell line, a human neuroblastoma cell line, ATCC: The Global Bioresource Center (CRL-2266 TM )(Manassas, Virgina 20108, USA). SH-SY5Y was cultured in a 75T-flask in a 5% CO₂ incubator at 37°C using DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin.

[0194]

[0195] 4-2. APPsw-transformed HeLa cell line culture

[0196] APPsw-transformed HeLa (APPsw HeLa) cell line is a cell line transformed with the APP swedish mutant (APPsw) gene and was provided by Professor Tae-Wan Kim (Department of Pathology, Columbia University Medical Center, New York, NY10032, USA). APPsw-transformed HeLa cells were cultured in 75T-flasks in DMEM medium containing 10% FBS, 1% penicillin / streptomycin, 0.4 mg / ml G418, and 260 μg / ml zeocin at 37°C in a 5% CO₂ incubator.

[0197]

[0198] Example

[0199] Example 1. Evaluation of cytotoxicity of Singa extract in SH-SY5Y and confirmation of LC3-Ⅱ increasing efficacy.

[0200] 1-1. Cytotoxicity test in SH-SY5Y cells

[0201] To confirm the cytotoxicity of the samples, SH-SY5Y was cultured in a 96-well plate at 5×10⁴cells / well for 24 hours under culture conditions, and each sample was cultured for an additional 24 hours at each concentration to measure cell viability.

[0202] To measure cell viability, 100 μl of EZ-Cytox (DoGenBio Co., Ltd, KR) diluted in medium to 1 / 10 the medium volume was dispensed per well and incubated for 30 minutes. The concentration of the reactant of EZ-Cytox and living cells was measured by absorbance at a wavelength of 450 nm using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[0203]

[0204] 1-2. Preparation of Western blot samples

[0205] SH-SY5Y was cultured in 12-well plates at 6 × 10 5After culturing cells / well for 24 hours, the samples were cultured for an additional 24 hours at different concentrations, the medium was removed, and Dulbecco's Phosphate Buffered Saline (DPBS, 1 ml) was added to each well to wash. Again, 1 ml of DPBS was added per well, and the cells collected with a scraper were placed in a 1.5 ml tube and centrifuged at 13,000 rpm for 10 minutes to obtain pellets. A solution (80 μl) containing a 200:1 mixture of PRO-PREP and phosphatase inhibitor cocktail set III was dispensed to the pellets to make a cell suspension. The cell suspension was left on ice for 30 minutes, and the supernatant centrifuged at 13,000 rpm for 15 minutes was used for protein quantification and western blot sample preparation. WIN-1001X was used as a positive control.

[0206] For protein quantification, the total protein concentration of the centrifuged supernatant was measured according to the enclosed BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA). Based on the quantified values, Western blot samples were prepared using a mixture of LDS Sample Buffer 4× / 2-mercaptoethanol (9:1) and the supernatant. Western blot samples were incubated at 100°C for 5 minutes before use in the experiment.

[0207]

[0208] 1-3. Western blot

[0209] The expression levels of LC3-Ⅰ and LC3-Ⅱ were measured by western blot.

[0210] The western blot samples of Example 1-2 were loaded onto a 10-12% Tris Glycine Gel so that the total protein amount was 20 μg, and electrophoresed at 100 V for 1 hour and 50 minutes to separate the proteins into bands according to size. The separated proteins were transferred from the gel to a PVDF (Millipore Corp., Birlington, MA) membrane using a semi-dry transfer device (Bio-rad, Hercules, CA) at 25 V for 50 minutes. The membrane to which the proteins were transferred was blocked with 5% skim milk in TBST (Tris-Buffered Saline, 1% Tween 20) for 1 hour at room temperature. After blocking, the membrane was reacted with the primary antibody listed in Table 1 below at 4°C overnight. The membrane reacted with the primary antibody was washed three times with TBST for 10 minutes each time. After washing, the membrane was reacted with a secondary antibody (Rabit anti-horseradish peroxidase (HRP)-linked IgG antibody) for 1 hour at room temperature. After reacting with the secondary antibody, the membrane was washed three times with TBST for 10 minutes each time, and then reacted with ECL (Invitrogen, Carlsbad, NY) solution for 30 seconds, and the protein expression level was measured using FUSION SOLO (Vilber, Paris, FR).

[0211] Primary antibodyRabbit anti-LC3B antibodyRabbit anti-GAPDH antibodySecondary antibodyRabbit anti-horseradish peroxidase (HRP)-linked IgG antibody

[0212]

[0213] 1-4. Cytotoxicity and LC3-Ⅱ increasing efficacy of Singa extract in SH-SY5Y cells

[0214] Cell viability was measured in SH-SY5Y with Singa extract (50, 75, 100 μg / ml).

[0215] As a result of the measurement, no toxicity was observed at concentrations below 100 μg / ml of the Singa extract (A in Figure 28), and the subsequent LC3-Ⅱ increasing efficacy experiment was conducted at 50, 75, and 100 μg / ml.

[0216] As a result, LC3-Ⅱ increased by 96.76±63.43%, 148.8±63.43%, and 530±63.43%, respectively, compared to the control group in the extracts of Singa (50, 75, and 100 μg / ml) (Fig. 28B). Therefore, the expression level of LC3-Ⅱ, which contributes to the formation of autophagosomes, increased, confirming the efficacy of activating autophagy.

[0217]

[0218] 1-5. Cytotoxicity and LC3-Ⅱ-increasing efficacy of the succinate-butanol solvent fraction in SH-SY5Y cells

[0219] The cell viability and LC3-Ⅱ increasing efficacy of the butanol solvent fractions (25, 50, and 75 μg / ml) of SH-SY5Y were measured.

[0220] As a result of the measurement, toxicity was observed at all three concentrations, but since the expression level of LC3-Ⅱ increased at 25 and 50 μg / ml (Fig. 29), the butanol solvent fractions were separated according to activity to separate the active and toxic fractions, and further experiments were conducted.

[0221]

[0222] 1-6. Cytotoxicity and LC3-Ⅱ-increasing efficacy of Bu1 to 6 fractions in SH-SY5Y cells

[0223] Cell viability was measured in SH-SY5Y with fractions Bu1 to 6 (40 μg / ml).

[0224] As the measurement results showed that no toxicity was observed in all samples (A in Figure 30), the LC3-Ⅱ increase efficacy experiment was then conducted at the same concentration.

[0225] As a result, LC3-Ⅱ increased by 1473±71.12% in the Bu4 fraction compared to the control group (Fig. 30B). In addition, when the Bu4 fraction, which showed the effect of increasing LC3-Ⅱ, was tested at concentrations of 30, 40, and 50 μg / ml, no toxicity was observed at any concentration (Fig. 30C), and the LC3-Ⅱ expression levels at 30, 40, and 50 μg / ml increased by 430.8%, 843.6%, and 1460% compared to the control group, respectively (Fig. 30D). Therefore, since the expression level of LC3-Ⅱ, which contributes to the formation of autophagosomes, increased, the effect of activating autophagy could be confirmed.

[0226]

[0227] 1-7. Cytotoxicity and LC3-Ⅱ-increasing efficacy of Bu4.1 fraction in SH-SY5Y cells

[0228] Cell viability of Bu4.1 fraction (30, 40, 50 μg / ml), a fraction of Bu4 in SH-SY5Y, was measured.

[0229] As a result of the measurement, no toxicity was observed at any concentration tested (A in Figure 31), so the LC3-Ⅱ increase efficacy experiment was subsequently conducted at the same concentration.

[0230] As a result, LC3-Ⅱ increased by 515.9±38.71%, 758.8±38.71%, and 1190±38.71%, respectively, compared to the control group in the Bu4.1 fraction (30, 40, and 50 μg / ml) (Fig. 31B). Therefore, the expression level of LC3-Ⅱ, which contributes to the formation of autophagosomes, increased, confirming the efficacy of activating autophagy.

[0231]

[0232] 1-8. LC3-Ⅱ-increasing efficacy of Bu4.1.8 fraction in SH-SY5Y cells

[0233] The LC3-Ⅱ increasing efficacy of the Bu4.1.8 fraction (30, 40, 50 μg / ml), a fraction of Bu4.1 in SH-SY5Y, was measured.

[0234] As a result of the measurement, LC3-Ⅱ increased by 95.83±68.03%, 106.6±68.03%, and 240.5±68.03%, respectively, compared to the control group in the Bu4.1.8 fraction (30, 40, and 50 μg / ml) (Fig. 32). Therefore, the expression level of LC3-Ⅱ, which contributes to the formation of autophagosomes, increased, confirming the efficacy of activating autophagy.

[0235]

[0236] Example 2. Effect of Singa extract on improving motor ability in MPTP-induced Parkinson's disease mice.

[0237] 2-1. Animal breeding and sample administration

[0238] Eight-week-old male C57BL / 6J mice (20–23 g) were purchased from Orient Bio (Seoul, KOR) and used in the experiment. The mice were housed in individual cages, six per group, in an automatically temperature- and humidity-controlled room (22±3°C, 50% humidity, 12-h light / 12-h dark cycle) with free feeding and feeding conditions. Animal experiments were conducted in accordance with the guidelines of the Sejong University Laboratory Animal Ethics Association (SJ-2022081001).

[0239] The mice were divided into six groups: control group (1), PBS administration group (2), MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) administration group (3), L-dopa and MPTP administration group (4), 50 mg / kg of 50% ethanol extract of Singa and MPTP administration group (5), 100 mg / kg and MPTP administration group (5), and 200 mg / kg and MPTP administration group (6, 200 mg / kg). Administration was started after the animals were acclimatized for one week under the breeding conditions. The samples were orally administered at each dose daily for five days, and on the third day of administration, MPTP dissolved in PBS was administered intraperitoneally four times at 2-hour intervals at a dose of 20 mg / kg.

[0240]

[0241] 2-2. Measuring exercise capacity using a rota-rod

[0242] To measure the motor function of MPTP-induced Parkinson's disease mice administered 50% ethanol extract of Singa (50, 100, 200 mg / kg), a rota-rod was used.

[0243] After administering the samples for 5 days, a behavioral experiment was conducted 2 days later using a rotarod. The mice underwent pre-training three times a day for 3 minutes each before the main experiment. The rotarod placed the mice on a rotating cylinder that gradually increased in speed from 2 to 24 rpm, and the time it took for them to fall was measured.

[0244] As a result of the measurement, compared to the control group, the negative control group fell 27.22±6.087 seconds faster, and in the groups administered 50% ethanol extract of Singa (50, 100, 200 mg / kg), the falling time was 16.50±6.087 seconds, 19.79±6.087 seconds, and 27.41±6.087 seconds later, respectively, compared to the negative control group (Fig. 33A). From this, it was confirmed that motor function was improved in a dose-dependent manner in mice administered Singa extract.

[0245]

[0246] 2-3. Measuring motor ability using a pole test

[0247] To measure the motor function of MPTP-induced Parkinson's disease mice administered 50% ethanol extract of Singa (50, 100, 200 mg / kg), a pole test was used.

[0248] After administering the sample for 5 days, a behavioral experiment using the pole test was conducted 2 days later. The mice were pre-trained three times a day for 3 minutes each before the main experiment. The pole test was conducted by placing a mouse on the top of an 8 mm diameter, 55 cm high iron bar that was erected vertically and measuring the time it took for the mouse to descend to the floor.

[0249] As a result of the measurement, the negative control group arrived 6.99±1.248 seconds later than the control group, and the groups administered 50% ethanol extract of Singa (50, 100, 200 mg / kg) arrived 3.890±1.248 seconds, 5.179±1.248 seconds, and 5.821±1.228 seconds faster than the negative control group, respectively (B in Figure 33). From this, it was confirmed that motor function was improved in a dose-dependent manner in mice administered Singa extract.

[0250]

[0251] Example 3. Evaluation of cytotoxicity of the extract of Singa in APPsw HeLa cells and confirmation of its inhibitory effect on amyloid beta 42 secretion.

[0252] 3-1. Cytotoxicity test in APPsw HeLa cells

[0253] To confirm the cytotoxicity of the samples used in the experiment, APPsw HeLa cells were cultured in a 96-well plate at 3 × 10⁴ cells / well for 24 hours under cell culture conditions, and each sample was cultured for an additional 8 hours at different concentrations to measure the cell viability. To measure the cell viability, 100 μl of EZ-Cytox (DoGenBio Co., Ltd, KR) diluted in medium to 1 / 10 of the medium volume was dispensed per well and cultured for 30 minutes. The concentration of the reactant of EZ-Cytox and living cells was measured by measuring the absorbance at a wavelength of 450 nm using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[0254]

[0255] 3-2. Enzyme-linked immunosorbent assay

[0256] To confirm the inhibition of Aβ42 secretion by the sample used in the experiment, APPsw HeLa cells were cultured in a 6-well plate at a density of 1.2Х10 6 After 24 h of culture at 10 cells / well, each sample was cultured for an additional 8 h at different concentrations, and 1 ml of the medium was centrifuged at 13,000 rpm for 10 min. The supernatant was used in the experiment. Justicidin A (1 μM) and Lanabecestat (0.5 μM) were used as positive controls. The amount of Aβ42 secretion was measured according to the experimental method enclosed with the Human Aβ42 ELISA Kit (invitrogen, Carlsbad, CA).

[0257]

[0258] 3-3. Cytotoxicity and inhibitory effect of Singa extract on amyloid beta-42 secretion in APPsw HeLa cells

[0259] The cell viability of the extract of Singa (25, 50, 75, and 100 μg / ml) was measured in APPsw HeLa cells. As a result of the measurement, no toxicity was observed at concentrations of Singa extract below 75 μg / ml (Fig. 34A), so the subsequent Aβ42 secretion inhibition efficacy experiment was performed at concentrations of 25, 50, and 75 μg / ml.

[0260] As a result, the amount of Aβ42 secretion was reduced by 9.312±2.441%, 14.46±2.228%, and 39.31±2.228%, respectively, compared to the control group in the extracts of Singa (25, 50, and 75 μg / ml) (B in Figure 34).

[0261]

[0262] 3-4. Cytotoxicity and inhibitory effect of the Singa fraction on amyloid beta-42 secretion in APPsw HeLa cells.

[0263] The cell viability of the solvent fractions of Singa (25, 50, 75, and 100 μg / ml) was measured in APPsw HeLa cells. As a result, no toxicity was observed at concentrations below 100 μg / ml of the hexane fraction, 50 μg / ml of the ethyl acetate fraction, 25 μg / ml of the butanol fraction, and 50 μg / ml of the water fraction (Fig. 35). Therefore, the Aβ42 secretion inhibitory effect experiment was performed with the hexane fraction (50, 75, and 100 μg / ml), the ethyl acetate and water fractions (12.5, 25, and 50 μg / ml), and the butanol fractions (6.25, 12.5, and 25 μg / ml).

[0264] As a result, the amount of Aβ42 secretion was reduced by 14.75±6.127% and 32.12±6.127% (Fig. 36A) compared to the control group in the hexane fraction (75 and 100 μg / ml), 8.961±3.226%, 19.76±4.081% and 47.18±4.081% (Fig. 36B) in the ethyl acetate fraction (12.5, 25 and 50 μg / ml), and 8.310±4.528%, 17.14±4.528% and 45.55±4.528% (Fig. 36C) in the butanol fraction (6.25, 12.5 and 25 μg / ml), respectively, while the amount of water fraction did not decrease at any of the concentrations tested (Fig. 36 D).

[0265]

[0266] 3-5. Cytotoxicity and inhibitory effect on amyloid beta-42 secretion of EA1-7 fractions in APPsw HeLa cells

[0267] The cell viability of EA1 to 7 fractions (10 and 20 μg / ml) was measured in APPsw HeLa cells. As a result of the measurement, EA1 to 7 fractions did not exhibit cytotoxicity at any concentration tested (Fig. 37A), so the subsequent Aβ42 secretion inhibition efficacy experiment was performed at 10 and 20 μg / ml.

[0268] As a result, the amount of Aβ42 secretion was reduced by 31.58±8.039% in the EA3 fraction (20 μg / ml) compared to the control group (B in Figure 37).

[0269]

[0270] 3-6. Cytotoxicity and inhibitory effect on amyloid beta-42 secretion of EA3.1-8 fractions in APPsw HeLa cells.

[0271] The cell viability of EA3.1 to EA3.8 fractions (10, 20, and 40 μg / ml) was measured in APPsw HeLa cells. As a result, no cytotoxicity was observed in the EA3.3 fraction starting from 20 μg / ml, but no cytotoxicity was observed in the remaining samples at any concentration used in the experiment (Fig. 38A). Therefore, the subsequent Aβ42 secretion inhibition efficacy experiment was performed at 10, 20, and 40 μg / ml.

[0272] As a result, the amount of Aβ42 secretion was reduced by 6.796±8.265%, 19.17±8.265%, and 66.00±8.265% compared to the control group in the EA3.8 fraction (10, 20, and 40 μg / ml), respectively (B in Figure 38).

[0273]

[0274] 3-7. Cytotoxicity and inhibitory effect on amyloid beta 42 secretion of a single substance in APPsw HeLa cells

[0275] The cell viability of compounds 1 to 6 (12.5, 25, 50, 75, and 100 μM) was measured in APPsw HeLa cells. Results showed that no cytotoxicity was observed for any of the six single compounds at any concentration used in the experiment (Fig. 39a). Subsequently, Aβ42 secretion inhibition was tested at concentrations of 12.5, 25, and 50 μM.

[0276] As a result of measuring Aβ42 secretion, when treated with compound 1, it decreased by 21.07±3.023% and 57.41±3.023% compared to the control group at concentrations of 25 and 50 μM, respectively (Fig. 21B). When treated with compound 2, it decreased by 34.69±4.585% compared to the control group at a concentration of 50 μM (Fig. 39B).

[0277]

[0278] Example 4. Inhibitory effect of compounds 1 and 2 on amyloid beta secretion in APPsw HeLa cells.

[0279] 4-1. Enzyme-linked immunosorbent assay

[0280] To confirm the inhibition of Aβ40 and sAPPβ secretion and increase of sAPPα secretion by the sample used in the experiment, APPsw HeLa cells were cultured in 6-well plates at a density of 1.2 X 10 6 After 24 hours of culture at cells / well, each sample was further cultured for 8 hours at different concentrations, and 1 ml of the medium was centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant used in the experiment. Justicidin A (1 μM) and Lanabecestat (0.5 μM) were used as positive controls.

[0281] Aβ40 secretion was measured using the Human Aβ40 ELISA Kit (invitrogen, Carlsbad, CA) according to the enclosed protocol. sAPPα and sAPPβ secretion was measured using the Human sAPPα and sAPPβsw high sensitive ELISA Kit (IBL, Fujioka, JP) according to the enclosed protocol.

[0282]

[0283] 4-2. Inhibition of amyloid beta 40 secretion by compounds 1 and 2

[0284] As a result of measuring Aβ40 secretion, when treated with compound 1, it decreased by 21.66±3.090% and 55.88±3.090% compared to the control group at concentrations of 25 and 50 μM, respectively (Figure 40). In addition, when treated with compound 2, it decreased by 19.97±3.825% and 37.98±3.825% compared to the control group at concentrations of 25 and 50 μM, respectively (Figure 40).

[0285]

[0286] 4-3. Changes in the secretion of sAPPα and sAPPβ by compound 1

[0287] sAPPα secretion increased by 3.235±7.091% and 28.95±7.091%, respectively, compared to the control group when treated with compound 1 (25 and 50 μM) (Fig. 41), and sAPPβ secretion decreased by 1.014±1.967%, 2.339±1.967%, and 13.98±1.967%, respectively, compared to the control group when treated with compound 1 (12.5, 25, and 50 μM) (Fig. 42).

[0288] Since the amount of sAPPα secreted through the non-amyloid pathway increased and the amount of sAPPβ secreted through the amyloid pathway decreased, the effect of reducing the amount of amyloid beta secretion could be confirmed.

[0289]

[0290] Example 5. Confirmation of expression and activity of enzymes related to amyloid beta production by compound 1 in APPsw HeLa cells.

[0291] 5-1. Preparation of Western blot samples

[0292] APPsw HeLa cells were cultured in 6-well plates at a density of 1.2Х10 6After culturing cells / well for 24 hours, compound 1 was additionally cultured for 8 hours at various concentrations, the medium was removed, and Dulbecco's Phosphate Buffered Saline (DPBS, 1 ml) was added to each well to wash. Again, 1 ml of DPBS was added per well, and the cells collected with a scraper were placed in a 1.5 ml tube and centrifuged at 13,000 rpm for 10 minutes to obtain pellets. A solution (80 μl) containing a 200:1 mixture of PRO-PREP and phosphatase inhibitor cocktail set III was dispensed to the pellets to resuspend the cells. The cell suspension was left on ice for 30 minutes, and the supernatant centrifuged at 13,000 rpm for 15 minutes was used for protein quantification and Western blot sample preparation. Justicidin A (1 μM) and Lanabecestat (0.5 μM) were used as positive controls.

[0293] For protein quantification, the total protein concentration of the centrifuged supernatant was measured according to the enclosed BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA). Based on the quantified values, Western blot samples were prepared using a mixture of LDS Sample Buffer 4× / 2-mercaptoethanol (9:1) and the supernatant. Western blot samples were incubated at 100°C for 5 minutes before use in the experiment.

[0294]

[0295] 5-2. Western blot

[0296] The expression levels of alpha-, beta-, and gamma-cleavage enzyme-related proteins were measured by western blot. The western blot samples of Example 5-1 and the primary and secondary antibodies shown in Table 2 below were used, and the specific experimental method was the same as in Example 1-3 to perform the western blot.

[0297] Primary antibodyRabbit anti-ADAM9 antibodyRabbit anti-ADAM10 antibodyRabbit anti-ADAM17 antibodyRabbit anti-BACE(D10E5) antibodyRabbit anti-Presenilin 1 antibodyRabbit anti-GAPDH antibodySecondary antibodyRabbit anti-horseradish peroxidase (HRP)-linked IgG antibody

[0298]

[0299] 5-3. Measurement of enzyme activity

[0300] Alpha- and beta-secretase activities were measured using the Innozyme TACE activity kit (Sigma-Aldrich, St. Louis, MO) and the β-secretase activity fluorometric assay kit (Sigma-Aldrich, St. Louis, MO). APPsw HeLa cells cultured in a 75 T-flask were washed with DPBS, 10 ml of DBPS was added, and the cells were collected with a scraper. The pellets were centrifuged at 1,500 rpm for 5 minutes, and protein extraction buffer was added to the pellets. The pellets were suspended and left on ice for 30 minutes. The supernatants were centrifuged at 13,000 rpm for 10 minutes, and the activity of compound 1 was measured according to the instructions enclosed with each kit.

[0301]

[0302] 5-4. Expression and activity of alpha-cleaving enzyme

[0303] As a result of measuring the expression levels of alpha-cleaving enzymes ADAM 9, ADAM 10, and ADAM 17, the expression levels of ADAM 9 and ADAM 17 did not significantly increase compared to the control group (Fig. 43A, B, E, and F), and the expression level of ADAM 10 showed a tendency to decrease compared to the control group (Fig. 43C and D). In addition, the activity of alpha-cleaving enzymes also did not significantly increase compared to the control group (Fig. 44).

[0304]

[0305] 5-5. Expression and activity of beta-cleaving enzyme

[0306] As a result of measuring the expression level of BACE 1, a beta-cleaving enzyme, the expression level of BACE 1 decreased by 45.26±4.725% compared to the control group when treated with compound 1 (50 μM) and by 34.62±9.576% when treated with compound 2 (50 μM) (Fig. 45).

[0307] Additionally, the activity of beta-cleavage enzyme was reduced by 12.66±2.626%, 28.01±2.626%, and 47.26±2.413%, respectively, compared to the control group when treated with compound 1 (12.5, 25, and 50 μM) (Fig. 46). Since the expression and activity of beta-cleavage enzyme involved in the amyloid pathway were reduced, the effect of reducing amyloid beta secretion was confirmed as a result.

[0308]

[0309] 5-6. Expression of gamma-cleavage enzyme

[0310] Among the four proteins that form the gamma-cleavage enzyme, the expression level of PS1 (presenilin 1), which acts as the active site, was reduced by 29.93±7.624% compared to the control group when treated with compound 1 (50 μM), and by 17.68±5.765% and 34.24±7.940% compared to the control group when treated with compound 2 (25 and 50 μM), respectively (Fig. 47). Compounds 1 and 2 exhibit the effect of reducing the amount of amyloid beta secretion by reducing the expression level of PS1, the active site of the gamma-cleavage enzyme that cleaves β-CTF generated by the beta-cleavage enzyme into Aβ and AICD.

Claims

1. A pharmaceutical composition for preventing or treating degenerative brain diseases, comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

2. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the singa extract in claim 1 is an extract of a first solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof.

3. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the fraction of the extract of the singa plant is a fraction of a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof.

4. A pharmaceutical composition for preventing or treating a degenerative brain disease according to claim 1, wherein the degenerative brain disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, cerebral apoplexy, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

5. A food composition for preventing or improving degenerative brain disease, comprising a Singa extract, a fraction thereof, or a compound of the following chemical formula 1 isolated therefrom or a food-related acceptable salt thereof: [Chemical Formula 1] (In the formula, R1 is H or OH, and R2 is CH3 or CH2OH).

6. A food composition for preventing or improving degenerative brain disease, wherein the singa extract in claim 5 is an extract of a first solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof.

7. A food composition for preventing or improving degenerative brain disease, wherein the fraction of the singa extract in claim 5 is a fraction of a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof.

8. A food composition for preventing or improving a degenerative brain disease according to claim 5, wherein the degenerative brain disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, cerebral apoplexy, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

9. A functional food composition for improving cognitive function or memory, comprising a singa extract or a fraction thereof.

10. A functional food composition for improving cognitive function or memory, wherein the singa extract is an extract of a first solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof, in claim 9.

11. A functional food composition for improving cognitive function or memory, wherein the fraction of the singa extract according to claim 9 is a fraction of a second solvent selected from the group consisting of water, hexane, ethyl acetate, butanol, methanol, acetonitrile, and mixed solvents thereof.

12. A functional food composition for improving cognitive function or memory, wherein the formulation of the food composition according to claim 9 is any one selected from the group consisting of powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

13. In claim 9, the food composition is a functional food composition for improving cognitive function or memory, which is any one selected from the group consisting of drinks, meat, sausage, bread, candy, snacks, noodles, ice cream, dairy products, soups, sports drinks, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

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