COMPOSITIONS CONTAINING PTEROSIN COMPOUND AND DERIVATIVES THEREOF ACTIVE INGREDIENTS FOR PREVENTION OR TREATMENT OF DEGENERATIVE BRAIN DISEASES

Abstract

The present invention relates to compositions containing a pterosin compound and derivatives thereof as active ingredients for the prevention or treatment of degenerative brain diseases and, more specifically, to a pharmaceutical composition for the prevention or treatment of degenerative brain diseases and a food composition for the prevention or alleviation of degenerative brain diseases, each of which contains a pterosin compound defined by chemical formula 1 or a derivative thereof as an active ingredient. A method of the present invention can be favorably used to provide a therapeutic agent for preventing or treating degenerative brain diseases, a food for alleviating degenerative brain diseases, or a functional food for the promotion of cognitive functions, by using a pterosin compound extracted from Pteridium aquilinum and derivatives thereof.

Claims

1. A pharmaceutical composition comprising a pterosin compound defined by the following Chemical Formula 1 or a derivative thereof as an effective ingredient for preventing or treating a degenerative brain disease: ##STR00033## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O -alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O -heterocycloalkyl, glucose, and O-glucose.

2. The pharmaceutical composition of claim 1, wherein the pterosin compound or the derivative is selected from the group consisting of pterosin A, pterosin B, pterosin C, pterosin D, pterosin J, pterosin M, pterosin P, pterosin S, pterosin Z, pteroside A, pteroside A.sub.2, pteroside B, pteroside C, pteroside D, pteroside N, pteroside P pteroside Z, and sulfated pterosin C.

3. The pharmaceutical composition of claim 1, wherein the pterosin compound or the derivative is purified from a bracken fern.

4. The pharmaceutical composition of claim 1, wherein the degenerative brain disease is one selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourettt's syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

5. A method for preparation of the pterosin compound or derivative of claim 1, the method comprising the steps of: (a) soaking a bracken fern in water or an organic solvent of 1 to 4 carbon atoms to obtain an extract; (b) fractionating the extract obtained in step (a) with ethyl acetate or butanol; and (c) isolating and purifying the ethyl acetate fraction or butanol fraction obtained in step (b) by concentration-gradient chromatography.

6. The method of claim 5, wherein the organic solvent in step (a) is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, dichloromethane, or petroleum ether.

7. A food composition comprising a pterosin compound defined by Chemical Formula 1 or a derivative thereof as an effective ingredient for preventing or alleviating a degenerative brain disease: ##STR00034## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O-alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O-heterocycloalkyl, glucose, and O-glucose.

8. The food composition of claim 7, wherein the pterosin compound or the derivative is selected from the group consisting of pterosin A, pterosin B, pterosin C, pterosin D, pterosin J, pterosin M, pterosin P, pterosin S, pterosin Z, pteroside A, pteroside A.sub.2, pteroside B, pteroside C, pteroside D, pteroside N, pteroside P pteroside Z, and sulfated pterosin C.

9. The food composition of claim 7, wherein the degenerative brain disease is one selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourettt's syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

10. A food composition comprising a pterosin compound defined by Chemical Formula 1 or a derivative thereof as an effective ingredient for enhancing a cognitive function: ##STR00035## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O-alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O-heterocycloalkyl, glucose, and O-glucose.

11. Use of a compound defined by Chemical Formula 1 or a salt thereof in preparing an agent for treating a degenerative brain disease; ##STR00036## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O-alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O-heterocycloalkyl, glucose, and O-glucose.

12. A method for treating a degenerative brain disease in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a compound defined by Chemical Formula 1 or a salt thereof as an effective ingredient: ##STR00037## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O-alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O-heterocycloalkyl, glucose, and O-glucose.

13. Use of a compound defined by Chemical Formula 1 or a salt thereof in preparing an agent for enhancing a cognitive function: ##STR00038## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O-alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O-heterocycloalkyl, glucose, and O-glucose.

14. A method for enhancing a cognitive function in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a compound defined by Chemical Formula 1 or a salt thereof as an effective ingredient: ##STR00039## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.8 are each independently H, OH, an alkyl or alcohol of 1 to 4 carbon atoms, or alkyl-O-glucose; and R.sub.7 is one selected from the group consisting of halogen, H, OH, O-alkyl, O-alkenyl, O-alkynyl, O-alcohol, O-carboxyl, O-ether, O-sulfonic acid (—SO.sub.3H), O-cycloalkyl, O-heterocycloalkyl, glucose, and O-glucose.

15. A novel compound,d defined by the followed Chemical Formula 2; ##STR00040##

16. The compound of claim 15, wherein the compound is purified from a bracken fern.

17. A method for preparation of the novel compound of claim 1, the method comprising the steps of: (a) soaking a bracken fern in water or an organic solvent of 1 to 4 carbon atoms to obtain an extract; (b) fractionating the extract obtained in step (a) with ethyl acetate; and (c) isolating and purifying the ethyl acetate fraction obtained in step (b) by concentration-gradient chromatography.

18. The method of claim 17, wherein the organic solvent in step (a) is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, dichloromethane, or petroleum ether.

19. A pharmaceutical composition, comprising a novel compound defined by the following Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an effective ingredient for preventing or treating a degenerative brain disease: ##STR00041##

20. The pharmaceutical composition of claim 19, wherein the degenerative brain disease is one selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourettt's syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

21. A food composition comprising a novel compound defined by Chemical Formula 2 as an effective ingredient for preventing or alleviating a degenerative brain disease: ##STR00042##

22. The food composition of claim 21, wherein the degenerative brain disease is one selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourettt's syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

23. A food composition comprising a novel compound defined by Chemical Formula 2 as an effective ingredient for enhancing a cognitive function: ##STR00043##

24. Use of a compound defined by Chemical Formula or a pharmaceutically acceptable salt thereof in preparing an agent for treating a degenerative brain disease. ##STR00044##

25. A method for treating a degenerative brain disease in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a compound defined by Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an effective ingredient: ##STR00045##

26. Use of a compound defined by Chemical Formula 1 or a salt thereof in preparing an agent for enhancing a cognitive function: ##STR00046##

27. A method for enhancing a cognitive function in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a compound defined by Chemical Formula 2 or a salt thereof as an effective ingredient: ##STR00047##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0147] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0148] FIG. 1 is a schematic view illustrating processes of isolating and purifying the compounds of the present disclosure and derivatives thereof.

[0149] FIG. 2 shows HPLC conditions for isolating single compounds from EA-2 and butanol fractions.

[0150] FIG. 3 shows NMR analysis data for the novel compound.

[0151] FIG. 4 shows a .sup.1H-NMR spectrum for pteroside N (Compound 18, comp.15).

[0152] FIG. 5 shows a .sup.13C-NMR spectrum for pteroside N (Compound 18, comp.15).

[0153] FIG. 6 shows a DEPT 1 spectrum for pteroside N (Compound 18, comp.15).

[0154] FIG. 7 shows HMBC analysis results for pteroside N (Compound 18, comp.15).

[0155] FIG. 8 shows BACE1 activity inhibition mechanisms and Ki values of (2S, 3R)-pterosin C cis-isomer (A, D), (2R, 3R)-pterosin C trans-isomer (B, E), and pterosin B (C, F) as evaluated by Dixon plots (X-axis: concentration of the novel compound, Y-axis: 1/initial reaction rate of enzyme) (a) and Lineweaver plots (X-axis: 1/substrate concentration, Y-axis: /initial reaction rate of enzyme), wherein graphs A to C represent Dixon plots and graphs D to F represent Lineweaver plots.

[0156] FIG. 9 shows AChE activity inhibition mechanisms and Ki values of (2S, 3R)-pterosin C cis-isomer (A, D), (2R, 3R)-pterosin C trans-isomer (B, E), and pterosin B (C, F) as evaluated by Dixon plots (X-axis: concentration of the novel compound, Y-axis: 1/initial reaction rate of enzyme) (A, B, C) and Lineweaver plots (X-axis: 1/substrate concentration, Y-axis: 1/initial reaction rate of enzyme) (D, E, F).

[0157] FIG. 10 shows BChE activity inhibition mechanisms and Ki values of (2S, 3R)-pterosin C cis-isomer (A, D), (2R, 3R)-pterosin C trans-isomer (B, E), and pterosin B (C, F) as evaluated by Dixon plots (X-axis: concentration of the novel compound, Y-axis: 1/initial reaction rate of enzyme) (A, B, C) and Lineweaver plots (X-axis: 1/substrate concentration, Y-axis: 1/initial reaction rate of enzyme) (D, E, F).

[0158] FIG. 11 shows BACE1 activity inhibition mechanisms and Ki values of the novel compound (Compound 2) as evaluated by Lineweaver plots (X-axis: 1/substrate concentration, Y-axis: 1/initial reaction rate of enzyme) (upper panels) and Dixon plots (X-axis: concentration of the novel compound, Y-axis: 1/initial reaction rate of enzyme) (lower pannels).

[0159] FIG. 12 is a table in which data of molecular docking simulations of Compound 16 and the positive controls quercetin and berberine for BACE1, AChE, and BChE are summarized, showing bonding energies and binding sites between each compound and the enzymes.

[0160] FIG. 13 illustrates the interaction of (2R, 3R)-pteroside C (Compound 16) with human BACE1 to inhibit the activity of BACE1 in a 3D image (13a) and a plane image (13b), wherein the green color accounts for a hydrogen bond, the pink for hydrophobic interaction, and the purple color for a n-sigma bond.

[0161] FIG. 14 illustrates the interaction of (2R, 3R)-pteroside C (Compound 16) with human AChE to inhibit the activity of AChE in a 3D image (14a) and a plane image (14b), wherein the green color accounts for a hydrogen bond, the pink for hydrophobic interaction, and the purple color for a n-sigma bond.

[0162] FIG. 15 illustrates the interaction of (2R, 3R)-pteroside C (Compound 16) with human BACE1 in a 3D image (15a) and a plane image (15b), wherein the green color accounts for a hydrogen bond, the pink for hydrophobic interaction, and the purple color for a n-sigma bond.

MODE FOR CARRYING OUT THE INVENTION

[0163] Hereinafter, the present invention will be described in detail.

[0164] However, the following Examples are set forth to illustrate, but not to limit the present invention.

Experimental Methods

Experimental Instruments

[0165] .sup.1H NMR and .sup.13C NMR spectra were obtained at 700 MHz for .sup.1H NMR and at 175 MHz for .sup.13C NMR by Bruker Ascend III 700 spectrophotometer (Bruker Biospin, Rheinstetten, Germany) using deuterium chloroform (CDCl.sub.3), methanol (CD.sub.3OD), and dimethyl sulfoxide (DMSO-dd.

[0166] Column chromatography was performed with silica gel (70-230 meshes; Merk, Darmstadt, Germany) using RP-18 (40-63mm; Merck) and Sephadex LH-20(20-100mm; Sigma, St. Louis, Mo., USA). Thin layer chromatography (TLC) was performed with pre-coated Kiesel gel 60 F254 plates(0.25 mm; Merck) and 25 RP-18 F 254s plate(5 -10 cm, Merk). A spray reagent was 50% H.sub.2SO.sub.4. All the chemicals and solvents used in column chromatography were of reagent grade and were used as received without further purification.

Reagents

[0167] Electric eel acetylcholinesterase (AChE, EC3.1.1.7), horse serum butyrylcholinesterase (BChE, EC3.1.1.8), acetyl thiocholine iodide (ACh), butyryl thiocholine chloride (BCh), 5,5′-dithiobis) [2-nitrobenzoic acid)](DTNB), quercetin, and berberine were purchased from Sigma-Aldrich Co. (St. Louis, Mo., USA). BACE1 (beta-secretase) FRET assay kit was purchased from Pan Vera Co. (Madison, Wis., USA). All the chemicals and solvents used in column chromatography were of reagent grade and were used as received without further purification.

MTT Assay

[0168] Mouse fibroblast cell line NIH3T3 and mouse melanoma cell line B16F10 were separately seeded at a density of 1×10.sup.3 cells/well into 96-well plates and grown in DMEM supplemented with 10% FBS (fetal bovine serum) at 37° C. in a 5% CO.sub.2 atmosphere. The cells in each cell were incubated for 24 hours with predetermined concentrations of each of the compounds (pterosin A, pterosin B, pterosin C, pterosin D, pterosin P, pterosin Z, pteroside A, pteroside A.sub.2, pteroside B, pteroside C isomers, pteroside D, pteroside N, pteroside P, and pteroside Z) and then for 2 hours with 100 μL of MTT (0.5 mg/ml PBS). After aspiration of the medium from each well, incubation was conducted for 10 min with 100 μL of DMSO. Absorbance at 570 nm was read using a microplate reader (SPCTRA MAX 340PC, Molecular Devices, USA). Absorbance is an index accounting for a number of viable cells and was used to calculate cell proliferation rates according to the following formula. All experiments were conducted three times for reproducibility.

[0169] Cell proliferation rate (%)=OD.sub.550 (sample)/OD.sub.550 (control)

In vitro BACE1 Activity Assay

[0170] A BACE1 fluorescence resonance energy transfer (FRET) assay kit (β-secretase, recombinant human enzyme) was purchased from Pan Vera Co. (Madison, Wis., USA). The assay was carried out according to the manual provided, with a modification made thereto as described by Jung et al. (Jung et al., Biol Pharm Bull, 2010, 33:267-272). Quercetin was used as a positive control.

In Vitro Cholinesterase Activity Assay

[0171] Inhibitory activity of the compounds against cholinesterases were assayed using the spectrophotometry developed by Elman et al. (Elman et al., Biochem Pharmcol, 1961, 7:88-95), with ACh and BCh serving as substrates. A reaction mixture in which 140 μL of sodium phosphate buffer (pH 8.0), 20 μL of a test sample solution (final concentration 125 μM), and 20 μL of AChE or BChE were mixed was incubated at room temperature for 15 min. All of the test samples and the positive control (berberine) were dissolved in 10% ethanol in analysis grade. The reaction started with the addition of 10 μL of DTNB and 10 μL of Ach or Bch.

[0172] The hydrolysis of Ach or BCh was analyzed using a microplate spectrophotometer (Molecular Devices, Inc., Sunnyvale, Calif., USA) at 412 nm for 15 min. Briefly, the basis for this assay is the hydrolysis of acetylthiocholine by acetylcholinesterase, producing thiocholine that reacts with DTNB. In this case, peak absorption of the thionitrobenzoate (yellow) produced was detected at 412 nm UV in 96-well microplates so as to measure enzyme activity. Degrees of inhibition were calculated according to 1≤S/E≤100 wherein E and S represent enzyme activities in the presence and absence of a test sample, respectively.

Molecular Docking

[0173] In order to examine the inhibitory activity of the compounds of the present disclosure against enzymes, molecular docking study was performed on BACE1, AChE, and BChE. 2-Amino-3-(1r)-1-cyclohexyl-2-[(cyclohexylcarbonyl)amino]ethyl -6-phenoxyquinazolin-3-ium (QUD) was allowed to combine with an X-ray crystal structure of human BACE1 to form a complex. Complexes were formed by combination between ChE and donepezil (E2020) (PDB code: 4EY7) and between BChE and N-[(3R)-1-(2,3-dihydro-1H-inden-2-yl)piperidin-3-yl]methyl-N-(2-methoxyethyl) naphthalene-2-carboxamide(3F9) (PDB code: 4TPK). The complexes thus formed were searched for in the RCSB Protein Databank (https://www.rcsb.org/). Discovery Studio 2017 R2 (BIOVIA, San Diego: Dassault Syst) was used to construct 3D structures of docked ligands in minimal energies.

[0174] Docking studies were carried out using Dock AutoDock 4.2.6 software. In order to evaluate molecular docking settings, re-docking experiments were performed for co-crystallized ligands of above-mentioned PDBs. Thereafter, confirmed docking protocols were applied to the docking of other compounds. In the docking process, the receptor protein was set as a rigid while the ligand was set in a completely flexible state. Prior to docking, protein and ligand structures were processed using AutoDock Tools (ADT) 1.5.6. Co-crystallized ligand and water molecules were removed from the original PDB file. Kollman and Gasteiger charges united with polar hydrogen atom were assigned for protein structures. For docking calculation, Gasteiger charges were added to the ligands.

[0175] The number of rotatable bonds was set and all torsions could be rotated. A grid box for covering a protein active site for the co-crystallized ligand was setup for the AutoGrid program. The grid of 40×40×40 Å.sup.3, with a spacing of 0.375 Å was centered on the co-crystallized ligand of each enzyme. Lamarckian genetic Algorithm (LGA) was used for searching for structures.

[0176] Each docking experiment was derived from 100 different runs that were set to terminate after a maximum of 250,000 energy evaluations or 27,000 generations, yielding 100 docked conformations

[0177] Docking protocol was set for 100 different runs with a maximum of 25×10.sup.5 energy evaluations and 27,000 repetitions. Other docking parameters were defaulted. Docked poses were selected with reference to the criteria of scoring functions and protein-ligand interaction. Discovery Studio 2017 R2 was used to visualize the protein-ligand interaction and generate interaction values.

EXAMPLE 1: PURIFICATION OF NOVEL COMPOUND

[0178] A hot-water extract of bracken fern was fractionated with ethyl acetate and butanol to yield seven fractions. Each fraction was subjected to HPLC to isolate pterosin compounds and the novel compound.

[0179] In brief, 200-250 g of bracken ferns collected from an area of Gapyeong-gun, Gyenggi-do, South Korea was cleansed and steamed for 24 hours with 1.5 L of water in a steaming vessel (OSK-2002, Hongsambaksa, Well sosana™, DaeWoong Pharmaceutical Co. Ltd.), after which 3.5 L of water was further added and then the bracken ferns were aged for 72 hours. The hydrothermal extract thus obtained was stored in a refrigeration manner until use. The hydrothermal extract was added and well mixed with an equal volume of ethyl acetate (EA) and the EA layer thus formed was dried using a rotary evaporator. The resulting EA extract was dissolved in a minimum amount of DMSO and then diluted in water. In this regard, the dilution was made up to 70% of the original volume of the hydrothermal extract, with the expectation of a yield of approximately 70%.

[0180] The EA extract was divided into seven fractions by silica gel column chromatography using chloroform (CHCl.sub.3) and methanol. Then, each fraction was analyzed by Prep-HPLC. Fractions 2, 4, 5, 6, and 7 were analyzed to contain the novel compound [Compound 2, comp.1] and pterosin and derivatives thereof, as listed in Table 1, below. The extraction procedure is as illustrated in FIG. 1.

[0181] Separately, the aqueous layer remaining upon treatment with EA was added and well mixed with an equal volume of butanol. The butanol layer thus formed was dried using a rotary evaporator. The resulting butanol extract (Bu extract) was dissolved in a minimum amount of DMSO and then diluted in water. In this regard, the dilution was made up to 70% of the original volume of the hydrothermal extract, with the expectation of a yield of approximately 70%.

[0182] The Bu extract was divided into nine fractions by silica gel column chromatography using chloroform (CHCl.sub.3) and methanol. Then, each fraction was analyzed by Prep-HPLC. Fraction 6 was analyzed to contain pterosin derivatives, as listed in Table 1, below. The extraction procedure is as illustrated in FIG. 1.

[0183] HPLC conditions for extracting single compounds from EA-2 fraction and Bu-6 fraction are given in FIG. 2.

TABLE-US-00001 TABLE 1 Compound # (FIG. 1 comp.) Structural Formula Name 2 (comp. 1) [00009] 3-[2-(1- Acetylcyclopropyl)- 1-propenyl)-5- methyl-2- cyclopenten-1-one 3 (comp. 2) [00010] (2S)-pterosin A 4 (comp. 3) [00011] (2R)-pterosin B 5 [00012] (2R,3S)-pterosin C 6 [00013] (2S,3S)-pterosin C 7 (comp. 5) [00014] (2R,3R)-pterosin C 8 (comp. 4) [00015] (2S,3R)-pterosin C 9 (comp. 6) [00016] (3R)-Pterosin D 10 (comp. 7) [00017] (2S)-pterosin P 11 (comp. 8) [00018] pterosin Z 12 (comp. 9) [00019] (2S)-pteroside A 13 (comp. 10) [00020] (2S)-pteroside A.sub.2 14 (comp. 11) [00021] (2R)-pteroside B 15 (comp. 12) [00022] (2S,3R)-pteroside C 16 (comp. 13) [00023] (2R,3R)-pteroside C 17 (comp. 14) [00024] (3S)-pteroside D 18 (comp. 15) [00025] (−)-pteroside N 19 (comp. 16) [00026] (2S)-pteroside P 20 (comp. 17) [00027] pteroside Z 21 [00028] (2R,3S)-sulfated pterosin C 22 [00029] (2S,3S)-sulfated pterosin C 23 [00030] pterosin J 24 [00031] pterosin M 25 [00032] pterosin S

EXAMPLE 2: IDENTIFICATION OF NOVEL COMPOUND And DERIVATIVES THEREOF

[0184] The structure of the novel compound isolated and purified in Example 1 was identified by nuclear magnetic resonance (NMR) and mass spectroscopy (MS).

[0185] On the basis of the NMR data shown in FIG. 3, the isolated and purified compound was identified to be 3-[2-(1-acetylcyclopropyl)-1-propenyl]-5-methyl-2-cyclopenten -1-one (3-[2-(1-acetylcycloproply)-1-propenyl]-5-methyl-2-cyclopenten-1-one), which corresponds to compound 2 in Table 1. It was found to be a novel compound that is first isolated from nature and has not yet been reported thus far.

EXAMPLE 3: ASSAY FOR CYTOTOXICITY OF COMPOUNDS (MTT Assay)

[0186] In order to examine whether the compounds of the present disclosure have toxicity to cells, the following experiment was conducted.

[0187] The normal cell line NIH3T3 (mouse embryo fibroblast cell-line) and the cancerous cell line B16F10 (mouse melanoma cell-line) were separately grown in 96-well plates and incubated for 48 hours with predetermined concentrations of each of the compounds. After additional incubation with MTT, absorbance at 550 nm was read using a microplate reader. Absorbance is an index accounting for a number of viable cells and was used to calculate cell proliferation rates according to the following formula. All experiments were conducted three times for reproducibility.

[0188] Cell proliferation rate (%)=OD.sub.550(sample)/OD.sub.550(control)

[0189] Then, LD.sub.50 (Lethal Dose 50) values were calculated on the basis of changes in the cell proliferation rate.

[0190] The results are given in Table 2. As shown, the LD.sub.50 of pterosin A for the cancerous cells was 522±25 μM, which was the lowest among those detected in the compounds. LD.sub.50 values were measured to be 3,110±130 μM for pterosin B and 553±37μM for pterosin Z in the normal cells and 618±71 μM for pterosin Z in the cancerous cells. Except for them, the other compounds were measured to have LD.sub.50 values higher than 5000 μM in both the normal and the cancerous cells.

[0191] These results indicate that pterosin Z (Compound 11) is slightly toxic to cells and pterosin A (Compound 3) has slight cytotoxicity against cancerous cells only, while the other compounds are toxic to none of the normal and the cancerous cells.

[0192] In addition, the novel compound (Compound 2) was measured to have LD.sub.50 values of 1,550 μM and 688 μM against the normal cells and the cancerous cells, respectively. Thus, the novel compound is identified to be almost non-toxic to normal cells and slightly toxic to cancerous cells.

TABLE-US-00002 TABLE 2 LD.sub.50 (μM) Normal cell Cancer cell Compounds NIH3T3 816F10 3-[2-(1-acetylcycloproply)-1-propenyl]-5- 1,550 ± 100 688 ± 17 methyl-2-cyclopenten-1-one (2S)-Pterosin A >5,000 522 ± 25 (2,R)-Pterosin B 3,110 ± 130 >5,000 (2R,3S)-Pterosin C >5,000 >5,000 (2S,3S)-Pterosin C >5,000 >5,000 (2R,3R)-pterosin C >5,000 >5,000 (2S,3R)-pterosin C >5,000 >5,000 (3R)-Pterosin D >5,000 >5,000 (2S)-Pterosin P >5,000 >5,000 PterosinZ 553 ± 37 618 ± 71 (2S)-Pteroside A >5,000 >5,000 (2S)-Pteroside A, >5,000 >5,000 (2R) Pteroside B >5,000 >5,000 (2S,3R)-Pteroside C >5,000 >5,000 (2R,3R)-Pteroside C >5,000 >5,000 (3S)-Pteroside D >5,000 >5,000 (—)-Pteroside N >5,000 >5,000 (2S)-Pteroside P >5,000 >5,000 Pteroside Z >5,000 >5,000

EXAMPLE 4: INHIBITORY EFFECT OF INVENTIVE COMPOUNDS ON BACE1

[0193] BACE1 is an enzyme promotive of the production of β-amyloid, which destroys neurons. In order to examine the effects on the compounds of Table 1 on the activity of BACE1, IC(inhibition concentrations).sub.50 values were determined by measuring BACE1 activity in vitro according to Experimental Methods. Each compound was dissolved in 10% DMSO and applied at various concentrations up to 125 μM.

[0194] The results are given in Table 3, below. As shown, Compounds 2, 6, 7, 8, 16, and 17 have IC.sub.50 values lower than or similar to that of quercetin.

[0195] Of them, the positive control quercetin and Compound 2 (novel compound), Compound 4 ((2R)-Pterosin B), Compound 7 ((2R, 3R)-Pterosin C), Compound 8 ((2S, 3R)-Pterosin C), Compound 14 ((2R)-Pteroside B), Compound 16 ((2R, 3R)-Pteroside C), and Compound 17 ((3S)-Pteroside D), which have low IC.sub.50 values, were examined for inhibiting mechanisms against BACE 1 enzyme activity and for Ki through enzyme kinetics experiments using Dixon plot and Lineweaver plot.

[0196] The results are given in Table 4 (enzymatic kinetics of the compounds of the present disclosure based on Dixon plot and Lineweaver plot). As shown, the BACE1 inhibition mechanism was identified to be of noncompetitive type for Compounds 4, 8, and 14 and of mixed type for Compounds 7, 16, and 17. Having very low Ki values, all of Compound 4, 7, 8, 14, 16, and 17 were observed to bind strongly to BACE1 and thus they are very potent inhibitors against BACE1.

[0197] In addition, an enzyme kinetics experiment was conducted using the Lineweaver plot and Dixon plot (see FIG. 7) in order to examine the BACE1 inhibition mechanism of the novel compound (Compound 2). As shown in Table 3, the novel compound has a Ki value of as low as 19.0 μM, which explains strong binding force to BACE1. The inhibition mechanism was found to be of mixed type. The results of the experiment indicate that the novel compound of the present disclosure is a very potent inhibitor against BACE1.

[0198] Consequently, the compounds extracted according to the method of the present disclosure have excellent inhibitor activity against BACE1, which is an enzyme producing β-amyloid causative of degenerative brain disease.

TABLE-US-00003 TABLE 3 IC.sub.50 (μM) Compounds BACE1 3-[2-(1-acetylcycloproply)-1-propenyl 19.4 ± 1.7 ]-5-methyl-2-cyclopenten-1-one (2S)-Pterosin A 64.9 ± 5.1 (2R)-Pterosin B 18.0 ± 2.0 (2S,3R)-Pterosin C 14.1 ± 1.6 (2R,3R)-Pterosin C 15.9 ± 3.5 (3R)-Pterosin D 56.3 ± 3.9 (2S)-Pterosin P 40.8 ± 4.1 Pterosin Z 48.7 ± 3.2 (2S)-Pteroside A 84.6 ± 6.0 (2S)-Pteroside A 94.4 ± 4.5 (2R)-Pteroside B 43.4 ± 3.0 (2S,3R)-Pteroside C 28.9 ± 2.2 (2R,3R)-Pteroside C 9.74 ± 1.9 (3S)-Pteroside D 10.7 ± 1.5 (—)-Pteroside N 30.6 ± 1.8 (2S)-Pteroside P 81.3 ± 3.9 Pteroside Z 60.0 ± 4.3 Quercetin.sup.a 18.8 ± 1.0 .sup.aQuercetin positive control for BACE1. indicates data missing or illegible when filed

TABLE-US-00004 TABLE 4 Ki and inhibition type BACE1 Acetylcholinesterase Butyrylcholinesterase Compounds Ki (μM) Inhibition type Ki (μM) Inhibition type Ki (μM) Inhibition type 3-[2-(1-acetylcycloproply)-1-propenyl]- 19.0 Mixed-type 5-methyl-2-cyclopenten-1-one (2R)-Pterosin B 38.3 Noncompetitive 12.1 Mixed-type 53.5 Noncompetitive (2S,3R)-Pterosin C 33.8 Noncompetitive 16.3 Mixed-type 29.9 Noncompetitive (2R,3R)-Pterosin C 27.6 Mixed-type 29.6 Noncompetitive 4.77 Mixed-type (2R)-Pteroside B 72.5 Noncompetitive 4.89 Mixed-type 22.6 Mixed-type (2R,3R)-Pteroside C 12.6 Mixed-type (3S)-Pteroside D 16.5 Mixed-type

EXAMPLE 5: INHIBITORY EFFECT OF PTEROSIN COMPOUNDS ON CHOLINESTERASES

[0199] The compounds of Table 1 were evaluated for anti-Alzheimer activity. In this regard, inhibitory activity against AChE (acetylcholinesterase) and BChE (butyrylcholinesterase), which lyse acetylcholine in the central nervous system, was measured according to the Experimental Method to calculate IC(inhibition concentrations).sub.50 values. Each compound was dissolved in 10% DMSO and applied at various concentrations up to 125 μM.

[0200] Cholinesterase is an enzyme that catalyzes the hydrolysis of acethylcholine, which is a neurotransmitter serving to improve thought and memory. Acetylcholine hydrolysis activity is much more potent in AChE than BChE.

[0201] As shown in FIG. 5, each of the compounds was measured to have an IC.sub.50 value higher than that of the positive control berberine. In the compounds, the IC.sub.50 value against AChE was measured to be the lowest for Compound 14 and the highest for Compound 2. In addition, the IC.sub.50 value against BChE was measured to be the lowest for Compound 16 and the highest for Compound 9.

[0202] Among the pterosin compounds extracted by the method of the present disclosure, Compound 14 (pteroside B), Compound 16 (pteroside C), and Compound 4 (Pterosin B) were found to have excellent AChE inhibition activity as proven by the results. In addition, Compound 6 (pterosin C trans-isomer), Compound 14 (pteroside B), and Compound 20 (pteroside Z) showed excellent BChE inhibition activity.

[0203] Of them, the positive control berberine and Compound 2 (novel compound), Compound 4 ((2R)-Pterosin B), Compound 7 ((2R, 3R)-Pterosin C), Compound 8 ((2S, 3R)-Pterosin C), and Compound 14 ((2R)-Pteroside B), which have low IC.sub.50 values, were examined for inhibiting mechanisms against AChE and BChE enzyme activity nd for Ki through enzyme kinetics experiments using Dixon plot and Lineweaver plot (see FIGS. 9 and 10).

[0204] The results are given in Table 4. As shown, the AChE inhibition mechanism was identified to be of mixed type for Compounds 4, 8, and 14 and of noncompetitive type for Compound 7 while the BChE inhibition mechanism was identified to be of noncompetitive type for

[0205] Compounds 4 and 8 and of mixed type for Compounds 7 and 14. Having very low Ki values, all of Compounds 4, 7, 8, and 14 were observed to bind strongly to both AChE and BChE and thus they are very potent inhibitors against AChE and BChE.

[0206] Consequently, the pterosin compounds extracted according to the method of the present disclosure were found to have the ability to maintain certain levels of acetylcholine, which is a neurotransmitter responsible for the cognitive functions of the brain, such as thinking, memory, etc., by inhibiting the activities of AChE and BChE, which are enzymes lysing acetylcholine.

TABLE-US-00005 TABLE 5 IC.sub.50 (uM) Acetylcholine Butyrylcholine esterase esterase Compounds Mean ± SEM Mean ± SEM SI.sup.b 3-[2-(1-acetylcycloproply)-1-propenyl 87.7 ± 1.6 72.9 ± 0.73 0.83 ]-5-methyl-2-cyclopenten-1-one (2S)-Pterosin A 88.7 ± 2.6 57.3 ± 3.3  12 (2R)-Pterosin B 16.2 ± 1.0 48.1 ± 0.59 3.0 (2S,3R)-Pterosin C  12.8 ± 0.79 44.3 ± 1.0  3.5 (2R,3R)-Pterosin C 23.2 ± 4.8 20.3 ± 0.88 0.88 (3R)-Pterosin D 68.7 ± 3.7 >12 (2S)-Pterosin P  17.8 ± 0.62 55.9 ± 6.6  3.1 Pterosin Z 46.5 ± 3.4 80.1 ± 6.8  1.7 (2S)-Pteroside A  110 ± 3.0  199 ± 0.25 (2S)-Pteroside A 39.9 ± 1.9 119 ± 2.5  3.0 (2R)-Pteroside B  2.55 ± 0.23 17. (2S, 3R)-Pteroside C  9.57 ± 0.32 12.0 ± 0.14 1.4 (2R,3R)-Pteroside C  3.77 ± 0.38  ± 0.22  1.4 (3S)-Pteroside D 27.4 ± 1.2 19.3 ± 0.17 0.70 (—)-Pteroside N  4.47 ± 0.29 7.39 ± 0.99 1.7 (2S)-Pteroside P 57.5 ± 3.2 33.2 ± 3.0  0.57 Pteroside Z  ± 2.2  5.31 ± 0.19 0.24 Berberine.sup.a  0.39 ± 0.01 3.32 ± 0.12 8.5 .sup.aBerberine positive control for cholinesterases (AChE and BChE). .sup.bSI selectivity index (BChE/AChE) indicating which of AChE and BChE is more specifically inhibited. indicates data missing or illegible when filed

EXAMPLE 6: DOCKING RESULTS OF COMPOUNDS TO ACTIVE SITES OF BACE1, AChE, and BChE

[0207] BACE1, AChE, and BChE have several crystalline structures. Of them, human PDB was selected in consideration of wild-type structures, co-crystallized ligands, and solvents of structures. Selection was made of X-ray crystal structures of BACE1 forming a complex with QUAD (PDB code: 2WJO); AChE forming a complex with E2020 (PDB code: 4EY7); and BChE forming a complex with 3F9 (PDB code: 4TPK).

[0208] The compounds given in Table 1 were investigated for binding patterns. Docking studies were conducted for compounds selected for examination of structure activity correlation. (2R, 3R)-Pteroside C was found to be a potent inhibitor against BACE1, AChE, and BChE as measured by the enzyme inhibition assay. Therefore, (2R, 3R)-pteroside C was selected as a representative compound for docking. In addition, quercetin and berberine, which were used as positive controls in the enzyme inhibition assay, were subjected to the docking simulation.

[0209] The molecular docking simulation results are summarized in FIG. 12. As shown, (2R, 3R)-pteroside C strongly interacts with the active site of BACE1, with the bonding energy detected at −6.77 kcal/mol which is lower than the quercetin bonding energy −5.68 kcal/mol.

[0210] In addition, the bonding energy of the compound was −6.85 kcal/mol for AChE, which is higher than the berberine bonding energy −8.61 kcal/mol, and −5.99 kcal/mol for BChE, which is slightly higher than the berberine bonding energy −6.67 kcal/mol.

[0211] These results show high affinities and strong binding potentials the compounds of the present disclosure for the active sites of BACE1, AChE, and BChE, indicating that the compounds bind to the enzymes and inhibit the enzymatic activities.

[0212] FIGS. 13 to 15 are images showing the binding of (2R, 3R)-pteroside C to BACE1, AChE, and BChE, respectively.

[0213] Consequently, pterosin and derivatives thereof have promising inhibitory potentials against BACE1, AChE, and BChE, as proven by the in vitro enzyme assay and the molecular docking simulation. Taken together, the data obtained above suggest that the compounds of the present disclosure can be used in therapeutic or preventive agents for dementia thanks to the inhibitory activity thereof against BACE1 and AChE.

INDUSTRIAL APPLICABILITY

[0214] As described hitherto, the method of the present disclosure can be advantageously used for using the pterosin compounds extracted from bracken ferns or derivatives thereof to provide a therapeutic agent for preventing or treating degenerative brain disease or a food composition for alleviating degenerative brain disease or enhancing a cognitive function.