TRICYCLIC DILACTONE COMPOUND, AND PRODUCTION METHOD AND USE THEREOF

Abstract

Provided are a novel tricyclic dilactone compound, a strain producing the same, a method of producing the tricyclic dilactone compound, and use of the tricyclic dilactone compound. The tricyclic dilactone compound has activity of inhibiting aggregation of amyloid-beta and tau proteins, activity of inhibiting apoptosis, and anti-inflammation activity, and thus may be used to prevent, treat, or improve various neurodegenerative brain diseases including Alzheimer's disease and cognitive impairment.

Claims

1. A compound represented by Formula 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof: ##STR00006## wherein R.sub.x, R.sub.y, and R.sub.z are each independently one or more substituents selected from hydrogen, a hydroxy group, a halogen atom, an amine group, a carbonyl group, a cyano group, a substituted or unsubstituted C.sub.1-C.sub.20 alkyl group, a substituted or unsubstituted C.sub.1-C.sub.20 alkoxy group, a substituted or unsubstituted C.sub.2-C.sub.20 alkenyl group, a substituted or unsubstituted C.sub.2-C.sub.20 alkynyl group, —C(═O)R.sub.a, —C(═O)OR.sub.a, —OCO(OR.sub.a), —C═N(R.sub.a), —SR.sub.a, —S(═O)R.sub.a, —S(═O).sub.2R.sub.a, —PR.sub.a, a C.sub.2-C.sub.20 alkylene oxide group, a substituted or unsubstituted C.sub.6-C.sub.30 aryl group, a substituted or unsubstituted C.sub.6-C.sub.30 aryloxy group, a substituted or unsubstituted C.sub.6-C.sub.30 heteroaryl group, a substituted or unsubstituted C.sub.3-C.sub.30 cyclic group, a substituted or unsubstituted C.sub.3-C.sub.20 heterocyclic group, or a combination thereof, wherein R.sub.a is hydrogen, a C.sub.1-C.sub.10 alkyl group, or a C.sub.6-C.sub.20 aryl group, and m and n are each independently an integer from 1 to 5.

2. The compound, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein the compound represented by Formula 1 is represented by Formula 2: ##STR00007## wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently one or more substituents selected from hydrogen, a hydroxy group, a halogen atom, an amine group, a carbonyl group, a cyano group, a substituted or unsubstituted C.sub.1-C.sub.20 alkyl group, a substituted or unsubstituted C.sub.1-C.sub.20 alkoxy group, a substituted or unsubstituted C.sub.2-C.sub.20 alkenyl group, a substituted or unsubstituted C.sub.2-C.sub.20 alkynyl group, —C(═O)R.sub.a, —C(═O)OR.sub.a, —OCO(OR.sub.a), —C═N(R.sub.a), —SR.sub.a, —S(═O)R.sub.a, —S(═O).sub.2R.sub.a, —PR.sub.a, a C.sub.2-C.sub.20 alkylene oxide group, a substituted or unsubstituted C.sub.6-C.sub.30 aryl group, a substituted or unsubstituted C.sub.6-C.sub.30 aryloxy group, a substituted or unsubstituted C.sub.6-C.sub.30 heteroaryl group, a substituted or unsubstituted C.sub.3-C.sub.30 cyclic group, a substituted or unsubstituted C.sub.3-C.sub.20 heterocyclic group, or a combination thereof, wherein R.sub.a is hydrogen, a C.sub.1-C.sub.10 alkyl group, or a C.sub.6-C.sub.20 aryl group.

3. The compound, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 2, wherein R.sub.1, R.sub.3, and R.sub.4 are each independently a substituted or unsubstituted C.sub.1-C.sub.10 alkyl group.

4. The compound, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 2, wherein R.sub.2 is hydroxy, a substituted or unsubstituted C.sub.1-C.sub.10 alkyl group, a substituted or unsubstituted C.sub.1-C.sub.10 alkoxy group, or a substituted or unsubstituted C.sub.6-C.sub.30 aryloxy group.

5. The compound, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 2, wherein the compound represented by Formula 2 is represented by Formula 3: ##STR00008##

6. The compound, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 5, wherein the compound represented by Formula 3 is represented by Formula 4: ##STR00009##

7. A Streptomyces sp. WON17 strain deposited under Accession No: KCTC (Korean Collection for Type Cultures) 14217BP for producing the compound, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1.

8. A method of producing the compound of claim 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, the method comprising: culturing a Streptomyces sp. WON17 strain deposited under Accession No: KCTC14217BP; and separating the compound of claim 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof from the culture.

9. A pharmaceutical composition for preventing or treating neurodegenerative brain disease, the pharmaceutical composition comprising: the compound of claim 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition of claim 9, wherein the neurodegenerative brain disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, amyloidosis, multiple system atrophy, multiple sclerosis, tauopathies, Pick's disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

11. A functional health food for preventing or improving neurodegenerative brain disease or cognitive impairment, the functional health food comprising: the compound of claim 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

12. A method of preventing or treating neurodegenerative brain disease, the method comprising: administering a pharmaceutical composition for preventing or treating neurodegenerative brain disease to a subject, the pharmaceutical composition comprising the compound of claim 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

13. A method of preventing or improving neurodegenerative brain disease or cognitive impairment, the method comprising: administering a functional health food to a subject, the functional health food comprising the compound of claim 1, derivative, stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 is a photograph of a medium on which Streptomyces sp. WON17 strain was cultured.

[0084] FIG. 2A is a schematic diagram of an experiment to test inhibitory activity of rhizolutin on formation of amyloid-beta aggregates,

[0085] FIG. 2B shows images of amyloid-beta aggregates stained with thioflavine S in brain tissue,

[0086] FIG. 2C is a graph showing quantification of plaques in hippocampus (Veh: vehicle, Rhi: rhizolutin, *: p<0.05), and

[0087] FIG. 2D shows images and merged images of amyloid-beta aggregates stained with GFAP and thioflavine S in brain tissue (ThS: thioflavine S, Merged: merged images).

[0088] FIG. 3A is a graph showing inhibition of formation of amyloid-beta aggregates in the presence of rhizolutin, and

[0089] FIG. 3B is a graph showing disaggregation of formed amyloid-beta aggregates in the presence of rhizolutin (y-axis: relative fluorescence intensity (%), Rhi: rhizolutin, *: p<0.05, ***: p<0.001).

[0090] FIG. 4 is a graph showing cell viability (%) of amyloid-beta-treated HT-22 mouse hippocampal neuronal cell line in the presence of rhizolutin (NT: negative control group, Rhi: rhizolutin, *: p<0.05, **: p<0.01).

[0091] FIG. 5A is an immunoblotting image showing expression of cleaved caspase-3 and Bcl-2 (B-cell lymphoma 2) in HT-22 and BV2 cells treated with amyloid-beta aggregates and rhizolutin, and

[0092] FIG. 5B is a graph showing amounts of interleukin-1β measured by a sandwich ELISA method (Rhi: rhizolutin, *: p<0.05, **: p<0.01).

[0093] FIG. 6A is a graph showing inhibition of formation of tau protein aggregates in the presence of rhizolutin, and

[0094] FIG. 6B is a graph showing disaggregation of formed tau protein aggregates in the presence of rhizolutin (y-axis: relative fluorescence intensity (%), Rhi: rhizolutin, **: p<0.01, ***: p<0.001).

DETAILED DESCRIPTION

[0095] Hereinafter, the present disclosure will be described in more detail with reference to Examples below. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

Example 1. Separation of Rhizolutin and Identification of Structure Thereof

[0096] 1-1. Isolation of Rhizolutin-Producing Strain

[0097] A rhizolutin-producing Streptomyces sp. WON17 strain was isolated from the soil for growing Panax ginseng. The WON17 strain was isolated from a solid medium (2 g of sodium caseinate, 0.1 g of asparagine, 4 g of sodium propionate, 0.5 g of dipotassium phosphate, 0.1 g of magnesium sulfate, 0.001 g of ferrous sulfate, and 15 g of agar powder per 1 L of sterile water). Based on 16S rDNA sequencing analysis results, the corresponding strain was identified to belong to Streptomyces species. The strain was named Streptomyces sp. WON17 strain, and was deposited in Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology on Jun. 17, 2020 under Accession No: KCTC14217BP.

[0098] 1-2. Culture of Streptomyces sp. WON17 Strain

[0099] The Streptomyces sp. WON17 strain was spread on a sterilized YEME solid medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 18 g of agar per 1 L of distilled water), and cultured at about 30° C. for about 4 weeks.

[0100] Spores of the cultured WON17 strain were inoculated into a 50 mL-volume YEME liquid medium (4 g of yeast extract, 10 g of malt extract, and 4 g of glucose per 1 L of distilled water), and cultured at 30° C. for 5 days while shaking at a rate of 200 rpm. 10 mL of the culture broth was inoculated into a 200 mL-volume modified YEME broth (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, 10 mL of olive oil, and 11 g of dried ginseng powder per 1 L of distilled water), and cultured for about 5 days under conditions of 170 rpm and 30° C.

[0101] 1-3. Separation and Purification of Rhizolutin

[0102] Culture broth of the WON17 strain cultured in Example 1-2 was obtained.

[0103] 1 L of the WON17 strain culture broth obtained at the end of the culturing and about 1.5 L volume of ethyl acetate (EtOAc, Daejung Chemicals & Metals Co., Ltd.) were added to a separatory funnel mounted on a stand. The funnel was stoppered and shaken in the up, down, right, and left directions for 3 minutes to conduct a first extraction process. Afterwards, the separatory funnel was mounted on a stand again to completely separate water layer and EtOAc layer. The valve of the separatory funnel was then opened to remove the water layer. New EtOAc was added to the water layer, and iterative extractions were performed in the same way. The EtOAc layers were stored separately in a clean flask, and anhydrous sodium sulfate (Daejung Chemicals & Metals Co., Ltd.) was added thereto to remove the water remaining in the culture broth. By passing through several layers of clean gauze, impurities were removed. To remove olive oil, 20% (v/v) aqueous methanol solution and aqueous hexane solution (at a volume ratio of 1:1) were used. Yield of 99% was confirmed in the 20% (v/v) aqueous methanol solution, and the finally treated EtOAc was transferred to a 3 L-volume round flask and dried under reduced pressure using a vacuum drying machine. A total of 700 L of the culture broth was used for extraction to obtain about 210 mg of crude extract.

[0104] To purify rhizolutin from the crude extract of the WON17 strain, a purification process was performed thereon using preparative high-performance-liquid-chromatography. After stabilizing a closed column filled with C.sub.18 resin with methanol (MeOH) 31% (v/v)/H.sub.2O 69% (v/v), separation was conducted under a concentration gradient condition for about 40 minutes until the methanol concentration reached 50% (v/v). Rhizolutin was detected along with other substances including naphthomycin at about 31 minutes.

[0105] To obtain pure rhizolutin from a 30-minute peak fraction dried under reduced pressure, semi-preparative HPLC was conducted under acetonitrile (ACN) 40% (v/v)/H.sub.2O 60% (v/v) isocratic conditions. Detailed HPLC conditions are as follows:

[0106] (1) A reverse-phase column (C.sub.18 (2) Phenomenex Luna, 5 μm 250 mm×21.2 mm) was used, and separation was performed under a concentration gradient condition of 31% (v/v) to 50% (v/v) methanol(MeOH)/aqueous solution(flow rate: 10 mL/min, detection: UV 254 nm). Rhizolutin was identified in a fraction at about 31 minutes.

[0107] (2) The fraction obtained at about 31 minutes and dried under reduced pressure was subjected to a reverse-phase column (C.sub.18 Phenomenex 250 mm×4.6 mm), and purification was performed under aq isocratic conditions using 40% (v/v) ACN/aqueous solution to which 0.1% (v/v) formic acid was added (flow rate: 1 mL/min, detection: UV 254 nm). Rhizolutin was identified in a fraction at about 28 minutes.

[0108] The same process was repeated to obtain about 100 mg of rhizolutin from about 5 g of crude extract.

[0109] 1-4. Identification of Chemical Structure of Rhizolutin

[0110] The structure of rhizolutin was identified based on 1D and 2D nuclear magnetic resonance (NMR) spectra. The NMR spectra (.sup.1H NMR, .sup.13C NMR) were obtained by using a 500 MHz NMR spectrometer available from Bruker Company, and acetone-d.sub.6 as a solvent.

[0111] Table 1 shows the structural positioning of rhizolutin by the NMR spectra.

[0112] [Rhizolutin]

[0113] (1) Molecular formula: C.sub.21H.sub.28O.sub.5

[0114] (2) Molecular weight: 360

[0115] (3) Color: white powder

[0116] (4).sup.1H-NMR (Pyridine-d.sub.5, 600 MHz): see Table 1

[0117] (5).sup.13C-NMR (Pyridine-d.sub.5, 150 MHz): see Table 1

TABLE-US-00001 TABLE 1 Major conformational isomer Minor conformational isomer 1H HZ Position δ.sub.H, mult (J in Hz) δ.sub.C δ.sub.H, mult (J in Hz) δ.sub.C Difference 1 — 174.6, s — 174.4, s 2 3.69, 1H, t (7.5) 41.1, d 3.75, 1H, t (7.5) 46.8, d 31.87 3 6.15, 1H, d (7.5) 125.0, d 5.92, 1H, d (7.5) 121.8, d 139.42 4 — 135.8, s — 138.9, s 5 6.12, 1H, m 139.4, d 6.14, 1H, m 138.7, d 10.78 6 5.30, 1H, dd (16.0, 10.0) 128.3, d 5.32, 1H, m 123.2, d 12.10 7 3.31, 1H, m 43.9, d 3.09, 1H, m 45.3, d 128.04 8 1.98, 1H, m 40.0, t 1.95, 1H, m 36.6, t 18.97 2.56, 1H, dt (14.5, 5.0) 2.56, 1H, m 9 4.66, 1H, m 62.5, d 4.64, 1H, m 62.2, d 15.38 10 3.47, 2H, m 44.2, t 3.38, 1H, m 44.5, t 58.33 11 — 172.9, s — 172.6, s 12 6.13, 1H, m 83.1, d 5.88, 1H, m 80.8, d 147.46 13 5.04, 1H, d (6.5) 134.9, d 5.37, 1H, m 123.5, d 195.31 14 — 134.7, q — 148.1, t 15 2.63, 1H, t (8.5) 47.4, s 2.23, 1H, m 46.1, d 237.55 16 1.71, 1H, m 33.1, t 1.95, 1H, m 35.8, d 145.80 1.85, 1H, m 2.05, 1H, m 119.85 17 4.42, 1H, m 78.4, d 4.53, 1H, m 78.3, d 67.65 18 1.50, 1H, m 28.8, t 1.60, 1H, m 28.6, t 55.9 1.59, 1H, m 1.70, 1H, m 63.9 19 0.89, 3H, t (7.5) 10.0, q 0.98, 3H, t (7.5) 10.4, q 51.61 20 1.76, 3H, s 13.3, q 1.74, 3H, s 24.2, q 19.31 21 1.92, 3H, s 20.5, q 1.91, 3H, s 20.7, q 11.30

[0118] The chemical structural formula of rhizolutin analyzed from the 1D and 2D NMR spectra is shown below:

##STR00005##

Example 2. Identification of Activity of Rhizolutin

[0119] 2-1. Inhibitory Activity of Rhizolutin on Formation of Amyloid-Beta Aggregates

[0120] It was to test whether rhizolutin was able to dissolve amyloid-beta aggregates.

[0121] First, APP/PS1 transgenic mice (7.5-month-old, male, Jackson Laboratory, 9 to 10 mice/group) to which the genes of AD patients were injected were prepared. In the APP/PS1 transgenic mice, human amyloid-beta is expressed at a high level, and Alzheimer-like plaques are formed in the brain.

[0122] The prepared mice were intraperitoneally administered daily with rhizolutin at a dose of 8 mg/body weight (kg)/day for 3 weeks. As a negative control group, the prepared mice were intraperitoneally administered with a vehicle only.

[0123] Afterwards, the mouse brain was removed, and changes in amyloid-beta aggregates (in the form of oligomer or fibril) as AD biomarkers in the brain tissue were measured.

[0124] FIG. 2B shows images of the amyloid-beta aggregates stained with thioflavine S (available from Sigma-Aldrich) in the brain tissue, and FIG. 2C is a graph showing the quantification of plaques in hippocampus (by unpaired t-test, mean±SEM). As shown in FIG. 2B and FIG. 2C, the group administered with rhizolutin showed decrease in the number of amyloid-beta plaques in the hippocampus of the brain, compared to the negative control group.

[0125] FIG. 2D shows images of the amyloid-beta aggregates stained with a glial fibrillary acidic protein (GFAP), which is a biomarker of astrocytosis, and thioflavine S in the brain tissue. As shown in FIG. 2D, it was found that the amount of the amyloid-beta aggregates identified by thioflavine S staining was decreased by the administration of rhizolutin, and that the GFAP, a biomarker of neuroinflammation also decreased to reflect significant reduction in the level of astrocytosis around the plaques.

[0126] 2-2. Activity of Inhibiting and Disaggregating Amyloid-Beta Aggregation

[0127] An amyloid-beta solution was prepared by dissolving human A131-42 monomer (UniProt KB-P05067, a.a. 672 to 713) in DMSO to have a concentration of 10 mM. Then, the amyloid-beta solution was diluted with distilled water to prepare a solution having a concentration of 100 μM.

[0128] Meanwhile, thioflavine-T (Sigma-Aldrich) was dissolved in 50 mM glycin buffer (pH 8.5) to have a concentration of 5 mM. Then, the resulting solution was diluted with 50 mM glycin buffer (pH 8.5) to have a concentration of 5 μM, and stored light-blocked in a dark room.

[0129] To verify the inhibitory activity of rhizolutin on the amyloid-beta aggregation, the amyloid-beta solution was added to 1.5 mL microtubes to a concentration of 25 μM, and rhizolutin was added thereto at a concentration of 1 mM, followed by incubation at 37° C. for 120 hours.

[0130] 25 μl of the reaction solution obtained at the end of reaction was added to each well of a 96-well plate, and 75 μl of the prepared thioflavine-T solution was also added to each well. After a reaction was allowed for 5 minutes at room temperature in the darkroom, fluorescence was measured at an excitation wavelength of 450 nm and an emission wavelength of 485 nm by using a multi-mode microplate reader.

[0131] Based on the fluorescence intensity of the group (control group) in which only amyloid-beta was treated and aggregated for 120 hours, the fluorescence intensity (%) of each group was calculated, and results are shown in FIG. 3A (one-way ANOVA test followed by Bonferroni's post-hoc comparison test, mean±SEM, ***: p<0.001).

[0132] In addition, to verify the activity of rhizolutin on disaggregation of the amyloid-beta aggregation, the amyloid-beta solution was added to a 1.5 mL microtube to a concentration of 25 μM and incubated at 37° C. for 72 hours. Then, 1 mM of rhizolutin was added to each microtube, followed by incubation at 37° C. for 72 hours. To compare the activity, a compound known to effectively disaggregate the amyloid-beta aggregate, i.e., 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS, CAS Number: 16052-06-5) was incubated at a concentration of 20 mM under the same conditions.

[0133] 25 of the reaction solution obtained at the end of reaction was added to each well of a 96-well plate, and 75 of the prepared thioflavine-T solution was added to each well. Then, fluorescence intensity was measured at an excitation wavelength of 450 nm and an emission wavelength of 485 nm by using a multi-mode microplate reader.

[0134] Based on the fluorescence intensity of the group (control group) in which only amyloid-beta was treated and aggregated for 72 hours, the fluorescence intensity (%) of each group was calculated, and results are shown in FIG. 3B (mean±SEM, *: p<0.05, ***: p<0.001).

[0135] As shown in FIGS. 3A and 3B, it was confirmed that rhizolutin not only significantly inhibited the formation of the amyloid-beta aggregates (in the form of oligomer or fibril), but also disaggregated the amyloid-beta aggregates (in the form of oligomer or fibril) and the disaggegation activity of rhizolutin was better than that of EPPS, a compound known to have the activity.

[0136] 2-3. Inhibitory Activity of Rhizolutin on Apoptosis

[0137] It was to test whether rhizolutin was able to inhibit apoptosis of neurons exposed to neurotoxicity of the amyloid-beta aggregates.

[0138] To measure cytotoxicity of rhizolutin, a predetermined number of cells of HT-22 mouse hippocampal neuronal cell line were inoculated into a plate. To the HT-22 cells, an amyloid-beta solution prepared in the same manner as in Example 2-2 was added to a final concentration of 10 μM, followed by incubation at 37° C. for 72 hours. Then, 20 μM of rhizolutin was added to each microtube, followed by incubation at 37° C. for 24 hours. As a negative control, a vehicle without the amyloid-beta protein and rhizolutin was used.

[0139] The viability of the HT-22 cells was measured by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Based on the viability (%) of the HT-22 cells to which the amyloid-beta was not applied (negative control group), the cell viability (%) of each group was calculated, and results are shown in FIG. 4 (one-way ANOVA, followed by Bonferroni's post-hoc comparison test, mean±SEM, NT: negative control group, *: p<0.05, **: p<0.01).

[0140] As shown in FIG. 4, it was found that the apoptosis of neurons exposed to the neurotoxicity of the amyloid-beta aggregates was inhibited by rhizolutin. Accordingly, rhizolutin was confirmed to have activity of inhibiting apoptosis of neurons caused by the amyloid-beta aggregates.

[0141] 2-4. Verification of Inhibitory Activity of Rhizolutin on Apoptosis

[0142] To verify the inhibitory activity of rhizolutin on the neurotoxicity of the amyloid-beta aggregates, changes in apoptosis-related biomarkers after exposing neurons to the neurotoxicity of the amyloid-beta aggregates were observed. Here, cleaved caspase-3, which is an apoptosis-inducing factor, and B-cell lymphoma 2 (Bcl-2), which inhibits apoptosis, were used as the biomarkers.

[0143] HT-22 mouse hippocampal neuronal cells and BV2 microglial cells were inoculated into a plate in a predetermined number. To each of the HT-22 cell line and the BV2 cell line, an amyloid-beta solution prepared in the same manner as in Example 2-2 was added at a concentration of 1 mM, followed by incubation at 37° C. for 24 hours. Afterwards, 20 μM of rhizolutin was added to each microtube, followed by incubation with amyloid-beta in a final concentration of 20 μM at 37° C. for 24 hours. As a negative control group, a group without rhizolutin and the amyloid-beta protein was used.

[0144] Cleaved caspase-3 and Bcl-2, which are apoptosis-related biomarkers, were identified by immunoblotting, and images thereof are shown in FIG. 5A. As shown in FIG. 5A, due to the amyloid-beta aggregates, the expression of Bcl-2 decreased while the expression of cleaved caspase-3 increased, thereby confirming that the apoptosis of the neurons was induced by the amyloid-beta aggregates. In addition, when treated with the amyloid-beta aggregates and rhizolutin together, the expression of Bcl-2 increased, while the expression of cleaved caspase-3 decreased. Therefore, it was found that the amyloid-beta-induced apoptosis of cells was inhibited by the treatment with rhizolutin.

[0145] Meanwhile, in the HT-22 and BV2 cells that were treated with the amyloid-beta aggregates and rhizolutin together, the amount of interleukin-1β (IL-1β), which is a biomarker of inflammation, was measured by sandwitch ELISA method (using unpaired t-test). FIG. 5B is a graph showing the amount of IL-1β depending on the duration of the treatment with the amyloid-beta aggregates and rhizolutin (mean±SEM, *: p<0.05, **: p<0.01). As shown in FIG. 5B, it was confirmed that, due to the treatment with rhizolutin, the amyloid-beta-induced neuroinflammation of cells was inhibited.

[0146] 2-5. Inhibitory Activity of Rhizolutin on Formation of Tau Aggregates

[0147] It was to test whether rhizolutin was able to inhibit aggregation of tau protein, which is another causative protein of Alzheimer's disease.

[0148] Tau protein was prepared at a concentration of 1 mg/mL, and as described in Example 2-3, the prepared tau protein was added to a 1.5 mL microtube at a concentration of 0.5 mg/mL, and 1 mM of rhizolutin and 20 mM of EPPS were added thereto, followed by incubation at 37° C. for 120 hours.

[0149] Meanwhile, the tau protein was added to a 1.5 mL microtube at a concentration of 0.5 mg/mL, and incubated at 37° C. for 48 hours. Then, rhizolutin was added at a concentration of 1 mM, and incubated at 37° C. for 72 hours. To compare the efficacy, EPPS was added at a concentration of 20 mM, and incubated under the same conditions.

[0150] Thioflavin T was added to the reaction, and the fluorescence intensity was measured at an excitation wavelength of 450 nm and an emission wavelength of 485 nm. As a negative control group, a vehicle without the tau protein and rhizolutin was used.

[0151] Based on the fluorescence intensity of the group in which only the tau protein was treated (positive control group), the fluorescence intensity (%) of each group was calculated, and results are shown in FIGS. 6A and 6B (mean±SEM, *: p<0.01, ***: p<0.001).

[0152] As shown in FIGS. 6A and 6B, it was confirmed that, when treated with rhizolutin, the formation of the tau aggregates was inhibited (see FIG. 6A), and at the same time, the existing tau aggregates were disaggregated (see FIG. 6B). When using EPPS, which is known to be effective in the disaggregation of the amyloid-beta aggregates, as a comparison group, it was confirmed that rhizolutin had better effects of inhibiting the formation of the tau aggregates and disaggregating the tau aggregates.

[0153] Therefore, it was confirmed that rhizolutin had activities of inhibiting the formation of the amyloid-beta aggregates, inhibiting the amyloid-beta-induced apoptosis, inhibiting the amyloid-beta-induced cell inflammation, and inhibiting the formation of the tau aggregates, and thus would be used to treat Alzheimer's disease.

[0154] [Accession No]

[0155] Name of Depositary Authority: Korea Research Institute of Bioscience and Biotechnology

[0156] Accession No: KCTC14217BP

[0157] Date of Receipt: 20200617