Patent ID: 12234257

BEST MODE

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following experimental examples are merely presented to exemplify the present invention, and the scope of the present invention is not limited thereto.

Experimental Procedure

Optical rotations were acquired with a Perkin-Elmer (Waltham, Mass., USA) 343 polarimeter. UV and IR spectra were acquired with a Perkin-Elmer Lambda 35 spectrophotometer and a Thermo (Waltham, Mass., USA) iS50 spectrometer. Electron circular dichroism (ECD) spectra were obtained with an Applied Photophysics (Leatherhead, England) Chirascan V100 spectrometer. NMR spectra were recorded on Varian (Palo Alto, Calif., USA) 500 MHz, Joel (Tokyo, Japan) 600 MHz, and Bruker (Billerica, Mass., USA) 850 MHz NMR spectrometers. High-resolution mass spectrometry (HRMS) data were collected on a Thermo Q-Exactive mass spectrometer. Preparative HPLC system utilized YMC (Kyoto, Japan) LC-Forte/R and an ELS detector with a Phenomenex Luna C18 column (10 μm, 250 mm×21.2 mm). Column chromatography was carried out using GE Healthcare (Chicago, Ill., USA) Sephadex LH-20 gel.

<Plant Materials>

The whole plant ofAster koraiensiswas collected in October 2016 after cultivation at Pyeongchang Wild Plant Nursery and Farming Corporation (Pyeongchang, Republic of Korea). A voucher specimen (No. BS0083A1) was deposited in the Korea Institute of Science and Technology Gangneung Institute.

<Substance>

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin were purchased from Gibco (Thermo Fisher Scientific). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride, 1-methyl-4-phenylpyridinium (MPP+) iodide, 3-methyladenine (3-MA), and ropinirole were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). Wortmannin (Wart) and bafilomycin A1 (Baf) were purchased from Abcam (MA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-AMPK, anti-phosphorylated AMPK (p-AMPK), anti-ULK, anti-phosphorylated ULK555 (p-ULK555), anti-Erk, anti-phosphorylated Erk (p-Erk), anti-mTOR, anti-phosphorylated mTOR (p-mTOR), anti-LC3B, anti-tyrosine hydroxylase (TH), rabbit-derived anti-α-synuclein primary antibody, anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody, Erk inhibitor U0126, AMPK siRNA, and AMPK siRNA control were purchased from Cell Signaling Technology (Boston, Mass., USA). An autophagy tandem RFP-GFP-LC3B kit was purchased from Thermo Fisher Scientific (MA, USA). An MTT assay kit (Z-Cytox) was purchased from DAEILLAB Co, Ltd, Seoul, Republic of Korea. A dopamine ELISA kit was purchased from Abnova (Taipei City, Taiwan), and a MAO-B assay kit was supplied by Promega (Woods Hollow Road, Madison, Wis., USA). All reagents of the highest grades were used and selected from commercially available products.

Preparation Example 1: Cell Cultivation

Human neuroblastoma (SH-SY5Y) cells were purchased from the American Type Culture Collection (Manassas, Va., USA) and cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. The SH-SY5Y cells were cultured at 37° C. in a humid atmosphere containing 5% CO2. First, the cells were seeded on a 6-well plate at a density of 80×104cells/well with 2 mL of the culture medium followed by, after 24 hours, treatment with samples (AKNS EtOH extract, an AKNS n-BuOH fraction, or AKNS-2) having desired concentrations. For conditions of the presence of 3-MA, Wart, Baf, or U0126, the cells were treated with these reagents for 30 minutes before treating the cells with the samples. The SH-SY5Y cells were transfected with AMPK siRNA and then administered with AKNS-2 at 36 hours after siRNA transfection. When MPP+treatment is needed, after 1 hour from treatment with the samples, the cells were treated with MPP+. After 24 hours from treatment with the samples, the SH-SY5Y cells were recovered and used for subsequent analysis.

Experimental Example 1: MTT Assay to Measure Cell Viability

Cell viability was detected using an MTT assay kit (Z-Cytox). Briefly, the SH-SY5Y cells were seeded on a 96-well plate at a density of 2×104cells/well with 100 μL of a culture medium. After 24 hours, the SH-SY5Y cells were treated with AKNS-2 and/or MPP+having desired concentrations to identify cytotoxicity of AKNS-2 and MPP+. After 24 hours, 10 μL of the MTT reagent was added to each well of the 96-well plate including the cells. Absorbance was measured at 450 nm using a microplate spectrophotometer (BioTek, Vt., USA). In order to prove protective effects of AKNS-2 against cytotoxicity induced by MPP+, the cells were treated with 2 mM MPP+at 1 hour after the AKNS-2 treatment. The next day, absorbance was measured at 1 hour after adding the MTT reagent thereto. In order to identify whether AKNS-2 has protective effects against MPP+-induced cytotoxicity by inducing autophagy, SH-SY5Y cells were treated with U0126 or AMPK siRNA and AKNS-2 (5 μM and 10 μM) was added to the cells after 30 minutes and 36 hours, respectively. At 1 hour after the AKNS-2 treatment, 2 mM MPP+was added to the cells.

Experimental Example 2: Measurement of RFP-GFP-LC3 by Fluorophotometry

SH-SY5Y cells were cultured in a 24-well plate at a density of 8×104cells/well on a glass cover slip. Transfection was performed in accordance with guidelines of the autophagy tandem RFP-GFP-LC3B kit. After culturing the cells for 24 hours, the cells were treated with an LC3B reagent. After the transfected cells were incubated for 24 hours, 50 nM Wart or 100 nM Baf was administered thereto, and then AKNS-2 (10 μM) was added thereto. The cells were immobilized with 4% paraformaldehyde and permeabilized with 0.1% Triton X100. Nuclei thereof were stained with 4,6-diamidino-2-phenylindole (DAPI, 25 μg/mL). Fluorescent signals were detected using a confocal microscope (Leica, Solms, Germany).

Preparation Example 2: Preparation of Cell Lysates

At 24 hours after drug treatment, the culture medium was removed from the 6-well plate, and the cells were gently washed once with cold saline. Subsequently, 1 mL of cold saline was added to each well, and cells attached to the bottom were floated by rubbing the bottom with a 1 mL pipette. The suspension was collected in a 1.5 mL tube and centrifuged at 4° C. at 13000×g for 5 minutes. After removing a supernatant, 50 μL of a RIPA lysis buffer including a protease inhibitor cocktail (Roche, Mannheim, Germany) and purchased from Cell Signaling Technology (Danvers, Mass., USA) was added to the cell pellet. After shaking the mixture at 4° C. for 30 minutes, the obtained cells were centrifuged at 4° C. at 13000×g for 20 minutes. A supernatant was collected therefrom, and a concentration of proteins was measured using a standard curve constructed using BSA by the Bradford method. Then, the supernatant was diluted with a loading buffer and heated at 99° C. for 5 minutes. Resultant cells were used in a subsequent western blot analysis.

Preparation Example 3: Animal Test

In this study, all animal management and experimental protocols were performed in compliance with guidelines of the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology. 40 mice (C57BL/6j, male, 8 weeks old) were purchased from SLC Inc. (Shizuoka, Japan). After arrival, every four mice were hosed in each cage (30 cm×18.5 cm×13 cm) and allowed free access to feed and water. All mice were housed under the following constant conditions: lights on from 6:00 to 18:00, a temperature of 23° C.±1° C., and a humidity of 50%±10%. After 7 days of a habituation period, the mice were used in the experiments according to standard protocols.

Preparation Example 4: Animal Classification and Sample Treatment

40 mice were classified into five groups such that eight mice belonged to each group. After the habituation period, all mice were orally administered with a drug every day. While the mice of Groups 1 and 2 were administered with saline (p.o.), the mice of Groups 3, 4, and 5 were administered with 5 mg/kg ropinirole (p.o.), 5 mg/kg AKNS-2 (p.o.), and 15 mg/kg AKNS-2 (p.o.), respectively. From the 5th day therefrom, the mice of Group 1 were intraperitoneally administered with a saline at 1 hour after gavage feeding of saline, and the mice of Groups 2, 3, 4, and 5 were administered with 30 mg/kg MPTP (i.p.) at 1 hour after gavage feeding of saline, ropinirole, AKNS-2 (5 mg/kg), and AKNS-2 (15 mg/kg), respectively. Each mouse was administered with a single dose of saline/MPTP injection every day for 8 days. At 7 days after the final MPTP injection, all mice were sacrificed by cervical dislocation, and then the whole brain, SNpc, and striatum (ST) were excised for biochemical analysis.FIG.22shows a schematic diagram of an animal test.

Experimental Example 3: Rotarod Test

A rotarod test was performed according to a known method with minimal modification (Borlongan C. V. et al.,Pharmacology Biochemistry and Behavior.1995, 52:225; Hu X. et al.,Neuropharmacology,2017, 117:352). Briefly, the test consisted of a pretraining section and a test section. The pretraining section was performed for consecutive 4 days. The mice were positioned in a cylinder of a rotarod apparatus such that tails of all mice faced an operator and trained for 300 seconds at a constant speed of 16 rpm. During the 300 seconds, mice falling to the floor were placed back on the cylinder by the operator. All mice were trained three times in total every day at an interval of about 30 minutes before administration of MPTP. The mice were treated with a single dose of 30 mg/kg MPTP at 1 hour after administration of AKNS-2 (5 mg/kg and 15 mg/kg) for consecutive 8 days starting on the next day of the last day of behavioral training. Latency to fall off the rotarod was recorded for each mouse. In the test section, performance of all mice was tested on the rotarod according to protocols applied during the pretraining section at three time points, i.e., 2 hours, 24 hours, and 48 hours after the last administration of MPTP. Latency to fall off the rotarod was measured for each mouse. Only mice that stayed on the cylinder for 60 seconds or more in the pretraining section were used for statistical analysis. An average time of three tests was calculated to evaluate balance, grip strength, and motor coordination.

Experimental Example 4: Pole Test

A pole test was performed according to a known method with minimal modification (Choi D. Y. et al.,Neurobiology of Disease,2013, 49:159). Briefly, on the day before MPTP administration, a wood pole (50 cm in length and 1 cm in diameter) with a rough surface was placed in a sound-proof chamber. Each mouse was placed on the top of the pole with the head raised up, and a time taken for the mouse to turn and descend the pole was recorded up to 120 seconds with a stopwatch. The same training was performed three times at an interval of 30 minutes. Performance of all mice on the pole was tested according to protocols applied during the pretraining section at three time points, i.e., 2 hours, 24 hours, and 48 hours after the last administration of MPTP. An average time of three tests was calculated to evaluate motor function.

Experimental Example 5: Wire Hanging Test

A wire hanging test was performed according to a known method with minimal modification (Zhu W. et al., Brain, Behavior, and Immunity, 2018, 69: 568). Briefly, a horizontal wire (1.5 mm in diameter, 50 cm in length, and 30 cm above a bedding material) was fixed between two poles. Fluffy bedding was placed under the wire. Each mouse was handled with its tail such that the mouse held the center of the wire with its front paws. Immediately after the mouse properly floated, a timer was started. A time taken for the mouse to fall off the wire was recorded up to 300 seconds. On the day before MPTP administration, the test was performed three times for each mouse, and an average hanging time of three tests was analyzed as an index to evaluate balance, myofunction, and coordination. Performance of all mice on the wire hanging test was tested at three time points, i.e., 2 hours, 24 hours, and 48 hours after the administration of MPTP. Average latency to fall off the wire onto the bedding material was calculated for each mouse.

Preparation Example 5: Preparation of Brain Tissue of Mouse

On the 7thday after the last administration of MPTP, SNpc and ST of each mouse were carefully excised and stored at −80° C. until use. Thereafter, brain tissue of SNpc and ST was homogenized in a PRO-PREP™ lysis buffer (iNtRON, Gyeonggi, Korea) containing a phosphatase inhibitor cocktail set I (Sigma-Aldrich, Mo., USA). After shaking at 4° C. for 30 minutes, homogenates were centrifuged at 4° C. at 13000×g for 20 minutes, and a supernatant obtained therefrom was collected and added to a new 1.5 mL tube. A concentration of proteins contained in the supernatant was measured by the Bradford method. A part of the supernatant was mixed with the same volume of a loading buffer and denatured in a heater at 99° C. for 5 minutes for western blot analysis. The remaining supernatant was stored at −80° C. for subsequent tests for MAO-B activity and DA level using an ELISA kit.

Experimental Example 6: Measurement of DA Level

DA levels in ST were measured using a competitive ELISA kit (Abnova, Taipei City, Taiwan) according to the manufacturer's instructions. Briefly, ST was homogenized in 0.01 N HCl in the presence of EDTA and sodium metabisulfite. Homogenates were centrifuged at 13000×g for 5 minutes. A supernatant was collected therefrom and used to measure DA levels. After determining a concentration of proteins contained in the supernatant, the DA level in each brain sample was detected twice using the ELISA kit. Absorbance was measured using a microreader (BioTek, Vt., USA) at 450 nm, and the intensity was inversely proportional to the DA level. The DA level was expressed as ng/mg protein.

Experimental Example 7: Determination of MAO-B Activity

A MAO-B assay kit of Promega Corporation (Woods Hollow Road, Madison, Wis., USA) was used to determine MAO-B activity of ST and SN according to the manufacturer's instructions. In order to detect MAO activity, the kit was used according to a homogeneous luminescent method. The assay includes two steps. First, a MAO-B substrate was added to a MAO-B enzyme-containing substance (ST and SN samples) to produce methyl ester luciferin. Next, the produced methyl ester luciferin was reacted with esterase and luciferase to generate light. MAO-B activity was directly proportional to an amount of generated light. Luminescence signals were measured using an Infinite M1000 multimode microplate reader (TECAN, Männedorf, Switzerland). MAO-B activity was expressed as relative light unit (RLU)/mg protein).

Experimental Example 8: Western Blot Analysis

Protein markers contained in lysates of the SH-SY5Y cells and brain tissue of the mice (SNpc and ST) were measured by western blot analysis. Briefly, after determining a concentration, a protein sample (20 μg) was separated by 8%, 10%, or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins separated on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane for 45 minutes using a Trans-Blot Turbo Transfer System (Bio-Rad, USA). The membrane was washed with tris-buffered saline with 0.1% Tween 20, TBST) for 5 minutes and then blocked by 5% skim milk dissolved in a TBST buffer. The membrane was further incubated with monoclonal primary antibodies derived from rabbits (anti-GAPDH, anti-AMPK, anti-p-AMPK, anti-ULK, anti-p-ULK555, anti-Erk, anti-p-Erk, anti-mTOR, anti-p-mTOR, anti-LC3B, anti-TH, and anti-α-synuclein antibodies). The primary antibodies were diluted in a blocking buffer at a ratio of 1:1000 and incubated overnight at 4° C. On the second day, the membrane was washed with TBST three times (10 minutes each) and incubated at room temperature in a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (diluted in a blocking buffer at a ratio of 1:2000). After incubating in the secondary antibody for 1 hour, the membrane was washed for 30 minutes. The protein blots on the membrane were developed using an ECL detection kit and visualized using an LAS-4000 mini system (Fujifilm, Japan). The intensity of the protein blots was quantified using Multi Gauge software (Fujifilm, Japan).

Experimental Example 9: Statistical Analysis

In the present invention, all data were analyzed with GraphPad Prism 7 Software (CA, USA) and expressed as mean±SEM. Statistical analysis was performed using a one-way analysis of variance (ANOVA) or Student's t-test. A p-value of less than 0.05 was regarded as statistically significant.

Example 1: Extraction, Isolation, and Identification

DriedAster koraiensis(A. koraiensis,15 kg) was ground and extracted with 95% ethanol at 65° C. for 3 hours. The extracted solution was evaporated in a vacuum to obtain a powdered extract (1.7 kg, yield: 11.3%). Subsequently, the extract was subjected to fractionation using n-hexane, ethyl acetate, and n-butanol to obtain three types of fractions (149 g, 175 g, and 190 g, respectively). According to biological evaluation, n-butanol fraction (1.4 g) was chromatographed using preparative HPLC under isocratic conditions (CH3CN/H2O, 7:18, flow rate: 10.0 mL/min) to obtain a bioactive fraction (tR=33.0 min). The obtained fraction was separated on a Sephadex LH-20 column (2.8 cm×100 cm, CH3OH, flow rate: 0.25 mL/min) to obtain astersaponin I (Compound 1, 34.6 mg, tR=800 min).

White powder;

[α]20D−18.0 (c 0.01, CH3OH);

IR umax(ATR) 3392, 2916, 2850, 1637, 1571, 1416, 1088 cm−1,

ECD (c 0.1 mM, CH3CN) Δε+3.1 (203);

1H and13C NMR, See Table 1-1 and 1-2 below;

HRESIMS m/z 1487.68823 [M+H]+(calcd for C68H111O35, 1487.69004).

TABLE 1-1δH(J in Hz)δH(J in Hz)PositionδCAglyconeIntensitiesPositionδCSugar moiety144.62.09(dd)J = 14.0 Hz, 2.0 Hz2HGlc1′105.34.49(d)J = 7.5 Hz1.18(dd)J = 14.0 Hz, 3.5 Hz2′74.73.48(m)271.34.33(m)1H3′88.13.52(m)384.23.63(m)1H4′71.13.51(m)443.35′77.53.31(m)548.51.33(m)1H6′62.33.81(m)618.91.50(m)2H3.71(m)734.01.67(m)2HXylI1′′106.24.51(d)J = 7.5 Hz1.35(m)2′′73.23.64(m)841.01H3′′76.33.23(m)948.71.63(m)1H4′′70.23.81(m)1037.75′′67.63.87(d)J = 11.5 Hz1124.82.00, (m)2H3.57(d)J = 11.5 Hz1.96(m)Ara1′′94.15.63(brd)J = 3.0 Hz12123.95.38(brt)J = 3.5 Hz1H2′′′75.63.78(dd)J = 5.0 Hz, 3.0 Hz13144.93′′′70.63.91(m)1443.14′′′66.83.84(m)1536.51.78(m)2H5′′′63.43.92(m)1.39(m)3.49(m)1674.84.49(d)J = 5.0 Hz1HRhaI1′′′′101.05.00(brd)J = 1.5 Hz1750.52′′′′72.34.07(m)1842.33.06(brdd)J = 14.0 Hz, 4.0 Hz1H3′′′′82.73.87(m)1947.82.28(brdd)J = 14.0 Hz, 12.5 Hz2H4′′′′78.93.69(m)1.04(brdd)J = 12.5 Hz, 3.5 Hz5′′′′69.23.71(m)2031.56′′′′18.51.27(d)J = 6.0 Hz

TABLE 1-22136.61.93(m)2HXylII1′′′′′105.04.74(d)J = 8.0 Hz1.16(m)2′′′′′75.43.29(m)2232.11.92(m)2H3′′′′′84.53.41(m)1.80(m)4′′′′′70.43.50(m)2366.03.63(m)2H5′′′′′67.13.86(m)3.24(m)3.20(m)2415.00.95(s)3HRhaII1′′′′′′102.85.14(brd)J = 1.5 Hz2517.81.31(s)3H2′′′′′′72.43.93(m)2618.20.80(s)3H3′′′′′′72.43.70(m)2727.51.38(s)3H4′′′′′′74.13.40(m)28177.25′′′′′′70.14.02(m)2925.30.98(s)3H6′′′′′′18.01.24(d)J = 6.0 Hz3033.50.89(s)3HXylIII1′′′′′′′106.34.50(d)J = 7.5 Hz2′′′′′′′75.43.28(m)3′′′′′′′77.83.32(m)4′′′′′′′71.13.50(m)5′′′′′′′67.33.91(m)3.25(m)

Astersaponin I was isolated as a white amorphous powder. The IR data exhibited absorbance bands at 3368 cm−1and 1657 cm−1, indicating hydroxy and carbonyl groups, respectively (FIG.1). The UV data displayed only terminal absorption at 205 nm (FIG.2), which was attributable to terpene with little conjugation. The molecular formula was deduced to be C68H110O35on the basis of HR-MS data, and the fragmentation patterns (m/z 1487, 1355, 1209, 1077, 945, 799, 667, and 505) suggested that the triterpene aglycone was present with several sugar moieties (FIG.3). The1H and13C NMR data exhibited characteristic signals for aglycone and sugar moieties (see Tables 1-1 and 1-2, andFIGS.4and5). Six distinct methyl singlets (δH1.38, 1.31, 1.27, 0.98, 0.95, 0.89, and 0.80) and an olefinic methine signal (δH5.38) were observed in the1H NMR spectrum, along with three resonances (δC180.1, 144.7, and 123.7) in the13C NMR spectrum, which were indicative of an oleanane-type triterpenoid (FIG.4). Furthermore, two oxymethine signals (δH4.49 and 4.33) and one oxymethylene signal (δH3.63 and 3.24) were observed, and HSQC, COSY, and HMBC correlations indicated that two oxymethine groups were located at C-2 and C-16, while an oxymethylene group was located at C-23 (FIGS.6to9). Consequently, the aglycone was determined to be polygalacic acid. The relative configuration of the aglycone was deduced by ROESY correlation and comparison with NMR data previously reported (FIGS.6and10).

The absolute configuration was determined by using ECD calculation. The measured CD spectrum of Compound 1 exhibited a positive cotton effect (CE) at 203 nm (Δε=+3.1). This cotton effect is similar to that (Δε=+1.84 at 209 nm) of 2β,3β,16β,23-tetrahydroxy-olean-12-en-28-oic acid methyl ester (methyl polygalacate). The measured circular dichroism (CD) spectrum of Compound 1 was fit well with that of the theoretical ECD spectrum (FIG.11). In addition, seven characteristic peaks for anomeric protons were observed in a range between 4.40 ppm and 5.70 ppm [δH5.63 (br d, J=3.0 Hz), 5.14 (br d, J=1.5 Hz), 5.00 (br d, J=1.5 Hz), 4.74 (br d, J=8.0 Hz), 4.51 (br d, J=7.5 Hz), 4.50 (br d, J=7.5 Hz), and 4.49 (br d, J=7.5 Hz)], which were correlated with seven anomeric carbons (δC93.8, 102.8, 101.3, 105.1, 106.2, 106.3, and 105.3). These coupling constants and chemical shifts suggest that seven sugar moieties were one α-arabinose (Ara), two α-rhamnose (Rha I and Rha II), three β-xylose (Xyl I, Xyl II, and Xyl III), and one β-glucose (Glc). The TOCSY and HSQC-TOCSY correlations enabled the grouping and overall assignment of the1H and13C NMR signals of each sugar moiety (FIGS.12and13). The approximate sequence of linkage of sugar moieties was deduced by HR-MS/MS data (FIG.3). The downfield shifts in the1H NMR spectrum and HMBC correlations from anomeric protons to relevant carbons confirmed the exact position and sequence of sugar moieties (FIG.6A). According to a previous report, the structure of Compound 1 was similar to that of conyzasaponin K, except the replacement of β-apiose with β-xylose. Acid hydrolysis and comparative studies with standard samples using HPLC demonstrate that these sugar units were L-arabinose, L-rhamnose, D-xylose, and D-glucose (FIG.14). Consequently, the structure was determined to be 3-O-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosylpolygalacic acid 28-O-α-L-rhamnopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-[β-D-xylopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester, which is called astersaponin I (FIG.15).

Example 3: ECD Computation

All computational methods including conformational distribution, optimization, and energy calculation were performed according to methods well known in the art (J. Nat. Prod.,2016, 79(6):1689-1693). Specifically, conformational searches were performed by employing the procedure implemented in the Spartan'14 software under the MMFF molecular mechanics force field, and the conformers were selected for geometry optimizations. Geometry optimizations were operated with DFT calculations at the B3LYP/6-31+G (d,p) level using the Gaussian 09 package. TDDFT ECD calculations for the optimized conformers were performed at the CAM-B3LYP/SVP level with a CPCM solvent model in MeCN. The calculated ECD spectra were simulated with a half bandwidth of 0.3 eV, and the ECD curves were generated using the SpecDis 1.64 software. The ECD spectra were weighted by Boltzmann distribution after UV correction.

Example 4: Autophagy Induction Assay

To investigate the autophagy inducing effect ofA. koraiensis, human neuroblastoma (SH-SY5Y) cells were cultivated in 6-well plates at a density of 8×105cells/well in 2 mL DMEM medium (Gibco) at 37° C. in a humidified atmosphere with 5% CO2. After incubation for 24 hours, the cells were treated with an ethanol extract ofAster koraiensis(12.5 μg/mL, 25 μg/mL, and 50 μg/mL), an n-BuOH fraction thereof (12.5 μg/mL, 25 μg/mL, and 50 μg/mL), and Compound 1 isolated from the fraction (5 μM, 10 μM, and 20 μM) prepared according to Example 1, respectively. After additional incubation for 24 hours, the cells were harvested and lysed using a RIPA lysis buffer (Cell Signaling). The protein expression of LC3-II in cell lysates was measured using western blot analysis. A rabbit anti-LC3B primary antibody and a goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (both from Cell Signaling) were used to detect LC3 expression. The immune blots were visualized using an ECL detection kit and analyzed using a LAS-4000 mini system (Fujifilm).

The extract, fractions, and astersaponin I (Compound 1) were assessed for effects thereof on autophagy by analyzing the LC3-II/LC3-I ratio in SH-SY5Y cells. The LC3-II/LC3-I ratio has extensively been used as an indicator of autophagy activation because conversion from LC3-I to LC3-II is a necessary process for autophagosome formation. As shown inFIG.16, treatment with the ethanol extract and n-BuOH fraction significantly increased the ratio of LC3-II/LC3-I in a dose-dependent manner, while n-hexane and ethyl acetate fractions did not show an effect on LC3 expression (FIG.16A). Interestingly, treatment of Compound 1 led to an increase in the LC3-II/LC3-I ratio in a dose-dependent manner (FIG.16B), indicating the extent of autophagosome formation and autophagy activation. Previous studies have shown that several triterpene saponins, including ginsenosides, may enhance autophagy in a few cell lines, which are mainly related to cancer. However, autophagy may also have an important role in modulating various neurodegenerative diseases like Parkinson's disease (PD). Therefore, further mechanistic studies will be needed to clarify whether Compound 1 exerts a protective effect on PD through autophagy induction.

Astersaponins are known to exert antitumor, expectorant, and antitussive activities, while no autophagy-inducing effects of astersaponins or conyzasaponins in tumor cells or neuronal cells have been reported. Furthermore, there is no previous literature on astersaponins isolated fromAster koraiensis. Astersaponins have been mainly reported fromAster tataricus(J. Nat. Prod.,2016, 79:1689-1693). Astersaponin I is the first reported saponin fromA. koraiensis. The autophagy-inducing constituent of this plant was also reported for the first time in the present invention.

Example 5: Effect of AKNS Sample on mTOR-Dependent Autophagy Signaling Pathway

In order to investigate autophagy-inducing effects of AKNS samples, SH-SY5Y cells were treated with various concentrations of the AKNS samples (AKNS extract: 12.5 μg/mL, 25 μg/mL, or 50 μg/mL, AKNS n-BuOH fraction: 12.5 μg/mL, 25 μg/mL, or 50 μg/mL, AKNS-2: 5 μM, 10 μM, or 20 μM; and a chemical structure of AKNS-2 is disclosed inFIG.15). After 24 hours of treatment, expression levels of autophagy-involved protein markers were measured by western blot analysis. As shown inFIG.17A, the AKNS extract significantly increased the expression level of LC3-II at concentrations of 12.5 μg/mL, 25 μg/mL, and 50 μg/mL. Upon comparison with the conditions of the control, when the cells were treated with the AKNS extract at concentrations of 25 μg/mL and 50 μg/mL, the expression level of p-AMPK significantly increased, and the expression level of p-mTOR significantly decreased. Additionally, significant increases in p-Erk and p-ULK were confirmed in the AKNS extract-treated group at a high dose of 50 μg/mL.

Similarly, as shown inFIG.17B, upon comparison with the conditions of the control, the AKNS n-BuOH fraction significantly increased the expression level of LC3-II in a dose-dependent manner at a concentration of 12.5 μg/mL, 25 g/mL, or 50 μg/mL. Significant increases in p-Erk and p-ULK were confirmed at a high dose of 50 μg/mL. Also, upon comparison with the conditions of the control, while the expression level of p-AMPK significantly increased, the expression level of p-mTOR significantly increased by AKNS n-BuOH fraction at the concentrations of 25 μg/mL and 50 μg/mL.

Subsequently, autophagy-inducing effects of a single compound (AKNS-2) were identified. As shown inFIG.17C, AKNS-2 treatment induced the expression of LC3-II, which is an important autophagy marker, in a dose-dependent manner in the SH-SY5Y cells. In addition, while the expression level of p-Erk significantly increased due to AKNS-2 treatment at concentrations of 10 μM and 20 μM, the expression levels of p-AMPK and p-ULK significantly increased by AKNS-2 treatment at concentrations of 5 μM, 10 μM, and 20 μM. The expression of p-mTOR was inhibited by AKNS-2 at concentrations of 10 μM and 20 μM. These results indicate that the AKNS samples activated the AMPK/mTOR pathway and/or the Erk/mTOR pathway, resulting in up-regulation of autophagy.

Example 6: Inhibitory Effect of Autophagy Inhibitor on AKNS-2-Activated Autophagy in SH-SY5Y Cells

3-Methyladenine (3-MA) inhibits formation of autophagosomes. In order to identify autophagy activation induced by AKNS-2, accumulation of autophagosomes was inhibited using 3-MA (5 mM) for 30 minutes before treating the SH-SY5Y cells with AKNS-2 (10 μM). As a result, although AKNS-2 (10 μM) significantly increased the expression level of LC3-II (FIG.18A), the increased expression was significantly decreased by 3-MA; and p62, which also has an important role in autophagy, binds to LC3 via a region called the LC3-interacting region (LIR) and may be degraded when autophagy is activated. Interestingly, AKNS-2 also induced a significant decrease in expression of p62 (FIG.18B), and 3-MA treatment remarkably blocked the inhibitory effects of AKNS-2 on the expression level of p62. In a tandem fluorescent protein quenching assay, accumulation of GFP-RFP-LC3-II puncta (FIG.18C) was evaluated using an autophagy sensor of Thermo Fisher Scientific (MA, USA). In the transfected cells treated with AKNS-2 alone, more cytoplasmic puncta stained with green and red fluorescence were observed compared to in the control cells. In order to identify whether AKNS-2 enhances autophagy, the transfected cells were treated with AKNS-2 and wortmannin (Wart; 50 nM) or bafilomycin A, (Baf; 100 nM). While the AKNS-2-enhanced accumulation of green and red LC3-II puncta decreased due to Wart, Baf increased green fluorescence and decreased red fluorescence. This indicates that AKNS-2 activates autophagy.

Example 7: Protective Effect on SH-SY5Y Cells Against MPP+-Induced Neurotoxicity by AKNS-2-Activated Autophagy

Protective effects of AKNS-2 were identified in an MPP+-induced in vitro PD model.FIG.19Ashows that AKNS-2 did not significantly affect cytotoxicity up to a high concentration of 40 μM in an MTT assay. Working concentrations of MPP+and AKNS-2 were also determined by the MTT assay.FIG.19Bshows that MPP+with a concentration of 2 mM significantly decreased cell viability when the SH-SY5Y cells were treated with MPP+having various concentrations. Accordingly, 2 mM MPP+was used to induce an in vitro PD model. Subsequently, protective effects of AKNS-2 against MPP+-induced neurotoxicity were tested. Briefly, the SH-SH5Y cells were treated with various concentrations of AKNS-2, and 1 hour later, 2 mM MPP+was added to the cells. At 24 hours after the treatment, cell viability was detected. As a result (FIG.19C), it was confirmed that AKNS-2 (5 μM and 10 μM) significantly improved cell viability damaged by 2 mM MPP+.

In order to identify whether AKNS-2 has protective effects on MPP+-impaired SH-SY5Y cells by activating autophagy, autophagy activated by AKNS-2 was blocked using the autophagy inhibitor 3-MA. Upon comparison with a group co-administered with AKNS-2 and MPP+, a 3-MA-treated group significantly inhibited the expression of LC3-II (FIG.19D), but p62 exhibited inhibition induced by MPP+(FIG.19E), and AKNS-2 was restored by 3-MA. Accordingly, autophagy activated by AKNS-2 was blocked by 3-MA. Tyrosine hydroxylase (TH) is expressed by the central nervous system. TH converts tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA) that may proceed to DA. TH is a rate-limiting enzyme of DA synthesis. Interestingly, in addition to inhibiting autophagy, the MPP+-induced decrease in expression of TH was reversed by AKNS-2 treatment (FIG.19F), and advantageous effects of AKNS-2 were canceled out by 3-MA treatment.

Example 8: Up-Regulation of Autophagy and Protective Effect Against MPP+Cytotoxicity in SH-SY5Y Cells by Activating Erk/mTOR Pathway by AKNS-2

It has been reported that deficiency of Erk may partially inhibit autophagy, and that activation of Erk activates binding to TSC and inhibits mTORC1, and thus autophagy is up-regulated (Int. J. Biochem. Cell. Biol. 2004, 36, 2491-2502). U0126 is an inhibitor of MEK1/2 kinases. The U0126 inhibits activation of Erk1/2 and may be used to study the role of ErK in autophagy induction. In order to identify whether AKNS-2 activates autophagy by regulating the Erk/mTOR signal-transmitting pathway, the SH-SY5Y cells were treated with AKNS-2 (5 μM and 10 μM) in the presence or absence of U0126 (10 μM). 24 hours of incubation, the expression of proteins involved in autophagy induction and the Erk-regulated autophagy pathway was evaluated by western blot analysis. The results are shown inFIG.20. The expression level of LC3-II was significantly increased by AKNS-2 treatment (5 μM and 10 μM) compared to the conditions of the control. When 10 μM U0126 was added 30 minutes before the AKNS-2 treatment, a significant decrease was observed in the expression of LC3-II (FIG.20A) in the groups treated with AKNS-2 (5 μM and 10 μM), indicating that AKNS-2-induced autophagy was inhibited by U0126. While significantly increased expression levels of p-Erk were observed in AKNS-2-treated groups (5 μM and 10 μM) compared to the control (FIG.20B), the increased expression of p-ErK in the cells treated with AKNS-2 (10 μM) was canceled out by administration of 10 μM U0126. Subsequently, expression of p-mTOR was measured (FIG.20D). Upon comparison with the conditions of the control, AKNS-2 (10 μM) significantly inhibited the expression p-mTOR which had been inhibited, and the expression of p-mTOR inhibited by AKNS-2 (5 μM and 10 μM) was considerably restored when the cells were administered with 10 μM U0126 30 minutes before AKNS-2 treatment. While AKNS-2 (5 μM and 10 μM) significantly increased expression of p-ULK555 as shown inFIG.20C, 10 μM U0126 canceled out the effects of AKNS-2 (10 μM) on an increase in expression of p-ULK. This indicates that AKNS-2 activates the Erk/mTOR pathway, resulting in up-regulation of autophagy in the SH-SY5Y cells.

TH is a rate-limiting enzyme of DA synthesis. α-Synuclein is a primary constituent of LB, one of the pathologic features of PD. The present invention is intended to study protective effects of AKNS-2 against MPP+-induced neurotoxicity in the SH-SY5Y cells. Cell viability was identified by MTT assay. The results ofFIG.20Eindicate that AKNS-2 (5 μM and 10 μM) significantly enhanced cell viability that had decreased by MPP+, but such enhancement was canceled out by treatment with U0126. Next, expression of LC3 was identified (FIG.20F). These results indicate that U0126 blocked the effects of AKNS-2 (5 μM and 10 μM) on increasing the expression of LC3-II. Similarly, the effects of co-administration of AKNS-2 and MPP+on increasing the expression of LC3-II were canceled out by administration of U0126. Based on these results, it was confirmed that AKNS-2 improved cell viability, and that AKNS-2 improved autophagy and cell viability by regulating the Erk signal transmission. Also, while expression of TH significantly decreased due to 2 mM MPP+(FIG.20G), the decreased expression of TH may be restored by AKNS-2 (5 μM and 10 μM). When U0126 was administered before treatment with AKNS-2 and MPP+, the effects of AKNS-2 on increasing the expression of TH were inhibited. With regard to expression of α-synuclein, 2 mM MPP+significantly increased the expression of α-synuclein in the SH-SY5Y cells (FIG.20H), and AKNS-2 treatment canceled out the effects of MPP+and inhibited the expression of α-synuclein. Interestingly, the protective effects were blocked by the presence of U0126. AKNS-2 reversed the changed expression of TH and α-synuclein and enhanced cell viability that had been reduced by MPP+treatment in the SH-SY5Y cells. However, such protective effects may be inhibited by disturbing autophagy by blocking the Erk signal-transmitting pathway. This indicates that AKNS-2 activates autophagy by regulating the Erk signal, thereby having protective effects against MPP+-induced cytotoxicity.

Example 9: Up-Regulation of Autophagy and Protective Effect Against MPP+Cytotoxicity by AKNS-2 Via Activation of AMPK/mTOR Pathway in SH-SY5Y Cells

Activation of AMPK induces activation of TSC1/2, and accordingly inhibits the activity of mTOR by activating ULK1, which activates autophagy, and inactivating a TOR activator Rheb. In some cells, knockout of ULK1 blocks autophagy induction, indicating that ULK1 is a factor in the progression of autophagy. In order to identify whether AKNS-2 up-regulates autophagy by regulating the AMPK/mTOR pathway, AMPK signal transmission was disturbed in the SH-SY5Y cells by using AMPK siRNA (50 nM), and then the SH-SY5Y cells were treated with AKNS-2 (5 μM and 10 μM). 24 hours after the AKNS-2 treatment 24, representative protein markers, including LC3, AMPK, mTOR, and ULK, involved in the AMPK/mTOR signal-transmitting pathway and regulating autophagy were measured by western blot analysis. Significantly increased expression of LC3-II (FIG.21A), p-AMPK (FIG.21B), and p-ULK555 (FIG.21C) was observed in the AKNS-2-treated groups compared to the control. Increased expression of LC3-II, p-AMPK, and p-ULK555 by AKNS-2 was significantly decreased in SH-SY5Y cells transfected by AMPK siRNA when compared with SH-SY5Y cells treated with normal AKNS-2. In addition, although AKNS-2 significantly inhibited the expression of p-mTOR (FIG.21D), the inhibition was canceled out in accordance with the disturbance of AMPK signal transmission.

Subsequently, it was identified whether AKNS-2 protects the SH-SY5Y cells against MPP+-induced cytotoxicity, and a role of AMPK-mediated autophagy in the protection was identified. The SH-SY5Y cells were transfected with 50 nM AMPK siRNA and then treated with AKNS-2 (5 μM and 10 μM) in the presence or absence of 2 mM MPP+. Cell viability was tested by the MTT assay, and the results are shown inFIG.21E. As a result, significantly reduced cell viability was observed due to MPP+treatment when compared with the control, but AKNS-2 (5 μM and 10 μM) reversed the effects of MPP+on cell viability. Cell viability of the AMPK siRNA-transfected SH-SY5Y cells was far lower than that of normal SH-SY5Y cells in the presence of ANKS-2 and MPP+. With regard to expression of the protein markers, although the expression of LC3-II significantly increased by AKNS-2 (5 μM and 10 μM) in the normal SH-SY5Y cells (FIG.21F), the expression level of LC3-II of the AMPK siRNA-transfected cells was far lower than that of the normal SH-SY5Y cells in the presence of AKNS-2. Similarly, the expression of LC3-II significantly decreased in the siRNA-transfected cells after co-administration with AKNS-2 and MPP+compared to the normal cells. Furthermore, the expression of TH significantly decreased by MPP+treatment (FIG.21G), and AKNS-2 treatment offset toxicity of MPP+and reversed the expression of TH. Interestingly, the AKNS-2-mediated increase in the expression of TH was canceled out by AMPK siRNA. MPP+significantly increased the expression of α-synuclein (FIG.21H), and the increased expression level of α-synuclein was decreased by AKNS-2 in the normal cells. However, in the AMPK siRNA-transfected cells, inhibitory effects of AKNS-2 on α-synuclein were canceled out. This indicates that AKNS-2 up-regulates autophagy by regulating the AMPK signal transmission, and that activated autophagy protects the SH-SY5Y cells against the MPP+-induced toxicity.

Example 10: Effect of AKNS-2 on Enhancing Behavior Performance of MPTP-Induced In Vivo PD Model

The present inventors tested protective effects of AKNS-2 in the MPP+-induced in vitro PD model (schematic diagram inFIG.22A). First, effects of AKNS-2 on MPTP-impaired behavioral performance were tested by using a rotarod test, a pole test, and a wire hanging test. MPTP was administered to mice for 8 consecutive days after training. At 2 hours, 24 hours, and 48 hours after the last administration of MPTP, behavioral performance was tested in the rotarod test, the pole test, and the wire hanging test. In the rotarod test, although behavioral performance of the mice was significantly impaired at 2 hours and 24 hours when compared with the normal group, the impairment was reversed by administering ropinirole (5 mg/kg) and AKNS-2 (15 mg/kg) at 2 hours and 24 hours. Treatment with AKNS-2 (5 mg/kg) significantly improved MPTP-impaired behavior performance in the rotarod test at 24 hours. At 48 hours, behavior performance of all mice was restored to a normal level (FIG.22B). MPTP treatment significantly impaired the behavior performance in the pole test at 2 hours after treatment. However, the impaired behavior performance was considerably restored by administering ropinirole and AKNS-2 (5 mg/kg and 15 mg/kg) after 2 hours. At 24 hours and 24 hours after MPTP injection, no significant differences were observed in all groups (FIG.22C). With regard to the wire hanging test, upon comparison with the conditions of the control, MPTP injection significantly deteriorated latency to fall off at 2 hours and 24 hours. At 48 hours, deterioration of latency induced by MPTP was restored up to a level observed during the pre-training. While ropinirole (5 mg/kg), as a positive control, improved latency deteriorated by MPTP at 2 hours, impaired behavior performance was significantly improved by AKNS-2 (15 mg/kg) at 2 hours and 24 hours in the wire hanging test (FIG.22D).

Example 11: Protective Effect of AKNS-2 on Mouse Damaged by MPTP Administration

DA is a neurotransmitter that transmits a signal from one neuron to another in the brain. In addition, dopaminergic neuron damage induces loss of DA, causing motor symptoms of PD. The DA level in ST was measured using an ELISA kit (Abnova, Taipei City, Taiwan). As a result,FIG.23Ashows that the DA level significantly decreased by 30 mg/kg MPTP administration in ST. Interestingly, the decrease in the DA level induced by MPTP was restored by ropinirole (5 mg/kg) and AKNS-2 (15 mg/kg). This indicates that AKNS-2 protected dopaminergic neurons against damage induced by MPTP.

MPTP may be converted into MPP+by MAO-B in glial cells, and MPP+is an actual toxin that damages dopaminergic neurons. A MAO-B inhibitor inhibits the metabolism of MPTP into MPP+by blocking the action of MAO-B. The present invention is intended to identify whether dopaminergic neurons may be protected from toxicity of MPTP by inhibiting the activity of MAO-B. In SN and ST, the activity of MAO-B was detected using a MAO-B assay kit (Promega, Woods Hollow Road, Madison, Wis., USA). The results show that MPTP (30 mg/kg) significantly increased the activity of MAO-B in both ST (FIG.23B) and SN (FIG.23C). However, AKNS-2 (5 mg/kg and 15 mg/kg) could not reduce the activity of MAO-B that had been increased by MPTP in both ST and SN. This indicates that AKNS-2 is not an efficient MAO-B inhibitor, and that protective effects of AKNS-2 in the MPTP-impaired mice are not caused by inhibition of MAO-B.

The MPTP-induced PD model is characterized by a decrease in TH in dopaminergic neurons. LBs are one of the pathological properties of PD, and α-synuclein is a primary constituent of LBs. In the present invention, expression of TH and α-synuclein were measured in ST and SN of the MPTP-impaired mice by western blot analysis.FIG.23Dshows representative immunoblots of TH and α-synuclein in ST and SN. The TH level significantly decreased in the MPTP-treated group (30 mg/kg) compared to in the normal control in both ST (FIG.23E) and SN (FIG.23F). Interestingly, the decreased TH level in both ST and SN was considerably reversed by ropinirole administration (5 mg/kg), as the positive control, and AKNS-2 administration (5 mg/kg and 15 mg/kg). As shown inFIGS.23G and23H, the α-synuclein level significantly increased due to MPTP administration in both ST and SN, and a significant decrease in the α-synuclein level was observed in the positive control treated with ropinirole (5 mg/kg) compared to in the MPTP-treated mice. Also, a significant decrease in the α-synuclein level was observed in the AKNS-2-treated mice (5 mg/kg and 15 mg/kg) in both ST and SN compared to in the MPTP-treated mice.

Example 12: Induction of Autophagy by AKNS-2 in MPP+-Induced In Vitro PD Model

AKNS-2 induces autophagy in SH-SY5Y cells and protects cells against MPP+-induced cytotoxicity due to autophagy activation. The present invention was intended to identify the effects of AKNS-2 on regulating autophagy in the MPP+-induced in vitro PD model. At 7 days after treatment with AKNS-2 and MPTP, autophagy-related protein markers were measured in ST and SN using western blot analysis.FIGS.23A and23Bshow representative immunoblots and relative intensities of the protein markers in ST and SN, respectively. This indicates that MPTP increased the expression of LC3-II to some extent, but no noticeable difference was observed in ST and SN. Compared to MPTP treatment, AKNS-2 treatment (15 mg/kg) induced a significant increase in the expression of LC3-II in ST. Although no noticeable difference was observed, increased expression of LC3-II was observed in SN of the AKNS-2-treated group (15 mg/kg). Upon comparison with the normal group, a significant decrease in the expression of p62 was observed in ST of the MPTP-treated group, significantly decreased p62 levels were observed in both ST and SN of the AKNS-2-treated groups (5 mg/kg and 15 mg/kg) compared to the MPTP-treated group. Additionally, MPTP increased the expression level of p-AMPK in both ST and SN. Compared to MPTP treatment, treatment with ropinirole and AKNS-2 induced a significant increase in p-AMPK in both ST and SN. Furthermore, upon comparison with the conditions of the control, expression of p-Erk in both ST and SN decreased due to MPTP treatment (30 mg/kg) and significantly increased due to AKNS-2 treatment (15 mg/kg) compared to MTPT treatment. Similar to p-Erk expression, the p-ULK level noticeably decreased by MPTP administration in ST and SN, and treatment with ropinirole and AKNS-2 (5 mg/kg and 15 mg/kg) canceled out the effects of MPTP and significantly increased the expression of p-ULK in ST and SN. Also, the present inventors measured the expression of p-mTOR in mice, and no significant difference was observed in ST or SN between the normal group and the MPTP-treated group. Although no noticeable decrease was observed, ropinirole and AKNS-2 clearly showed a tendency to decrease the expression of p-mTOR in ST. In SN, a significant decrease in p-mTOR was confirmed in the groups treated with ropinirole and AKNS-2.