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Meroterpenoids with BACE1 Inhibitory Activity from the Fruiting Body of Boletinus asiaticus Genki Yatsu,† Yoshihiro Kino,‡ Hiroaki Sasaki,§ Jun-ichi Satoh,‡ Kaoru Kinoshita,† and Kiyotaka Koyama*,†

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†

Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan ‡ Department of Bioinformatics and Molecular Neuropathology, Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose-shi, Tokyo 204-8588, Japan § School of Pharmacy, Shujitsu University, Nishigawara 1-6-1, Naka-ku, Okayama-shi, Okayama 703-8516, Japan S Supporting Information *

ABSTRACT: BACE1 inhibitory activity-guided fractionation of an extract of the fruiting body of Boletinus asiaticus yielded five novel meroterpenoids (1−5) and one known compound (6; asiaticusin A). The structures of these compounds were determined by interpretation of NMR, MS, and IR spectral data. The five new compounds contain 4-hydroxybenzoic acid and geranylgeranoic acid units. Compounds 4−6 possessed BACE1 inhibitory activity (IC50 values: 14.7, 11.4, and 2.0 μM, respectively).

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A

RESULTS AND DISCUSSION Dry fruiting bodies of B. asiaticus were extracted with CHCl3 and MeOH at room temperature. Each extract showed BACE1 inhibitory activity at 100 μg/mL. The MeOH extract was subjected to silica gel column chromatography, ODS column chromatography, and preparative HPLC using bioactivityguided isolation to obtain six compounds including five new meroterpenoids (1−5). 14′,15′-Dihydroasiaticusin A methyl ester (1) was obtained as a colorless oil. Its molecular formula was established to be C28H40O5 on the basis of HREIMS. The IR spectrum of 1 suggested the presence of hydroxy (3258 cm−1) and carbonyl (1713 and 1683 cm−1) groups. The 1H NMR spectrum of 1 showed three aromatic protons at δH 6.85 (d, J = 8.9 Hz, H-5), 7.87 (d, J = 8.9 Hz, H-6), and 7.89 (s, H-2), three olefinic protons at δH 5.06 (m, H-6′), 5.08 (m, H-10′), and 5.33 (t, J = 7.1 Hz, H-2′), one methyl ester proton at δH 3.68 (s, −COOCH3), four methyl protons at δH 1.14 (d, J = 7.1 Hz, H17′), 1.55 (s, H-19′), 1.59 (s, H-18′), and 1.78 (s, H-20′), seven methylene protons at δH 1.34−1.40 (m, H-13′), 1.92− 2.18 (m, H-4′, 5′, 8′, 9′, 12′), 2.45 (m, H-14′), and 3.41 (d, J = 7.1 Hz, H-1′), and one methine proton at δH 1.92−2.18 (m, H-15′). The 13C NMR and DEPT spectra showed two carbonyl carbons at δC 171.2 (C-1-COOH) and 177.8 (C-16′), six aromatic carbons at δC 115.7 (C-5), 121.5 (C-1), 127.0 (C-

lzheimer’s disease (AD), the most common type of dementia, is a progressive neurodegenerative disorder that causes various symptoms including loss of memory and cognitive ability.1 There are over 40 million people worldwide who live with dementia, and AD accounts for 50−60% of them.2,3 Neuronal cell death caused by the accumulation of amyloid beta (Aβ) peptide, the amyloid cascade hypothesis,4 is proposed as a pathogenesis of AD. Aβ is produced by cleavage of the amyloid precursor protein (APP) by two proteases, βsecretase (BACE1) and γ-secretase. Aβ generated from APP aggregates and forms senile plaques in the brain. BACE1 plays an essential role in generating Aβ; thus inhibiting BACE1 may contribute to disease-modifying therapy.5 Previously, biflavonoids,6,7 triflavonoids,8 p-terphenyls,9 and dimethylorsellinic acid-based meroterpenoids10,11 have been reported as natural BACE1 inhibitors. Boletinus asiaticus in the family Suillaceae is a mushroom that grows in far eastern Asia. 4-Hydroxybenzoic acid-based meroterpenoids, asiaticusin A (6) and its geometric isomer asiaticusin B, were isolated from this mushroom.12 Their bioactivity has not been reported. In this study, we report the isolation, structure determination, and BACE1 inhibitory activity of five novel 4hydroxybenzoic acid-based meroterpenoids, named 14′,15′dihydroasiaticusin A methyl ester (1), asiaticusinols A−C (2− 4), and asiachromenic acid (5), along with the known compound asiaticusin A (6). © 2019 American Chemical Society and American Society of Pharmacognosy

Received: December 25, 2018 Published: June 25, 2019 1797

DOI: 10.1021/acs.jnatprod.8b01092 J. Nat. Prod. 2019, 82, 1797−1801

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Chart 1

Table 1. 1H and

13

C NMR Spectroscopic Data for 1−3

14′,15′-dihydroasiaticusin A methyl ester (1)a position

δC

1 2 3 4 5 6 1-COOH 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ 16′-OCH3

121.5 132.5 127.0 159.5 115.7 130.4 171.2 29.5 121.0 139.0 39.7 26.2 124.6 134.5 39.4 26.5 123.6 135.5 25.4 33.4 39.6 39.4 177.8 17.0 16.0 15.7 16.3 51.6

δH (J in Hz) 7.89, s

6.85, d (8.9) 7.87, d (8.9) 3.41, d (7.1) 5.33, t (7.1) 1.92−2.18, m 1.92−2.18, m 5.06, m 1.92−2.18, m 1.92−2.18, m 5.08, m 1.92−2.18, m 1.34−1.40, m 2.45, m 1.92−2.18, m 1.14, 1.59, 1.55, 1.78, 3.68,

d (7.1) s s s s

asiaticusinol A (2)b δC

δH (J in Hz)

122.6 132.6 129.3 161.1 115.2 130.4 171.1 28.9 123.5 137.2 40.8 27.6 126.1 135.1 43.5 127.7 138.8 73.2 42.3 24.8 144.1 128.7 171.8 12.4 28.2 16.2 16.3

7.76, d (2.1)

6.78, d (8.5) 7.74, dd (8.5, 2.1) 3.32, m 5.33, t (6.4) 2.01−2.24, m 2.01−2.24, m 5.19, t (5.9) 2.66, d (6.4) 5.22, td (15.5, 6.4) 5.52, d (15.5) 1.55−1.58, m 2.01−2.24, m 6.75, t (6.2)

1.78, 1.24, 1.59, 1.71,

s s s s

asiaticusinol B (3)b δC 123.0 132.6 128.9 160.6 115.2 130.3 171.4 29.1 124.2 136.3 43.6 127.0 139.4 73.5 43.8 23.9 126.6 135.1 39.4 28.2 139.4 129.6 172.6 12.6 15.9 28.0 16.4

δH (J in Hz) 7.75, d (2.0)

6.77, d (8.5) 7.70, dd (8.5, 2.0) 3.34, mc 5.40, t (6.2) 2.76, d (6.3) 5.57, dt (15.6, 6.3) 5.63, d (15.6) 1.48−1.52, m 1.96−2.10, m 5.14, t (7.3) 1.96−2.10, m 2.28, br q (7.5) 6.73, t (7.5)

1.79, 1.56, 1.24, 1.72,

s s s s

a

Measured in CDCl3. bMeasured in methanol-d4. cOverlapped signal with methanol-d4.

asiaticusin A (6).12 Therefore, 1 might contain a prenylated 4hydroxybenzoic acid skeleton. The NMR assignment of 1 was achieved using the HMBC method and comparison of the NMR data with compound 6. The prenylated benzoic acid unit of 1 was assigned by HMBC correlations observed from H-2 to C-4, C-6, and C-1-COOH, H-5 to C-6, and H-1′ to C-2, C-3, C-4, and C-2′. The carbomethoxy group at C-16′ was identified by HMBC correlations from H-16′-OCH3 to C16′ and H-17′ to C-16′. The reduced olefinic carbons at C-14′ and C-15′ were also confirmed by HMBC correlations from H-

3), 130.4 (C-6), 132.5 (C-2), and 159.5 (C-4), six olefinic carbons at δC 121.0 (C-2′), 123.6 (C-10′), 124.6 (C-6′), 134.5 (C-7′), 135.5 (C-11′), and 139.0 (C-3′), one methyl ester carbon at δC 51.6 (C16′-OCH3), four methyl carbons at δC 15.7 (C-19′), 16.0 (C-18′), 16.3 (C-20′), and 17.0 (C-17′), eight methylene carbons at δC 25.4 (C-12′), 26.2 (C-5′), 26.5 (C-9′), 29.5 (C-1′), 33.4 (C-13’), 39.4 (C-8′), 39.6 (C-14′), and 39.7 (C-4′), and one methine carbon at δC 39.4 (C-15′) (Table 1). The spectroscopic data for 1 were similar to those of the known 4-hydroxybenzoic acid-based meroterpenoid 1798

DOI: 10.1021/acs.jnatprod.8b01092 J. Nat. Prod. 2019, 82, 1797−1801

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Figure 1. HMBC (blue arrows) and 1H−1H COSY (red lines) correlations of 1−5.

an olefinic moiety at δC 127.7 (C-9′), 138.8 (C-10′), and δH 5.22 (td, J = 15.5, 6.4 Hz, H-9′), 5.52 (d, J = 15.5 Hz, H-10′). The HMBC correlations from H-8′ to C-9′ and C-10′, H-9′ to C-10′, H-10′ to C-11′, H-12′ to C-10′ and C-11′, and H-18′ to C-11′ revealed the presence of a shifted double bond and an oxygenated prenyl unit.13 The configuration of the shifted double bond was determined to be the E form by the coupling constant between H-9′ and H-10′ (J = 15.5 Hz). From the above, the structure of 2 was determined as shown in Figure 1. Since the ECD spectrum showed no Cotton effect, 2 was obtained as a racemic mixture. Asiaticusinol B (3) was obtained as a colorless oil. The molecular formula of 3 was determined to be C27H36O6 on the basis of HRFABMS. The NMR data of 3 were almost identical to those of 2. In the 13C NMR spectrum, the shifted olefinic methine carbon at δC 127.0 (C-5′), 139.4 (C-6′) and one sp3 carbon at δC 73.5 (C-7′) were confirmed as in 2. The assignment of the shifted double bond was determined at C-5′ and C-6′ using HMBC correlations from H-2′ to C-4′, H-4′ to C-5′, H-5′ to C-4′, H-6′ to C-4′, H-8′ to C-6′ and C-7′, H-19′ to C-7′, and H-20′ to C-4′. The assignment of the 1H and 13C NMR data was performed by 2D NMR analysis. On the basis of the optical rotation and the ECD spectrum, 3 was obtained as a racemic mixture. Asiaticusinol C (4) was obtained as a colorless oil. The molecular formula of 4 was determined to be C27H36O6 on the basis of HRFABMS. The NMR data for 4 were similar to those of 1, indicating that 4 also has a prenylated 4-hydroxybenzoic acid moiety. The 1H NMR spectrum revealed the presence of three aromatic protons at δH 6.77 (d, J = 8.5 Hz, H-5), 7.70 (dd, J = 8.5, 2.1 Hz, H-6), and 7.76 (d, J = 2.1 Hz, H-2), three olefinic protons at δH 5.19 (t, J = 6.8 Hz, H-10′), 5.37 (t, J = 7.3 Hz, H-2′), and 6.73 (t, J = 7.3 Hz, H-14′), two exomethylene protons at δH 4.83 (br s, H-19′) and 5.01 (br s, H-19′), one methine proton at δH 4.01 (t, J = 6.3 Hz, H-6′), seven methylene protons at δH 1.98−2.20 (m, H-4′, 5′, 8′, 9′, 12′), 2.29 (br q, J = 7.3 Hz, H-13′), and 3.34 (overlapped, H1′), and three methyl protons at δH 1.62 (s, H-18′), 1.73 (s, H20′), and 1.79 (s, H-17′). The 13C NMR and DEPT spectra showed two carboxyl carbons at δC 170.4 (C-1-COOH) and 171.7 (C-16′), six aromatic carbons at δC 115.0 (C-5), 122.5 (C-1), 129.0 (C-3), 130.2 (C-6), 132.4 (C-2), and 160.8 (C4), seven olefinic carbons at δC 123.5 (C-2′), 126.1 (C-10′), 128.8 (C-15′), 135.1 (C-11′), 139.6 (C-3′), 143.3 (C-14′), and 152.5 (C-7′), one exomethylene carbon at δC 110.1 (C19′), one tertiary carbon at δC 75.3 (C-6′), seven methylene carbons at δC 27.5 (C-9′), 28.0 (C-13′), 28.8 (C-1′), 32.1 (C-

14′ to C-17′ and H-17′ to C-15′. Optical rotation of 1 indicated a specific rotation ([α]25D −8.8), although the electronic circular dichroism (ECD) spectrum did not show any Cotton effect. These data suggested that 1 was isolated as a scalemic mixture. Asaiticusinol A (2) was obtained as a colorless oil. The molecular formula of 2 was determined to be C27H36O6 on the basis of HRFABMS. The NMR data for 2 were similar to those of 1, indicating that 2 might also have a prenylated 4hydroxybenzoic acid moiety. The 1H and 13C NMR spectra revealed the presence of one sp3 carbon at δC 73.2 (C-11′) and Table 2. 1H and

13

C NMR Spectroscopic Data for 4 and 5

asiaticusinol C (4)b position

δC

1 2 3 4 5 6 1-COOH 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′ 19′

122.5 132.4 129.0 160.8 115.0 130.2 170.4 28.8 123.5 139.6 36.7 34.6 75.3 152.5 32.1 27.5 126.1 135.1 39.1 28.0 143.3 128.8 171.7 12.3 15.8 110.1

20′

16.1

δH (J in Hz) 7.76, d (2.1)

6.77, d (8.5) 7.70, dd (8.5, 2.1) 3.34, mc 5.37, t (7.3) 1.98−2.20, m 1.98−2.20, m 4.01, t (6.3) 1.98−2.20, m 1.98−2.20, m 5.19, t (6.8) 1.98−2.20, m 2.29, br q (7.3) 6.73, t (7.3)

1.79, 1.62, 4.83, 5.01, 1.73,

s s br s br s s

asiachromenic acid (5)a δC 121.5 128.7 120.5 158.2 116.0 131.9 171.5 125.2 129.9 80.1 41.6 22.5 123.9 135.2 39.4 26.3 125.2 133.6 38.0 27.4 144.8 127.0 173.2 12.1 15.9 15.9 27.3

δH (J in Hz) 7.69, s

6.77, d (8.5) 7.84, d (8.5) 6.37, d (10.1) 5.60, d (10.1) 1.62−1.79, m 1.98−2.14, m 5.08−5.11, m 1.98−2.14, m 1.98−2.14, m 5.08−5.11, m 1.98−2.14, m 2.27, br q (7.3) 6.87, t (7.3)

1.81, s 1.59, s 1.55, s 1.43, s

a

Measured in CDCl3. bMeasured in methanol-d4. cOverlapped signal with methanol-d4. 1799

DOI: 10.1021/acs.jnatprod.8b01092 J. Nat. Prod. 2019, 82, 1797−1801

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Fraction 2.3 (456.6 mg) was fractionated by three chromatography steps: (1) silica gel column chromatography eluted with CHCl3− MeOH−H2O (70:1:0.1, 0:100:0, 0:0:100), (2) ODS column chromatography with MeOH−H2O (7:3, 8:2, 9:1, 10:0), and (3) preparative HPLC eluted with MeCN−H2O (7:3) to obtain 1 (8.5 mg, tR 40 min) and 5 (4.8 mg, tR 24 min). Fraction 2.5 (1.2 g) was subjected to ODS column chromatography with MeCN−H2O (4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0) to provide 6 (129.3 mg). Compound 6 was eluted with MeCN−H2O (8:2). Fraction 2.6 (806.7 mg) was fractionated using silica gel column chromatography with n-hexane−EtOAc−MeOH (10:1:0, 7:1:0, 5:1:0, 3:1:0, 1:1:0, 0:100:0, 0:0:100) to yield seven fractions (2.6.1−2.6.7). Fraction 2.6.3 was subjected to two chromatography steps: (1) silica gel column chromatography eluted with CHCl3−MeOH−H2O (50:1:0.1, 40:1:0.1, 30:1:0.1, 0:100:0) and (2) preparative HPLC eluted with MeCN−H2O (1:1) to provide 4 (3.8 mg, tR 20 min). Fraction 2.6.4 (50.0 mg) was subjected to two chromatography steps: (1) ODS column chromatography with MeOH−H2O (5:5, 6:4, 10:0) and (2) preparative HPLC eluted with MeCN−H2O (1:1) to obtain 3 (1.5 mg, tR 18 min). Fraction 2.6.7 (147.8 mg) was subjected to three chromatography steps: (1) ODS column chromatography with MeOH−H2O (8:2, 10:0), (2) silica gel column chromatography with CHCl3−MeOH− H2O (70:1:0.1, 60:1:0.1, 50:1:0.1, 40:1:0.1, 30:1:0.1, 0:100:0), and (3) preparative HPLC eluted with MeCN−H2O (1:1) to yield 2 (4.3 mg, tR 18 min). 14′,15′-Dihydroasiaticusin A methyl ester (1): colorless oil; [α]25D −8.8 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 206 (4.38), 253 (4.00) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 3258, 1713, 1683 cm−1; 1H and 13C NMR (CDCl3), see Table 1; HREIMS m/z 456.2862 [M]+ (calcd for C28H40O5, 456.2874). Asiaticusinol A (2): colorless oil; [α]20D ±0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.61), 254 (4.12) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 3174, 2923, 1679 cm−1; 1H and 13C NMR (methanol-d4), see Table 1; HRFABMS m/z 455.2444 [M − H]− (calcd for C27H35O6, 455.2434). Asiaticusinol B (3): colorless oil; [α]20D ±0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (4.54), 254 (4.06) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 2926, 1683, 1652 cm−1; 1H and 13C NMR (methanol-d4), see Table 1; HRFABMS m/z 455.2427 [M − H]− (calcd for C27H35O6, 455.2434). Asiaticusinol C (4): colorless oil; [α]29D −0.8 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 206 (4.38), 253 (4.00) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 2926, 1683 cm−1; 1H and 13C NMR (methanol-d4), see Table 2; HRFABMS m/z 455.2443 [M − H]− (calcd for C27H35O6, 455.2434). Asiachromenic acid (5): colorless oil; [α]25D −3.4 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 206 (4.32), 292 (3.41) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 2925, 1683, 1644 cm−1; 1H and 13C NMR (CDCl3), see Table 2; HREIMS m/z 438.24014 [M]+ (calcd for C27H34O5, 438.2406). BACE1 FRET Assay. BACE1 assays of isolated compounds 1−6 and material extracts were performed using the BACE1 FRET assay kit, Red (Thermo Fisher Scientific, USA). Test samples were dissolved in the assay buffer (50 mM sodium acetate, pH 4.5). A 9 μL amount of test sample, BACE1 substrate (750 nM of RhEVNNLDAEFK-Quencher in 50 mM ammonium bicarbonate), and BACE1 enzyme (1 U/mL) were mixed on a 384-well black plate and incubated 3 h at room temperature. The fluorescence intensities were measured by a SYNERGY HTX multimode reader (BioTek, USA) for excitation at 540 nm and emission at 590 nm. The inhibition rate was calculated by the following equation: inhibition (%) = [1 − {(S − S0) − (B − B0)/(C − C0) − (B − B0)}] × 100, where C is the fluorescence of a DMSO control [enzyme, substrate, and assay buffer with DMSO (final concentration: 10%)] after 3 h of incubation, C0 is the fluorescence of the DMSO control at 1 h after incubation, B is the fluorescence of a no-enzyme control [substrate and assay buffer with DMSO (final concentration: 10%)] after 3 h of incubation, B0 is the fluorescence of the no-enzyme control at 1 h after incubation, S is the fluorescence of the tested samples (enzyme, sample solution, and

8′), 34.6 (C-5′), 36.7 (C-4′), and 39.1 (C-12′), and three methyl carbons at δC 12.3 (C-17′), 15.8 (C-18′), and 16.1 (C20′). The exomethylene carbon at δC 110.1 (C-19′) and oxygenated methine carbon at δC 75.3 (C-6′) were determined by HMBC correlations from H-2′ to C-4′ and C-20′, H-6′ to C-4′, C-5′, C-7′, C-8′, and C-19′, H-10′ to C-8′ and C-9′, H19′ to C-7′ and C-8′, and H-20′ to C-2′, C-3′, and C-4′. The assignment of the 1H and 13C NMR data was achieved using HMBC spectral data. The optical rotation and ECD spectrum suggested that 4 was also a racemic mixture. Asiachromenic acid (5) was obtained as a colorless oil. The molecular formula of 5 was determined to be C27H34O5 on the basis of HREIMS. 1H and 13C NMR spectra indicated the presence of one coupled olefin at δC 125.2 (C-1′), 129.9 (C2′), and δH 6.37 (d, J = 10.1 Hz, H-1′) 5.60 (d, J = 10.1 Hz, H2′) and a carbon at δC 80.1 (C-3′). The HMBC correlations from H-1′ to C-2, C-3, and C-3′, H-4′ to C-3′ and C-20′, and H-20′ to C-2′ and C-3′ and a COSY correlation between H-1′ and H-2′ suggested that 5 might possess a chromene skeleton. Since the other NMR spectral data were similar to those of 1, we assumed that 5 also had a prenylated benzoic acid moiety. The assignment of the 1H and 13C NMR data was performed using HMBC spectral data. The ECD spectrum of 5 did not show any Cotton effect, suggesting that 5 was isolated as a racemic mixture. BACE1 inhibitory activity was evaluated using the BACE1 FRET assay.6 β-Secretase inhibitor IV (Merck, Germany) was used as a positive control (IC50 15 nM). Isolated compounds 1−6 were tested for their BACE1 inhibitory activity, and 4−6 inhibited BACE1 activity with IC50 values of 14.7, 11.4, and 2.0 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Horiba SEPA-300 polarimeter. IR spectra were recorded with a Thermo FT-IR Nicolet iS5 spectrophotometer (ATR). UV spectra were recorded with a Thermo Genesys 10S UV− vis spectrophotometer. ECD measurements were carried out on a Jasco 820 spectropolarimeter. 1D and 2D NMR spectra of 1−6 were measured with a JEOL JNM-AL400 MHz spectrometer, using tetramethylsilane as the internal standard. Low- and high-resolution EI and FABMS spectra were measured with a JEOL JMS-700 spectrometer. Column chromatography was performed using silica gel 60N (63-210 μm, Kanto Chemical) and ODS silica gel (YMC-GEL ODS-A, YMC). Preparative HPLC was performed on an SSC-3461 equipped with an SSC-5410 UV detector at 254 nm (Senshu Scientific) at a flow rate of 3.0 mL/min and a Jasco PU-2080 Plus equipped with a Jasco PU-2075 Plus UV detector at 254 nm at a flow rate of 3.0 mL/min. The HPLC column was an Inertsustain C18 column (10⦶ × 250 mm, 5 μm, GL Sciences). Fungal Material. Fruiting bodies of Boletinus asiaticus were collected in Yamanashi Prefecture, Japan, in October 2002. A voucher specimen (BA-2002) was deposited at the Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University. Species identification was confirmed by one of the authors (K. Koyama). Extraction and Isolation. Dried and fractured fruiting bodies of B. asiaticus (200 g) were extracted three times each with CHCl3 and MeOH at room temperature. The MeOH extract (34.3 g) was fractionated by silica gel column chromatography with CHCl3− MeOH (100:1, 50:1, 30:1, 7:1, 5:1, 1:1, 0:100) to yield six fractions (1−6). Fraction 2 (9.7 g) was subjected to two chromatography steps: (1) silica gel column chromatography with CHCl3−MeOH−H2O (30:5:0.5, 30:10:1, 6:4:1, 0:100:0) and (2) silica gel column chromatography with CHCl3−MeOH (50:1, 25:1, 10:1, 5:1, 3:1, 0:100), to yield seven fractions (2.1−2.7). 1800

DOI: 10.1021/acs.jnatprod.8b01092 J. Nat. Prod. 2019, 82, 1797−1801

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substrate) after 3 h of incubation, and S0 is the fluorescence of the tested samples at 1 h after incubation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01092. 1 H and 13C NMR, 2D NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-42-495-8911. Fax: +81-42-495-8912. E-mail: [email protected]. ORCID

Kiyotaka Koyama: 0000-0001-9884-0080 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a grant from the Dementia Drug Resource Development Center (DRC) Project, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (S1511016).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b01092 J. Nat. Prod. 2019, 82, 1797−1801