3.3. Extraction and Isolation
mycelium was extracted and isolated as previously reported with slight modifications [41
]. Briefly the MeOH extract (45 g) was suspended in water and then partitioned with EtOAc and n-BuOH. The EtOAc fraction (4.41 g) was subjected to silica gel column chromatography (CC) using CHCl3
–MeOH (20:1, 9:1, 7:3, 5:5) to give four fractions (E1–E4).
Fraction E1 (116 mg) was separated by silica gel CC eluted with hexane-EtOAc (4:1, 2:1, 1:1) to give five fractions (E11–E15). Fraction E11 was separated by ODS column chromatography (CC) using 90% MeOH in water and then purified by preparative TLC using hexane-EtOAc (5:1) to obtain compound 1 (8 mg). Fraction E2 (982 mg) was subjected to silica gel CC eluted with CHCl3-EtOAc (2:1, 1:1) to give ten fractions (E21–E30). Compound 2 (29.6 mg) was obtained from fraction E24 (80 mg) by silica gel CC using CHCl3-EtOAc (9:1). Fraction E29 (82 mg) was separated by ODS CC using MeOH-H2O (3:7, 5:5, 7:3) and then purified by RP-HPLC using MeOH-H2O (9:1) to obtain 3 (6 mg). Fraction E3 (1.215 g) was separated by silica gel CC eluted with CHCl3-MeOH-H2O (7:3:0.5) to give two fractions (E31, E32). Fraction E31 (956 mg) was separated by silica gel CC using hexane-EtOAc (1:4, 0:1) to afford four fractions (E311–E314). Fraction 314 (276 mg) was separated on Sephadex LH-20 CC eluted with MeOH to give three fractions (E3141–E3143). Fraction 3142 (71 mg) was further separated by ODS CC eluted with MeOH-H2O (1:2) to give three fractions (E31421–E31423). Compound 4 (5 mg) was obtained from fraction E31421 (32 mg) by RP-HPLC using 5% acetonitrile in water. Compound 5 (10 mg) was obtained from fraction E32 (215 mg) by RP-HPLC using MeOH-H2O (4:6). Fraction E4 (1.08 g) was separated by ODS CC using MeOH-H2O (5:5, 7:3) to give three fractions (E41–E43). Fraction E41 (459 mg) was separated by silica gel CC eluted with EtOAc-MeOH (9:1) to give two fractions (E411, E412). Fraction E412 (112.2 mg) was separated by silica gel CC using CHCl3-MeOH (9:1) and then purified by RP-HPLC using 10% MeOH in water to obtain 6 (1 mg). Fraction E42 (384 mg) was separated by silica gel CC using a gradient of CHCl3-MeOH (20:1, 10:1) to afford four fractions (E421–E424). Fraction 421 (116.3 mg) was further separated by ODS CC using MeOH-H2O (1:1) to give three fractions (E4211–E4213). Compound 7 (2 mg) was obtained from fraction E4213 (24.8 mg) by silica gel CC eluted with CHCl3-EtOAc (1:5).
Compound 2 (=3-(hydroxymethyl)-2-furaldehyde):yellow oil; 1H NMR (CD3OD, 500 M Hz) δ 4.72 (2H, s, H-7), 6.52 (1H, d, J = 3.5 Hz, H-4), 7.22 (1H, d, J = 3.5 Hz, H-5),9.58 (1H, s, H-6); 13C NMR (CD3OD, 125 M Hz) δ 57.8 (C-7), 110.2 (C-4), 123.0 (C-5), 152.5 (C-2), 160.8 (C-3), 177.9 (C-6); EIMS (rel int) m/z 126[M]+ (100); HREIMS m/z 126.0315 [M]+ (calcd for C6H6O3, 126.0317).
Acetylation of compound 5. To a solution of 5 (3 mg) in pyridine (1 mL) was added acetic anhydride (1 mL), and the mixture stood at RT for 24 h. The reaction mixture was concentrated to dryness, the residue were purified on silica gel CC using EtOAc, affording a new diacetate 5a (2.1 mg) as an off-white gum. 1H NMR (CDCl3, 500 M Hz) δ 1.50 (3H, d, J = 6.0 Hz), 2.10 (3H, d, J = 3.0 Hz), 2.16 (3H, d, J = 1.5 Hz), 4.90 (2H, s), 5.90 (1H, dd, J = 6.5, 1 Hz), 6.36 (1H, s), 7.79 (1H, d, J = 0.5 Hz); 13CNMR (CDCl3, 125 M Hz) δ 19.9, 20.6, 21.2, 61.1, 65.7, 114.7, 129.6, 151.9, 162.2, 169.6, 169.9, 176.8; HRCIMS m/z 255.0874 [M + H]+(calcd for C12H15O6, 255.0863).
: amorphous, white powder; IR (film) νmax
3354 (OH, NH), 1659 (C=O), 1626, 1541, 1507, 1370, 1215, 1067 cm−1
H NMR (CDCl3
, 600 M Hz) and 13
C NMR (CDCl3
, 150 M Hz) data, Table 1
; CIMS m
168 [M + H]+
; HRCIMS m
168.0658 [M + H]+
(calcd for C8H10NO3, 168.0655).
3.7. Western Blotting Analysis
PC12 cells (1 × 106 cells/mL) were seeded in poly-l-lysine-coated six-well plates in normal serum medium for 24 h, then shifted to low serum medium for 14 h prior to exposure to vehicle (0.1% DMSO) or indicated reagents. Cells were lysed on ice for 30 min in RIPA buffer, 10 mM PMSF (Beyotime Institute of Biotechnology, Shanghai, China), 1% protein inhibitor cocktail (Sigma-Aldrich China Inc.: Shanghai, China). Lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. The protein concentration was measured by the BCA protein assay kit (Solarbio Science and Technology Co., Ltd., Beijing, China). Each sample (30 μg) was separated by SDS-PAGE in 8–12% gels, transferred to a PVDF membrane (0.45 μm, Solarbio Science and Technology Co., Ltd.: Beijing, China). The membranes were blocked at room temperature in 5% BSA in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20 (Polysorbate 20)) for 1 h. Blots were incubated with the appropriate primary antibodies (Cell Signaling Technology, Inc., Beverly, MA) overnight at 4 °C, detected with HRP-conjugated secondary antibodies (Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at room temperature and finally visualized by ECL chemiluminescence reagents (Beyotime Institute of Biotechnology: Shanghai, China).