3.1. General Experimental Procedures
The used HPLC was composed of dual Shimadzu LC-10AT pumps and a Shimadzu SPD-10A UV-vis detector (Shimadzu Inc., Kyoto, Japan), as well as a Waters Atlantis T3 RP 150 × 4.6 mm, 5 μm preparative column (Waters Corp., Milford, CT, USA) or a Thermo ODS Hypersil 250 × 4.6 mm, 5 μm preparative column (Thermo Fisher Scientific Inc., Rockford, IL, USA). UV spectra were obtained using a JASCO UV-530 ultraviolet spectrophotometer (JASCO Inc., Tokyo, Japan). IR spectra were obtained on a Mattson Genesis II infrared spectrophotometer (Thermo Fisher Scientific Inc., Tokyo, Japan). Optical rotations were measured with JASCO DIP 370 and P-1020 digital polarimeters (JASCO Inc., Tokyo, Japan). NMR (400 MHz and 600 MHz) spectra were obtained on a Varian Unity 400 MHz FT-NMR and a Varian Unity 600 MHz FT-NMR (Varian Inc., Palo Alto, CA, USA). ESI-MS data were collected on a VG Biotech Quattro 5022 mass spectrometer (VG Biotech, Altrincham, UK). High-resolution ESI-MS data were obtained on a Bruker APEX II spectrometer (FT-ICR/MS, FTMS) (Bruker Daltonics Inc., Billerica, MA, USA).
Column chromatography (CC) was performed on silica gel (40–63 and 63–200 μm, Merck KGaA, Darmstadt, Germany). Preparative TLC and TLC analyses were performed on silica gel 60 F254 plates (Merck KGaA) and spots were visualized by UV and by spraying 50% H2SO4-ethanol solution followed by heating for 5 min.
Eagle’s phenol-red free minimum essential medium, fetal bovine serum, l-glutamine, and the antibiotic mixture (penicillin-streptomycin) were purchased from Invitrogen Co. (Invitrogen Co., Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Na3PO4, EDTA, X-Gluc, and Triton X-100, and doxorubicin [(D1515-98.0%–102.0% (HPLC)] were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), K3Fe(CN)6, K4Fe(CN)6, were purchased from Merck (Merck KGaA). The purity of the isolated compounds used for biological assays was determined by HPLC (>95%). 17β-Estradiol (E2) was purchased from TCI (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan).
3.3. Extraction and Isolation
The dried roots of F. macrophylla
(7.7 kg) were extracted with 95% aqueous MeOH (10 L × 5) at room temperature and then concentrated under reduced pressure. The crude extract (654.0 g) was partitioned with H2
O (2 L × 3) and EtOAc (2 L × 3) to yield a H2
O layer (524.0 g), an insoluble portion (1.7 g) and an EtOAc layer (127.4 g). The insoluble portion was recrystallized from MeOH to yield genistin (4
, 1.2 g). The EtOAc layer was then partitioned between n-hexane (2 L) and 90% aqueous MeOH solution (2 L × 3) to provide an n-hexane layer (35.5 g) and a MeOH layer (91.9 g). The MeOH layer exhibited the most potent estrogenic activity (Figure S1
) and was selected for further bioactivity-guided fractionation (Figure S2
). The MeOH layer was isolated by silica gel column chromatography (63–200 μM, 8.5 × 35 cm) under a gradient elution of n-hexane:EtOAc:MeOH (1:0:0→0:0:1) to yield 14 fractions.
Fraction one (FM-1, 243.8 mg) was loaded on a silica gel column (40–63 μM, 2.5 × 30 cm), and eluted with gradient mixtures of n-hexane:CHCl3 (1:0→5:1→1:1) to yield 11 fractions. A mixture of β-sitosterol and stigmasterol (23 and 24) (4.7 mg) was separated from FM-1-10 (16.1 mg) by preparative TLC (CHCl3:EtOAc, 25:1).
Fraction two (FM-2, 546.9 mg) was loaded on a silica gel column (40–63 μM, 3.0 × 33 cm) eluted with gradient solvent mixtures of n-hexane:CHCl3:MeOH (3:1:0→0:1:0→0:30:1→0:20:1→0:10:1), followed by purification using reversed phase solid–phase extraction (RP-SPE, C18 gel) to yield six fractions and fleminone (16) (20.6 mg). FM-2-3-SP2 (10.8 mg) was selected to be purified on RP-HPLC (MeOH:H2O, 3:1) yielding flemiflavanone A (20) (5.0 mg).
Fraction three (FM-3, 397.0 mg) was loaded on a silica gel column (40–63 μM, 3.0 × 32 cm) eluted with gradient solvent mixtures of CHCl3:EtOAc (1:0→20:1→15:1) to yield seven fractions. FM-3-4 (104.6 mg) was purified by RP–HPLC (MeOH:H2O, 7:3) to yield flemiphilippinin F (3) (15.9 mg) and isoderrone (15) (3.1 mg) and also two subfractions (FM-3-4-L1 and FM-3-4-L2) were obtained. Subfraction FM-3-4-L2 (10.9 mg) was purified by RP-HPLC (MeCN:H2O, 7:3) to yield 7-(3,3-dimethylallyl)genistein (9) (6.4 mg).
Fraction six (FM–6, 1.5 g) was chromatographed on a silica gel column (40–63 μM, 2 × 29 cm), which was eluted with CHCl3:EtOAc (8:1) to yield five fractions (FM-6-1 to FM-6-5). FM-6-1 (105.3 mg) was loaded on a silica gel column eluted with CHCl3:MeOH (30:1) to yield prunetin (10) (2.5 mg) and olmelin (11) (1.0 mg). FM-6-3 (308.1 mg) was selected for a silica gel column chromatography to yield three fractions. FM-6-3-1 (214.7 mg) was purified by RP-SPE with MeOH:H2O (3:2→1:0) to yield six fractions. FM-6-3-1-SP3 (11.2 mg) was purified by RP-HPLC (MeOH−:H2O, 4:1) to yield flemichin E (2) (1.5 mg). FM-6-3-1-SP5 (10.4 mg) was purified by RP-HPLC (MeOH:H2O, 4:1) to yield a mixture of 17 and 18 (2.5:1) (5.5 mg). FM-6-3-2 was purified by RP-SPE with MeOH:H2O (3:2→1:0) to yield six fractions. FM-6-3-2-SP3 (74.0 mg) was similarly purified by RP-HPLC (Waters, C18, 150 × 4.6 mm; MeOH:H2O = 3:1, HPLC–Shimadzu LC-10AT; UV-vis 254 nm; flow rate: 2 mL/min) to yield flemingin (1) (3.6 mg), 8 (48.0 mg) and 13 (1.2 mg). FM-6-5 (259.4 mg) was chromatographed on silica gel column chromatography, eluted with CHCl3:MeOH (30:1) to yield four fractions (FM-6-5-1 to FM-6-5-4). From FM-6-5-3, compound 21 (74.1 mg) was separated. FM-6-5-2 (110.9 mg) was isolated using the same solvent system (CHCl3:MeOH, 30:1) to yield 21 (92.3 mg).
Fraction seven (FM-7, 7.2 g) was recrystallized from n-hexane–EtOAc to render genistein (5) (898.0 mg). The remaining material of FM-7 was then separated by silica gel column chromatography and eluted with CHCl3-MeOH (25:1→20:1) to yield seven fractions (FM-7-1 to FM-7-7). From FM-7-6, compound 6 (2′-hydroxygenistein, 241.4 mg) was separated. FM-7-2 (1.1 g) was chromatographed on silica gel column (CHCl3–MeOH, 35:1) and purified by RP-SPE (MeOH:H2O, 40:1→1:0) and RP-HPLC (MeOH:H2O, 7:3) to yield cajanin (7) (11.3 mg), neoraufurane (14) (1.7 mg), erythrinin B (12) (3.2 mg), and flemiphilippinin D (19) (4.0 mg).
Fraction nine (FM–9, 5.2 g) was loaded on a silica gel column (63–200 μM, 4.5 × 21 cm) eluted with CHCl3:MeOH (6:1) to yield eight fractions. FM-9-7 (3.1 g) and FM-9-8 (583.4 mg) were combined and chromatographed on silica gel column eluted with gradient solvent mixtures of CHCl3:MeOH (10:1→6:1→5:1) to yield 4′-O-methyl-gallocatechin (22) (1.7 g). The isolated compounds were identified by NMR and the spectra were compared with the previously published data.
), 5,2′,4′-trihydroxy-7,8-(1,2,2-trimethyl-dihydrofurano)isoflavone: yellow amorphous solid;
: −13.45° (c
); UV λmax
(MeOH) nm (log ɛ
): 263 (4.41), 295 (3.99), 335 (4.52); IR (neat) Vmax
: 3377, 1651, 1615, 1506; HRESIMS m
377.1001 [M + Na]+
(calculated for C20
: 377.1005); 1
H and 13
C NMR data: Table 1
Flemichin E (2), (2S)-(5,2′-dihydroxy-6,7-(1,1-dimethylpyrano)-8-(3-methylbut-2-enyl)-4′,5′-(2- hydroxy-1,1-dimethylpyrano)flavanone: yellow amorphous solid; UV λmax (MeOH) nm (log ɛ): 266 (4.63), 273 (4.65), 292 (4.30), 311 (4.18), 364 (3.65), 335 (4.52); IR(neat) Vmax cm−1: 3366, 1642, 1601, 1507; HRESIMS m/z 529.2202 [M+Na]+ (calculated for C30H34O7: 529.2199).