Journal of Natural Products
Article
Ltd., Suzhou, China), and a chiral HPLC column (Chiralcel OD-H,
150 × 4.6 mm i.d.; Daicel Chemical Industries, Ltd., Tokyo, Japan)
were used for analytical purposes. A YMC-Pack ODS-A HPLC column
(C18, 250 × 10 mm i.d.; YMC, Tokyo, Japan) was used for
semipreparative purposes. Gas chromatography was conducted on an
Agilent 7820A system. An OV-17 capillary column (30 m × 0.32 mm
× 0.5 μm; Lanzhou Zhongke Antai Analysis Technology Co., Ltd.,
Lanzhou, China) was used for the GC analysis. The DPPH, FRAP, and
MTT assays were all performed on a BioTek Synergy 2 multimode
microplate reader. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazo-
lium bromide (MTT), amyloid β-protein fragment 25−35 (Aβ25−35),
2,2-diphenyl-1-picrylhydrazyl, L-ascorbic acid (vitamin C), α-tocopher-
ol (vitamin E), L-cysteine methyl ester hydrochloride, hexamethyl
disilazane, trimethylchlorosilane, D-glucose, L-glucose, D-arabinose, and
L-arabinose were purchased from Aladdin Industry Corporation,
Shanghai, China. β-Cellulase (from Trichoderma viride, activity ≥35
u/mg) was manufactured by Shanghai YuanYe Biotechnology Co.,
Ltd., Shanghai, China. Trifluoroacetic acid was purchased from
Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. A FRAP
assay kit was purchased from the Beyotime Institute of Biotechnology,
Nantong, China.
8.65 × 10−3, CH3OH) λmax (θ) 220 (−10817, tr), 240 (730, pk), 265
(−1415, tr), 281 (−167, pk), 298 (−1183, tr) nm; IR (KBr) νmax 3423,
2963, 2921, 1714, 1595, 1513, 1443, 1366, 1259, 1158, 1106, 1068,
1033, 806, 763, 679 cm−1; 1H and 13C NMR data, see Table 1;
HRESIMS m/z 409.1256 [M + Na]+ (calcd for C21H22O7Na,
409.1263).
(8R,8′R)-9-O-(6′-O-α-L-Arabinofuranosyl)-β-D-glucopyranosyldi-
hydrocubebin (3): white, amorphous powder; [α]2D0 −44 (c 0.2,
CH3OH); UV (CH3OH) λmax (log ε) 236 (3.97), 288 (3.96) nm;
ECD (c 1.63 × 10−3, CH3OH) λmax (θ) 234 (−1852, tr), 260 (−218,
pk), 275 (−380, tr), 296 (344, pk) nm; IR (KBr) νmax 3392, 2928,
2892, 1633, 1610, 1489, 1442, 1360, 1317, 1247, 1190, 1038, 927, 861,
810, 776, 719, 636 cm−1; 1H and 13C NMR data, see Table 2;
HRESIMS m/z 675.2252 [M + Na]+ (calcd for C31H40O15Na
675.2265).
(8R,8′R)-9-O-β-D-Glucopyranosylpiperphilippinin VI (4a) and
(8R,8′R)-9′-O-β-D-glucopyranosylpiperphilippinin VI (4b): white,
amorphous powder; [α]2D0 −18 (c 0.2, CH3OH); UV (CH3OH) λmax
(log ε) 231 (4.10), 284 (3.90) nm; ECD (c 1.578 × 10−3, CH3OH)
λmax (θ) 214 (1729, tr), 218 (2312, pk), 232 (−1290, tr), 243 (−690,
pk), 244 (−693, tr), 258 (−271, pk), 283 (−599, tr), 312 (−101, pk)
nm; IR (KBr) νmax 3417, 2920, 2852, 1644, 1608, 1513, 1490, 1443,
1369, 1247, 1157, 1124, 1076, 1035, 926, 864, 811, 739 cm−1; 1H and
13C NMR data, see Table 3; HRESIMS m/z 545.1984 [M + Na]+
Plant Material. The fruit of S. glaucescens was collected in the
Shennongjia Mountains of Hubei Province, China, in September 2011
and was identified by Mr. Shi-Gui Shi (Shennongjia Institute for Drug
Control). A voucher specimen (ID 20110905) was deposited in the
Herbarium of Materia Medica, Faculty of Pharmacy, Tongji Medical
College of Huazhong University of Science and Technology, Wuhan,
China.
(calcd for C26H34O11Na, 545.1999) and 1067.4086 [2 M + Na]+
(calcd for C52H68O22Na, 1067.4100).
ECD Calculations. The conformational spaces for the 7′R,8′S and
7′S,8′R isomers of compounds 1 and 2 were explored using the
BALLOON program with a genetic algorithm.33 The conformations
generated by BALLOON were subjected to semiempirical PM3
quantum mechanical geometry optimizations using the Gaussian 09
program.34 Duplicate conformations were identified and removed
when the root-mean-square (RMS) distance was less than 0.5 Å for
any two geometry-optimized conformations. The remaining con-
formations were further optimized at the B3LYP/6-31G* level of
theory in MeOH with the IEFPCM solvation model using Gaussian
09,35 and the duplicate conformations emerging after these
calculations were removed according to the same RMS criteria
above. The harmonic vibrational frequencies were calculated to
confirm the stability of the final conformers (Figures S69 and S70,
Supporting Information). The oscillator strengths and rotational
strengths of the 20 weakest electronic excitations of each conformer
were calculated using the TDDFT methodology at the B3LYP/6-
311G** level of theory with MeOH as the solvent with the IEFPCM
solvation model implemented in Gaussian 09. The ECD spectra for
each conformer were simulated using a Gaussian function with a
bandwidth σ of 0.45 eV. The spectra were combined after Boltzmann
weighting according to their population contributions (Tables S1 and
S2, Supporting Information).
Enzymatic Hydrolysis. To obtain the aglycones without the
occurrence of structural changes, compounds 3 and 4 (each 5 mg)
were treated with β-cellulase (from Trichoderma viride, 0.5 mg for both
3 and 4) in aqueous solution (2 mL for both 3 and 4) at 50 °C for 3 h.
After extraction with EtOAc (5 mL × 5), the combined EtOAc layer
was evaporated to dryness and analyzed by TLC and HPLC.
Semipreparative HPLC was used to isolate the aglycones of the two
compounds.
Acid Hydrolysis and GC Analysis. GC was utilized to determine
the absolute configurations of the saccharides of compounds 3 and 4,
according to a slightly modified literature procedure.36 Briefly, 3 and 4
(each 2 mg) were heated at 90 °C with 4 mol/L aqueous TFA (2 mL)
for 3 h. The reaction mixture was freeze-dried overnight and diluted
with H2O (5 mL). After extraction with EtOAc (5 mL × 4), the
aqueous layer was evaporated to dryness, and the residue was dissolved
in pyridine (1 mL) and reacted with 5 mg of L-cysteine methyl ester
hydrochloride at 60 °C for 1 h. Subsequently, 0.9 mL of hexamethyl
disilazane−trimethylchlorosilane (2:1) was added, and the mixture was
heated at 60 °C for 1 h. The mixture was washed with H2O (5 mL ×
2) and extracted with n-hexane, and the n-hexane layer was analyzed
by GC-FID using an OV-17 capillary column (30 m × 0.32 mm × 0.5
Extraction and Isolation. The air-dried fruit of S. glaucescens (15
kg) was extracted with 95% ethanol at room temperature, and the
solution was concentrated in vacuo to yield a crude extract (750 g).
The extract was added to 1.5 L of deionized H2O and sequentially
partitioned with petroleum ether (PE), EtOAc, and n-BuOH. The
petroleum ether-soluble fraction (210 g) was chromatographed on a
silica gel column eluting with PE−EtOAc (from 1:0 to 6:4) to afford
10 fractions. Fraction 6 (18.5 g) was chromatographed on silica gel
(CHCl3−EtOAc, from 1:0 to 3:1) and Sephadex LH-20 (CH2Cl2−
MeOH, 1:1) followed by semipreparative HPLC (MeOH−H2O,
80:20) to yield compounds 1 (11 mg) and 2 (8 mg). The EtOAc-
soluble fraction (150 g) was chromatographed on a silica gel column
eluting with PE−EtOAc (from 99:1 to 1:2) to afford 14 fractions.
Fraction 2 (12.5 g) was chromatographed on Sephadex LH-20
(CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, from 15:1 to 10:1)
followed by semipreparative HPLC (MeOH−H2O, 85:15) to yield
compounds 6 (16 mg), 8 (560 mg), 9 (18 mg), and 14 (26 mg).
Fraction 3 (4.8 g) was chromatographed on Sephadex LH-20
(CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, 6:1) columns to
yield compound 13 (70 mg). Fraction 4 (15.3 g) was chromato-
graphed on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel
(PE−EtOAc, from 9:1 to 3:1) columns to yield compounds 7 (240
mg) and 12 (110 mg). Fraction 6 (5.3 g) was chromatographed on
Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc,
4:1) followed by semipreparative HPLC (MeOH−H2O, 77.5:22.5) to
yield compounds 10 (15 mg) and 11 (22 mg). Fraction 10 (19.7 g)
was chromatographed on MCI gel (MeOH−H2O, from 40:60 to
100:0) and silica gel (CH2Cl2−MeOH, from 85:15 to 60:40) followed
by semipreparative HPLC (MeOH−H2O, 55:45) to yield compounds
3 (42 mg), 4 (11 mg), and 5 (350 mg).
(7′R,8′S)-3,4-Dimethoxy-3′,4′-methylenedioxy-7,8-seco-7,7′-ep-
oxylignan-7,8-dione (1): colorless oil; [α]2D0 −39 (c 0.9, CHCl3); UV
(CH3OH) λmax (log ε) 228 (4.73), 257 (4.35), 288 (4.03) nm; ECD (c
9.48 × 10−3, CH3OH) λmax (θ) 208 (−5231, tr), 234 (3957, pk), 259
(−1864, tr), 281 (697, pk), 294 (−550, tr) nm; IR (KBr) νmax 3433,
2922, 1713, 1598, 1512, 1446, 1416, 1270, 1248, 1221, 1175, 1137,
1
1100, 1037, 814, 764, 678 cm−1; H and 13C NMR data, see Table 1;
HRESIMS m/z 409.1258 [M + Na]+ (calcd for C21H22O7Na,
409.1263).
(7′R,8′S)-3,4-Methylenedioxy-3′,4′-dimethoxy-7,8-seco-7,7′-ep-
oxylignan-7,8-dione (2): colorless oil; [α]2D0 −73 (c 1.5, CHCl3); UV
(CH3OH) λmax (log ε) 226 (4.56), 259 (4.13), 288 (3.85) nm; ECD (c
H
dx.doi.org/10.1021/np4010536 | J. Nat. Prod. XXXX, XXX, XXX−XXX