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M. Li et al. / Fitoterapia 99 (2014) 48–55
same computational level. Then ECD calculations were carried
6″-O-(3R-1-N-β-D-glucopyranosyl-2-oxo-3-hydroxy-in-
dole-3-acetyl)spinosin (6)
out in the methanol solvent medium using time-dependent
density functional theory (TDDFT) with B3LYP functional and
DGDZVP basis set.
Yellow solid; [α]D27 −42.0 (c 0.25, MeOH); UV (MeOH) λmax
(log ε): 212 (4.70), 268 (4.38), 335 (4.46) nm; IR (KBr) νmax
:
3398, 2882, 1733, 1608, 1351, 1075, 581 cm−1; CD (MeOH)
2.7. ORAC assay
λ
max (Δε) 213 (+12.0), 240 (−13.4), 263 (+1.4) nm; 1H NMR
and 13C NMR, see Tables 2 and 3. HR-ESI-MS m/z 960.2777 [M
+ H]+ (calcd. for C44H50NO23, 960.2774).
Automated ORAC assay was carried out on a GENios
luciferase-based microplate reader (TECAN, Switzerland) with
an excitation/emission filter pair of 485/527 nm as previously
described [12]. Fluorescein was used as a fluorescence probe,
and the reaction was initiated with the addition of AAPH.
Trolox was used as a standard. The final results were calculated
on the basis of the difference in the area under the fluorescence
decay curve between the AAPH control and each sample.
Samples were prepared in stock solutions of 5 μM, 2.5 μM and
1.25 μM respectively. Each sample at a scheduled concentration
was done in quadruplicate. Data are expressed as micromoles
of Trolox equivalents (TE) per microliter of sample (μmol TE/
mL or U/mL).
6″-O-(3S-1-N-β-D-glucopyranosyl-2-oxo-3-hydroxy-in-
dole-3-acetyl)-6‴-feruloylspinosin (7)
Yellow solid; [α]D27 −59.2 (c 0.25, MeOH); UV (MeOH) λmax
(log ε): 212 (4.76), 283 (4.45), 330 (4.61) nm; IR (KBr) νmax
:
3412, 2882, 1721, 1607, 1354, 1077, 572 cm−1; CD (MeOH)
max (Δε) 208 (−16.4), 240 (+10.9), 262 (−6.2), 308 (+9.9),
347 (−11.1) nm; 1H NMR and 13C NMR, see Tables 2 and 3. HR-
ESI-MS m/z 1136.3250 [M + H]+ (calcd. for C54H58NO26
1136.3247).
λ
,
6″-O-(3R-1-N-β-D-glucopyranosyl-2-oxo-3-hydroxy-in-
dole-3-acetyl)-6‴-feruloylspinosin (8)
3. Results and discussion
Yellow solid; [α]D27 −28.0 (c 0.25, MeOH); UV (MeOH) λmax
(log ε): 212 (4.68), 283 (4.35), 330 (4.52) nm; IR (KBr) νmax
:
The crude extract of the seeds of Z. jujuba var. spinosa was
subjected to macroporous resin, silica gel and HW-40 column,
followed by repeated RP-HPLC to afford eight new compounds
1–8.
3410, 2882, 1721, 1607, 1354, 1076, 581 cm−1; CD (MeOH)
λmax (Δε) 215 (+9.9), 234 (−8.4), 262 (+6.2), 308 (+9.8),
346 (−11.8) nm; 1H NMR and 13C NMR, see Tables 2 and 3. HR-
ESI-MS m/z 1136.3229 [M + H]+ (calcd. for C54H58NO26
,
Compounds 1 and 2 were isolated as colorless solids. The
HRESIMS of 1 showed the molecular ion peak at m/z 392.0966
[M + Na]+, corresponding to the molecular formula C16H19NO9,
with eight degrees of unsaturation. The 1H NMR spectrum of 1
revealed the presence of 1,2-disubstituted benzene ring, as well
as one sugar moiety with the anomeric proton at δ 5.40 (1H, d,
J = 9.0 Hz). In the 13C NMR spectrum, only 15 carbon signals
were observed, including one carbonyl carbon (δ 179.3), one
quaternary carbon (δ 74.3) and one methylene (δ 43.7), as well
as signals for benzene ring and sugar moiety. Acid hydrolysis of
1 yielded D-glucose, which was detected by HPLC analysis [11].
The large coupling constant (9.0 Hz) of the anomeric proton
indicated a β configuration. The complete assignments of the
benzene and glycosidic signals were achieved by 2D-NMR
experiments. The planar structure was established according to
the HMBC correlations from H-1′ to C-2/C-8, from H-4 to C-3, and
from H-10 to C-2/C-3/C-9/C-11 (Fig. 2). The molecular formula
of compound 2 was also determined as C16H19NO9 by HRESIMS
at m/z 392.0963 [M + Na]+. The NMR data of 2 was similar to
those of 1 (Table 1). Comprehensive analysis of 2D-NMR spectra
established the planar structure of 2, which was the same as 1.
Compounds 3–8 were all obtained as yellow amorphous
powders. The molecular formula of 3 was established to be
C44H49NO23 by HRESIMS at m/z 960.2764 [M + H]+, with
21 degrees of unsaturation. The UV absorption bands at 211,
268, and 336 nm were recorded, which were the characteristic
absorptions of a flavone skeleton. The 1H NMR and 13C NMR
spectra showed a doubling of many of the signals, which were
in a nearly 1:1 ratio. This phenomenon has been reported
previously in flavone C-glycosides from Ziziphi Spinosae
Semen, such as spinosin (9) and 6‴-feruloylspinosin (10). It
was deemed that there were two stable conformers produced
by rotational barrier 7-OCH3 in flavone-6-C-glycoside at low
1136.3247).
2.4. Acid hydrolysis and sugar identification
The compounds 1–6 (each
1 mg) were respectively
hydrolyzed by 2 M HCl (2 mL) at 90 °C for 2 h. The reaction
mixture was concentrated to dryness. After extraction with
CH3Cl–H2O, the H2O layer was dried and reacted with L-
cysteine methyl ester hydrochloride (1 mg) and o-tolyl
isothiocyanate (5 μL) in pyridine (1 mL) at 60 °C, as Tanaka
et al. already reported [11]. The mixture was analyzed by HPLC
[Cosmosil 5C18-MS-II, 250 × 4.6 mm; mobile phase, 25% CH3CN
(0.1% CH3COOH); UV 250 nm]. Compared with the derivatives
of standard sugars, D-glucose were detected from derivatives of
1–6.
2.5. Mild alkaline hydrolysis
Solutions of compounds 5 and 6 (each 10 mg) were
respectively hydrolyzed by 0.05 mol/L NH4OH (10 ml) at room
temperature for 1.5 h [4]. The reaction mixture was neutralized
with formic acid, was and then extracted with EtOAc. The H2O
layer was further purified by HPLC to afford 5a (2.5 mg) and 6a
(2.2 mg), respectively [YMC–Pack ODS-A, 250 × 10 mm; mobile
phase, 5% CH3OH (0.1% HCOOH); UV 254 nm].
2.6. Quantum chemical ECD calculation method
In theoretical calculations, the geometry of the molecules
was optimized with Gaussian 09 package at B3LYP/6-
31G(d) computational level. The minimum nature of the
structure was confirmed by frequency calculations at the
Li, Min
