72
B. Wu et al. / Carbohydrate Research 351 (2012) 68–73
control (50 plants) and stressed groups (50 plants). To elicit the
stress, plants were sprayed with 2% aq solution of CuCl2. After
48 h, leaves of the control and sprayed plants were collected and
dried at 60 °C, and finely powdered in an electronic blender and
kept in separate containers for extraction.
The mixture was stirred at 60 °C for 1.5 h, then hexamethyldisilaz-
ane–trimethylchlorosilane (2:1) was added, and stirred for 0.5 h.32
After centrifugation, the supernatant was directly subjected to GC
analysis. The sugar derivatives obtained from 1–3 were detected in
each case by co-injection of the D-glucose derivatives.
3.3. Extraction and isolation
3.6. Determination of antioxidative activities
The dried, powdered CuCl2 treated leaves and stems (708 g) of
P. oleracea and untreated leaves (771 g) were extracted at room
temperature with MeOH (3 ꢁ 5 L), respectively. The extracts were
evaporated in vacuo to afford a gummy residue (68 g) for treated
and a gummy residue (72 g) for the corresponding control. The res-
idues were partitioned in H2O (500 mL) and extracted with EtOAc
(4 ꢁ 500 mL) and n-butanol (4 ꢁ 500 mL), successively. The EtOAc
and n-butanol extracts of treated and the corresponding control
were subjected to TLC examination on aluminium sheets pre-
coated with Si Gel 60 F 254 (Merck). The spots were applied in
as equal amounts as possible. The plates were developed in the fol-
lowing developing solvent systems: benzene–acetone (6:1), ben-
zene–EtOAc (5:1) petroleum ether–EtOAc (5:1) for the EtOAc
extract; CHCl3–MeOH (3:1), CH2Cl2–MeOH (4:1) and benzene–
CHCl3–MeOH (1:3:1) for the n-butanol extract. After development,
the plates were examined under UV light (254 nm) to locate any
additional spots in the different extracts of the treatments in com-
parison with that of the corresponding control extracts. The spots
on the plates were also visualized by spraying with an EtOH–
H2SO4 solution. Several prep-TLC plates were prepared and the
compounds were separated by preparative TLC in different solvent
systems. The crude compounds were applied to a Sephadex LH-20
column (1 ꢁ 80 cm, 38 g, Amersham), and eluted with MeOH to
yield pure compounds 1 (1.4 mg), 2 (1.2 mg), 3 (10.9 mg), 4
(8.1 mg) and 5 (10.3 mg). The extract of untreated leaves were sep-
arated by the same methods to afford 3 (12.0 mg), 4 (12.5 mg) and
5 (11.9 mg).
The antioxidative activities of all the compounds were screened
by DPPH assay as previously described.23 The purities of com-
pounds used for the assay were above 95% checked by HPLC and
1H NMR experiments. In brief, 100
ll test samples at different con-
centrations in MeOH and 8.0 ꢁ 10ꢀ5 M DPPH in MeOH (300
ll)
were added to a 96-well microtiter plate. The plate was shaken
for 1 min on a plate shaker, and incubated for 30 min at room tem-
perature in the dark. After incubation, the absorbance was re-
corded at 517 nm. The tested samples at different concentrations
without DPPH solution were used as a blank control to eliminate
the influence of sample color. Vitamin E was used as a positive con-
trol. All tests were independently performed in triplicate and the
definition of IC50 values in the tested compounds is the concentra-
tion required to scavenge 50% DPPH free radicals. The DPPH radical
scavenging activity was calculated according to the following
equation: DPPH radical scavenging activity (%) = [{Ab-
sC ꢀ (AbsS ꢀ AbsB)/AbsC}] ꢁ 100.
Acknowledgments
This work was supported by the NSFC of China (No. 30801430),
and the Science Foundation of Zhejiang Province (No. J20110305).
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Each compound (2.0 mg) in 10% HCl was stirred at 90 °C for 4 h.
The reaction mixture was filtered, and examined by TLC together
with authentic
D
-glucose. The dried filtrate was dissolved in dry pyr-
-cysteine methyl ester hydrochloride.
idine,31 to which was added
L