CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1583
Acid Hydrolysis and Determination of Sugar Components. Compounds 3 and 4 (each 2.0 mg) were
hydrolyzed in 1m aq. HCl soln. (1.0 ml) for 2 h at 858. The mixture was cooled and partitioned between
CHCl (2.0 ml) and H O (2.0 ml). The aq. layer was washed with CHCl (3Â3.0 ml), neutralized with
3
2
3
Ba(OH) , filtered, and evaporated under reduced pressure. The residue was dissolved in pyridine
2
(
1.0 ml) and 0.1m l-cysteine methyl ester hydrochloride in pyridine (2.0 ml) was added. The mixture was
heated at 608 for 1 h. An equal volume of Ac O was added and heating was continued for 1 h. The
2
acetylated thiazolidine derivatives were analyzed by GC using authentic samples as standards.
Temperatures of injector and detector were both 2808. A temp. gradient system was used for the oven,
starting at 1608 and increasing up to 1958 at a rate of 58/min. Identification of d-xylose from 3 and d-
xylose and d-glucose from 4 present in the aq. layer was carried out by comparison of t values with those
R
of authentic samples (NICPBP, P. R.China; tR1 10.2 min (d-xylose), tR2 13.9 min (d-glucose)).
Determination of NO Production and Cell Viability Assay. Mouse monocyte-macrophage RAW264.7
cells (ATCC TIB-71) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai,
P. R. China). RPMI 1640 medium, penicillin, streptomycin, and fetal bovine serum (FBS) were
purchased from HyClone (N.Y., USA). LPS, DMSO, MTT, and hydrocortisone were purchased from
À1
Sigma Co. The cells were suspended in RPMI 1640 medium supplemented with penicillin (100 U ml ),
À1
streptomycin (100 U ml ), and 10% heat-inactivated FBS under a humidified atmosphere of 5% CO at
2
3
78. The cells were harvested with trypsin until they attained confluence and were used for assays during
the exponential growth phase.
4
The cells were seeded in 96-well plates with 6Â10 cells/well. After 1 h incubation, cells were treated
À1
with 1 mg ml LPS and various concentrations of test compounds for 24 h [28]. Each compound was
dissolved in DMSO, which was applied at a final concentration of 0.1% (v/v) in cell culture supernatants.
Control groups received an equal amount of DMSO. NO Production was determined by adding 100 ml
Griess reagent (1% sulfanilamide and 0.1% N-(naphthalen-1-yl)ethane-1,2-diamine in 5% H PO ) to
3
4
1
00 ml supernatant from LPS or the compound-treated cells in triplicate. After 5 min of incubation, the
absorbance was measured at 540 nm with a microplate reader. Cytotoxicity was determined by MTT
À
colorimetric assay, after 24 h of incubation with test compounds. Concentrations of NO2 in the
supernatant were calculated by a working line from 0, 1, 2, 5, 10, 20, 50, and 100 mm NaNO solns. The
2
inhibitory rate was calculated according to the formula:
À
À
½
NO À ½NO
À À
2
LPS
2 LPSþsample
Inhibitory rate ½% ¼ 100 Á
½
NO À ½NO
2
LPS
2 untreated
Every experiment was performed in triplicate; data are expressed as meanÆSD of three
independent experiments.
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