JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
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(CC) eluted with aqueous EtOH (0, 20, 40, and 60%) to afford four fractions (FA–FD). FB
was further purified on a Sephadex LH-20 column to give 8 (980.2 mg). FC (15.2 g) was
further fractionated on an ODS column (3 × 30 cm), eluted with a constant gradient of
MeOH/H2O (20%), to obtain five portions (FC1–FC5). FC1 (1.2 g) was further fractionated
on an ODS column with a constant gradient of MeOH /H2O (30%), followed by preparative
RP-C18 HPLC, to give 5 (30.3 mg). FC2 (2.5 g) was further fractionated on an ODS column
with a constant gradient of MeOH /H2O (32%), followed by preparative RP-C18 HPLC
(flow rate 2 ml/min, UV detection at λ = 300 nm), to give 1 (5.8 mg, tR = 22.3 min) and 3
(34.0 mg, tR = 31.5 min). FC3 (450 mg) was further fractionated on an ODS column with a
gradient of MeOH/H2O (from 30 to 50%), followed by preparative RP-C18 HPLC, to give
2 (6.8 mg, tR = 19.6 min) and 7 (6.8 mg, tR = 21.5 min). FC4 (4.5 g) was further purified on
a Sephadex LH-20 column to give 4 (1.9 g) and 6 (10.5 mg).
3.3.1. Bletilloside A (1)
Amorphous power; [ꢀ]2D5 − 40.8 (c 0.05, MeOH); UV (MeOH) λ
(log ε) 223 (4.26)
max
304 (4.24) nm; IR (KBr) vmax 3422, 2923, 1701, 1637, 1509, 1458, 1384, 1258, 1234, 1162,
1072 cm−1; 1H and 13C NMR spectral data, see Table 1; HRESIMS: m/z 647.1939 [M + Na]+
(calcd for C29H36O15Na, 647.1952).
3.4. Acid hydrolysis of 1
Compound 1 (1.0 mg) was heated in 2 mol/L trifluoroacetic acid (TFA) (1 ml) for 2 h in
screen-cap vial. Afer cooling, 1 ml of EtOAc was added into the hydrolysate. e aque-
ous layer was evaporated to dryness with ethanol under vacuum until TFA was removed.
e residues were determined by comparison with D-glucose using TLC (CHCl3: MeOH:
H2O = 3: 2:0.2, visualization with ethanol-5% H2SO4 spraying). In addition, the specific
rotation of sugar derived from 1 ([ꢀ]2D5 + 79.8) was in accordance with that of D-glucose
([ꢀ]2D5 + 96.8) and opposite to that of L-glucose [ꢀ]2D5 − 79.8), indicating the sugar of 1 to
be in the D-form.
3.5. Cell viability
Cell viability was evaluated by MTT assay. RAW 264.7 macrophage cell was seeded at
2 × 105 cells/ml in 96-well plates containing 100 μl of DMEM (Dulbecco’s modified Eagle’s
medium) (supplemented with 10% FBS (Fetal bovine serum), 100 units/ml penicillin,
and 100 μg/ml streptomycin sulfate), and cultured in a 37 °C humidified atmosphere of
5% CO2. Afer overnight incubation, the cells were treated with LPS (1 μg/ml) in the absence
or presence of the test compounds at different concentrations and the plates were incubated
for 24 h. e cells were then incubated with an MTT solution for 4 h at 37 °C under 5% CO2.
e medium was removed and DMSO (150 μl) was added to each well. e absorbance was
measured at 492 nm by a Microplate Reader (Multiskan MK3, ermo).
3.6. NO inhibition assay
Using the Griess method, the nitrite concentration was determined in the culture superna-
tants. e cells were planted at 2 × 105 cells/ml in 96-well plates and incubated overnight.