Journal of Asian Natural Products Research
773
Bruker AVANCE III-500 NMR spec-
trometer (Bruker). Preparative HPLC was
carried out on an Agilent 1100 Series with
Shimpak RP-C18 column (20 £ 200 mm,
i.d.) and 1100 Series Multiple Wavelength
detector. Analytical HPLC was carried out
on an Agilent 1260 Infinity Chromatograph
equipped with an Agilent ZORBAX SB-C18
column (5mm, 4.6 mm £ 250 mm). GC-MS
was carried out on a Varian CP-3800 Gas
Chromatograph (Varian, Palo Alto, CA,
USA) with a Saturn 2200 Mass detector.
Column chromatography (CC) was taken on
a silica gel (200–300 mesh, Qingdao
Haiyang Chemical Co. Ltd, Qingdao,
China), macroporous resin D101 (pore size
B 13–14nm, H&E Co., Beijing, China),
MCI-gel CHP20P (75–150 mm, Mitsubishi
Chemical, Tokyo, Japan), ODS-C18 (40–
63mm, Fuji Silysia Chemical Ltd, Aichi,
Japan), and Sephadex LH-20 (20–100 mm,
Pharmacia, Uppsala, Sweden). Fractions
were monitored by TLC with silica gel
GF254 (Qingdao Haiyang Chemical Co.
Ltd). Detection of spots was done by heating
silica gel plates sprayed with 1% vanillin–
10% H2SO4 in EtOH.
successively. Then, the 30% ethanol elution
fraction (22 g) was subjected to MCI
column using a MeOH–H2O gradient
(0:100, 10:90, 30:70, 50:50, v/v) to afford
four fractions A–D. Fraction C (4 g) was
applied on ODS-C18 column (MeOH–H2O,
30:70, v/v) to give three fractions C1–C3.
Fraction C3 was separated by sephadex LH-
20 (MeOH) and silica gel column chroma-
tography eluted with CHCl3–MeOH (4:1,
v/v) to give compound 1 (13.1 mg). Fraction
C2 was successively purified over sephadex
LH-20 (MeOH) and preparative HPLC
(ODS column: 20 £ 200 mm, i.d., 5 mm;
flow rate: 10ml/min) using MeOH–H2O
(40:60, v/v) to afford compounds 2 (9.2mg,
tR ¼ 11.7min), 3 (2.6mg, tR ¼ 11.4min), 4
(4.3 mg, tR ¼ 9.9 min), and 5 (3.8 mg,
tR ¼ 10.9min).
3.3.1 Compound 1
17
White amorphous powder; ½aꢀD 213.8
(c ¼ 0.12, MeOH); UV (MeOH) lmax (nm)
(log 1): 205 (4.28), 231 (3.49); IR nmax
(KBr) cm21: 3364, 2914, 1640, 1453, 1380,
1
1074, 771; for H and 13C NMR spectral
data, see Table 1; ESI-MS m/z 562.8
[M 2 H]2, 238.7 [M 2 H-162-162]2;
HR-ESI-MS m/z 587.0465 [M þ Na]þ
(calcd for C20H27Cl3O12Na, 587.0460).
3.2 Plant material
The air-dried bulbs of L. brownii var.
viridulum were purchased from Anhui
Bozhou Crude Drug Market in November
2010 and were identified by Prof. Mian
Zhang, from the Department of Medicinal
Plants, China Pharmaceutical University.
A voucher specimen (No. L20101101)
has been deposited in the Department
of Natural Medicinal Chemistry, China
Pharmaceutical University.
3.3.2 Compound 2
17
White viscous syrup; ½aꢀD 247.3
(c ¼ 0.14, MeOH); UV (MeOH) lmax
(nm) (log1): 201 (4.39), 223 (3.75); IR
n
max (KBr) cm21: 3417, 2925, 1677, 1591,
1452, 1340, 1050, 757; for 1H and 13C NMR
spectraldata, seeTable1;ESI-MSm/z465.2
[M 2 H]2, 156.8 [M 2 H-146-162]2; HR-
ESI-MS m/z 489.1131 [M þ Na]þ (calcd
for C19H27ClO11Na, 489.1134).
3.3 Extraction and isolation
The dried bulbs of L. brownii var. viridulum
(6kg) were extracted three times with 95%
EtOH under reflux. After evaporation, the
residue was dissolved in H2O. The H2O
solution was passed through a macroporous
resin D101 column, eluted with EtOH–
H2O (0:100, 30:70, 70:30, 95:5, v/v),
3.4 Acid hydrolysis
Eachofcompounds1and2(5 mg)dissolved
in MeOH (3ml) was treated with 2 M HCl–
MeOH (tube, 6 h, 508C) [14,15]. The
hydrolysate was concentrated to dryness,
then partitioned with H2O and CHCl3. The