Journal of Asian Natural Products Research
3
mined to be icaruralin A. The HR-ESI-MS,
1H and 13C NMR spectra of 3 were
identical to those of 2, indicating that they
share the same planar structure. However,
the different retention time in same
chromatographic condition by HPLC anal-
ysis (Figure S1) and optical rotation in
same concentration indicated the structural
isomer of 2 and 3, which means the
difference in the absolute configuration of
C-200 for these two compounds. Thus, the
structure of 3 was established as icaruralin
B. The oxidation of double bond in prenyl
moiety led to the creation of new chiral
center (C-200), so the following cyclization
generated two products. There is not any
chromophore group attaching to C-200
directly, which means that the absolute
configuration of C-200 has little effect on the
Cotton effect in CD spectrum. So these two
compounds share the similar CD spectrum,
and their absolute configuration of C-200
remains unresolved.
meter (PerkinElmer, Inc., Waltham, MA,
USA). IR spectra were carried out on a
Nicolet 5700 FT-IR microscope spec-
trometer (Thermo Electron Scientific
Instrument Crop., Madison, WI, USA).
The CD spectra were recorded on a JASCO
J-815 spectropolarimeter (JASCO Corp.,
Tokyo, Japan). The 1H and 13C NMR
spectra were recorded on a Mercury Plus-
400 and VNS-600 spectrometer (Varian,
Inc., Palo Alto, CA, USA) using acetone-d6
and methanol-d4 as solvent and internal
reference. Chemical shifts (d) were given in
ppm, and coupling constants (J) were given
in hertz (Hz). HR-ESI-MS and ESI-MS
were obtained on an LCQ-Fleet mass
spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA). Semi-preparative
reversed-phase HPLC was performed on a
Shimadzu LC-20AT instrument and a
Shimadzu RID-10A detector (Shimadzu
Corp., Tokyo, Japan). Analytical HPLC
was carried out on an Agilent 1200 (Agilent
Technologies, Santa Clara, CA, USA), the
UV detector was set at 270 nm, and the
column was operated at 308C. A C18
column (250 mm £ 4.6 mm i.d., 5 m;
Apollo Silica, Alltech Co. Ltd, Chicago,
IL, USA) was used for HPLC analysis and a
C18 column (250 mm £ 10 mm i.d., 5 m;
Grace Adsorbosphere, W.R. Grace Co.,
Columbia, MD, USA) was used for semi-
preparative reverse-phase HPLC. Column
chromatography (CC) was performed
using Sephadex LH(20 (Pharmacia Fine
Chemical Co., Ltd, Uppsala, Sweden) and
silica gel (200(300 mesh, Qingdao Marine
Chemical Factory, Qingdao, China). Pre-
coated silica gel GF254 plates (Qingdao
Marine Chemical Factory) were used for
analytical TLC. Spots were detected on
TLC under UV light or by heating after
spraying with 10% H2SO4 in EtOH (v/v).
Compound 4 was isolated as a yellow
powder which gives an ESI-MS ion peak at
m/z 514.9 [M þ H]þ. Its molecular weight
is 162 amu less than that of 1, suggesting
the losing of one glucose moiety in 1. The
1H and 13C NMR spectra of 4 were similar
to those of 1 except for the absence of one
glucose. By comparing its spectroscopic
data with those reported in the literature
[13], 4 was identified as baohuoside I.
The occurred reactions of biotransform-
ation of 1 by G. uralensis cell suspension
cultures included deglycosylation, hydroxy-
lation, and cyclization. Icariin (1) was
transformed through regio-selective deglu-
cosylation to give baohuoside I (4). Further
hydroxylation on the double bond of C-200/
C-300 and non-stereo-specific intramolecular
cyclization with C-7 and C-200 by an ether
linkage led to the generation of 2 and 3.
3. Experimental
3.2 Tissue and cell culture
3.1 General experimental procedures
The seeds of G. uralensis and M. alba
(identified by Associate Professor Lin
Yang, Minzu University of China) were
Optical rotations were measured on a
Pekin-Elmer Model-343 digital polari-