1656 The Journal of Natural Products, 2007, Vol. 70, No. 10
Notes
UPLC was performed on a Waters Acquity UPLC system, which was
equipped with a binary solvent delivery system and a sample delivery
system. MS was performed on a Waters Micromass Quattro Premier
tandem quadrupole mass spectrometer. The LC/MS system control was
achieved by using MassLynx 4.0.
UPLC Conditions. The UPLC separation was performed on a
Waters Acquity ethylene-bridged (BEH) C18 column (1.7 µm, 2.1 ×
100 mm) at 38 °C with a flow rate of 0.3 mL/min. The analytes were
eluted from the column with a mixed solvent of 20% aqueous MeOH
with 0.1% AcOH (solvent A) and MeOH with 0.1% AcOH (solvent
B) using a linear gradient mode. From 0 s to 0.2 min, the combination
of solvents A and B was 20:80, and from 0.2 min to 3.1 min, the
combination of solvents A and B was linearly converted from 20:80
to 0:100. The column was finally eluted with solvent A:B (0:100) from
3.1 min to 4.0 min.
MS Conditions. All MS optimization experiments were performed
in both the MS scan mode and the product scan mode. All quantifica-
tions were performed in the multiple reaction monitoring (MRM) mode.
The tune page parameters and conditions for each of the MRM
transitions were optimized by infusing the neat standard solution into
the mass spectrometer at 10 µg/mL. To ensure that the tune page
parameters were compatible with the UPLC flow during the tuning, an
UPLC flow of 0.6 mL/min at solvent A:B (1:1) was introduced into
the mass spectrometer at the same time by utilizing a T unit (Upchurch
Scientific, Oak Harbor, WA). For MRM data collection during the LC
experiments, the capillary voltage was 3.0 kV, the source temperature
was 120 °C, the desolvation temperature was 350 °C, the desolvation
gas flow was 800 L/h, and the cone gas flow was 50L/h. During each
LC injection, the mass spectrometer was set to collect data in MRM
mode using electrospray ionization (ESI) in the negative-ion mode.
The MRM transitions for the analytes for bruceine J (2) in ESI- were
as follow: 506.90 f 100.4 using cone voltage 46.00 and collision
energy 46.00, 506.90 f 110.4 using cone voltage 46.00 and collision
energy 36.00, and 506.90 f 422.7 using cone voltage 46.00 and
collision energy 28.00.
Plant Material. The plant materials were purchased from the Bandar
Jaya traditional market, Lampung, Indonesia, in April 2005. The plants
were identified by Aris Winarso at the Herbal Medicine Research and
Education Centre of “Karya Tama”, Lampung, Indonesia. Voucher
specimens are deposited at the Laboratory of Bioorganic Chemistry,
Graduate School of Agriculture, Hokkaido University, Japan. The names
of these medicinal plants, their families, local name, parts used, and
voucher specimen numbers for the experiments are listed in Table S1
(Supporting Information).
Bruceantinol B (1): amorphous solid; [R]20 -41.0 (c 0.60,
D
pyridine); IR (KBr) νmax 3407 (OH), 1722 (δ-lactone and ester CdO),
1685, 1647 (R,ꢀ-unsaturated CdO) cm-1; 1H NMR (CD3OD, 500 MHz)
and 13C NMR (CD3OD, 125 MHz) data, see Table 1; FABMS (negative)
m/z 591 [M - H]-; HRFABMS m/z 591.2073 [M - H]- (calcd for
C29H35O13, 591.2077).
Bruceine J (2): amorphous solid; [R]20 -73.0 (c 0.6, pyridine);
D
IR (KBr) νmax 3411 (OH), 1724 (δ-lactone and ester CdO), 1686, 1649
1
(R,ꢀ-unsaturated CdO) cm-1; H NMR (CD3OD, 500 MHz) and 13C
NMR (CD3OD, 125 MHz) data, see Table 1; FABMS (negative) m/z
507 [M - H]-; HRFABMS m/z 507.1864 [M - H]- (calcd for
C25H31O11, 507.1866).
Methylation of 1 and 2. An excess of an Et2O solution of CH2N2,
which was prepared from N-methyl-N-nitrosourea, was added to a
MeOH (1 mL) solution of 1 (10 mg, 0.0169 mmol) at 0 °C. After the
reaction mixture was stirred at 0 °C for 2.5 h, the solvent was evaporated
to give a crude product. Purification of the crude product by preparative
TLC (hexane–EtOAc, 1:4) afforded 3 (8.4 mg, amorphous solid, [R]20
D
-7.3 (c 1.2, pyridine). Methylation of 2 (10 mg, 0.0197 mmol) was
carried out in the same manner as described above and afforded 4 (8.0
mg, amorphous solid, [R]20D -80.3 (c 0.8, pyridine). The physical and
spectroscopic data of 3 and 4 were identical with those reported for
bruceantinol and bruceine A, respectively.15,16
Extraction for MRM Experiment. The dried fruits of Br. jaVanica
(0.1 kg) were extracted with 70% aqueous MeOH. The 70% aqueous
MeOH layer was filtered to give a dark blown solution, which was
evaporated and extracted with EtOAc to give aqueous and EtOAc layers.
The volatile components of the EtOAc layer were removed under
reduced pressure, and the residue was dissolved with MeOH (2 mL)
and placed on a Bond Elut DEA cartridge column. The column was
successively washed with MeOH (2 mL × 2) and 1 mol/L AcOH–
MeOH (2 mL × 3). The volatile components of the AcOH–MeOH
eluents were removed, and a portion of the residue was subjected to
the UPLC/MS/MS experiment.
Antibabesial Assay. The in vitro assay against B. gibsoni was
described in detail in a previous paper.19 In this study, diminazene
aceturate (Ganaseg) was used as control.
Acknowledgment. The authors are grateful to Mr. Kenji Watanabe
and Dr. Eri Fukushi for measuring the NMR and mass spectra. Financial
support and a postdoctoral fellowship (to Subeki) from the Japan Society
for the Promotion of Science and financial support provided by grants-
in-aid for scientific research (grant 17-05213 to Ken Katakura) from
the Ministry of Education, Culture, Sports, Science, and Technology
of Japan are very much appreciated.
Plant Extract Preparation. For preliminary testing, 10 g samples
of dried material of each plant part were boiled twice in 200 mL of
H2O for 30 min. The boiling solution was cooled, filtered, and freeze-
dried to give a powder. The powdered extract was then tested for
antibabesial activity against B. gibsoni in vitro.
Supporting Information Available: Tables listing plants collected
and preliminary screening data and figure showing UPLC/photodiode
array and MS/MS MRM chromatograms for bruceine J. This informa-
Extraction and Isolation. Extraction and isolation of the active
compounds were monitored by the assay of antibabesial activity. The
dried fruits of Br. jaVanica (1 kg) were boiled twice in 5 L of H2O for
30 min. The boiling solution was filtered and extracted with EtOAc to
give aqueous and EtOAc layers. The EtOAc layer (60.6 g) was
chromatographed on a silica gel column, eluted successively with CHCl3
(2 L), MeOH–CHCl3 (3:97, 2 L), MeOH–CHCl3 (1:4, 2 L),
MeOH–CHCl3 (7:3, 2 L), and MeOH (2 L). The MeOH–CHCl3 (1:4)
eluate was evaporated to yield a residue (10.8 g), which was subjected
to column chromatography on silica gel, eluted with hexane–EtOAc
(1:1), to give 10 fractions (A–J). Fraction E was recrystallized from
methanol to give bruceine A (4, 362 mg). Recrystallization of fractions
G, H, and I from hexane–EtOAc (9:1) gave bruceantinol (3, 247 mg),
bruceine B (5, 533 mg), and bruceine C (6, 362 mg), respectively. The
MeOH–CHCl3 (7:3) eluate was evaporated to yield a residue (4.9 g),
which was subjected to column chromatography on silica gel, eluted
with MeOH–EtOAc (1:19), to give six fractions (K–P). Fraction M
was purified by preparative TLC, eluted with MeOH–EtOAc (1:19),
to yield bruceine D (7, 159 mg). Fraction O was purified by preparative
TLC using MeOH–EtOAc (1:9) to yield yadanziolide A (8, 44 mg).
After removal of the solvent, fraction P (832 mg) was further subjected
to column chromatography on silica gel using CHCl3–MeOH–H2O (60:
36:4) to give four subfractions (K–N). When rechromatograped on a
silica gel column using CHCl3–MeOH–H2O (60:36:4), fractions L (67
mg) and M (48 mg) afforded bruceantinol B (1, 41 mg) and bruceine
J (2, 36 mg), respectively.
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