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Vol. 58, No. 10
230 mesh and 230—400 mesh, Merck, Whitehouse Station, NJ, U.S.A.) or
substrate, hippuryl-histidyl-leucine (HHL) and ACE from rabbit lung (EC
YMC RP-18 resins (30—50 mm, Fujisilisa Chemical Ltd., Kasugai, Aichi, 3.4.15.1) were purchased from Sigma (St. Louis, MO, U.S.A.). Aliqouts
Japan), and thin layer chromatography (TLC) using pre-coated silica-gel 60
F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck).
Plant Material The leaves of M. esculenta were collected in Hoabinh
Province, Vietnam in June, 2006, and identified by Dr. Ninh Khac Ban (In- were incubated at 37 °C for 1 h. The reaction was stopped with 150 ml of
stitute of Ecology and Biological Resources, VAST). A voucher specimen 0.5 N HCl. The hippuric acid formed was detected and quantified by HPLC.
(50 ml) of each sample were incubated with 100 ml of 1.0 M NaCl-borate
buffer (pH 8.3) containing 2.0 mU ACE solution at 37 °C for 10 min. Then
100 ml of 5.0 mM HHL was added to the reaction mixture. Test solutions
(ME0606) was deposited at the Herbarium of Institute of Marine Biochem- A volume of 5 ml of each sample was injected using Agilent an ALS 1100
istry, VAST, Vietnam.
Extraction and Isolation The dried leaves of M. esculenta (2.0 kg)
were extracted with MeOH three times under reflux for 15 h to yield 240 g of
autosampler into an Agilent 1100 series HPLC (Agilent Technologies)
equipped with DAD 1100 diode array detector. The solvents used for gradi-
ent were 10 mM phosphoric acid (pH 2.5) and 100% methanol. The methanol
a dark solid extract, which was then suspended in water and successively concentration was increased to 60% for the first 8 min and to 100% for the
partitioned with chloroform (CHCl3) and ethyl acetate (EtOAc) to obtain next 5 min, then decreased to 0% for last 5 min (total run time: 18 min). The
CHCl3 (ME1, 79.4 g), EtOAc (ME2, 60.3 g), and water (ME3, 72.5 g) ex- analytical column used was a Nucleosil 100-5C18, 250ϫ4.6 mm i.d., using
tracts after removing solvent in vacuo. ME1 was chromatographed on a sil- with packing material with a particle size of 5 mm at a flow rate 1 ml/min at
ica gel column and eluted with n-hexane-acetone gradient (100 : 1-1 : 1, v/v)
to obtain four subfractions, ME1A (30.5 g), ME1B (16.4 g), ME1C (18.0 g),
and ME1D (10.1 g). The ME1B fraction was chromatographed on a silica
gel column eluting with CHCl3-MeOH (30 : 1, v/v) to obtain 9 (8.0 mg) and
10 (6.4 mg). The ME1C fraction was further chromatographed on a silica gel
column eluting with CH2Cl2-MeOH (20 : 1, v/v) to give three smaller frac-
tions, ME1C1—ME1C3. The ME1C2 fraction was chromatographed on an
YMC RP-18 column eluting with acetone–water (5 : 1, v/v) to yield 5
(8.6 mg) and 7 (6.0 mg). ME2 was chromatographed on a silica gel column
and eluted with CHCl3-MeOH gradient (50 : 1-1 : 1, v/v) to obtain four sub-
fractions, ME2A—ME2D. The ME2B fraction was chromatographed on a
silica gel column eluting with acetone–MeOH (8 : 1, v/v) to yield 11
ambient temperature. During each run the chromatogram was recorded at
228 nm and integrated using Agilent Chemstation enhanced integrator to de-
tect liberated hippuric acid. Pure hippuric acid (purchased from Sigma
Chemical Co.) was used to calibrate the standard curve and retention time.
The percent inhibition was calculated as follows:
⎛
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⎜
⎝
⎞
⎟
⎟
⎠
⎡
⎢
⎤
⎥
E
ControlϪ ESample
% inhibitionϭ
ϫ100
EControlϪ EBlank
⎢
⎣
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Acknowledgments This study was supported by the Priority Research
Center Program through the National Research Foundation of Korea (NRF)
(8.0 mg). The ME2C fraction was chromatographed on a silica gel column funded by the Ministry of Education, Science and Technology (2009-
eluting with CHCl3-MeOH (6 : 1, v/v) to give three smaller fractions, 0093815), Republic of Korea. The authors would like to thank the Korean
ME2C1—ME2C3. Fraction ME2C1 was chromatographed on an YMC RP-
Basic Science Institute (KBSI) for performing the NMR experiments.
18 column eluting with MeOH-water (3 : 1, v/v) to yield 6 (7.0 mg) and 12
(8.5 mg). The water soluble fraction ME3 was chromatographed on a Diaion References
HP-20P column (Mitsubishi Chem. Ind. Co., Tokyo, Japan) eluting with
water containing increasing concentrations of MeOH (100% H2O, 25%
MeOH, 50% MeOH, 75% MeOH, 100% MeOH) to obtain four fractions,
ME3A—ME3D. Fraction ME3B was chromatographed on a silica gel col-
umn eluting with CHCl3-MeOH-H2O (5 : 1 : 0.1, v/v/v) to give three frac-
tions, ME3B1—ME3B3. The ME3B2 fraction was chromatographed on a
silica gel column eluting with CH2Cl2-MeOH-H2O (5 : 1 : 0.1, v/v/v) to ob-
tain compound 8 (14.0 mg). The ME3B3 fraction was chromatographed on a
silica gel column eluting with CH2Cl2-acetone-H2O (1 : 2.5 : 0.1, v/v/v) to
obtain the new compound 1 (50.0 mg) and compound 2 (18.5 mg). Fraction
ME3D was chromatographed on a silica gel column using CH2Cl2-MeOH-
H2O (6 : 1 : 0.05, v/v/v) to give four fractions ME3D1—ME3D4. The
ME3D4 fraction was further separated on a YMC RP-18 column eluting
with acetone-MeOH-H2O (1 : 2 : 2, v/v/v) to obtain compounds 3 (5.0 mg)
and 4 (8.0 mg).
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organic layer was analyzed by GC: Column: column of SPB-1 (0.25 mmϫ
30 m); detector FID, column temp 210 °C, injector temp. 270 °C, detector
temp. 300 °C, carrier gas He (2.0 ml/min). The retention times of persily-
lated glucose and apiose were founded to be 14.11 and 6.70 min, respec-
tively, when compared with the standard solutions prepared by the same re-
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