740
Vol. 58, No. 5
Table 2. Inhibition of Tyrosinase and Anti-melanogenesis Activity of 1—3
Anti-tyrosinase activity (%)
Anti-melanogenesis activity (%)
50 mM
25 mM
12.5 mM
100 mM
50 mM
25 mM
1
2
3
N.A.a)
79
N.A.a)
59
N.A.a)
33
N.D.b)
65
N.D.b)
45
N.D.b)
40
N.A.a)
84
N.A.a)
55
N.A.a)
31
N.D.b)
N.D.b)
N.D.b)
Kojic acid
Arbutin
N.D.b)
32%
20%
a) N.A., not active at 50 mM. b) N.D., not determined.
(ꢁ1.9), 240 (ꢁ7.6), 208 (ꢀ21.1); IR (film) nmax 3375, 1716, and 1627 cmꢀ1
;
1H- and 13C-NMR data (Table 1); ESI-MS m/z 753 (MꢀH)ꢀ; HR-ESI-TOF-
MS m/z 753.1280 [(MꢀH)ꢀ, Calcd for C35H29O19, 753.1298, D ꢀ1.75
mmu].
Papuabalanol B (2): Yellow amorphous solid; [a]D27 ꢁ24 (cꢂ1.0, MeOH);
UV (MeOH) lmax (log e) 280 (5.2), 221 (5.5); CD (MeOH) lmax (De) 370
(ꢁ1.5), 240 (ꢁ6.7), 206 (ꢀ19.1); IR (film) nmax 3401, 1723, 1709, and
1626 cmꢀ1 1H- and 13C-NMR data (Table 1); ESI-MS m/z 737 (MꢀH)ꢀ;
;
HR-ESI-TOF-MS m/z 737.1335 [(MꢀH)ꢀ, Calcd for C35H29O18, 737.1348,
D ꢀ1.34 mmu].
Acid Hydrolysis of 1 and 2 A solution of papuabalanol A (1: 1.0 mg) in
2 N HCl was heated at 100 °C for 2.5 h. The reaction mixture was evaporated
in vacuo. The hydrolysate was dissolved in H2O, subsequently to HPLC
analysis (Capcell Pak NH2 UG80, 4.6ꢃ250 mm; eluted with H2O/acetoni-
trile (15 : 85); flow rate, 1.0 ml/min) with optical rotation detector. The hy-
drolysate was shown to be D-glucose by HPLC retention time (tR 9.5 min),
and sign (positive) of the optical rotation. Papuabalanol B (2) was also in-
vestigated in the same manner, and the sugar unit was identified as D-glucose
Fig. 2. Typical Recording of the Vasorelaxation Effects of 1 and 3 (Each
1ꢃ10ꢀ4 M) on the Rat Aortic Rings Precontracted with 3ꢃ10ꢀ7 M PE
to the endothelial cells to release nitric oxide (NO).
Experimental
General Experimental Procedures Optical rotations were measured on
a JASCO DIP-1000 automatic digital polarimeter. UV spectra were obtained by HPLC analysis.
on an Ultrospec 2100 pro spectrophotometer, CD spectra were measured on
Anti-melanogenesis Assay B16 cells were cultured in DMEM medim
a JASCO J-820 spectropolarimeter, and IR spectra were recorded on a supplemented with 10% FBS and penicillin/streptmycin. B16 cells at 5000
JASCO FT/IR-4100 spectrophotometer. 1H and 2D NMR spectra were cells in 100 ml per well were seeded onto 96-well microtiter plate, and were
recorded on a JEOL ECA600 and Bruker AV 400 spectrometers, and chemi-
preincubated for 24 h at 37 °C in a humidified atmosphere containing 5%
cal shifts were referenced to the residual acetone-d6 (dH 2.04 and dC 29.8). CO2. The cells were cultured in the medium containing 3-isobutyl-1-
Standard pulse sequences were employed for the 2D NMR experiments.
methylxanthine (IBMX) (100 mM) with or without the test sample of differ-
ent concentrations for 48 h, and then the medium were replaced the same
1H–1H COSY spectrum was measured with spectral widths of both dimen-
sions of 4800 Hz, and 32 scans with two dummy scans were accumulated condition fresh medium, further incubation for 48 h. The cells were dis-
into 1K data points for each of 256 t1 increments. For HSQC spectra in the
solved in 50 ml of 1 N NaOH, and incubated at 90 °C for 1 h. Relative
phase sensitive mode and HMBC spectra, a total of 256 increments of 1K melanin content was determined spectrophotometrically by absorbance at
data points were collected. For HMBC spectra with Z-axis PFG, a 50 ms 400 nm. Arbutin was used as a positive control.
delay time was used for long-range C–H coupling. Zero-filling to 1K for F1
Cell Viability Assay The cell viability was determined by 3-(4,5-di-
and multiplication with squared cosine-bell windows shifted in both dimen- methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fifteen
sions were performed prior to 2D Fourier transformation. High-resolution
ESI-MS were obtained on a LTQ Orbitrap XL (Thermo Scientific).
Plant Material A voucher specimen of Balanophora papuana was
identified by Dr. Noramly Muslim, UKM, Malaysia and a voucher specimen
was deposited at the herbarium of UKM, Malaysia.
Extraction and Isolation The fresh tubers of aerial part of Bal-
anophora papuana (300 g) were extracted with ethyl acetate (3ꢃ500 ml, 3 d)
at room temperature. After filtration and removal the solvent by evaporation
in vacuo, a residue (31.2 g) was obtained. A part of the ethyl acetate extracts
(1.82 g) was applied to a ODS column (Column A: 33ꢃ180 mm) and eluted
with H2O/MeOH gradient system (1 : 1 and 3 : 7) and MeOH to afford 6
fractions. A fraction (Fr. 2) of column A eluted with H2O/MeOH (1 : 1) was
further purified with an ODS HPLC (YMC-Pack ProC18, 10ꢃ250 mm;
eluted with H2O/acetonitrile (70 : 30); flow rate, 2.0 ml/min; UV detection at
microliters of MTT solution (5 mg/ml) was added into each well of the cul-
tured medium. After further 2 h incubation, the medium was removed, and
then 50 ml of DMSO were added to resolve the formazan crystals. The opti-
cal density measurements were made using a microplate reader equipped
with a two wavelengths system at 550 nm and 700 nm. In each experiment,
three replicates were prepared for each sample. The ratio of the living cells
was determined on the basis of the difference of the absorbance between
those of samples and controls.
Tyrosinase Activity Assay Tyrosinase activity was spectrophotometri-
cally determined as L-tyrosine oxidase activity of mushroom tyrosinase with
same modifications. Briefly, 500 ml of 0.1 M phosphate buffer (pH 6.8) with
or without different concentrations of test samples, 300 ml of 2.5 mM L-tyro-
sine were mixed in an eppentube. Two hundred microliters of the mixtures
were transferred to 96-well microtiter plate. The absorbance at 475 nm was
254 nm) to afford ferulaldehyde (0.7 mg, tR 19 min) and (ꢀ)-pinoresinol measured with microplate reader as a blank. Then, 50 ml of 200 units/ml ty-
(15.8 mg, tR 28 min). A fraction (Fr. 3) of column A eluted with H2O/MeOH rosinase was added to each well. After incubation at 37 °C for 15 min, the
(1 : 1) was further purified with an ODS HPLC (Mightysil RP-18,
20ꢃ250 mm; eluted with H2O/acetonitrile (65 : 35); flow rate, 5.0 ml/min;
UV detection at 254 nm) to afford papuabalanol A (1: 13.6 mg, tR 36 min). A
amount of dopachrome produced in the reaction mixture was determined
spectrophotometrically at 475 nm on a microplate reader. The tyrosinase ac-
tivity of each test sample was calculated as follow: % inhibitionꢂ100ꢀ
fraction (Fr. 4) of column A eluted with H2O/MeOH (3 : 7) was separated [(AꢀB)/C]ꢃ100 where A is the OD475 with enzyme and test sample; B the
with a Sephadex LH-20 column (23ꢃ500 mm, eluted with MeOH), and the
OD475 with test sample, but without enzyme; C the OD475 with enzyme, but
second fraction was further purified with an ODS HPLC (YMC-Pack without test sample. Kojic acid was used as a positive control.
ProC18, 10ꢃ250 mm; eluted with H2O/acetonitrile (68 : 32); flow rate,
Vasodilation Assay A male Wistar rat weighting 260 g was sacrificed
2.0 ml/min; UV detection at 254 nm) to afford papuabalanol B (2: 7.8 mg, tR by bleeding from carotid arteries under anesthesia. A section of the thoracic
30 min).
Papuabalanol A (1): Yellow amorphous solid; [a]D27 ꢁ39 (cꢂ1.0, MeOH);
UV (MeOH) lmax (log e) 280 (5.4), 222 (5.6); CD (MeOH) lmax (De) 379
aorta between the aortic arch and the diaphragm was removed and placed in
oxygenated, modified Krebs–Henseleit solution (KHS: 118.0 mM NaCl,
4.7 mM KCl, 25.0 mM NaHCO3, 1.8 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM
Hosoya, Takahiro
