J Nat Med (2013) 67:369–374
373
140
control cells treated with the solvent DMSO/water without
sample analyte.
L-tyrosin
L-DOPA
117
115
120
Identification of compounds 1 and 2
Compounds 1 and 2 were identified by 1H NMR, 13C
100 100
100
99
98
1
1
NMR, H H COSY, HMQC, HMBC and UPLC-TOFMS.
Methanol-d4 was used as the NMR solvent. NMR mea-
surements were performed by using Bruker Biospin
AVANCE III 800 MHz NMR and JEOL EC600 MHz
NMR. UPLC-TOFMS (Waters WatersÒXevoTM QTof MS)
was performed using column C18 (2.1 mm u 9 100 mm l)
with MeOH/water = 5/95 (0 min), 100/0 (10 min), 100/0
(13 min) as eluent. The data were collected in negative
ionization mode. The capillary voltage was 3.0 kV. Cone
and desolvation gas flow rates were set at 50 and 1000 L/h,
respectively, and the source and desolvation temperature
was 150 and 500 °C, respectively.
80
60
40
20
The NMR data of the compounds isolated from H. ze-
ylanica roots are shown in Tables 1 and 2.
Compound 1: yellow powder, UPLC–TOFMS ES-
:
[M–1]- 787.2029, 625.1410, 463.0890, 301.0372 (m/z).
0
control
compound 1
compound 2
20
: 207, 265, 345 (nm). ½aꢁD : -33.4° (c = 0.84,
MeOH
max
UV k
10µM
MeOH:H2O 1:1 v/v). IR (KBr): 3402, 1655, 1612, 1499,
1457, 1365, 1260, 1205, 1072 (cm-1).
Fig. 3 Tyrosinase activity of compounds 1 and 2. Black bars
tyrosinase activity using L-tyrosine as substrate. White bars tyrosinase
activity using L-DOPA as substrate
Compound 2: yellow powder, UPLC–TOFMS ES-
:
[M–1]- 949.2466 (m/z).
MeOH
20
UV k
: 207, 265, 342 (nm). ½aꢁD : -41.0° (c = 0.83,
max
0.25 % trypsin/EDTA. The cells were placed in 10-cm
Petri dishes (1.0 9 105 cells/dish) and allowed to adhere at
37 °C for 24 h. After adding samples, cells were incubated
for 72 h and then washed with PBS, followed by lysis in
200 ll of 2 M NaOH with 40 min heating at 80 °C to
solubilize the melanin. The resulting lysate (150 ll) was
placed in a 96-well microplate, and the absorbance was
measured at 405 nm with a microplate reader. Each
experiment was repeated twice. Enhancement of melanine
production is expressed as percentage of that of control
cells treated with the solvent DMSO/water without sample
materials.
H2O). IR (KBr): 3402, 1642, 1615, 1519, 1466, 1343,
1312, 1162, 1087 (cm-1).
Acid hydrolysis
Acid hydrolysis was performed according to the previous
method [15]: 5 ml of 1 N HCl was added to 10 mg of com-
pound 1, and the mixture was stirred at 80 °C for 4 h. After
cooling down to room temperature, the solution was parti-
tioned between H2O and EtOAc. The H2O layer was freeze-
dried, and the EtOAc layer was analyzed by JEOL
EC600 MHz NMR. The freeze-dried product from the H2O
layer was dissolved in 1-(trimethylsilyl)-imidazole 0.4 ml and
pyridine5.0 ml to trimethylsilylate. Thesolutionwas stirred at
60 °Cfor5 min.AfterdryingthesolutioninastreamofN2, the
residue was partitioned between H2O and CHCl3. The CHCl3
layer was analysed by GC–MS (Shimadzu GCMS-QP 5050A)
using a DB-5 MS column (J&W Scientific 0.25 mm u 9
30 ml). Temperatures of the injector and detector were
250 °C. The temperature gradient of the column oven started
at 80 °C for 2 min and increased up to 250 °C over a period of
9 min. ThehydrolysisofstandardmonosaccharidesD-glucose,
D-galactose, D-arabinose and L-rhamnose were performed by
the same method as with compound 1.
Cell viability
Cell viability was determined using a hemocytometer
(Erma, Tokyo, Japan). B16 cells were cultured and samples
added as described in ‘‘Extraction and fractionation of
H. zeylanica root powder’’. After 72 h incubation, B16
melanoma cells were removed using 0.25 % trypsin/EDTA
solution, and then 10 ll trypan blue solution was added to
200 ll of the solution in a 1.5-ml tube. The cell number of
10 ll of mixed solution was counted in the hemocytometer.
Each experiment was repeated twice. Enhancement of
melanine production is expressed as percentage of that of
123
Yamauchi, Kosei
