Journal of Agricultural and Food Chemistry
Article
room with the specific activity of the enzyme of about 6680 U/mg was
also the product of Sigma-Aldrich. Trypsin−EDTA, penicillin,
streptomycin, and RPMI-1640 (Medium 1640 has a wide range of
applications for mammalian cells, developed by Roswell Park
Memorial Institute; 1640 is medium code) were purchased from
Gibco (Germany). Diethyl pyrocarbonate (DEPC), dimethyl sulfoxide
protein contents were determined by using the BCA protein assay kit
(Pierce, Rockford, IL). The reaction mixture, consisting of cell extract
supernatant (0.1 mL) and 0.9 mL of L-DOPA (3.8 mM) in 25 mM
phosphate buffer (pH 6.8), was mixed, and the tyrosinase activity was
read at 475 nm for 30 min. The reaction was performed at 25 °C.
Determination of Intracellular Melanin Levels. Melanin assay
was performed using procedures described previously by Tsuboi et
(
DMSO), Triton X-100, and 3-(4,5-dimethylthiazol-2-yl)-2.5-diphe-
nyltetrazolium bromide (MTT) were purchased from Amresco
USA). All other reagents were of analytical grade. The water used
1
9
al. Samples were added to the cell culture at different concentrations
0.05−1 mg/mL). After culturing for 72 h, cells were harvested. An
(
(
aliquot was used for protein determination, and the remaining cells
were centrifugation at 10000g for 15 min. The protein contents were
determined by using the BCA protein assay kit, and the remaining cells
were lysed in 0.5 mL of 5 M NaOH at 100 °C for 1 h; 0.2 mL portions
of crude cell extract were transferred to 96-well plates. Melanin
concentrations were calculated by comparison of the OD value at 405
nm.
was redistillated and ion-free.
Synthesis. Compounds were prepared by the reaction of
corresponding benzaldehyde with thiosemicarbazide in an acidic
1
6
solution of ethanol, as previous described.
A mixture of
corresponding benzaldehyde (20 mmol) with thiosemicarbazide (20
mmol) in 60 mL of ethanol with 4 mL of acetic acid solution was
refluxed for 2−5 h and then cooled to room temperature. The
precipitates were collected and washed with cold ethanol. The
products were purified by recrystallization from ethanol and were
identified by ESI-MS and NMR analyses. ESI-MS data were obtained
on a Bruker ESQUIRE-LC (Germany), and NMR data were acquired
on a 400 MHz NMR spectrometer (AV400) from Bruker (Germany).
Assay of the Enzyme Activity and Inhibitory Effects. The
monophenolase activity and the diphenolase activity assays were
Measurement of Cell Viability. Cell viability was determined
using the MTT assay. MTT is a tetrazolium salt and is converted to
insoluble formazan by mitochondrial dehydrogenase of living cells.
Briefly, after cells were incubated with samples (0.05−1 mg/mL) for
7
2 h, 20 μL of MTT (5 mg/mL stock solution) was added to each
well. After 1 h of incubation, the reaction was terminated by the
addition of dimethyl sulfoxide. The optical density of each well was
measured at 570 nm. Each treatment was performed in triplicate, and
each experiment was repeated three times.
1
2
performed as previously reported with modification. In this
investigation, L-Tyr was used as the substrate for the monophenolase
activity assay and L-DOPA as the substrate for the diphenolase activity
assay. The reaction media (3 mL) for activity assay contained 0.6 mM
RESULTS AND DISCUSSION
■
L-Tyr or 0.5 mM L-DOPA in 50 mM Na HPO −NaH PO buffer (pH
2
4
2
4
Chemical Synthesis of HBT and MBT. HBT and MBT
were synthesized and crystallized with cold ethanol. The
products obtained were white acicular crystals with yields of
6
.8) and 0.1 mL of different concentrations of inhibitor (dissolved in
DMSO as previously). They were first incubated at 30 °C for 10 min,
and then 0.1 mL of the aqueous solution of mushroom tyrosinase was
added to the mixture. The final concentrations of mushroom
tyrosinase were 222.66 U/mL for monophenolase activity and 44.56
U/mL for diphenolase activity. The enzyme activity was determined
by following the increasing absorbance at 475 nm accompanying the
oxidation of the substrates with a molar absorption coefficient of 3700
8
4.6 and 89.5%, respectively. The products were identified by
1
ESI-MS and H NMR. HBT has the characters m/z 196 (M +
H , DMSO); H NMR (DMSO-d , TMS, 400 MHz) δ 11.25
+
1
6
(HN, s), 9.89 (OH, s), 7.95 (CH, s), 8.08, 7.82 (NH
(C H , 2H, d), 6.76 (C H , 2H, d). MBT has the characters m/
, d), 7.62
2
6
2
6
2
−1
−1
+
1
(
M
cm ) by using a Beckman UV-650 spectrophotometer. The
z 210 (M + H , DMSO); H NMR (DMSO-d , TMS, 400
6
temperature was controlled at 30 °C. The extent of inhibition by the
addition of the sample was expressed as the percentage necessary for
MHz) δ 11.31 (HN, s), 8.00 (CH, s), 8.13, 7.92 (NH , d), 7.72
2
(
C H , 2H, d), 6.95 (C H , 2H, d), 3.76 (OCH , 3H, s). Their
6 2 6 2 3
5
0% inhibition (IC ). Controls, without inhibitor but containing 3.3%
50
structures are shown in Figure 1.
DMSO, were routinely carried out.
Determination of the Inhibition Type and Inhibition
Constant. The inhibition type was assayed by Lineweaver−Burk
plot, and the inhibition constant was determined by the second plots
of the apparent K /V
or 1/Vmapp versus the concentration of the
m
mapp
inhibitor.
Radical-Scavenging Activities. Radical-scavenging activities of
the samples on DPPH were estimated according to the modified
17
procedure described by Iwai et al. First, 1 mL of 100 mM acetate
buffer (pH 5.5), 1.80 mL of ethanol, and 0.1 mL of ethanolic solution
of 3 mM DPPH were put into a test tube. Then, 0.1 mL of the
different concentration of the sample solution (dissolved in DMSO)
was added to the tube and incubated at 25 °C for 20 min. As control,
Figure 1. Chemical structures of HBT (a) and MBT (b).
Effect of HBT and MBT on the Monophenolase
Activity of Tyrosinase. The kinetic courses of the oxidation
of L-Tyr by mushroom tyrosinase in different concentrations of
HBT or MBT were tested, and the results are shown in panels
A and B, respectively, of Figure 2. When the monophenolase
activity of tyrosinase was assayed using L-Tyr as substrate, a lag
time, characteristic of monophenolase activity, was observed
simultaneously with the appearance of the first stable product,
dopachrome. The system reached a constant rate after the lag
time, which was estimated by extrapolation of the linear portion
of the product accumulation curve to the abscissa. After the
reaction system reached the steady state, the curve of the
product increased linearly with increasing reaction time. The
slope of the line denoted the steady-state rate and the lag time.
The steady-state rate was influenced by HBT as shown in
Figure 2A-I. With increasing inhibitor concentration, the
steady-state rate descended distinctly and dose-dependently.
0.1 mL of DMSO was added to the tube. From the decrease of the
absorbance, the scavenging activity was calculated.
Cell Culture. B16 mouse melanoma cells were obtained from
Shanghai Institute of Biochemistry and Cell Biology (IBCB, Shanghai,
China). The cells were cultured in DMEM containing 10% heat-
inactivated fetal bovine serum, 2 mM glutamine, 100 units/mL
penicillin, and 100 μg/mL streptomycin in a humidified 5% CO /95%
2
air controlled incubator at 37 °C. Cell viability was detected with MTT
assay.
Intracellular Tyrosinase Activity Assay in B16 Cells. Tyrosinase
activity in B cells was detected as previously described by Mallick et
16
18
al. Cells were plated in 6-well plates at a density of 150 cells/mL.
After B16 cells were incubated in the presence or absence of samples
(
0.05−1 mg/mL) for 72 h, cells were washed with PBS and then
treated with lysis buffer (containing 1% Nonidet P-40, 0.01% SDS, and
.02% proteinase inhibitor cocktail). Cellular lysates were centrifuged
at 10000g at 4 °C for 20 min. The supernatants were collected, and the
0
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dx.doi.org/10.1021/jf204420x | J. Agric.Food Chem. 2012, 60, 1542−1547