Tyrosinase-inhibitory constituents from the twigs of Cinnamomum cassia

Article abstract of DOI:10.1021/np900031q

A methanol extract of the twigs of Cinnamomum cassia was found to possess inhibitory activity against tyrosinase. Purification of the MeOH extract afforded four new phenolics, cassiferaldehyde (6), icariside DC (9), cinnacassinol (10), and dihydrocinnacasside (13), together with 10 known compounds (1-5, 7-12, and 14). The structures of the new compounds were determined by spectroscopic data interpretation. Compounds 1-6 and 8-13 showed strong inhibitory activity against tyrosinase, with IC₅₀ values ranging from 0.24 to 0.94 mM.

Full text of DOI:10.1021/np900031q

J. Nat. Prod. 2009, 72, 1205–1208
1205
Tyrosinase-Inhibitory Constituents from the Twigs of Cinnamomum cassia
Tran Minh Ngoc,† IkSoo Lee,† Do Thi Ha,† HongJin Kim,† ByungSun Min,‡ and KiHwan Bae*,†
College of Pharmacy, Chungnam National UniVersity, Daejeon 305-764, South Korea, and College of Pharmacy, Catholic UniVersity of
Daegu, Gyeongsan 712-702, South Korea
ReceiVed January 19, 2009
A methanol extract of the twigs of Cinnamomum cassia was found to possess inhibitory activity against tyrosinase.
Purification of the MeOH extract afforded four new phenolics, cassiferaldehyde (6), icariside DC (9), cinnacassinol
(10), and dihydrocinnacasside (13), together with 10 known compounds (1-5, 7-12, and 14). The structures of the
new compounds were determined by spectroscopic data interpretation. Compounds 1-6 and 8-13 showed strong
inhibitory activity against tyrosinase, with IC50 values ranging from 0.24 to 0.94 mM.
Cinnamomum cassia Blume (Lauraceae) is distributed in the
southern part of mainland China, Myanmar, Laos, and Vietnam.
The twigs are used in traditional Chinese medicine for treating
dyspepsia, gastritis, blood circulation disturbances, diabetes, and
inflammatory diseases.1 Chemical and pharmacological investiga-
tions on C. cassia have resulted in the isolation of several bioactive
compounds such as cinnamaldehyde and cinnamic acid, as well as
coumarins, diterpenoids, and polyphenols,2,3 which have been found
to exhibit antifungal, cytotoxic, antipyretic, antioxidant, and
antimicrobial activities.4-9 In the course of a program to screen
for tyrosinase inhibitors from plants, it was found that a MeOH
extract of the twigs of C. cassia exhibited a strong inhibitory activity
(>85% inhibition at 100 µg/mL). Since it has been reported that
the tyrosinase inhibitors may have antimelanin synthesis activity
chromatography of these fractions resulted in the purification
of compounds 1-14. The known compounds were identified as
cinnamaldehyde (1),11 2-methoxycinnamaldehyde (2),12 2-hydroxy-
cinnamaldehyde (3),13 cinnamic acid (4),14 coniferaldehyde (5),15
cinnamic alcohol (7),16 o-coumaric acid (8),17 dihydomelilotoside
(11),18 methyl dihydromelilotoside (12),19 and rosavin (14).20
Compound 6 was obtained as a colorless, amorphous powder,
and its positive HREIMS showed a molecular ion peak [M]+ at
m/z 178.0631, consistent with the molecular formula C10H10O3. Its
IR spectrum showed absorption bands for a hydroxy group and a
carbonyl aldehyde, at νmax 3280 and 1673 cm-1, respectively. The
1H NMR spectrum displayed a singlet methoxy resonance and
signals for two trans-olefinic protons and three aromatic protons.
The 13C and DEPT NMR data (Table 1) of 6 were very similar to
those of coniferaldehyde (5)15 except for signals indicating the
locations of a methoxy group at C-2 (δC 148.9) and a hydroxy group
at C-3 (δC 152.1). This was supported by the long-range correlations
in the HMBC spectrum from the methoxy protons and the H-7
olefinic proton to C-2 and from the aromatic protons H-4 and H-5
to C-3 (Figure 1). Therefore, 6 was determined as a new cinna-
maldehyde derivative and has been named cassiferaldehyde.
Compound 9 was obtained as a colorless gum, with [R]25D +22.5.
Its IR spectrum showed an absorption band at 1458 cm-1 due to
an aromatic function and strong absorption bands at 3399 and 1075
cm-1 suggestive of a glycosidic structure. The molecular formula
was established as C19H28O10 from a molecular ion peak at m/z
416.1672 [M]+ in the HREIMS. The 1H and 13C NMR data (Table
1) of 9 indicated the presence of a ꢀ-D-glucopyranosyl moiety, a
ꢀ-D-apiofuranosyl moiety, and ethylphenyl resonances. The 1H and
13C NMR data were very similar to those of icariside D1,21 with a
downfield shift of C-4′ (δC 78.8) indicating the linkage to C-1′′ of
an apiofuranosyl moiety. Analysis of the HBMC spectrum of 9
showed long-range correlations between the H-4′ proton and C-1′′
carbon and between the H-1′′ proton and the C-4′ carbon (Figure
1). Consequently, the structure of icariside DC (9) was determined
as phenylethyl-1-O-ꢀ-D-apiofuranosyl-(1f4)-ꢀ-D-glucopyranoside.
Compound 10 was purified as an amorphous solid. The molecular
formula, C39H54O20, was observed from a molecular ion peak at
m/z 842.8362 [M]+ in the HREIMS. Combined analysis of the 1H,
13C, DEPT, and HMQC NMR spectra (Table 2) of 10 revealed the
presence of an o-hydroxycinnamic alcohol skeleton possessing an
oxygenated methylene signal, two trans-olefinic resonances, and
and offer a potential treatment for Parkinson’s disease,10
a
phytochemical investigation was carried out. The present report
describes the isolation of 14 phenolics (1-14) from the MeOH
extract of the twigs of C. cassia, including the characterization of
four new compounds (6, 9, 10, and 13). The inhibitory activity of
compounds 1-14 against tyrosinase has been evaluated.
1
o-hydroxyphenyl and phenyl methanol moieties. The H and 13C
The MeOH extract of the twigs of C. cassia was partitioned into
hexane-, EtOAc-, and BuOH-soluble fractions. Repeated column
NMR spectra also suggested the presence of four sugar moieties
of two ꢀ-D-glucopyranosyl units, one ꢀ-D-apiofuranosyl unit, and
one R-L-rhamnopyranosyl unit. The HMBC spectrum displayed key
correlations of the H-1′ aromeric proton to the C-2 carbon and the
H-7′′′′′ oxymethine protons to the C-1′′ carbon, showing the
connectivities of the ꢀ-D-glucopyranosyl to the cinnamic alchohol
* To whom correspondence should be addressed. Tel: 82-42-821-5925.
Fax: 82-42-823-6566. E-mail: baekh@cnu.ac.kr.
† Chungnam National University.
‡ Catholic University of Daegu.
10.1021/np900031q CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/21/2009

1206 Journal of Natural Products, 2009, Vol. 72, No. 6
Notes
Table 1. NMR Spectroscopic Data for 6, 9, and 13 (in MeOD)
6
9
13
position
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
2
3
4
5
6
7
129.5, qC
148.9, qC
152.1, qC
120.9, CH
125.9, CH
120.1, CH
150.1, CH
140.2, qC
129.5, CH
130.1, CH
127.3, CH
130.1, CH
129.6, CH
71.7, CH2
131.5, qC
157.1, qC
123.5, CH
128.8, CH
116.4, CH
131.1, CH
27.1, CH2
7.26 d (1.6, 7.6)
7.26 d (1.6, 7.6)
7.17 m
7.26 d (1.6, 7.6)
7.26 d (1.6, 7.6)
3.74 dt (9.6, 7.2)
4.09 dt (9.6, 7.2)
2.92 t (7.2)
6.91 dt (2.8, 7.2)
7.14 d (7.2) overlap
7.13 d (7.2) overlap
7.12 d (7.2) overlap
2.97 m
6.95 dd (1.5, 7.8)
7.00t (7.8)
7.17 dd(1.5, 7.8)
7.90 d (16.2)
8
9
130.6, CH
196.7, CHO
6.79 dd (7.8, 16.2)
9.67 d (7.8)
37.4, CH2
35.5, CH2
177.7, qC
102.7, CH
75.1, CH
78.3, CH
71.5, CH
78.2, CH
62.6,CH2
2.62 t (7.6)
Glc-1′
103.4, CH
78.7, CH
78.0, CH
78.8,CH
78.0, CH
62.8, CH2
4.35 d (7.6)
3.51 m
3.42 m
3.41 m
3.49 m
3.96 d (12.0)
3.85 dd (2.4, 12.0)
5.38 d (1.6)
3.93 d (1.6)
4.92 d (7.6)
3.51 m
3.42 m
3.41 m
3.49 m
2′
3′
4′
5′
6′
3.89 d (12.0) 3.71 dd (5.2, 12.0)
Apio-1′′
110.6, CH
77.9, CH
80.8, qC
2′′
3′′
4′′
75.4, CH2
3.96 d (9.6)
3.68 d (9.6)
5′′
66.3, CH2
3.59 dd (11.2, 14.2)
OCH3
61.8, CH3
3.85 s
at C-2, and another ꢀ-D-glucopyranosyl to phenylmethanyl at
C-7′′′′′. The correlations from H-1′′′′ to C-6′ and from H-1′′′ to
C-6′′ indicated the linkages of the R-L-rhamnopyranosyl and the
allowed the assignment of the esterification location of the ꢀ-D-
glucopyranosyl group in 13 at C-9, which was supported by the
key HMBC correlation between the H-1′ and the carbonyl carbon
C-9 (δC 177.7) (Figure 1). Accordingly, the structure of dihydro-
cinnacasside (13) was elucidated as o-hydroxyphenylpropanoyl-
O-ꢀ-D-glucopyranoside.
Compounds 1-14, together with kojic acid and hydroquinone
as positive controls, were examined for their inhibitory effects on
tyrosinase activity in an in vitro assay.22 The results are presented
in Table 3, and it may be seen that compounds 1-6 and 8-13
exhibited strong inhibition of tyrosinase with IC50 values ranging
from 0.24 to 0.94 mM. Of the positive controls used, hydroquinone
is used as a commercial whitening agent in comestics.23 The other
compounds (7 and 14) were inactive (IC50 >10 mM).
3
ꢀ-D-apiofuranosyl to C-6′ and C-6′′, respectively. The JC,H cor-
relations from H-2′ to C-2′′ and H-2′′ to C-2′ were supportive of
the (2-2) linkage of two the ꢀ-D-glucopyranosyl units (Figure 1).
Accordingly, the structure of cinnacassiol (10) was assigned as 2-[1-
O-(phenylmethanyl)-O-ꢀ-D-apiofuranosyl-(1-6)-ꢀ-D-glucopyrano-
syl]-(2-2)(O-R-L-rhamnopyranosyl)-(1-6)-ꢀ-D-glucopyranosyl-
cinnanic alcohol.
Compound 13 was isolated as a white amorphous powder, with
[R]25 +29.1, and showed a [M]+ ion at m/z 328.1168 in the
D
HREIMS, corresponding to the molecular formula C15H20O8. The
IR spectrum of 13 showed a glycosidic nature from absorptions at
3354 and 1087 cm-1, and a band at 1735 cm-1 for a carbonyl ester
group. The 1H NMR spectrum displayed the following representa-
tive signals: a propanoyl proton, an o-hydroxyphenyl resonance,
Experimental Section
1
General Experimental Procedures. Specific rotations were mea-
sured on a JASCO DIP-370 polarimeter. UV spectra were obtained
using a Beckman DU-650 recording spectrophotometer. FT-IR spectra
were carried out using a JASCO Report-100 infrared spectrometer. FT-
NMR spectra were recorded on a Bruker DRX-300 spectrometer (1H
NMR, 300 MHz; 13C NMR, 75 MHz) using CD3OD as the solvent
and tetramethylsilane (TMS) as an internal standard. Chemical shifts
(δ) are expressed in ppm with reference to the TMS signals. Two-
dimensional (2D) NMR (HMQC, HMBC) experiments were performed
on a Bruker Avance 500 spectrometer. HRMS was measured on a JMS-
700 Mstation mass spectrometer. Semipreparative HPLC was conducted
on Trilution LC with a UV/vis-151 detector, a 321 pump, a 402 syringe
pump, and a GX-271 liquid handler (Gilson, Inc.), using a YMC-pack
Pro C18 (250 × 20 mm, i.d.) column. Column chromatography was
performed using silica gel (Kieselgel 60, 70-230 mesh and 230-400
mesh, Merck). Thin-layer chromatography (TLC) was performed on
Merck precoated silica gel 60 F254 and/or RP-18 F254s plates (0.25 mm),
and compounds were observed under UV light at 254 and 365 nm or
visualized by spraying the dried plates with 10% H2SO4, followed by
heating at 180 °C. Optical density (OD) values in the tyrosinase
inhibitory activity assays were read on an Emax Precision microplate
reader.
and an aromeric proton of a ꢀ-D-glucopyranosyl moiety. The H
and 13C NMR data (Table 1) of 13 were closely comparable with
those of dihydromelilotoside (11).18 Analysis of the 2D NMR data
Plant Material. The dried twigs of C. cassia were purchased from
a pharmacy in Daejeon, Korea, in November 2006, and were identified
by one of us (K.B.). A voucher specimen (CN 95125) was deposited
at the herbarium in the College of Pharmacy, Chungnam National
University.
Figure 1. Selected HMBC correlations of compounds 6, 9, 10, and
13.

Notes
Journal of Natural Products, 2009, Vol. 72, No. 6 1207
Table 2. NMR Spectroscopic Data for 10 (in MeOD)
position
δC, mult.
δH (J in Hz)
position
δC, mult.
δH (J in Hz)
4.97 d (2.5)
3.90 d (2.5)
1
2
3
4
128.5, qC
156.3, qC
117.4, CH
129.9, CH
1′′′
2′′′
3′′′
4′′′
111.7, CH
78.2, CH
80.6, qC
75.3, CH
7.18 dd (1.5, 7.5)
7.21 dd (1.5, 7.5)
3.95 d (10.0)
3.74 d (10.0)
3.58 dd (1.5, 11.0)
4.78 d (1.0)
3.87 m
3.70 m
3.39 m
3.70 m
1.27 d (6.3)
5
6
7
8
123.7,CH
127.4, CH
126.6, CH
130.6, CH
64.2, CH2
103.0, CH
75.1, CH
78.2, CH
71.9, CH
77.2, CH
68.3, CH2
6.98 br t (7.5)
7.48 dd (1.5, 7.5)
7.07 d (16.0)
6.34 dd (5.5, 16.0)
4.25 dd (1.5, 5.5)
4.48 d (8.0)
3.51 m
3.45 m
3.29 m
3.58 m
3.67 m
5′′′
65.8, CH2
102.5, CH
72.4, CH
72.6, CH
74.2, CH
70.0, CH
18.2, CH3
139.0, qC
129.5, CH
129.4, CH
128.9, CH
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
1′′′′′
2′′′′′
3′′′′′
4′′′′′
9
1′
2′
3′
4′
5′
6′
7.42 brd (7.5)
7.33 d (7.5)
7.27 br t (7.5)
4.01 dd (2.0, 11.5)
4.33 d (7.5)
3.23 m
1′′
2′′
3′′
103.3, CH
75.1, CH
78.3, CH
5′′′′′
6′′′′′
7′′′′′
129.4, CH
129.5, CH
71.9, CH2
7.33 d (7.5)
7.42 brd (7.5)
4.87 d (12.5)
4.46 d (12.5)
3.30 m
4′′
5′′
6′′
71.8, CH
77.1, CH
68.9, CH
3.36 m
3.38 m
3.61 m
4.02 dd (1.5, 11.0)
matographed on a silica gel column, with mixtures of hexane-EtOAc
(10:1 to 2:1) as the eluting solvent systems, and semipreparative HPLC
(YMC-ODS, 10 × 250 mm, 5 µm), eluted with MeOH-H2O (40:60),
to afford 5 (516 mg) and 6 (4.2 mg). Compound 7 (120 mg) was purified
from fraction E4 using silica gel column chromatography, eluted with
hexane-EtOAc (3:1), and then purified by MPLC (YMC-ODS 15 ×
300 mm, 5 µm), with MeOH-H2O (60:40) as mobile phase. Subfraction
E5 was subjected to silica gel column chromatography, eluted with
CHCl3-MeOH-H2O (3:1:0.1), to afford 8 (52 mg).
Table 3. Inhibitory Activity of Isolated Compounds 1-14
against Tyrosinase
inhibitory activity
compound
IC50 (mM)a
cinnamaldehyde (1)
2-methoxy cinnamaldehyde (2)
2-hydroxy cinnamaldehyde (3)
cinnamic acid (4)
coniferaldehyde (5)
cassiferaldehyde (6)
cinnamic alcohol (7)
O-coumaric acid (8)
icariside DC (9)
0.52 ( 0.03
0.42 ( 0.02
0.47 ( 0.03
0.41 ( 0.01
0.24 ( 0.03
0.26 ( 0.04
>10
The n-BuOH-soluble fraction (135 g) was suspended in H2O and
then applied to a Diaion HP-20 column with a H2O and MeOH gradient
eluent to give fractions B1-B14. Fraction B5 was subjected to passage
over silica gel, using CH3Cl-MeOH-H2O (15:1:0.1 to 1:1:0.1) for
elution, to yield nine subfractions, B5.1-B5.9. Subfraction B5.5 was
applied to a Sephadex LH-20 column, eluted with MeOH-H2O (1:5),
and then YMC-ODS column chromatography with the mobile phase
system MeOH-H2O (1:4) to give 9 (87 mg) and 10 (2.7 mg). Fraction
B6 was chromatographed on a Sephadex LH-20 column with
MeOH-H2O (1:5) as solvent and then purified by reversed-phase HPLC
(YMC-ODS, 10 × 250 mm, 5 µm), with MeOH-H2O (1:5) as mobile
phase, yielding 11 (15 mg), 12 (10 mg), 13 (12 mg), and 14 (43 mg).
Cassiferaldehyde (6): colorless, amorphous powder; mp 78-80 °C;
UV (MeOH) λmax (log ꢀ) 214 (3.30) and 350 (3.24) nm; IR (KBr) νmax
3280, 1673, 1586, 1331, 1206, 1106, and 727 cm-1; 1H and 13C NMR
data, see Table 1; HREIMS m/z 178.0631 [M]+ (calcd for C10H10O3,
178.0630).
0.67 ( 0.03
0.71 ( 0.03
0.94 ( 0.04
0.57 ( 0.01
0.63 ( 0.02
0.67 ( 0.03
>10
cinnacassinol (10)
dihydromelilotoside (11)
methyl dihydromelilotoside (12)
dihydrocinnacasside (13)
rosavin (14)
kojic acidb
0.14 ( 0.02
4.21 ( 0.05
hydroquinoneb
a Values present mean ( SD of triplicate experiments. b Positive
control.
Extraction and Isolation. The sliced twigs of C. cassia (30 kg)
were extracted with hot MeOH (60 L × 3 times) for two days. The
MeOH extracts were filtered, combined, and concentrated in vacuo,
resulting in the production of a residue (1650 g). This residue was
suspended in H2O and then fractionated successively with hexane,
EtOAc, and n-BuOH, producing a hexane-soluble (410 g), EtOAc-
soluble (230 g), and n-BuOH-soluble (135 g) fraction, respectively.
The hexane-soluble fraction (410 g) was subjected to silica gel
column chromatography, eluted with hexane and EtOAc gradient
mixtures (100:1-0:100), to give seven fractions, H1-H7. Fraction
H1 was chromatographed on silica gel, eluting with hexane-EtOAc
(25:1), as well as ODS silica gel with MeOH-H2O (55:45), to afford
1 (6.0 g) and 2 (1.4 g). Fraction H6 was subjected to silica gel
column chromatography, eluting with hexane-EtOAc (10:1), and
then purification by medium-pressure liquid chromatography (MPLC)
(YMC-ODS 15 × 30 mm, 5 µm), eluted with MeOH-H2O (60:
40), to give 3 (1.2 g). Compound 4 (2.2 g) was isolated and purified
from fraction H7 using YMC-ODS column chromatography, with
MeOH-H2O (60:40) as eluant, and MPLC (YMC-ODS 15 × 300
mm, 5 µm), eluted with MeOH-H2O (50:50).
Icariside DC (9): colorless gum; [R]25 +22.5 (c 0.24, MeOH);
D
UV (MeOH) λmax (log ꢀ) 214 (3.00) nm; IR (KBr) νmax 3399, 2936,
1
1458, and 1066 cm-1; H and 13C NMR data, see Table 1; HREIMS
m/z 416.1672 [M]+ (calcd for C19H28O10, 416.1682).
Cinnacassiol (10): white, amorphous powder; [R]25D -57.2 (c 0.35,
MeOH); UV (MeOH) λmax (log ꢀ) 204 (2.31), 248 (1.82), and 284 (1.33)
1
nm; IR (KBr) νmax 3506, 3354, 2900, 1735, 1240, and 1087 cm-1; H
and 13C NMR data, see Table 2; HREIMS m/z 842.8362 [M]+ (calcd
for C39H54O20, 842.8368).
Dihydrocinnacasside (13): colorless, amorphous powder; mp
174-175 °C; [R]25 -29.1 (c 0.4, MeOH); UV (MeOH) λmax (log ꢀ)
D
214 (2.55) nm; IR (KBr) νmax 3354, 2900, 1735, 1240, and 1087 cm-1
;
1H and 13C NMR data, see Table 1; HREIMS m/z 328.1168 [M]+ (calcd
for C19H28O10, 328.1158).
Acid Hydrolysis of Compounds 9, 10, and 13. Solutions of 9 (5.2
mg), 10 (1.2 mg), and 13 (2.5 mg) in 0.5 N H2SO4 (dioxane-H2O,
1:1, 2 mL) were each heated at 100 °C for 2 h. Each reaction mixture
was diluted with H2O (3 mL) and extracted with CHCl3 (5 mL × 3
times). The H2O layer was dried in vacuo after neutralization with 1 N
NaOH and passed through a Sep-Pak C18 cartridge (Waters, Milford,
The EtOAc-soluble fraction (230 g) was chromatographed on a silica
gel column with a stepwise gradient of CHCl3 and MeOH (60:1 to
0:1), to separate eight fractions, E1-E8. Subfraction E2 was chro-

1208 Journal of Natural Products, 2009, Vol. 72, No. 6
Notes
(3) Yazaki, K.; Okudu, T. Phytochemistry 1990, 29, 1559–1562.
(4) Singh, H. B.; Srivastava, M.; Singh, A. B.; Srivastava, A. K. Allergy
1995, 50, 995–999.
MA). Sugars were identified as glucose (Rf 0.16), rhamnose (Rf 0.32),
and apiose (Rf 0.38) by TLC in CHCl3-MeOH-H2O (12:6:1), using
the authentic samples D-glucose, L-rhamnose, and D-apiose. The
remaining eluate was concentrated to dryness, and the residue was
stirred with D-cysteine methyl ester hydrochloride, hexamethyldisila-
zane, and trimethylsilylchloride in pyridine using the same procedures
as in a previous report.24 After the reaction, the supernatant was
analyzed by GC [column: capillary column BD-5, 0.25 mm × 30 m,
detector: FID, detector temperature: 300 °C, injector temperature: 270
°C, carrier gas, N2; column temperature, 210 °C]. The peaks corre-
sponding to the D-glucosyl, L-rhamnosyl, and D-apiosyl derivatives
appeared with tR 8.55, 5.31, and 4.03 min, respectively.
(5) Kwon, B. M.; Lee, S. H.; Choi, S. U.; Park, S. H.; Lee, C. O.; Cho,
Y. K.; Sung, N. D.; Bok, S. H. Arch. Pharm. Res. 1998, 21, 147–152.
(6) Kurokawa, M.; Kumeda, C. A.; Yamamura, T.; Shiraki, K. Eur.
J. Pharmacol. 1998, 348, 45–51.
(7) Lin, C. C.; Wu, S. J.; Chang, C. H.; Ng, L. T. Phytother. Res. 2003,
17, 726–730.
(8) Kim, H. O.; Park, S. W.; Park, H. D. Food Microbiol. 2004, 21, 105–
110.
(9) Cheng, S. S.; Liu, J. Y.; Tsai, K. H.; Chen, W. J.; Chang, S. T. J.
Agric. Food Chem. 2004, 52, 4395–4400.
Tyrosinase Inhibitory Activity Assay. Mushroom tyrosinase and
L-dopa used for the bioassay were purchased from Sigma Chemical
Co. Antityrosinase activity was measured by spectrophotometry,
according to the method of Mason and Peterson25 with minor
modifications. Each test substance was dissolved in 0.1 mL of 10%
DMSO in aqueous solution and incubated with 0.1 mL of 135 U/mL
mushroom tyrosinase in phosphate buffer solution (PBS, pH 6.8) at 25
°C for 10 min, and then 0.1 mL of L-dopa (0.5 mM, PBS pH 6.8) was
added. The reaction mixture was incubated for 5 min. The amount of
dopachrome in the mixture was determined by the optical density (OD)
at 475 nm using an Emax Precision microplate reader. Kojic acid and
hydroquinone (Sigma) were used as positive control agents. The
inhibitory percentage of tyrosinase was calculated as follows: %
inhibition ) {[(A - B) - (C - D)]/(A - B)} × 100 (A: OD at 475 nm
without test substance; B: OD at 475 nm without test substance and
tyrosinase; C: OD at 475 nm with test substance; D: OD at 475 nm
with test substance, but without tyrosinase).
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Acknowledgment. This work was supported by a grant from the
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1
Supporting Information Available: H, 13C, HMQC, and HMBC
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available free charge via the Internet at http://pubs.acs.org.
References and Notes
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