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Tyrosinase Inhibitors from the Wood

of Artocarpus heterophyllus
Ngan Bui

Journal of Natural Products

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pubs.acs.org/jnp

Tyrosinase Inhibitors from the Wood of Artocarpus heterophyllus

Nhan Trung Nguyen, Mai Ha Khoa Nguyen, Hai Xuan Nguyen, Ngan Kim Nguyen Bui,
and Mai Thanh Thi Nguyen*
Faculty of Chemistry, University of Science, Vietnam National University−HoChiMinh City, 227 Nguyen Van Cu Street, District 5,
HoChiMinh City, Vietnam
*
S Supporting Information

ABSTRACT: From the methanolic-soluble extract of the

wood of Artocarpus heterophyllus, four new flavones,
artocarmins A−D (1−4), and three new chalcones,
artocarmitins A−C (5−7), have been isolated together with
13 known compounds. Their structures were determined on
the basis of the spectroscopic data. Compounds 1−4, 6, 7, 9−
16, and 20 displayed significant tyrosinase inhibitory activity.
The most active compound, morachalcone A (12) (IC50, 0.013
μM), was 3000 times more active as a tyrosinase inhibitor than
a positive control, kojic acid (IC50, 44.6 μM).

Artocarpus heterophyllus Lam. (Moraceae), known as “jackfruit”,
is distributed widely in tropical and subtropical regions of Asia.
In Vietnam, A. heterophyllus is known as “Mit”, and this plant is
commonly cultivated for its edible fruits, while its wood has
been used as an anti-inflammatory, antioxidant, and antiaging
agent.1 Previously, prenylflavonoids, stilbenes, triterpenes, and
sterols have been isolated from the roots and stems of this
plant.2 As part of a search for biologically active compounds
with utility against overproduction of melanin in human skin
epidermal layers, a screen has been initiated to evaluate natural
product extracts for the inhibition of tyrosinase. A methanolic
extract of the wood of A. heterophyllus showed significant
tyrosinase inhibitory activity with an IC50 value of 2.3 μg/mL.
Thus, the constituents of this plant were examined, and four
new flavones (1−4) and three new chalcones (5−7) were
isolated, together with 13 known compounds (8−20). In this
paper, we report the isolation and structure elucidation of the
new compounds using spectroscopic techniques, together with
their tyrosinase inhibitory activity.
■ RESULTS AND DISCUSSION
The MeOH extract of the wood of A. heterophyllus was
partitioned between EtOAc and water to give an EtOAc-soluble
fraction. The EtOAc fraction was subjected to a series of
chromatographic separation and preparative TLC steps to
afford four new flavones, artocarmins A−D (1−4), and three
new chalcones, artocarmitins A−C (5−7), together with 13
known compounds. The known compounds were identified by
analysis of their spectroscopic data and comparison with
literature data as 3′-[γ-hydroxymethyl-(Z)-γ-methylallyl]-
4,2′,4′-trihydroxychalcone (8),3 gemichalcone B (9),3 gem-
ichalcone A (10),3 isogemichalcone B (11),3 morachalcone A
(12),4 norartocarpin (13),5 cudraflavone C (14),6 albanin A
(15),7 p-hydroxybenzoic acid (16),8 β-resorcylic acid (17),9
© 2012 American Chemical Society and
American Society of Pharmacognosy
vanillic acid (18),10 goldfussinol (19),11 and p-coumaric acid
(20).12
Artocarmin A (1) showed a quasimolecular ion at m/z
377.0998 [M + Na]+, corresponding to the molecular formula
C20H18O6 in the HRESIMS. Its IR spectrum displayed the
absorbance of hydroxy (3400 cm−1), phenyl (1605, 1450
cm−1), and carbonyl (1660 cm−1) groups. The 1H NMR
spectrum of 1 (Table 1) exhibited signals due to two sets of
ortho-coupled aromatic protons at δ 7.90 (2H, d, J = 9.0 Hz)
and 6.92 (2H, d, J = 9.0 Hz), an isolated aromatic proton at δ
6.54 (1H, s), and two isolated olefinic protons at δ 6.75 (1H, s)
and 5.40 (1H, t, J = 7.0 Hz), together with one methyl, one
methylene, one oxymethylene, and a characteristic signal of a
hydrogen-bonded hydroxy proton (δ 13.21). On the other
hand, the 13C NMR spectrum (Table 1) showed 20 carbon
signals including a carbonyl carbon, two sp2 carbons, an sp2
quaternary carbon, an sp2 oxygenated quaternary carbon, a
methyl carbon, a methylene carbon, an oxymethylene carbon,
and 12 aromatic carbons. These data were similar to those of
apigenin,13 a common flavone found in plants, except for the
Received: August 21, 2012
Published: October 31, 2012
1951 dx.doi.org/10.1021/np300576w | J. Nat. Prod. 2012, 75, 1951−1955
Journal of Natural Products Article

Table 1. 1H and 13C NMR Data (δ, 500 and 125 MHz) for Compounds 1−4 in DMSO-d6
1 2 3 4
position δH δC δH δC δH δC δH δC
2 163.5 163.9 163.7 161.5a
3 6.75, s 102.8 6.76, s 103.0 6.75, s 102.8 6.97, s 106.7
4 181.8 182.0 181.8 181.9
4a 103.5 103.8 103.6 103.3
5 158.4 158.8 158.5 158.6b
6 110.8 110.2 110.0 110.4
7 161.8 162.1 161.9 161.7a
8 6.54, s 93.2 6.55, s 93.4 6.55, s) 93.3 6.51, s 93.0
8a 155.1 155.5 155.5 155.1
1′ 121.3 121.6 121.3 108.7
2′ 7.90, d (9.0) 128.4 7.91, d (8.8) 128.5 7.90, d (9.0) 128.5 158.3b
3′ 6.92, d (9.0) 116.0 6.92, d (8.8) 116.1 6.92, d (9.0) 116.0 6.50, d (2.3) 103.3
4′ 161.1 161.3 161.1 161.6a
5′ 6.92, d (9.0) 116.0 6.92, d (8.8) 116.1 6.92, d (9.0) 116.0 6.44, dd (8.8, 2.3) 108.0
6′ 7.90, d (9.0) 128.4 7.91, d (8.8) 128.5 7.90, d (9.0) 128.5 7.73, d (8.8) 129.6
1″ 3.25, d (7.0) 20.5 3.30, d (7.1) 20.9 3.29, d (7.0) 20.8 3.25, d (7.2) 20.4
2″ 5.40, t (7.0) 121.1 5.56, t (7.1) 126.5 5.56, t (7.0) 126.4 5.40, t (7.2) 121.2
3″ 135.2 130.3 130.2 135.1
4″ 3.75, d (5.0) 66.3 4.52, s 69.2 4.51, s 69.1 3.75, d (5.5) 66.3
5″ 1.69, s 13.6 1.80, s 13.9 1.80, s 14.0 1.69, s 13.5
1‴ 125.4 125.7
2‴ 7.54, d (8.6) 130.3 7.30, d (1.5) 111.3
3‴ 6.78, d (8.6) 115.9 148.0
4‴ 160.0 149.4
5‴ 6.78, d (8.6) 115.9 6.77, d (8.0) 115.6
6‴ 7.54, d (8.6) 130.3 7.10, dd (8.0, 1.5) 123.2
7‴ 7.54, d (16.0) 144.9 7.52, d (16.0) 145.2
8‴ 6.39, d (16.0) 114.3 6.47, d (16.0) 114.5
9‴ 166.6 166.6
OH-5 13.21, s 13.27, s 13.26, s 13.29, s
OH-4″ 4.64, t (5.0) 4.62, t (5.5)
OMe-3‴ 3.79, s 55.8
a,b
These signals may be interchanged.

presence of signals of a set of resonances due to a 4-
hydroxyprenyl unit, which was confirmed by the HMBC
correlations of protons of H-2″ and H-5″ with C-4″ (Figure 1)
and the downfield shift of C-4″ (δH 3.75, δC 66.3). The location
Figure 1. Connectivities (bold lines) deduced by the COSY spectrum,
significant HMBC correlations (arrows), and NOESY correlations
(dashed arrows) observed for 1−3.
1952
of the 4-hydroxyprenyl group was determined at C6 on the
basis of the HMBC correlations of H-1″ with C-5, C-6, and C-7
and of a NOESY correlation of a proton of OH-5 with H-1″.
Finally, the NOESY correlations of H-1″ with H-5″ and of H-
2″ with H-4″ indicated an E-configuration for the 2″, 3″ double
bond (Figure 1). From this spectroscopic evidence, the
structure of artocarmin A was concluded to be 1.
Artocarmin B (2) and C (3) were obtained as pale yellow,
amorphous solids. The 1H and 13C NMR spectra of 2 and 3
were similar to those of 1, but signals were displayed due to one
more trans-p-coumaroyl unit in 2 and one more trans-feruloyl
moiety in 3, which were confirmed by the COSY, HSQC, and
HMBC spectra (Figure 1). The locations of these moieties
were determined at C-4″ by HMBC correlation of H-4″ with
the C-9‴ ester carbonyl carbon of these moieties. The NOESY
correlations of H-1″ with H-5″ and of H-2″ with H-4″
indicated the configurations of 2 and 3 to be the same as that of
1 (Figure 1). Thus, the structures of artocarmins B and C (3)
were concluded to be 4″-trans-p-coumaroylartocarmin A (2)
and 4″-trans-feruloylartocarmin A (3), respectively.
The molecular formula of artocarmin D (4) was determined
as C20H18O7 by HRESIMS. The 1H and 13C NMR spectra of 4
were similar to those of 1, except for the presence of signals due
to an ABX system accompanied by the disappearance of signals
due to two sets of ortho-coupled aromatic protons in 1 (Table
dx.doi.org/10.1021/np300576w | J. Nat. Prod. 2012, 75, 1951−1955
Journal of Natural Products Article

1). Analysis of the COSY, HSQC, and HMBC spectra of 4
indicated a hydroxy group is present at C-2′. The configuration
of 4 was confirmed to be the same as 1 based on the results of
NOESY spectrum. From the above finding, the structure of
artocarmin D (4) was determined to be 2′-hydroxyartocarmin
A.
The HRESIMS of artocarmitin A (5) showed a quasimo-
lecular ion at m/z 363.1216 [M + Na]+, consistent with the
molecular formula C20H20O5. The 1H NMR spectrum of 5
(Table 2) displayed signals due to three sets of ortho-coupled
aromatic protons at δ 7.72 (2H, d, J = 8.5 Hz), 6.92 (2H, d, J =
8.5 Hz), 6.54 (1H, d, J = 9.0 Hz), and 7.97 (1H, d, J = 9.0 Hz),
two trans olefinic protons at δ 7.75 (1H, d, J = 15.5 Hz) and
7.83 (1H, d, J = 15.5 Hz), an isolated olefinic proton at δ 5.52
(1H, t, J = 7.5 Hz), and one methyl, one methylene, one
oxymethylene, and a characteristic signal of a hydrogen-bonded
hydroxy proton (δ 13.99). The 13C NMR spectrum (Table 2)
showed 20 carbon signals including those corresponding to the
above groups, as well as a ketone carbonyl carbon (δ 193.2).
These data suggested 5 is a chalcone bearing a prenyl group.
Analysis of the COSY, HMQC, and HMBC spectra indicated
the chalcone to be 4,2′,4′-trihydroxychalcone. Similar to 1, the
1
H and 13C NMR spectra of 5 showed characteristic peaks for a
4-hydroxyprenyl group. The location of the 4-hydroxyprenyl
group was determined to be at C-3′ on the basis of the HMBC
correlations of H-1″ with C-2′, C-3′, and C-4′ (Figure 2). The

Table 2. 1H and 13C NMR Data (δ, 500 and 125 MHz) for
Compounds 5−7 in Acetone-d6

5 6 7
position δH δC δH δC δH δC
1 127.7 127.5 127.5
2 7.72, d 131.7 7.73, d 131.7 7.72, d 131.7
(8.5) (8.5) (8.0)
3 6.92, d 116.8 6.93, d 116.8 6.92, d 116.8
(8.5) (8.5) (8.0)
4 161.1 161.1 161.1
5 6.92, d 116.8 6.93, d 116.8 6.92, d 116.8
(8.5) (8.5) (8.0)
6 7.72, d 131.7 7.73, d 131.7 7.72, d 131.7
(8.5) (8.5) (8.0)
CO 193.2 193.1 193.0 Figure 2. Connectivities (bold lines) deduced by the COSY spectrum,
α 7.75, d 118.6 7.76, d 118.4 7.74, d 118.4
significant HMBC correlations (arrows), and NOESY correlations
(15.5) (15.5) (15.0) (dashed arrows) observed for 5 and 7.
β 7.83, d 145.0 7.84, d 145.0 7.83, d 145.0
(15.5) (15.5) (15.0)
1′ 114.5 114.4 114.4
NOESY correlations of H-1″ with H-5″ and of H-2″ with H-4″
2′ 165.3 165.2 165.1
indicated that the double bond of this prenyl group has the E-
3′ 115.9 115.1 115.1
configuration (Figure 2). Therefore, compound 5 was the
4′ 162.9 162.9 162.9
geometric isomer of 8, isolated from the same extract, which
5′ 6.54, d 108.1 6.56, d 108.1 6.56, d 108.1
possesses a Z-configuration. From these data, the structure of
(9.0) (9.0) (8.5) artocarmitin A was concluded to be 5.
6′ 7.97, d 130.4 8.00, d 130.5 7.99, d 130.5 Artocarmitin B (6) was isolated as an amorphous powder.
(9.0) (9.0) (8.5) The HRESIMS indicated its molecular formula to be C30H28O8.
1″ 3.41, d 21.9 3.47, d 22.0 3.50, d 22.1 The 13C NMR data of 6 (Table 2) showed 15 carbons for a
(7.5) (7.5) (7.5)
4,2′,4′-trihydroxychalcone unit together with a 4-hydroxyprenyl
2″ 5.52, t 123.1 5.68, t 127.7 5.58, t 128.5
(7.5) (7.5) (7.5) group similar to 5. The remaining 10 carbons were accounted
3″ 136.2 131.2 131.0 for by a trans-feruloyl substituent. The location of the 4-
4″ 3.89, s 68.5 4.55, s 70.2 1.75, s 21.6 hydroxyprenyl group was deduced at C-3′, the same as 5, from
5″ 1.80, s 13.9 1.88, s 14.2 4.96, s 63.6 the HMBC correlations of H-1″ with C-2′, C-3′, and C-4′. A
1‴ 127.4 126.2 HMBC correlation of proton H-4″ with the C-9‴ ester
2‴ 7.34, d 111.3 7.04, s 106.9 carbonyl carbon of the feruloyl moiety indicated that the
(1.5) feruloyl moiety is connected with the prenyl unit at C-4″. The
3‴ 148.8 149.0 configuration of the 2″, 3″ double bond of 6 was determined to
4‴ 150.1 139.5 be the same as that of 5 from the NOESY spectrum, with 6
5‴ 6.86, d 116.0 149.0 being a geometric isomer of 10, isolated from the same extract.
(8.5) Thus, the structure of artocarmitin B (6) was concluded to be
6‴ 7.34, d 124.0 7.04, s 106.9 4″-trans-feruloylartocarmitin A.
(8.5, 1.5)
7‴ 7.58, d 145.7 7.61, d 145.9
Artocarmitin C (7) was found to possess a molecular formula
(16.0) (16.0) of C31H30O9 on the basis of its HRESIMS and NMR data. The
1
8‴ 6.40, d 115.8 6.46, d 116.2 H and 13C NMR spectra of 4 (Table 2) also closely resembled
(16.0) (16.0) those of artocamitin B (6), but they showed the presence of
9‴ 167.3 167.6 one more methoxy group (δH 3.89, δC 56.7) and a set of meta-
OH-2′ 13.99 s 14.06, s 14.07, s coupled aromatic protons instead of signals due to an ABX
OMe-3‴ 3.91, s 56.3 3.89, s 56.7 system in 6. The methoxy group was placed at C-5‴ by analysis
OMe-5‴ 3.89, s 56.7 of the COSY, HSQC, and HMBC spectra (Figure 2), indicating
1953 dx.doi.org/10.1021/np300576w | J. Nat. Prod. 2012, 75, 1951−1955
Journal of Natural Products
Table 3. Tyrosinase Inhibitory Activity of the Isolated Compounds
compound IC50, μMa compound
1 18.7 ± 0.1 8 >100
2 8.4 ± 0.1 9 55.3
3 40.0 ± 0.4 10
4 47.3 ± 0.4 11
5 >100 12
6 66.2 ± 0.6 13
7 20.6 ± 0.2 14
a
Each value represents the mean ± SD of three determinations.
the presence of a trans-sinapoyl moiety in 7. The location of
this sinapoyl substituent was determined at C-5″ of the prenyl
unit by the HMBC correlation of H-5″ with C-9‴ of the
sinapoyl moiety. The 13C NMR spectrum of the prenyl
chalcone moiety in 7 showed identical chemical shifts to those
of the corresponding carbon signals for 5 and 6, except for the
chemical shifts of C-4″ and C-5″, which were shifted to high
field and low field, respectively, compared with 5 and 6. This
indicated that the configuration of the 2″, 3″ double bond of 7
is Z, which was confirmed by the NOESY correlations of H-1″
with H-5″ and of H-2″ with H-4″ (Figure 2). Thus, the
structure of artocarmitin C was concluded to be 7.
These compounds were examined for their tyrosinase
inhibitory activity (Table 3). The assay was carried out at
four different concentrations ranging from 0.01−100 μM, and
compounds 1−4, 6, 7, 9−16, and 20 displayed significant
concentration-dependent inhibition. Compounds 1−3, 7, 12−
16, and 20 showed more potent inhibitory activities (IC50
values 0.013 to 40.0 μM) than a positive control, kojic acid
(IC50, 44.6 μM), a well-known tyrosinase inhibitor currently
used as a cosmetic skin-whitening agent. The most active
compound, morachalcone A (12) (IC50, 0.013 μM), was 3000
times more active in tyrosinase inhibitory activity than kojic
acid. Therefore, the traditional use of A. heterophyllus for
antioxidant and skin-care agents in Vietnam might be
attributable to the tyrosinase inhibitory activity of its flavonoid,
chalcone, and phenolic constituents.
■ EXPERIMENTAL SECTION
General Experimental Procedures. IR spectra were measured
with a Shimadzu IR-408 spectrophotometer in CHCl3 solution. NMR
spectra were taken on a Bruker Avance III 500 spectrometer (Bruker
Biospin) with tetramethylsilane as an internal standard, and chemical
shifts are expressed in δ values. HRESIMS measurements were carried
out on a Bruker microTOF-QII spectrometer. Column chromatog-
raphy was performed with BW-820MH Si gel (Fuji Silisia, Aichi,
Japan). Analytical and preparative TLC were carried out on precoated
Merck Kieselgel 60F254 or RP-18F254 plates (0.25 or 0.5 mm
thickness).
Plant Material. The wood of A. heterophyllus was collected at the
Seven-Mountain area, An Giang Province, Vietnam, in August 2010,
and identified by Ms. Hoang Viet, Faculty of Biology, University of
Science, Vietnam National University−HoChiMinh City. A voucher
sample of the wood part (AN-2985) has been deposited at the
Department of Analytical Chemistry, Faculty of Chemistry, University
of Science, Vietnam National University−HoChiMinh City.
Extraction and Isolation. The dried powdered wood of A.
heterophyllus (5.8 kg) was extracted with MeOH (15 L, reflux, 3 h × 3)
to yield a MeOH extract (310 g; IC50 2.3 μg/mL). The extract was
partitioned between EtOAc and water to give an EtOAc-soluble
fraction (64.2 g; IC50 1.0 μg/mL). The EtOAc-soluble fraction was
subjected to silica gel column chromatography with acetone−hexane
to give six fractions: fr. 1 (0.3 g; IC50 > 100 μg/mL), fr. 2 (2.3 g; IC50
1954
Article
IC50, μMa compound IC50, μMa
15 1.01 ± 0.05
± 0.5 16 9.3 ± 0.1
73.6 ± 0.7 17 >100
82.2 ± 0.8 18 >100
0.013 ± 0.002 19 >100
17.3 ± 0.1 20 2.3 ± 0.1
21.4 ± 0.2 kojic acid 44.6 ± 0.4
1.8 μg/mL), fr. 3 (0.3 g; IC50 11.5 μg/mL), fr. 4 (1.2 g; IC50 0.5 μg/
mL), fr. 5 (1.3 g; IC50 1.4 μg/mL), and fr. 6 (42.3 g; IC50 < 0.1 μg/
mL).
Fraction 2 was subjected to silica gel column chromatography,
eluted with EtOAc−hexane (0−30%), to afford two subfractions (2-1,
450 mg; 2-2, 1.5 mg). Subfraction 2-1 was recrystallized with acetone−
hexane to give 15 (50.0 mg), while subfraction 2-2 was rechromato-
graphed with 2% MeOH−CHCl3, followed by preparative TLC with
3% MeOH−CHCl3, to give 10 (6.8 mg), 11 (5.7 mg), 17 (5 mg), and
18 (6.7 mg).
Fraction 3 was chromatographed further using MeOH−CHCl3 (0−
30%) to afford two subfractions (3-1, 110 mg; 3-2, 130 mg).
Subfraction 3-1 was separated by preparative TLC with 2% MeOH−
CHCl3 to give 1 (7.0 mg) and 4 (8.3 mg). Subfraction 3-2 was
rechromatographed on silica gel with 2% MeOH−CHCl3, followed by
final purification using preparative TLC with 40% acetone−hexane, to
give 2 (5.0 mg), 3 (6.0 mg), and 8 (8.5 mg).
Fraction 4 was subjected to silica gel column chromatography,
eluted with MeOH−CHCl3 (0−50%), to afford three subfractions (4-
1, 450 mg; 4-2, 150 mg; 4-3, 400 mg). Subfraction 4-1 was
chromatographed further using a MeOH−CHCl3 gradient system,
followed by reversed-phase preparative TLC with 70% acetone−H2O,
to give 13 (9.0 mg), 14 (7.5 mg), and 16 (6.5 mg). Subfraction 4-2
was recrystallized using acetone−hexane to give 9 (12.0 mg).
Fraction 5 was chromatographed further with a MeOH−CHCl3
gradient system, with additional separation by reversed-phase
preparative TLC with 50% MeOH−H2O, to give 5 (5.0 mg), 6 (8.5
mg), 7 (5.5 mg), and 12 (7.8 mg).
Fraction 6 was chromatographed further using a MeOH−CHCl3
gradient system, with final purification effected by preparative TLC
with 10% MeOH−CHCl3, to give 19 (6.5 mg) and 20 (20.8 mg).
Artocarmin A (1): pale yellow, amorphous solid; IR νmax (CHCl3)
3400, 1660, 1605, 1450 cm−1; 1H and 13C NMR (DMSO-d6, 500
MHz), see Table 1; HRESIMS m/z 377.0998 (calcd for C20H18O6Na
[M + Na]+, 377.1001).
Artocarmin B (2): pale yellow, amorphous solid; IR νmax (CHCl3)
3398, 1650, 1610, 1400 cm−1; 1H and 13C NMR (DMSO-d6, 500
MHz), see Table 1; HRESIMS m/z 523.1353 (calcd for C29H24O8Na
[M + Na]+, 523.1369).
Artocarmin C (3): pale yellow, amorphous solid; IR νmax (CHCl3)
3410, 1680, 1604, 1440 cm−1; 1H and 13C NMR (DMSO-d6, 500
MHz), see Table 1; HRESIMS m/z 553.1446 (calcd for C30H26O9Na
[M + Na]+, 553.1475).
Artocarmin D (4): pale yellow, amorphous solid; IR νmax (CHCl3)
3450, 1670, 1600, 1450 cm−1; 1H and 13C NMR (DMSO-d6, 500
MHz), see Table 1; HRESIMS m/z 371.1110 (calcd for C20H19O7 [M
+ H]+, 371.1131).
Artocarmitin A (5): yellow, amorphous solid; IR νmax (CHCl3)
3420, 1680, 1605, 1460 cm−1; 1H and 13C NMR (acetone-d6, 500
MHz), see Table 2; HRESIMS m/z 363.1216 (calcd for C20H20O5Na
[M + Na]+, 363.1208).
Artocarmitin B (6): yellow, amorphous solid; IR νmax (CHCl3)
3400, 1670, 1600, 1450 cm−1; 1H and 13C NMR (acetone-d6, 500
MHz), see Table 2; HRESIMS m/z 539.1674 (calcd for C30H28O8Na
[M + Na]+, 539.1682).
Artocarmitin C (7): yellow, amorphous solid; IR νmax (CHCl3)
3425, 1650, 1605, 1460 cm−1; 1H and 13C NMR (acetone-d6, 500
dx.doi.org/10.1021/np300576w | J. Nat. Prod. 2012, 75, 1951−1955
 Journal of Natural Products Article

MHz), see Table 2; HRESIMS m/z 569.1750 (calcd for C31H30O9Na

[M + Na]+, 569.1788).
Tyrosinase Inhibitory Assay. All the samples were first dissolved
in DMSO and used at concentrations of 100, 50, 25, and 10 μg/mL
(or μM for pure compounds). The tyrosinase inhibitory activity assay
was performed as previously described by Arung et al.14 The assay
mixtures consisting of 1900 μL of test solution in 0.1 M phosphate
buffer pH 6.8 and 100 μL of enzyme solution (15 U/mL in 0.1 M
phosphate buffer pH 6.8) was prepared immediately before use. After
preincubation at room temperature for 30 min, the reaction was
initiated by the addition of 1000 μL of substrate solution (1.5 mM L-
dihydroxyphenylalanine in 0.1 M phosphate buffer pH 6.8). The assay
mixture was incubated at room temperature for 7 min, and the
absorbance at 475 nm was measured with a Shimadzu UV-1800
spectrophotometer. Kojic acid, a known tyrosinase inhibitor, was used
as positive control.

■ ASSOCIATED CONTENT
*
S Supporting Information
1
H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY NMR
and MS spectra of new compounds (1−7) are available free of
charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION
Corresponding Author
*Tel: (84)-907426331. Fax: (84)-838-350-096. E-mail:
nttmai@hcmus.edu.vn.
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
This work was supported by grant 104.01-2010.44 from
Vietnam’s National Foundation for Science and Technology
Development (NAFOSTED).

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