Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211

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Journal of the Taiwan Institute of Chemical Engineers

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Bioconstituents from stems of Synsepalum dulcicum Daniell (Sapotaceae) inhibit

human melanoma proliferation, reduce mushroom tyrosinase activity and have
antioxidant properties
Hui-Min Wang a, Yi-Ting Chou b, Zi-Ling Hong c, Hsi-An Chen a, Yu-Chen Chang a, Woei-Ling Yang c,
Hou-Chien Chang d, Chao-Ting Mai a, Chung-Yi Chen e,*
a
Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b
Department of Physiology and Masters Program, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c
Department of Medical Technology, Fooyin University, Kaohsiung County 831, Taiwan
d
Department of Chemical Engineering, National Chung Hsing University, Taichung 250, Taiwan
e
School of Medical and Heath Science, Fooyin University, Kaohuing County 831, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: A new amide, dihydro-feruloyl-5-methoxytyramine (1), along with 13 known compounds, including (+)-
Received 8 March 2010 syringaresinol (2), (+)-epi-syringaresinol (3), 4-acetonyl-3,5-dimethoxy-p-quinol (4), cis-p-coumaric
Received in revised form 12 May 2010 acid (5), trans-p-coumaric acid (6), p-hydroxybenzoic acid (7), syringic acid (8), vanillic acid (9), veratric
Accepted 16 May 2010
acid (10), N-cis-feruloyltyramine (11), N-trans-feruloyltyramine (12) and N-cis-caffeoyltyramine (13),
were isolated from the stems of Synsepalum dulcicum Daniell (Sapotaceae). The structures of these
Keywords: compounds were established on the basis of spectroscopic analysis. One of the purposes of this study was
Synsepalum dulcicum Daniell (Sapotaceae)
to survey the antioxidant properties of 13 pure constituents. The radical scavenging and antioxidant
(+)-Syringaresinol
(+)-epi-Syringaresinol
activities were investigated by: scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals; metal
Melanoma cell chelating power was completed by ferrous ions; and reducing power was demonstrated by iron ions
Tyrosinase inhibitor reduction reaction. In addition, all compounds were evaluated for their cell proliferation inhibition
Antioxidant activities on human skin melanoma cells and tyrosinase inhibitions. The anti-tyrosinase effects were to
calculate the hydroxylation of L-tyrosine to L-dopa according to in vitro mushroom tyrosinase assay. In
sum, the inhibition effects of compounds (23) on human melanoma cells were signicant. Besides,
DPPH, ABTS radical scavenging, metal chelating and reducing power were found to be moderate
compared with the positive controls.
2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction
Synsepalum dulcicum Daniell (Sapotaceae) is an evergreen
shrub native of tropical West Africa, and the fruits, red berries,
have the property of remarkably altering the sour taste into
sweet taste (Kurihara and Beidler, 1969). The methanol extract of
its stems was subjected to solvent partitioning and chro-
matographic separation to afford six fractions. The chemical
constituents in the stems of S. dulcicum were separated with
column chromatography. Thirteen compounds, including dihy-
dro-feruloyl-5-methoxytyramine (1), (+)-syringaresinol (2), (+)-
epi-syringaresinol (3) (Chen et al., 1998a,b), 4-acetonyl-3,5-
dimethoxy-p-quinol (4) (Luo et al., 2008), cis-p-coumaric acid (5)
(Kort et al., 1996), trans-p-coumaric acid (6), p-hydroxybenzoic
* Corresponding author. Tel.: +886 7 7811151x6200; fax: +886 7 7863667.
E-mail address: xx377@mail.fy.edu.tw (C.-Y. Chen).
acid (7) (Hsieh et al., 2003), syringic acid (8), vanillic acid (9)
(Chen et al., 1999), veratric acid (10), N-cis-feruloyltyramine (11),
N-trans-feruloyltyramine (12) (Lo et al., 2004) and N-cis-
caffeoyltyramine (13) (Chen et al., 1998a,b) were isolated from
the methanolic extract. Among them, 1 was a new compound; 2
6, 10 and 13 were isolated for the rst time from this plant. The
present paper deals with the isolation and characterization of the
13 pure components.
To prevent some diseases, the importance of diet, especially
with plants with antioxidant properties is well recognized. Over
the past decade, the interest in antioxidants has remarkably
increased due to the related protective effects in opposition to
various diseases, including anti-aging, inammatory, and neuro-
logical, cardiovascular diseases, as well as cancers (Lu and Foo,
1997; Scalbert et al., 2005; Sur et al., 2001). Antioxidant constitutes
are essential in foods and cosmetic products due to their ability to
reduce free radical-mediated degradation of cells and organs in
human beings (Jin et al., 2004; Wongkham et al., 2001; Yao et al.,

1876-1070/$ see front matter 2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jtice.2010.05.008
 H.-M. Wang et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211
Nomenclature
(1) dihydro-feruloyl-5-methoxytyramine
(2) (+)-syringaresinol
(3) (+)-epi-syringaresinol
(4) 4-acetonyl-3,5-dimethoxy-p-quinol
(5) cis-p-coumaric acid
(6) trans-p-coumaric acid
(7) p-hydroxybenzoic acid
(8) syringic acid
(9) vanillic acid
(10) veratric acid
(11) N-cis-feruloyltyramine
(12) N-trans-feruloyltyramine
(13) N-cis-caffeoyltyramine
A375.S2 melanoma cells
DPPH 1,1-diphenyl-2-picrylhydrazyl
1998). The mechanism generally accepted is that free radical
scavenging activities would reduce oxidative stress to prevent the
resulting diseases. In addition to whole-grain cereals, oats,
legumes, fruits and vegetables, there are still many other
antioxidant natural sources (Karakaya and Kavas, 1999; Nihal
et al., 2005).
Human skin is frequently contacted with oxidative stress which
is produced by intrinsic and external sources induced by free
radicals and ultraviolet (UV) radiation (Baldea et al., 2009; Wang
et al., 2001). There are many reports have studied that skin exposed
to oxidative stress and UV are responsible for photoaging and
tumor generation (Briganti and Picardo, 2003; Frank et al., 2001;
Mauro et al., 1996). Solar UV exposure has been associated with
over two-thirds of the cases and may be a major etiological factor
in skin cancer (Halachmi and Gilchrest, 2001; Weinstock, 2000).
Occurrences of cutaneous malignant melanomas have increased in
the past several decades since it has a strong propensity to
metastasize so is one of the most aggressive among skin cancers
(Jemal et al., 2001; Tran et al., 2003). Unlike other cancers,
metastatic melanoma is not easy to treat by methods such as
surgery, radiotherapy or chemotherapy (Burnstock et al., 2009). A
good chemo preventive agent shall be a naturally occurring agent
that can induce apoptosis in cancer cells without signicant side
effects.
Hyper-pigmentations, such as senile lentigo, melasma, freckles
and pigmented acne scars, are of particular concern to women
(Hirobe, 2005; Kim et al., 2008; Tripathi et al., 1992). Their
treatment usually involves the use of medicines or medicinal
cosmetics containing de-pigmenting agents or skin whitening
agents. Inhibition of tyrosinase is one of the major strategies to
treat hyper-pigmentation. However, due to issues on safety, only a
few effective natural and synthetic regulators that act to minimize
skin pigmentation abnormalities are used as therapeutic agents.
Recently, much attention has been drawn to the application of
tyrosinase inhibitors to medical treatments or cosmetic business.
Tyrosinase catalyzes two distinct signicant reactions in melanin
synthesis: the hydroxylation of L-tyrosine to 3,4-dihydroxy-L-
phenylalanine (L-dopa) and the oxidation of L-dopa to dopaqui-
none, followed by further conversion to melanin production
(Marles et al., 2003; Solano et al., 2006). Therefore, in clinical usage,
tyrosinase inhibitors are being taken for dermatological disorder
treatments related to melanin hyper-accumulation, and are thus
essential in cosmetics for de-pigmentation (Gillbro et al., 2004;
Schallreuter et al., 2009).
205
2. Materials and methods
2.1. Reagents and materials
Mushroom tyrosinase, vitamin C, dimethyl sulfoxide (DMSO),
kojic acid, L-tyrosine, 1,1-diphenyl-2-picrylhydrazyl (DPPH), ethyl-
ene diamine tetra-acetic acid (EDTA), 3-tert-butyl-4-hydroxyanisole
(BHA), potassium ferricyanide [K3Fe(CN)6], trichloroacetic acid,
FeCl3, FeCl24H2O, and 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT), penicillin, streptomycin, and ampho-
tericin B were purchased from Sigma Chemical (St. Louis, MO). Fetal
bovine serum (FBS) and minimum essential medium (MEM) were
obtained from GIBCO BRL (Gaithersburg, MD). All buffers and other
reagents were of the highest purity commercially available.
2.2. General experimental procedures
Melting points were determined using a Yanagimoto micro-
melting point apparatus, and are uncorrected. Optical rotations
were measured with a JASCO DIP-370 digital polarimeter. UV
spectra were obtained in MeCN, using a JASCO V-530 spectropho-
tometer. The IR spectra were measured on a Hitachi 260-30
spectrophotometer. 1H (400 MHz, using CDCl3 as solvent), 13C
(100 MHz), DEPT, and NOESY NMR spectra were obtained on a
Unity Plus Varian spectrometer. LRFABMS and LREIMS spectra
were obtained with a JEOL JMS-SX/SX 102A mass spectrometer, or
a Quattro GCMS spectrometer with a direct inlet system.
HRFABMS and HREIMS were measured on a JEOL JMS-HX 110
mass spectrometer. Silica gel 60 (Merck, 230400 mesh) was used
for column chromatography. Precoated silica gel plates (Merck,
Kieselgel 60 F-254, 0.20 mm) were used for analytical TLC, and
precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.50 mm)
were used for preparative TLC. Spots were detected by spraying
with 50% H2SO4 and then heating on a hot plate.
2.3. Plant materials
The specimen of S. dulcicum was collected from Kaohsiung
County, Taiwan, October 2007. A voucher specimen was identied
by Dr. Fu-Yuan Lu (Department of Forestry and Natural Resources
College of Agriculture, National Chiayi University) and was
deposited in the School of Medical and Heath Science, Fooyin
University, Kaohsiung County, Taiwan.
2.4. Extraction and isolation
The stems (6.14 kg) of S. dulcicum were extracted repeatedly
with MeOH at room temperature for 2448 h. The MeOH extract
was dried and evaporated to leave a viscous residue (425.8 g). The
residue was placed on a silica gel column and eluted with CH2Cl2
gradually enriched with MeOH to afford 6 fractions. Fraction 2
(4.15 g) eluted with n-hexaneEtOAc (1:1) was further puried by
silica gel CC using the same solvent system to obtain (+)-
syringaresinol (2) (11 mg), (+)-epi-syringaresinol (3) (9 mg) and
4-acetonyl-3,5-dimethoxy-p-quinol (4) (16 mg), respectively.
Fraction 3 (8.61 g) eluted with CH2Cl2MeOH (50:1) was further
separated using silica gel CC and prep. TLC (n-hexaneacetone
(3:1)) and gave cis-p-coumaric acid (5) (4 mg), trans-p-coumaric
acid (6) (4 mg), p-hydroxybenzoic acid (7) (3 mg), syringic acid (8)
(6 mg), vanillic acid (9) (13 mg) and veratric acid (10) (3 mg),
respectively. Fraction 4 (4.83 g) was puried by silica gel
chromatography (CH2Cl2MeOH, 30:1) to give N-cis-feruloyltyr-
amine (11) (5 mg) and N-trans-feruloyltyramine (12) (16 mg).
Fraction 5 (9.02 g) was puried by silica gel chromatography
(CH2Cl2MeOH, 20:1) to give dihydro-feruloyl-5-methoxytyra-
mine (1) (5 mg) and N-cis-caffeoyltyramine (13) (7 mg).
206 H.-M. Wang et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211

2.5. Determination of DPPH radical scavenging capacity
Most cosmetics and food compounds have free radical
scavenging abilities. The antioxidant activity of our target
compound was measured in terms of hydrogen donating or
radical scavenging ability using the stable DPPH method modied
(Chen et al., 2009a,b; Wang et al., 2005). Different concentrations of
the samples were added to 0.2 ml of DPPH (60 mM) solution. When
DPPH reacts with an antioxidant compound that donates hydro-
gen, it is reduced, resulting in a decrease in the absorbance at
520 nm. The absorbance was recorded at 5 min intervals up to
30 min using a UVvis spectrophotometer, and evaluated at the
end point (30 min). Vitamin C was used as a positive control. The
percentages of remaining DPPH were plotted against the sample to
obtain the amount of antioxidant required to reduce the initial
concentration of DPPH. Scavenging activity (%) was determined as
ODcontrol  ODsample
100% 
ODcontrol
IC50 value of the inhibitor was determined by tting the initial
rates versus inhibitor concentrations using the following equation.
  
I
EI E0  1 
I IC50
In this equation, E(I) is the scavenging activity with inhibitor
concentration I; E(0) is the scavenging activity without inhibitor; I
is the inhibitor concentration; and IC50 is the concentration of
inhibitor which caused half the reaction rate. The data were
presented in Table 1.
2.6. Metal chelating activity
The ferrous ion-chelating potential of chlorophyll was investi-
gated according to a method described previously (Decker and
Welch, 1990; Wang et al., 2010). Briey, various testing
concentrations of samples dissolved in DMSO were added to a
solution of 2 mM FeCl24H2O (0.05 ml). The reaction was initiated
by the addition of 5 mM ferrozine (0.2 ml) and the mixture was
vigorously shaken and left standing at room temperature for
10 min. And then, the absorbance of the mixture was read at
560 nm against a blank. EDTA was used as a positive control and
the IC50 of chelating activity calculation formula was similar to
Eq. (2) and demonstrated in Table 1.
2.7. Reducing power assay
The reducing power of ()-N-formylanonaine was determined
according to a previously described method (Elmastas et al., 2006).
Various concentrations of testing samples in 0.063 mL of methyl
alcohol were mixed with sodium phosphate buffer (0.1 mL 0.2 M,
pH 6.8) and 2.5 mL of 20% potassium ferricyanide [K3Fe(CN)6]. The
mixture was incubated at 50 8C for 20 min, and then 0.16 mL of
trichloroacetic acid (10%) was added to the mixture, which was
then centrifuged for 10 min at 3000  g. The upper layer of solution
(75 mL) was mixed with distilled water (25 mL) and FeCl3 (25 mL,
2%), and the absorbance was measured with a 96-well plate
spectrophotometer at 650 nm. BHA was used as a positive control.
A higher absorbance demonstrates a higher reductive capability
and showed the data in Table 1.
(1)
2.8. Cell culture
The human melanoma cell line A375.S2 (BCRC 60263) was used
in this study. It was maintained in monolayer culture at 37 8C and
(2) 5% CO2 in MEM supplemented with 10% fetal bovine serum, 2 mM
glutamine, 0.1 mM non-essential amino acids, 1.0 mM sodium
pyruvate and 100 U/ml of penicillin, 100 mg/ml of streptomycin,
and 0.25 mg/ml of amphotericin B. And the cells were incubated at
37 8C under a humidied atmosphere of 5% CO2 in air and routine
passage by trypsinization.
2.9. Cell viability assay
The effects of compounds on cell viabilities were according to
the MTT assay procedures (Chen et al., 2009a,b; Lin et al., 2007).
Briey, cells were seeded in 96-well plates at a density of 4  103
cells/well. The medium was then changed and cells were
maintained in either solvent alone (control cells) or in the presence
of the indicated drug concentrations in a nal volume of 150 ml in
10% FBS culture medium. The testing samples were dissolved in
sterile DMSO to treat a working concentration of 10, 50 and
100 mM. Each concentration was added to the plates in three
replicates and incubated under the same conditions as above for

Table 1
IC50 values of mushroom tyrosinase inhibition and antioxidant effects of S. dulcicum compounds. Data were expressed as a mean value of three independent experiments.

DPPH+ scavenging Metal chelating Reducing power Mushroom tyrosinase

IC50 (mM) IC50 (mM) (100 mM, OD700) IC50 (mM)

Dihydro-feruloyl-5-methoxytyramine (1) 148.4 ns 0.141 ns

(+)-Syringaresinol (2) 99.8 218.6 0.361 ns
(+)-epi-Syringaresinol (3) 100.2 245.7 0.499 200.5
4-Acetonyl-3,5-dimethoxy-p-quinol (4) ns 178.5 0.069 208.1
cis-p-Coumaric acid (5) ns ns 0.095 197.9
trans-p-Coumaric acid (6) ns ns 0.072 168.7
p-Hydroxybenzoic acid (7) ns ns 0.074 358.6
Syringic acid (8) 162.1 142.3 0.128 ns
Vanillic acid (9) ns 153.8 0.161 174.4
Veratric acid (10) ns ns 0.116 ns
N-cis-Feruloyl-tyramine (11) 154.7 ns 0.122 215.5
N-trans-Feruloyl-tyramine (12) 152.2 ns 0.146 ns
N-cis-Caffeoyltyramine (13) 164.2 ns 0.102 ns
Vitamin Ca 26.9
EDTAb 10.2
BHAc 0.407
Kojic acidd 68.2

() no testing; (ns) no signicance.

a
Vitamin C was used as a positive control on DPPH assay.
b
EDTA was used as a positive control on metal chelating ability.
c
BHA was used as a positive control on reducing power at 100 mM, and so did testing samples.
d
Kojic acid was used as a positive control of mushroom tyrosinase assay.
 H.-M. Wang et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211
48 h. After 48 h of incubation, the medium was replaced with
100 ml fresh medium included 0.5 mg/ml MTT. The plate was
cultured in a 37 8C incubator lled with 5% CO2 for 2 h.
Each precipitate in a specic dish was dissolved in 100 mL of
DMSO to dissolve the purple formazan crystals. After the dishes
were gently shaken for 20 min in the dark to ensure maximal
dissolution of formazan crystals, and the absorbance (A) values of
the supernatant were measured at 595 nm. Cell viability was
calculated as
Asample  Ablank
 100%
Acontrol  Ablank
2.10. Assay on mushroom tyrosinase activity
Tyrosinase activity inhibition was determined spectrophoto-
metrically according to the method described previously (Chen
et al., 2010; Likhitwitayawuid and Sritularak, 2001), with minor
modications. Assays were conducted in a 96-well micro-plate, an
ELSA plate reader being used to determine the absorbance at
490 nm (Molecular Devices). Kojic acid was used as a positive
control on the tyrosinase inhibitory assay. The testing substance
was dissolved in aqueous DMSO, and incubated with L-tyrosine
(2.5 mg/ml) in 50 mM phosphate buffer (pH 6.8). All samples were
dissolved in DMSO which did not affect tyrosinase activity when
DMSO was less than 1% of the total volume. Then, 25 U/ml of
mushroom tyrosinase in the same buffer was added, and the
mixture was incubated at 37 8C for 30 min (Table 1). Tyrosinase
inhibitory activity was determined by the following equation:
 
A  B  C  D
% inhibition 100% 
A  B
where A is the optical density (OD490) without testing substance; B
is the OD490 without testing substance, but with tyrosinase; C is the
OD490 with testing substance; and D is the OD490 with testing
substance, but without tyrosinase. The calculation formula of IC50
value was similar to Eq. (2) and demonstrated in Table 1.
2.11. Statistical analysis
Results are presented as a mean value of the data obtained from
triplicate experiments. Students t-test was used to determine the
level of signicance.
3. Results and discussion
3.1. Structure identications of S. dulcicum pure compounds
Dihydro-feruloyl-5-methoxytyramine (1) was obtained as a
white powder. Its molecular formula was established as C19H23NO5
by HREI mass spectrometry (found 345.1578, calcd 345.1576) and
13
C NMR. Two IR bands at 3300 and 1650 cm1 and a signal
appearing at d 175.4 (s) in the 13C NMR spectrum suggested that
hydroxyl groups and an amide group might be present. The UV
absorption maxima at 205, 255sh and 277 underwent bath-
ochromic shifts on adding base, suggesting that 1 should be a
phenolic derivative of an amide. The 1H NMR spectrum revealed an
ABX pattern at d 6.89 (1H, d, J = 8.0 Hz), 6.78 (1H, dd, J = 8.0, 2.0 Hz)
and 6.72 (1H, d, J = 2.0 Hz) for H-8, H-9 and H-5, two coupled
triplets of methylene protons at d 2.54 and 2.83 (each 2H, t,
J = 7.6 Hz) for H-2 and H-3 in the dihydro-ferulic acid moiety,
respectively. Another ABX pattern was observed at d 6.40 (1H, dd,
J = 8.0, 2.0 Hz), 6.62 (1H, d, J = 8.0 Hz) and 6.80 (1H, d, J = 2.0 Hz) for
H-80 , H-70 and H-40 in the methoxytyramine moiety, respectively.
Two coupled triplets of methylene protons at d 2.71 and 3.43 (each
207
2H, t, J = 7.2 Hz) were ascribed to H-20 and H-10 in the
methoxytyramine moiety, respectively. 13C NMR and DEPT
experiments on 1 showed 18 resonance lines consisting of two
methyls, four methylenes, six methines and seven quaternary
carbons (including a carbonyl signal at d 175.4). The mass, UV, IR
and 1H NMR data suggested that 1 is a type of phenolic acid amide
and that the position of methoxyl and hydroxyl groups should be
located on the skeleton.
In order to elucidate the position of aromatic substitution, a
NOESY experiment was performed on 1. This revealed that the
methoxyl protons were at d 3.85 on the dihydro-ferulic acid
(3)
moiety, with a signicant degree of NOE enhancement being
observed for H-5 (at d 6.72) with a negligible degree of NOE
enhancement being observed for H-9 (at d 6.78) (Fig. 1). The other
methoxyl protons at d 3.81 on the methoxytyramine moiety
revealed a signicant degree of NOE enhancement for H-40 (at d
6.80) with a negligible degree of NOE enhancement being observed
for H-80 (at d 6.40) (Fig. 1). These observations conrmed the
structure of 1 as the new phenolic acid amide, dihydro-feruloyl-5-
methoxytyramine. Comparison of the spectral data with a known
acid amide, N-trans-feruloyl-methoxytyramine revealed that both
of the compounds were similar, except that the double bond of N-
trans-feruloyl-methoxytyramine was replaced by one pair of
methylenes in 1.
3.1.1. Dihydro-feruloyl-5-methoxytyramine (1)
White powder (CH2Cl2), UV lmax 205 (2.71), 255 (sh, 3.62), 277
(2.35) nm, IR nmax 3300 (OH), 1650 (C5 5O) cm1, 1H NMR
(400 MHz, CDCl3) dihydro-feruloyl moiety: d 2.54 (2H, t,
J = 7.6 Hz, H-2), 2.83 (2H, t, J = 7.6 Hz, H-3), 3.85 (3H, s, OMe),
6.78 (1H, dd, J = 8.0, 2.0 Hz, H-9), 6.72 (1H, d, J = 2.0 Hz, H-5), 6.89
(4) (1H, d, J = 8.0, H-8); methoxytyraminel moiety: d 2.71 (2H, t,
J = 7.2 Hz, H-20 ), 3.43 (2H, t, J = 7.2 Hz, H-10 ), 3.81 (3H, s, OMe), 6.40
(1H, dd, J = 8.0, 2.0 Hz, H-80 .), 6.62 (1H, d, J = 8.0 Hz, H-70 ), 6.80 (1H,
d, J = 2.0, H-40 ), 13C NMR (100 MHz, CDCl3) dihydro-feruloyl moiety:
d 32.4 (C-3), 39.2 (C-2), 56.3 (OMe), 113.3 (C-5), 116.5 (C-8), 121.7
(C-9), 133.9 (C-4), 148.2 (C-6), 149.1 (C-7), 175.4 (C-1); methox-
ytyraminel moiety: d 36.3 (C-20 ), 42.4 (C-10 ), 56.4 (OMe), 113.2 (C-
40 ), 116.5 (C-70 ), 122.2 (C-80 ), 128.1 (C-30 ), 145.9 (C-60 ), 149.1 (C-50 ).
HREIMS m/z: 345.1578 [M]+ (345.1576 calcd for C19H23NO5).
3.1.2. (+)-Syringaresinol (2)
White powder (CH2Cl2), UV lmax 220, 238, 280 nm, IR nmax
3400, 1610, 1505 cm1, 1H NMR (400 MHz, CDCl3): d 3.09 (2H, m,
H-1 and H-5), 3.91 (2H, dd, J = 3.6, 5.4 Hz, H-4axia. and H-8axia.), 3.88
(12H, s, OMe), 4.25 (2H, dd, J = 9.2, 6.8 Hz, H-4equ. and H-8equ.), 4.72
(2H, d, J = 4.4 Hz, H-2 and H-6), 5.52 (2H, s, OH), 6.56 (4H, s, H-20 , H-
200 , H-60 and H-600 ), EI-MS m/z: 418 [M]+.
3.1.3. (+)-epi-Syringaresinol (3)
White powder (CH2Cl2), UV lmax 220, 235, 273 nm, IR nmax 3400,
1610, 1500 cm1, 1H NMR (400 MHz, CDCl3): d 2.91 (1H, m, H-1),
3.15 (1H, m, H-5), 3.20 (1H, m, H-4axia.), 3.70 (2H, m, H-4equ. and H-
8axia.), 3.90 (12H, s, OMe), 4.25 (1H, m, H-8equ.), 4.42 (1H, d, J = 6.8 Hz,
H-6), 4.86 (1H, d, J = 5.2 Hz, H-2), 5.47 (2H, s, OH), 6.59 (2H, s, H-60
and H-600 ), 6.61 (2H, s, H-20 and H-200 ), EI-MS m/z: 418 [M]+.
3.1.4. 4-Acetonyl-3,5-dimethoxy-p-quinol (4)
Colorless needles (CH2Cl2), IR nmax 3350, 1660, 1595 cm1, 1H
NMR (400 MHz, CDCl3): d 2.19 (3H, s, H-30 ), 3.01 (2H, s, H-10 ), 3.76
(6H, s, OMe), 5.43 (2H, s, H-2 and H-6), EI-MS m/z: 226 [M]+.
3.1.5. cis-p-Coumaric acid (5)
Colorless needles (CH2Cl2), UV lmax 230, 260, 315 nm, IR nmax
3350, 1610, 1510 cm1, 1H NMR (400 MHz, CDCl3): d 5.91 (1H, d,
J = 16.0 Hz, H-2), 6.89 (2H, d, J = 8.8 Hz, H-6 and H-8), 6.95 (1H, d,
[()TD$FIG]
208 H.-M. Wang et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211
Fig. 1. The compound structures (113) from S. dulcicum. NOESY experiments of dihydro-feruloyl-5-methoxytyramine (1).
J = 16.0 Hz, H-3), 7.54 (2H, d, J = 8.8 Hz, H-5 and H-9), EI-MS m/z:
164 [M]+.
3.1.6. trans-p-Coumaric acid (6)
Colorless needles (CH2Cl2), UV lmax 230, 260, 315 nm, IR nmax
3350, 1610, 1510 cm1, 1H NMR (400 MHz, CDCl3): d 6.32 (1H, d,
J = 16.0 Hz, H-2), 6.88 (2H, d, J = 8.7 Hz, H-6 and H-8), 7.61 (1H, d,
J = 16.0 Hz, H-3), 7.53 (2H, d, J = 8.7 Hz, H-5 and H-9), EI-MS m/z:
164 [M]+.
3.1.7. p-Hydroxybenzoic acid (7)
Brown powder (CH2Cl2), UV lmax 250, 285, 290 nm, IR nmax
3500, 1660, 1510, 1280 cm1, 1H NMR (400 MHz, CDCl3): d 6.85
(2H, d, J = 8.6 Hz, H-3 and H-5), 7.96 (2H, d, J = 8.6 Hz, H-2 and H-6),
EI-MS m/z: 138 [M]+.
3.1.8. Syringic acid (8)
Brown needles (CH2Cl2), UV lmax 212, 235, 308 nm, IR nmax
3255, 1670, 1514, 1330 cm1, 1H NMR (400 MHz, CDCl3): d 3.83
(6H, s, C3-OMe and C5-OMe), 7.37 (2H, s, H-2 and H-6), EI-MS m/z:
198 [M]+.
3.1.9. Vanillic acid (9)
Colorless needles (acetone), UV lmax 220, 265, 300 nm, IR nmax
3550, 1680, 1510, 1280 cm1, 1H NMR (400 MHz, acetone): d 3.90
(3H, s, OCH3), 6.89 (1H, d, J = 8.4 Hz, H-5), 7.55 (1H, d, J = 2.0 Hz, H-
2), 7.57 (1H, dd, J = 8.4, 2.0 Hz, H-6), EI-MS m/z: 168 [M]+.
3.1.10. Veratric acid (10)
Colorless needles (acetone), UV lmax 220, 261 nm, IR nmax 3444
(OH), 1716 (C5 5O) cm1, 1H NMR (400 MHz, CDCl3): d 3.88 (3H, s, 3-
OCH3), 3.94 (3H, s, 4-OCH3), 6.94 (1H, d, J = 8.4 Hz, H-5), 7.55 (1H, d,
J = 1.6 Hz, H-2), 7.65 (1H, dd, J = 8.4, 1.6 Hz, H-6), EI-MS m/z: 168
[M]+.
3.1.11. N-cis-feruloyl-tyramine (11)
Yellow oil, UV lmax 220, 284, 312 nm, IR nmax 3347 (OH), 1647
(C55O) cm1, 1H NMR (400 MHz, acetone): d 2.71 (2H, t, J = 7.4 Hz,
 H.-M. Wang et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211
H-2), 3.43 (2H, t, J = 7.4 Hz, H-1), 3.83 (3H, s, OCH3), 5.83 (1H, d,
J = 12.8 Hz, H-20 ), 6.55 (1H, d, J = 12.8 Hz, H-30 ), 6.74 (2H, d,
J = 8.4 Hz, H-5 and H-7), 6.77 (1H, d, J = 8.0 Hz, H-80 ), 7.03 (2H, d,
J = 8.4 Hz, H-4 and H-8), 7.11 (1H, dd, J = 8.0, 2.0 Hz, H-90 ), 7.87 (1H,
d, J = 2.0 Hz, H-50 ), EI-MS m/z: 313 [M]+.
3.1.12. N-trans-feruloyl-tyramine (12)
Colorless crystals (MeOH), UV lmax 220, 288, 318 nm, IR nmax
3345 (OH), 1655 (C5 5O) cm1, 1H NMR (400 MHz, CD3OD): d 2.75
(2H, t, J = 7.4 Hz, H-2), 3.46 (2H, t, J = 7.4 Hz, H-1), 3.88 (3H, s,
OCH3), 6.42 (1H, d, J = 15.6 Hz, H-20 ), 6.72 (2H, d, J = 8.6 Hz, H-5 and
H-7), 6.80 (1H, d, J = 8.4 Hz, H-80 ), 7.04 (1H, dd, J = 8.4, 2.0 Hz, H-90 ),
7.06 (2H, d, J = 8.6 Hz, H-4 and H-8), 7.12 (1H, d, J = 2.0 Hz, H-50 ),
7.45 (1H, d, J = 15.6 Hz, H-30 ), EI-MS m/z: 313 [M]+.
3.1.13. N-cis-caffeoyltyramine (13)
Yellow oil, UV lmax 220, 280, 310 nm, IR nmax 3400 (OH), 1650
(C55O) cm1, 1H NMR (400 MHz, CD3OD): d 2.68 (2H, t, J = 6.8 Hz,
H-20 ), 3.38 (2H, t, J = 6.8 Hz, H-10 ), 5.77 (1H, d, J = 12.6 Hz, H-2), 6.56
(1H, d, J = 12.6 Hz, H-3), 6.68 (2H, d, J = 8.4 Hz, H-50 and H-70 ), 6.70
(1H, s, J = 8.2 Hz, H-8), 6.83 (1H, dd, J = 8.2, 2.0 Hz, H-9), 6.99 (2H, d,
J = 8.4 Hz, H-40 and H-80 ), 7.04 (1H, d, J = 2.0 Hz, H-5), EI-MS m/z:
300 [M]+.
3.2. DPPH free radical scavenging activity
Since, too much formation and accumulation of free radicals
would accelerate the oxidation of lipids in cosmetics and foods,
natural antioxidant properties, especially free radical scavenging
abilities are essential in skin care and functional foods (Gulcin et al.,
2007). The inhibition IC50 values for S. dulcicum 13 compounds to
DPPH were listed in Table 1. In DPPH free radical scavenging
system, antioxidants act to inhibit oxidation products, so it forms
an accepted mechanism and the scavenging of the DPPH radical
was then used in this investigation. In this assay, antioxidants were
able to reduce the stable radical DPPH to the yellow-colored
diphenyl-picrylhydrazine. (+)-syringaresinol (2) and (+)-epi-syr-
ingaresinol (3) showed a middle-high inhibitory effect on DPPH
assay compared to vitamin C. Natural S. dulcicum compounds
might be active as chain break antioxidants or be united with the
free radical ions to inhibit the free radical accumulations.
3.3. Ferrous ions chelating capacity
The ferrous ion-chelating activities of S. dulcicum 13 com-
pounds were shown in Table 1. Ferrozine could form complexes
with Fe2+ quantitatively. With the existence of chelating agents,
the complex construction was disrupted, resulting in a lightening
of the red color of the complex. The IC50 value of compounds (24,
89) showed a minor level of Fe2+ scavenging effect when EDTA
presented a strong scavenging ability.
3.4. Reducing capability
In this assay, the color of the testing solutions altered from
yellow to different shades of green and blue depending upon their
reducing power of these antioxidants. The presence of antioxidant
substance induces the reduction of the Fe3+/ferricyanide complex
to the ferrous form. Table 1 illustrated the reducing power of S.
dulcicum 13 compounds at 100 mM. The reducing power of
compounds (23) exhibited a high level potential comparing to
BHA at the same dose.
It is assumed that the formations of free radicals have been
associated with human aging and consumers tend to prefer fresher,
non-articial and more natural products. Therefore, the usage of
antioxidants as natural preservatives in cosmetics or foods has
209
great potential. Excessive formation and accumulation of free
radicals accelerate the oxidation of lipids in cosmetics and foods
while decreasing product quality and consumer acceptance. The
antioxidant property may be directly inuenced by their redox
properties, which can play an important role in absorbing and
neutralizing free radicals, quenching singlet and triplet oxygen, or
decomposing peroxides. In other words, it perhaps may be
inuenced by the supply of hydrogen combining with radicals,
and produced a stable radical to cease the radical chain reaction
(Wang and Ballington, 2007). It is also possible that to join with the
radical ions required for the radical chain reaction, followed by the
termination of the chain. Therefore, there are many natural
antioxidant reports closely related to their bio-functions such as
the reduction of chronic diseases and the prevention of cancer,
which are often linked to the termination of free radical
propagation in biological systems (Mates et al., 2009; Park et al.,
2008).
3.5. Anti-proliferation by S. dulcicum compounds on A375.S2 cells
To date, the major cause of ineffective treatment resulting in the
death of a patient is due to tumor proliferation and metastasis.
Hence, to improve the efcacy of cancer treatment, it is important
and valuable to develop novel medicinal components in anti-
cancer therapies (Wang et al., 2009). Cell proliferation in normal
mammalian physiological conditions plays a role in homeostasis
and is a multiple process involving growth factors binding to
membrane receptors, stimulating signaling pathways and result-
ing in stimulation of cellular motility machinery. However, the
deregulated cellular proliferation is responsible for the metastasis
and invasion of malignant tumor cells. The MTT cell viability assay
showed that the anti-cell proliferation of 13 constitutes on A375.S2
cell line for 48 h treatments (Fig. 2). Cells were incubated in the
presence of serially diluted concentrations (i.e., 100, 50, and
10 mM), and the cell growth rates were determined for each test.
The proliferation of A375.S2 cells was inhibited by (+)-syringar-
esinol (2) and (+)-epi-syringaresinol (3) in a dose-dependent
manner from 10 to 100 mM.
Many anti-cancer therapies killed cancer cells by generating
high reactive oxygen species (ROS) (Ding et al., 2009). However,
accumulated ROS often detected various cancer cells. It is well
known that low concentrations of intrinsic ROS induces activation
and release of MMP-2 and MMP-9, which are responsible for
cancer migration and metastasis (Belkhiri et al., 1997). Moreover, a
recent study showed a natural antioxidant, silybin inhibited
cellular proliferation and metastasis of a human hepatocellular
carcinoma cell by eliminating ROS generation in the glucose-
oxidase (Huber et al., 2008). We demonstrated in this study that
the proliferation of A375.S2 cells was markedly inhibited by (+)-
syringaresinol (2) and (+)-epi-syringaresinol (3) (Fig. 2), and might
be because of antioxidant activities. As mentioned before, two
compounds might against oxidative deterioration to prevent
carcinogenesis as well as act as pharmaceutical applications for
cancer chemotherapy. To the best of our knowledge, this is the rst
demonstration that S. dulcicum pure compounds could inhibit
proliferation of human A375.S2 cells. Our further study will shed
light on the mechanism underlying the anti-proliferation of S.
dulcicum against human melanoma cells.
3.6. Mushroom tyrosinase inhibition
As initially stated, we measured the inhibitory effects of pure
compounds from S. dulcicum on in vitro mushroom tyrosinase
inhibition in the hope of nding a novel and effective substance for
skin whitening and the prevention of hyper-pigmentation;
successfully, we found that seven compounds (37, 9, 11) could
[()TD$FIG]
210 H.-M. Wang et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 204211
Fig. 2. Viability of A375.S2 cells measured by the MTT assay. Cells were treated for 48 h with 10, 50 and 100 mM of S. dulcicum compounds, respectively. Control with no
testing sample, and taxol with 0.1 and 0.5 mM (slash and horizontal lines) respectively. The data represented the mean  SD of triplicate values for three independent
experiments.
reduce mushroom tyrosinase activity with dose-dependent trends
(Table 1). These compounds present potential applications to
inhibit melanin synthesis. Tyrosinase catalyzes two distinct
reactions in melanin synthesis: the hydroxylation of L-tyrosine
to L-dopa and the oxidation of L-dopa to dopaquinone (Tripathi
et al., 1992). After spontaneous conversion of dopaquinone to
dopachrome, dopachrome tautomerase (tyrosinase-related pro-
tein-2, DCT/TRP-2) catalyzes the conversion of dopachrome to 5,6-
dihydroxyindole-2-carboxylic acid (DHICA). Subsequently, DHICA
is converted to indolequinonecarboxylic acid by DHICA oxidase
(TRP-1) (Maeda et al., 1997). The tyrosinase-related proteins, TRP-
1, and TRP-2 catalyze distal melanin biosynthesis steps that control
the type of melanin produced. Although three enzymes are known
to be involved in melanin biosynthesis in mammals, tyrosinase is
the focus point as it directly regulates the amount of melanin
produced, whereas the other enzymes simply modify the type of
melanin synthesized (Hirobe, 2005; Kobayashi et al., 1994; Sturm
et al., 1998). Tyrosinase inhibitors are not only used in medical
treatment for hyper-pigmentation but are usually used in cosmetic
business purposes.
4. Conclusion
Various biochemical characterization properties of S. dulcicum
were demonstrated for the rst time in this work. The antioxidant
assays revealed that (+)-syringaresinol (2) and (+)-epi-syringar-
esinol (3) had approximately moderate antioxidant abilities in
DPPH scavenging activity, reducing power and metal chelating
abilities in total. It is interesting that two compounds (23) have
bi-functionality, not just containing the anti-aging and anti-
oxidative abilities, but also had the inhibitory effects on human
skin cancer cells. Through the in vitro mushroom tyrosinase
inhibition screening, we found compounds (37, 9, 11) inhibit the
target protein activities with moderate IC50 values. These
constitutes from S. dulcicum exhibited potential applications in
medical cosmetology and food supplementation, simultaneously.
Acknowledgments
This investigation was supported by a grant from the National
Science Council of Republic of China (NSC97-2221-E-037-002-,
NSC98-2221-E-037-005- and NSC-97-2320-B-242-002-MY3);
Ministry of Economic Affairs (98-EC-17-A-10-S2-0066); and Li-
Tek Corporation Company. The authors would also like thank Min-
Yii Julie Wang for the English editing and Ya-Ling Yeh for the
assistance.
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