SUPPLEMENTARY MATERIAL
Inhibition effect of phenyl compounds from the Oryza sativa roots
on melanin production in murine B16-F10 melanoma cells
Jin-Gyeong Choa, Jeongran Huhb, Rak-Hun Jeonga, Byeong-Ju Chaa, Sabina
Shresthaa, Dong-Geol Leeb, Hee-Cheol Kangb, Ji-Young Kima and Nam-In
Baeka*
a
Graduate School of Biotechnology and Department of Oriental Medicine Biotechnology,
Kyung Hee University, Yongin 446-701, Republic of Korea
b
R&D center, GFC Co., Ltd., Suwon 443-813, Republic of Korea
*Corresponding Author. E-mail: nibaek@khu.ac.kr
Abstract
Five phenyl compounds, vanillin (1), methyl trans-ferulate (2), trans-p-coumaric acid methyl
ester (3), N-benzoyltryptamine (4), and N-(trans-cinnamoyl)tryptamine (5), were isolated
from the roots of Oryza sativa L. and identified on the basis of spectroscopic data.
Compounds 3 and 5 showed strong inhibition effect on melanin production in murine B16F10 melanoma cells and tyrosinase activity. Also the quantitative analysis of the compounds
were carried out using LC/MS/MS experiment. Compounds 3 and 5 could be used as skinwhitening agents.
Keywords: anti-melanogenic effect, N-(trans-cinnamoyl)tryptamine, trans-p-coumaric acid
methyl ester, phenyl compound, rice root
EXPERIMENTAL
1. Plant materials
The roots of O. sativa (Chucheongbyeo) were collected from Ganghwa-gun (Incheon) in
November 2011, and were identified by Dr. Jun Hyun Jo of Functional Crop Resource
Development Division, National Institute of Crop Sciences, Rural Development
Administration, Milyang, Korea. The voucher specimen (KHU111113) is deposited in the
Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.
2. Instruments and chemicals
For instrumental and chemicals, see previous papers (Wu et. al., 2013; Song et. al., 2013).
3. Extraction of O. sativa roots and isolation of phenyl compounds
The dried roots of O. sativa (8.35 kg) was extracted with 80% MeOH (280 L × 3) at room
temperature for 12 hours. The extract was filtered through filter paper and concentrated in a
rotary vacuum evaporator (N-1000, EYELA, Tokyo, Japan) to yield a brownish residue (284
g). The residue was suspended in 4 L water and successively partitioned with EtOAc (4 L × 6)
and n-BuOH (3.2 L × 3), yielding concentrated extracts of EtOAc (ORE, 38 g), n-BuOH
(ORB, 26 g), and H2O, sequentially.
The EtOAc fraction (ORE, 38 g) was fractionated to the SiO2 c.c. (11.5 × 13.0 cm) eluting
with n-hexane-EtOAc (7:1, 8 L → 5:1, 7 L → 3:1, 17 L → 1:1, 10 L) and CHCl 3-MeOH
(17:1, 4 L → 15:1, 7 L → 12:1, 3 L → 9:1, 7 L → 6:1, 2 L → 3:1, 4 L → 1:1, 6 L) to yield 32
fractions (ORE-1 to ORE-32). ORE-10 (1.3 g, Ve/Vt 0.13 to 0.17) was subjected to an ODS
c.c. (4.5 × 8.0 cm) and eluted with acetone-acetonitrile-H2O (2:2:7, 3.0 L), yielding 18
fractions (ORE-10-1 to ORE-10-18). ORE-10-6 (250 mg, Ve/Vt 0.06 to 0.11) was applied to
the SiO2 c.c. (3.4 × 11.0 cm) and eluted with CHCl3-MeOH (38:1, 1.5L), yielding 12
fractions (ORE-10-6-1 to ORE-10-6-12) and ultimately gave compound 1 [ORE-10-6-3, 57
mg, Ve/Vt 0.21 to 0.29, TLC (SiO2 F254) Rf 0.55, CHCl3-MeOH = 23:1] and compound 2
[ORE-10-6-8, 132 mg, Ve/Vt 0.63 to 0.71, TLC (SiO2 F254) Rf 0.40, CHCl3-MeOH = 23:1].
ORE-12 (692 mg, Ve/Vt 0.19 to 0.21) was separated by the ODS c.c. (4.0 × 8.0 cm) and
eluted with acetone-acetonitrile-H2O (1:1:4, 1.0 L → 1:1:2, 1.0 L) to obtain 25 fractions
(ORE-12-1 to ORE-12-25). ORE-12-2 (18 mg, Ve/Vt 0.02 to 0.05) was separated by the SiO2
c.c. (2.0 × 13.0 cm) and eluted with CHCl3-MeOH (45:1, 0.7 L), yielding seven fractions
(ORE-12-2-1 to ORE-12-2-7) and ultimately gave compound 3 [ORE-12-2-2, 2.7 mg, Ve/Vt
0.15 to 0.25, TLC (SiO2 F254) Rf 0.55, CHCl3-MeOH = 19:1]. ORE-17 (177 mg, Ve/Vt 0.29 to
0.34) was separated by the ODS c.c. (3.0 × 6.0 cm) and eluted with acetone-acetonitrile-H2O
(1:1:4, 1.2 L), yielding 12 fractions (ORE-17-1 to ORE-17-12) and isolated compound 4
[ORE-17-5, 5.4 mg, Ve/Vt 0.20 to 0.34, TLC (RP-18 F254s) Rf 0.33, acetone-acetonitrile-H2O
= 2:2:3]. ORE-20 (596 mg, Ve/Vt 0.34 to 0.41) was subjected to ODS c.c. (3.0 × 5.0 cm) and
eluted with acetone-acetonitrile-H2O (2:2:7, 1.8 L), yielding 17 fractions (ORE-20-1 to ORE20-17) and ultimately gave compound 5 [ORE-20-6, 19 mg, Ve/Vt 0.33 to 0.39, TLC (RP-18
F254s) Rf 0.23, acetone-acetonitrile-H2O = 2:2:3].
Vanillin (1): White solid (MeOH); m.p. 80-81°C; EI/MS m/z 152 [M]+, 151, 137, 123, 81; IR
(CaF2, ν, cm-1) 3192, 1669; 1H NMR (400 MHz, CDCl3, δH) 9.80 (1H, s, H-aldehyde), 7.41
(1H, dd, J = 8.0, 2.4 Hz, H-6), 7.39 (1H, d, J = 2.4 Hz, H-2), 7.02 (1H, d, J = 8.0 Hz, H-5),
3.94 (3H, s, 3-OCH3).
13
C NMR (100 MHz, CDCl3, δC) 190.8 (C-aldehyde), 151.6 (C-4),
147.1 (C-3), 129.8 (C-1), 127.6 (C-6), 114.3 (C-2), 108.7 (C-5), 56.1 (3-OCH3).
Methyl trans-ferulate (2): White crystals (MeOH); m.p. 68°C; ESI/MS m/z 209 [M+H]+; IR
(KBR, ν, cm-1) 3395, 1693; 1H NMR (400 MHz, CDCl3, δH) 7.58 (1H, d, J = 16.0 Hz, H-7),
7.02 (1H, dd, J = 8.4, 2.0 Hz, H-6), 6.97 (1H, d, J = 2.0 Hz, H-2), 6.87 (1H, d, J = 8.4 Hz, H5), 6.25 (1H, d, J = 16.0 Hz, H-8), 3.86 (3H, s, 3-OCH3), 3.76 (3H, s, 9-OCH3).
13
C NMR
(100 MHz, CDCl3, δC) 167.7 (C-9), 147.9 (C-4), 146.7 (C-3), 144.9 (C-7), 126.7 (C-1), 122.9
(C-6), 114.9 (C-8), 114.7 (C-5), 109.3 (C-2), 55.8 (3-OCH3) , 51.5 (9-OCH3).
trans-p-Coumaric acid methyl ester (3): Colorless solid (MeOH); m.p. 139-140°C; EI/MS
m/z 178 [M]+, 147, 119, 91, 65; IR (CaF2, ν, cm-1) 3357, 3028, 1694, 1642, 1606, 1517, 1445,
1172, 834; 1H NMR (400 MHz, C5D5N, δH) 8.17 (1H, d, J = 15.6 Hz, H-7), 7.85 (2H, d, J =
8.4 Hz, H-2, 6), 7.41 (2H, d, J = 8.4 Hz, H-3, 5), 6.83 (1H, d, J = 15.6 Hz, H-8), 4.03 (3H, s,
9-OCH3). 13C NMR (100 MHz, C5D5N, δC) 167.2 (C-9), 160.9 (C-4), 144.7 (C-7), 130.1 (C-2,
6), 125.5 (C-1), 116.3 (C-3, 5), 114.3 (C-8), 50.7 (9-OCH3).
N-Benzoyltryptamine (4): White crystals (MeOH); m.p. 131°C; EI/MS m/z 264 [M]+, 143,
121, 105; IR (KBr, ν, cm-1) 3407, 3308, 1649, 1484; 1H NMR (400 MHz, CDCl3, δH) 7.64
(2H, d, J = 7.6 Hz, H-2', 6'), 7.63 (1H, d, J = 7.6 Hz, H-4), 7.44 (1H, dd, J = 7.6, 7.6 Hz, H4'), 7.36 (1H, d, J = 7.6 Hz, H-7), 7.35 (2H, dd, J = 7.6, 7.6 Hz, H-3', 5'), 7.20 (1H, dd, J =
7.6, 7.6 Hz, H-6), 7.11 (1H, dd, J = 7.6, 7.6 Hz, H-5), 7.06 (1H, d, J = 2.0 Hz, H-2), 3.79 (1H,
q, J = 6.4 Hz, H-11), 3.08 (1H, t, J = 6.4 Hz, H-10). 13C NMR (100 MHz, CDCl3, δC) 167.4
(C-7'), 136.4 (C-8), 134.6 (C-1'), 131.3 (C-4'), 128.4 (C-3', 5'), 127.3 (C-9), 126.8 (C-2', 6'),
122.3 (C-2), 122.1 (C-6), 119.5 (C-4), 118.7 (C-5), 113.0 (C-3), 111.3 (C-7), 40.2 (C-11),
25.3 (C-10).
N-(trans-Cinnamoyl)tryptamine (5): White ctystals (MeOH); 1H NMR (400 MHz, CDCl3, δH)
7.60 (1H, dd, J = 8.0, 1.2 Hz, H-4), 7.58 (1H, d, J = 15.6 Hz, H-7'), 7.42 (2H, m, H-2', 6'),
7.36 (1H, dd, J = 8.0, 1.2 Hz, H-7), 7.31 (3H, m, H-3', 4', 5'), 7.18 (1H, ddd, J = 8.0, 8.0, 1.2
Hz, H-6), 7.10 (1H, ddd, J = 8.0, 8.0, 1.2 Hz, H-5), 7.03 (1H, d, J = 2.4 Hz, H-2), 6.27 (1H, d,
J = 15.6 Hz, H-8'), 3.71 (1H, q, J = 6.8 Hz, H-11), 3.02 (1H, t, J = 6.8 Hz, H-10). 13C NMR
(100 MHz, CDCl3, δC) 165.9 (C-9'), 140.8 (C-7'), 136.4 (C-8), 134.7 (C-1'), 129.5 (C-4'),
128.7 (C-3', 5'), 127.7 (C-2', 6'), 127.3 (C-9), 122.2 (C-2), 122.1 (C-6), 120.7 (C-8'), 119.4
(C-5), 118.7 (C-4), 112.8 (C-3), 111.3 (C-7), 39.9 (C-11), 25.2 (C-10).
4. Quantitative analysis of phenyl compounds using RRLC-ESI-MS-MS
EtOAc fraction was dissolved in MeOH (16.5 mg/ml) and filtered through a 0.2 μm syringe
filter. The assay was performed on an Agilent 6410B triple quadrupole LC–MS system
(Agilent Corporation, MA, USA) consisting of an Agilent 1200 RRLC system (Agilent Co.)
connected to a triple quadrupole MS analyzer with an electrospray ionization (ESI) interface
usable in either positive-ionization or negative-ionization mode. The LC analysis was
performed on an Agilent Eclipse Plus C18 column (1.8 μm, 100 × 2.1 mm) at a temperature
of 30°C. The mobile phase consisted of (A) water (0.1% formic acid) and (B) acetonitrile
(0.1% formic acid) using a gradient elution of B was 30-30% at 0-5 min, 30-45% at 5-20 min,
45-60% at 20-21 min, 60-60% at 21-30 min, 60-100% at 30-31 min, 100-100% at 31-35 min,
100-30% at 35-37 min, and 30-30% at 37-40 min. The solvent flow rate was 0.4 mL/min, a
photodiode array detector was used at 225 nm, and the sample injection volume was 5 μL.
The optimum operating parameters of the ESI interface in positive mode were: flow rate 10
L/min N2; drying gas temperature 350°C; nebulizer pressure 40 psi N2; capillary voltage 4000
V. High purity nitrogen was used as collision gas. Quantitation was achieved in multiple
reaction monitoring (MRM) mode. For quantification of individual phenyl compounds in rice
root, the standard curves of individual phenyl compounds that relate various concentrations
of authentic standard solutions to areas of their corresponding peaks were utilized.
5. Cell culture
Murin B16-F10 melanoma cells were obtained from Korean cell line bank (LCLB, Korea),
and the cells were grown in DMEM supplemented with 10% FBS, penicillin (100 units/ml),
and streptomycin (100 μg/ml) at 37°C in 5% CO2/air. Cells were harvested by incubation in
phosphate-buffered saline (PBS) containing EDTA 1 mM and 0.25% trypsin for 2 min at
37°C.
6. Cell viability by EX-CyTox
B16-F10 melanoma cells were seeded at 4 × 103 cells/well in 96-well flat-bottomed plates
(SPL, Korea). After 24 hours at 37°C, the medium was replaced with media containing
compounds diluted to the appropriate concentrations. After 48 hours, the media containing
the compounds were replaced with media containing 10% EX-CyTox (Daeil Lab Service,
Korea). The cells were then incubated at 37°C for 2 hours, and cell proliferation was
monitored using a microplate reader at a wavelength of 450 nm. All assays were performed in
triplicate. The cytotoxic effect of each treatment was expressed as a percentage of cell
viability relative to the untreated control cells and is defined as [(A450 nm treated
cells)/(A450 nm untreated cells)] x 100.
7. Determination of melanin content in B16-F10 melanoma cells
Melanin content was measured as previously described (Huang et. al., 2013), but with a slight
modification. Briefly, the cells were treated with the samples in DMEM containing 10% FBS
without phenol-red for 3 days. Media was collected, and 100 microliter aliquots of media
were then placed in 96-well plates and the optical density of each culture well was measured
using a microplate reader at 450 nm. After concentration by centrifugation, the cell pellets
were dissolved in 140 μl of 1 N NaOH at 80°C for 30 minutes and centrifuged again for 30
minutes at 16,000 g. The optical densities of the supernatants were measured at 450 nm using
a microplate reader. α-Arbutin was used as a positive control. Data represent the mean values
± SD of three experiments. The Statistical significance among the test groups was determined
by Student’s t-test (*P≤0.05, **P≤0.005).
8. Assay of cellular tyrosinase activity
Cellular Tyrosinase activity in B16-F10 cells was examined by measuring the rate of
oxidation of L-DOPA (Chiang et. al., 2013; Huang et. al., 2013). B16-F10 melanoma cells
were seeded at a density of 2×105 cells/60mm dish. After 24 h, a single substance was
replaced by media conditioned with various concentrations, and medium were removed after
50 h and washed with PBS. Cells were harvested and homogenized with lysis buffer. The
lysates were centrifuged at 17,000 g for 30 min at 4°C, and protein content was determined
using Pierce BCA protein assay kit (Thermo Scientific, PA, USA). The 50 μL of sample (50
μg of protein) and 50 μL of L-DOPA (50 mM in pH 6.4 0.1 M sodium phosphate buffer)
were placed in a 96-well plate, and the absorbance at 450 nm was read every 10 min for 1 h
at 37°C using an ELISA plate reader. The inhibition rate of tyrosinase activity was calculated
using the following equation: Activity (%) = (A450 nm treated cells)/(A450 nm untreated
cells)*100
References
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Table S1. ESI-MS data for phenyl compounds in rice roots EtOAc fraction
Compound
RT
(min)
Precursor
UVmax
ion [M+H]
(m/z)
Product ion
(m/z)
(1) vanillin
3.3
226, 278, 309
153
125, 93, 65
(3) trans-p-coumaric acid
8.3
310, 227, 211
179
147, 119, 91, 65
(2) methyl trans-ferulate
9.1
324, 218, 235
209
177, 145, 117, 89
(4) N-benzoyltryptamine
16.4
223
265
144, 105, 77
(5) N-(trans-cinnamoyl)
21.3
218, 272
291
144, 131, 103
methyl ester
tryptamine
Figure S1. Cytotoxic effect of phenyl compounds on B16-F10 melanoma cells treated with
different concentrations of each compound (up to 50 μM). After 1 h of incubation, cell
viability was monitored using a microplate reader at a 450 nm. Data are represented as the
mean ± SD of triplicate. The statistical significance among the test groups was determined by
Student’s t-test (*P<0.05).
Figure S2. Cytotoxic effect of compounds 3 and 5 on B16-F10 melanoma cells treated with
different concentrations of each compound (up to 200 μM). After 1 h of incubation, cell
viability was monitored using a microplate reader at a 450 nm. Data are represented as the
mean ± SD of triplicate. The statistical significance among the test groups was determined by
Student’s t-test (*P<0.05).
(a)
(b)
Figure S3. Effect of phenyl compounds 2-5 on (a) extracellular and (b) intracellular melanin
contents in α-MSH-stimulated B16-F10 cells. α-Arbutin was used as a positive control. Data
represent the mean values ± SD of three experiments. The Statistical significance among the
test groups was determined by Student’s t-test (*P≤0.05, **P≤0.005).
(a)
(b)
Figure S4. Effect of compounds 3 and 5 on (a) extracellular and (b) intracellular melanin
contents in α-MSH-stimulated B16-F10 cells. Data represent the mean values of three
experiments ± SD. The Statistical significance among the test groups was determined by
Student’s t-test (*P≤0.05, **P≤0.005).
Figure S5. Tyrosinase inhibitory activity of compounds 3 and 5 compared with α-arbutin.
Data represent the mean values ± SD of three experiments. The Statistical significance among
the test groups was determined by Student’s t-test (**P≤0.05, ***P≤0.005).