Extracellular melanogenesis inhibitory activity and the structure-activity relationships of ugonins from Helminthostachys zeylanica roots

Article abstract of DOI:10.1016/j.fitote.2015.05.006

Ugonin J, K, and L, which are luteolin derivatives, were isolated from Helminthostachys zeylanica roots by a series of chromatographic separations of a 50% ethanol/water extract. They were identified using nuclear magnetic resonance (NMR), ultraviolet (UV) spectra, and ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS). In this study, the intra and extracellular melanogenic activity of the ugonins were determined using B16 melanoma cells. The results showed that ugonin J at 12.5, 25, and 50 μM reduced extracellular melanin contents to 75, 16, and 14%, respectively, compared to the control. This indicates that ugonin J showed a stronger activity than arbutin, used as the positive control. Moreover, ugonin K showed a more potent inhibition with 19, 8, and 9% extracellular melanin reduction at the same concentrations, than that shown by ugonin J. In contrast, ugonin L did not inhibit intra- or extracellular melanogenic activity. Furthermore, in order to investigate the structure-activity relationships of the ugonins, the intra- and extracellular melanogenic activity of luteolin, methylluteolin, quercetin, eriodictyol, apigenin, and chrysin were determined. Consequently, it was suggested that the catechol and flavone skeleton of ugonin K is essential for the extracellular melanogenic inhibitory activity, and the low polarity substituent groups on the A ring of ugonin K may increase the activity.

Full text of DOI:10.1016/j.fitote.2015.05.006

Extracellular melanogenesis inhibitory activity and the structure-activity
relationships of ugonins from Helminthostachys zeylanica roots
Kosei Yamauchi, Tohru Mitsunaga, Yuki Itakura, Irmanida Batubara
PII:
DOI:
Reference:
S0367-326X(15)30001-0
doi: 10.1016/j.fitote.2015.05.006
FITOTE 3180
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
7 April 2015
7 May 2015
10 May 2015
Please cite this article as: Kosei Yamauchi, Tohru Mitsunaga, Yuki Itakura, Irmanida
Batubara, Extracellular melanogenesis inhibitory activity and the structure-activity re-
lationships of ugonins from Helminthostachys zeylanica roots, Fitoterapia (2015), doi:
1
0.1016/j.fitote.2015.05.006
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ACCEPTED MANUSCRIPT
Extracellular melanogenesis inhibitory activity and the structure-activity
relationships of ugonins from Helminthostachys zeylanica roots
a
a,
b
c
Kosei Yamauchi , Tohru Mitsunaga *, Yuki Itakura , Irmanida Batubara
Affiliation
a
The united graduate school of agricultural science, Gifu University, 1-1 Yanagido,
01-1193, Gifu, Japan.
5
b
Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, 501-1193 Gifu
Japan.
c
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Biopharmaca
Research Center, Bogor Agricultural University Jl. Taman Kencana No. 3, Kampus IPB
Taman Kencana, Bogor 16151 Indonesia, e-mail: ime@ipb.ac.id
*
Correspondence
Prof. Dr. Tohru Mitsunaga, The united graduate school of agricultural science, Gifu
University, 1-1 Yanagido, 501-1193, Gifu, Japan. E-mail: mitunaga@gifu-u.ac.jp
Phone.: +81 58 293 2920 Fax : +81 58 293 2920

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Abbreviations
ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinase; L-DOPA,
L-3,4-dihydroxyphenylalanine; MITF, microphthalmia-associated transcription factor;
MAPK, mitogen-activated protein kinase.

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Abstract
Ugonin J, K, and L, which are luteolin derivatives, were isolated from
Helminthostachys zeylanica roots by a series of chromatographic separations of a 50%
ethanol/water extract. They were identified using nuclear magnetic resonance (NMR),
ultraviolet (UV) spectra, and ultra-performance liquid chromatography coupled to
time-of-flight mass spectrometry (UPLC-TOF-MS). In this study, the intra and
extracellular melanogenic activity of the ugonins were determined using B16 melanoma
cells. The results showed that ugonin J at 12.5, 25, and 50 μM reduced the extracellular
melanin contents to 75, 16, and 14%, respectively, compared to the control. This
indicates that ugonin J showed a stronger activity than arbutin, used as the positive
control. Moreover, ugonin K showed a more potent inhibition with 19, 8, and 9%
extracellular melanin reduction at the same concentrations, than that shown by ugonin J.
In contrast, ugonin L did not inhibit intra- or extracellular melanogenic activity.
Furthermore, in order to investigate the structure-activity relationships of the ugonins,
the intra- and extracellular melanogenic activity of luteolin, methylluteolin, quercetin,
eriodictyol, apigenin, and chrysin were determined. Consequently, it was suggested that
the catechol and flavone skeleton of ugonin K is essential for the extracellular
melanogenic inhibitory activity, and the low polarity substituent groups on the A ring of
ugonin K may increase the activity.
Keywords
Helminthostachys zeylanica; Tyrosinase; B 16 melanoma cell; Ugonin J; Ugonin K

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1
. Introduction
Melanin is a pigment found in the skin and is thought to play an important role in
protecting skin from ultraviolet (UV) rays, thereby preventing skin cancer [1,2].
However, excessive accumulation of melanin in the skin surface results in mottled skin;
mottles are unfavorable to esthetics and health. Melanin is biosynthesized in melanocyte
cells from L-tyrosine. The key enzyme involved in melanin biosynthesis is tyrosinase,
which contains copper and catalyzes two reactions in this process [3]. In the cells, the
first key step in melanin biosynthesis is the hydroxylation of L-tyrosine to
L-3,4-dihydroxyphenylalanine (L-DOPA), and the second is the oxidation of L-DOPA to
L-DOPA-quinone by the enzymatic reaction of tyrosinase. This results in the production
of the red-orange-colored pheomelanin and blackish-brown-colored eumelanin [3]. The
melanin biosynthesized in melanocytes is transported to the keratinocytes present on the
skin surface. Skin pigmentation subsequently occurs by the cornification of
keratinocytes and then, the mottles form from the melanin released outside of the
melanocytes. Intracellular melanin status is usually determined using B16 melanoma
cells to screen compounds, which elicit melanogenic controlling activity [4-6].
Extracellular melanin was the focus of this study. Therefore, the levels of both intra- and
extracellular melanin were determined using B16 melanoma cells to clarify the
importance of the extracellular melanin in the development of skin whitening agents.
Furthermore, in this study, the melanogenic activity of components of the
Helminthostachys zeylanica root extract was investigated by determining the
absorbance of intra- and extracellular melanin following extract treatment.
H. zeylanica, belongs to the Ophioglossaceae family, and is used as a traditional folk

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medicine in the treatment of ailments as an antipyretic, antiphlogistic, anodyne [7],
antimalarial agent, and for the treatment of sciatica and ulcer boils [8]. Previous studies
have isolated ugonins A–T from H. zeylanica extracts and investigated their antioxidant
and anti-inflammatory activity [9-12]. Furthermore, a novel quercetin glycoside, which
stimulated intercellular melanogenesis, was isolated and identified from the H.
zeylanica root extracts [13]. In this study, we further investigated the intra- and
extracellular melanogenic activity of the extract components. Moreover, this is the first
study to report the importance of determining extracellular melanogenesis in the search
for useful ingredients for whitening agents. This was achieved by determining the
melanogenic inhibitory effect and structure-activity relationship of the ugonins isolated
from the H. zeylanica root extracts. In addition, we determined the levels of intra- and
extracellular melanin.
2
. Materials and methods
2
.1. General experimental procedures
1
13
The proton ( H) and carbon-13 ( C) nuclear magnetic resonance (NMR) spectra
were recorded in methanol-d using a Jeol EC600M Hz NMR spectrometer. The
4
coupling constants were expressed in Hz, and the chemical shifts were determined on a
δ (ppm) scale. The ultra-performance liquid chromatography time-of-flight mass
R
TM
spectrometry (UPLC-TOF-MS, Waters Xevo TQ mass spectrometer) was performed
using a C column (2.1 mm φ × 100 mm L, Waters, Milford, MA). The
1
8
UPLC-TOF-MS data were collected in negative ionization modes. The capillary voltage
was 3.0 kV. The cone and desolvation gas flow rates were set at 50 and 1000 L/h,

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respectively, and the source and desolvation temperatures were 150 and 500°C,
respectively. The UV spectra were recorded on a Shimadzu SPD-M20A diode array
detector. The preparative high-performance liquid chromatography (HPLC, Jasco) was
performed using an Inertsil ODS-3 column (10 mm φ × 250 mm L, GL Sciences, Tokyo,
Japan). Synthetic melanin was purchased from Sigma-Aldrich (St. Louis, MO). The
other products were commercially available and purchased from Wako Chemicals
(Richmond, VA).
2
.2. Materials
The H. zeylanica root powder was collected from Samarinda, Indonesia in 2006.
The sample and voucher specimen (number WAN0017365) were deposited at the
Herbarium at Wanariset, Samboja, East Kalimantan, Indonesia.
2
.3. Isolation and identification of ugonins from H. zeylanica root powder extract
The H. zeylanica root powder (about 100 g) was extracted with a 50% ethanol and
water mixture. The extract was separated using Sephadex LH20 gel column (25 mm φ ×
8
1
20 mm L) chromatography and eluting with a solvent mix of water:methanol (MeOH,
:1 v/v), to obtain fractions LHFr.1–6. The LHFr.6 fraction was further separated using
the same chromatographic procedure except that the eluting solvent was a mixture of
water:MeOH at a ratio of 2:3 (v/v) and subfractions LHFr.6A–6G were obtained.
Ugonin J, K, and L were isolated from subfractions LHFr.6E, 6F, and 6G respectively,
R
using preparative HPLC (inertsil ODS-3V, 10 mm φ × 250 mm L, isocratic elution with
MeOH:0.05% aqueous trifluoroacetic acid, TFAaq., 95:5).
1
13
The isolated ugonins were identified using H NMR, C NMR, and UV spectral
analysis, as well as UPLC-TOF-MS. The UPLC-TOFMS measurements were

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performed using a C (2.1 × 100 mm) column with MeOH:water as the eluent with the
1
8
following schedule: 5:95 (0 min), 100:0 (10 min), 100:0 (13 min). The physical
descriptions and spectral data are as follows:
1
Ugonin J: Yellow powder; UV λ max 214, 274, 345 nm; H NMR (CD OD, 600 MHz) δ
3
H
0
.91 (3H, s, H-13), 1.03 (3H, s, H-12), 1.22 (1H, m, H-14), 1.47 (1H, m, H-15), 1.57
(1H, m, H-15), 1.66 (1H, m, H-14), 1.92 (1H, m, H-16), 2.37 (1H, dd, J = 12.1, 4.08 Hz,
H-10), 2.50 (1H, m, H-16), 2.68 (1H, dd, J = 13.2, 4.07 Hz, H-9), 2.91 (1H, t, J = 11.7
Hz, H-9), 4.17 (1H, br s, H-18), 4.39 (1H, d, J = 2.04 Hz, H-18), 6.37 (1H, s, H-8), 6.47
(1H, s, H-3), 6.86 (1H, d, J = 6.90 Hz, H-5’), 7.31 (1H, dd, J = 6.90, 2.04 Hz, H-6’),
1
3
7
.32 (1H, d, J = 2.04 Hz, H-2’); C NMR (CD OD,150 MHz) δ 21.0 (C-9), 23.2
3 C
(C-15), 27.1 (C-13), 27.3 (C-12), 30.9 (C-16), 34.1 (C-14), 34.5 (C-11), 52.1 (C-10),
9
2.6 (C-8), 102.4 (C-3), 103.6 (C-4a), 108.3 (C-18), 112.1 (C-6), 112.8 (C-2’), 115.4
(
(
[
C-5’), 118.8 (C-6’), 122.5 (C-1’), 145.8 (C-3’), 149.3 (C-4’), 149.7 (C-17), 155.7
C-8a), 159.0 (C-5), 162.8 (C-7), 164.5 (C-2), 182.6 (C-4); UPLC-TOFMS m/z 421.165
-
M-H] .
1
Ugonin K: Yellow powder; UV λ max 215, 275, 344 nm; H NMR (CD OD, 600 MHz)
3
δH 0.91 (3H, s, H-13), 1.04 (3H, s, H-12), 1.23 (1H, m, H-14), 1.49 (1H, m, H-15), 1.58
(1H, m, H-15), 1.59 (1H, m, H-14), 1.94 (1H, m, H-16), 2.25 (1H, dd, J = 11.7, 4.14 Hz,
H-10), 2.47 (1H, m, H-16), 2.72 (1H, dd, J = 13.1, 4.14 Hz, H-9), 2.91 (1H, t, J = 12.4

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Hz, H-9), 3.88 (3H, s, 7OCH ), 4.08 (1H, d, J = 1.20 Hz, H-18), 4.37 (1H, d, J = 2.04
3
Hz, H-18), 6.54 (1H, s, H-3), 6.61 (1H, s, H-8), 6.88 (1H, d, J = 8.22 Hz, H-5’), 7.36
1
3
(
1H, d, J = 2.76 Hz, H-2’), 7.39 (1H, dd, J = 8.28, 2.10 Hz, H-6’); C NMR (CD OD,
3
1
50 MHz) δ 20.8 (C-9), 23.0 (C-15), 27.3 (C-12, 13), 30.7 (C-16), 34.1 (C-14), 34.2
C
(
C-11), 52.8 (C-10), 55.0 (7OCH ), 89.3 (C-8), 102.8 (C-3), 104.6 (C-4a), 108.3 (C-18),
3
112.7 (C-6), 112.8 (C-2’), 115.4 (C-5’), 119.0 (C-6’), 122.6 (C-1’), 143.3 (C-3’), 145.7
(C-4’), 149.7 (C-17), 155.3 (C-8a), 158.0 (C-5), 163.4 (C-7), 164.1 (C-2), 182.7 (C-4);
-
UPLC-TOFMS m/z 435.186 [M-H] .
1
Ugonin L: Yellow powder; UV λ max 215, 271, 336 nm; H NMR (CD OD, 600 MHz)
3
δH 0.94 (3H, s, H-13), 1.02 (3H, s, H-12), 1.37 (1H, dd, J = 14.4, 6.18 Hz, H-14), 1.50
(
1H, br d, J = 13.1 Hz, H-14), 1.63 (1H, dd, J = 13.1, 4.80 Hz, H-10), 1.70 (3H, m,
H-15,16), 2.11 (1H, br d, J = 12.4 Hz, H-16), 2.32 (1H, dd, J = 16.5, 13.0 Hz, H-9), 2.73
1H, dd, J = 17.2, 4.80 Hz, H-9), 3.96 (3H, s, 7OCH ), 6.48 (1H, s, H-3), 6.70 (1H, s,
(
3
H-8), 6.88 (1H, d, J = 8.22 Hz, H-5’), 7.34 (1H, d, J = 2.10 Hz, H-2’), 7.36 (1H, dd, J =
1
3
8
.22, 2.04 Hz, H-6’); C NMR (CD OD, 150 MHz) δ 17.8 (C-9), 18.4 (C-12), 19.5
3 C
(
C-18), 19.6 (C-14), 31.1 (C-11), 39.1 (C-13), 41.2 (C-16), 46.7 (C-10), 55.3 (7OCH3),
8.8 (C-17), 90.4(C-8), 105.5 (C-3), 108.0 (C-4a), 108.7 (C-6), 112.6 (C-2’), 115.6
C-5’), 118.5 (C-6’), 124.2 (C-1’), 143.8 (C-3’), 149.1 (C-4’), 153.6 (C-5), 158.2 (C-8a),
7
(

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-
1
62.4 (C-7), 162.5 (C-2), 185.7 (C-4); UPLC-TOFMS m/z 435.185 [M-H] .
2
.4. Tyrosinase activity assay
The tyrosinase activity measurements were performed using a previously described
method [14,15]. The sample (60 μL) was placed in a 96-well plate and 30 µL of
mushroom tyrosinase (333 U/mL in phosphate buffer, 50 mM, pH 6.5) and 110 μL of
substrates (2 mM L-tyrosine or 2 mM L-DOPA) were added. After incubation at 37°C
for 30 min, the absorbance was measured at 510 nm using a microplate reader. The
tyrosinase activity was expressed as the half-maximal inhibitory concentration (IC ),
5
0
which is the concentration of the samples producing 50% inhibition.
2
.5. Cell culture
The mouse melanoma B16-F0 cells (DS Pharma Biomedical, Osaka, Japan) were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere
of 5% CO2.
2
.6. Measurement of cellular melanin content
The measurement of cellular melanin was performed according to a previously
described method [16]. In brief, confluent cultures of B16 melanoma cells were rinsed
in phosphate-buffered saline (PBS) and detached using 0.25% trypsin/
ethylenediaminetetraacetic acid (EDTA). The cells were placed in a 24-well plate (5.0 ×
4
1
0 cells/well) and allowed to adhere at 37°C for 24 h. The samples were added to the
cells followed by incubation for 72 h, and then 200 μL of the culture medium was added

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to a 96-well plate. The extracellular melanin content was determined by measuring the
absorbance of the medium at 510 nm using a microplate reader. The cells were then
washed with PBS following by lysis in 600 μL of 1 M sodium hydroxide (NaOH) with
heating at 100°C for 30 min to solubilize the melanin. The resulting lysate (250 μL) was
placed in a 96-well microplate, and the intracellular melanin contents were determined
by measuring the absorbance at 405 nm using a microplate reader. Each experiment was
repeated twice. The melanin production activity was expressed as a percentage of that of
the control cells treated with dimethyl sulfoxide (DMSO) alone.
2
.7. Cell viability
The cell viability measurement was performed according to a previously described
method [17]. The cell viability was determined using the microculture tetrazolium
technique (MTT). Cell cultures were initiated in 24-well plates at a density of 5.0 × 10
cells/well. After incubation, 50 μL of the MTT reagent
4
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in PBS, 5 mg/mL)
was added to each well. The plates were incubated in a humidified atmosphere of 5%
CO at 37°C for 4 h. After the medium was removed, 1.0 mL isopropyl alcohol
2
(containing 0.04N hydrochloric acid, HCl) was added to the plate, and 150 μL was
added to a 96-well plate. The absorbance was measured at 590 nm using a microplate
reader. Each experiment was repeated twice. The cell viability was expressed as a
percentage of that of the control cells treated with the DMSO vehicle alone.
2
.8. Quantitative determination of the intra- and extracellular melanin
The intra- and extracellular melanin content of the control cells treated with the
DMSO vehicle alone was determined using the standard curve method. Similar to the

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procedure for determining the intracellular melanin content, the standard synthesized
melanin was dissolved in 1N NaOH and heated for 30 min at 100°C. Then, 250 μL of
melanin solution was placed in a 96-well microplate, and the absorbance was measured
at 405 nm with a microplate reader to obtain the standard curve. To determine the
extracellular melanin quantity, the standard synthesized melanin was dissolved in
DMEM and 200 μL of the melanin solution was placed in a 96-well microplate, and the
absorbance was measured at 510 nm with a microplate reader to obtain a standard curve
to quantify the extracellular melanin contents.
2
2
.9. Synthesis of methylluteolins
.9.1. Synthesis of 7,4’-O-dimethylluteolin
Luteolin (100 mg, 0.350 mmol), potassium carbonate (K CO , 193 mg, 1.40 mmol),
2
3
and dimethyl sulfate (133 μL, 1.40 mmol) were added to 10 mL of dimethylformamide
DMF) and the mixture was stirred for 3 h at 60–65°C. The resulting mixture was then
(
diluted with 50 mL of ethyl acetate (EtOAc) and washed with water (2 × 50 mL). The
EtOAc phase was dried using sodium sulfate (Na SO ), and the reactant was obtained
2
4
by evaporating the solvent. Then 30 mL of MeOH was added to the reactant, and the
precipitate was obtained as 7,4’-O-dimethylluteolin with a yield of 20.9% by filtration.
The structure of the synthesized 7,4’-O-dimethylluteolin was confirmed using NMR and
UV spectra as well as UPLC-TOF-MS.
2
.9.2. Synthesis of 7-O-methylluteolin and 4’-O-methylluteolin
Luteolin (100 mg, 0.350 mmol), K CO (48.3 mg, 0.350 mmol), and dimethyl
2
3
sulfate (33.3 μL, 0.350 mmol) were added to 10 mL of DMF and the mixture was then
stirred for 3 h at 60–65°C. The resulting mixture was diluted with 50 mL of EtOAc and

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washed with water (2 × 50 mL). The EtOAc phase was dried using Na SO , and the
2
4
reactant was obtained by evaporating the solvent. The 7-O-methylluteolin and
’-O-methylluteolin obtained from luteolin were yellow powders with yields of 10.0
4
and 20.2% respectively. They were purified using preparative HPLC with an ODS-3
column (10 mm φ × 250 mm L) eluted using a linear gradient of MeOH:0.05% TFA aq.,
and the following schedule: 60:40 (0 min), 100:0 (50 min), and 100:0 (60 min). The
structures of the synthesized 7-O-methylluteolin and 4’-O-methylluteolin were
confirmed using NMR and UV spectral analysis, as well as UPLC-TOF-MS. The
physical descriptions and spectral data are as follows:
1
7
6
6
=
,4’-O-dimethylluteolin: Yellow powder; UV λ max 216, 262, 350 nm; H NMR (CD OD,
3
00 MHz) δ 3.83 (6H, s, 4’OCH , 7OCH ), 6.34 (1H, br s, H-6), 6.70 (1H, br s, H-8),
H
3
3
.77 (1H, s, H-3), 7.06 (1H, d, J = 8.2 Hz, H-5’), 7.42 (1H, br s, H-2'), 7.53 (1H, br s, J
1
3
8.9 Hz, H-6’); C NMR (CD OD, 150 MHz) δ 56.3(7OCH ), 56.6 (4’OCH ), 93.2
3
C
3
3
(C-8), 98.5 (C-6), 104.2 (C-3), 105.2 (C-4a), 112.6 (C-5’), 113.6 (C-2’), 119.4 (C-6’),
1
23.4 (C-1’), 147.3 (C-3’), 151.8 (C-4’), 157.8 (C-8a), 161.7 (C-5), 164.4 (C-2), 165.7
-
(
C-7), 182.4 (C-4); UPLC-TOFMS m/z 313.058 [M-H] .
1
4
6
’-O-methylluteolin: Yellow powder; UV λ max 206, 252, 346 nm; H NMR (CD OD,
3
00 MHz) δ 3.92 (3H, s, 4’OCH ), 6.23 (1H, br s, H-6), 6.53 (1H, br s, H-8), 6.62 (1H,
H
3
s, H-3), 7.10 (1H, d, J = 8.2 Hz, H-5’), 7.47 (1H, d, J = 2.1 Hz, H-2’), 7.53 (1H, dd, J =
1
3
8
.2, 2.0 Hz, H-6’); C NMR (CD OD, 150 MHz) δ 55.6 (4’OCH ), 93.9 (C-8), 98.9
3 C 3

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(C-6), 103.9 (C-3), 104.5 (C-4a), 111.6 (C-5’), 112.8 (C-2’), 118.9 (C-6’), 124.0 (C-1’),
1
47.0 (C-3’), 150.9 (C-4’), 158.0 (C-8a), 162.5 (C-5), 164.0 (C-2), 164.1 (C-7), 182.4
-
(
C-4); UPLC-TOFMS m/z 299.053 [M-H] .
1
7
6
-O-methylluteolin: Yellow powder; UV λ max 207, 254, 349 nm; H NMR (CD OD,
3
00 MHz) δ 3.91 (3H, s, 7OCH ), 6.29 (1H, br s, H-6), 6.60 (1H, br s, H-3), 6.66 (1H,
H
3
s, H-8), 6.99 (1H, d, J = 8.2 Hz, H-5’), 7.46 (1H, br d, J = 9.3 Hz, H-6’), 7.52 (1H, br s,
1
3
H-2’); C NMR (CD OD, 150 MHz) δ 55.6 (7OCH ), 92.4 (C-8), 97.8 (C-6), 103.4
3
C
3
(C-3), 105.1 (C-4a), 113.5 (C-2’), 115.9 (C-5’), 119.3 (C-6’), 122.7 (C-1’), 145.7 (C-3’),
1
49.4 (C-4’), 157.6 (C-8a), 157.9 (C-2), 162.2 (C-5), 165.7 (C-7), 182.3 (C-4);
-
UPLC-TOFMS m/z 299.052 [M-H] .
3
. Results and discussion
3
.1. Identification of compounds
The isolated ugonin J, K, and L were identified by comparing the data obtained in
this study to previous results obtained by Huang et al. (2003) [18]. The chemical shifts
1
of the B and C rings observed in the H NMR spectra were almost similar for all three
compounds. In contrast, three singlet protons were observed around 3.9 ppm in the
1
spectra of ugonin K and L, which were not observed in the H NMR spectra of ugonin J.
This suggests the existence of a methoxyl group in the structures of ugonin K and L,

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which was further confirmed by the hetero-nuclear multiple-bond correlation (HMBC)
data (not shown). The differences between ugonin K and L were shown by the
differences in the chemical shift of C-17 and C-18, which were observed at 149.7 and
1
08.3 ppm, respectively for ugonin K, and 78.8 and 19.5 ppm, respectively, for ugonin
L. This result indicated that the C-17 and C-18 of ugonin L were not linked by a double
bond. Moreover, the chemical shift of C-17 corroborated the ring structure of ugonin L
(Fig. 1). The UPLC-TOF-MS data also corroborated each structure.
The synthesized methylluteolins were also identified using the same analytical
instruments, and the position of their methoxyl groups were clarified using HMBC. As
shown in Fig. 2, the protons of the methoxyl group in 7-O-methylluteolin correlated
with the carbon binding to the methoxyl group. The location of this group was identified
as position 7 on the luteolin structure, using the other key HMBC correlations between
the H-6 and C-7, or H-8 and C-7. Similarly, the HMBC correlations were used to clarify
the exact position of the methoxyl groups in the other synthesized methylluteolins.
3
.2. Inhibition of melanogenesis by ugonins and their tyrosinase activity
The quantitative determination of the intra- and extracellular melanin of the B16
melanoma cells revealed that the control levels were 6.2 and 140 μg, respectively, per
well. This result shows that the level of the extracellular melanin is approximately
2
3-fold higher than that of the intracellular melanin, which provided us with convincing
evidence to develop the whitening agent. There are numerous reports that have only
determined intracellular melanin in the process of investigating compounds that are
suspected to regulate melanogenesis. However, the result of our quantitative
determination shows that most of the melanin synthesized by the B16 melanoma cells

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was released outside the cells. Therefore, to elucidate the regulation of melanogenesis
by the compounds under investigation, it is essential to determine not only the
intracellular but also the extracellular activity of the compounds.
The results of both the intra- and extracellular melanogenic activity of the ugonins
isolated from the 50% ethanol/water extract of the roots of H. zeylanica are shown in
Table 1. The results revealed that the extracellular melanogenic activities of ugonin J at
1
2.5, 25, and 50 μM were 75, 16, and 14%, respectively, compared to the control, which
had 100% melanogenic activity. This result suggests that the effect of ugonin J was
stronger than that of arbutin, which was used as the positive control. Moreover, the
inhibitory activity of ugonin K was more potent than that shown by ugonin J, with 19, 8,
and 9% extracellular melanogenic activity at the same concentrations. Interestingly,
ugonin J and K showed no inhibition of intracellular melanogenesis, suggesting that
they may inhibit the transportation or release of melanosome or both, in B16 melanoma
cells as well as inhibit melanin biosynthesis. In contrast, ugonin L did not show intra- or
extracellular inhibition of melanogenesis. Comparing the melanogenic activity of
ugonin J, K, and L, the C-6 substituent cyclogeranyl group of ugonin J and K may play
an important role in their inhibitory effects. This is because the inhibitory activity
appears to have been abrogated by the formation of a ring with the 5-hydroxyl group as
was observed with the structure and lack of activity of ugonin L. Furthermore, the
presence of the 7-methoxyl group in ugonin K may increase its inhibitory effect on
extracellular melanogenesis.
To further investigate the importance of the C-6 substituent cyclogeranyl and the
7
-methoxyl group for the inhibition of extracellular melanin biosynthesis, luteolin and
the synthesized 7-O-methylluteolin were used as targets for comparison. Consequently,

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the inhibition of extracellular melanogenesis by luteolin was lower than that exerted by
7
7
-O-methylluteolin, ugonin J, and K at 25 and 12.5 μM. In addition, luteolin and
-O-methylluteolin showed higher cytotoxicity than ugonin J and K. These results
confirm that the 7-methoxyl and C-6 substituent cyclogeranyl groups of ugonin K are
important for the inhibitory activity against extracellular melanin biosynthesis.
Additionally, it was shown that the substituent groups that bind to the A ring of ugonin J
and K may increase the cell viability.
Next, to clarify the role of the B and C rings of ugonin J and K in their
structure-activity relationships, the melanogenic activities of the commercially available
quercetin, eriodictyol, apigenin, and chrysin and the synthesized 4’-O-methylluteolin
and 7,4’-O-dimethylluteolin were determined. Quercetin is known as the intracellular
melanogenesis inhibitor [19]. This study also shows that quercetin reduces the
intracellular melanogenesis with 56% at 50μM. However, quercetin and eriodictyol,
which have substituents on the C ring that differ from that of luteolin, showed no
extracellular melanogenesis inhibitory activity. In addition, apigenin, chrysin,
4
’-O-methylluteolin, and 7,4’-O-dimethylluteolin, which have substituents on the B ring
that differ from those of luteolin or 7-O-methylluteolin, also showed no inhibitory
activity. Furthermore, luteolin and its 7-O-methylate suppressed the extracellular
melanin levels at 25 and 50 μM. Conversely, apigenin stimulated intra- and extracellular
melanin biosynthesis. These results suggest that the flavone skeleton on the C and
catechol skeleton on B rings of ugonin J, K, luteolin, and 7-O-methylluteolin may play a
fundamental role in their inhibition of extracellular melanognesis.
The IC values of the tyrosinase activity of ugonin J, K, L, and the target agents
5
0
with which they were compared are shown in Table 2. Ugonin J, K, luteolin, and

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7
-O-methylluteolin, which inhibited extracellular melanogenesis, did not inhibit
tyrosinase. Therefore, these compounds may suppress the expression of tyrosinase,
which is transcriptionally regulated by the microphthalmia-associated transcription
factor (MITF). Furthermore, MITF expression is activated or suppressed by kinases
such as the p38 mitogen-activated protein kinase (MAPK) cascade, extracellular
signal-regulated kinases (ERK), and c-Jun N-terminal kinase (JNK)
0
]. Some melanogenic regulating agents were discovered by determining their
effects on the expression of tyrosinase, p38, JNK, ERK, and MITF as well as tyrosinase
activity [21-23]. In particular, the determination of the downregulation of the expression
of activated p38 and MITF is commonly used to investigate the mechanism by which
compounds inhibit melanogenesis [21, 24]. In the case of ugonin K, it is noteworthy that
previous studies have reported that p38 MAPK stimulation also plays an important role
in osteoblastic differentiation [25]. In addition, studies suggest that the osteoblastic
differentiation was promoted by ugonin K, which accelerates p38 MAPK in mouse
MC3T3-E1 cells [26]. This study reported that ugonin K stimulates p38 MAPK, which
upregulates the expression of tyrosinase. This result may be contrary to the results
showing the inhibition of melanin biosynthesis by ugonin K in this study. Therefore,
regarding the expression of tyrosinase, ugonin K may suppress extracellular melanin
levels by a cascade other than that involving p38 MAPK. In addition, ugonin K may be
involved in the transportation of melanosome because it reduced the extracellular
melanin levels despite its lack of inhibitory effects on intracellular melanogenesis.
Melanosome is transported by a wide variety of proteins in melanocyte such as actin,
myosin Va, Rab27A, Slac2-a, and others. The mature melanosomes interact with myosin
Va, which is a motor protein, via Rab27A localized on the mature melanosomes and

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Slp2-a [27, 28]. The complex is transported by actin to the periphery of the cell from the
perinuclear region of melanocytes [29]. Ugonin J and K may inhibit the transportation
of the melanosomes by suppressing the expression or the activity of these proteins or
both.
4
. Conclusions
In this study, ugonin J, K, and L were isolated from H. zeylanica roots and identified
using NMR and UV spectral analysis, as well as UPLC-TOF-MS. Ugonin J and K
showed high inhibition of extracellular melanogenesis. The result of the
structure-activity relationship investigation revealed that the flavone and catechol
skeleton of ugonin J, K may be critical for its activity. In addition, the substituent groups
that bind to the A ring of ugonin J and K may play an important role in increasing its
activity. The level of extracellular melanin was considerably higher than that of
intracellular melanin. In addition, skin pigmentation is caused by the melanin released
outside from the melanocytes. Therefore, ugonin J and K may be useful lead
compounds for the development of new whitening agents. Additional investigations are
required to elucidate the mechanism of the activity of ugonin J and K. This may be
achieved by determining the expression and activity of the proteins involved in the
expression of tyrosinase or transportation of melanosomes.
Acknowledgement
This research supported by Japan Society for the Promotion of Science (JSPS) for

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Research Fellow.
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OH
OH
3'
OH
3'
3
'
'
OH
4
4' OH
OH
2
'
2'
4' OH
5'
2
'
3
'
4' R1
5'
8
1'
8
1
'
HO
9
8a
4a
O
5'
H CO
3
O
8
1'
2'
1'
8a
4a
5' H CO
3
O
8a
4a
7
0
2
6'
7
2
6'
8
7
2
6'
R2
O
8a
4a
3
9
9
7
2
6'
6
5
OH
4
O
3
6
3
6
5
OH
4
O
5
O
1
2
4
O
1
2
1
0
1
12
6
3
1
3
18
10
5
OH
4
O
1
3
1
8
11 17
1
1
17
16
13
18
1
1
1
7
1
4
16
1
4
1
4
16
15
Luteolin:R
-O-methylluteolin:R =OH R =OCH
3
1 2
=R =OH
1
5
1
5
7
1
2
4
’-O-methylluteolin:R =OH R =OCH
Ugonin J
Ugonin K
Ugonin L
1 2 3
7
,4’-O-dimethylluteolin:R =R =OCH
1 2 3
OH
OH
3'
3
'
3
'
OH
OH
4' OH
5'
3'
4
'
4'
5'
4'
5'
2
'
2'
1'
2'
1'
2'
1'
8
1'
8
8
HO
8a
4a
O
5'
HO
8a
4a
O
HO
8a
4a
O
8
HO
8a
4a
O
7
2
6'
7
2
6'
7
2
6'
7
2
6'
6
3
OH
6
3
6
3
5
4
5
4
5
OH
4
O
6
3
5
OH
4
O
OH
O
OH
O
Apigenin
Chrysin
Quercetin
Eriodictyol
Fig.1. Structures of ugonins and compounds used for investigation of structure-activity
relationships

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OH
OH
OH
H
OH
H
H
O
H
H
H
O
H
H
CH3
H
CH3
O
H
O
O
HO
H
O
O
H C
O
H
O
O
3
H C
3
H
H
H
H
H
H
OH
OH
OH
7
-O-methylluteolin
4’-O-methylluteolin
7,4’-O-dimethylluteolin
Fig.2. Key HMBC correlations of synthesized methylluteolins

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Table 1.
Intra- and extracellular melanogenesis activities and cell
viabilities of ugonins and compounds used for investigation of
structure-activity relationships
Compounds name and
concentration (μM)
Cell viability
(%)
Intracellular
melanogeneis
activity (%)
Extracellular melanogenesis
activity (%)
Ugonin J
50.0
25.0
12.5
62±5.4**
100±1.0
105±0.6
106±13.6
88±7.7
99±5.9
14±1.6**
16±1.6**
75±36.7
Ugonin K
50.0
25.0
12.5
79±15.5
95±7.1
110±1.9
93±4.1
107±10.7
122±18.0
9±2.0**
8±0.8**
19±2.0**
Ugonin L
50.0
25.0
12.5
111±0.1
114±0.1
106±0.0
110±0.4
96±6.3
113±1.8
81±9.6
78±5.3
92±2.7
Luteolin
5
2
1
7
5
2
1
0.0
5.0
2.5
69±0.6
75±3.5
88±0.1
52±3.7
60±5.1
90±7.5
13±0.1**
70±11.2
132±3.5
-O-methylluteolin
0.0
5.0
2.5
80±5.8
64±8.6*
70±0.9*
56±10.5
103±2.6
118±1.5
15±0.0**
22±2.7*
57±0.4
4
5
2
1
7
5
2
1
’-O-methylluteolin
0.0
5.0
2.5
88±1.7
104±8.8
105±6.7
124±15
100±8.5
102±7.4
78±1.1
78±3.0
78±10.1
,4’-O-dimethylluteolin
0.0
5.0
2.5
97±1.3
93±0.0
90±5.4
114±16.2
107±2.7
115±10.6
101±7.1
105±7.8
110±9.6
Quercetin
50.0
25.0
12.5
94±1.7
105±1.0
110±1.3
56±1.8
84±3.5
94±7.0
104±7.0
109±1.8
99±2.1
Eriodictyol
50.0
25.0
12.5
71±1.3**
93±7.2
92±3.5
193±73.4
117±2.3
124±3.4
135±11.8
106±5.2
112±8.6
Apigenin
50.0
25.0
12.5
48±1.8**
70±1.1**
94±2.7
515±94.2*
157±6.1**
106±2.6
215±4.6**
153±0.5**
108±5.1
Chrysin
50.0
25.0
12.5
94±1.7
105±1.0
110±1.3
100±0.2
77±3.1
72±0.3
127±1.2
116±0.1
114±0.3
Arbutin
30.0
7
105±0.7
86±7.2
52±12.7*
All data were expressed as the mean ± S.D. Differences were examined for statistical significance using
Student’s t-test.
n=2. * p<0.05 and ** p<0.01 compared with respective control values

ACCEPTED MANUSCRIPT
Table 2. Tyrosinase inhibitory activities of ugonins and compounds used for
investigation of structure-activity relationships
substrates
L-tyrosine
L-DOPA
IC50 (μg/ml)
IC50 (μg/ml)
Ugonin J
Ugonin K
Ugonin L
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
21.5
>100
>100
>100
>100
>100
>100
>100
34.8
Luteolin
-O-methylluteolin
’-O-methylluteolin
,4’-O-dimethylluteolin
Quercetin
7
4
7
Eriodictyol
Apigenin
Chrysin
Kojic acid
Arbutin
67.3
>100
>100
0.030
>100
>100

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Graphical abstract