Novel quercetin glucosides from Helminthostachys zeylanica root and acceleratory activity of melanin biosynthesis

Article abstract of DOI:10.1007/s11418-012-0672-9

Two novel quercetin glucosides, namely 4′-O-β-d-glucopyranosyl- quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (compound 1) and 4′-O-β-d-glucopyranosyl-(1→2)-β-d- glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1→4) -β-d-glucopyranoside (compound 2), were isolated from Helminthostachys zeylanica root 50 % EtOH extract. Structural analysis of isolated compounds was achieved mainly by 600 MHz and 800 MHz NMR, UPLC-TOFMS and GC-MS. Of the two quercetin glucosides, compound 1 showed a high melanogenic acceleratory effect, 2.7 times higher than control, at 10 μM concentration in murine B16 melanoma cells, with no cytotoxic effect.

Full text of DOI:10.1007/s11418-012-0672-9

J Nat Med (2013) 67:369–374
DOI 10.1007/s11418-012-0672-9
NOTE
Novel quercetin glucosides from Helminthostachys zeylanica root
and acceleratory activity of melanin biosynthesis
•
Kosei Yamauchi Tohru Mitsunaga
•
Irmanida Batubara
Received: 13 February 2012 / Accepted: 19 April 2012 / Published online: 13 May 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract Two novel quercetin glucosides, namely 40-O-
b-D-glucopyranosyl-quercetin-3-O-b-D-glucopyranosyl-(1?
4)-b-D-glucopyranoside (compound 1) and 40-O-b-D-glu-
copyranosyl-(1?2)-b-D-glucopyranosyl-quercetin-3-O-b-
D-glucopyranosyl-(1?4)-b-D-glucopyranoside (compound
2), were isolated from Helminthostachys zeylanica root
50 % EtOH extract. Structural analysis of isolated com-
pounds was achieved mainly by 600 MHz and 800 MHz
NMR, UPLC–TOFMS and GC–MS. Of the two quercetin
glucosides, compound 1 showed a high melanogenic
acceleratory effect, 2.7 times higher than control, at 10 lM
concentration in murine B16 melanoma cells, with no
cytotoxic effect.
anodyne [1], and to treat sciatica, boils, ulcers and malaria
[2]. Four flavonoids, ugonins A–D, were isolated from the
rhizomes of H. zeylanica [3, 4]. Moreover, Huang et al.
isolated ugonins E-T from the root of H. zeylanica and
identified their antioxidant and anti-inflammatory activity
[5, 6].
Melanin is a pigment which is biosynthesized from tyro-
sine by enzymatic oxidation of tyrosinase. It is widely dis-
tributed in skin, retina, nigra of brain, adrenal medullae, etc.
It plays an important role in skin cancer prevention by pro-
tecting cells from ultraviolet rays, and inhibition of grey hair
formation by stress or aging. Melanogenesis enhancing
agents are thus valuable for skin care and anti-grey hair
formation. The present study reports the isolation and iden-
tification of novel compounds from H. zeylanica root extract
and their potent melanogenesis enhancement activity.
Keywords Quercetin glucoside ꢀ B16 melanoma cells ꢀ
Tyrosinase ꢀ Helminthostachys zeylanica
Introduction
Result and discussion
Helminthostachys zeylanica belongs to the family Ophio-
glossaceae, and is used as an antipyretic, antiphlogistic and
Chemical structures of compounds 1 and 2
Compound 1 (Fig. 1) was obtained from a water/EtOH
extract of H. zeylanica root using LH-20 gel column
chromatography, and the structure was analyzed by NMR
and UPLC–TOFMS. In addition, compound 1 was hydro-
lyzed with acid to determine the constitution of sugars and
aglycon. The sugars were analyzed using GC–MS after
trimethylsilylation, and the aglycon was determined using
NMR. The total ion chromatograms of trimethylsilylated
D-glucose, L-rhamnose, D-arabinose, D-galactose and
hydrolysates of compound 1 were determined. Peaks of
trimethylsilylated D-glucose in the standard were observed
at 24.5, 25.5 and 27.7 min of retention time, exactly the
same retention times of hydrolysates of compound 1.
K. Yamauchi ꢀ T. Mitsunaga (&)
Faculty of Applied Biological Science,
Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan
e-mail: mitunaga@gifu-u.ac.jp
I. Batubara
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
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OH
3'
Table 1 1H and 13C-NMR chemical shifts and key HMBC corre-
lations of compound 1
C6
OH
C5
O
O
2'
1'
Position Compound 1
dH (ppm)
J (Hz)
dC
(ppm)
HMBC
C4
OH
OH
C3
4'
C1
8
C2
OR
HO
O
O
8a
4a
7
5'
2
156.8
134.6
178.1
104.4
161.7
2
6'
3
6
4
3
O
A6
4
OH
A5
5
4a
5
O
OH
A4
OH
A3
B6
A1
A2
OH
B5
6
6.18 d
6.36 d
2.04
2.10
98.6 C-5, C-7
164.9
O
O
7
OH
B4
OH
Compound 1 : R=H
OH
B3
8
93.4 C-7, C-8a
157.1
B1
B2
8a
10
20
30
40
50
60
D6
OH
D5
OH
121.5
116.6 C-2, C-40
O
Compound 2 : R=
D4
OH
OH
D3
7.67 d
2.04
8.94
D1
D2
OH
146.2
147.6
115.8 C-30, C-40
Fig. 1 Structures of compounds 1 and 2
7.25 d
7.62 dd
3-Glucose
5.28 d
8.94, 2.04 121.5 C-2, C-40
Moreover, the mass spectrum of compound 1 showed four
major peaks at 787.2029 [M–1]-, 625.1410 [M–1-162]-,
463.0890 [M–1-324]- and 301.0372 [M–1-466]- (m/z) by
UPLC–TOFMS. Considering that the 162 m/z differences
of each peak depend on the fragment of glucose, compound
1 contained three molecules of glucose. Additionally, the
specific optical rotations of compounds 1 and 2 were
-33.4° (c = 0.84, MeOH:H2O 1:1 v/v) and -41.0°
(c = 0.83, H2O), respectively, which are the negative
rotations of another quercetin D-glucoside according to the
previous paper. Furthermore, the specific optical rotations
of sugars from compounds 1 and 2 were positive rotations
?83.0° (c = 0.23, H2O) and ?86.7° (c = 0.19, H2O),
respectively. Compounds 1 and 2 were therefore confirmed
as D-glucosides.
A1
A2
A3
A4
A5
A6
7.56
8.22
102.6 C-3, C-A5
75.0
3.49 t
3.57 m
76.4 C-A2, C-A4
79.0 C-A5, C-A6, C-B1
75.6 C-A4
3.33 m
3.33 m
3.70 (2H) m
(1?4)-Glucose
4.37 d
60.3 C-A4
B1
B2
B3
B4
B5
B6
7.56
8.94
103.2 C-A4, C-B5
3.19 t
73.5
3.30 m
76.7
3.31 m
69.9
3.29 m
76.4
3.63 (2H) m
40-Glucose
4.91 d
61.0 C-B4
C1
C2
C3
C4
C5
C6
7.56
8.94
101.9 C-40
73.4
The NMR data of aglycon accorded with that of quer-
cetin reported by Lasse et al. [7] and Moon et al. [8]. In
order to elucidate the binding position of three glucoses of
compound 1, HMBC correlations (Table 1) were deter-
mined using 800 MHz NMR. HMBC of compound 1 gave
correlations between H-C1 and C-40, and between H-A1
and C-3. These correlations show the presence of C-C1 to
C-40 and C-A1 to C-3 bonds, respectively, via oxygen.
Additionally, correlation between H-B1 and C-A4 was
observed, and thus compound 1 was identified as 40-O-b-D-
glucopyranosyl-quercetin-3-O-b-D-glucopyranosyl-(1?4)-
b-D-glucopyranoside.
Compound 2 (Fig. 1) was identified by comparison with
the NMR and UPLC–TOFMS data of compound 1. The
NMR data of compound 2 shown in Table 2 was related to
that of compound 1. According to the HMBC data, the
proton signal at 4.75 ppm of compound 2 which did not
3.56 t
3.68 m
74.3
3.52 m
69.9
3.57 m
76.7
3.90, 3.86 m, m
61.2
appear in compound 1 correlated with C-C2. The coupling
constant of H-D3 was more than 7 Hz, so H-D2, H-D3 and
H-D4 were therefore shown to be in axial positions. Fur-
thermore, UPLC–TOFMS data of compound 2 was 949
[M–1]- m/z, 162 m/z higher than that of compound 1 (787
m/z). Compound 2 is therefore identified as 40-O-b-D-glu-
copyranosyl-(1?2)-b-D-glucopyranosyl-quercetin-3-O-b-
D-glucopyranosyl-(1?4)-b-D-glucopyranoside.
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371
Table 2 1H and 13C-NMR chemical shifts and key HMBC corre-
lations of compound 2
Melanogenesis and tyrosinase enhancement activities
The melanogenesis enhancement activity of compounds 1
and 2 at 10 lM concentration is shown in Figure 2. The
melanogenesis activity and cell viability of compound 1
were 270 and 93 %, respectively, while compound 2 did not
exhibit melanogenesis enhancement activity. Quercetin, the
aglycon of compound 1 and 2, was reported to have high
melanogenesis inhibitory activity. However, quercetin-40-O-
b-D-glucoside, quercetin-3-O-b-D-glucoside, quercetin-3,40-
O-b-D-glucoside and rutin showed a lower inhibitory activity
than quercetin [9]. Furthermore, it was demonstrated that
quercetin-3-O-b-D-glucoside enhances melanogenesis by
accelerating the expression of tyrosinase-related protein-1
and -2 [10]. Our results extended knowledge of the mela-
nogenesis activities of quercetin glycosides, especially gly-
coside attached to C-3 and 40 of quercetin which had no
melanogenesis inhibitory activity. Compound 1 showed
melanogenesis acceleration activity of 2.7 times that of
control, while interestingly compound 2 had no melano-
genesis enhancement activity in spite of the similarity in the
structure. This result means the number of sugars connecting
C-40 may play an important role in melanogenesis activity.
Since melanogenesis is related to tyrosinase activity, we
also determined the tyrosinase activity of compounds 1 and
2. The activity of compounds 1 and 2 at 10 lM concen-
tration is presented in Figure 3. The tyrosinase activity of
compound 1 was 115 and 117 % using L-tyrosine and
L-DOPA as substrate, respectively. No enhancement
activity of tyrosinase was showed for compound 1 in spite
of the dramatic activity of melanin biosynthesis.
Position Compound 2
dH (ppm)
J (Hz)
dC (ppm) HMBC
2
156.8
134.7
178.1
104.4
161.7
3
4
4a
5
6
6.18 d
6.36 d
2.04
2.04
98.8
165.1
93.5
C-5, C-7
7
8
C-7, C-8a
8a
10
20
30
40
50
60
157.2
125.6
116.3
146.6
147.7
116.3
7.66 d
2.04
8.28
C-2, C-40
7.29 d
C-30, C-40
C-2, C-40
7.60 dd
3-Glucose
5.29 d
8.22, 2.04 121.5
A1
A2
A3
A4
7.56
8.94
102.5
74.4
76.5
79.0
C-3
3.50 t
3.57 m
3.33 m
C-A2, C-A4
C-A5, C-A6,
C-B1
A5
A6
3.33 m
75.6
60.5
C-A4
C-A4
3.71 (2H) m
(1?4)-Glucose
4.37 d
B1
B2
B3
B4
B5
B6
8.22
103.2
73.5
76.5
70.0
76.5
61.1
C-A4, C-B5
3.19 dd
8.94, 7.56
3.28 m
Tyrosinase is transcriptionally regulated by micro-
phthalmia-associated transcription factor (MITF) and its
expression is activated by the p38 MAPK cascade and
extracellularly responsive kinase (ERK). On the other hand,
thec-JunN-terminalkinase (JNK) pathway hasbeen reported
toberelatedtothedown-regulationofmelaninsynthesis[11].
Some melanogenesis-enhancing agents have been examined
at several points of melanogenesis such as expression of
tyrosinase, p38, JNK, ERK and MITF as well as tyrosinase
activity. Astudysuggestedthatp38MAPKwasstimulatedby
cubebin, which enhanced melanogenesis activity in murine
B16 melanoma cells [12]. Compound 1 may also enhance the
activity or expression of MITF by regulating kinase as
mentioned above, because it did not show tyrosinase accel-
eration activity in spite of its high melanogenesis stimulation
activity in B16 melanoma cells. Moreover, considering the
structures of the two novel quercetin glucosides, the glucoses
connecting quercetin C40 may play an important role in
melanogenesis stimulation activity. In the next paper, we
need to elucidate the mechanism of the melanogenesis
enhancement activity of compound 1 by determining the
tyrosinase and MITF expression in B16 melanoma cells.
3.30 m
3.29 m
3.68 (2H) m
40-Glucose
4.97 d
C-B4
C1
C2
C3
C4
C5
C6
7.56
7.56
101.2
81.8
74.1
69.9
76.8
61.1
C-40, C-C2
3.80 t
3.68 m
3.52 m
3.59 m
3.90, 3.86 m, m
(1?2)-Glucose
4.75 d
D1
D2
D3
D4
D5
D6
8.22
104.2
75.0
76.3
69.6
77.0
60.9
C-C2
3.27 t
8.94
3.39 dd
13.7, 8.93
3.47 m
3.31 m
3.68, 3.65 m, m
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Hz, and chemical shifts were given on a d (ppm) scale.
UPLC–TOFMS (Waters WatersÒXevoTM QTof MS) was
performed using column C18 (2.1 mm u 9 100 mm l,
Waters). GC–MS (Shimadzu GCMS-QP 5050A) was per-
formed using a DB-5 MS column (0.25 mm u 9 30 ml,
J&W Scientific). Column chromatography was performed
with Sephadex LH-20 (18–111 lm, GE Healthcare). IR
spectra were taken on a Perkin Elmer Spectrum 100 FT-IR.
UV spectra were recorded on a Simadzu SPD-M20A diode
array detector. Optical rotations were measured on a JASCO
P-2300.
300
250
200
150
100
50
270
melanogenesis activity (%)
cell viability (%)
Materials
The sample was collected from Samarinda, Indonesia in
2006. The sample and the voucher specimen were depos-
ited in Herbarium Wanariset, Samboja, East Kalimantan,
Indonesia, identification no. WAN0017365
100 100
100
96
93
Extraction and fractionation of H. zeylanica
root powder
H. zeylanica root powder c. 100 g was extracted with 50 %
ethanol. The 50 % ethanol extract was separated with
Sephadex LH-20 gel column (25 mm u 9 820 mm l)
chromatography eluting with the solvent (water:
MeOH = 1:1 v/v) to obtain compound 1 (yellow powder
52.5 mg) and 2 (yellow powder 771 mg).
0
control
compound 1
10µM
compound 2
Tyrosinase activity assay
Fig. 2 Melanogenesis activity and cell viability of compounds 1 and
2. Black bars melanogenesis activity of B16 melanoma cells treated
with samples. White bars cell viability of B16 melanoma cells treated
with samples
The tyrosinase activity assay was performed based on
Batubara et al. [13]. The sample (70 ll) was placed in a
96-well plate. Tyrosinase 30 ll (333 U/ml in phosphate
buffer 50 mM pH 6.5) and 110 ll of substrates (L-tyrosine
2 mM or L-DOPA 2 mM) were added. After incubation at
37 °C for 30 min, the absorbance at 510 nm was measured
using a microplate reader. IC50 is expressed as the con-
centration of inhibitor showing 50 % inhibition.
Conclusions
In this study, two novel quercetin glucosides were isolated
from H. zeylanica root extract. Of them, compound 1
showed high melanogenesis enhancement activity, and
may also enhance the activity or expression of MITF by
regulating kinase in B16 melanoma cells. Moreover, the
glucoses connecting quercetin C40 may play an important
role in melanogenesis stimulation activity.
Cell culture
Murine melanoma B16-F0 cells (DS Pharma Biomedical,
USA) grown in DMEM medium supplemented with 10 %
fetal bovine serum and 1 % penicillin/streptomycin were
cultured at 37 °C in a humidified atmosphere of 5 % CO2.
Experimental
Measurement of cellular melanin contents
General
Measurement of cellular melanin content was performed
according to the method of Arung et al. [14]. In brief,
confluent cultures of B16 melanoma cells were rinsed in
phosphate-buffered saline (PBS) and removed using
1H and 13C NMR spectra were recorded in methanol-d4 with
Bruker Biospin AVANCE III 800 MHz NMR and JEOL
EC600 MHz NMR. Coupling constants were expressed in
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J Nat Med (2013) 67:369–374
373
140
control cells treated with the solvent DMSO/water without
sample analyte.
L-tyrosin
L-DOPA
117
115
120
Identification of compounds 1 and 2
Compounds 1 and 2 were identified by 1H NMR, 13C
100 100
100
99
98
1
1
NMR, H H COSY, HMQC, HMBC and UPLC-TOFMS.
Methanol-d4 was used as the NMR solvent. NMR mea-
surements were performed by using Bruker Biospin
AVANCE III 800 MHz NMR and JEOL EC600 MHz
NMR. UPLC-TOFMS (Waters WatersÒXevoTM QTof MS)
was performed using column C18 (2.1 mm u 9 100 mm l)
with MeOH/water = 5/95 (0 min), 100/0 (10 min), 100/0
(13 min) as eluent. The data were collected in negative
ionization mode. The capillary voltage was 3.0 kV. Cone
and desolvation gas flow rates were set at 50 and 1000 L/h,
respectively, and the source and desolvation temperature
was 150 and 500 °C, respectively.
80
60
40
20
The NMR data of the compounds isolated from H. ze-
ylanica roots are shown in Tables 1 and 2.
Compound 1: yellow powder, UPLC–TOFMS ES-
:
[M–1]- 787.2029, 625.1410, 463.0890, 301.0372 (m/z).
0
control
compound 1
compound 2
20
: 207, 265, 345 (nm). ½aꢁD : -33.4° (c = 0.84,
MeOH
max
UV k
10µM
MeOH:H2O 1:1 v/v). IR (KBr): 3402, 1655, 1612, 1499,
1457, 1365, 1260, 1205, 1072 (cm-1).
Fig. 3 Tyrosinase activity of compounds 1 and 2. Black bars
tyrosinase activity using L-tyrosine as substrate. White bars tyrosinase
activity using L-DOPA as substrate
Compound 2: yellow powder, UPLC–TOFMS ES-
:
[M–1]- 949.2466 (m/z).
MeOH
20
UV k
: 207, 265, 342 (nm). ½aꢁD : -41.0° (c = 0.83,
max
0.25 % trypsin/EDTA. The cells were placed in 10-cm
Petri dishes (1.0 9 105 cells/dish) and allowed to adhere at
37 °C for 24 h. After adding samples, cells were incubated
for 72 h and then washed with PBS, followed by lysis in
200 ll of 2 M NaOH with 40 min heating at 80 °C to
solubilize the melanin. The resulting lysate (150 ll) was
placed in a 96-well microplate, and the absorbance was
measured at 405 nm with a microplate reader. Each
experiment was repeated twice. Enhancement of melanine
production is expressed as percentage of that of control
cells treated with the solvent DMSO/water without sample
materials.
H2O). IR (KBr): 3402, 1642, 1615, 1519, 1466, 1343,
1312, 1162, 1087 (cm-1).
Acid hydrolysis
Acid hydrolysis was performed according to the previous
method [15]: 5 ml of 1 N HCl was added to 10 mg of com-
pound 1, and the mixture was stirred at 80 °C for 4 h. After
cooling down to room temperature, the solution was parti-
tioned between H2O and EtOAc. The H2O layer was freeze-
dried, and the EtOAc layer was analyzed by JEOL
EC600 MHz NMR. The freeze-dried product from the H2O
layer was dissolved in 1-(trimethylsilyl)-imidazole 0.4 ml and
pyridine5.0 ml to trimethylsilylate. Thesolutionwas stirred at
60 °Cfor5 min.AfterdryingthesolutioninastreamofN2, the
residue was partitioned between H2O and CHCl3. The CHCl3
layer was analysed by GC–MS (Shimadzu GCMS-QP 5050A)
using a DB-5 MS column (J&W Scientific 0.25 mm u 9
30 ml). Temperatures of the injector and detector were
250 °C. The temperature gradient of the column oven started
at 80 °C for 2 min and increased up to 250 °C over a period of
9 min. ThehydrolysisofstandardmonosaccharidesD-glucose,
D-galactose, D-arabinose and L-rhamnose were performed by
the same method as with compound 1.
Cell viability
Cell viability was determined using a hemocytometer
(Erma, Tokyo, Japan). B16 cells were cultured and samples
added as described in ‘‘Extraction and fractionation of
H. zeylanica root powder’’. After 72 h incubation, B16
melanoma cells were removed using 0.25 % trypsin/EDTA
solution, and then 10 ll trypan blue solution was added to
200 ll of the solution in a 1.5-ml tube. The cell number of
10 ll of mixed solution was counted in the hemocytometer.
Each experiment was repeated twice. Enhancement of
melanine production is expressed as percentage of that of
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