Isolation of tyrosinase and melanogenesis inhibitory flavonoids from Juniperus chinensis fruits

Article abstract of DOI:10.1080/09168451.2018.1511367

A new biflavonoid, amentoflavone-7-O-β-D-glucoside, and thirteen known flavonoids were isolated from the fruits of Juniperus chinensis using a bioactivity-guided method and their tyrosinase inhibitory effects were tested using a mushroom tyrosinase bioassay. Two isolates, hypolaetin-7-O-β-D-glucoside and quercetin-7-O-α-L-rhamnoside, were found to reduce tyrosinase activity at a concentration of 50 μM. Quercetin-7-O-α-L-rhamnoside attenuated cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells. Molecular docking simulation revealed that quercetin-7-O-α-L-rhamnoside inhibits tyrosinase activity by hydrogen bonding with residues His85, His244, Thr261, and Gly281 of tyrosinase.

Full text of DOI:10.1080/09168451.2018.1511367

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Isolation of tyrosinase and melanogenesis
inhibitory flavonoids from Juniperus chinensis fruits
Sang-a Park, Jonghwan Jegal, Ki Wung Chung, Hee Jin Jung, Sang Gyun Noh,
Hae Young Chung, Jongmin Ahn, Jinwoong Kim & Min Hye Yang
To cite this article: Sang-a Park, Jonghwan Jegal, Ki Wung Chung, Hee Jin Jung, Sang Gyun
Noh, Hae Young Chung, Jongmin Ahn, Jinwoong Kim & Min Hye Yang (2018): Isolation of
tyrosinase and melanogenesis inhibitory flavonoids from Juniperusꢀchinensis fruits, Bioscience,
Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2018.1511367
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2018.1511367
Isolation of tyrosinase and melanogenesis inhibitory flavonoids from
Juniperus chinensis fruits
Sang-a Parka*, Jonghwan Jegala*, Ki Wung Chunga, Hee Jin Junga, Sang Gyun Noha, Hae Young Chunga,
Jongmin Ahnb, Jinwoong Kimb and Min Hye Yanga
b
aCollege of Pharmacy, Pusan National University, Busan, South Korea; College of Pharmacy and Research Institute of Pharmaceutical
Sciences, Seoul National University, Seoul, South Korea
ABSTRACT
ARTICLE HISTORY
Received 17 July 2018
Accepted 1 August 2018
A new biflavonoid, amentoflavone-7-O-β-D-glucoside, and thirteen known flavonoids were
isolated from the fruits of Juniperus chinensis using a bioactivity-guided method and their
tyrosinase inhibitory effects were tested using a mushroom tyrosinase bioassay. Two isolates,
hypolaetin-7-O-β-D-glucoside and quercetin-7-O-α-L-rhamnoside, were found to reduce tyrosi-
nase activity at a concentration of 50 μM. Quercetin-7-O-α-L-rhamnoside attenuated cellular
tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells.
Molecular docking simulation revealed that quercetin-7-O-α-L-rhamnoside inhibits tyrosinase
activity by hydrogen bonding with residues His85, His244, Thr261, and Gly281 of tyrosinase.
KEYWORDS
Juniperus chinensis fruit;
amentoflavone-7-O-β-D-
glucoside; quercetin-7-O-α-
L-rhamnoside; tyrosinase;
melanogenesis
Abbreviations: EtOH, ethanol; CH2Cl2, dichloromethane; EtOAc, ethylacetate; n-BuOH,
n-butanol; MeOH, metanol; CHCl3,chloroform; DMSO, dimethylsulfoxide; DMEM, Dulbecco’s
modified Eagle’s medium; FBS, fetal bovine serum; α-MSH, α-melanocyte stimulating hor-
mone; L-DOPA, L-3, 4-dihydroxyphenylalanine
Visible skin pigmentation primarily depends on the func-
widely distributed in Korea, China, and Japan [9,10]. The
heartwood of J. chinensis has been traditionally used to
treat the common cold and inflammatory diseases, such
as, urinary infections and rheumatic arthritis, whereas its
fruit is known to treat convulsion, hyperhidrosis, and
hepatitis [11,12]. Analyses of the chemical compositions
of Juniperus fruits have shown that phenolics, including
phenolic acids and flavonoids, constitute the main class of
non-volatile metabolites [13,14], and natural phenolics
that effectively inhibit tyrosinase are of interest to those
designing and developing skin-whitening cosmetics
[15,16]. On the other hand, the flavonoids are naturally
occurring antioxidants, and are also considered poten-
tially useful skin-depigmenting agents and tyrosinase-
inhibitors [17,18]. In the present study, we undertook to
isolate and identify flavonoids from the fruits of J. chi-
nensis with tyrosinase inhibitory activities using cellular
and non-cellular systems and to investigate the mechan-
isms involved by molecular docking.
tions of melanocytes, the pigment melanin-producing
cells that specialize in the synthesis and distribution of
melanin [1,2]. Melanin production in melanocytes is
triggered by a variety of physiological factors, such as,
melanosomal enzymes and melanogenic regulators.
Pigment production in murine melanoma cells is mainly
regulated by the enzymatic components of melanosomes,
such as, tyrosinase, tyrosinase-related protein 1, and
dopachrome tautomerase [3]. Tyrosinase is a copper-
dependent, rate-limiting factor that catalyzes melanin
synthesis in melanocytes [4], which when excessive can
cause pathologic and esthetic problems, such as, freckles,
age spots, and melisma [5]. Diverse skin-whitening
agents like kojic acid and hydroquinone, act as strong
tyrosinase inhibitors, but their topical toxicities and sto-
rage stabilities are topics of concern [6,7]. Natural sub-
stances are widely used to treatment skin problems
because of their proven safeties and therapeutic proper-
ties, which including anti-inflammatory, antimicrobial,
and cell-stimulating effects [8]. Thus, plant resources
including extracts, pure compounds, and phytochemical
combinations continue to attract the interests of those
seeking to find new ways of preventing and treating
conditions related to skin hyperpigmentation.
Materials and methods
General
1H and 13C NMR, COSY, HMQC, HMBC, and NOESY
spectral data were obtained using an Agilent
Superconducting FT-NMR 400 or 500 MHz
Juniperus chinensis L. (Cupressaceae, common name
Chinese juniper) is an evergreen coniferous tree that is
CONTACT Min Hye Yang
mhyang@pusan.ac.kr
*These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
© 2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
S.-A. PARK ET AL.
Spectrometer. HR-ESI mass spectra were recorded on
an Agilent Technologies, 6530 Accurate-Mass Q-TOF
LC/MS. The HPLC unit (Shimadzu, Japan) was
equipped with a pump (Model LC-20AT), an UV detec-
tor (Model SPD-20A) at 254 nm, and a data station
(Model CBM-20A). Silica gel (230–400 mesh, Merck,
Germany) and Sephadex LH-20 gel (25–100 μM mesh,
Pharmacia, Sweden) were used for column chromato-
graphy. L-tyrosine, mushroom tyrosinase, kojic acid, α-
MSH (α-melanocyte stimulating hormone), and MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) were purchased from Sigma (St. Louis,
MO, USA).
JChE8 (CH2Cl2:MeOH = 5:1, 798.1 mg) was fractio-
nated into thirteen subfractions (Frs. JChE8-
1~ JChEN8-13) by Sephadex LH-20 (MeOH).
Subfraction JChE8-6 (178.3 mg) was separated by RP
HPLC (Watchers 120 ODS-BP, S-10 μm, 150 × 10 mm;
detection, UV at 254 nm; flow rate, 2 mL/min) and
eluted with a MeOH-H2O gradient (4:6 → 9:1,
40 min) to yield compound 2 (1.2 mg, tR 19 min) and
compound 1 (12.5 mg, tR 20 min). Subfraction JChE8-8
(14.1 mg) was subjected to RP HPLC (Watchers 120
ODS-BP, S-10 μm, 150 × 10 mm; detection, UV at
254 nm; flow rate, 2 mL/min) and eluted with a
MeOH-H2O gradient (3:7 → 9:1, 40 min) to yield
compound 12 (3.6 mg, tR 29 min). The subfraction
JChE8-9 (4.9 mg) was also subjected to RP HPLC
(Watchers 120 ODS-BP, S-10 μm, 150 × 10 mm; detec-
tion, UV at 254 nm; flow rate, 2 mL/min) and eluted
with a MeOH-H2O gradient (3:7 → 9:1, 40 min) to yield
compound 9 (1.8 mg, tR 32 min).
Plant material
Fresh fruits of J. chinensis were collected from Muju
(Jeollanam-Do) in South Korea and identified by Prof.
Eun Ju Jeong at the Department of Agronomy and
Medicinal Plant Resources, Gyeongnam National
University of Science and Technology. A voucher speci-
men (PNU-0022) was deposited at the Medicinal Herb
Garden, Pusan National University.
Amentoflavone-7-o-β-d-glucoside (9)
Yellowish powder; UV (MeOH) λmax (log ε) at 336 nm
(4.06), 269 nm (4.17); IR (neat) νmax 3357, 1653, 1608,
1494 cm−1; 1H NMR (DMSO, 600 MHz) and 13C NMR
(DMSO, 150 MHz) see Table 1; HRESIMS m/z
699.1386 (calcd. 700.1428 for C36H28O15).
Extraction and isolation
Dried fruits of J. chinensis (3.9 kg) were extracted with
95% EtOH (30 L × 3) and the extract obtained was then
evaporated under reduced pressure. This extract
(824.2 g) was then suspended in distilled water (1 L)
and successively partitioned with CH2Cl2 (6 L), EtOAc
(6 L), and n-BuOH (6 L). The active EtOAc soluble
fraction (14.6 g) was chromatographed on a Silica gel
CC using CH2Cl2-MeOH (10:1 → 100% MeOH, gra-
dient system) as eluent to yield ten fractions
(JChE1~ JChE10). Fraction JChE3 (329.7 mg) was
applied to Sephadex LH-20 (MeOH), to afford com-
pound 14 (2.4 mg), fraction JChE4 (CH2Cl2:
MeOH = 10:1, 544.7 mg) yielded compound 8
(7.0 mg), and fraction JChE5 (CH2Cl2:MeOH = 10:1,
977.3 mg) yielded compound 10 (22.1 mg) and com-
pound 7 (22.6 mg), sequentially. Fraction JChE6
(CH2Cl2:MeOH = 5:1, 626.8 mg) was suspended in
MeOH and compound 13 (31.4 mg) was obtained by
filtration. The resulting filtrate of JChE6 was fractio-
nated into six subfractions (Frs. JChE6-1~ JChE6-6) by
Sephadex LH-20 (MeOH). Subfractions JChE6-4 and
JChE6-5 contained compound 3 (5.9 mg) and 11
(4.1 mg), respectively. Subfraction JChE6-3 (150.0 mg)
was subjected to RP HPLC (Watchers 120 ODS-BP,
S-10 μm, 150 × 10 mm; detection, UV at 254 nm; flow
rate, 2 mL/min) using a MeOH-H2O gradient (5:5 →
9:1 over 40 min) to yield compound 6 (53.2 mg, tR
20 min). Compound 4 (30.4 mg) was obtained as pure
powder by filtering fraction JChE7 (CH2Cl2:
MeOH = 5:1, 1306.6 mg) and compound 5 (36.5 mg)
by recrystallizing JChE7 filtrate from MeOH.. Fraction
1
Table 1. H (600 MHz) and 13C (150 MHz) NMR spectral data
of compound 9 in DMSO-d6.
Compound 9
Position
δH (J in Hz)
δC
2
3
4
5
6
7
8
163.54
103.18
182.66
161.93
99.76
165.11
95.48
6.87 (s)
6.41 (s)
6.72 (s)
9
10
1’
2’
3’
4’
5’
6’
2”
3”
4”
5”
157.62
103.66
121.34
128.27
122.08
161.59
116.45
121.34
164.13
103.18
182.60
161.59
99.37
8.12 (s)
7.01 (d, 8.0)
7.97 (d, 8.0)
6.72 (s)
6”
6.41 (s)
7”
8”
9”
10”
1’’’
2’’’ (6’’’)
3’’’ (5’’’)
4’’’
161.76
105.93
155.16
103.66
122.08
128.83
116.35
161.16
100.13
73.71
7.60 (d, 8.1)
6.64 (d, 8.2)
Glc 1
5.02 (d, 7.3)
3.33
2
3
4
5
6
3.33
3.15
3.33
3.64,
77.02
70.06
77.70
61.13

JUNIPERUS CHINENSIS FRUITS
3
Acid hydrolysis of compound 9
melanin. Absorbance at 405 nm converted to concen-
trations using a standard curve prepared with syn-
thetic melanin. Relative melanin levels were
calculated with respect to protein contents, which
were determined using BCA protein assay reagent
(Thermo Scientific, Rockford, IL, USA). Results are
presented as means of three individual experiments.
Acid hydrolysis was determined as described pre-
viously [19]. Briefly, compound 9 (1 mg) in 50%
MeOH was hydrolyzed with 2 M HCl for 3 h and
concentrated under reduced pressure. The residue
was dissolved in H2O and extracted with CHCl3.
The collected aqueous layer was concentrated and
glucose was identified by the TLC with an authentic
sample of amentoflavone using CHCl3–MeOH (10:1).
Cellular tyrosinase activity assay
B16F10 cells were plated in 6-well dishes at a density
of 2.5 × 104 cells/well, incubated with or without 0.5
μM α-MSH and 200 μM IBMX, and then treated for
48h with compounds 1 to 14 at concentrations of 0 to
50 μM. Cells were then washed and lysed in 100 μL of
50 mM sodium phosphate buffer (pH 6.5) containing
1% Triton X-100 and 0.1mM PMSF (phenylmethyl-
sulfonyl fluoride), frozen at – 80°C for 30 min,
thawed, mixed, and cellular extracts were the clarified
by centrifuging at 12,000 rpm for 30min at 4°C. In
each case an 8 μL sample of supernatant and 20 μL of
L-DOPA (2 mg/mL) were placed in a 96-well plate,
and the absorbance was read at 492 nm every 10 min
for 1h at 37°C using an ELISA reader. Final activities
were expressed as Δ OD/min.
In vitro mushroom tyrosinase assay
A mixed solution containing 170 μL of 1 mM
L-tyrosine in 50 mM phosphate buffer (pH 6.5) and
10 μL of test sample was prepared for in vitro mush-
room (Agaricus bisporus) tyrosinase experiment. The
reaction conducted by adding 20 μL of 1000 U/mL of
tyrosinase and incubating the mixture at 25°C for
30 min. DMSO (0.5%) was added as a negative con-
trol and kojic acid (50 μM) was used as a positive
control. The amount of dopachrome produced was
monitored against a blank (no tyrosinase addition) at
492 nm using a microplate reader (GENios; Tecan
Instruments, Salzburg, Austria).
Cell culture. Mouse melanoma B16F10 cells were
obtained from the Korea Cell Line Bank, cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% FBS and 1% penicillin/streptomycin,
and then incubated at 37°C in a 5% CO2 atmosphere.
Docking simulation of tyrosinase and compound 5
AutoDock 4.2 was used for the in silico protein-ligand
docking simulation. Successful binding was obtained
between a protein and a ligand. The 3D structure of
tyrosinase used was as previously described for the
tyrosinase of Agaricus bisporus (PDB ID: 2Y9X), and
the defined binding site of tyrosine was used as the
docking pocket. Docking simulations were performed
between tyrosinase and compound 5 or kojic acid. To
prepare compounds for docking simulation, (1) 2D
structures were converted into 3D structures, (2)
charges were calculated, and (3) hydrogen atoms
were added using the ChemOffice program (http://
www.cambridgesoft.com). LigandScout 3.0 was used
to predict possible residues responsible for hydrogen
bonding between the 14 compounds and tyrosinase.
Cell viability
Cell viabilities were assessed using the EZ-Cytox
assay. Cells were seeded at 5 × 103 cells/well in
DMEM containing 10% FBS and incubated for 24 h.
Cells were then treated with 1–100 μM one of the 14
compounds for 24 h or 48 h. Cells treated with 0.1%
DMSO were used as controls. After treatments, 10 μL
of EZ-Cytox solution was added to each well, and
cells were incubated for additional 2 h. Well absor-
bance were measured at 450 nm using an ELISA
reader. All determinations were performed in tripli-
cate and results were averaged.
Results and discussion
Melanin content assay
Preliminary screening of plant materials for anti-tyr-
osinase activity showed the 95% EtOH extract of J.
chinensis fruits inhibited mushroom tyrosinase activ-
ity. The 95% EtOH extract of J. chinensis fruit was
suspended in distilled water and partitioned sequen-
tially against CH2Cl2, EtOAc, and n-BuOH. The
active EtOAc soluble fraction (14.6 g) was subjected
to silica gel column chromatography, Sephadex LH-
20 column chromatography, and RP HPLC to yield a
novel biflavonoid, amentoflavone 7-O-β-D-glucoside
(9) and 13 known flavonoids; apigenin 7-O-β-D-
B16F10 cells were seeded in 6-well culture plates at
2.5 × 104 cells/well in a 5% CO2 atmosphere and
cultured for 24 h. To determine the inhibitory effect
of MHY773 on melanogenesis, fresh medium was
replaced with medium containing one of the 14 com-
pounds at 10, 20, or 50 µM or with 10 μM kojic acid
(positive control), and 0.5 μM α-MSH and 200 μM
IBMX for 48 h. Cells were then washed twice with
PBS, centrifuged, disrupted in 100 μL of 1 N NaOH,
incubated at 60°C for 1 h, and mixed to solubilize the

4
S.-A. PARK ET AL.
glucoside (1) [20], luteolin 7-O-β-D-glucoside (2)
[21], hypolaetin 7-O-β-D-xyloside (3) [22], isoscutel-
larein 7-O-β-D-xyloside (4) [22], quercetin 7-O-α-L-
rhamnoside (5) [23], kaempferol 3-O-α-L-rhamno-
side (6) [24], amentoflavone (7) [25], 7-O-methyla-
mentoflavone (8) [26], cupressuflavone (10) [27],
moghatin (11) [27], cupressuflavone 4′-O-β-D-gluco-
side (12) [28], hinokiflavone (13) [25], and isocryp-
tomerin (14) [25], (Figure 1). The structures of these
14 compounds were elucidated by 2D-NMR and
HR-MS.
Compound 9 was obtained as a yellowish pow-
der, [α]D – 66.9 (c 0.1, MeOH) of molecular for-
mula C36H28O15, as determined by a negative-ion
HRESIMS peak at m/z 699.1386. The MS fragmen-
tation pattern of 9 showed fragments characteristic
of amentoflavone [29]. Acid hydrolysis of 9
resulted in the isolation of amentoflavone (7) and
glucose, both of which were identified by direct
TLC comparisons with authentic materials. The IR
spectrum of compound 9 displayed three significant
absorption bands at 3357, 1653, and 1494 cm−1,
which were indicative of hydroxyl and carbonyl
groups and an aromatic ring, respectively. UV
spectrum maxima at 336 nm (log ε 4.06) and
269 nm (log ε 4.17) indicated that 9 was a fla-
vone-like compound. MS, IR, and UV data indi-
cated
9 contained a biflavone monoglycoside
moiety containing a hexosyl residue.
1
The H NMR spectrum of compound 9 (Table 1)
revealed the presence of 1,4,5-trisubstituted benzene
ring [δ 8.12 (1H, s, H-2′), 7.01 (1H, d, J = 8.6 Hz,
H-5′), and 7.97 (1H, d, J = 8.6 Hz, H-6′)], ortho-
coupled protons were assignable to two p-substituted
phenyl moieties [δ 7.60 (2H, d, J = 8.8 Hz, H-2′′′ and
H-6′′′) and 6.64 (2H, d, J = 8.8 Hz, H-3′′′ and H-5′′′)],
and three aromatic singlets [δ 6.87 (1H, H-3), 6.41
(2H, H-6 and 6′′), and 6.72 (2H, H-3′′ and H-8)].
Detailed analyses of HMQC and HMBC spectra
1
allowed all H- and 13C-NMR signals to be assigned
Figure 1. Structures of flavonoids isolated from J. chinensis fruit extract.

JUNIPERUS CHINENSIS FRUITS
5
Table 2. Inhibitory effects of flavonoids isolated from J.
chinensis fruit on mushroom tyrosinase.
to glucosyl and aglycone moieties. HMBC correla-
tions between H-8 and C-6, H-2′ and C-6′, H-6′′
and C-5′′/C-7′′′, and H-3′′/C-3′′′ and C-1′′′ were con-
sistent with 3′,8″-coupled dimers of apigenin (5,7,4′-
trihydroxyflavone), indicating amentoflavone was the
common aglycone in compound 9. Furthermore,
NOESY correlations between anomeric H and H-6
and H-8 (Figure 2) confirmed a β-D-glucopyranosyl
moiety at OH-7 in amentoflavone. Therefore, com-
pound 9 was designated amentoflavone-7-O-β-D-
glucoside.
The tyrosinase inhibitory effects of all compounds
from J. chinensis fruits were investigated using non-cel-
lular mushroom tyrosinase. Of the 14 flavonoids,
hypolaetin-7-O-β-D-glucoside (3) and quercetin-7-O-α-
L-rhamnoside (5) effectively reduced mushroom tyrosi-
nase activity to < 50% at a concentration of 50 μM
(Table 2). Compound 3 was isolated from J. chinensis
for the first time and most potently inhibited mushroom
tyrosinase, which concurs with our previous report on
the activity of hypolaetin-7-O-β-D-glucoside isolated
from J. communis [30]. Quercetin-7-O-α-L-rhamnoside
(5) effectively inhibited mushroom tyrosinase with an
IC50 value of 56.75 μM using tyrosine as a substrate
(Figure 3). It has been already reported that quercetin,
Relative enzyme
activity (%)a
Compounds
50 μM
Apigenin-7-O-β-D-glucoside (1)
Luteolin-7-O-β-D-glucoside (2)
Hypolaetin-7-O-β-D-xyloside (3)
Isoscutellarein-7-O-β-D-xyloside (4)
Quercetin-7-O-α-L-rhamnoside (5)
Kaempferol-3-O-α-L-rhamnoside (6)
Amentoflavone (7)
7-O-Methylamentoflavone (8)
Amentoflavone-7-O-β-D-glucoside (9)
Cupressuflavone (10)
75.86 1.07***
71.96 2.27***
26.71 0.71***
74.58 3.70***
46.81 2.47***
81.59 6.68*
83.40 5.21*
74.46 1.84***
85.86 6.98*
75.69 6.01**
88.81 0.12**
79.54 5.38**
65.46 0.10***
76.80 2.15***
35.18 2.38***
Moghatin (11)
Cupressuflavone-4′-O-β-D-glucoside (12)
Hinokiflavone (13)
Isocryptomerin (14)
Kojic acidb
a Relative enzyme activities were calculated as percentages of the activity
of the control.
b
Positive control.
an aglycone form of compound 5, also exhibited a potent
inhibitory effect on dopa oxidase activity of mushroom
tyrosinase [31]. This study suggested both quercetin and
its 7-rhamnoside derivate can be utilized in skin-light-
ening attempts as strong tyrosinase inhibitors. The depig-
menting effect of compound 5 was investigated using
B16F10 melanoma cells. Both tyrosinase activity and
melanin content in B16F10 cells were markedly increased
by 8.2-fold and 1.7-fold of the vehicle control by co-
treating cells with compound 5 and α-MSH/IBMX. As
shown in Figure 4(a), 5 down-regulated tyrosinase activ-
ity in both cell types at 50 μM in vitro. α-MSH and IBMX-
induced melanin production was also reduced in B16F10
cells by 92.8% by 5 at 10 μM (Figure 4(b)). Structural and
molecular docking simulations showed the best ranked
energy docking result with lowest binding energy (−6.4
kcal/mol) (Figure 5(a)). The tyrosinase docking abilities
of compound 5 and kojic acid (−5.7 kcal/mol, positive
control) were similar. Compound 5 was found to bind to
Figure 2. Selected HMBC and NOESY correlations of amento-
flavone-7-O-β-D-glucoside (9).
Figure 3. Dose-dependent inhibition of mushroom tyrosinase activity by quercetin-7-O-α-L-rhamnoside.
Results are presented as the means standard errors of triplicate assays.

6
S.-A. PARK ET AL.
Figure 4. Inhibitory effects of quercetin-7-O-α-L-rhamnoside from J. chinensis fruit on cellular tyrosinase activities (a) and
melanin contents (b).
Results are presented as means SDs of triplicate experiments; #p < 0.001 vs. the control and *p < 0.05 and ***p < 0.001 vs. α-MSH and IBMX-treated
cells.
Figure 5. In silico molecular docking with tyrosinase.
(a) Computational docking simulation images for tyrosinase with quercetin-7-O-α-L-rhamnoside and kojic acid. The geometry of whole mushroom
tyrosinase is shown on the left. The active site of tyrosinase is magnified on the right. The two brown spheres indicate copper ions at the active site.
Cyan denotes tyrosine binding sites predicted by Autodock, and magenta and yellow represent kojic acid and quercetin-7-O-α-L-rhamnoside binding
sites, respectively, as predicted by Autodock. Amino acids in the cavity are shown as stick models. (b) Possible hydrogen-bonding interactions of
compound 5.

JUNIPERUS CHINENSIS FRUITS
7
Table 3. Interaction and Autodock scores of quercetin-7-O-α-L-rhamnoside for tyrosinase.
Compound
Docking score kcal/mol
Interacting residue
Kojic acid
Quercetin-7-O-α-L-rhamnoside
−5.7
−6.4
HIS259, HIS263, and MET280
HIS85, HIS244, GLY281, and VAL283
His85 near the active site of tyrosinase mainly by hydro-
gen bonding (Table 3 and Figure 5(b)). In silico docking
results suggested that compound 5 (quercetin-7-O-α-L-
rhamnoside) from J. chinensis competitively bound to the
active site of tyrosinase.
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In conclusion, bioactivity-guided isolation of the
95% ethanol extract of the fruits of J. chinensis
afforded a new biflavonoid amentoflavone-7-O-β-D-
glucoside and thirteen known compounds. Cell-free
and cell-based assay systems and molecular docking
indicated quercetin-7-O-α-L-rhamnoside from J. chi-
nensis is a promising candidate inhibitor of tyrosinase
and melanin formation for cosmetic applications, and
that J. chinensis fruit extracts or quercetin-7-O-α-L-
rhamnoside might be useful for the treatment of skin
pigmentary disorders.
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Flavonoids and biflavonoids in Tuscan berries of
Juniperus communis L.: detection and quantitation
Authors contribution
S. Park, J. Jegal, and M.H. Yang conceived and designed the
research. S. Park, J. Jegal, K.W. Chung, H.J. Jung, S.G. Noh,
and J. Ahn performed experiments. S. Park, J. Jegal, H.Y.
Chung, J. Kim and M.H. Yang drafted manuscript. All
authors read and approved the final manuscript.
by HPLC/DAD/ESI/MS.
J Agric Food Chem.
2007;55:6596–6602.
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lation of traditional and folk medicine: Northeast
Asia–part II. Vol. 2. Singapore: World Scientific;
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cea Labill. berries from Turkey. Food Chem Toxicol.
2011;49:2600–2608.
Disclosure statement
No potential conflict of interest was reported by the
authors.
[15] Lee SY, Baek N, Nam TG. Natural, semisynthetic
and synthetic tyrosinase inhibitors. J Enzyme Inhib
Med Chem. 2016;31:1–13.
Funding
[16] Kim YJ, Uyama H. Tyrosinase inhibitors from nat-
ural and synthetic sources: structure, inhibition
mechanism and perspective for the future. Cell Mol
Life Sci. 2005;62:1707–1723.
[17] Pandey KB, Rizvi SI. Plant polyphenols as dietary
antioxidants in human health and disease. Oxid Med
Cell Longev. 2009;2:270–278.
This work was supported by the Korean National
Research Foundation (NRF) funded by the Korean gov-
ernment
(MSIP)
(grant
nos.
NRF-2016R1C1B
2007694 and NRF-2016K1A1A8A01938595) and the
“Cooperative Research Program for Agriculture Science
and Technology Development (Project No. PJ01282301)”
Rural Development Administration, Republic of Korea.
[18] Miller NJ, Ruiz-Larrea MB. Flavonoids and other
plant phenols in the diet: their significance as anti-
oxidants. J Nutr Environ Med. 2002;12:39–51.
[19] Ma LY, Ma SC, Wei F, et al. Uncinoside A and B,
two new antiviral chromone glycosides from
Selaginella uncinata. Chem Pharm Bull.
2003;51:1264–1267.
[20] Zapesochnaya GG, Pangarova TT. Hypolaetin 7-glu-
coside from Caryopteris monolica. Chem Nat
Compd. 1973;9:521.
[21] Gohari AR, Ebrahimi H, Saeidnia S, et al Flavones
and flavone glycosides from Salvia macrosiphon
Boiss. Iran J Pharm Res. 2011;10:247–251.
[22] Nakanishi T, Iida N, Inatomi Y, et al Neolignan and
flavonoid glycosides in Juniperus communis var.
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