Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene

Article abstract of DOI:10.1080/09168451.2016.1240606

The stilbene compound resveratrol was glycosylated to give its 4′-O-β-D-glucoside as the major product in addition to its 3-O-β-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4′-O-β-D-glucoside, and converted pinostilbene to its 4′-O-β-D-glucoside as a major product and its 3-O-β-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-β-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-β-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.

Full text of DOI:10.1080/09168451.2016.1240606

Bioscience, Biotechnology, and Biochemistry
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Synthesis, oxygen radical absorbance capacity,
and tyrosinase inhibitory activity of glycosides of
resveratrol, pterostilbene, and pinostilbene
Daisuke Uesugi, Hiroki Hamada, Kei Shimoda, Naoji Kubota, Shin-ichi Ozaki
& Naoki Nagatani
To cite this article: Daisuke Uesugi, Hiroki Hamada, Kei Shimoda, Naoji Kubota, Shin-ichi
Ozaki & Naoki Nagatani (2016): Synthesis, oxygen radical absorbance capacity, and tyrosinase
inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene, Bioscience,
Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2016.1240606
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Date: 31 October 2016, At: 04:19

Bioscience, Biotechnology, and Biochemistry, 2016
Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory
activity of glycosides of resveratrol, pterostilbene, and pinostilbene
Daisuke Uesugi1, Hiroki Hamada1,*, Kei Shimoda2, Naoji Kubota2, Shin-ichi Ozaki3 and
Naoki Nagatani4
2
1Faculty of Science, Department of Life Science, Okayama University of Science, Okayama, Japan; Faculty of
3
Medicine, Department of Biomedical Chemistry, Oita University, Oita, Japan; Faculty of Agriculture, Department of
4
Biological Sciences, Yamaguchi University, Yamaguchi, Japan; Department of Applied Chemistry, Graduate School
of Engineering, Okayama University of Science, Okayama, Japan
Received July 20, 2016; accepted September 2, 2016
http://dx.doi.org/10.1080/09168451.2016.1240606
The stilbene compound resveratrol was glycosy-
lated to give its 4′-O-β-D-glucoside as the major
product in addition to its 3-O-β-D-glucoside by a
plant glucosyltransferase from Phytolacca americana
expressed in recombinant Escherichia coli. This
enzyme transformed pterostilbene to its 4′-O-β-D-glu-
coside, and converted pinostilbene to its 4′-O-β-D-glu-
coside as a major product and its 3-O-β-D-glucoside
as a minor product. An analysis of antioxidant capac-
ity showed that the above stilbene glycosides had
lower oxygen radical absorbance capacity (ORAC)
values than those of the corresponding stilbene agly-
cones. The 3-O-β-D-glucoside of resveratrol showed
the highest ORAC value among the stilbene glyco-
sides tested, and pinostilbene had the highest value
among the stilbene compounds. The tyrosinase inhibi-
tory activities of the stilbene aglycones were improved
by glycosylation; the stilbene glycosides had higher
activities than the stilbene aglycones. Resveratrol
3-O-β-D-glucoside had the highest tyrosinase inhibi-
tory activity among the stilbene compounds tested.
hand, the water-insolubility of resveratrol and its sensi-
tivity to air and light represent a serious impediment to
its bioavailability in the formulation of functional
foods.
Plant-cultured cells have been studied with respect to
biotransformation reactions because of their biochemi-
cal potential to produce specific secondary metabolites
such as flavors, pigments, and agrochemicals. The reac-
tions that are associated with the biotransformation of
organic compounds by cultured plant cells include oxi-
dation, reduction, hydroxylation, esterification, methyla-
tion, isomerization, hydrolysis, and glycosylation.
Particularly, glycosylation by cultured plant cells has
been the subject of increasing attention, since a one-
step enzymatic glycosylation by cultured plant cells is
more convenient than chemical glycosylation, which
can require tedious steps such as protection-deprotec-
tion of the hydroxyl groups of sugar moieties. Addi-
tionally, the glycosylation of bioactive compounds can
enhance their water-solubility, physicochemical stabil-
ity, intestinal absorption, and biological half-life, and
improve their bio- and pharmacological properties.11–18)
We recently reported that cultured cells of Phytolacca
americana had high potential for the glucosylation of
resveratrol, and that the glucosyltransferase from P.
americana expressed in recombinant Escherichia coli
catalyzed the glucosylation of stilbene compounds such
as resveratrol.19)
Key words: resveratrol; pterostilbene; pinostilbene;
glycoside
Resveratrol (trans-3,5,4′-trihydroxystilbene) is
a
natural polyphenol that is classified as a stilbene. It is
found in fruits, such as grapes and berries, peanuts, and
some medicinal plants, in which it is produced under
conditions of environmental stress. It exhibits anticar-
cinogenic, anti-inflammatory, and anti-aging activities.
These activities of resveratrol have been attributed to
its antioxidant properties, which help to control the
intracellular redox balance by inhibiting the formation
of reactive oxygen species (ROS).1–10) On the other
Recently, an assay to measure hydrophilic antioxi-
dant activity against ROS was developed based on the
detection of chemical damage to β-phycoerythrins
(PEs) through the decrease in their fluorescence
emission. In this assay, PEs are exposed to either
peroxyl radicals, which are generated by the thermal
decomposition of 2,2′-azobis(2-methylpropionamidine)
dihydrochloride (AAPH), or hydroxy radicals, which
*Corresponding author. Email: hamada@dls.ous.ac.jp
Abbreviations: ROS, reactive oxygen species; PE, phycoerythrin; AAPH, 2,2′-azobis(2-methylpropionamidine) dihydrochloride; AUC, area under
the curve; ORAC, oxygen radical absorbance capacity; FABMS, fast atom bombardment mass spectroscopy; NMR, nuclear magnetic resonance;
HMQC, heteronuclear multiple quantum correlation; HMBC, heteronuclear multiple bond correlation; DMSO, dimethyl sulfoxide; TMS, tetram-
ethylsilane; MS, Murashige and Skoog; fr. wt, fresh weight; EtOAc, ethyl acetate; MeOH, methanol; BuOH, butanol; HPLC, high performance liq-
uid chromatography.
© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
D. Uesugi et al.
are generated at copper-binding sites on macro-
molecules in the presence of ascorbate and Cu2+. The
decay in the fluorescence of PE follows a zero-order
kinetic in the presence of peroxyl radicals, and the
antioxidant capacity of a compound is related to the
increase observed in the lag phase and/or the decrease
in the rate constant. The protective effect of an antioxi-
dant is evaluated by assessing the area under the fluo-
rescence decay curve (area under the curve (AUC)) of
the sample using β-phycoerythrin as a fluorescent probe
and AAPH as a free radical initiatorin comparison to
that of the blank, where no antioxidant is present. The
results are expressed as Trolox equivalents. This
method is called the oxygen radical absorbance capac-
ity (ORAC) assay.20–22) There have been few reports
on the ORAC of stilbene compounds.
Tyrosinase is involved in melanoma biosynthesis and
the abnormal accumulation of melanin pigments lead-
ing to hyperpigmentation disorders that can be treated
with depigmenting agents. Little attention has been
paid to the tyrosinase inhibitory effects of stilbenes and
their glycosides.
We report here the syntheses of glucosides of
stilbene compounds, i.e. resveratrol, pterostilbene, and
pinostilbene, by biocatalytic glycosylation using
glucosyltransferase from P. americana expressed in
recombinant E. coli. In addition, we individually
examined the ORAC values and tyrosinase inhibitory
activities of the glycosides of resveratrol, pterostilbene,
and pinostilbene.
was dialyzed with 50 mM Tris–HCl (pH 7.2) contain-
ing 5 mM dithiothreitol, and stored at –80 °C.
Glucosylation reactions were performed at 35 °C for
24 h in 5 mL of 50 mM potassium phosphate buffer
(pH 7.2) supplemented with 50 μM substrate, 100 μM
UDP-glucose, and 5 μM enzyme. The incubation was
stopped by adding 1.5% trifluoroacetic acid; and the
reaction mixture was analyzed by high performance liq-
uid chromatography (HPLC). The reaction mixture was
extracted with n-BuOH. The n-BuOH fraction was con-
centrated by evaporation and the residue was dissolved
in water. The water fraction was applied to Diaion
HP20, washed with water, and eluted with methanol.
The methanol solution was subjected to preparative
HPLC.
The NMR data of compounds 4–6 were in good
agreement with those of the corresponding glucosides
reported previously.29) The 13C NMR data of com-
pounds 7 and 8 are as follows.
Pinostilbene 3-O-β-D-glucoside (7): NMR δC
(CD3OD): 55.8 (MeO), 62.7 (C-6″), 71.6 (C-4″), 75.0
(C-2″), 78.1 (C-3″), 78.3 (C-5″), 102.6 (C-4, C-1″),
107.2 (C-6), 107.9 (C-2), 116.6 (C-3′, C-5′), 126.5
(C-7), 129.0 (C-2′, C-6′), 130.2 (C-8), 130.4 (C-1′),
141.5 (C-1), 158.7 (C-4′), 160.5 (C-3), 162.3 (C-5).
Pinostilbene 4′-O-β-D-glucoside (8): NMR δC
(CD3OD): 55.7 (MeO), 62.5 (C-6″), 71.4 (C-4″), 74.9
(C-2″), 78.0 (C-3″), 78.2 (C-5″), 101.7 (C-4), 102.2
(C-1″), 104.5 (C-2), 106.9 (C-6), 117.9 (C-3′, C-5′),
128.5 (C-7), 128.7 (C-2′, C-6′), 129.2 (C-8), 133.2 (C-
1′), 141.0 (C-1), 158.8 (C-4′), 159.8 (C-3), 162.6 (C-5).
Materials and methods
ORAC assay. All samples were diluted to a con-
centration of 24 μM. The sample to be tested was
mixed with a fluorescent molecule (Fluorescein) and an
oxygen radical generator (AAPH). The level of fluores-
cence was measured every 2 min over a 120-min per-
iod to create fluorescence decay curves. Experimental
samples were run in the same plate as a standard
(Trolox) and control samples (Fluorescein alone and
Fluorescein plus AAPH). Samples were run in a BMG
FLUOstar Optima plate reader with 485 nm emission
and 590 nm excitation filters. Samples were run in six
different plates with 12 replicates per plate for each
compound.
Analyses.
The structures of products were deter-
mined based on the FABMS and NMR spectroscopic
1
analyses. The H- and 13C-NMR spectra were recorded
using a JNM-ECS400 spectrometer in CD3OD or
dimethyl sulfoxide (DMSO)-d6 solutions, and the
chemical shifts are expressed in δ (ppm) with reference
to TMS. The FABMS spectra were measured using a
JMS-700 MStation in CH3OH solution.
Glycosylation of resveratrol, pterostilbene, and
pinostilbene by glucosyltransferase from Phytolacca
Americana.
P. americana cells were subcultured at
four-week intervals for two months on solid Murashige
and Skoog medium (MS; 100 mL in a 300-mL conical
flask) containing 3% sucrose, 10 mM 2,4-dichlorophe-
noxyacetic acid, and 1% agar (adjusted to pH 5.7) at
25 °C.23–29) A suspension culture was started by trans-
ferring the cultured cells to 100 mL of liquid medium
in a 300-mL conical flask, and incubated on a rotary
shaker (120 rpm) at 25 °C. Prior to use in this work,
some of the callus tissue (fresh weight (fr. wt) 20 g)
was transplanted to freshly prepared MS medium
(100 mL in a 300-mL conical flask) and grown with
continuous shaking for 2 days on a rotary shaker
(120 rpm).
Tyrosinase assay.
Mushroom tyrosinase (EC
1.14.18.1) (Sigma Chemical Co.) was used for the
tyrosinase assay, with either L-DOPA or L-tyrosine as
a substrate. In spectrophotometric experiments, enzyme
activity was taken as the initial velocity (Vi) monitored
by observing dopachrome formation at 475 nm with a
UV spectrophotometer at 30 °C. All samples were dis-
solved in ethanol at 10 mM. First, 200 μL of a 2.7 mM
L-tyrosine or 5.4 mM L-DOPA aqueous solution was
mixed with 2687 μL of 0.25 M phosphate buffer (pH
6.8). Next, 100 μL of the sample solution and 13 μL of
the same phosphate buffer solution containing mush-
room tyrosinase (144 units) were added to the mixture.
The inhibitor concentration that gave a 50% loss of
activity (IC50) was obtained by fitting the experimental
data to the logistic curve.
cDNA of glucosyltransferase from P. americana
(PaGT) was cloned into pQE30, and the resulting plas-
mids were transformed into E. coli M15 cells. The
expression and purification of PaGT were performed as
described previously.19) The purified enzyme solution

Synthesis and evaluation of stilbene glucosides
3
Table 1. Oxygen radical absorbance capacity (ORAC) of
compounds 1–8.
Results and discussion
The glucosides of resveratrol, pterostilbene, and
pinostilbene were synthesized by their glucosylation
with glucosyltransferase from P. americana (PaGT),
and subjected to the ORAC assay and tyrosinase assay.
The glucosyltransferase from P. americana (PaGT) was
expressed in E. coli as described in the Materials and
methods. PaGT was purified using a His-accept column
and used as a biocatalyst. This enzyme transformed the
substrate, resveratrol (1), into two products, 4 and 5.
The chemical structures of the products were deter-
mined on the basis of their FABMS and NMR spectro-
scopic analyses. Compounds 4 and 5 were identified as
resveratrol 3-O-β-D-glucoside (4, 29%) and resveratrol
4′-O-β-D-glucoside (5, 68%). Pterostilbene (2) was
converted into compound 6 (95%), which was identi-
fied as pterostilbene 4′-O-β-D-glucoside. On the other
hand, pinostilbene (3) was glucosylated to pinostilbene
3-O-β-D-glucoside (7, 18%) and pinostilbene 4′-O-β-D-
glucoside (8, 79%) in the same enzymatic transforma-
tion (Fig. 1).
Next, the antioxidant capacities of stilbenes and their
glucosides were examined using the ORAC assay
(Table 1). Among the three stilbene aglycone com-
pounds, resveratrol showed the highest ORAC value of
5.26 Trolox equivalents/μM, followed by pinostilbene
(5.01 Trolox equivalents/μM) and pterostilbene (2.70
Trolox equivalents/μM). On the other hand, glucosyla-
tion of resveratrol decreased the ORAC value: the
ORAC value of resveratrol 3-O-β-D-glucoside was
4.01 Trolox equivalents/μM and that of resveratrol 4′-
O-β-D-glucoside was 4.89 Trolox equivalents/μM.
These resveratrol glucosides had higher ORAC values
than pterostilbene. Also, the glucosylation of pterostil-
bene lowered its ORAC value; the ORAC value of
pterostilbene 4′-O-β-D-glucoside was 0.68 Trolox
equivalents/μM. The ORAC values of pinostilbene
3-O-β-D-glucoside (1.89 Trolox equivalents/μM) and
pinostilbene 4′-O-β-D-glucoside (3.13 Trolox equiva-
lents/μM) were lower than that of pinostilbene. Vegeta-
bles and fruits contain many antioxidants, including
ORAC
Trolox equivalents/μM
Compound
1
2
3
4
5
6
7
8
5.26 0.26
2.70 0.30
5.01 0.27
4.89 0.79
4.01 0.71
0.68 0.15
3.13 0.12
1.89 0.25
Notes: The results are shown as the mean standard deviation from triplicate
experiments. The assay system for determining the ORAC value is described
in the Materials and methods.
carotenoids, tocopherols, ascorbic acid, and flavones,
and have been reported to have high antioxidant capac-
ity.20–22) Recently, stilbenes such as resveratrol, pteros-
tilbene, and piceatannol, which are the principal
polyphenols in grapes, have been reported to show
high antioxidant capacity.30) However, there have been
no reports on the antioxidant capacities of stilbene gly-
cosides using the ORAC assay. This is the first report
on the antioxidant capacities of stilbene glucosides.
As shown in Table 2, the IC50 value of resveratrol
for tyrosinase inhibitory activity was 565 μM. The glu-
coside of resveratrol, resveratrol 3-O-β-D-glucoside,
showed high inhibitory activity against tyrosinase with
an IC50 value of 14 μM. This result indicates that the
glucosylation of resveratrol improved its tyrosinase
inhibitory activity. In addition, the glucosylation of
resveratrol at its 4′-position greatly enhanced its tyrosi-
nase inhibitory activity: the IC50 value of resveratrol
4′-O-β-D-glucoside for tyrosinase inhibitory activity
was 29 μM. The glucosylation of pterostilbene (IC50
value for tyrosinase inhibitory activity = 653 μM) at its
4′-position increased its tyrosinase inhibitory activity:
the IC50 value of pterostilbene 4′-O-β-D-glucoside was
237 μM. On the other hand, glucosylation of pinostil-
bene at the 3- and 4′-positions enhanced its tyrosinase
inhibitory activity: the IC50 values of pinostilbene,
pinostilbene 3-O-β-D-glucoside, and pinostilbene 4′-O-
β-D-glucoside for tyrosinase inhibitory activity were
594, 66, and 85 μM, respectively. Arbutin, which is a
useful skin-whitening agent in cosmetics, exhibits
Table 2. Tyrosinase inhibitory activities of compounds 1–8.
IC50 (μM)
Compound
Tyrosinase inhibitory activity
1
2
3
4
565 42
653 126
594 139
14 0.5
5
29
6
6
7
8
237 46
66 26
85 18
145 11
Arbutin
Notes: Tyrosinase inhibitory activity is expressed as the 50% inhibitory
concentration (IC50). The results are shown as the mean standard deviation
from triplicate experiments. The assay system for determining tyrosinase
inhibitory activity is described in the Materials and methods.
Fig. 1. Chemical structures of substrates 1–3 and products 4–8.
Note: The synthetic procedure is described in the Materials and
methods.

4
D. Uesugi et al.
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tyrosinase inhibitory activity with an IC50 value of
145 μM. Thus, the present results suggest that stilbene
glucosides, resveratrol 3-O-β-D-glucoside, resveratrol
4′-O-β-D-glucoside, pterostilbene 4′-O-β-D-glucoside,
pinostilbene 3-O-β-D-glucoside, and pinostilbene 4′-O-
β-D-glucoside, may also be useful as effective
skin-whitening agents. Stilbene glucosides might fit the
substrate binding pocket of tyrosinase more readily
rather than the corresponding stilbenes.
In conclusion, the stilbenes resveratrol, pterostilbene,
and pinostilbene were transformed to the correspond-
ing glucoside products by glucosyltransferase from
P. americana expressed in recombinant E. coli. This
enzyme catalyzed the glucosylation of both the 3- and
4′-positions of stilbenes. An analysis of antioxidant
capacity showed that pinostilbene had the highest
ORAC value among the stilbene aglycones tested. The
ORAC values of the stilbene glycosides were high, but
lower than those of the stilbene aglycones. The 3-O-β-
D-glucoside of resveratrol had the highest ORAC value
among the stilbene glycosides tested. The tyrosinase
inhibitory activities of the stilbene glycosides were
higher than those of the stilbene aglycones. Resveratrol
3-O-β-D-glucoside had the highest tyrosinase inhibitory
activity among the stilbene compounds tested.
Author contributions
[15] Shimoda K, Kondo Y, Nishida T, et al. Biotransformation of
thymol, carvacrol, and eugenol by cultured cells of Eucalyptus
perriniana. Phytochemistry. 2006;67:2256–2261.
Conceived and designed the experiments: D. Uesugi,
H. Hamada, K. Shimoda, N. Kubota, S. Ozaki, N.
Nagatani. Analyzed the data: D. Uesugi. Wrote the first
draft of the manuscript: D. Uesugi. Contributed to the
writing of the manuscript: D. Uesugi, H. Hamada, K.
Shimoda. Agree with manuscript results and conclu-
sions: D. Uesugi, H. Hamada, K. Shimoda, N. Kubota,
S. Ozaki, N. Nagatani. Jointly developed the structure
and arguments for the paper: D. Uesugi, H. Hamada,
K. Shimoda, N. Kubota, S. Ozaki, N. Nagatani. Made
critical revisions and approved final version: D. Uesugi,
H. Hamada, K. Shimoda. All authors reviewed and
approved of the final manuscript.
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detoxification of 2-phenylenediamine by Bacillus cereus strain
PDA-1. J. Biosci. Bioeng. 2006;102:21–27.
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[19] Iwakiri T, Imai H, Hamada H, et al. Synthesis of 3, 5, 3′, 4′-te-
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(ORAC) and phenolic and anthocyanin concentrations in fruit
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Disclosure statement
[21] Bangalore DV, McGlynn W, Scott DD. Effect of β-cyclodextrin
in improving the correlation between lycopene concentration and
ORAC values. J. Agric. Food Chem. 2005;53:1878–1883.
[22] Bellido GG, Beta T. Anthocyanin composition and oxygen
radical scavenging capacity (ORAC) of milled and pearled pur-
ple, black, and common barley. J. Agric. Food Chem. 2009;57:
1022–1028.
[23] Kondo Y, Shimoda K, Kubota N, et al. Biotransformation of
monofluorophenols by cultured cells of Eucalyptus perriniana.
Plant Biotechnol. 2006;23:329–331.
[24] Kondo Y, Shimoda K, Takimura J, et al. Glycosylation of vita-
min E homologue by cultured plant cells. Chem. Lett. 2006;
35:324–325.
[25] Kondo Y, Shimoda K, Miyahara K, et al. Regioselective hydrox-
ylation, reduction, and glycosylation of diphenyl compounds by
cultured plant cells of Eucalyptus perriniana. Plant Biotechnol.
2006;23:291–296.
No potential conflict of interest was reported by the authors.
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