Synthesis of daidzein glycosides, α-tocopherol glycosides, hesperetin glycosides by bioconversion and their potential for anti-allergic functional-foods and cosmetics

Article abstract of DOI:10.3390/molecules24162975

Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation, anti-allergy, and anti-aging. α-Tocopherol is the tocopherol isoform with the highest vitamin E activity including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor absorption after oral administration. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. They were transformed by cultured Nicotiana tabacum cells to 7-β-glucoside and 7-β-gentiobioside of daidzein, and 3⁰- and 7-β-glucosides, 3⁰,7-β-diglucoside, and 7-β-gentiobioside of hesperetin. Daidzein and α-tocopherol were glycosylated by galactosylation with β-glucosidase to give 4⁰- and 7-β-galactosides of daidzein, which were new compounds, and α-tocopherol 6-β-galactoside. These nine glycosides showed higher anti-allergic activity, i.e., inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the corresponding aglycones. Glycosylation of daidzein, α-tocopherol, and hesperetin greatly improved their biological activities.

Full text of DOI:10.3390/molecules24162975

molecules
Article
Synthesis of Daidzein Glycosides, α-Tocopherol
Glycosides, Hesperetin Glycosides by Bioconversion
and Their Potential for Anti-Allergic
Functional-Foods and Cosmetics
Yuya Fujitaka 1, Hiroki Hamada 1,*, Daisuke Uesugi 1, Atsuhito Kuboki 2, Kei Shimoda 3,
Takafumi Iwaki 4, Yuya Kiriake 5 and Tomohiro Saikawa 6
1
Department of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku,
Okayama 700-0005, Japan
2
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku,
Okayama 700-0005, Japan
Department of Biomedical Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi,
3
Oita 879-5593, Japan
4
Department of Biophysics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
Faculty of Medicine and Health Sciences, Yamaguchi University, 1-1-1 Minamikogushi, Ube-shi,
5
Yamaguchi 755-8505, Japan
Department of Nursing, Junshin Gakuen University, 1-1-1 Tikushigaoka, Fukuoka-shi, Minami-ku,
6
Fukuoka 815-8510, Japan
*
Correspondence: hamada@dls.ous.ac.jp; Tel.: +81-86-256-9473
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Francesco Epifano and Serena Fiorito
Received: 22 July 2019; Accepted: 15 August 2019; Published: 16 August 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation,
anti-allergy, and anti-aging. -Tocopherol is the tocopherol isoform with the highest vitamin E activity
α
including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent
anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor
absorption after oral administration. The glycosylation of bioactive compounds can enhance their
water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve
their bio- and pharmacological properties. They were transformed by cultured Nicotiana tabacum cells
to 7-
and 7-
with
and
β
-glucoside and 7-
-gentiobioside of hesperetin. Daidzein and
-glucosidase to give 40- and 7-
-galactosides of daidzein, which were new compounds,
-tocopherol 6- -galactoside. These nine glycosides showed higher anti-allergic activity, i.e.,
β
-gentiobioside of daidzein, and 30- and 7-
β β-diglucoside,
-glucosides, 30,7-
β
α
-tocopherol were glycosylated by galactosylation
β
β
α
β
inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective
aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the
corresponding aglycones. Glycosylation of daidzein,
their biological activities.
α-tocopherol, and hesperetin greatly improved
Keywords: daidzein;
α-tocopherol; hesperetin; β-glycoside; anti-allergic activity; tyrosinase
inhibitory activity
1. Introduction
Daidzein is the most widely studied isoflavone and is found in beans such as soybean, sweet
red bean, and kidney bean. It exhibits anti-oxidative, anti-inflammatory, and anti-aging action
and has chemopreventive effects in several biological systems, such as cancer prevention [
1
–
10].
α
-Tocopherol is found in wheat germ. It has chemopreventive activities against cancer such as breast
Molecules 2019, 24, 2975; doi:10.3390/molecules24162975
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2975
2 of 11
and prostate cancers [11
–
15]. Hesperetin, which occurs in citrus fruits and flowers, is a bioactive
flavonoid (vitamin P) that has been well documented for its medicinal properties as an important
Chinese traditional medicine [16]. Available clinical information on hesperetin includes its effects on
the blood–brain barrier, signal transduction pathway, and certain kinds of cancer [17–21]. Hesperetin
has been used in cosmetics as it has antioxidant and anti-allergic activities. Despite the bio- and
physiological activities of these compounds, their use as medicines, functional-foods, and cosmetics
has been limited due to their insolubility in water and poor absorption after oral administration.
Several flavonoids have been reported to possess a histamine release-inhibitory activity on rat
peritoneal mast cells [22]. However, comparative studies on the anti-allergic activity of many kinds of
flavonoid glycosides have not yet been done. The release of inflammatory mediators, such as histamine,
from mast cell or basophils, is mediated by cell-surface receptors for immunoglobulin E (IgE), and
stimulation by antigen results in allergic reactions called immediate-type hypersensitivity. In vitro
anti-allergic screening has generally been performed by using rat peritoneal mast cells [22]. On the
other hand, melanogenesis is a physiological process resulting in the production of melanin pigment,
which plays an important role in the prevention of sun-induced skin injury. Although the melanin
production in human skin is a major defense mechanism against UV light, the accumulation of an
excess of epidermal pigmentation can cause various hyperpigmentation disorders, such as melasma,
age spots, and sites of actinic damage. Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme widely
distributed in nature. It catalyzes two distinct reactions involving molecular oxygen in the melanin
synthesis, the hydroxylation of l-tyrosine to l-dopa and the oxidation of l-dopa to dopaquinone. This
dopaquinone is highly reactive and can polymerize spontaneously to form melanin in a series of
reaction pathways. Accordingly, the regulation of melanin synthesis by the inhibition of tyrosinase to
prevent hyperpigmentation has been a recent subject of many studies [23].
Plant cell cultures are ideal systems for propagating rare plants and for studying the biosynthesis
of secondary metabolites such as flavors, pigments, and agrochemicals, except for a very limited
number of compounds (e.g., pyrethrins, bialaphos, and nicotin). Furthermore, the bioconversion of
various organic compounds has been investigated as a target in the biotechnological application of
plant cell culture systems. Plant cultured cells can be used to convert organic molecules to more
useful compounds by catalyzing hydrolysis, oxidation, reduction, esterification, isomerization, and
glycosylation reactions. A recent paper reported that alkyl esters of caffeic acid, especially propyl
caffeate, have stronger antioxidant activity than caffeic acid [24]. On the other hand, the glycosylation of
bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption,
biological half-life, and improve their bio- and pharmacological properties [25–33].
We report here the syntheses of glycosides of daidzein,
bioconversion with cultured plant cells and -glucosidase. In addition, we report the physiological
properties of glycosides of daidzein, -tocopherol, and hesperetin such as their histamine-release
α-tocopherol, and hesperetin (Figure 1) by
β
α
inhibitory activity (anti-allergic activity) and tyrosinase inhibitory activity.
Figure 1. Chemical structures of daidzein, α-tocopherol, and hesperetin.

Molecules 2019, 24, 2975
3 of 11
2. Results
2.1. Synthesis of Daidzein Glycosides and Hesperetin Glycosides
Daidzein (
products, and
Based on the analyses of mass and nuclear magnetic resonance (NMR) analyses, the products were
1) was subjected to a bioconversion system using cultured cells of N. tabacum. Two
2
3, were isolated from the cells by extraction with MeOH after two days of incubation.
determined to be daidzein 7-β-glucoside (2) and daidzein 7-β-gentiobioside (3), which were known
compounds (Figure 2) [34].
On the other hand, only a small amount of the glycosylation products in the MeOH extracts
of N. tabacum cells treated with α-tocopherol (6) was obtained. It was not enough for analyses of
biological activities.
Furthermore, biotransformation of hesperetin (
12 were isolated from the MeOH extracts from cultured N. tabacum cells. The structure of
compounds -glucoside ( ), hesperetin 7- -glucoside (10),
12 was identified as hesperetin 30-
hesperetin 30,7-
-diglucoside (11), and hesperetin 7- -gentiobioside (12), which have been reported
8) with N. tabacum cells was carried out. Products
9
–
9
–
β
9
β
β
β
before [35], (Figure 2), based on analyses of mass and nuclear magnetic resonance (NMR) spectra.
1
O
8
HO
RO
O
O
7
9
2
3
2'
5'
6
1'
N. tabacum
4
3'
4'
10
5
O
6'
OH
OH
2
Daidzein 7- -glucoside ( ): R=Glc
1
Daidzein ( )
3
Daidzein 7- -gentiobioside ( ): R=Glc-Glc
Figure 2. Synthetic schemes of glycosides of daidzein and hesperetin.
2.2. Enzymatic Galactosylation with β-Glucosidase Isolated from Sweet Almond
2.2.1. Synthesis of Daidzein Galactosides and α-Tocopherol Galactoside
Galactosylation of daidzein (1) was performed by enzymatic synthetic procedure. β-Glucosidase
isolated from sweet almond, which can catalyze glycosylation, i.e., reverse hydrolysis, such as
glucosylation and galactosylation [36], was used for the synthesis of daidzein galactosides and the
conditions were optimized at pH 7, over 72 h, 110 AU of enzyme. Based on the analyses of HRESIMS,
1H- and 13C-NMR, and HMBC spectra, the products were determined to be daidzein 40-
(4) and daidzein 7-β-galactoside (5) (Figure 3). Products 4 and 5
β
-galactoside
were two new compounds. No
glucosylation of the substrates occurred, because d-galactose was used as the galactose donor.

Molecules 2019, 24, 2975
4 of 11
Figure 3. Synthetic schemes of galactosides of daidzein and α-tocopherol.
On the other hand,
described above. Product
was identified as
13C-NMR, and HMBC spectra.
α
-tocopherol (
was isolated by preparative HPLC. The chemical structure of the product
-galactoside (Figure 3) [36], based on the analyses of ESIMS, 1H- and
6) was subjected to the same galactosylation procedures as
7
7
α
-tocopherol 6-
β
No galactosylation products were isolated from the reaction mixture of enzymatic transformation
of hesperetin (8). It might be explained by the substrate specificity of sweet almond β-glucosidase.
2.2.2. Determination of Chemical Structure of New Daidzein Galactosides
The HRESIMS spectrum of
consistent with a molecular formula of C21H20O9 (calcd. 439.368 for C21H20O9Na). The 1H-NMR
spectrum of had a signal at 4.91 (1H, d, J = 8.0 Hz) corresponding to its attachment to the anomeric
carbon (C-1”) (Figure S1). The 13C-NMR spectrum of
exhibited the anomeric carbon signal at
103.0 (Figure S2). This 13C chemical shift of the anomeric carbon at
103.0 indicates the presence
of O-galactoside in the structure of 36]. From the coupling pattern of the proton signals and the
chemical shifts of the carbon resonances due to the sugar moiety, the sugar component in was
determined to be -d-galactopyranose. Hydrolysis of using -galactosidase gave daidzein ( ) as the
product. This finding shows that the product has O- -galactosylation moiety. The HMBC correlation
4
showed a pseudomolecular ion [M + Na]+ peak at m/z (439.265),
4
δ
4
δ
δ
4
[
4
β
4
β
1
β
(Figure 4) was observed between the anomeric proton signal at
δ
4.91 (H-1”) and the carbon signal at
δ
159.1 (C-40) to establish that the galactopyranosyl residue was attached to the 40-hydroxy group of
1
(Figure S3). Thus, the structure of 4 was determined to be daidzein 40-β-galactoside.
The HRESIMS spectra of
5
included a pseudomolecular ion [M + Na]+ peak at m/z (439.248),
indicating that the product consisted of one substrate and one hexose. The sugar component in the
product was determined to be galactose on the basis of the chemical shifts of their carbon signals
(Figure S5) [36]. The 1H-NMR spectrum showed a proton signal at
δ
5.05 (1H, d, J = 7.2 Hz), indicating
-orientation (Figure S4). The HMBC spectra of
included the correlation between the proton signal at 5.05 (H-1”) and the carbon signal at 163.6
(C-7) (Figure S6). These data indicate that was -galactosyl analogue of , the sugar moiety of which
that the galactoside linkage in the compound had
β
5
δ
δ
5
β
1

Molecules 2019, 24, 2975
5 of 11
was attached to the 7-position of
1
(Figure 4). Thus, the structure of
5
was determined to be daidzein
7-β-galactoside.
Figure 4. HMBC correlations of daidzein galactosides.
2.3. Biological Effects of Daidzein Glycosides, α-Tocopherol Glycoside, and Hesperetin Glycosides
2.3.1. Suppression for Histamine Release from Rat Peritoneal Mast Cells
The anti-allergic activities of daidzein and its glycosides were examined to clarify the effects
of glycosylation of daidzein on its physiological activities. The inhibitory activities of daidzein (
daidzein 7- -glucoside ( ), daidzein 7- -gentiobioside ( -galactoside ( ), and daidzein
), daidzein 40-
7- -galactoside ( ) for compound 48/80-induced histamine release from rat peritoneal mast cells
were investigated (Table 1). Compound 48/80-induced histamine release from rat peritoneal mast
cells was inhibited by daidzein ( ) with a %inhibition of 58%. On the other hand, the inhibitory
activity of daidzein 7- -glucoside ( ) toward histamine release from rat peritoneal mast cells was
higher than that of the aglycone daidzein ( ). The inhibitory activity of daidzein 7- -gentiobioside
) was higher than that of the daidzein glucoside -galactoside ( ) and
. Additionally, daidzein 40-
daidzein 7- -galactoside ( ) showed higher inhibitory activities toward histamine release than daidzein
1),
β
2
β
3
β
4
β
5
1
β
2
1
β
(3
2
β
4
β
5
gentiobioside 3.
In addition, α-tocopherol 6-β-galactoside (7) had stronger inhibitory activity toward histamine
release than α-tocopherol (6).
Table 1. Anti-allergic activities of compounds 1–5.
Compound
Histamine Release-Inhibiting Activity / %inhibition 1
Kaempferol
80
58
62
67
70
76
61
82
38
40
45
63
69
1
2
3
4
5
6
7
8
9
10
11
12
1
Histamine release-inhibiting activity is expressed as %inhibition.
The %inhibition of hesperetin 30-
than that of hesperetin (
). Furthermore, hesperetin 30,7-
activity toward histamine release than hesperetin glucosides
β
-glucoside (
9
) and hesperetin 7-
-diglucoside (11) showed higher inhibitory
and 10. The inhibitory activity toward
β-glucoside (10) was higher
8
β
9
histamine release of hesperetin 7-β-gentiobioside (12) was stronger than hesperetin diglucoside 11.
The anti-allergic activities were examined at pH 7.4 and 37 ◦C according to the reported methods [37].
The pH and temperature of the reaction mixture may have an effect on anti-allergic activities. Effects of

Molecules 2019, 24, 2975
6 of 11
pH and temperature on anti-allergic activities of the obtained glycosides should be investigated and
reported on in the near future.
2.3.2. Tyrosinase Inhibitory Activity
The IC50 value of daidzein (
of daidzein, daidzein 7- -glucoside (
indicates that the glucosylation of daidzein improved its tyrosinase inhibitory activity. In addition,
the glucosylation of daidzein glucoside to daidzein 7- -gentiobioside ( ) enhanced its tyrosinase
inhibitory activity. Overall, daidzein 7- -galactoside ( ) showed the highest inhibitory activity against
1
) for tyrosinase inhibitory activity was 392
µM (Table 2). The glucoside
β
2), showed high inhibitory activity against tyrosinase. This result
2
β
3
β
5
tyrosinase among daidzein compounds tested.
A similar tendency was found in the case of
α
-tocopherol and its glycoside. The tyrosinase
inhibitory activity of α-tocopherol 6-β-galactoside (7) was higher than that of α-tocopherol (6).
Additionally, tyrosinase inhibition by hesperetin 30-
-glucoside ( ) and hesperetin 7- -glucoside
). Hesperetin 30,7-
10) was stronger than that by hesperetin ( -diglucoside (11) had stronger tyrosinase
inhibitory activity than hesperetin glucosides and 10. Particularly, hesperetin 7- -gentiobioside (12)
β
9
β
(
8
β
9
β
showed the strongest tyrosinase inhibition among the hesperetin compounds tested. Effects of pH and
temperature on tyrosinase inhibitory activity of the obtained glycosides should be investigated and
reported in the near future.
Table 2. Tyrosinase inhibitory activities of compounds 1–5.
1
Compound
Tyrosinase Inhibitory Activity IC50 / µM
Kojic acid
35 ± 15
392 ± 88
303 ± 45
280 ± 33
125 ± 41
102 ± 39
510 ± 108
54 ± 25
1
2
3
4
5
6
7
8
9
437 ± 76
355 ± 32
318 ± 27
176 ± 35
139 ± 14
10
11
12
1
Tyrosinase inhibitory activity is expressed as the 50% inhibitory concentration (IC50). The results are shown as the
mean ± standard deviation from triplicate experiments.
3. Discussion
Thus, the glycosylation derivatives of daidzein,
bioconversion with cultured N. tabacum and -glucosidase. Cultured N. tabacum cells glycosylated
daidzein and hesperetin to 7- -glucoside and 7- -glucosides,
-gentiobioside of daidzein, and 30- and 7-
30,7-
-diglucoside, and 7- -gentiobioside of hesperetin. A recent paper reported that cultured
E. perriniana cells converted daidzein into 7- -glucoside and 7- -gentiobioside of daidzein [34]. The
bioconversion system of N. tabacum cells is the same as E. perriniana cells. It was reported that E. perriniana
cells glycosylated hesperetin to hesperetin 30-O- -glucoside, hesperetin 30,7-O-
-diglucoside,
hesperetin 7-O- -gentiobioside, hesperetin 5-O- -glucoside, hesperetin 7-O- -glucoside, and
hesperetin 7-O- -rutinoside [35]. The bioconversion pathway of hesperetin by cultured N. tabacum cells
is quite different from that of cultured E. perriniana cells. Daidzein and -tocopherol were galactosylated
by -galactosides of daidzein, which were new compounds, and
-glucosidase to give 40- and 7-
α-tocopherol 6-β-galactoside.
Recently, biocatalytic synthesis of daidzein glycoside has been reported. The cell culture of
Staphylococcus saprophyticus CQ16 glycosylated daidzein to daidzein 7-O- -glucoside [38]. In addition,
α
-tocopherol, and hesperetin were prepared by
β
β
β
β
β
β
β
β
β
β
β
β
β
β
α
β
β
β

Molecules 2019, 24, 2975
7 of 11
the biocatalytic glucosylation of 8-hydroxydaidzein to its 7-O-β-glucoside and 8-O-β-glucoside has been
reported. 8-Hydroxydaidzein has been proven to possess some important bioactivities, however, the
low aqueous solubility and stability of 8-hydroxydaidzein limit its pharmaceutical and cosmeceutical
applications. The glucosylation of 8-hydroxydaidzein by glucosyltransferase from Bacillus subtilis
ATCC 6633 improved its drawbacks in solubility and stability [39]. These microbiological glycosylation
of isoflavones gave only mono glucoside as the product. Compared with microbiological glycosylation,
bioconversion of isoflavone by plant cells is a convenient method to prepare diverse glycosides, such
as mono- and di-glycosides including gentoibioside.
Recently, it was reported that anti-allergic activity of stilbene compounds, such as resveratrol,
pterostilbene, and piceatannol, were enhanced by glycoside modification [40
inhibitory activities of daidzein, daidzein glycosides, -tocopherol, -tocopherol glycoside, hesperetin,
and hesperetin glycosides toward histamine release from rat peritoneal mast cells showed that
glycosylation of the isoflavone daidzein, vitamin E ( -tocopherol), and flavone hesperetin improved
their anti-allergic activities. -Tocopherol 6- -galactoside had the highest inhibitory activity toward
histamine release among the compounds tested. Kaempferol was used as a positive control, the
inhibitory activity toward histamine release of which was 80%. -Tocopherol 6- -galactoside showed
,41]. An analysis of the
α
α
α
α
β
α
β
higher anti-allergic activity than kaempferol. Improvement of anti-allergic activity was achieved by
glycosylation for not only stilbene compounds but also daidzein, α-tocopherol, and hesperetin.
Uesugi and his co-workers recently reported that glycosylation of stilbene compounds, such as
resveratrol, pterostilbene, and pinostilbene, increased their tyrosinase inhibitory activity [41]. The
tyrosinase inhibitory activities of the daidzein glycosides,
glycosides were higher than those of their respective aglycones.
α
-tocopherol glycoside, and hesperetin
-Tocopherol 6- -galactoside
α
β
showed the strongest tyrosinase inhibitory activity among the compounds tested. Kojic acid was
used as a positive control, the tyrosinase inhibitory activity of which was IC50 = 35. Although the
tyrosinase inhibitory activity of
α-tocopherol 6-β-galactoside was slightly lower than that of kojic acid,
α
-tocopherol 6- -galactoside acts as a strong tyrosinase inhibitor. The present results suggest that the
β
galactosylated compounds might be useful as effective skin-whitening agents with strong tyrosinase
inhibitory and anti-allergic activities. Tyrosinase inhibitory activity of not only stilbene compounds but
also daidzein,
describes the anti-allergic activity of daidzein galactosides, which are new, and the inhibitory activity
of -tocopherol galactoside toward tyrosinase. The structure–activity relationship of the glycosides is
α-tocopherol, and hesperetin, was enhanced by glycosylation. This is the first report that
α
now in progress in our laboratory.
4. Materials and Methods
4.1. Analyses
The structures of products were determined based on the analysis of HRESIMS, 1H- and 13C-NMR,
and HMBC spectra. The 1H- and 13C-NMR, and HMBC spectra were recorded using a JNM-ECS400
spectrometer (JEOL Ltd., Tokyo, Japan) in CD3OD solutions, and chemical shifts are expressed in
δ
(ppm) with reference to TMS. The HRESIMS spectra were measured using a JMS-700 MStation (JEOL)
in CH3OH solution.
4.2. Glycosylation by Cultured Plant Cells
The substrate was transformed by using plant cultured cells of N. tabacum as biocatalysts. The
cultured plant cells of P. americana were sub-cultured at 4-week intervals on solid medium containing
2% glucose, 1 ppm 2,4-dichlorophenoxyacetic acid, and 1% agar (adjusted to pH 5.7) in the dark.
A suspension culture was started by transferring 20 g of the cultured cells to 300 mL of liquid MS
medium in a 500 mL-conical flask. The cultured cells in the stationary growth phase have been used
for experiments. To a 300 mL flask containing 100 mL of the culture medium and suspension cultured
cells (25 g) was added 15 mg of substrate, i.e., daidzein, α-tocopherol, or hesperetin. The culture

Molecules 2019, 24, 2975
8 of 11
◦
was incubated at 25 C for 2 days on a rotary shaker (120 rpm). After the incubation period, the
cells and medium were separated by filtration with suction. The filtered medium was extracted with
ethyl acetate (AcOEt). The cells were extracted by homogenization with MeOH, and the resulting
extract was concentrated. The residue was partitioned between H2O and AcOEt. The AcOEt layer was
evaporated and the residue was re-dissolved in MeOH and purified by preparative high-performance
liquid chromatography (HPLC) (column: CrestPak C18S; flow rate: 1.0 mL/min; column temperature:
40 ◦C).
4.3. Galactosylation by Enzyme
A typical galactosylation procedure was performed as follows. Syntheses of daidzein galactosides
involved refluxing daidzein (
the presence of 25–210 AU
1) (0.25 mmol) with 0.5 mmol d-galactose in 100 ml di-isopropyl ether in
β
-glucosidase and 0.01 M pH 4–8 buffer for 72 h at 68 ◦C. After the reaction,
the solvent was evaporated and the enzyme denatured at 100 ◦C by holding in a boiling water-bath for
10 min. The product glycoside was dissolved in water, extracted with ethylacetate, concentrated, and
subjected to preparative HPLC to afford daidzein 40-
(5), respectively.
β-galactoside (4) and daidzein 7-β-galactoside
The spectral data of products 4 and 5, which were new compounds, are as follows.
Daidzein 40- ): HRESIMS [M + Na]+: m/z 439.265 (439.368 calcd. for C21H20O9Na);
β
-galactoside (
4
1H-NMR (400 MHz, CD3OD): 3.58–3.92 (6H, multiplet, H-2”-H-6”), 4.91 (1H, doublet, J = 8.0 Hz,
H-1”), 6.69 (1H, doublet, J = 2.4 Hz, H-8), 6.83 (1H, double-doublet, J = 8.4, 2.0 Hz, H-6), 7.16–7.18 (2H,
multiplet, H-30, H-50), 7.46–7.48 (2H, multiplet, H-20, 60), 7.97 (1H, doublet, J = 8.8 Hz, H-5), 8.10 (1H,
singlet, H-2); 13C-NMR (100 MHz, CD3OD): 62.4 (C-6”), 70.3 (C-4”), 72.3 (C-2”), 74.9 (C-3”), 77.0 (C-5”),
103.0 (C-1”), 103.8 (C-8), 116.2 (C-10), 117.7 (C-30, C-50), 119.0 (C-6), 125.3 (C-3), 127.7 (C-10), 128.0 (C-5),
131.4 (C-20, 60), 154.5 (C-2), 159.1 (C-40), 160.6 (C-9), 169.9 (C-7), 178.0 (C-4).
Daidzein 7-β-galactoside (5
): HRESIMS [M + Na]+: m/z 439.248 (439.368 calcd. for C21H20O9Na);
1H-NMR (400 MHz, CD3OD): 3.62–3.94 (6H, multiplet, H-2”-H-6”), 5.05 (1H, doublet, J = 7.2 Hz, H-1”),
6.83–6.85 (2H, multiplet, H-30, H-50), 7.21 (1H, double-doublet, J = 8.8, 2.4 Hz, H-6), 7.25 (1H, doublet,
J = 2.4 Hz, H-8), 7.36–7.38 (2H, multiplet, H-20, 60), 8.13 (1H, doublet, J = 8.8 Hz, H-5), 8.18 (1H, singlet,
H-2); 13C-NMR (100 MHz, CD3OD): 62.5 (C-6”), 70.2 (C-4”), 72.1 (C-2”), 74.8 (C-3”), 77.3 (C-5”), 102.5
(C-1”), 105.0 (C-8), 116.5 (C-30, C-50), 117.1 (C-6), 120.2 (C-10), 123.8 (C-10),126.3 (C-3), 128.3 (C-5), 131.4
(C-20, 60), 155.0 (C-2), 159.3 (C-40, C-9), 163.6 (C-7), 178.1 (C-4).
4.4. Inhibition of Histamine Release from Rat Peritoneal Mast Cells
The effects of daidzein, α-tocopherol, hesperetin, and their glycosides on compound 48/80-induced
histamine release from rat peritoneal mast cells were examined as follows. Peritoneal mast cells
were collected from the abdominal cavities of rats (Male Wistar rats, Nippon SLC) and purified
to a level higher than 95%. The purified mast cells were suspended in a physiological buffered
solution (PBS) containing 145 mM NaCl, 2.7 mM KCl, 1.0 mM CaCl2, 5.6 mM glucose, and 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4) to give approximately 104 mast
cells/mL. Cell viability was always greater than 90% as judged by the trypan blue exclusion test. Mast
cells were preincubated with the test compound (1
to compound 48/80 at 0.35 g/mL. Histamine release was determined by a fluorometric assay, and was
µ
M) for 15 min at 37 ◦C, and subsequently exposed
µ
expressed as a percentage of total histamine [37].
4.5. Tyrosinase Assay
Mushroom tyrosinase (EC 1.14.18.1) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) 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 dissolved 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

Molecules 2019, 24, 2975
9 of 11
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 mushroom 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.
5. Conclusions
The glycosides of daidzein,
procedures. These two bioconversion systems prepared nine glycosides of these compounds,
i.e., daidzein 7- -glucoside, daidzein 7- -glucoside, hesperetin
-gentiobioside, hesperetin 30-
7- -diglucoside, hesperetin 7- -galactoside,
-glucoside, hesperetin 30,7- -gentiobioside, daidzein 40-
daidzein 7- -galactoside, and -tocopherol 6- -galactoside and daidzein
-galactoside. Daidzein 40-
7- -galactoside were two new compounds. Glycosylation of daidzein, -tocopherol, and hesperetin
α-tocopherol, and hesperetin were synthesized by bioconversion
β
β
β
β
β
β
β
β
α
β
β
β
α
much improved their biological activities such as suppression activity toward histamine release from
rat peritoneal mast cells and tyrosinase inhibitory activity. The diglucoside derivatives showed
stronger activity than glucoside derivatives. The gentiobioside derivative had stronger activity than
the diglucoside derivative. The galactoside derivative exerted the strongest physiological activity
among the glycosides tested.
Supplementary Materials: The following are available online, Figure S1: 1H NMR of daidzein 40-
Figure S2: 13C NMR of daidzein 40- -galactoside, Figure S3: HMBC of daidzein 40-
-galactoside, Figure S5: 13C NMR of daidzein 7-
β
-galactoside,
1
β
β
-galactoside, Figure S4: H
-galactoside, Figure S6: HMBC of daidzein
NMR of daidzein 7-
β
β
7-β-galactoside.
Author Contributions: Conceptualization, Y.F., H.H., D.U., A.K., K.S., T.I., Y.K. and T.S.; methodology, Y.F.;
validation, Y.F. and H.H.; formal analysis, Y.F.; investigation, Y.F.; resources, H.H.; data curation, D.U.;
writing—original draft preparation, Y.F.; writing—review and editing, H.H.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Sak, K. Current epidemiological knowledge about the role of flavonoids in prostate carcinogenesis. Exp. Oncol.
2017, 39, 98–105. [CrossRef]
Yu, B.; Tang, D.Z.; Li, S.Y.; Wu, Y.; Chen, M. Daidzein promotes proliferation and differentiation in osteoblastic
OCT1 cells via activation of the BMP-2/Smads pathway. Pharmazie 2017, 72, 35–40. [PubMed]
Zhang, F.; Ru, N.; Shang, Z.H.; Chen, J.F.; Yan, C.; Li, Y.; Liang, J. Daidzein ameliorates spinal cord
ischemia/reperfusion injury-induced neurological function deficits in Sprague-Dawley rats through PI3K/Akt
signaling pathway. Exp. Ther. Med. 2017, 14, 4878–4886. [CrossRef] [PubMed]
4.
Ono, M.; Ejima, K.; Higuchi, T.; Takeshima, M.; Wakimoto, R.; Nakano, S. Equol enhances apoptosis-inducing
activity of genistein by increasing Bax/Bcl-xL expression ratio in MCF-7 human breast cancer cells. Nutr. Cancer
2017, 69, 1300–1307. [CrossRef] [PubMed]
5.
6.
Sak, K.; Lust, H.; Kase, M.; Saar, M.; Jaal, J. Suppression of taxanes cytotoxicity by citrus flavonoid hesperetin
in PPC-1 human prostate cancer cells. Anticancer Res. 2018, 38, 6209–6215. [CrossRef] [PubMed]
Kühn, G.; Pallauf, K.; Schulz, C.; Rimbach, G. Flavonoids as putative modulators of
6-desaturases: Studies in cultured hepatocytes, myocytes, and adipocytes. Biofactors 2018, 44, 485–495.
[CrossRef] [PubMed]
∆4-, ∆5-, and
∆
7.
8.
Hua, F.; Li, C.H.; Chen, X.G.; Liu, X.P. Daidzein exerts anticancer activity towards SKOV3 human ovarian
cancer cells by inducing apoptosis and cell cycle arrest, and inhibiting the Raf/MEK/ERK cascade. Int. J.
Mol. Med. 2018, 41, 3485–3492. [CrossRef] [PubMed]
Kaushik, S.; Shyam, H.; Sharma, R.; Balapure, A.K. Dietary isoflavone daidzein synergizes centchroman
action via induction of apoptosis and inhibition of PI3K/Akt pathway in MCF-7/MDA MB-231 human breast
cancer cells. Phytomedicine 2018, 40, 116–124. [CrossRef] [PubMed]

Molecules 2019, 24, 2975
10 of 11
9.
Sivonˇov
á, M.K.; Kaplán, P.; Tatarková, Z.; Lichardusová, L.; Dušenka, R.; Jurecˇekov
á, J. Androgen receptor
and soy isoflavones in prostate cancer. Mol. Clin. Oncol. 2019, 10, 191–204. [CrossRef]
10. Grainger, E.M.; Moran, N.E.; Francis, D.M.; Schwartz, S.J.; Wan, L.; Thomas-Ahner, J.; Kopec, R.E.; Riedl, K.M.;
Young, G.S.; Abaza, R.; et al. A Novel tomato-soy iuice induces a dose-response increase in urinary and
plasma phytochemical biomarkers in men with prostate cancer. J. Nutr. 2019, 149, 26–35. [CrossRef]
11. Fontana, F.; Raimondi, M.; Marzagalli, M.; Moretti, R.M.; Marelli, M.M.; Limonta, P. Tocotrienols and cancer:
From the state of the art to promising novel patents. Recent Pat. Anticancer Drug Discov. 2019, 14, 5–18.
[CrossRef] [PubMed]
12. Liese, J.; Hinrichs, T.M.; Lange, M.; Fulda, S. Cotreatment with sorafenib and oleanolic acid induces reactive
oxygen species-dependent and mitochondrial-mediated apoptotic cell death in hepatocellular carcinoma
cells. Anticancer Drugs 2019, 30, 209–217. [CrossRef] [PubMed]
13. Wei, C.W.; Yu, Y.L.; Chen, Y.H.; Hung, Y.T.; Yiang, G.T. Anticancer effects of methotrexate in combination with
α
-tocopherol and α-tocopherol succinate on triple-negative breast cancer. Oncol Rep. 2019, 41, 2060–2066.
[CrossRef] [PubMed]
14. Dong, K.; Lei, Q.; Qi, H.; Zhang, Y.; Cui, N.; Wu, X.; Xie, L.; Yan, X.; Lu, T. Amplification of oxidative stress in
MCF-7 cells by a novel pH-responsive amphiphilic micellar system enhances anticancer therapy. Mol. Pharm.
2019, 16, 689–700. [CrossRef] [PubMed]
15. Lee, S.Y.; Cho, H.J. Mitochondria targeting and destabilizing hyaluronic acid derivative-based nanoparticles
for the delivery of lapatinib to triple-negative breast cancer. Biomacromolecules 2019, 20, 835–845. [CrossRef]
[PubMed]
16. Formica, J.V.; Regelson, W. Review of the biology of quercetin and related bioflavonoids. Food Chem. Toxicol.
1995, 33, 1061–1080. [CrossRef]
17. So, F.V.; Guthrie, N.; Chambers, A.F.; Carroll, K.K. Inhibition of proliferation of estrogen receptor-positive
MCF-7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen. Cancer Lett.
1997, 112, 127–133. [CrossRef]
18. Mitsunaga, Y.; Takanaga, H.; Matsuo, H.; Naito, M.; Tsuruo, T.; Ohtani, H.; Sawada, Y. Effect of bioflavonoids
on vinicristine transport across blood-brain barrier. Euro. J. Pharm. 2000, 395, 193–201. [CrossRef]
19. O’Prey, J.; Brown, J.; Fleming, J.; Harrison, P.R. Effects of dietary flavonoids on major signal transduction
pathways in human epithelial cells. Biochem. Pharm. 2003, 66, 2075–2088. [CrossRef]
20. Youdim, K.A.; Dobbie, M.S.; Kuhnle, G.; Proteggente, A.R.; Abbott, N.J.; Rice-Evans, C. Interaction between
flavonoids and the blood-brain barrier: In vitro study. J. Neurochem. 2003, 85, 180–192. [CrossRef]
21. Cooray, H.C.; Janvilisri, T.; Veen, H.W.; Hladky, S.B.; Barrand, M.A. Interaction of the breast cancer resistance
protein with plant polyphenols. Biochem. Biophys. Res. Commun. 2004, 317, 269–275. [CrossRef] [PubMed]
22. Kawasaki, M.; Toyoda, M.; Teshima, R.; Sawada, J.; Hayashi, T.; Arisawa, M.; Shimizu, M.; Morita, N.;
Inoue, S.; Saito, Y. In vitro antiallergic activity of flavonoids in histamine release assay using rat basophilic
leukemia (RBL-2H3) cells. J. Food Hyg. Soc. Jpn. 1994, 35, 495–503. [CrossRef]
23. Lee, Y.S.; Park, J.H.; Kim, M.H.; Seo, S.H.; Kim, H.J. Synthesis of tyrosinase inhibitory kojic acid derivative.
Arch. Pharm. Chem. Life Sci. 2006, 339, 111–114. [CrossRef] [PubMed]
24. Liu, W.; Han, L. Lipophilisation of caffeic acid through esterification with propanol using water-tolerable
acidic ionic liquid as catalyst. J. Oleo Sci. 2015, 64, 1297–1305. [CrossRef] [PubMed]
25. Furuya, T.; Ushiyama, M.; Ashida, Y.; Yoshikawa, T. Biotransformation of 2-phenylpropionic acid in root
culture of Panax ginseng. Phytochemistry 1989, 28, 483–487. [CrossRef]
26. Kamel, S.; Brazier, M.; Desmet, G.; Fliniaux, M.-A.; Jacquin-Dubreuil, A.A. Glucosylation of butyric acid by
cell suspension culture of Nicotiana plumbaginifolia. Phytochemistry 1992, 31, 1581–1583. [CrossRef]
27. Morand, C.; Crespy, V.; Manach, C.; Besson, C.; Demigné, C.; Rémésy, C. Plasma metabolites of quercetin
and their antioxidant properties. Am. J. Physiol. 1998, 275, R212–R219. [CrossRef] [PubMed]
28. Moriguchi, Y.; Kita, M.; Hasegawa, S.; Omura, S. Molecular approach to citrus flavonoid and limonoid
biosynthesis. J. Food Agri. Environ. 2003, 1, 22–25.
29. Shimoda, K.; Kondo, Y.; Nishida, T.; Hamada, H.; Nakajima, N.; Hamada, H. Biotransformation of thymol,
carvacrol, and eugenol by cultured cells of Eucalyptus perriniana. Phytochemistry 2006, 67, 2256–2261.
[CrossRef]

Molecules 2019, 24, 2975
11 of 11
30. Takenaka, S.; Mulyono; Sasano, Y.; Takahashi, Y.; Murakami, S.; Aoki, K. Microbial transformation of aniline
derivatives: Regioselective biotransformation and detoxification of 2-phenylenediamine by Bacillus cereus
strain PDa-1. J. Biosci. Bioeng. 2006, 102, 21–27. [CrossRef]
31. Yang, G.; Zhang, Z.; Bai, H.; Gong, J.; Wang, Y.; Li, B.; Li, J. Biotransformation of beta-amyrin acetate by
Rhodobacter sphaeroides. J. Biosci. Bioeng. 2008, 105, 558–561. [CrossRef] [PubMed]
32. Imai, H.; Kitagawa, M.; Ishihara, K.; Masuoka, N.; Shimoda, K.; Nakajima, N.; Hamada, H. Glycosylation of
trans-resveratrol by plant-cultured cells. Biosci. Biotechnol. Biochem. 2012, 8, 1552–1554. [CrossRef] [PubMed]
33. Iwakiri, T.; Imai, H.; Hamada, H.; Nakayama, T.; Ozaki, S. Synthesis of 3,5,30,40-tetrahydroxy-
trans-stilbene-40-O-beta-d-glucopyranoside by glucosyltransferases from Phytolacca americana.
Nat. Prod. Commun. 2013, 8, 119–120. [PubMed]
34. Shimoda, K.; Sato, N.; Kobayashi, T.; Hamada, H.; Hamada, H. Glycosylation of daidzein by the Eucalyptus
cell cultures. Phytochemistry 2008, 69, 2303–2306. [CrossRef] [PubMed]
35. Shimoda, K.; Hamada, H.; Hamada, H. Glycosylation of hesperetin by plant cell cultures. Phytochemistry
2008, 69, 1135–1140. [CrossRef] [PubMed]
36. Ponrasu, T.; Charles, R.E.; Sivakumar, R.; Divakar, S. Syntheses of α-tocopheryl glycosides by glucosidases.
Biotechnol. Lett. 2008, 30, 1431–1439. [CrossRef] [PubMed]
37. Akagi, M.; Katakuse, Y.; Fukuishi, N.; Kan, T.; Akagi, R. Superoxide anion-induced histamine release from
rat peritoneal mast cells. Biol. Pharm. Bull. 1994, 17, 732–734. [CrossRef] [PubMed]
38. Szeja, W.; Grynkiewicz, G.; Rusin, A. Isoflavones, their glycosides and glycoconjugates. Synthesis and
biological activity. Curr. Org. Chem. 2017, 21, 218–235. [CrossRef]
39. Chiang, C.-M.; Wang, T.-Y.; Yang, S.-Y.; Wu, J.-Y.; Chang, T.-S. Production of new isoflavone glucosides from
glycosylation of 8-hydroxydaidzein by glycosyltransferase from Bacillus subtilis ATCC 6633. Catalysts 2018,
8, 387. [CrossRef]
40. Sato, D.; Shimizu, N.; Shimizu, Y.; Akagi, M.; Eshita, Y.; Ozaki, S.; Nakajima, N.; Ishihara, K.; Masuoka, N.;
Hamada, H.; et al. Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant,
anti-allergic, and neuroprotective activities. Biosci. Biotechnol. Biochem. 2014, 78, 1123–1128. [CrossRef]
41. Uesugi, D.; Hamada, H.; Shimoda, K.; Kubota, N.; Ozaki, S.; Nagatani, N. Synthesis, oxygen radical
absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and
pinostilbene. Biosci. Biotechnol. Biochem. 2017, 81, 226–230. [CrossRef] [PubMed]
Sample Availability: Not available.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).