Isolation, synthesis, and biological activities of a bibenzyl from Empetrum nigrum var. Japonicum

Article abstract of DOI:10.1080/09168451.2019.1662279

4-(2-Hydroxyphenethyl)-2,6-dimethoxyphenol, a bibenzyl, was isolated from the leaves of Empetrum nigrum var. japonicum, collected from Mount Tateyama. Japanese rock ptarmigans frequently eat the leaves and fruits of this plant. The structure of the bibenz

Full text of DOI:10.1080/09168451.2019.1662279

Bioscience, Biotechnology, and Biochemistry
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Isolation, synthesis, and biological activities of a
bibenzyl from Empetrum nigrum var. japonicum
Sayuki Oka, Ryo Kuniba, Nozomi Tsuboi, Sayaka Tsuchida, Kazunari Ushida,
Shusuke Tomoshige & Kouji Kuramochi
To cite this article: Sayuki Oka, Ryo Kuniba, Nozomi Tsuboi, Sayaka Tsuchida, Kazunari Ushida,
Shusuke Tomoshige & Kouji Kuramochi (2019): Isolation, synthesis, and biological activities of a
bibenzyl from Empetrumꢀnigrum var. japonicum, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2019.1662279
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1662279
Isolation, synthesis, and biological activities of a bibenzyl from Empetrum
nigrum var. japonicum
Sayuki Okaa*, Ryo Kunibaa*, Nozomi Tsuboia*, Sayaka Tsuchida b,c, Kazunari Ushida
,
b,c
a
a
Shusuke Tomoshige
and Kouji Kuramochi
b
aDepartment of Applied Biological Science, Tokyo University of Science, Chiba, Japan; Academy of Emerging Sciences, Chubu
c
University, Kasugai-shi, Aichi, Japan; Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
ABSTRACT
ARTICLE HISTORY
Received 1 August 2019
Accepted 23 August 2019
4-(2-Hydroxyphenethyl)-2,6-dimethoxyphenol, a bibenzyl, was isolated from the leaves of
Empetrum nigrum var. japonicum, collected from Mount Tateyama. Japanese rock ptarmigans
frequently eat the leaves and fruits of this plant. The structure of the bibenzyl was confirmed
by NMR spectroscopic analysis and fully characterized. A synthesis of this compound was
accomplished by coupling 2-hydroxyphenylacetic acid with syringaldehyde, decarboxylation
of the resultant isoaurones, and hydrogenation of the double bond in the corresponding
stilbene. This compound displayed cytotoxic activity against human cancer cells (HCT116 and
Hela cells) and leukemia cells (HL-60 cells). The present study suggests that this plant serves
as a source of biologically active natural products. Also, our findings provide information on
the secondary metabolites in the diet of Japanese rock ptarmigans.
KEYWORDS
Bibenzyl; isolation; synthesis;
biological activity
Empetrum nigrum var. japonicum is an evergreen plant
that retains its leaves all year round [1]. This plant
mainly grows at high altitudes and in cold climates,
such as hilly and mountainous areas. In Japan, it is
distributed mainly in the Japan Alps and Hokkaido.
Japanese rock ptarmigans (Lagopus muta japonica) in
the Northern Japan Alps eat the leaves and fruits of
Empetrum nigrum var. japonicum [2]. The chemical
constituents of related plant species such as Empetrum
nigrum L. var. nigrum and Empetrum nigrum subsp.
asiaticum have been reported [3–12]. Chalcones, dihy-
drochalcones, phenanthrenes, dihydrophenanthrenes,
bibenzyls, and terpenoids were isolated from the leaves
of Empetrum nigrum L. var. nigrum [3−7].
Dihydrochalcones, flavonols, and anthocyanins were
isolated from the black crowberry (fruits) of this plant
[8,9]. However, little is known about the chemical con-
stituents of Empetrum nigrum var. japonicum [13,14].
In this study, 4-(2-hydroxyphenethyl)-2,6-dimethoxy-
phenol, a bibenzyl, was isolated from the leaves of
Empetrum nigrum var. japonicum. The structure of
the bibenzyl was determined by 1D and 2D NMR
spectroscopic analysis. Synthesis of this compound
was achieved and allowed its complete structural con-
firmation and biological evaluation. Evaluation of the
biological activities revealed that the bibenzyl exhibited
cytotoxic activity against human cancer cells such as
human colon carcinoma HCT116 cells, human cervix
epitheloid carcinoma Hela cells, and human promyelo-
cytic leukemia HL-60 cells.
Materials and methods
General information
All solvents and reagents were used without further
purification unless otherwise noted. Analytical TLC
was performed using Silica gel 60 F254 plates
(0.25 mm) (Merck, Darmstadt, Germany). Flash col-
umn chromatography was performed using silica gel
(SiliaFlash F60, particle size 40–63 μm, 230–400 mesh
ASTM) (SiliCycle, Québec, Canada). Melting point
(Mp) data were determined using a MM-2 instru-
ment (Shimadzu corp., Kyoto, Japan) and were
uncorrected. IR spectra were recorded on a FT-720
spectrometer (Horiba, Kyoto, Japan), using NaCl
(neat) or KBr pellets (solid). 1H and proton-
decoupled 13C (13C{1H}) NMR spectra were recorded
on a Bruker Avance 400 spectrometer (400 and 100
MHz, respectively) (Bruker, Billerica, MA), using
chloroform-d (CDCl3), acetone-d6 or methanol-d4
(CD3OD) as a solvent. Chemical shift values are
expressed in δ (ppm) relative to tetramethylsilane
(TMS, δ 0.00 ppm) or the residual solvent resonance
(CDCl3, δ 77.0 ppm for 13C NMR; acetone-d6, δ 2.04
ppm for 1H NMR and δ 29.4 ppm for 13C NMR; CD3
1
OD, δ 3.30 ppm for H NMR and δ 49.0 ppm for
13C NMR). Data are reported as follows: chemical
shift, multiplicity (s = singlet, d = doublet, m = multi-
plet), coupling constants (J; Hz), and integration.
Mass spectra were obtained by a JEOL JMS-700 dou-
ble-focusing mass spectrometer (JEOL, Tokyo, Japan)
CONTACT Kouji Kuramochi
kuramoch@rs.tus.ac.jp
*These authors contributed equally to this work.
The supplemental data for this article can be accessed here.
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
S. OKA ET AL.
using fast atom bombardment (FAB). FAB mass
spectra of compounds 1 and 5 were measured using
a 2:3 (v/v) mixture of glycerol and 3-nitrobenzyl
alcohol as a matrix in positive ion mode. The pH
was adjusted with a glass electrode type hydrogen ion
concentration indicator HM-30R (DKK-TOA cor-
poration). The optical density at 550 nm (OD550) of
each bacterial cell suspension was measured by
Ultrospec 2100 pro (GE healthcare). Colorimetric
changes were measured using a Spectra Max 190
microplate reader (Molecular Devices).
chromatography with hexane/EtOAc (2/1) to give ben-
zoic acid (51.6 mg) and compound 1 (23.7 mg).
Physical properties of 4-(2-hydroxyphenethyl)-
2,6-dimethoxyphenol (1)
Mp = 111–114°C; IR (KBr) νmax = 3395, 2957, 2934,
2852, 2835, 1615, 1592, 1521, 1507, 1464, 1459 cm−1;
1H and 13C{1H} NMR data in acetone-d6, see Table 1;
1H NMR (400 MHz, CD3OD) δ 6.99 (dd, J = 8.0, 1.7
Hz, 1H), 6.94 (ddd, J = 8.0, 7.4, 1.7 Hz, 1H), 6.74 (dd,
J = 8.0, 1.2 Hz, 1H), 6.68 (ddd, J = 8.0, 7.4, 1.2 Hz,
1H), 6.40 (s, 2H), 3.76 (s, 6H), 2.85–2.81 (m, 2H),
2.80–2.75 (m, 2H); 13C{1H} NMR (100 MHz, CD3
OD) δ 156.3, 148.9 (2C), 134.6, 134.3, 131.4, 129.4,
127.9, 120.4, 115.8, 106.7 (2C), 56.6 (2C), 37.2, 33.9;
HRMS (FAB/double-focusing MS) m/z: [M]+ Calcd
for C16H18O4 274.1205; Found 274.1208.
Collection of Empetrum nigrumvar. japonicum
The leaves and stems of Empetrum nigrum var. japo-
nicum were collected in Mt. Tateyama with permis-
sions from Ministry of the Environment of Japan
(No. 1,605,111), Forestry Agency of Japan (No.172
and No. 320 in 2016), and Toyama prefecture (No.
117) in accordance with the Environment Research
and Technology Development Fund (4–1604).
Synthesis of (E)-4-(2-hydroxystyryl)-
2,6-dimethoxyphenol (5)
A solution of 2-hydroxyphenylacetic acid (2) (2.28 g, 15.0
mmol), syringaldehyde (3) (2.73 g, 15.0 mmol) in
a mixture of triethylamine (3.8 mL) and acetic anhydride
(4.2 mL) was stirred at 110°C for 5 h. The mixture was
concentrated. The resulting residue was passed through
the short pad of silica gel to yield a crude isoaurone 4
(3.10 g). A solution of the crude isoaurone 4 (3.10 g) and
potassium hydroxide (85%, 5.80 g, 87.9 mmol) in ethy-
lene glycol (58 mL) was stirred at 180°C for 1.5 h. The
reaction was quenched by the addition of 1 M aqueous
Extraction and purification of compounds
The leaves (3 g) of Empetrum nigrum var. japonicum
were pulverized and suspended with methanol (100 mL).
The suspension was stirred at room temperature for
1 day. The mixture was filtered. The filtrate was concen-
trated to afford a crude extract (803 mg). This crude
extract was separated by silica gel column chromatogra-
phy with hexane/EtOAc (4/1 to 1/1) to give fractions 1–6.
Fraction 6 (116.2 mg) was separated by silica gel column
1
Table 1. H and 13C NMR spectroscopic data for compound 1.a
position
δC
δH (mult; J in Hz)
HMBC
1, 3, 4
1
2
3
4
5
6
α
β
133.6
106.7
148.4
134.9
148.4
106.7
36.8
6.48 (s)
6.48 (s)
2.82 − 2.77 (m)
2.90 − 2.83 (m)
1, 4, 5
1, 2, 6, 1ʹ
1, 1ʹ, 2ʹ, 3ʹ
33.4
1ʹ
2ʹ
3ʹ
4ʹ
5ʹ
6ʹ
3-OCH3
5-OCH3
129.0
155.9
131.0
120.2
127.7
115.7
56.5
7.04 (dd, 7.4, 1.7)
6.72 (ddd, 7.7, 7.4, 1.2)
7.00 (ddd, 8.0, 7.7, 1.7)
6.84 (dd, 8.0, 1.2)
3.76 (s)
2ʹ, 5ʹ
1ʹ, 6ʹ
2ʹ, 3ʹ
1ʹ, 4ʹ
3
56.5
3.76 (s)
5
aNMR spectra were measured in acetone-d6 as a solvent.

ISOLATION, SYNTHESIS, AND BIOLOGICAL ACTIVITY OF A BIBENZYL
3
HCl solution. The mixture was diluted with EtOAc. The
layers were separated, the organic layer was washed with
water, brine, dried over Na2SO4, and concentrated. The
residue was purified by silica gel chromatography (hex-
ane/EtOAc = 3/1) to yield 5 (1.58 g, 39% over two steps).
Mp = 89–90°C; IR (KBr) νmax = 3481, 3415, 3008, 2964,
2840, 1612, 1599, 1585, 1456 cm−1; 1H NMR (400 MHz,
CDCl3, TMS) δ 7.51 (d, J = 7.1 Hz, 1H), 7.21 (d, J = 16.3
Hz, 1H), 7.14 (dd, J = 7.8, 7.1 Hz, 1H), 7.04 (d, J= 16.3 Hz,
1H), 6.95 (dd, J = 7.8, 7.1 Hz, 1H), 6.82 (d, J = 7.8 Hz,
1H), 6.77 (s, 2H), 5.57 (s, 1H), 5.03 (s, 1H), 3.95 (s, 6H);
13C{1H} NMR (100 MHz, CDCl3) δ 152.8, 147.2 (2C),
134.7, 130.3, 129.2, 128.4, 127.1, 124.7, 121.2, 121.1,
115.9, 103.3 (2C), 56.3 (2C); HRMS (FAB/double-
focusing MS) m/z: [M]+ Calcd for C16H16O4 272.1049;
Found 272.1048.
blank wells (0 cells/100 μL) and control wells (5000
cells/100 μL). Adherent HCT116 and Hela cells were
preincubated for 24 h before exposure to the test
compounds. The plates were incubated with various
concentrations of each compound for 48 h. At the
end of incubation, 10 μL of the WST-8 solution was
then added, and the resulting mixture was incubated
for 2 h at 37°C. The absorbance values were then
measured at 450 nm with a 96-well plate reader.
Cell growth inhibition was evaluated as the ratio of
the absorbance of the sample to that of the control.
Evaluation of antimicrobial activity
The broth microdilution method [16] was used to
determine the minimum inhibitory concentration
(MIC) of the compounds against Escherichia coli
(NBRC 3301) and Bacillus subtilis (NBRC 3134).
Triplicate twofold serial dilutions of the test samples
(90 µL/well) were prepared in sterile flat-bottom 96-well
plates by dilution with Cation-adjusted Mueller-Hinton
broth (CMHB) (Kyokuto Pharmaceutical Industrial Co.
Ltd, Tokyo, Japan). After the bacterial cell suspension
was adjusted to 1.5 × 108 CFU/mL, which is comparable
to 0.5 McFarland turbidity standard [optical density at
550 nm (OD550) = 0.125], and diluted 30 times with
phosphate-buffered saline (PBS) (Nacalai tesque,
Kyoto, Japan) (5.0 × 106 CFU/mL), the adjusted cell
suspension (10 µL) was added into the wells, except for
the blank. The final concentration of bacteria in the
assay was 5 × 105 CFU/mL, and that of DMSO was
1.28% v/v. The plates were incubated at 37°C for 18
h. After incubation, the MIC was determined as the
lowest concentration at which no growth was observed
Synthesis of 4-(2-hydroxyphenethyl)-
2,6-dimethoxyphenol (1)
A suspension of 5 (706 mg, 2.59 mmol) and 10%
palladium on carbon (90.1 mg) in THF (10 mL) was
stirred under an H2 atmosphere at room temperature
for 18 h. The mixture was filtered through a short pad
of Celite and washed with EtOAc. The filtrate was
concentrated to yield pure 1 (681 mg, 96%). The
1H NMR spectrum of synthetic 1 in CD3OD was
identical with that of natural 1.
Cell culture
HCT116, Hela, HL-60, and Jurkat cells were pur-
chased from the Riken Cell Bank (Tsukuba, Japan).
BALL-1 cells were obtained from the Cell Resource
Center for Biomedical Research, Tohoku University
(Sendai, Japan). HCT116 and Hela cells were cultured
at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s
medium (DMEM) (Nacalai tesque, Kyoto, Japan)
supplemented with 10% fetal bovine serum (FBS)
(BioWest, Nuaillé, France). HL-60, Jurkat, and
BALL-1 cells were cultured at 37°C with 5% CO2 in
Roswell Park Memorial Institute (RPMI) 1640 med-
ium (Nacalai tesque, Kyoto, Japan) supplemented
with 10% FBS.
in triplicate wells, visually and by measuring OD550
.
Ampicillin (Nacalai tesque, Kyoto, Japan) was used as
a positive control.
Results
Isolation and structural characterization of
4-(2-hydroxyphenethyl)-2,6-dimethoxyphenol (1)
Separation of the extract from the leaves of
Empetrum nigrum var. japonicum using silica gel
yielded compounds 1 and benzoic acid. The mole-
cular formula of C16H18O4 for compound 1 was
determined by HRMS (FAB). The peak that corre-
sponds to the molecular ion [M]+ was observed
at m/z 274 in the FAB mass spectrum of 1 (Figure
Evaluation of cytotoxic activity
Cell growth was evaluated using cell counting kit SF
(Nacalai tesque, Kyoto, Japan) according to the man-
ufacturer’s instructions based on the WST-8
[4-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-
2H-tetrazol-3-ium-5-yl)-3-sulfobenzenesulfonate,
sodium salt] assay [15]. For the assay, the cells were
cultured in a 96-well plate, with each well containing
5000 cells in a total volume of 100 μL. The concen-
tration of dimethyl sulfoxide (DMSO) in the cell
cultures was 0.1% (v/v). The plates also included
1
S10 in Supplementary Material) [17]. The H and
13C NMR data for 1 are summarized in Table 1.
The HMBC spectrum suggested that 1 is a bibenzyl,
which has a 4-hydroxy-2,6-dimethoxylbenzyl moi-
ety and a 2-hydroxybenzyl moiety. Taken together,
the structure of 1 was determined to be 4-(2-hydro-
xyphenethyl)-2,6-dimethoxyphenol. Although the

4
S. OKA ET AL.
isolation of 1 from diseased Discorea mangenotiana
[18] and Dioscorea opposite [19] was reported pre-
viously, only 1H NMR data for 1 was reported.
A structural assignment and characterization for 1
was not made. In order to determine the structure
of 1 unambiguously and to obtain larger quantities
for biological experiments, a synthetic route toward
1 was examined.
below 100 μM. On the other hand, this compound
did not affect the proliferation of Jurkat and BALL-1
cells. The antibacterial activity of 1 against Gram-
positive B. subtilis and Gram-negative E. coli was
evaluated by broth microdilution assay [16].
However, compound 1 did not show antibacterial
activity against B. subtilis and E. coli at concentra-
tions below 128 μg/mL (Figure S11 in the
Supplementary Information).
Synthesis of 4-(2-hydroxyphenethyl)-
2,6-dimethoxyphenol (1)
Conclusion
The
synthesis
of
4-(2-hydroxyphenethyl)-
In this study, the isolation, structural determination,
synthesis, and biological evaluation of 4-(2-hydroxy-
phenethyl)-2,6-dimethoxyphenol (1) are reported. The
structure of this compound was fully characterized by
1D and 2D NMR spectroscopy. The synthesis of 1 was
accomplished in only three steps from commercially
available 2-hydroxyphenylacetic acid and syringalde-
hyde. The synthesis enabled unambiguous structural
confirmation and biological studies. The biological eva-
luation revealed that 1 exhibits cytotoxic activity against
HCT116, Hela and HL-60 cells at concentrations below
100 μM. However, this compound is not toxic against
Jurkat and BALL-1 cells at concentrations below 100
μM, nor is it toxic against B. subtilis and E. coli at
concentrations below 128 μg/mL. Jurkat and BALL-1
cells are derived from T-cell and B-cell acute lympho-
blastic leukemia, respectively. These results suggest that
this compound might exert the growth inhibitory activ-
ity against cancer cells without affecting the growth of
lymphocytes and enteric bacteria. Interesting biological
activities and structure–activity relationships of struc-
turally related natural products such as resveratrol [21]
and combretastatins [22] have been reported. Thus,
further biological studies including elucidation of the
structure-activity relationships and the mechanism of
action are currently underway. In addition, the present
study gives useful information on the diet of Japanese
2,6-dimethoxyphenol (1) starts from 2-hydroxyphe-
nylacetic acid (2) and syringaldehyde (3) (Figure 1).
A coupling of 2 with 3 using triethylamine and acetic
anhydride gave a mixture of E and Z-aurone 4 [20].
Without purification, crude 4 was treated with potas-
sium hydroxide in ethylene glycol at 180°C.
Hydrolysis of 4 and decarboxylation of the resultant
carboxylate occurred to give E-stilbene 5 in 39% yield
over two steps. Finally, hydrogenation of 5 gave 1 in
1
99% yield. The H NMR spectrum of synthetic 1 was
identical with that of natural 1, indicating that the
structure of 1 was unambiguously determined.
Biological activities of 4-(2-hydroxyphenethyl)-
2,6-dimethoxyphenol (1)
The cytotoxic activity of 1 against five cancer cells
(HCT116, Hela, HL-60, Jurkat and BALL-1 cells) was
evaluated via the WST-8 method [15] after treatment
with test compounds for 48 h (Table 2 and Figure S10
in the Supplementary Information). SN-38, the active
metabolite of irinotecan, was used as a positive con-
trol in this study. The 50% inhibitory concentrations
(IC50s) of cell viability of 1 are summarized in Table
2. Compound 1 showed cytotoxic activity against
HCT116, Hela, and HL-60 cells with IC50 values
Figure 1. Synthesis of compound 1.

ISOLATION, SYNTHESIS, AND BIOLOGICAL ACTIVITY OF A BIBENZYL
5
Table 2. Cytotoxic activity of 1 against human cancer cells.a
chemical structures and exudate localization. Bot
Acta. 1992;105:300–305.
IC50 value (μM)
[4] Arriaga-Giner FJ, Wollenweber E, Dörr M. Bibenzyl
compound
HCT116
88.4 10.4 70.4 3.9 63.2 1.3 > 100 > 100
< 3 < 3 < 3 < 3 < 10
Hela
HL-60
Jurkat BALL-1
form
crowberry
leaves.
Phytochemistry.
1
SN-38b
1993;33:725–726.
aFifty percent inhibitory concentrations (IC50s) of cell viability were
determined using the WST-8 assay. IC50 values are expressed as the
mean SD of triplicate experiments. bSN-38 was used as a positive
control.
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This research was supported by the Environment Research
and Technology Development Fund [ERTDF/No. 4-1604
and 4-1903] of the Environmental Restoration and
Conservation Agency of Japan.
Author contributions
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Empetrum nigrum. J Nat Prod. 2015;78:2837–2840.
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14786419.2018.1542390
S.Tsuchida, K.U. and K.K. designed the research; S.O., R.
K., N.T., S.Tsuchida, and K.U. performed the research. S.
Tomoshige and K.K. analyzed the data; N.T., S.Tsuchida,
K.U., S.Tomoshige and K.K. wrote the article.
Disclosure statement
No potential conflict of interest was reported by the
authors.
[13] Zhang YL, Mei RQ, Liu X, et al. Chemical constitu-
ents of Empetrum nigrum var. japonicum. Chin
Tradit Herbal Drugs. 2014;49:2293–2298.
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Funding
This research was supported by the Environment Research
and Technology Development Fund [ERTDF/No. 4-1604
and 4-1903] of the Environmental Restoration and
Conservation Agency of Japan.
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ORCID
Sayaka Tsuchida
Kazunari Ushida
Shusuke Tomoshige
5809
http://orcid.org/0000-0003-3753-990X
http://orcid.org/0000-0002-3155-4538
http://orcid.org/0000-0002-4948-
Kouji Kuramochi
http://orcid.org/0000-0003-0571-9703
[18] Kaganda NG, Adesanya SA. A new dihydrostilbene
from diseased Dioscorea mangenotiana. J Nat Prod.
1990;53:1345–1346.
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