Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides

Article abstract of DOI:10.1080/09168451.2018.1530970

Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC₅₀ 7.0 μM) among the tested compounds.

Full text of DOI:10.1080/09168451.2018.1530970

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Chemoenzymatic synthesis of hydroxytyrosol
monoesters and their suppression effect
on nitric oxide production stimulated by
lipopolysaccharides
Ayaka Sakakura, Martin Pauze, Atsuhiro Namiki, Megumi Funakoshi-Tago,
Hiroomi Tamura, Kengo Hanaya, Shuhei Higashibayashi & Takeshi Sugai
To cite this article: Ayaka Sakakura, Martin Pauze, Atsuhiro Namiki, Megumi Funakoshi-
Tago, Hiroomi Tamura, Kengo Hanaya, Shuhei Higashibayashi & Takeshi Sugai (2018):
Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric
oxide production stimulated by lipopolysaccharides, Bioscience, Biotechnology, and Biochemistry,
DOI: 10.1080/09168451.2018.1530970
To link to this article: https://doi.org/10.1080/09168451.2018.1530970
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2018.1530970
Chemoenzymatic synthesis of hydroxytyrosol monoesters and their
suppression effect on nitric oxide production stimulated by
lipopolysaccharides
Ayaka Sakakuraa, Martin Pauzea,b, Atsuhiro Namikia, Megumi Funakoshi-Tagoa, Hiroomi Tamuraa,
a
Kengo Hanayaa, Shuhei Higashibayashia and Takeshi Sugai
aFaculty of Pharmacy, Keio University, Tokyo, Japan; bDepartment of Chemistry, Graduate School of SIGMA Clermont, Aubiere Cedex, France
ABSTRACT
ARTICLE HISTORY
Received 20 August 2018
Accepted 25 September 2018
Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in
two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase
B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic
hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to
the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric
oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxy-
tyrosol butyrate exhibited the highest inhibition (IC50 7.0 μM) among the tested compounds.
KEYWORDS
Hydroxytyrosol; fatty acid
monoesters; chemoselective
acylation; site-selective
hydroxylation;
anti-inflammatory effect
Oleuropein (1a) and its hydrolysate, hydroxytyrosol
(1b, Scheme 1) are representative antioxidative cate-
chols in olives [1]. A variety of physiological activities
of 1b have been reported [2–15]. For example, 1b
exhibited anti-inflammatory activity by preventing
nitric oxide (NO) production stimulated by lipopoly-
saccharides (LPS) in macrophages [15]. Recently, one
of the authors (M. F.-T.) has investigated the protec-
tive effect of 1b and its ester derivatives 1c and 1d
against neuronal cells apoptosis induced by the
Parkinson’s disease-related neurotoxin 6-hydroxydo-
pamine (6-OHDA) in SH-SY5 cells, with butyrate 1d
showing the strongest inhibition. Only 1d among
three compounds induced the expression of the tran-
scription factor, NF-E2-related factor-2 (Nrf2) and
enhanced its transcriptional activation in those cells
[16]. Independently, they found that Nrf2 suppressed
the LPS-induced transcriptional up-regulation of the
proinflammatory cytokines and the inducible NO
synthase (iNOS) [17]. We became interested in the
inhibitory effect of esters 1c–f in NO generation in
the murine macrophage RAW 264.7 cell line. For
longer chain fatty acid esters 1e (stearate) and 1f
(oleate), which are known as antioxidants [18–22],
the esterification with long chain fatty acids was
expected to increase the effectiveness [23,24].
However, 1b itself is an expensive starting mate-
rial, although it can be supplied either by the extrac-
tion and purification of wastewater from the
manufacture of olive oil [12,27–30]. Chemists’ efforts
have been devoted to the synthesis of 1b [30–33].
Among them, the site-selective hydroxylation of tyr-
osol (2a) with 2-iodoxybenzoic acid (IBX) as shown
in Scheme 1 is straightforward. The reported yield,
however, was only 30% due to the difficulty in isola-
tion of the desired product 1b. Those authors insisted
that either increased lipophilicity of substrates or use
of the elaborated polymer-supported IBX derivative
[33] is necessary, to acquire an enhanced yield.
To solve above-mentioned problems, we herein
describe alternative routes for the synthesis of 1c–f
from readily available 2a, by exchanging the order of
hydroxylation and acylation.
Results and discussion
Candida antarctica lipase B-catalyzed acylation of 2a
has so far been reported [22,34]. In our case, the
desired reaction proceeded smoothly by employing a
short chain fatty acid vinyl ester to give the corre-
sponding acetate 2b and butyrate 2c in a quantitative
manner. Hydroxylation adjacent to the phenolic
hydroxy group was performed by IBX-mediated oxi-
dation [21] and subsequent reduction with sodium
borohydride [35]. The desired products 1c (at 88%
conversion) and 1d (at 91% conversion) were isolated
in 62% and 58% yields, respectively (Scheme 2).
Due to limited availability of long chain fatty
acid vinyl esters with high purity, we adopted free
For the biological assay, the efficient preparation
of fatty acid esters 1c–f was demanded. In the pre-
vious study [16], butyrate 1d was synthesized by
lipase-mediated chemoselective acylation of primary
aliphatic hydroxy group over phenolic hydroxy
groups in commercially available 1b [25,26] as
shown in Scheme 1.
CONTACT Takeshi Sugai
sugai-tk@pha.keio.ac.jp; Megumi Funakoshi-Tago
tago-mg@pha.keio.ac.jp
© 2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
A. SAKAKURA ET AL.
fatty acids for the lipase-catalyzed acylation [25] in
the synthesis of stearate 2d and oleate 2e.
Molecular sieves 4A were added to remove water
that was concomitantly formed during the esterifi-
cation. The reactions were slow even under such
forced conditions and required prolonged reaction
times (48 h) at room temperature, compared with
the reactions of the short chain vinyl esters. At a
conversion of around 50%, the reaction was worked
up, and the desired products were purified by med-
ium-pressure liquid chromatography. Monoesters
2c and 2d were isolated in 39% and 42% yields,
respectively (Scheme 2). Although the hydroxyla-
tion yields were somewhat lower, stearate 1e and
oleate 1f were isolated in 40% and 48% yields in
sufficient amounts for the subsequent evaluation of
inhibitory effects on NO production stimulated by
LPS in RAW264.7 cells.
The results for the suppression effects of 1b-f on
NO production are summarized in Figure 1(a–e).
Judging from the bar charts, butyrate 1d exhibited
the strongest inhibitory effect (Figure 1(c)) among the
tested compounds. The estimated IC50 values (μM)
for each compound were 18.5 for 1b, 12.7 for 1c, 7.0
for 1d, 14.5 for 1e, and 11.2 for 1f, respectively. It
should be noted that none of the compounds used in
this study caused cell death, indicating that their
inhibitory effects on NO production were not due
to their cytotoxicity.
probably ascribable to the cell permeability
depending upon the lipophilicity of compounds.
The synthesis and evaluation of acylated forms
with fatty acids of intermediate chain length
between 1d and 1e may provide a clue to clarify
this phenomenon.
Conclusion
We synthesized four fatty acid monoesters (1c-f)
of hydroxytyrosol (1b) from inexpensive tyrosol
(2a) by combining lipase-catalyzed chemoselective
acylation and subsequent hydroxylation. By arran-
ging the order of these two reactions in this way,
the total yield of the products and the handling of
the intermediates were improved from those in the
originally reported procedure [25,26]. All products
showed inhibitory effects on NO production sti-
mulated by LPS. Among them, butyrate 1d was
the most potent inhibitor with IC50 value of 7.0
μM. In this way, we found a new property of
hydroxytyrosol monoesters other than so-far
reported anti-apoptosis effect.
Experimental
General
Candida antarctica lipase B (Novozym 435) was pur-
chased from Novozymes Japan. 2-(4-Hydroxyphenyl)
ethanol (tyrosol, H0720) was purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan).
RAW264.7 cells were purchased from the Riken Cell
Bank (Ibaraki, Japan). LPS (E. coli 055:B5) was pur-
chased from Sigma-Aldrich Co. (St, Louis, MO,
USA). Dulbecco’s Modified Eagle’s Medium
We state here only a brief comment for the pro-
posed mechanism of the inhibition. As mentioned in
the introduction [16,17], butyrate 1d induced the
expression of Nrf2 and enhanced its transcriptional
activation in the cells. Also, Nrf2 interfered with the
LPS-induced transcriptional up-regulation of the
proinflammatory cytokines and inducible NO
synthase (iNOS) in RAW264.7 cells. From these find-
ings, we suppose that butyrate 1d effectively inhibited
NO production by activating Nrf2.
(DMEM) and
a penicillin (10,000 units/mL)-
Streptomycin (1 mg/mL) mixed solution were pur-
chased from Nacalai Tesque Inc. (Kyoto, Japan). Fetal
bovine serum (FBS, Gibco®) was purchased from
Thermo Fisher Scientific Inc. (Waltham MA, USA).
(Nacalai Tesque). Column chromatography was per-
formed with silica gel (Kanto Chemical Co. Silica Gel
The action of intracellular short-chain fatty acid
esterases on 1b–d would promptly converge them
into the identical free form 1b. The difference in
inhibitory effect among those of compounds is

HYDROXYTYROSOL MONOESTERS
3
Scheme 1. Catechols involved in olives 1a and 1b, examples of fatty acid monoesters 1c to 1f derived from 1a, and the
previous synthesis of 1d.
Scheme 2. Chemoenzymatic synthesis of 1c - f starting from readily available tyrosol 2a.
60 N 37560-79, spherical and neutral, 40–50 µm).
Medium-pressure liquid chromatography was per-
formed using Silica Gel 60 (spherical and neutral;
100–210 µm, 37560-79, Kanto Chemical Co.) and
the Isolera One flash purification system (Biotage,
Sweden). Preparative TLC was performed with
Merck Silica Gel 60 F254 plates (0.5 mm thickness,
Tokyo Japan) was used for the measurement of
absorbance.
2-(4-Hydroxylphenyl)ethyl acetate (2b)
To a solution of 2a (2.8 g, 20 mmol) in tetrahydrofuran
(THF, 37 mL) was added C. antarctica lipase B (409 mg)
and vinyl acetate (2.8 mL, 30 mmol). The mixture was
stirred for 4 h at room temperature. The mixture was
diluted with chloroform and filtered with a pad of Celite.
The combined filtrate and washings were dried in vacuo
to give 2b as colorless solid (3.84 g, quantitative). M.p.
59–60°C. 1H-NMR (400 MHz, DMSO- d6) δ: 1.96 (3H,
s), 2.73 (2H, t, J = 7.0 Hz), 4.10 (2H, t, J = 7.0 Hz), 6.66
(2H, d, J = 8.4 Hz), 7.00 (2H, d, J = 8.4 Hz), 9.19 (s, OH).
13C-NMR (100 MHz, DMSO-d6) δ: 21.2, 34.0, 65.2, 115.6,
128.3, 130.2, 156.3, 170.8. IR νmax cm–1: 3361, 3022, 2957,
1705, 1614, 1596, 1515, 1444, 1388, 1364, 1227, 1173,
1107, 1030, 975, 830. HR-MS [ESI+, (M+Na)+] calculated
for C10H12O3Na, 203.0684; found, 203.0707.
1
No. 1.05744.0001). H NMR spectra were measured
at 400 MHz on a VARIAN 400-MR or at 500 MHz
on an Agilent INOVA-500 spectrometer and 13C
NMR spectra were measured at 100 MHz on a
VARIAN 400-MR or at 125 MHz on an Agilent
INOVA-500 spectrometer. DMSO-d6 and CDCl3
were used as a solvent and the residual peaks were
used as an internal standard (1H NMR: DMSO-d6
2.48 ppm, CHCl3 in CDCl3 7.26 ppm; 13C NMR:
DMSO-d6 39.9 ppm, CDCl3 77.0 ppm). IR spectra
were measured as ATR on a Jasco FT/IR-4700 FT-IR
spectrometer. High resolution mass spectra (HRMS)
were measured by a on Jeol JMS-T100LP AccuTOF.
Microplate reader Infinite M1000 (Tecan Group Ltd.

4
A. SAKAKURA ET AL.
Figure 1. Suppression effect of 1b - f on the NO production stimulated by LPS in RAW264.7 cells. Shaded (left) bars and open
(right) bars represent concentration of NO without (control experiments) or with LPS, respectively. Values are given as the
mean S.D. from four independent experiments. Multiple group comparisons were made using one- or two-way analysis of
variance (ANOVA) followed by Tukey’s test. *P < 0.05; **P < 0.01; ***P < 0.001 significantly different from the control group
stimulated with LPS. For detail, see experimental section.
2-(4-Hydroxylphenyl)ethyl butyrate (2c)
J = 8.4 Hz). Due to the broadening of signal, proton
on phenolic hydroxy group was not detected. 13C-
NMR (125 MHz, CDCl3) δ: 14.1, 22.7, 24.9, 29.1,
29.3, 29.4, 29.5, 29.6, 29.7, 29.7, 31.9, 34.3, 34.4, 65.0,
115.3, 130.0, 154.2, 174.0. Some signals of methylene
carbons were overlapped. IR νmax cm–1: 3252, 2915,
2847, 2359, 1731, 1615, 1519, 1464, 1392, 1251, 1234,
1214, 1193, 1163, 826, 719, 564, 521. HR-MS [ESI+, (M
+Na)+] calculated for C26H44O3Na, 427.3188; found,
427.3171.
In a similar manner as described for the synthesis of 2b
in the previous section, diol 2a was treated with C.
antarctica lipase B and vinyl butyrate. The workup
and purification furnished 2c as pale yellow oil (5.36 g,
1
quantitative). H-NMR (400 MHz, DMSO-d6) δ: 0.84
(3H, t, J = 7.4 Hz), 1.51 (2H, dq, J = 7.3, 7.4 Hz), 2.23
(2H, t, J = 7.3 Hz), 2.75 (2H, t, J = 7.0 Hz), 4.14 (2H, t,
J = 7.0 Hz), 6.67 (2H, d, J = 8.6 Hz), 7.02 (2H, d,
J = 8.6 Hz), 9.21 (OH, s). 13C-NMR (125 MHz,
DMSO-d6) δ: 13.8, 18.4, 34.0, 35.8, 65.0, 115.6, 128.3,
130.2, 156.3, 173.1. IR νmax cm−1 3380, 2963, 2935, 2875,
1704, 1614, 1596, 1515, 1445, 1389, 1356, 1308, 1185,
1171, 1105, 1046, 985, 828. HR-MS [ESI+, (M+Na)+]:
calculated for C12H16O3Na 231.0997; found, 231.1001.
2-(4-Hydroxylphenyl)ethyl oleate (2e)
In a similar manner as described for the synthesis of
2d in the previous section, diol 2a was treated with C.
antarctica lipase B and oleic acid. The workup and
purification furnished 2e as colorless amorphous
1
2-(4-Hydroxylphenyl)ethyl stearate (2d)
solid (180 mg, 42%). H-NMR (500 MHz, CDCl3) δ:
0.81 (3H, t, J = 6.9 Hz), 1.16–1.34 (20H), 1.43–1.63
(2H, m), 1.87–2.01 (4H, m), 2.21 (2H, t, J = 7.5 Hz),
2.78 (2H, t, J = 7.1 Hz), 4.17 (2H, t, J = 7.1 Hz), 5.10–
5.44 (2H, m), 6.68 (2H, d, J = 8.4 Hz), 7.00 (2H, d,
J = 8.4 Hz). Due to the broadening of signal, proton
on phenolic hydroxy group was not detected. 13C-
NMR (125 MHz, CDCl3) δ: 14.1, 22.7, 24.9, 27.2,
27.2, 29.1, 29.2, 29.3, 29.5, 29.7, 29.8, 31.9, 34.3,
34.4, 65.0, 115.3, 129.8, 130.0, 130.0, 154.3, 174.0.
Some signals of methylene carbons were overlapped.
IR νmax cm–1: 3389, 3004, 2923, 2852, 2359, 2342,
1737, 1710, 1615, 1596, 1517, 1456, 1354, 1225,
1172, 1006, 830, 724. HR-MS [ESI+, (M+Na)+] calcu-
lated for C26H42O3Na, 425.3032; found, 425.3035.
To a solution of 2a (150 mg, 1.1 mmol) in THF
(1.5 mL) was added C. antarctica lipase B (15 mg),
molecular sieves 4A (50 mg), and stearic acid (460 mg,
1.6 mmol). The mixture was stirred for 48 h at room
temperature. The mixture was diluted with chloro-
form and filtered with a pad of Celite. The combined
filtrate and washings were dried in vacuo. The residue
was purified by medium-pressure liquid chromatogra-
phy. Elution with hexane-ethyl acetate (10:1) furn-
ished 2d as colorless amorphous solid (172 mg, 39%).
1H-NMR (500 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.0 Hz),
1.16–1.34 (28H, m), 1.51–1.65 (2H, m), 2.28 (2H, t,
J = 7.6 Hz), 2.86 (2H, t, J = 7.1 Hz), 4.24 (2H, t,
J = 7.1 Hz), 6.66 (2H, d, J = 8.4 Hz), 7.00 (2H, d,

HYDROXYTYROSOL MONOESTERS
5
2-(3,4-Dihydroxylphenyl)ethyl acetate (1c)
2-(3,4-Dihydroxylphenyl)ethyl stearate (1e)
IBX (3.34 g, 45% purity, 5.4 mmol) was sonicated in
N,N-dimethylformamide (DMF, 128 mL). The result-
ing suspension was stirred at – 15°C under Ar, and to
that was added a solution of 2b (724 mg, 4.0 mmol)
in DMF (12 mL) in one portion. The mixture was
further stirred for 22 h at – 15°C under Ar. To the
mixture, a pre-cooled solution of sodium borohy-
dride (NaBH4, 120 mg) in ethanol (8.0 mL) was
slowly added at – 15°C, and the color of the mixture
turned from brown to yellow. The mixture was
diluted with ethyl acetate (180 mL) and to that was
added a solution of 0.1 M phosphate buffer with 3.5%
of sodium dithionite and 35% of sodium chloride.
The mixture was stirred at room temperature for
1.5 h. The organic layer was separated, and the aqu-
eous layer was extracted with ethyl acetate twice. The
combined extract was washed with aqueous solution
including sodium hydrogen carbonate (10%) and
sodium dithionite (5%) and brine, dried over anhy-
drous sodium sulfate and concentrated in vacuo. The
residue was purified by silica gel column chromato-
graphy. Elution with hexane-ethyl acetate (5:1 to 3:1)
furnished 1c as a pale yellow oil (489 mg, conversion:
In a similar manner as described for the synthesis
of 1c in the previous section, stearate 2d (221 mg,
0.55 mmol) was treated with IBX then NaBH4. The
workup and purification furnished 1e as colorless
amorphous solid (92 mg, yield: 40%). 1H-NMR
(500 MHz, CDCl3) δ: 0.81 (3H, t, J = 7.0 Hz),
1.07–1.32 (28H, m), 1.51–1.65 (2H, m), 2.22 (2H,
t, J = 7.6 Hz), 2.75 (2H, t, J = 7.1 Hz), 4.16 (2H, t,
J = 7.1 Hz), 5.13 (1H, broad), 5.30 (1H, broad),
6.57 (1H, dd, J = 2.0, 8.0 Hz), 6.67 (1H, d,
J = 2.0 Hz), 6.72 (1H, d, J = 8.0 Hz). 13C-NMR
(125 MHz, CDCl3) δ: 14.1, 22.7, 24.9, 29.1, 29.3,
29.4, 29.7, 29.7, 31.9, 34.4, 34.4, 64.8, 115.3, 116.0,
121.4, 130.9, 142.1, 143.5, 174.0. Some signals of
methylene carbons were overlapped. IR νmax cm–1:
3321, 2954, 2915, 2848, 1737, 1598, 1520, 1462,
1330, 1275, 1254, 1234, 1213, 1192, 1173, 1119,
961, 822, 728. HR-MS [ESI+, (M+Na)+] calculated
for C26H44O4Na, 443.3137; found, 443.3159.
2-(3,4-Dihydroxylphenyl)ethyl oleate (1f)
In a similar manner as described for the synthesis
of 1c in the previous section, oleate 2f (120 mg,
0.3 mmol) was treated with IBX then NaBH4. The
workup and purification furnished 1f as a colorless
oil (60 mg, yield: 48%). 1H-NMR (500 MHz,
CDCl3) δ: 0.88 (3H, t, J = 7.0 Hz), 1.07–1.32
(20H, m), 1.51–1.63 (2H, m), 1.92–2.10 (4H, m),
2.29 (2H, t, J = 7.6 Hz), 2.81 (2H, t, J = 7.1 Hz),
4.24 (2H, t, J = 7.1 Hz), 5.23–5.45 (2H, m), 6.64
(1H, dd, J = 1.5, 8.0 Hz), 6.73 (1H, d, J = 1.5 Hz),
6.78 (1H, d, J = 8.0 Hz). Due to the broadening of
signal, proton on phenolic hydroxy group was not
detected. 13C-NMR (125 MHz, CDCl3) δ: 14.1,
22.7, 24.9, 27.2, 29.1, 29.2, 29.3, 29.5, 29.7, 29.8,
31.9, 34.4, 34.4, 64.9, 115.3, 115.9, 121.3, 129.8,
130.0, 130.8, 142.1, 143.5, 174.1. Some signals of
methylene carbons were overlapped. IR νmax cm–1:
3334, 3004, 2918, 2849, 2360, 2341, 1732, 1706,
1604, 1520, 1463, 1276, 1252, 1214, 1179, 1113,
813, 720, 504. HR-MS [ESI+, (M+Na)+] calculated
for C26H44O4Na, 441.2981; found, 441.2990.
1
85%; yield: 62%). H-NMR (500 MHz, DMSO-d6) δ:
1.96 (3H, s), 2.67 (2H, t, J = 7.1 Hz), 4.08 (2H, t,
J = 7.1 Hz), 6.45 (1H, dd, J = 2.2, 8.0 Hz), 6.59 (1H, d,
J = 2.2 Hz), 6.62 (1H, d, J = 8.0 Hz), 8.67 (OH, s),
8.75 (OH, s). 13C-NMR (100 MHz, DMSO-d6) δ:
21.2, 34.2, 65.2, 116.0, 116.6, 119.9, 129.0, 144.2,
145.5, 170.8. IR νmax cm−1 3360, 3035, 2959, 1703,
1605, 1519, 1444, 1387, 1363, 1241, 1192, 1149, 1113,
1031, 978, 956, 864, 807, 782. HR-MS [ESI+, (M
+Na)+]: calculated for C10H12O4Na, 219.0633;
found, 219.0671.
2-(3,4-Dihydroxylphenyl)ethyl butyrate (1d)
In a similar manner as described for the synthesis
of 1c in the previous section, butyrate 2c (418 mg,
2.0 mmol) was treated with IBX then NaBH4. The
workup and purification furnished 1d as colorless
1
oil (261 mg, conversion: 91%; yield: 58%). H-NMR
(400 MHz, DMSO-d6) δ: 0.85 (3H, t, J = 7.3 Hz),
1.51 (2H, tq, J = 7.3, 7.3 Hz), 2.24 (2H, t,
J = 7.3 Hz), 2.69 (2H, t, J = 7.1 Hz), 4.12 (2H, t,
J = 7.1 Hz), 6.46 (1H, dd, J = 2.2, 7.8 Hz), 6.60
(1H, d, J = 2.2 Hz), 6.63 (1H, d, J = 7.8 Hz), 8.69
(OH, s), 8.76 (OH, s). 13C-NMR (125 MHz,
DMSO-d6) δ: 13.8, 18.4, 34.3, 35.8, 65.0, 115.9,
116.6, 119.9, 129.0, 144.2, 145.5, 173.2. IR νmax
cm−1 3378, 3035, 2963, 2934, 2875, 1703, 1605,
1520, 1444, 1384, 1346, 1279, 1256, 1184, 1112,
1044, 986, 956, 923, 865, 807, 782. HR-MS [ESI+,
(M+Na)+]: calculated for C12H16O4Na, 247.0946;
found, 247.0938.
Examination of the effect of 1b–1f on the
production of NO mediated by LPS
The murine macrophage cell line, RAW264.7 was
cultured at 37°C under 5% CO2/95% air in DMEM
supplemented with 10% FBS and penicillin-strepto-
mycin mixed solution. RAW264.7 cells (2 × 105
cells) were cultured in a 24-well plate and pre-incu-
bated with various concentrations of hydroxytyrosol
and its derivatives (1b-f) or a solution of dimethyl
sulfoxide (0.1%) at 37°C for 1 h prior to the

6
A. SAKAKURA ET AL.
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in the culture supernatants was estimated with
microplate reader at the absorbance at 540 nm,
after coloration using Griess reagent, which con-
–
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a phenolic
N-naphthylethylenediamine (0.1%), and H3PO4
(2.5%). IC50 values were calculated from the concen-
tration of 1b-f which was required for 50% inhibi-
tion of NO production in the experiments. The
results were expressed as the bar charts in Figure 1
(a–e).
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Acknowledgments
M.P. thanks both of Graduate School of SIGMA Clermont
and Graduate School of Pharmaceutical Sciences, Keio
University, to participate in this study as a visiting research
student under agreement from May to September in 2017.
Disclosure statement
[10] Fabiani R, De Bartolomeo A, Rosignoli P, et al
Cancer chemoprevention by hydroxytyrosol isolated
from virgin olive oil through G1 cell cycle arrest and
apoptosis. Eur J Cancer Prev. 2002;11:351–358.
[11] Palmerini CA, Carlini E, Saccardi C, et al Activity of
olive oil phenols on lymphomonocyte cytosolic cal-
cium. J Nutr Biochem. 2005;16:109–113.
[12] Schaffer S, Podstawa M, Visioli F, et al
Hydroxytyrosol-rich olive mill wastewater extract
protects brain cells in vitro and ex vivo. J Agric
Food Chem. 2007;55:5043–5049.
[13] Fabiani R, De Bartolomeo A, Rosignoli P, et al
Virgin olive oil phenols inhibits proliferation of
human promyelocytic leukemia cells (HL60) by
inducing apoptosis and differentiation. J Nutr.
2006;136:614–619.
[14] Guichard C, Pedruzzi E, Fay M, et al
Dihydroxyphenylethanol induces apoptosis by activat-
ing serine/threonine protein phosphatase PP2A and pro-
motes the endoplasmic reticulum stress response in
human colon carcinoma cells. Carcinogenesis.
2006;27:1812–1827.
No potential conflict of interest was reported by the
authors.
Funding
This work was supported by Japan society for the
Promotion of Science KAKENHI (17K08286) for M.F.-T.
and by a grant from a public interest incorporated founda-
tion “Amano Institute of Technology” for T.S. and are
gratefully acknowledged with thanks.
ORCID
Takeshi Sugai
http://orcid.org/0000-0003-4416-6435
Author contributions
T.S and M.F-T. designed this study; A.S. and M.P.
mainly carried out the experiments in chemistry; A.N.
mainly carried out the experiment in biology; T.S. and
M.F.-T. wrote the manuscript with assistance from all
authors; and K.H., S.H. and H.T. occasionally discussed
on the results.
[15] Takeda Y, Bui VN, Iwasaki K, et al. Influence of olive-
derived hydroxytyrosol on the toll-like receptor 4-
dependent inflammatory response of mouse peritoneal
macrophages. Biochem Biophys Res Commun.
2014;446:1225–1230.
[16] Funakoshi-Tago M, Sakata T, Fujiwara S, et al
Hydroxytyrosol butyrate inhibits 6-OHDA-induced
apoptosis through activation of the Nrf2/HO-1 axis in
SH-SY5Y cells. Eur J Pharmacol. 2018;834:246–256.
[17] Niino T, Tago K, Yasuda D, et al A 5-hydroxyox-
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[18] Trujillo M, Mateos R, Dollantes de Teran L, et al
Lipophilic hydroxytyrosyl esters. Antioxidant activity
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