Lipophilic hydroxytyrosol esters: Fatty acid conjugates for potential topical administration

Article abstract of DOI:10.1021/np200405s

Hydroxytyrosol is a potent antioxidant natural molecule isolated from olive leaves and fruits. The presence of three hydroxy groups in its structure poses a limit for the topical application of this lead compound. A set of hydroxytyrosol conjugates with fatty acids at different molecular weights were synthesized under mild conditions. The topical delivery features of this new set of antioxidant molecules were evaluated as a function of their permeation profiles through the human stratum corneum and viable epidermis membranes. A dependence on their partition coefficients, their molecular weights, and their isometric configurations was then postulated. Encouraging results prompt further investigations on the polyfunctional role that hydroxytyrosol conjugates could have as agents in both anti-inflammatory and antioxidant therapies.

Full text of DOI:10.1021/np200405s

Article
pubs.acs.org/jnp
Lipophilic Hydroxytyrosol Esters: Fatty Acid Conjugates for Potential
Topical Administration
Antonio Procopio,*,† Christian Celia,† Monica Nardi,‡ Manuela Oliverio,† Donatella Paolino,§
and Giovanni Sindona‡
†Dipartimento Farmacobiologico, Universita
(CZ), Italy
̀
Magna Græcia di Catanzaro Complesso Nini Barbieri, 88021, Roccelletta di Borgia
̀
‡Dipartimento di Chimica, Universita
̀
della Calabria, Ponte Bucci, 87030, Arcavacata di Rende (CS), Italy
§Dipartimento di Medicina Sperimentale e Clinica, Universita
Catanzaro, Italy
̀
Magna Græcia di Catanzaro, Viale Europa, Loc. Germaneto, 88100,
S
* Supporting Information
ABSTRACT: Hydroxytyrosol is a potent antioxidant natural molecule
isolated from olive leaves and fruits. The presence of three hydroxy
groups in its structure poses a limit for the topical application of this
lead compound. A set of hydroxytyrosol conjugates with fatty acids at
different molecular weights were synthesized under mild conditions.
The topical delivery features of this new set of antioxidant molecules
were evaluated as a function of their permeation profiles through the
human stratum corneum and viable epidermis membranes. A dependence on their partition coefficients, their molecular weights,
and their isometric configurations was then postulated. Encouraging results prompt further investigations on the polyfunctional
role that hydroxytyrosol conjugates could have as agents in both anti-inflammatory and antioxidant therapies.
he topical treatment of diseases has attracted great interest
in the pharmaceutical sciences.1,2 Natural and synthetic
tion,14 inhibits several lipoxygenases,15 and scavenges peroxyl
radicals.16 In addition, hydroxytyrosol alters L-iso-aspartine
residues induced by UVA irradiation and counteracts the
cytotoxic effects of reactive oxygen species (ROS) in various
human cellular systems.17 These findings have suggested that
hydroxytyrosol may be proposed as a potent natural antioxidant
and anti-inflammatory agent for several therapeutic applica-
tions,10,18 and the topical route might represent an efficacious
pathway for its administration.
These properties have prompted various procedures for
chemical19 and enzymatic synthesis20 or extraction of
hydroxytyrosol.21 However, the physicochemical features of
hydroxytyrosol suggest this compound will undergo a low
topical permeation through the human stratum corneum and
viable epidermis (SCE) membranes. In recent years, the
conjugation of hydroxytyrosol with fatty acids of different
molecular weights has been suggested to increase the
lipophilicity of the molecule without modifying its antioxidant
properties.22−25 There is still a need for new methods for the
synthesis of these derivatives since the few reported examples
are in the patent literature23−26 or involve the use of enzymes,27
with the yields having been obtained for only two reported
examples.28 Our group has developed environmentally friendly
catalytic methods for the strategic protection/deprotection
steps of functional groups,29−31 and some of them concern the
acylation of alcohols and phenols using lanthanoid salts as
T
products are generally proposed for their therapeutic activities,
and their effects are evaluated extensively through different
formulations.3,4 Even if a systemic pathway represents the best
option for these drugs, side effects, frequently related to the
formulation excipients or due to metabolic products, may occur
after administration.5 Recently, different groups have proposed
the topical administration route as an alternative for drug
activity.6 Both natural and synthetic anti-inflammatory and
antioxidant drugs have been applied topically to the skin, and
their effects have been evaluated using “in vitro”7 and “in vivo”8
models. Natural products, as free or entrapped formulations,
have been considered in these models as an efficient alternative
to synthetic drugs. In particular, olive leaves (Olea europea L.,
Oleaceae) and olive oil extracts have been considered as
therapeutic agents, and various examples of these derivatives are
reported in the literature.9 Experimental findings showed that,
although a complex matrix effect is involved in the therapeutic
activity of olive oil, phenolic derivatives are generally associated
with its efficacy. To this purpose, our research group has
described phenolic derivates, such secoiridoid derivatives of 3,4-
dihydroxyphenylethanol (hydroxytyrosol), as potential cyclo-
oxygenase inhibitors7 and antitumor drugs.10 In particular,
peracetylated derivatives of hydroxytyrosol show an efficacy due
to their antioxidant properties if applied both in “in vitro”7,11
and “in vivo”12 models. Furthermore, hydroxytyrosol exerts a
protective effect on UVA-irradiated cells, thus preventing the
overexpression of typical oxidative stress markers, and delays
LDL (low-density lipoprotein),13 prevents platelet aggrega-
Received: May 12, 2011
Published: October 20, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
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Article
Lewis acid catalysts. Herein, a simple and efficient catalytic
method to obtain acylated hydroxytyrosol derivatives with a
long alkyl chain is reported. Butyryl, decanoyl, elaidyl, linoleyl,
oleyl, stearyl, and palmitoyl moieties have been conjugated to
hydroxytyrosol, and permeation studies carried out in “in vitro”
modeling using Franz diffusion cells.
Table 1. Experimental and Calculated log Kow Values of
Compounds 1a−g
a
b
c
compound
hydroxytyrosol
log Kow
cLog Kow
miLog Kow
0.809
1.1
0.516
2.645
7.697
5.35
hydroxytyrosol butanoate (1b)
hydroxytyrosol palmitate (1a)
hydroxytyrosol decanoate (1c)
hydroxytyrosol sterate (1d)
hydroxytyrosol oleate (1e)
hydroxytyrosol elaidate (1f)
hydroxytyrosol linoleate (1g)
1.64
2.52
8.09
5.35
9.02
9.12
8.38
8.668
d
>3.3
d
d
d
d
d
>3.3
>3.3
>3.3
>3.3
>3.3
RESULTS AND DISCUSSION
■
8.976
9.75
Several derivatives of hydroxytyrosol were prepared by
synthesis as shown in Scheme 1. Several methods of
derivatization were attempted, as is summarized in Table S1,
Supporting Information.
8.38
8.668
a
Experimental log Kow values were determined by HPLC.6
b
Theoretical log Kow values were determined by Actelion Property
Scheme 1. Synthesis of Hydroxytyrosol Fatty Esters
c
Explorer. Theoretical log Kow values were determined by Molinispira-
tion Property Explorer. The reported value of 3.3 is the higher log
d
Kow value determined by the RP-HPLC method, and it is referred to
naphthalene used as standard in the calibration curve.
Kow values much higher than hydroxytyrosol and hydroxytyr-
osol butanoate (1b).35
Permeation through the skin represents a very important
prerequisite for the topical delivery of bioactive compounds,
especially in the case of pro-drugs,36 synthetic conjugates,37 or
colloidal devices2 applied to the skin. As previously reported,
the efficacy of new formulations containing natural or synthetic
products is based on their ability to penetrate the SCE
membrane without any modification.38 Overcoming this barrier
allows the drugs to be delivered into the site of action, thus
improving their therapeutic activity. Another important aspect
to be considered for the topical delivery of bioactive
compounds is their repartition between the external surface
of the skin and lipids of the SCE membrane.39
In order to test the topical application of the hydroxytyrosol
fatty esters compared to free hydroxytyrosol, an “in vitro”
permeation study using Franz diffusion cells was performed on
all the conjugates previously synthesized. Figure 1 (panels A
and B) shows the percutaneous permeation profile of
hydroxytyrosol and conjugates 1a−g through the SCE
membranes. No lag time was observed during the experiments,
and zero-order kinetics were observed for various formulations
(Figure 1A). The permeation profiles of the hydroxytyrosol
conjugates show that different compounds increased the
percentage of hydroxytyrosol permeated through the SCE
membranes (Figure 1A). Only elaidate conjugate 1f showed a
profile similar to that obtained for hydroxytyrosol, probably
depending on the trans configuration of the double bond on the
carbon chain.
These findings demonstrated also that the hydroxytyrosol
butanoate (1a), decanoate and linoleate (1c and 1g), oleate
(1e), and stearate and palmitate (1d and 1e) permeation
profiles were increased 1.8-fold, 2.2-fold, 3.6-fold, and 1.7-fold,
respectively, compared to hydroxytyrosol (Figure 1A). The
differences recorded for the different hydroxytyrosol conjugates
probably depend on their partition coefficients, their molecular
weights, and the isomeric configuration of their double bonds.
In fact, in spite of the behavior of the cis-unsaturated oleate 1e
and linoleate 1g, no significant permeation increase regarding
the simple hydroxytyrosol was obtained for the trans isomer
elaidate 1f (Figure 1A), despite this derivative containing 18
carbon atoms in its side chain. The trans chemical structure of
elaidate conjugate 1f is responsible for a side chain folding that
could decrease the molecular radius, thus reducing its contact
The biological activity of phenolic compounds depends on
their antioxidant properties as much as their lipophilicity. As
mentioned in a previous report, the inclusion of a lipophilic
chain in the hydroxytyrosol molecule enhanced its antioxidant
capacities in the biological model investigated.22 Furthermore,
lipophilic hydroxytyrosyl esters such as 1a−g show a high free-
radical-scavenging capacity, preventing protein oxidation and
lipid peroxidation when ex vivo cells are exposed to active-
oxygen substances and/or free radicals. The same hydroxytyr-
osol esters showed greater antioxidant activity than α-
tocopherol or BHT to protect proteins and lipids against
oxidation caused by peroxyl radicals in a brain homogenate.22
Conventionally, the standard measure of the hydrophobicity
of a compound is its octanol−water partition coefficient, Kow,
which is the equilibrium ratio of the concentration of the
compound in an octanol phase and its concentration in an
aqueous phase. Kow is frequently used to estimate drug
partitioning between aqueous and lipid phases32 and is closely
related to aqueous solubility.33 The experimental determination
of the partition coefficient is based on a correlation of Kow with
reversed-phase (RP) HPLC retention times, which depends on
a similarity of the retention mechanism to octanol−water
partitioning but does not require that the reactions be identical.
Klein et al.34 validated and included a HPLC method as one of
the standard Kow determination methods. In a previous paper,
the application of a modified HPLC method was used to
evaluate the Kow for hydroxytyrosol and an acetylated
derivative.6 Herein, we applied the same method to the Kow
determination of the hydroxytyrosol lipophilc esters 1a−g (see
Supporting Information). As shown in Table 1, all the
hydroxytyrosol fatty esters with more than four carbon atoms
in their chain were considerably more lipophilic than natural
hydroxytyrosol (so that it was not practical to determine exactly
their Kow by this HPLC method). However, it was possible to
conclude that hydroxytyrosol palmitate (1a), decanoate (1c),
stearate (1d), oleate (1e), elaidate (1f), and linoleate (1g) have
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Figure 1. “In vitro” percutaneous permeation through an SCE membrane of various hydroxytyrosol conjugates 1a−g as a function of percentage (A)
and amount (B) of compounds delivered through the skin. The chemical structures of different compounds are reported in Scheme 1. Experimental
findings are the average of three different measurements ± standard deviations.
Perkin-Elmer UV WinLab 2.8 acquisition software (Perkin-Elmer
surface with the SCE membrane (Figure 1A, B). This effect
became more evident in comparison with the oleate derivative
(1e). Although hydroxytyrosol oleate (1e) is characterized by
the same number of atoms in the side carbon chain, the
opposite double-bond geometry compared to hydroxytyrosol
elaidate (1f) gives rise to a difference in their SCE permeation
profiles (Figure 1A, B). In fact, the amount of hydroxytyrosol
that permeated through the SCE membrane in the case of the
oleate conjugate 1e after 12 h was 3334.88 μg/cm2 h−1,
compared to 1045.82 μg/cm2 h−1 obtained in the case of
elaidate conjugate 1f (Figure 1B).
The palmitate (1a), butanoate (1b), decanoate (1c), and
stearate (1d) conjugates showed permeation profiles similar to
one another (Figure 1A, B). These permeation profiles seem to
be slightly dependent on the side carbon chain dimension and
strongly influenced by the double-bond geometry. Percentages
of 64.18% and 60.52% permeation (Figure 1A) were obtained
for the decanoate (1c) and the linoleate (1g), respectively,
while the amounts of compounds permeating through the skin
were 2672.05 μg/cm2 h−1 (1c) and 2297.39 μg/cm2 h−1 (1g),
respectively (Figure 1B).
In conclusion, a new mild catalytic system is proposed for the
synthesis of a range of lipophilic hydroxytyrosol fatty esters.
These conjugates offer the potential of being administered as
topical therapeutic agents for the treatment of cutaneous
diseases. Almost all the compounds produced permeated
efficaciously through the SCE membrane when applied in an
“in vitro” Franz cell model. The skin permeation of
hydroxytyrosol conjugates generally correlated to their chemical
structure and to the configuration of the unsaturated fatty acids
conjugated to hydroxytyrosol.
GmbH, Uberlingen, Germany). 1H and 13C NMR spectra were
̈
recorded on a Bruker WM 300 NMR spectrometer on samples
dissolved in CDCl3. Chemical shifts are given in parts per million
(ppm) from tetramethylsilane as the internal standard (0.0 ppm).
Coupling constants (J) are given in hertz. ESIMS were performed on a
Applied Biosistem hybrid Q-STAR XL spectrometer working in a
positive mode. The RP-HPLC system used in the log Kow
determination was made by a JASCO PU 1580 unit that included
an Intellgent HPLC pump with an external column heater and
multiwavelength JASCO MD detector 1540. An Altech 4.6 × 150 mm
Adsobosphere C18 column with 5 μm particles of bonded silica gel
with a guard column (4.6 × 7.4 mm Adsobosphere C18) was used.
Absorbance chromatograms were obtained at 254 nm. TLC was
performed using 60-F264 silica plates on alumina (Merk). Flash
chromatography was performed on a Supelco Versa Flash HTFP
station on silica gel cartridges. Fetal calf serum (FCS) was purchased
from Gibco [Invitrogen Corporation, Giuliano Milanese (Mi), Italy].
Resorcinol, catechol, benzoic acid, and naphthalene (≥99% purity)
were purchased from Sigma-Aldrich. Polysorbate 80 (Tween 80) was
obtained from ACEF Spa (Piacenza, Italy). Double-distilled pyrogen-
free water was from Sifra SpA (Verona, Italy). All other chemical
reagents used in this investigation were of analytical grade (Carlo Erba,
Milan, Italy).
Synthesis of Hydroxytyrosol. Hydroxytyrosol was synthesized
using a procedure reported in the literature.6 3,4-Dihydroxyphenyl-
acetic acid (28.73 mmol, 1 equiv) was reacted with ethanol (EtOH)
(15.0 mL) in the presence of 10% v/v H2SO4 at reflux temperature for
3 h and reduced with a solution of NaBH4 (7−10 equiv) in water (40
mL) at room temperature for 8 h, as reported in the literature.6 1H
NMR (300 MHz, CDCl3) δ 2.53 (3H, t, J = 7.4 Hz, H-2), 3.17 (1H, s,
OH), 3.49 (2H, t, J = 7.4 Hz, H-3), 6.66−6.41 (1H, m), 6.60−6.58
(2H, m); EIMS m/z 136 [M − H2O]+, 43 [MeCO]+.
Synthesis of Hydroxytyrosol Fatty Esters. To a solution of
hydroxytyrosol (1.62 mmol) in dry THF (8 mL) were added 1
equiv of each fatty acid chloride (1.62 mmol) and 1 mol % of
Er(OTf)3 (0.0162 mmol) under stirring. Each mixture was reacted for
12 h at room temperature under nitrogen. After completion the
mixture was poured in water saturated with NaHCO3 and extracted
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV−vis quantification was
performed using a Perkin-Elmer Lambda 20 instrument connected to
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with CHCl3 (3 × 10 mL). The organic layers were combined and
dried on Na2SO4 and filtered, and the solvent was evaporated under
vacuum. The crude product was purified by flash chromatography
(CHCl3/MeOH, 9.5/0.5, as eluent).
derivative dissolved in the receptor chamber solution (4 mg/mL), and
a steady-state condition was maintained during experimental
investigations. The experiments were carried out in nonocclusive
conditions for 24 h at a thermostatted temperature of 36 ± 1 °C. A
minimum of three diffusion cells were used simultaneously for each
formulation, and 1 mL of each sample was withdrawn every 1 h up to
12 h of incubation using an FC 204 fraction collector [Gilson Italia
S.r.l., Cinisello Balsamo (MI), Italy] connected to a Minipuls 3
peristaltic pump (Gilson Italia S.r.l.). The volume withdrawn was
replaced by the same volume of fresh receptor phase. Samples
collected from the receiving compartment were analyzed immediately
using a UV−vis spectrophotometer as described below. Each
formulation was analyzed in triplicate, and the results are expressed
as mean values ± standard deviation.
UV−Vis Spectrophotometer Characteristics and Calibra-
tion. UV−vis quantification of the lipophilic hydroxytyrosol deriva-
tives was performed with the zero-order spectrum, and the first
derivative spectrum was recorded for each compound (see Supporting
Information). The absorbances of different samples were recorded at
the λmax measured for each compound, and the measurements were
carried out in triplicate as a function of their specific calibration curve
(see Supporting Information).
Standard Sample Preparation for UV Calibration Curve.
Calibration curves for UV−vis analysis were carried out using different
lipophilic hydroxytyrosol derivates. Standard solutions were obtained
by dissolving 1 mg of single derivatives in 1 mL of EtOH. A linear
correlation was obtained in the concentration range between 25 and
0.5 μg/mL. Six different readings (25, 10, 5, 2.5, 1, and 0.5 μg/mL)
were measured for each single agent by adding the standard solution to
a blank fetal calf serum sample. The samples obtained were then
extracted in the following manner and immediately submitted to UV−
vis analysis. FCS solution was used as blank during the analysis.
Sample Preparation for UV−Vis Analysis. Hydroxytyrosol
lipophilic derivatives (4 mg/mL) were collected from a receptor
chamber and extracted using ethanol (100 μL). Organic solvent was
added to 100 μL of sample and transferred into an Eppendorf tube of
1.5 mL. Each mixture was vortex-mixed at 700 rpm for 10 s (MS1
minishaker, Ika-Werke Gmbh, Staufen, Germany), combined with
hexane (500 μL), and further vortex-mixed for additional 30 s to
obtain a complete homogenization of samples. Organic phases were
centrifuged in a Mini Spoin Eppendorf centrifugue for 15 min at
13.400 rpm and further separated through a gravimetric process. The
supernatant-containing hexane solution (450 L) was transferred in a
clean tube, dried under nitrogen (N2) flux, and diluted with 1 mL of
EtOH in a glass tube for UV−vis analysis.
Hydroxytyrosol palmitate (1a): 1H NMR (300 MHz, CDCl3) δ
0.88 (3H, t, J = 6.7 Hz), 1.28 (24H, m), 1.64 (2H, m), 2.32 (2H, t, J =
7.2 Hz), 2.88 (2H, t, J = 7.0 Hz), 4.38 (2H, t, J = 7.0 Hz), 6.73 (3H,
m); HRMS (FAB) m/z 415.28 [M + Na]+.
Hydroxytyrosol butyrrate (1b): 1H NMR (300 MHz, CDCl3) δ
0.90 (3H, t, J = 6.8 Hz), 1.79 (2H, m), 2.32 (2H, t, J = 7.2 Hz), 2.96
(2H, t, J = 7.1 Hz), 4.41 (2H, t, J = 7.1 Hz), 7.05 (3H, m); HRMS
(CI) m/z 224.10 [M]+.
Hydroxytyrosol decanoate (1c): 1H NMR (300 MHz, CDCl3) δ
0.87 (3H, t, J = 6.8 Hz), 1.26 (12H, m), 1.61 (2H, m), 2.28 (2H, t, J =
7.2 Hz), 2.92 (2H, t, J = 7.1 Hz), 4.28 (2H, t, J = 7.1 Hz), 7.05 (3H,
m); ESIMS (positive mode) m/z 323.21 [M + H]+.
Hydroxytyrosol stearate (1d): 1H NMR (300 MHz, CDCl3) δ 0.88
(3H, t, J = 6.7 Hz), 1.28 (28H, m), 1.64 (2H, m), 2.32 (2H, t, J = 7.2
Hz), 2.88 (2H, t, J = 7.0 Hz), 4.38 (2H, t, J = 7.0 Hz), 6.73 (3H, m);
HRMS (CI) m/z 420.32 [M]+.
Hydroxytyrosol oleate (1e): 1H NMR (300 MHz, CDCl3) δ 0.87
(3H, t, J = 7.0 Hz), 1.30 (14H, m), 1.61 (2H, m), 1.98 (4H, m), 2.32
(2H, t, J = 7.6 Hz), 2.80 (2H, t, J = 7.2 Hz), 4.28 (2H, t, J = 7.3 Hz),
5.35 (2H, m), 6.60 (1H, dd, J = 3.1, 1.9 Hz), 6.79 (2H, dd, J = 8.0, 1.9
Hz); HRMS (CI) m/z 419.21 [M + H]+.
Hydroxytyrosol elaidate (1f): 1H NMR (300 MHz, CDCl3) δ 0.87
(3H, t, J = 6.6 Hz), 1.30 (14H, m), 1.61 (2H, m), 1.98 (4H, m), 2.32
(2H, dt, J = 17.9, 6.4 Hz), 2.70 (2H, t, J = 7.1 Hz), 4.28 (2H, t), 5.35
(2H, m), 6.79 (3H, m); 13C NMR (300 MHz, CDCl3) δ 13.9, 22.6,
25.4, 28.9, 29.0, 29.1, 29.1, 29.2, 29.4, 29.6, 29.7, 31.8, 32.5, 32.5, 33.7,
115.5, 116.0, 121.3, 130.2, 130.5, 130.8, 142.4, 143.8, 174.1; ESIMS
(positive mode) m/z 419.53 [M + H]+, 441.52 [M + Na]+.
Hydroxytyrosol linoleate (1g): 1H NMR (300 MHz, CDCl3) δ
0.89 (3H, t, J = 6.6 Hz), 1.30 (14H, m), 1.61 (2H, t, J = 7.1 Hz), 1.98
(4H, m, J = 7.5 Hz), 2.32 (2H, dt, J = 18.2, 7.4 Hz), 2.75 (2H, m), 2.77
(2H, t, J = 7.1 Hz), 4.25 (2H, t, J = 7.1 Hz), 5.35 (4H, m), 6.79 (3H,
m); HRMS (CI) m/z 417.29 [M]+.
HPLC Operations, Materials, Solutions, and Chromato-
graphic Conditions. The HPLC system calibration and the log
Kow determination were performed as previously described.6 The
organic compounds with known log P used as standards were
hydroquinone, vanillin, p-cresol, and p-chlorophenol (Supporting
Information). All the standard substances were 99% pure and were
obtained from Sigma-Aldrich. The mobile phase was chosen according
to the literature40 (30% water/CF3COOH, pH 2.4, 70% methanol), in
order to correlate linearly the known log Kow values of the standards
with their HPLC retention times and to estimate the log Kow of the
unknown compounds, 1a−g, by linear regression analysis. For those
compounds too lipophilic to be analyzed by this method, the
assumption was made that their log Kow is higher than the most
lipophilic standard analyzed (naphthalene, log Kow = 3.3) by the same
method.
Percutaneous Permeation of Lipophilic Hydroxytyrosol
Derivates. Franz diffusion cells were used to evaluate “in vitro” the
percutaneous permeation of different liphophilic hydroxytyrosol
derivates through SCE membranes, which were prepared using fresh
abdominal human skin obtained from plastic surgery from a group of
volunteers fully informed about the nature of the study and the
procedures involved and who gave a written consent. The stratum
corneum and viable epidermis were separated from subcutaneous fat
tissue according to a method previously reported.8 Experimental
investigations were carried out within 24 h of surgical removal of the
skin. Franz diffusion cells were characterized by a 0.75 cm2 diffusion
surface area and a nominal receptor volume of 4.75 mL. The receptor
chamber was filled with a mixture of double-distilled pyrogen-free
water/ethanol/FCS (4/1/5 v/v) and maintained under continuous
stirring at 600 rpm. SCE membranes were placed between the donor
and receptor compartment with the stratum corneum side up, and the
system was equilibrated for 6 h before the experiment. The donor
compartment was filled with 200 μL of each lipophilic hydroxytyrosol
ASSOCIATED CONTENT
■
S
* Supporting Information
A description of the unsuccessful attempts made in preparing
the hydroxytyrosol esters as well as the yields recorded with the
optimized synthetic procedure (Table S1), the experimental
details of bioassays and synthetic procedures, UV and MS,
ESIMS, and NMR spectra of hydroxytyrosol conjugates, HPLC
chromatogram of hydroxytyrosol and hydroxytyrosol butanoate
(1b), and their UV calibration curves. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (39) 3694120. Fax: (39) 961395713. E-mail: procopio@
unicz.it.
ACKNOWLEDGMENTS
■
Financial support from MIUR PRIN 2008 “A Green Approach
to Process Intensification in Organic Synthesis” is gratefully
acknowledged.
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(33) Han, S. Y.; Qiao, J. Q.; Zhang, Y. Y.; Yang, L. L.; Lian, H. Z.;
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