Octadecyl ferulate behavior in 1,2-Dioleoylphosphocholine liposomes

Article abstract of DOI:10.1016/j.saa.2015.08.009

Octadecyl ferulate was prepared using solid acid catalyst, monitored using Supercritical Fluid Chromatography and purified to a 42% yield. Differential scanning calorimetry measurements determined octadecyl ferulate to have melting/solidification phase transitions at 67 and 39 °C, respectively. AFM imaging shows that 5-mol% present in a lipid bilayer induced domains to form. Phase behavior measurements confirmed that octadecyl ferulate increased transition temperature of phospholipids. Fluorescence measurements demonstrated that octadecyl ferulate stabilized liposomes against leakage, maintained antioxidant capacity within liposomes, and oriented such that the feruloyl moiety remained in the hydrophilic region of the bilayer. Molecular modeling calculation indicated that antioxidant activity was mostly influenced by interactions within the bilayer.

Full text of DOI:10.1016/j.saa.2015.08.009

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
☆
Octadecyl ferulate behavior in 1,2-Dioleoylphosphocholine liposomes
a
Kervin O. Evans a, , David L. Compton , Nathan A. Whitman a,1, Joseph A. Laszlo a, Michael Appell b,
⁎
Karl E. Vermillion c, Sanghoon Kim d
a
Renewable Products Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture,
1815 N. University Street, Peoria, IL 61604, USA
b
Bacterial Foodborne Pathogens and Mycology, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture,
1815 N. University Street, Peoria, IL 61604, USA
c
Functional Foods Research, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University Street, Peoria,
IL 61604, USA
d
Plant Polymer Research, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University Street, Peoria, IL 61604, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Octadecyl ferulate was prepared using solid acid catalyst, monitored using Supercritical Fluid Chromatography
and purified to a 42% yield. Differential scanning calorimetry measurements determined octadecyl ferulate to
have melting/solidification phase transitions at 67 and 39 °C, respectively. AFM imaging shows that 5-mol%
present in a lipid bilayer induced domains to form. Phase behavior measurements confirmed that octadecyl
ferulate increased transition temperature of phospholipids. Fluorescence measurements demonstrated that
octadecyl ferulate stabilized liposomes against leakage, maintained antioxidant capacity within liposomes, and
oriented such that the feruloyl moiety remained in the hydrophilic region of the bilayer. Molecular modeling
calculation indicated that antioxidant activity was mostly influenced by interactions within the bilayer.
Published by Elsevier B.V.
Received 27 February 2015
Received in revised form 17 July 2015
Accepted 4 August 2015
Available online 11 August 2015
Keywords:
Fluorescence
Antioxidation
Octadecyl ferulate
Partition coefficient
Liposomes
1. Introduction
to assess their potential as cosmeceutical, nutraceutical, and pharma-
ceutical ingredients [3,7–15]. One of the features sought in these cosme-
Plants and fungi containing bioactive compounds have been used for
centuries for their medicinal benefits. In recent years, identification and
exploration of these bioactive compounds have been of particular inter-
est to researchers. One such compound is ferulic acid (FA), which itself
and its derivatives, are readily found in foods such as tomatoes, grain
foods, citrus fruits, and coffee [1]. FA has been shown to inhibit oxidative
stress in human lymphocytes [2] and its derivatives have been shown to
have anti-cancer activity [3].
Octadecyl ferulate (ODF) is one of many FA derivatives that are
found throughout the plant and fungi kingdoms. For instance, ODF can
be found naturally in fungi such as Annulohypoxylon squamulosum [4],
in plants such as potato tubers [5], and in herbs [6]. Despite the natural
sources, ODF is not particularly abundant in nature and thus the need
for de novo synthesis of ODF and other phenolic derivatives in order
ceutical, nutraceutical and pharmaceutical ingredients is their ability to
retard lipid oxidation.
Lipid peroxidation is damaging to cells because the radical products
can propagate to polyunsaturated lipids creating a chain reaction within
a cell. Nature counters lipid peroxidation through use of both enzymatic
and non-enzymatic antioxidants [16]. Non-enzymatic antioxidants
include a large variety of antioxidants that are either aqueous-based
or lipophilic like ODF, which is readily produced by plants. We recently
demonstrated the antioxidant capacity of the lipophilic feruloyl glycer-
ide derivative, feruloyl dioleoylglycerol [17]. We further explore feruloyl
derivatives for antioxidant capacity by demonstrating the antioxidant
capacity of ODF. Specifically, we used a Trolox-based assay because
Trolox can readily incorporate into liposomes [18]. Peroxyl radicals
play a substantial role in peroxidation of polyunsaturated fatty acids of
cells which leads to membrane structural loss [19]. The use of liposomes
is important because they can readily serve as model cellular systems
and can be used as delivery systems of active agents like antioxidants
in formulations [20,21]. Thus, it is important to characterize the behav-
ior of ODF within liposomes.
☆
Mention of trade names or commercial products in this publication is solely for the
purpose of providing specific information and does not imply recommendation or
endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity
provider and employer.
⁎
Corresponding author.
In this work, we report the chemical synthesis of ODF from ferulic
acid and octadecanol using a solid acid catalyst as monitored by Super-
critical Fluid Chromatography (SFC), the phase transition temperature
E-mail address: Kervin.Evans@ars.usda.gov (K.O. Evans).
Current address: Department of Chemistry, University of North Carolina — Chapel Hill,
1
Chapel Hill, NC 27599, USA.
http://dx.doi.org/10.1016/j.saa.2015.08.009
1386-1425/Published by Elsevier B.V.

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K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
of ODF and its behavior in a lipid matrix of phospholipid liposomes.
We compare it to that of slightly less lipophilic ethyl ferulate (EF) in
liposomes.
Yield: 9.77 g, 42.2% (based on ferulic acid). Analytical calculated for
C28H46O4: C, 75.29 and H, 10.38; found: C, 73.36 and H, 10.86. ESI-MS
(m/z) calculated for C28H47O4 [M + H]+: 447.34; found: 447.34749.
1H NMR (500 MHz, d6-acetone): δ 8.12 (bs, 0.60 H, \\OH), 7.60
(d, J = 16.0 Hz, 1.00 H, α), 7.35 (d, J = 1.90, 1.00 H, A2), 7.15 (dd,
J = 8.12, 2.03, 1.99 Hz, 1.00 H, A6), 6.88 (d, J = 8.16 Hz, 0.98 H, A5),
6.40 (d, J = 15.90 Hz, 1.01 H, β), 4.16 (t, 2.04 H, 1), 3.93 (s, 2.99 H,
A7), 1.63 (p, 2.07 H, 2), 1.37 (bm, 30.97 H, 3–17), and 0.89 (t, 3.15 H,
18) ppm. 13C NMR (125.77 MHz, d6-acetone): δ 166.6 (γ), 149.2 (A3),
147.9 (A4), 144.6 (α), 126.6 (A1), 123.0 (A6), 115.2 (A5), 115.1 (β),
110.4 (A2), 63.8 (1), 55.4 (A7), 31.7 (16), 29.4 (11 C, 5–15), 29.2 (4),
28.7 (2), 25.8 (3), 22.4 (17), and 13.4 (18) ppm (Fig. 1).
2. Materials and methods
2.1. Reagents and materials
Ferulic acid (4-hydroxy-3-methoxy cinnamic acid), 1-octadecanol,
p-toluenesulfonic acid monohydrate (p-TSA), anhydrous toluene, mag-
nesium sulfate, cobalt (II) chloride hexahydrate, ethylenediaminetetra-
acetic acid disodium dehydrate (EDTA), 2,2′-azobis(2-amidinopropane)
dihydrochloride (AAPH), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),
and all common solvents were purchased from Sigma-Aldrich. Ferulic
acid ethyl ester (ethyl ferulate) was obtained from Sinova Corporation
(Ningbo, China). Ethanol (200 proof) was purchased from Decon Labo-
ratories, Inc. (King of Prussia, PA). Supercritical Fluid Chromatography
(SFC)/supercritical fluid extraction (SFE) grade CO2 was purchased
from Airgas Products Co. (Radnor Township, PA). 1,2-Dioleoyl-sn-
glycero-3-phosphocholine (DOPC) was bought from Avanti Polar
Lipids (Alabaster, AL). High purity calcein, 4,4-difluoro-5-(4-
phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic
acid (C11-Bodipy ® 581/591), 1,6-diphenylhexatriene (DPH), and
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-
toluenesulfonate (TMA-DPH) were purchased from Invitrogen
(Carlsbad, CA). Tris(hydroxymethyl)aminomethane hydrogen chloride
(Tris–HCl), potassium phosphate monobasic, potassium phosphate
dibasic, Sephadex G-75 column beads, columns and sodium chloride
were obtained from Fisher Scientific. Octaethylene glycol monododecyl
ether (C12E8) was purchased from Sigma-Aldrich.
2.3. Supercritical fluid chromatography (SFC)
The synthesis of ODF was monitored by SFC using a Selerity Technol-
ogies Series 4000 chromatograph. Samples (100 μL) taken from the
reduction reactions were diluted in 1.0 mL of ethanol and 10-μL aliquots
were used for injection. The samples were developed with CO2 on
a Selerity Technologies SB-methyl-100 SFC column (PN AE002,
50 μm × 10 m × .25 μm film) starting at 100 atm and 100 °C. The starting
pressure was held for 5 min and ramped at 15 atm/min to a final pres-
sure of 310 atm, which was held for 11 min. The column temperature
was isothermal for the duration of the analysis. The FID was set at
350 °C. Under these analytical SFC conditions octadecanol, ferulic acid,
and ODF resulted in Rt of 11.96, 12.52, and 15.00 min, respectively.
2.4. NMR
1H and 13C NMR spectra were obtained on a Bruker Avance 500 spec-
trometer (500 MHz 1H/125.77 MHz 13C) using a 5 mm BBI probe. All
samples were dissolved in d6-acetone, and all spectra were acquired
at 27 °C. Chemical shifts are reported as ppm from tetramethylsilane
calculated from the lock signal (ΞD = 15.350609%). Noteworthy: The
reaction as monitored by SFC showed nearly 85% conversion of ferulic
acid to ODF, which belies the reported yield of 40%. Much of the product
was lost in optimizing the flash chromatography and in the precipita-
tions. The ODF is N95% pure by SFC and 1H NMR.
2.2. Octadecyl ferulate (ODF) synthesis
The procedure was adapted from Taniguchi et al. [22]. Ferulic acid
(10.07 g, 520 mmol), octadecanol (21.10 g, 780 mmol), and p-TSA
(1.05 g, 5.5 mmol) were combined in a 500-mL Schlenk flask under a
dry, nitrogen atmosphere using standard Schlenk line techniques. An-
hydrous toluene (250 mL) was added via syringe, and the Schlenk
flask was fitted with a reflux condenser. The slurry was heated to
85 °C with stirring. Octadecanol and p-TSA dissolved after 24 h, but
not the ferulic acid. The entire slurry became a clear, yellow solution
after stirring and heating for an additional 30 h (54 h total). The solution
was then cooled to ambient temperature for 18 h, resulting in the
formation of an off-white precipitate (confirmed by SFC analysis to be
unreacted ferulic acid). MgSO4 (1.00 g) was added and the solution
stirred for 15 min. The solid was removed by filtration through a fine
frit, and the solvent removed from the filtrate resulting in ~30.0 g of
off-white solid.
The crude product was purified in 10-g batches by flash chromatog-
raphy. A 10-g fraction of the crude product was placed in a 1-L round
bottom flask and dissolved in 500 mL of acetone. Silica gel (20 g, Silica
Gel 60, 70–230 mesh ASTM) was added and the slurry thoroughly
mixed. The acetone was then removed by rotor-evaporation. The
product-loaded silica was slurried in hexane and poured into a Pyrex,
600-mL, glass frit (10–15 ASTM) fitted on top of a 1-L Erlenmeyer filtra-
tion flask. A vacuum was applied and the resultant 5-cm bed of product-
loaded silica was developed with 3.5 L of 2% acetone/hexane, 2.5 L of 5%
acetone/hexane, 0.5 L of 10% acetone/hexane, and finally 0.5 L of 100%
acetone, respectively. Fractions containing ODF (as monitored by SFC)
were placed on ice and a white precipitate formed. The precipitates
were collected by filtration (Whatman #2, 4.25 cm filter paper) and
combined. The supernatants were combined and placed on ice, and a
second precipitant formed. The second precipitant was collected by
filtration and combined with the first, and the white precipitate was
dried at 35 °C in vacuo.
2.5. Differential scanning calorimetry (DSC)
The thermal properties of ODF were characterized using a Q2000
MDSC™ (TA Instruments; New Castle, DE). ODF (2–3 mg) was weighed
into aluminum pans and hermetically sealed. The pans were heated
from 10 °C to 100 °C at a heating rate of 1.0 °C/min, followed by cooling
from 100° to 10 °C at the same rate (1.0 °C/min). The heating/cooling
cycle was repeated twice per sample to determine if ODF exhibited
any property changes. All measurements were conducted under a
nitrogen atmosphere and run in quadruplicate. Universal Analysis
2000 software (TA Instruments) was used to conduct analysis of the
thermographs.
2.6. Fluorescence measurements
Fluorescence emission measurements were conducted using a
Jobin-Yvon Horiba Fluorolog 3-21 spectrofluorometer (Edison, NJ)
equipped with a 450-W xenon lamp and four-position cuvette holder.
Constant temperature was maintained using a Thermo Neslab RTE-7
refrigerated water circulator. Fluorescence measurements were conduct-
ed in a 10 mm × 10 mm quartz cuvette (Starna Cells, Inc., Atascadero, CA)
with constant stirring. All spectra were corrected and background
subtracted using a solution containing only buffer or buffer/liposomes
with the corresponding amount of ODF only. Excitation and emission
slits were 5 and 10 nm, respectively (unless stated otherwise).

K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
335
Fig. 1. Octadecyl ferulate structure.
2.7. Liposome preparation
(0.6 mM CoCl2, 0.25 mM calcein, 2.3 mM potassium phosphate,
pH 7.4) was added to resuspend the dried lipid film. Liposomes were
created as described above. Liposomes were passed down a G-75
Sephadex column equilibrated with 5 mM NaCl, 2.3 mM potassium
phosphate 10 mM EDTA, and pH 7.4 (column buffer used to osmotically
balance liposomes) to remove unencapsulated cobalt–calcein complex.
Liposomes were maintained on ice in the dark until just prior to the
measurement; this reduces any significant leakage. Immediately before
making the measurement, liposomes were diluted in column buffer
which had been preheated to 37 °C in a cuvette inside the fluorometer.
Fluorescent intensity was measured at 520 nm (excitation at 490 nm)
for 30 min in 5 s intervals. Leakage percentage (L), was calculated
Liposomes were generated as previously described [14,17,21]. Brief-
ly, appropriate amounts of DOPC and ODF in chloroform were added to
4-mL amber vials to give the following DOPC/ODF mole ratios: 100/0,
99/1 and 95/5. The vials were gently swirled to give a homogenous
mix of lipids which were subsequently dried to a thin film under a slight
stream of argon and placed under vacuum overnight to remove residual
solvent. The lipid film was resuspended in the appropriate buffer
and periodically mixed over 60 min. Afterwards, the newly formed
multilamellar liposomes were subjected to freeze/thaw cycles using
dry ice in 2-propanol and a 60 °C water bath, respectively. Multilamellar
liposomes were then extruded 11-times through two stacked polycar-
bonate filters of the appropriate pore size using a LiposoFast extruder
(AVESTIN, Inc., Ottawa, Canada) at ambient temperature.
100ꢁðFt −Fo
Þ
as L ¼
with Ft being the fluorescent signal over time, Fo
ðFmax−Fo
Þ
the extrapolated initial signal at time zero, and Fmax the fluorescent sig-
nal at the end of the experiment after rupture by addition of C12E8
solution. Regression analysis within Sigma Plot software was used to
2.8. Partitioning into DOPC liposomes measuring via fluorescence
spectroscopy
nt
fit the leakage data to the general equation L ¼ ∑Lnð1−e−k Þ where
n was 1 or 2 for single or double-exponential, respectively.
DOPC liposomes without either EF or ODF were extruded through
100-nm pore filters in 10 mM HEPES, 100 mM NaCl, and pH 7.4 buffer
as described above. Resulting liposomes were diluted to the appropriat-
ed concentrations and incubated for an hour with 40 μM of either EF or
ODF. Fluorescence measurement was repeated in triplicate; excitation
was at 330 nm.
Fitting Eq. (1) below at a fixed wavelength for the partitioning of EF
or ODF into DOPC liposomes resulted in calculating the apparent parti-
tion coefficient KP,
2.10. Atomic force microscope (AFM)
AFM images were taken using a Nanoscope IV controller (Bruker
Nano Surface, Santa Barbara, CA) in tapping mode via a fluid cell. The
fluid cell was cleaned via sonication in 1/1 (v/v) mix of methanol and
water for 5 min, rinsed thoroughly and dried under a stream of argon.
Image scan rate was 1 Hz. Fresh cleaved mica was exposed for 15 min
to liposomes that were extruded at 50-nm (it has been our experience
that larger liposomes can adsorb unfused and lead to misinterpretation
of the image). The fluid cell was then rinsed with buffer. The buffer
contained 2 mM CaCl2, 10 mM HEPES, 100 mM NaCl, and pH 7.4.
Kp½Lꢀ
FðLÞ ¼ F0 þ Fmax
;
ð1Þ
½Wꢀ þ Kp½Lꢀ
where F0 is the initial fluorescence signal of EF or ODF in water with no
liposomes present [23]; Fmax is the maximal fluorescence signal when
full partition has occurred; [L] and [W] are respectively, the molar
concentration of lipid and the molar concentration of water (taken as
55.3 M); and Kp is the molar fraction partition coefficient. Alternatively,
partitioning was determined using the n-octanol/water method based
on the description given by Turina et al. [24]. Briefly, an aliquot of EF
or ODF in chloroform was dried under argon in a vial and equal amounts
of nanopure water and n-octanol (6 mL each) were added to give a final
concentration of 40 μM of EF or ODF in a 1/1 n-octanol/water n-octanol.
The mix was stirred and equilibrated overnight in the dark. The solution
was centrifuged at 1000 ×g for 10 min to ensure phase separation. EF
(or ODF) absorbance was measured for the water (Aw) and n-octanol
(Ao) phases. Partitioning was determined by taking the logarithm of
the ratio Ao/Aw at 327 nm (the peak of absorbance in n-octanol).
2.11. Phase behavior monitored with DPH and TMA–DPH
Liposomes composed of 1,2-dielaidoylphosphocholine (DEPC) were
made as described earlier (DEPC was chosen because it has the same
diacyl chain lipid as DOPC but the double bond is 18:1, Δ9 trans versus
18:1, Δ9 cis, allowing us to measure the phase transition temperature
above 0 °C). DPH or TMA–DPH at 1 μM was added to liposomes at
100 μM to maintain a 100:1 lipid:probe ratio. Liposomes were incubated
for an hour to allow maximum incorporation of probe. Excitation and
emission were at 350 nm and 428 nm, respectively. Liposomes were
equilibrated for 10 min at each temperature prior to measurement.
The steady-state anisotropy, r, was monitored as a function of tempera-
ture to determine phase behavior of DEPC liposomes containing ODF at
various concentrations. Anisotropy (r) was determined as follows:
2.9. Liposome leakage (Calcein–Cobalt)
ðIVV−G ꢂ IVH
Þ
IHV
IHH
r ¼
; where G ¼
ð2Þ
ðIVV þ 2G ꢂ IVH
Þ
Liposomes containing 0–5-mol% of either EF or ODF were monitored
for membrane integrity via a leakage assay [17,25]. Briefly, the assay is
designed such that liposomes entrapped calcein–cobalt complexes
which exhibit a quenched fluorescent signal. Leakage in the membrane
results in the complex passing through the bilayer where EDTA chelates
the cobalt, resulting in increased fluorescence. The leakage buffer
and VV, VH, HV, and HH denote the excitation/emission polarizers in the
vertical/vertical, vertical/horizontal, horizontal/vertical and horizontal/
horizontal positions, respectively; G is the correction factor for polariza-
tion sensitivity of the detector.

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K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
2.12. Lipid peroxidation
EF and ODF abilities to inhibit lipid peroxidation were measured
using an assay incorporating the aqueous free-radical initiator AAPH
and the oxidation-sensitive reporter lipid C11-Bodipy [26]. C11-Bodipy,
which resides well within the bilayer because of lipophilicity, shifts its
fluorescence from 595 nm to nearly 520 nm when it is oxidized [27].
It is assumed that the affinity for generated radicals is the same for
C11-Bodipy and phospholipids in the bilayer; thus, it is believed that
C11-Bodipy reports the oxidation of all lipids in the bilayer. Sample
conditions used were similar as detailed previously [17]. Briefly,
DOPC liposomes containing 36 μM C11-Bodipy and either EF, ODF
(each at 0-, 1- or 5-mol% of total lipids), or Trolox were generated as
previously described above [17,25,28]. Liposomes without C11-Bodipy
were used as background subtraction. Samples were diluted to a final
concentration of 2.5 mM DOPC (2.4 μM C11-Bodipy) and equilibrated
to 37 °C for 15 min prior to the introduction of AAPH. Fluorescence in-
tensity (excitation at 540 nm, emission at 595 nm) was measured
every minute for 180 min from the moment of AAPH addition at a
final concentration of 40 mM. Experiments were conducted in triplicate
at least. The relative fluorescence intensity was calculated by dividing
the reading at each minute by the initial fluorescence at the beginning
of the experiment (Ft/Fo). Background subtraction was accomplished
using the appropriate liposomes without C11-Bodipy.
Fig. 2. Representative DSC scan of ODF from 10 to 100 °C at a 10 °C/min scan rate. Sample
replicates were scanned through two cycles of heating and cooling, monitoring for prop-
erty changes. Positive heat flow values represent exothermic transitions.
(12DOXYL-PC), Lcl is the distance between the “shallow” quencher
and the center of the bilayer, C is the concentration of quenchers per
area (mole fraction of nitroxide-labeled lipids/area of lipid), and L21 is
the difference in depth between quenchers. The area of the phosphati-
dylcholine lipids was taken to be 70.1 Å2 per molecule [33] and the
distances from the bilayer center for the nitroxide group were taken
from Kachel et al. where TEMPO-PC was located at 19.5 Å, 5DOXYL-PC
at 12.2 Å, and 12DOXYL-PC at 5.9 Å [32].
The lipid peroxidation inhibition ability of ODF and EF were com-
pared to Trolox. Trolox, being lipophilic and somewhat water-soluble
[18], was incorporated along with C11-Bodipy within DOPC liposomes
in the same manner as EF or ODF, but in the final range of 10 to
125 μM. The peroxyl radical scavenging capacity (i.e., the relative
antioxidation activity) was calculated accordingly:
ΔAUCsample
½sampleꢀ
2.14. Molecular modeling
Relative‐Antioxidant‐CapacityðRACÞ ¼
=
;
ð3Þ
ΔAUCTrolox ½Troloxꢀ
Molecular models were constructed using Hyperchem v8.0.10 (Hy-
percube Inc. Gainesville, Florida). Models of EF, ODF, and DOPC were ini-
tially optimized in vacuo using Charmm27 molecular mechanics force
field and the PM3 semi-empirical method with Polak–Rebiere conjugate
where ΔAUC is the difference in the area under the fluorescence curve
(AUC) of liposomes containing antioxidant or standard curve minus
empty liposomes [29]. The AUC was calculated as described by Cao
and et al. [30].
gradient method and the convergence criteria of 0.01 kcal mol−1
.
The conformational preferences of ODF were studied by geometry
optimization using the B3LYP three parameter density functional at
the 6–31 + G* level as implemented in Spartan'10. Areas and volumes
were calculated using the QSAR properties module in Hyperchem on
B3LYP/6–31 + G* optimized geometries.
2.13. Bilayer location using nitroxide-labeled lipids
The location of the feruloyl group of EF or ODF within the phospho-
lipid bilayer of liposomes was determined using the parallax analysis
[31]. This is based on the expectation that ODF fluorescence intensity
is reduced by collisional quenching with nitroxide-labeled lipids as
described by Kachel et al. [32]. The nitroxide-labeled lipids used had
the nitroxide quencher positioned either in the headgroup region
(TEMPO-PC), just below the headgroup region (5DOXYL-PC), or farther
down the acyl chain (12DOXYL-PC). It is expected that the most
quenching would occur from the nitroxide group closest to the feruloyl
group. Nitroxide-labeled lipids were incorporated at 15 mol% into the
membrane in conjunction with ODF at various concentrations. Mea-
surements were conducted in potassium phosphate buffer (PPB) at
pH 7.4. Fluorescence emission spectra were recorded using an excita-
tion wavelength of 330 nm and band pass slits of 5 nm by 5 nm with
the appropriate background subtracted.
3. Results and discussion
3.1. Thermal properties of ODF
The thermal properties of ODF were evaluated over a 10 to 100 °C
temperature range employing DSC. Fig. 2 exhibits a representative
heating and cooling thermograph scan of ODF. DSC measurements
determined that ODF has a melting phase transition at 67 °C. This is ap-
proximately 9–18° above the range determined for other synthetic
octadecyl esters [34–37] and lyso-stearoylphosphatidylcholine [38],
indicating that the feruloyl moiety contributes significantly to the
thermotropic characteristics.
The location of the feruloyl group was determined (Chattopadhyay
and London, 1987; Kondo et al., 2008) as follows:
Table 1
h
i
Thermal properties of ODF measured by DSC at 10 °C/min.
zcf ¼ Lcl þ − lnðF1=F2Þ=πC−L221 =2L21
:
ð4Þ
Melting
Solidification
Onset temp Peak temp Latent heat
Onset temp Peak temp Latent heat
where zcf is the distance the fluorophores are from the center of
the membrane, F1 is the normalized emission fluorescence intensity
(normalization done with respect to liposomes with EF or ODF only)
in the presence of the “shallow” quencher (TEMPO-PC), F2 is the nor-
malized fluorescence intensity in the presence of the “deep” quencher
(°C)
(°C)
(J/g)
(°C)
(°C)
(J/g)
62.9 0.5
66.7 0.5 127.5 1.7 38.9 0.3
38.5 0.3 100.2 8.7
Reported results are the average of 4 measurements of 2 heating/cooling cycles per
measurement.

K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
337
The solidification transition was determined to occur at 38.5 °C. The
onset temperatures for both melting and solidification phase transitions
were roughly 63 and 39 °C, respectively (Table 1). The latent heat was
determined to be 128 and 100 J/g for the melting and solidification
phase transitions, respectively. Both phase transitions exhibited sharp
peaks which indicate pure material. A pre-transition phase occurred
around 39 °C during the second cycle (data not shown), which may
indicate formation of an ordered, tilted gel state [39]. However, the
pre-transition phase was not reproducible and thus may not be thermo-
dynamically stable for ODF. It should be noted that a tilted gel-state was
readily found in the thermotropic behavior of C16 and C18 saturated
diacylphospholipids like dipalmitoylphosphocholine (C16) [40] and
distearoylphosphocholine (C18) [41], but not found for C18 monoacyl
ester lipids [34,37] or a C18-lysophospholipid [38]. The possible
presence of a pre-transition state further indicates the influence of the
feruloyl moiety on the packing behavior of ODF.
3.2. AFM
The data from the thermal properties of ODF indicates that ODF
would form a gel state within a phospholipid bilayer at temperatures
below 63 °C. AFM imaging was undertaken to explore the possible
presence of gel state of 5-mol% ODF within a DOPC supported bilayer.
Fig. 3a confirms that a supported bilayer formed on mica with two
areas of two different materials (demonstrated by phase behavior
imaging; not shown). One area was dark and the other one was light.
The dark area was consistent with a uniform bilayer formed on the
mica (not shown) and thus classified. The light regions, or domains
(Fig. 3a) extended at least 2 nm above the uniform bilayer (based on
bearing analysis, not shown) and as much as 4 nm (demonstrated by
typical height profile in Fig. 3b). Fig. 3c shows that the domains are
mobile in that several of them coalesced into larger units or became
more spherical (see symbols in Fig. 3c). Thus, the light domains were
interpreted as the result of gel state formations with the bilayer that
were likely ODF-enriched.
3.3. Phase behavior
Further investigation of the effect that ODF has on liposomes was
done by monitoring the phase behavior of liposomes composed of
DEPC which has a phase transition temperature of 12 °C [42]. The
presence of domains as revealed by AFM is expected to raise the
phase transition (Tm) of DEPC. Fig. 4 depicts the phase transition of
the acyl chain region (as determined by DPH anisotropy) and the
phase transition of the headgroup regions (as determined by TMA–
DPH anisotropy) of DEPC liposomes as a function of ODF concentration.
It is clear that the presence of ODF steadily increased the phase transi-
tion temperature of DEPC liposomes in both acyl chains and headgroup
regions, further suggesting that ODF caused a more “gel” state within
the liposomes.
3.4. Log P — Partition coefficient
ODF has potential use in cosmeceutical, nutraceutical, and pharma-
ceutical ingredients. This means that ODF may have to be formulated
within a lipid matrix as lipid matrices are regularly used to deliver
water insoluble ingredients [43,44]. Then the question becomes, how
well does ODF associate with a lipid matrix? The answer to this question
was pursued using DOPC liposomes as the chosen lipid matrix (because
of the abundance of DOPC and the simplicity of its liposomes) to explore
the partitioning of ODF. It was previously reported that molecular
Fig. 3. Topographical AFM images of a supported bilayer of DOPC containing 5-mol% ODF
showing (a) that domains formed and (b) a height profile across two of the regions (solid
black line); (c) domains shown 3 h, 10 min, and 20 s later where some nearest neighbors
have migrated together (star). Dimensions for both images are 5 μm × 5 μm.

338
K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
Fig. 4. Phase behavior temperature (Tm) as a function of ODF mole percent within DEPC li-
posomes. The circles represent the Tm of the acyl chains as measured by the anisotropy of
DPH; the inverted triangles represent the Tm of the headgroups as measured by TMA–DPH.
partitioning into the membrane can be determined by monitoring the
fluorescence signal of a molecule as it is titrated with liposomes [45].
The determination of a partitioning coefficient for ODF and EF was
attempted using this approach, but reliable measurements were possi-
ble only for EF (Supplemental Data). As an alternative approach, the af-
finity of EF and ODF for a 1/1 n-octanol/water mixture was determined
and compared. Table 2 shows the values of n-octanol/water partitioning
coefficient indicating that EF and ODF partitioned into n-octanol simi-
larly with ODF partitioning slightly more. This correlated well with the
difference in hydrophobicity expressed by the difference in longer acyl
chain length [39].
3.5. Liposome stability
It was shown earlier in this work that the thermotropic properties of
ODF have a phase transition temperature at approximately 67 °C, indi-
cating that ODF would exist in a gel state at 37 °C. AFM measurements
and phase behavior demonstrate a more “gel-like” state with ODF
present within liposomes. It is thus important to understand how ODF
affects liposomal stability at pharmaceutical/nutraceutical/cosmeceuti-
cal concentrations. EF and ODF effects on liposome stability were stud-
ied using the calcein–cobalt content leakage assay described previously
[17,25]. Fig. 5a and b displays the results of the content leakage studies
EF and ODF, respectively. It is clear that EF and ODF had distinctively
different effects on the leakage of DOPC liposomes. EF (Fig. 5a) at both
concentrations explored displayed a two-step leakage process where
there a rapid release of entrapped contents (fast component of leakage)
followed by a slow release (slow component), whereas liposomes
empty of EF displayed a single-step leakage process. More specifically,
the fast component of leakage induced by EF (not shown) was compa-
rable to the single-exponential leakage observed for FDOG [17]
and lipoyl dioleoylglycerol [28] lipids in DOPC liposomes. The slow
Fig. 5. (a) Content leakage as a function of time for EF incorporated at 0-, 1-, and 5-mol% in
DOPC liposomes. (b) ODF effect on DOPC liposome content leakage.
component, on the other hand, dominated the leakage process and in-
creased in percentage as a function of EF concentration (Table 3). This
suggests that lipids with EF present retard the leakage rate as the extent
of leakage approaches that of the control liposomes.
A more straight-forward analysis was possible using the initial rates
or “velocities” of leakage at the start (t = 0 s) of the experiment
(Table 3). Leakage initial rates showed that overall leakage induced by
1- and 5-mol% EF was twice and 1.5, respectively, as fast as control lipo-
somes (0-mol% EF). The indication was that despite any lipid movement
Table 3
Leakage properties of DOPC liposomes with ethyl and octadecyl ferulates incorporated.
Ferulate
Amount
(mol%)
Initial ratesa, b, c
Slow component
percentd
(%*s−1 × 10−2
)
EF
0
1
5
1
5
2.96 ( 0.33)
6.22 ( 0.94)
4.33 ( 0.52)
1.72 ( 0.72)
2.18 ( 0.83)
–
Table 2
87.7 ( 1.8)
94.3 ( 0.6)
–
–
Log P partitioning determine via experiments.
Log Kp
ODF
Liposomes
an-octanol/water
a
Leakage initial rates reported as the mean and standard deviation of determinations
EF
ODF
b4.85 0.13
–
2.45 0.12
2.71 0.21
conducted in 3–12 replicates.
b
Statistical analysis was done using one-way ANOVA where each was statistically dif-
a
Determined using 1/1 n-octanol/water mixture.
ferent (P b 0.05) from the control sample (zero percent antioxidant).
b
c
n t
Calculated from least-squares fit of experimental data for partitioning between lipo-
somes and aqueous buffer.
L was obtained by fitting data to the equation:L ¼ ∑Lnð1−e−k Þ; where n ¼ 1 or 2.
d
L2
1
Fraction was determined by L
ꢁ 100.
¼L2

K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
339
Table 4
ODF-loaded liposomes displayed a single-exponential leakage pro-
file. Statistical analysis indicated that the initial rates, though similar in
magnitude, were significantly different and less that either the control
sample or the EF-loaded liposomes. The indication here was that leak-
age is slower in ODF-loaded liposomes than non-loaded (control) or
EF-loaded liposomes. Additionally, each leakage profile for ODF-loaded
liposomes resulted in nearly one-third of the percent leakage exhibited
by unloaded liposomes (control). This suggests that ODF stabilized the
liposomes against leakage. Based on the facts that the liposomes were
in a fluid state under the current experimental conditions at 37 °C
(DOPC has a phase transition temperature at −20 °C), ODF lipids have
phase transition temperature of 67 °C, and the neutrally charged lipids
had a minimum electrostatic interaction with negatively charged
calcein [46], it is interpreted that content leakage correlated with the
fluidity of the membrane. Therefore, it stands to reason to conclude
that ODF reduced membrane fluidity by altering the membrane packing
density. This is consistent with an earlier report that a similarly struc-
tured lyso-lipid altered the fluidity of phosphatidylcholine membranes
Relative antioxidant capacity of ethyl and octadecyl ferulates in DOPC liposomes.
Antioxidant
Concentration
(μM)
Relative antioxidant
capacitya
EF
25
62.5
125
25
62.5
125
1.70 0.07
1.43 0.02
1.40 0.02
0.89 0.04
0.65 0.02
0.53 0.01
ODF
a
Data is reported as the mean and standard deviation of values calculated. Experiments
were conducted in triplicate at least. Values are relative to 50 μM Trolox. One-way ANOVA
was applied to the data. All are considered to be statistically different (P b 0.05).
within the bilayer, the leakage profile in each case was quite similar (as
borne out by the traces overlapping and reaching similar %leakage
values). Thus, it was interpreted that EF did not induce any bilayer
changes that resulted in significant bilayer destabilization.
Fig. 6. Antioxidation behavior of (a) ethyl ferulate — EF and (b) octadecyl ferulate — ODF in DOPC liposomes.

340
K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
Fig. 6 (continued).
[47] and consistent with the thermotropic behavior described earlier in
this work. The ability of ODF to reduce membrane fluidity is, however, in
contrast to the effect exhibited by FDOG on the same lipid system [17]
where FDOG had no effect on liposome stability (leakage remained
unchanged).
The liposomal leakage exhibited by ODF can be explained by the
structural of ODF. ODF has a feruloyl moiety esterified to a single
octadecyl group, which resulted in ODF being a solid at room tempera-
ture and up to 67 °C. This suggests that ODF would form a more rigid re-
gion within the bilayer, presenting a barrier to local content leakage
unlike FDOG which was a “fluid state” lipid (the result transesterification
of ethyl ferulate to triolein in the sn−1 or sn−3 position) and did not af-
fect leakage [17]. Thus, ODF can limit permeability due to increased local
packing stress in the membrane, unlike local structural changes that
resulted in greater permeability of 1-palmitoyl-3-oleoyl phosphocholine
liposomes under shear flow [48].
3.6. Lipid peroxidation inhibition
Ferulic acid has well-known antioxidant characteristics [49]. It is
unclear, though, about the extent of antioxidant properties for ODF,
especially within phosphatidylcholine liposomes. Therefore, the antiox-
idant properties of ODF in DOPC liposomes were tested and compared
to EF, a smaller feruloyl derivative (Table 4). The method utilized
AAPH, a free-radical generator and C11-Bodipy as the radical identifier.
AAPH thermally decomposed into two peroxyl radicals which, upon
interaction with C11-Bodipy molecules, caused a spectral shift from
595 nm. This spectral shift was seen as a fluorescence signal reduction
when detected at 595 nm. Experiments were conducted at roughly a
1042 to 1 lipid-to-probe molar ratio.
DOPC liposomes incorporated with EF demonstrated significant an-
tioxidant behavior, as displayed by the lag plateau present in all concen-
trations explored (Fig. 6a, top). Clearly, there was linearity between

K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
341
Fig. 7. B3LYP/6–31 + G* conformations of ODF, EF, and FDOG rendered in space filling overlapping spheres.
ΔAUC and EF concentration (Fig. 6a, bottom). Linearity between ΔAUC
and concentration was also present for ODF (Fig. 6b), demonstrating
that ODF maintain antioxidant capability. It is noteworthy to mention
that ODF did not exhibit any lag plateau and the ΔAUC values for ODF
were 2–3 times less than values for EF at corresponding concentration.
This suggests that ODF was less effective as an antioxidant in liposomes
than EF. However, the relationship between ΔAUC and antioxidant con-
centration may rely on multiple factors and thus make interpretation
complex. It therefore may be simpler to normalize the ΔAUC with
respect to Trolox, the standard used.
Normalization of the ΔAUC values based on one concentration of the
standard gave an antioxidant capacity number that was relative to
Trolox. Table 4 summarizes that although there was a slight decrease
in relative antioxidant capacity with concentration for EF and ODF,
ODF maintained antioxidant capacity but was less than Trolox and EF.
It should be noted that ODF proved to have nearly the same antioxidant
capacity as FDOG. It is believed that this similarity in relative antioxida-
tion capacity was primarily due to the fact that both have 18-carbon acyl
chains and less due to the presence of multiple acyl chains and chain
saturation/unsaturation.
ODF has tendency to form a spherical micelle and partition into mem-
branes [51]. In contrast, the EF has a much shorter acyl chain, smaller
molecular volume and smaller headgroup-to-acyl chain area ratio
than ODF. Despite this, the similar intrinsic curvature of EF still suggests
the formation of possible micelles. However, structurally EF may be bet-
ter described as a hydrotrope [52] since EF as an aromatic ring attached
to a short chain. Thus, described as a hydrotrope EF likely will form
sphere-like aggregates due to a conical molecular shape. This similarity
in molecular shape explains the resemblance in portioning coefficients
of ODF and EF. In fact, the similarities between aggregates formed by
short-chained hydrotropes, such as EF, and more traditional long tail
surfactants similar to ODF have been extensively investigated [53].
FDOG, on the other hand, has a headgroup-to-acyl chain area ratio less
than one which gives FDOG a cylindrical shape with negative intrinsic
curvature and thus likely forming bilayers [51] and expected to have a
much higher (presently immeasurable) partition coefficient. Surpris-
ingly, ODF, EF and FDOG have similar frontier molecular orbitals
(εHOMO and εLUMO) and band gap energies despite their significant
differences in acyl chain sizes and volumes, suggesting that the feruloyl
moiety was the main contributor to orbitals. Furthermore, modeling
shows ODF, EF and FDOG to possess similar chemical reactivity and
electron accepting/donating properties to free ferulic acid [54], leading
to the conclusion that interaction within the lipid bilayer exerts the
greatest influence on antioxidant activity.
3.7. Modeling
Molecular modeling was undertaken to better understand the struc-
tural nature of ODF and how its intrinsic curvature affects partitioning
into a DOPC membrane. Intrinsic curvature properties calculated on
single molecules have been shown to be related to partitioning and
micelle/bilayer formation [49–52]. ODF molecular model was optimized
using SPARTAN'10 and the B3LYP density functional and the 6–31 + G*
basis set. Molecular modeling shows that energetically preferred ODF
confirmation has the feruloyl moiety angled with respect to the acyl
chain tail (Fig. 7). This resulted in a positive headgroup-to-acyl chain
area ratio (Table 5), suggesting that ODF has a conical shape or positive
intrinsic curvature [50]. This positive intrinsic curvature indicates that
3.8. ODF Position in DOPC bilayer
ODF at 5-mol% was incorporated into DOPC liposomes to determine
membrane depth of the feruloyl group using nitroxide labeled phospho-
lipids as fluorescent quenchers. ODF presented a strong fluorescent
signal within DOPC liposomes which was quenched the most by
12DOXYL-PC, the deepest quencher employed (data not shown).
Table 6 summarizes the results of EF and ODF locations within the
DOPC bilayer. The finding was the feruloyl group of EF and ODF were
found on average approximately 12 Å from the bilayer center, which
is right at or above the carbonyl groups of the phospholipids that are
at the lower limit of bilayer hydrophilic region, just slightly deeper
than the feruloyl moiety for FDOG [17].
Table 5
Parameters from B3LYP/6–31 + G* calculations on three conformations of ODF.
ODF 1
0
ODF 2
1.29
ODF 3
8.28
EF
FDOG
0
ΔE (kcal mol−1
)
0
Table 6
Dipole (Debye)
1.55
5.55
−5.96
−1.86
−4.10
932.3
3.76
−6.05
−2.03
−4.02
968.9
1.76
1.87
Approximate location of feruloyl moiety in phosphocholine bilayer.
ε
ε
HOMO (eV)
LUMO (eV)
−5.93
−1.85
−4.08
980.9
1602.8
1.42
−5.94
−1.87
−4.07
453.7
710.8
1.80
−6.23
−2.06
−4.17
1389.9
2587.5
0.33
Sample
Approximate distance from
the bilayer center, zcF (Å)
Band gap (eV)
Area (Å2)
Volume (Å3)
1581.6
2.16
1578.3
1.99
EF
ODF
12.38
12.15
Areaheadgroup/Areaacyl chain

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K.O. Evans et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 333–343
[18] J. Zhang, R.A. Stanley, L.D. Melton, Lipid peroxidation inhibition capacity assay for
4. Conclusions
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In summary, ODF was synthesized and investigated for its thermo-
tropic properties and behavior within DOPC liposomes using differential
scanning calorimetry, fluorescence methods and molecular modeling. It
was ascertained that ODF has melting and solidification temperatures of
67 and 39, respectively. It was demonstrated that DOPC supported
bilayer containing 5-mol% ODF formed domains on mica and the pres-
ence of ODF increased liposomal phase transition temperature. Studies
of ODF behavior show readily partitioning into n-octanol and the ability
ODF to induce an increase in lipid packing density. Further studies
revealed that ODF retained antioxidation behavior and resided within
a DOPC similar to FDOG. We expect this work to deepen the basic
understanding of increasing the lipophilicity of phenolic compounds.
Supplementary data to this article can be found online at http://dx.
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We graciously thank Leslie Smith, Judy Blackburn, Ray Holloway and
Jason Adkins for their technical assistance.
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