Journal of Agricultural and Food Chemistry
Article
several times and dried over sodium sulfate to remove traces of water.
Then, the remaining alcohol and phenolic acid were eliminated by
flash chromatography (CombiFlash Companion system, Teledyne Isco
Inc., Lincoln, NE, USA). Separation was carried out on a silica column
(pore size 60 Å, Acros organics, Geel, Belgium) using an elution
gradient of two solvents with different polarities. Because of the
different polarities of the phenolic acids and their alkyl esters, a
different elution gradient was applied for caffeates (chloroform and
methanol, 0−100%), ferulates, and coumarates (dichloromethane and
ethyl acetate, 5−100%), respectively. For some phenolipids, alcohol
was still present after flash chromatography. In these cases, preparative
TLC with different migration solvents was applied (caffeates, 96%
chloroform and 4% methanol; ferulates, 95% dichloromethane and 5%
ethyl acetate; and coumarates, 90% dichloromethane and 10% ethyl
acetate).
Antioxidant Properties. The antioxidative properties of the
phenolic acids and their synthesized alkyl esters were evaluated using
three different in vitro antioxidant assays: radical scavenging (DPPH),
iron chelating activity, and reducing power. All antioxidants were
evaluated in 4 concentrations (final concentrations: 50, 100, 200, and
400 μM) and were measured in triplicate (n = 3). The concentrations
were selected based on earlier antioxidant property analysis and
storage experiments with similar compounds.14
were also diluted in PBS (pH 7.2). The final concentration of
antioxidant was 100 μM. Iron, as FeSO4, was solubilized in 0.50 M
HCl and buffer (1:1, v/v) and added to pure buffer and to the
antioxidant−buffer solution. The final concentration of iron was 100
μM. Each solution was measured using a spectrophotometer (UV-
1800, Shimadzu Scientific Instruments, Columbia, MD, USA), and a
spectrum was recorded in the UV−vis region (200−800 nm).
Conjugated Autoxidizable Triene (CAT) Assay. Stock solutions
of the different hydroxycinnamic acids, their alkyl esters, and trolox
were prepared in methanol. Various volumes of these antioxidant
methanolic solutions were added to 1.8 mL of PBS (pH 7.2) and then
filled up to 2 mL with pure methanol. These buffered solutions with
antioxidant (50 μL) were transfered into a microtiterplate, (UV-Star
96-well microplate, Greiner, Frickenhausen, Germany). The micro-
plate was then preheated (37 °C) and stirred in the microplate reader
(Synergy 2, BioTek, Winooski, VT, USA) for 5 min at medium speed.
Brij 35-PBS (34 μM, 25 mL) was added to 5 mg of stripped tung oil in
a brown glass bottle. Brij 35−PBS−tung oil was premixed for at least
10 s using a vortex apparatus before the mixture was homogenized
with a POLYTRON PT1200E (Kinematica AG, Lucerne, Switzerland)
at high speed for 4 min. Each well was then filled with 100 μL of the
tung oil-in-PBS microemulsion; the plate was again preheated and
stirred for 1 min. The oxidation of tung oil was initiated by adding 50
μL of 4 mM AAPH in PBS. Finally, each microplate well contained
200 μL of the following mixture: 115 μM stripped tung oil, 17 μM Brij
35, 1 mM AAPH, and various concentrations of phenolics,
phenolipids, or trolox (0.2−0.8 μM). The progress of lipid oxidation
was immediately followed by measuring the decrease in absorbance at
273 nm. Measurements were performed each minute for 6 h at 37 °C
with 5 s stirring before each measurement. Each antioxidant
concentration was measured in triplicate on the plate and via
independent measurements (three different microplates), n = 9.
Results were expressed as CAT value (mean SD). This method was
developed by Laguerre et al.16 For further details about the
calculations, see refs 7 and 16 by Laguerre et al.
Radical Scavenging. Antioxidants were solubilized in methanol in
different concentrations, and BHT was included in this assay as a
positive control in the same concentrations. The antioxidant solution
(150 μL) was diluted 1:1 (v/v) with 0.1 mM DPPH in ethanol (96%,
150 μL). Then, 200 μL of this mixture was transferred to a well in the
microplate.15 The absorbance at 517 nm was measured after 30 min
(RT, darkness) in a microplate reader (Synergy 2 BioTek, Winooski,
VT, USA). Results are expressed as inhibition percentages.
⎛
⎞
⎟
⎠
As − A0
Inhibition [%] = 1 −
× 100
⎜
Ab
⎝
Removal of Tocopherols from Tung Oil. Tung oil was stripped
from tocopherols using an alumina packed glass column. A glass
column was packed with 25 g of alumina oxide in hexane. The excess
of hexane was removed. Tocopherols were removed from the tung oil
by passing 25 mL (200 mg/mL) of tung oil in hexane through the
packed column. Pure hexane (50 mL) was then loaded to the column
to get the loaded tung oil through the column. After passing the tung
oil through the column, hexane in the stripped tung oil was removed
using a vacuum rotary evaporator. Finally, a trace of hexane in the
stripped tung oil was removed under nitrogen. The stripped tung oil
was bottled (5 mg/bottle) in brown glass tubes, flushed with nitrogen,
and stored at −80 °C until use (CAT Assay). Furthermore, the
absence of tocopherols in the stripped tung oil was checked by HPLC
according to the AOCS method.17
Data Treatment. All measurements were performed in triplicate
and reported as average SD. In the separate method descriptions,
calculations are described, or there are references to data treatment.
Statistics. The obtained results were analyzed by one-way ANOVA
(GraphPad Prism, version 4.03, GraphPad Software Inc.). Bonferroni
multiple comparison post-test was used to test differences between
samples. For statistics, a significance level of p < 0.05 was used. When a
significant difference was observed between samples, they are denoted
with different lowercase letters in the text, table and figures.
where As is the measured absorbance after the antioxidant-DPPH
reaction, A0 is the measured absorbance of antioxidant in ethanol
without DPPH (sample control), and Ab is the absorbance measured
of DPPH without the antioxidant added.
Reducing Power. Antioxidants were solubilized in methanol in
different concentrations, and ascorbic acid was included in this assay as
a positive control in the same concentrations. Antioxidant methanolic
solutions (200 μL) were transferred to test tubes, and 0.2 M
phosphate buffer (200 μL) and 1% potassium ferricyanide (200 μL)
were added. These mixtures were incubated at 50 °C for 20 min. After
incubation, 10% TCA (trichloroacetic acid, 200 μL) was added and
mixed. This reaction mixture (228 μL) was transferred to an
Eppendorf tube and mixed with an equal amount of water (228
μL). Ferric chloride (0.1%, 46 μL) was added to the reaction mixture,
and it was incubated for 10 min at RT. After incubation, the reaction
mixtures (200 μL) were transferred to the wells in the microplate,15
and absorbance was measured at 700 nm in a microplate reader
(Synergy 2 BioTek, Winooski, VT, USA). Results are shown relative to
ascorbic acid (AbsSample/AbsAscorbic acid).
Iron Chelating Ability. Antioxidants were solubilized in warm water
in different concentrations. Because of solubility problems with long
alkyl chain phenolipids, only caffeic acid, ferulic acid, coumaric acid,
and their methyl esters were evaluated for their chelating ability of
ferrous chloride. EDTA was included in this assay as a positive control
in the same concentrations as the phenols and phenolipids. For further
details, refer to Sørensen et al.14 The absorbance was measured at 562
nm (UV-1800 Shimadzu, Columbia, MD, USA), and results are
expressed as chelating activities.
RESULTS
■
Antioxidant Properties. All the synthesized antioxidants
were evaluated for different antioxidant properties as radical
scavenger (DPPH), reducing power, and iron chelation in in
vitro assays.
Antioxidant (Hydroxycinnamic Acids and Their Alkyl
Esters)−Iron Interactions. Interactions between iron and the
antioxidant were evaluated using a spectrophotometer according to
the method developed by Sørensen et al.9 Methanolic solutions of
caffeic acid, ferulic acid, coumaric acid, and their methyl esters were
diluted in a buffer solution: 10 mM sodium acetate−imidazole (pH 7).
Moreover, methanolic solutions of caffeic acid and methyl caffeate
Radical Scavenging Properties. The phenolics and
synthesized phenolipids were all able to scavenge free radicals
measured by the DPPH assay (Figure 2A−C). Caffeic acid and
caffeates had similar radical scavenging activities in the
12555
dx.doi.org/10.1021/jf500588s | J. Agric. Food Chem. 2014, 62, 12553−12562