Novel caffeic acid amide antioxidants: Synthesis, radical scavenging activity and performance under storage and frying conditions

Article abstract of DOI:10.1016/j.foodchem.2011.08.021

Twelve novel dihydro-caffeic acid amides were synthesised in good yields and fully characterised by ¹H NMR, ¹³C NMR, and MS. Their radical scavenging activities were assessed by DPPH assay. Additionally, their abilities to protect polyunsaturated oils under accelerated storage and frying conditions were evaluated. All the new compounds possessed significantly higher radical scavenging activities than α-tocopherol and butylated hydroxytoluene (BHT). The radical scavenging activity of N-decyl-N-(3,5- dimethoxy-4-hydroxybenzyl)-3-(3,4-dihydroxyphenyl) propanamide was 1.7 and 4 times higher than α-tocopherol and BHT, respectively. At the end of the storage period, the respective amounts of hydroperoxides in canola oil triacylglycerols (CTAG) fortified with α-tocopherol and BHT was 6.1 and 1.4 times higher, respectively, than CTAG containing the amide. The frying test showed that CTAG containing N-decyl-N-benzyl-3-(3,4-dihydroxybenzyl) propanamide was 1.3, 1.4, and 1.6 times more stable compared to oil fortified with dihydro-caffeic acid, α-tocopherol and BHT, respectively, as assessed by the amounts of the total polar compounds. Moreover, these compounds were remarkably thermally stable, making them suitable for frying applications.

Full text of DOI:10.1016/j.foodchem.2011.08.021

Food Chemistry 130 (2012) 945–952
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Novel caffeic acid amide antioxidants: Synthesis, radical scavenging activity
and performance under storage and frying conditions
⇑
Felix Aladedunye, Yohann Catel, Roman Przybylski
Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K3 M4
a r t i c l e i n f o
a b s t r a c t
Twelve novel dihydro-caffeic acid amides were synthesised in good yields and fully characterised by 1H
NMR, 13C NMR, and MS. Their radical scavenging activities were assessed by DPPH assay. Additionally,
their abilities to protect polyunsaturated oils under accelerated storage and frying conditions were eval
Article history:
Received 24 March 2011
Received in revised form 7 June 2011
Accepted 5 August 2011
Available online 12 August 2011
uated. All the new compounds possessed significantly higher radical scavenging activities than
erol and butylated hydroxytoluene (BHT). The radical scavenging activity of N-decyl-N-(3,5-dimethoxy-
4-hydroxybenzyl)-3-(3,4-dihydroxyphenyl) propanamide was 1.7 and 4 times higher than -tocopherol
and BHT, respectively. At the end of the storage period, the respective amounts of hydroperoxides in
canola oil triacylglycerols (CTAG) fortified with -tocopherol and BHT was 6.1 and 1.4 times higher,
a-tocoph
a
Keywords:
Phenolic acids
Caffeic amides
Antioxidants
Frying
a
respectively, than CTAG containing the amide. The frying test showed that CTAG containing N-decyl-
N-benzyl-3-(3,4-dihydroxybenzyl) propanamide was 1.3, 1.4, and 1.6 times more stable compared to
oil fortified with dihydro-caffeic acid,
a-tocopherol and BHT, respectively, as assessed by the amounts
DPPH
Storage
of the total polar compounds. Moreover, these compounds were remarkably thermally stable, making
them suitable for frying applications.
Radicals scavenging
Polar components
Peroxide value
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
offer good protection during storage, however their use has been
limited because of the potential detrimental effects on the human
Recent global awareness campaigns for the health benefits of
essential fatty acids have led to a considerable increase in human
consumption of polyunsaturated oils. However, these oils are
susceptible to oxidative degradation leading to the generation of
free radicals and other toxic products. Free radicals have been
implicated in the pathogenesis of inflammatory and cardiovascular
diseases, cancer and ageing (Reuter, Gupta, Chaturvedi, & Aggar-
wal, 2010). Under storage and frying conditions, polyunsaturated
oils rapidly degrade, negatively impacting the physical, flavour,
nutritional and functional qualities of the oils and the foods that
contain them. It is imperative, therefore, that these oils are
protected against oxidative and thermal degradation. One such
measure is the application of antioxidants. Phenolic antioxidants
are particularly potent and many of them occur naturally in food.
They slow the degradation of food lipids by inhibiting the propaga-
tion of oxidation, and many of them are known to possess a variety
of interesting biological activities (Jiang et al., 2005; Ou & Kwok,
2004; Priscilla & Prince, 2009). On the other hand, synthetic antiox-
idants such as butylated hydroxytoluene (BHT), butylated
hydroxyanisole (BHA) and tertiary butyl hydroquinone (TBHQ)
health (Frankel, 2007, Chap. 1). As a consequence, there is a grow-
ing interest in the development of new antioxidants exhibiting low
toxicity, high antioxidant capacity, good thermal stability and pre-
pared from natural precursors.
Recently, Rajan et al. (2001) described the synthesis of new caf-
feic acid amides exhibiting good antioxidant activities. In their
study, the amide group was chosen for enhanced metabolic stabil-
ity. Jung et al. (2002) also reported the synthesis of 4-hydroxyphe-
nylacetic acid amide possessing potent analgesic activity. The
synthesis of caffeic acid amides possessing antimicrobial activities
was recently reported by Fu, Cheng, Zhang, Fang, and Zhu (2010).
The amide derivative of N-acetylcysteine (NACA), a commercially
available pharmaceutical drug and nutritional supplement, was
found to more efficiently permeate cell membranes than the par-
ent form (Ates, Abraham, & Ercal, 2008). Several hydroxycinnamic
acid amides occur in different parts of plants and a number of them
possess interesting physiological activities (Chen, Chang, Yen, &
Wu, 1998; Dalby-Brown, Barsett, Landbo, Meyer, & Mølgaard,
2005; Fagerlund, Sunnerheim, & Dimberg, 2009).
In this paper, we synthesised a variety of new N-alkyl-N-aryl-3-
(3,4-dihydroxyphenyl) propane-amides and assessed their radical
scavenging activities using the DPPH assay. Recognising that there
is a lack of direct relation between the radical scavenging activity
measurements by DPPH or ORAC assays and the real antioxidant
⇑
Corresponding author. Tel.: +1 403 317 5055; fax: +1 403 329 2057.
E-mail
addresses:
felix.aladedunye@uleth.ca
(F.
Aladedunye),
roman.przybylski@uleth.ca (R. Przybylski).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.08.021

946
F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
activity in a food system (Alamed, Chaiyasit, Mc Clements, & Deck-
er, 2009), we assessed the effectiveness of the novel antioxidants
using standard accelerated storage and frying tests. Canola oil tria-
cylglycerols, which are typical polyunsaturated oils, were used as
the substrate. We chose to prepare derivatives of dihydro-caffeic
acid due to its higher radical scavenging and antioxidant activity
compared to caffeic acid (Nenadis, Boyle, Bakalbassis, & Tsimidou,
2003).
Utilising an ethyl acetate/hexane mixture (3:2) as eluant,
the crude N-propyl-N-benzyl-3-(3,4-dihydroxyphenyl) propana-
mide (3a); N-decyl-N-benzyl-3-(3,4 dihydroxyphenyl) propana-
mide (3c); N-hexyl-N-(4-hydroxybenzyl)-3-(3,4-dihydroxyphe
nyl ) propanamide (3e) and N-decyl-N-(4-hydroxybenzyl)-3-(3,4-
dihydroxyphenyl) propanamide (3f) were purified by a flash chro-
matography. The crude N-hexyl-N-benzyl-3-(3,4-dihydroxy-
phenyl) propanamide (3b) was purified in the same manner
using an ethyl acetate/hexane mixture (1:1). Crude N-propyl-N-
(4-hydroxybenzyl)-3-(3,4-dihydroxyphenyl) propanamide (3d);
N-propyl-N-(3-methoxy-4-hydroxybenzyl)-3-(3,4-dihydroxyphen
yl) propanamide (3g); N-hexyl-N-(3-methoxy-4-hydroxybenzyl)-
3-(3,4-dihydroxyphenyl) propanamide (3h); N-decyl-N-(3-meth-
2. Materials and methods
2.1. Reagents
oxy-4-hydroxybenzyl)-3-(3,4-dihydroxyphenyl)
(3i); N-propyl-N-(3,5-dimethoxy-4-hydroxybenzyl)-3-(3,4-dihy
propanamide
Benzaldehydes; 4-hydroxybenzaldehyde; vanillin (3-methoxy-
4-hydroxybenzaldehyde); syringaldehyde (3,5-dimethoxy-4-hydr
oxybenzaldehyde); propylamine; hexylamine; decylamine; dihy-
dro-caffeic acid (DCA); 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and
silica gel were purchased from Sigma–Aldrich (St. Louis, MO, USA).
Benzotriazole-1-yl-oxy-tris (dimethylamino) phosphonium hexa-
fluorophosphate (BOP), butylated hydroxytoluene (BHT) and
droxyphenyl) propanamide (3j); N-hexyl-N-(3,5-dimethoxy-4-
hydroxybenzyl)-3-(3,4-dihydroxyphenyl) propanamide (3k) and
N-decyl-N-(3,5-dimethoxy-4-hydroxybenzyl)-3-(3,4-dihydroxy-
phenyl) propanamide (3L) were purified by a flash chromatogra-
phy using an ethyl acetate/hexane mixture (7:3).
a
-tocopherol were from Calbiochem-Novabiochem (San Diego, CA,
2.3. Instruments
USA). Dry dichloromethane (DCM) was purified using a MBraun Sol-
vent Purification System (M. Braun Incorporated, Stratham, NH). All
other solvents were HPLC grade.
1H NMR and 13C NMR were recorded on a 300 MHz Bruker
Avance II spectrometer (Bruker Daltonics, Billerica, MA) with tetra-
methylsilane (TMS) as internal standard. HPLC analyses for the
residual antioxidants were performed on a Finnigan Surveyor LC
system (Thermo Electron, Waltham, MA, USA) with a Finnigan Sur-
veyor Autosampler Plus, Finnigan Surveyor FL Plus fluorescence
detector and a Finnigan Surveyor photodiode array detector
(PDA). The column was a normal phase Diol column (5
250 ꢀ 4.6 mm; Monochrom, Varian, CA). Column chromatography
with EMD silica gel Si 60 (40–63 m) was used for separation
and purification. Melting points (Mp) were measured with an Elec-
trothermal MEL-TEMP 3.0 (Barnstead, Dubuque, IA, USA). A Beck-
man DU-65 spectrophotometer (Beckman Instruments, Inc.,
Fullerton, CA) was used in the DPPH assay as well as for the deter-
mination of PV. High-resolution mass spectra (HRMS) were ob-
tained with a QSTAR Elite mass spectrometer (AB SCIEX, Concord,
ON, Canada) equipped with an electrospray operating in positive
ion mode.
2.2. Synthesis
2.2.1. General procedure for the synthesis of amines 2a–2L
The aldehyde (10 mmol) and the amine (15 mmol) were dis-
solved in methanol (10 ml) and the mixture was stirred for 3 h,
then NaBH4 (10 mmol) was added in small portions. The mixture
was again stirred for 3 h following by solvent removal under re-
duced pressure using a rotary evaporator. Distilled water (10 ml)
was then added and the solution extracted three times with ethyl
acetate (10 ml each). The organic layers were combined and dried
on magnesium sulphate followed by solvent removal under re-
duced pressure in a rotary evaporator and the desired crude prod-
uct was obtained.
Propylbenzylamine (2a); 4-[(propylamino) methyl] phenol (2d)
and 2-methoxy-4-[(propylamino) methyl] phenol (2g) were used
in the next step without purification. The crude hexylbenzylamine
(2b) and decylbenzylamine (2c) were purified by a flash chromatog-
raphy using ethyl acetate as an eluant. 4-[(decylamino) methyl]
phenol (2f); 2-methoxy-4-[(hexylamino) methyl] phenol (2h) and
2-methoxy-4-[(decylamino) methyl] phenol (2i) were similarly
purified using ethyl acetate/methanol (9:1) as an eluant. The crude
4-[(hexylamino) methyl] phenol (2e) was purified in the same
manner using an ethyl acetate/methanol (9.5:0.5). Whereas 2,
6-dimethoxy-4-[(propylamino)methyl]phenol (2j); 2,6-dimethoxy
-4-[(hexylamino) methyl] phenol (2k) and 2,6-dimethoxy-4-
[(decylamino) methyl] phenol (2L) were purified by recrystallisa-
tion in hexane.
lm;
l
2.2.2. General procedure for the synthesis of new DCA amides (3a-3L)
DCA (200 mg, 1.1 mmol) and triethylamine (1.1 mmol) were
added to dimethylformamide (2 ml) and the mixture was stirred
at 0 °C for 30 min. Afterwards, amine (2.2 mmol), dissolved in a
minimum of DCM, and BOP (486 mg, 1.1 mmol), dissolved in
2 ml of DCM, were added and stirred for 2 h at room temperature.
The solution was concentrated under reduced pressure using a ro-
tary evaporator. Ten milliliters of water was added to the residue
and the solution was extracted three times with 10 ml of ethyl ace-
tate. The organic layers were combined and dried on magnesium
sulphate and the solvent was removed under reduced pressure
using a rotary evaporator.
Fig. 1. Reactions and structures of the novel antioxidants 3a–3L. For details see the
text.

F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
947
2.4. DPPH radical scavenging assay
decrease in absorbance at 516 nm was measured at 25 °C after
20 min of reaction. The blank solution contained the same amount
The DPPH assay was performed according to a method de-
scribed by Nenadis et al. (2003). Briefly, an ethanolic DPPH solution
of DPPH reagent and 40 ll of ethanol and each test was performed
in triplicate. The results were expressed as the percentage of DPPH
(0.1 mM; 2960 ll) was added to the 40 ll of antioxidant solution in
inhibition, calculated as follows:
ethanol at the following concentrations: 0.74 mM, 1.11 mM,
1.85 mM, 3.7 mM, providing the ratios of the molar amounts of
antioxidant to DPPH at 0.10, 0.15, 0.25 and 0.5, respectively. The
ðAcontrol ꢁ AtestÞ100
% DPPH ¼
ð1Þ
Acontrol
Table 1
Characteristics of synthesised compounds.
Compound
Description
Mp (°C)
Yield (%)
[M + H]+
Rotamers ratio
Calculated
Found
2d
2e
2f
2g
2h
2i
Light yellow, viscous oil
Light orange solids
Light orange solids
Light orange solids
Light yellow solids
White solids
White solids
White solids
White solids
Colourless, highly viscous oil
Light yellow, highly viscous oil
Light yellow, highly viscous oil
White solids
Light green, highly viscous oil
Light yellow, highly viscous oil
White solids
Orange, highly viscous oil
Light orange, highly viscous oil
White solids
Orange, highly viscous oil
Orange, highly viscous oil
NA
98
89
85
98
86
80
90
68
76
58
68
56
64
55
47
60
56
53
54
54
61
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
NA
NA
NA
NA
NA
61–62
61–63
56–58
65–67
77–78
139–141
83–85
90–92
NA
NA
NA
62–64
NA
NA
59–61
NA
NA
60–62
NA
NA
2j
2k
2L
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3L
ND
ND
NA
314.1751
356.2220
412.2846
330.1700
375.2169
428.2795
360.1805
402.2275
458.2901
390.1911
432.2381
488.3007
314.1754
356.2231
412.2854
330.1706
372.2168
428.2785
360.1805
402.2286
458.2907
390.1909
432.2378
488.3020
55:45
55:45
57:43
55:45
55:45
60:40
59:41
60:40
60:40
57:43
59:41
60:40
NA = Not Applicable; ND = Not determined; Compounds 2d–2L are intermediates used in synthesis; Mp = Melting point.
Table 2
1H and 13C NMR of the amine precursors synthesised in the study.
2d*
2e
2f
2g*
2h
2i
2j*
2k
2L
Hydrogen
CH3 (t)
CH2 (m)
CH2CH2N
0.90, J = 5
0.88, J = 6.9
1.20–1.38
0.90, J = 6.9
1.22–1.38
1.49–1.62(m)
0.94, J = 6.9
0.89, J = 6.9
1.22–1.38
0.88, J = 6.9
1.20–1.38
1.43–1.58(m)
0.88, J = 7.2
0.88, J = 6.9
1.24–1.38
1.43(sx), J = 7.2 1.46–
1.58(m)
0.86, J = 6.9
1.14–1.38
1.42–
1.58(m)
2.62, J = 7.2
3.71
1.55(sx), J = 7.5 1.50–1.61(m)
1.56,sx, J = 7.5 1.45–1.57(m)
CH2CH2N (t) 2.63, J = 7.5
2.70, J = 7.5
3.70
2.69, J = 7.5
3.71
2.63, J = 7.2
3.73
3.87
2.62, J = 7.5
3.71
3.88
2.62, J = 7.5
3.71
3.88
2.44, J = 7.2
3.57
3.74
2.63, J = 7.2
3.71
3.88
ArCH2 N (s)
OCH3 (s)
CHAr
CHAr (d)
CHAr (s)
OH, NH (s)
3.67
3.88
6.56(s)
6.57(d), J = 8.4
7.05, J = 8.4
6.59(d), J = 8.4 6.63(d), J = 8.4 6.79(d), J = 8.1 6.77(d), J = 7.8 6.77(d), J = 7.8 6.59(s)
7.08, J = 8.4
6.56(s)
7.09, J = 8.4
6.85, J = 8.1
6.88
6.05
6.84, J = 7.8
6.87
6.84, J = 7.8
6.87
4.50
12.3
5.12
6.05
Carbon
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2CH2N
CH2CH2N
ArCH2N
OCH3
CHAr
CHAr
CHAr
CHAr
CHAr
CHAr
14.0
22.5
27.0
29.3
14.2
22.7
27.3
29.2
29.4
29.5
29.6
31.9
49.2
53.3
11.8
14.0
22.6
27.0
29.3
14.1
22.7
27.4
29.3
29.5
29.6
29.7
31.9
49.3
53.8
55.7
145.2
114.7
111.2
121.0
131.3
147.1
12.4
14.1
22.6
27.1
30.0
14.1
22.7
27.4
29.3
29.5
29.6
30.0
31.9
49.6
54.4
56.2
147.2
147.2
104.8
104.8
131.4
133.7
23.0
51.0
53.1
31.6
49.2
53.3
22.8
51.1
53.8
55.6
145.2
114.7
111.2
121.0
132.9
147.1
31.7
49.0
53.6
55.4
145.8
115.2
111.6
121.1
130.3
147.6
23.1
51.1
53.7
56.3
148.2
148.2
105.6
105.6
131.6
134.4
31.8
49.5
54.4
56.2
147.2
147.2
104.8
104.8
131.4
133.7
115.3
115.3
129.5
129.5
131.5
156.5
116.0
116.0
129.3
129.4
129.8
156.5
116.0
116.0
129.3
129.3
129.8
156.6
*
Solvent = DMSO-d6; s, singlet; d, doublet; t, triplet; sx, sextet; m, multiplet. J, coupling constant in Hertz. For details see the text.

948
F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
Fig. 2. 1H NMR spectrum of the novel antioxidant 3a. See the text for details.
Where: Acontrol and Atest are the absorbances of the control and test
samples, respectively. All standard deviations (SD) for DPPH tests
were below 3.0%. BHT,
ences. Calculated IC50 represents the concentration of antioxidant
required to decrease the DPPH amount by 50%.
a-tocopherol and DCA were used as refer-
2.5
2.0
1.5
1.0
0.5
2.5. Antioxidant activity under storage and frying conditions
2.5.1. Purification of canola oil
Canola oil was stripped of its endogenous minor components,
including antioxidants via adsorption chromatography, following
the method described by Lampi and Kamal-Eldin (1998).
0.0
60
2.5.2. Accelerated storage – Schaal oven test
The ability of the novel antioxidants 3a–3L to protect oil from
oxidative degradation was determined using the Schaal oven test.
50
40
30
20
10
Canola triacylglycerols (1.0 g), fortified with 350 lg/g of the tested
antioxidant, were introduced in the vials (National Scientific Target
DP Vials; 2 ml, 12 ꢀ 32 mm). The ratio of surface area to volume
was 0.78. The uncapped vials were stored in darkness at 60 °C for
up to 7 days. Samples were examined at 24 h intervals by collect-
ing individual vials at the particular period. The oxidative stability
of the samples was evaluated by peroxide value (PV). The effective-
ness of the new compounds was compared with
a-tocopherol, as
natural antioxidant, and butylated hydroxytoluene (BHT), as syn-
thetic antioxidant. Experiments were set up in two repetitions
for each tested antioxidant, and samples from each repetition were
analysed in triplicate.
3a 3b 3c 3d 3e 3f 3g
3i
3k 3L αT BHT DCA
Anti3ohxidant3j
Fig. 3. DPPH radicals scavenging and IC50 for
a
-tocopherol, BHT, DCA and the novel
antioxidants. All antioxidants were tested at a concentration of 1.11 mM.
tocopherol; BHT – butylated hydroxytoluene; DCA – dihydro-caffeic acid. See the
text for details.
aT – a-
2.5.3. Determination of peroxide value
PV was assessed according to procedure described by Arkadi-
´
usz, Roszko, Sosinska, Derewiaka, and Lewicki (2010). Briefly,
200 mg of oil was dissolved in 5 ml of hexane. Two hundred micro-
liters of the solution was mixed with 5 ml of methanol/chloroform/
developed in our laboratory (Aladedunye & Przybylski, in press).
Briefly, 12 g of CTAG’s fortified with 500 lg/g of the tested antiox-
HCl solution (1:1:0.012, v/v). Thereafter, 100
ll of FeCl2 (0.4%
idant, was weighed into a glass beaker (Pyrex, USA). An octagonal
stir bar (ThermoFischer Scientific, USA) was placed into the vessel,
forming the final surface to volume ratio of 0.42. Afterwards, vessel
with the content was heated at 185 5 °C for 10 min and 1.2 g of
formulated food containing a mixture of gelatinised potato starch,
glucose and silica gel (4:1:1w/w) was added. The heating was con-
tinued for another 20 min and the mixture stirred at 500 rpm.
Heating and stirring were maintained for another 90 min. About
water solution) and 100 l of NH4SCN (30% water solution) were
l
added. The reaction was run at room temperature for 5 min, and
the absorbance measured at 480 nm using all reagents for the
blank sample.
2.5.4. Frying test
The effectiveness of the developed antioxidants to protect
CTAG’s under frying conditions was assessed using a frying test

F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
949
Table 3
1H and 13C NMR of the novel antioxidants 3a–3L.
3a
3b
3c
3d⁄
3e⁄
3f
3g
3h
3i
3j
3k
3L
Hydrogen
CH3
0.85(t)
0.85(t)
0.84-
0.77(t)
0.81-
0.83-
0.84(t)
0.83-
0.84-
0.85(t)0.87(t)
0.80–
0.83–
J = 7.5
0.89(m)
0.92(m)
0.87(t)
J = 7.5
0.87(t)
J = 7.5
1.18–
0.92(m)
J = 7.5
0.88(m)
0.92(m)
0.85(t)
J = 7.2
0.93(m)
0.92(m)
CH2 (m)
CH2CH2N
CH2CO
1.13–
1.10–
1.11–
1.15–
1.16–
1.14–
1.32
1.39–
1.55(m)
2.61(t)
2.67(t)
J = 7.5
2.83(t)
2.89(t)
J = 7.5
1.13–
1.35
1.41–
1.57(m)
2.62(t)
2.68(t)
J = 7.5
2.85(t)
2.90(t)
J = 7.5
1.31
1.42–
1.35
1.40–
1.13
1.31–
1.48(m)
2.47–
2.73(m)
1.35
1.37–
1.32
1.40–
1.34
1.38–
1.55(sx)
J = 7.5
2.63(t)
2.71(t)
J = 7.5
1.33–
1.49(m)
2.48–
1.46–
1.45–1.60(m)
1.58(m)
2.65(t)
2.71(t)
J = 7.5
2.86(t)
2.93(t)
J = 7.5
1.56(m)
2.60(t)
2.69(t)
J = 7.5
2.84(t)
2.91(t)
J = 7.5
1.52(m)
2.57(t)
2.65(t)
J = 7.5
2.78(t)
2.86(t)
J = 7.5
1.63(m)
2.62(t)
2.67(t)
J = 7.8
2.82(t)
2.87(t)
J = 7.5
1.57(m)
2.64(t)
2.69(t)
J = 7.2
2.85(t)
2.90(t)
J = 7.2
1.56(m)
2.61(t)
2.66(t)
J = 7.5
2.85(t)
2.90(t)
J = 7.5
2.62(t)
2.67(t)
J = 7.5
2.83(t)
2.89(t)
J = 7.5
2.80(m)
CH2CH2CO 2.87(t)
2.93(t)
2.48–
2.80(m)
2.47–
2.73(m)
J = 7.5
CH2CH2N
(t)
OCH3 (s)
ArCH2 N
(s)
3.12, 3.32
J = 7.5
3.13, 3.34 3.11, 3.32 3.07, 3.16 3.10, 3.18 3.09, 3.30 3.11, 3.30 3.15, 3.33 3.12, 3.32 3.13, 3.31
3.14, 3.33 3.15, 3.34
J = 7.5 J = 7.8
3.80, 3.81 3.81, 3.82
4.35, 4.49 4.37, 4.50
J = 7.8
J = 7.8
J = 7.8
J = 7.8
J = 7.8
J = 7.8
3.79, 3.81 3.79, 3.83 3.80, 3.81 3.80, 3.81
4.35, 4.46 4.36, 4.50 4.38, 4.51 4.36, 4.49 4.36, 4.50
J = 7.8
J = 7.8
J = 7.5
4.45, 4.61
4.45, 4.60 4.43, 4.58 4.38
4.38
CHAr
6.52(d)
6.63(d)
J = 7.8
6.52(d)
6.62(d)
J = 8.1
6.74–
6.85
6.50(d)
6.60(d)
J = 8.1
6.72–
6.83
6.40(d)
6.48(d)
J = 8.1
6.54–
6.40(d)
6.47(d)
J = 8.0
6.54–
6.47(d)
6.55(d)
J = 7.8
6.67–
6.49-
6.64 m)
6.49-
6.65(m)
6.50-
6.64(m)
6.28(d)
6.41(d)
J = 8.1
6.27(d)
6.41(d)
J = 8.1
6.70–
6.29(d)
6.42(d)
J = 8.1
6.71–
CHAr (m)
CHAr
6.75–6.83
6.71–
6.79
6.82(d)
6.87(d)
J = 8.1
6.72–
6.82
6.84(d)
6.88(d)
J = 8.1
6.71–
6.79
6.82(d)
6.87(d)
J = 8.1
6.70–6.81
6.76
6.75
6.80
6.81
6.81
7.05–7.15
7.24–7.39
7.05–
7.16
7.03–
7.14
6.94(d)
6.98(d)
J = 8.4
6.93(d)
6.98(d)
J = 8.4
6.86(d)
6.90(d)
J = 8.4
6.49(d)
6.57(d)
J = 8.1
6.48(d)
6.56(d)
J = 8.1
6.51(d)
6.59(d)
J = 8.1
CHAr (m)
OH
7.21–
7.38
7.19–
7.37
8.61–
8.62(s)
8.74(m)
8.64(s)
OH (s)
OH (s)
8.69, 8.72
9.29, 9.36 9.29, 9.36
Carbon
CH2
CH2
CH2
CH2
CH2
CH2
CH2
27.4, 28.5 27.4, 28.5
31.1, 31.3 29.2, 29.3
31.4, 31.5 29.3, 29.4
27.4, 28.5 27.4, 28.5
30.9, 31.0 29.3
31.4, 31.5 29.4
29.5
27.4, 28.4 27.5, 28.5
31.2 29.3
31.4, 31.5 29.3, 29.4
27.5, 28.5 27.6, 28.5
31.0, 31.1 29.3
31.4, 31.5 29.3, 29.4
29.5
29.5
29.6
31.2, 31.3
31.9
29.5
29.6
31.1, 31.2
31.9
29.6
31.0, 31.2
31.9
29.6
31.0, 31.1
31.9
CH2CH2CO 34.6, 35.1
34.9, 35.3 35.0, 35.4 34.7, 35.1 34.7, 35.2 34.8, 35.4 35.1, 35.5 35.2, 35.5 35.1, 35.5 35.1, 35.3
46.9, 47.6 46.9, 47.6 47.3, 47.4 45.7, 46.8 46.9, 47.8 48.4, 48.7 46.8, 47.5 46.8, 47.5 48.5, 49.0
48.8, 51.3 48.8, 51.3 48.4, 50.3 47.4, 50.2 48.5, 51.0 49.1, 51.2 48.7, 51.2 48.7, 51.2 49.1, 51.4
35.1, 35.3 35.1, 35.3
46.9, 47.4 46.9, 47.5
49.0, 51.4 49.0, 51.4
CH2CH2N
ArCH2N
OCH3
CAr
47.8, 48.0
48.9, 50.8
55.9
55.9
55.9
56.4
56.4
56.3
115.8
116.3
119.3
126.8
127.5
127.8
128.8
132.6
138.4
138.8
143.7
144.4
172.1
115.0
115.7
119.9
126.2
127.3
128.6
129.0
132.8
136.4
137.0
142.9
144.1
173.4
115.0
115.6
119.9
126.2
127.3
128.6
132.8
132.8
136.4
137.0
142.9
144.1
173.5
115.5
115.8
116.2
117.1
121.8
128.1
128.9
130.0
132.7
143.4
144.7
156.7
171.7
115.5
115.7
116.2
119.3
121.2
128.9
129.3
131.6
132.6
143.7
145.4
156.7
171.7
115.5
115.7
115.9
119.1
120.9
128.3
129.1
129.9
132.6
142.7
143.9
155.4
173.8
108.9
114.4
115.4
115.7
119.0
120.1
128.1
132.9
142.8
144.2
145.0
147.2
173.7
108.9
114.4
115.4
115.7
119.1
120.1
128.1
132.8
142.8
144.3
145.1
147.0
173.6
108.8
114.3
115.3
115.7
119.1
120.1
128.1
132.8
142.8
144.2
145.1
146.9
173.6
102.7
102.8
115.1
115.6
120.0
127.6
133.0
134.1
142.7
143.9
143.9
147.1
173.3
102.8
102.8
115.1
115.6
120.0
127.6
132.9
134.1
142.8
144.0
144.0
147.1
173.3
102.8
102.8
115.1
115.6
119.9
127.6
132.9
134.1
142.8
144.1
144.1
147.1
173.3
CAr
CAr
CAr
CAr
CAr
CAr
CAr
CAr
CAr
CAr
CAr
CO
*
Solvent = DMSO-d6; s, singlet; d, doublet; t, triplet; sx, sextet; m, multiplet; J, coupling constant in Hertz. See the text for details.
0.5 g of sample fat was withdrawn at the 60th, 80th, 100th and
120th minutes of heating. Selected sampling points are equivalent
to 1, 3, 5 and 7 days of actual frying time using an institutional
fryer (General Electric Company, NY, USA). At the selected
intervals, oil accumulated similar amounts of polar components
compared to actual frying. Frying performance of oils was assessed
by measuring an amount of the total polar components (TPC) and
2.5.5. Total polar compound
TPC were determined by the gravimetric method following
AOAC Method 982.27 (AOAC, 1990) with Schulte modification
(2004). Briefly, oils were separated into non-polar and polar frac-
tions using a hydrated silica gel in a column chromatography.
2.5.6. Analyses of residual antioxidants
by assessing the amount of antioxidant retained in the oil. BHT, a-
Tocopherol was analysed according to AOCS Official Method Ce
8–89 (AOCS, 1999). Briefly, 50 mg of oil samples were weighed di-
rectly into the autosampler vials and dissolved in 1 ml hexane. The
mobilephase consisted of 7% methyl-tert-butyl-ether in hexane with
tocopherol and DCA were used as references. Samples from two
repetitions of frying test were analysed in duplicate for TPC and
residual antioxidant.

950
F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
160
120
80
26
3a
24
22
20
18
16
14
12
10
8
3b
40
3a
3c
3b
15
10
5
3d
3c
CTG
3d
CTG
BHT
a-T
α
-T
6
4
2
0
0
60
40
24
22
20
18
16
14
12
10
8
3e
3f
3e
3f
16
12
8
3g
3g
3h
3h
BHT
BHT
a-T
6
4
4
2
0
0
60
40
18
16
14
12
10
8
3i
3j
3i
16
12
8
3k
3j
3L
DCA
3k
3L
BHT
a-T
6
4
4
2
0
0
0
1
2
3
4
5
6
7
0
10
20
30
40
50
60
70
80
90 100 110 120
Storage Time (days)
Frying Time (min)
Fig. 4. Changes in peroxide value during accelerated storage of canola triacylgly-
cerols fortified with -tocopherol, BHT, and the novel antioxidant 3a–3L at 350 g/
g. CTAG – canola triacylglycerols; DCA – dihydro-caffeic acid; BHT – butylated
hydroxytoluene; T – -tocopherol. See the text for details.
Fig. 5. Formation of polar components during the frying test of canola triacylgly-
cerides with added -tocopherol, BHT, DCA and the novel antioxidants 3a–3L at
500 g/g. CTAG – canola triacylglycerols; T – -tocopherol. See the text for details.
a
l
a
l
a
a
a
a
prepared from DCA and the corresponding amines 2a–2L using
BPO as a coupling reagent (Rajan et al., 2001). After purification
by flash chromatography, compounds 3a–3L were obtained in mod-
erate to good yields, each as a mixture of the two rotamers (Table 1).
The structure of the novel antioxidants were confirmed by 1H
NMR, 13C NMR and HRMS (Tables 1 and 3). As an example, the
1H NMR spectrum of antioxidant 3a is reported in Fig. 2. The 1H
NMR spectrum is characterised by a methyl group at 0.85 and
0.87 ppm (2t, one for each rotamer), five methylene groups at
1.55 ppm (sx), 2.63 and 2.71 ppm (2t, one for each rotamer), 2.87
and 2.93 ppm (2t), 3.12 and 3.32 ppm (2t) and at 4.45 and
4.61 ppm (2t). The protons of the aromatic rings are identified by
two doublets at 6.52 and 6.53 ppm, as well as by three multiplets
at 6.75–6.83 ppm, 7.06–7.15 ppm and 7.24–7.39 ppm. The calcu-
lated molecular mass agreed very well with the mass established
by mass spectrometry (Table 1).
a flow rate of 0.6 ml/min and the fluorescence detector set for excita-
tion at 292 nm and emission at 325 nm were used. For BHT, DCA and
thenovelantioxidants the mobilephase was changed to 50% methyl-
tert-butyl-ether in hexane and a flow rate to 0.3 ml/min using PDA at
281 nm for quantification, and injecting 10 ll of each sample.
2.6. Statistical analysis
Data were analysed by single factor analysis of variance (ANO-
VA) and regression using Minitab 2000 statistical software (Mini-
tab Inc., PA, ver. 13.2). Statistically significant differences
between means were determined by Duncan’s multiple range tests
for p < 0.05.
3. Results and discussion
3.1. Synthesis
3.2. DPPH radical scavenging assay
New antioxidants, 3a–3L, were prepared in two steps synthesis
from the corresponding aldehydes 1a–1d (Fig. 1). Amines 2a–2L
were obtained by reductive amination. Reaction of aldehydes 1a–
1d with an excess of alkylamine in methanol, yielded the expected
imines. The imines were subsequently reduced with sodium boro-
hydride and the desired amines were isolated in moderate to good
yields (Table 1). The physical and spectroscopic characteristics of
the amines are presented in Tables 1 and 2, respectively. The spec-
tral results for compounds 2a, 2b and 2c were in agreement with
the data reported in literature (Guino, Brule, & de-Miguel, 2003;
Lai, Lee, & Liu, 2008; Sato et al., 2004). Antioxidants 3a–3L were
Results indicate that all the developed compounds displayed a
considerable concentration dependent radical scavenging activity
(Fig. 3; data for 1.11 mM concentration are presented). At the
tested concentrations, all the novel antioxidants exhibited signifi-
cantly better (p < 0.05) radical scavenging capacity than a-tocoph-
erol (Results not included). At the concentration of 1.11 mM, 3L,
the most active of the new antioxidants was four times more effec-
tive than BHT (Fig. 3). Interestingly, no significant differences were
observed in the radical scavenging capacity between DCA and the
following new antioxidants: 3a, 3b, 3c, 3d, 3e and 3f, indicating

F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
951
that the dihydro-caffeoyl group is mainly responsible for their rad-
ical scavenging activities. However, the presence of one or more
methoxy groups in the ortho position to the hydroxyl group
seemed to make the benzyl amine moiety more efficient in radicals
scavenging. This is consistent with the general knowledge that an
electron donating group in the ortho or para position increases the
radical scavenging activity of some compounds (Fagerlund et al.,
2009). Consequently, antioxidants 3g, 3h, 3i, 3j, 3k and 3L dis-
played radical scavenging activity significantly higher than DCA
(Fig. 3). The superior radical scavenging activity of the new antiox-
idants was also confirmed by assessing their IC50, demonstrating
(Fig. 4). BHT and the novel antioxidants: 3a, 3b, 3c, 3d, 3e and 3f
protected CTAG’s with the same efficiency during accelerated stor-
age (Fig. 4), however, antioxidants 3g, 3h, 3i, 3j, 3k, and 3L offered
much better protection than BHT. These results are consistent with
observations from the DPPH assay, suggesting that more hydroxyl
and methoxy groups activate the benzyl amine moiety and im-
prove antioxidant efficiency. Consistently, as was observed within
each group, the antioxidant activity decreases when the alkyl chain
length was shorter in the following order n = 9 < n = 5 < n = 2, how-
ever, this trend was statistically not significant at p < 0.05.
significantly lower values than
(2.55 mM) and DCA (1.31 mM) (Fig. 3).
a-tocopherol (1.75 mM), BHT
3.4. Protection under frying conditions
Furthermore, the results implied that the length of alkyl chain
did not have an effect on the radical scavenging activities and no
significant differences were observed for the following compounds
(chain length): 3a (n = 2), 3b (n = 5) and 3c (n = 9). Our results are
in agreement with data published by Silva et al. (2000) and Hsu,
Chen, Chang, and Chang (2009).
The effectiveness of the novel antioxidants to protect CTAG’s
from oxidative degradation under frying conditions was deter-
mined using a frying test developed in our laboratory (Catel, Alad-
edunye, & Przybylski, 2010). CTAG’s fortified with 500 lg/g of
antioxidant were tested under frying conditions and the perfor-
mance was assessed by the measurement of the amount of the to-
tal polar components (TPC) formed as a function of frying time. The
effectiveness of the new antioxidants was compared to BHT,
a-
3.3. Antioxidant activity under accelerated storage
tocopherol and DCA. The results showed that all novel antioxidants
(3a–3L) were more efficient in protecting CTAG’s during frying. At
the end of the frying time, the amounts of TPC formed in CTAGs
fortified with the novel antioxidants were significantly lower
(p < 0.05) compared to CTAG alone (Fig. 5). Furthermore, the novel
The results indicated that all new antioxidants outstandingly
protected CTAG’s from oxidative degradation and the formation
of rancidity (Fig. 4). At the end of the accelerated storage time
the amount of hydroperoxides formed in unprotected CTAG’s was
10 times higher than in samples fortified with the novel antioxi-
antioxidants were more efficient than
a-tocopherol and BHT
(Fig. 5). Amongst the synthesised antioxidants, compound 3c was
the most efficient and better protected oil than the parent DCA.
Interestingly, the group of antioxidants in which the benzyl
amine moiety was not hydroxylated, (compounds 3a, 3b, and 3c),
were the most effective in the protection of CTAG’s during frying.
This observation is contrary to the results from the radical scaveng-
ing and accelerated storage stability tests, suggesting that the nat-
ure of the reactions under frying conditions are different from
dants. Compared to a-tocopherol, a natural lipophilic antioxidant,
CTAG’s fortified with the new antioxidants were significantly more
stable (p < 0.05). The amounts of hydroperoxides formed in CTAG’s
containing 350 lg/g of compound 3L was 6.1 times lower com-
pared to CTAG’s containing the same amount of
a-tocopherol
100
90
80
100
3a
70
3a
3b
3c
3d
3e
3f
80
60
40
20
3b
60
3c
50
40
30
3d
3e
aT
3f
BHT
20
100
0
100
90
80
70
60
50
40
30
20
80
60
40
20
0
3g
3h
3i
3g
3h
3i
3j
3j
3k
3k
3L
3L
BHT
DCA
α
-T
0
20
40
60
80
100
120
0
1
2
3
4
5
6
7
Storage Time (days)
Frying Test Time (min)
Fig. 6. Changes of the antioxidants during accelerated storage of canola triacylgly-
cerols fortified with BHT, -tocopherol and the novel antioxidants 3a–3L added at
350 g/g. See the text for details.
Fig. 7. Changes of antioxidants during the frying tests of canola triacylglycerols
fortified with BHT, -tocopherol, DCA and the novel antioxidants 3a–3L added at
500 g/g. See the text for details.
a
a
l
l

952
F. Aladedunye et al. / Food Chemistry 130 (2012) 945–952
Arkadiusz, S. M., Roszko, E., Sosin´ ska, D., Derewiaka, P., & Lewicki, P. (2010).
Chemical composition and oxidative stability of selected plant oils. Journal of
the American Oil Chemists’ Society, 87, 637–645.
Ates, B., Abraham, L., & Ercal, N. (2008). Antioxidant and free radical scavenging
properties of N-acetylcysteine amide (NACA) and comparison with N-
acetylcysteine (NAC). Free Radical Research, 42, 372–377.
Catel, Y., Aladedunye, F., & Przybylski, R. (2010). Synthesis, radical scavenging
activity, protection during storage, and frying by novel antioxidants. Journal of
Agricultural and Food Chemistry, 58, 11081–11089.
Chen, C.-Y., Chang, F.-R., Yen, H.-F., & Wu, Y.-C. (1998). Amides from stems of Annola
cherimola. Phytochemistry, 49, 1443–1447.
those happening during low temperature applications (Frankel,
2007, Chap. 1). The increase in alkyl chain length presumably en-
hanced the lipophilicity of the antioxidants, however, this trend
was observed, but without statistical significance in their perfor-
mance (Fig. 5).
3.5. Residual antioxidants
In addition to being effective, an antioxidant should also be
thermally stable so that it can provide lasting protection to food
and biological systems at ambient and frying temperatures. This
is particularly important during frying where a temperature of
185 °C is generally utilised. In the present study, the stability of
the new antioxidants under both accelerated storage and frying
were investigated and the amounts of residual antioxidants
remaining at storage and frying in Figs. 6 and 7 are presented,
respectively. Evidently, the novel antioxidants were significantly
Dalby-Brown, L., Barsett, H., Landbo, A.-K. R., Meyer, A. S., & Mølgaard, P. (2005).
Synergistic antioxidative effects of alkamides, caffeic acid derivatives, and
polysaccharide fractions from Echinacea purpurea on in vitro oxidation of
human low-density lipoproteins. Journal of Agricultural and Food Chemistry, 53,
9413–9423.
Fagerlund, A., Sunnerheim, K., & Dimberg, L. H. (2009). Radical-scavenging and
antioxidant activity of avenanthramides. Food Chemistry, 113, 550–556.
Frankel, E. D. (2007). Antioxidants in food and biology. Facts and fiction. Bridgwater:
The Oily Press.
Fu, J., Cheng, K., Zhang, Z., Fang, R., & Zhu, H. (2010). Synthesis, structure and
structure-activity relationship analysis of caffeic acid amides as potential
antimicrobials. European Journal of Medicinal Chemistry, 45, 2638–2643.
Guino, M., Brule, E., & de-Miguel, Y. R. (2003). Recycling and reuse of a polymer-
supported scavenger for amine sequestration. Journal of Combinatorial
Chemistry, 5, 161–165.
Hsu, F.-L., Chen, P.-S., Chang, H.-T., & Chang, S.-T. (2009). Effects of alkyl chain length
of gallates on their antifugal property and potency as an environmentally
benign preservative against wood-decay. International Biodeterioration and
Biodegradation, 63, 543–547.
more stable than
CTAG’s during accelerated storage at 60 °C (Fig. 6). At the fifth
day of storage, the remaining amounts of -tocopherol, BHT and
a-tocopherol, BHT and DCA when tested with
a
DCA were at 39%, 65%, and 54%, respectively, compared to a mini-
mum of 75% for the novel antioxidants. The same superior stability
was observed during frying (Fig. 7). At all stages of frying signifi-
cantly higher amounts of the novel antioxidants were present in
Jiang, R.-W., Lau, K.-M., Hon, P.-M., Mak, T. C. W., Woo, K.-S., & Fung, K.-P. (2005).
Chemistry and biological activities of caffeic acid derivatives from Salvia
miltiorrhiza. Current Medicinal Chemistry, 12, 237–246.
the oil compared to a-tocopherol, BTH and DCA (Fig. 7).
Jung, Y.-S., Kang, T.-S., Yoon, J.-H., Joe, B.-Y., Lim, H.-J., Seong, C.-M., et al. (2002).
Synthesis and evaluation of 4-hydroxyphenylacetic acid amides and 4-
4. Conclusions
hydroxycinnamamides as antioxidants. Bioorganic
Letters, 12, 2599–2602.
& Medicinal Chemistry
Lai, R.-Y., Lee, C.-I., & Liu, S.-T. (2008). One-pot reductive amination of aldehydes
catalyzed by a hydrio-iridium (III) complex in aqueous medium. Tetrahedron,
64, 1213–1217.
The novel antioxidants 3a–3L have been conveniently prepared
in good yield from DCA. DPPH assays demonstrated their signifi-
cantly higher radical scavenging activities than
a-tocopherol,
Lampi, A. M., & Kamal-Eldin, A. (1998). Effect of
a- and c-tocopherols on thermal
polymerization of purified high-oleic sunflower triacylglycerols. Journal of
American Oil Chemists’ Society, 75, 1699–1703.
Nenadis, N., Boyle, S., Bakalbassis, E. G., & Tsimidou, M. (2003). An experimental
approach to structure–activity relationships of caffeic and dihydrocaffeic acids
and related monophenols. Journal of the American Oil Chemists’ Society, 80,
451–458.
Ou, S., & Kwok, K.-C. (2004). Ferulic acid: Pharmaceutical functions, preparation and
applications in foods. Journal of the Science of Food and Agriculture, 84,
1261–1269.
Priscilla, D. H., & Prince, P. S. M. (2009). Cardioprotective effect of gallic acid on
cardiac troponin-T, cardiac marker enzymes, lipid peroxidation products and
antioxidants in experimentally induced myocardial infarction in Wistar rats.
Chemico-Biological Interaction, 179, 118–124.
Rajan, P., Vedernikova, I., Cos, P., Berghe, V. D., Augustyns, K., & Haemers, A. (2001).
Synthesis and evaluation of caffeic acid amides as antioxidants. Bioorganic &
Medicinal Chemistry Letters, 11, 215–217.
Reuter, S., Gupta, S. C., Chaturvedi, M. M., & Aggarwal, B. B. (2010). Oxidative stress,
inflammation, and cancer: How are they related? Free Radical Biology and
Medicine, 49, 1603–1616.
BHT and DCA. Furthermore, when compared to -tocopherol and
a
BHT, the new compounds offered better protection to polyunsatu-
rated oil both under storage and frying conditions. Beside their
superior antioxidant activities, the higher lipophilicity and thermal
stability make them more desirable than the precursor phenolic
acid.
Further investigations are underway to develop a better under-
standing of the relationship between structure and antioxidant
activity, the nature of the novel antioxidants degradation and
products formed. This study was designed to prepare novel antiox-
idants and assess their activity in an oil system; however their po-
tential application in various foods and regulatory acceptance will
be a matter of further development.
References
Sato, S., Sakamoto, T., Miyazawa, E., & Kikugawa, Y. (2004). One-pot reductive
amination of aldehydes and ketones with
water, and in neat conditions. Tetrahedron, 60, 7899–7906.
a-picoline-borane in methanol, in
Aladedunye, F. A., & Przybylski, R. Rapid assessment of frying performance using
small size samples of oils/fats. Journal of the American Oil Chemists’ Society, in
press.
AOAC. (1990). Official methods of analysis of the AOAC international (15th ed.). VA,
USA: Arlington.
AOCS. (1999). Official methods and recommended practices of the American Oil
Chemists’ Society (5th ed.). Champaign, IL, USA: American Oil Chemists’ Society.
Alamed, J., Chaiyasit, W., Mc Clements, D. J., & Decker, E. A. (2009). Relationships
between free radical scavenging and antioxidant activity in foods. Journal of
Agricultural and Food Chemistry, 57, 2969–2976.
Schulte, E. (2004). Economical micromethod for determination of polar components
in frying fats. European Journal of Lipid Science and Technology, 106, 772–776.
Silva, F. M., Borges, F., Guimaraes, J. J., Lima, F. C., Matos, C., & Reis, C. (2000).
Phenolic acids and derivatives: Studies on the relationship among structure,
radical scavenging activity, and physicochemical parameters. Journal of
Agricultural and Food Chemistry, 48, 2122–2126.