Radical scavenging activity and performance of novel phenolic antioxidants in oils during storage and frying

Article abstract of DOI:10.1007/s11746-011-1889-6

Novel phenolic antioxidants: 2a (6′-hydroxy-2′,5′, 7′,8′-tetramethylchroman-2′-yl)methyl 3-methoxy-4- hydroxycinnamate, 2b (6′-hydroxy-2′,5′,7′,8′- tetramethylchroman-2′-yl)methyl 3,5-dimethoxy-4-hydroxycinnamate, 2c (6′-hydroxy-2′,5′,7′,8′-tetramethylchr

Full text of DOI:10.1007/s11746-011-1889-6

J Am Oil Chem Soc (2012) 89:55–66
DOI 10.1007/s11746-011-1889-6
ORIGINAL ARTICLE
Radical Scavenging Activity and Performance of Novel Phenolic
Antioxidants in Oils During Storage and Frying
•
Yohann Catel Felix Aladedunye Roman Przybylski
•
Received: 25 November 2010 / Revised: 25 May 2011 / Accepted: 15 June 2011 / Published online: 30 June 2011
Ó AOCS 2011
Abstract Novel phenolic antioxidants: 2a (60-hydroxy-20,
50,70,80-tetramethylchroman-20-yl)methyl
3-methoxy-4-
hydroxycinnamate, 2b (60-hydroxy-20,50,70,80-tetramethylch-
roman-20-yl)methyl
3,5-dimethoxy-4-hydroxycinnamate,
its flavor and forms free radicals which may stimulate and/
or initiate development of some health problems [1]. Free
radicals are also involved in inflammatory and cardiovas-
cular diseases, cancer and aging [2–5]. To prevent devel-
opment of rancidity in foods, antioxidants have been
utilized for years. Antioxidants are efficient scavengers of
radicals, the later are formed during the initiation or the
propagation stages of oxidative degradation [3].
2c (60-hydroxy-20,50,70,80-tetramethylchroman-20-yl)methyl
3,4-dihydroxycinnamate, and 3 (6-hydroxy-2,5,7,8-tetra-
methylchroman-2-yl)methyl (60-hydroxy-20,50,70,80-tetrame-
thylchroman-20-carboxylate) have been prepared in good
1
yields and fully characterized by H and 13C NMR, and
Phenolic compounds including phenolic acids, tocoph-
erol and flavonoids are widely distributed in the plants and
are the efficient cell antioxidants [6]. Besides their high
antioxidant activity, some phenolic derivatives present
interesting biological properties. Caffeic acid and its ana-
logues provide antiviral, anti-thrombosis, anti-hyperten-
sion, anti-fibrosis and antitumor properties [7]. Ferulic acid
and its derivatives, the most abundant phenolic acid in
plants, has been shown to possess antioxidant, antimicro-
bial, anti-inflammatory, anti-thrombosis and anti-cancer
activities [8].
HRMS. Their radical scavenging activities have been eval-
uated by DPPH and ORAC assays. Each of the synthesized
antioxidants exhibited significantly higher radical scaveng-
ing activities than trolox and a-tocopherol. These novel
antioxidants efficiently protected canola oil triacylglycerides
(CTG) during accelerated storage and frying. Compounds 2c
and 3 were significantly more efficient than a-tocopherol
protecting CTG under accelerated storage. All new antioxi-
dants were more efficient than a-tocopherol under frying
conditions and present significantly higher thermal stability.
Synthetic antioxidants such as BHT, BHA and TBHQ
have been used in some food applications; however, their
safety has been challenged due to possible negative effect
on human health [9, 10]. Consequently, the synthesis of
new antioxidants derived from natural ingredients, offering
low toxicity, interesting biological properties as well as
high antioxidant effectiveness are of current interest. In this
context, our group recently took an interest in the prepa-
ration of new phenolic compounds presenting higher anti-
oxidant efficiency than those of the common natural
antioxidants [11]. We focused our work on the preparation
of esters of common phenolic acids and components
bearing a chromanol ring, the later is responsible for the
antioxidant activity.
Keywords Phenolic antioxidants ꢀ Synthesis ꢀ
Canola oil ꢀ Frying ꢀ Storage stability ꢀ Trolox ꢀ
Cinnamic acid ꢀ Caffeic acid
Introduction
Oxidation of polyunsaturated lipids is one of the main
causes of food degradation [1]. Degraded lipids deteriorate
Y. Catel ꢀ F. Aladedunye ꢀ R. Przybylski (&)
Department of Chemistry and Biochemistry,
University of Lethbridge, Lethbridge,
AB T1K 3M4, Canada
Phenolic antioxidants break the free radical chain and a
stable phenoxyl radical is formed, stability of it is
e-mail: roman.przybylski@uleth.ca
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J Am Oil Chem Soc (2012) 89:55–66
achieved by the delocalization of unpaired electrons on
the aromatic ring [12]. It is well known that an electron
donating groups such as hydroxyl and methoxy present in
the ortho or para position to hydroxyl substituent on the
aromatic ring increases the antioxidant effectiveness.
Placing an electron acceptor substituent in the same
position, such as an ester group, is lowering antioxidant
potency of a compound [12]. Therefore, in order to
improve the antioxidant capacities of synthesized phe-
nolic compounds, we focused on the preparation of the
new derivatives fulfilling discussed structural functions
(Fig. 1).
N,N0-dicyclohexylcarbodiimide (DCC), dimethylamino-
pyridine (DMAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
fluorescein disodium salt, trolox and phenolic acids were
purchased from Sigma–Aldrich (St. Louis, MO). THF and
dichloromethane (DCM) were purified using MBraun
Solvent Purification System (M. Braun Incorporated,
Stratham, NH). Dimethylformamide (DMF) and other
solvents used in this work were of HPLC grade and were
obtained from VWR (Edmonton, Canada).
Methods
According to structure activity principles, the presence
of an alkyl chain between the ester function and the aro-
matic ring should increase the radical scavenging activity
of the compounds [7, 8]. The high activity of a-tocopherol
is defined by the substituents present on the chromanol
ring, we applied this consideration when preparing com-
pound 3, where two chromanol rings were joined by alkyl
chain (Fig. 1).
Synthesis of Benzyl 3-Methoxy-4-benzyloxycinnamate (5a)
and Benzyl 3,5-Dimethoxy-4-benzyloxycinnamate (5b)
(Scheme 1)
Potassium carbonate (10.7 g, 77.2 mmol, 3.0 equiv) and
benzyl bromide (9.2 mL, 77.2 mmol, 3.0 equiv) were
added to a solution of the desired cinnamic acid derivative
(25.7 mmol), the later dissolved in 100 mL of dry DMF,
and kept under an argon blanket. The mixture was stirred
for 15 h and then transferred into distilled water (150 mL).
The compound of interest was extracted thrice with diethyl
ether (100 mL). The combined extracts were washed with
distilled water (100 mL), dried on magnesium sulfate, and
concentrated under vacuum using a rotary evaporator.
The synthesis and radical scavenging activities of
the novel antioxidants: 2a, 2b, 2c, and 3 is reported.
Furthermore, the ability of these compounds to protect
CTG under accelerated storage and frying has been
investigated.
Materials and Methods
Data of Compounds 5a and 5b
Materials
Benzyl 3-methoxy-4-benzyloxycinnamate (5a): The crude
ester was purified by crystallization in hexane. Aspect:
White solids. Mp = 89–90 °C. Yield = 86%. The spectral
results were in agreement with data published by Leschot
et al. [13].
Refined, bleached and deoderized regular canola oil was
obtained from Richardson Oilseeds (Lethbridge, Canada).
2,20-azobis (2-amidinopropane) hydrochloride (AAPH),
Fig. 1 Chemical structure of
the novel antioxidants 2a, 2b, 2c
and 3
R1
HO
OH
HO
R2
R3
O
O
O
O
O
O
O
3
2a: R1 = OCH3; R2 = OH; R3 = H
2b: R1 = R3 = OCH3; R2 = OH
2c: R1 = R2 = OH; R3 = H
Scheme 1 Reagents and
conditions used for the synthesis
of cinnamic acid derivatives.
(i) BnBr, K2CO3, DMF;
(ii) KOH, H2O/EtOH, reflux.
For details see the text
O
O
O
R1
R2
(i)
R1
R2
R1
R2
(ii)
OH
OBn
OH
R3
R3
R3
4a: R1 = OMe; R2 = OH; R3 = H
4b: R1 = R3 = OMe; R2 = OH
4c: R1 = R2 = OH; R3 = H
5a: R1 = OMe; R2 = OBn; R3 = H
5b: R1 = R3 = OMe; R2 = OBn
5c: R1 = R2 = OBn; R3 = H
6a: R1 = OMe; R2 = OBn; R3 = H
6b: R1 = R3 = OMe; R2 = OBn
6c: R1 = R2 = OBn; R3 = H
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J Am Oil Chem Soc (2012) 89:55–66
57
Benzyl 3,5-dimethoxy-4-benzyloxycinnamate (5b): The
crude oily product was purified by washing three times with
hot hexane (100 mL). Aspect: Yellow oil. Yield = 92%.
1H NMR (300.0 MHz, CDCl3): d: 3.84 (s, 6H, OCH3);
5.05 (s, 2H, OCH2Ph); 5.25 (s, 2H, OCH2Ph); 6.40
sulfuric acid until white solids were formed. The suspen-
sion was then extracted thrice with ethyl acetate (200 mL).
The combined extracts were washed with distilled water,
dried on magnesium sulfate, and finally concentrated under
vacuum using a rotary evaporator.
3
(d, JHH = 15.9 Hz, 1H, CH=CH–CO); 6.74 (s, 2H, CHAr);
3
7.26–7.49 (m, 10H, CHAr); 7.63 (d, JHH = 15.9 Hz, 1H,
Data for Compound 6a–6c
CH=CH–CO). 13C NMR (300.0 MHz, CDCl3): d: 56.1
(OCH3); 66.4 (OCH2Ph); 75.1 (OCH2Ph); 105.3 (CAr); 117.1
(CH=CH–CO); 128.0 (CAr); 128.2 (CAr); 128.4 (CAr); 128.5
(CAr); 128.7 (CAr); 130.0 (CAr); 136.1 (CAr); 137.6 (CAr);
139.0 (CAr); 145.3 (CH=CH–CO); 153.8 (CAr); 166.8 (C=O).
HRMS (m/z): calculated for C25H24O5 = 405.1697; found
405.1692 [M ? H]?.
3-Methoxy-4-benzyloxycinnamic acid (6a): Aspect: White
solids. Mp = 193–194 °C. Yield = 86%. The spectral
results matched the published results by Muller et al. [14].
3,5-Dimethoxy-4-benzyloxycinnamic acid (6b): Aspect:
1
White solids. Mp = 114–115 °C. Yield = 98%. H NMR
(300.0 MHz, DMSO-d6): d: 3.83 (s, 6H, OCH3); 4.95
(s, 2H, OCH2Ph); 6.66 (d, JHH = 15.9 Hz, 1H, CH=CH–
3
Synthesis of Benzyl 3,4-Dibenzyloxycinnamate 5c
(Scheme 1)
CO); 7.06 (s, 2H, CHAr); 7.31–7.48 (m, 5H, CHAr); 7.55 (d,
3JHH = 15.9 Hz, 1H, CH=CH–CO); 12.32 (s, 1H, COOH).
13C NMR (300.0 MHz, DMSO-d6): d: 56.4 (OCH3); 74.5
(OCH2Ph); 106.1 (CAr); 119.0 (CH=CH–CO); 128.2 (CAr);
128.4 (CAr); 128.5 (CAr); 130.5 (CAr); 138.2 (CAr); 138.6
(CAr); 144.7 (CH=CH–CO); 153.7 (CAr); 168.3 (C=O).
HRMS (m/z): calculated for C18H18O5 = 315.1232; found
315.1256 [M ? H]?.
Potassium carbonate (15.3 g, 111.0 mmol, 4.0 equiv) and
benzyl bromide (13.2 mL, 111.0 mmol, 4.0 equiv) were
added to a solution of caffeic acid (5.0 g, 27.7 mmol)
dissolved in 100 mL of dry DMF. The mixture was stirred
for 15 h under an argon blanket and then transferred into
distilled water (150 mL). The compound of interest was
extracted thrice with diethyl ether (100 mL). The com-
bined extracts were washed with distilled water (100 mL),
dried on magnesium sulfate, and concentrated under vac-
uum using a rotary evaporator. The crude product was
purified by crystallization in hexanes. The desired benzoic
ester was isolated as white solids with 91% yield.
3,4-Dibenzyloxycinnamic acid (6c): Aspect: White sol-
ids. Mp = 202–203 °C. Yield = 90%. The spectral results
matched those reported by Percec et al. [15].
Compounds 7 and 8 were synthesized according to a
procedure described by Muller et al. [14].
Synthesis of (60-Benzyloxy-20,50,70,80-tetramethylchroman-
20-yl) Methyl 3-Methoxy-4-benzyloxycinnamate (9a),
(60-Benzyloxy-20,50,70,80-tetramethylchroman-20-yl)
Methyl 3,5-Dimethoxy-4-benzyloxycinnamate (9b),
and (60-Benzyloxy-20,50,70,80-tetramethylchroman-20-yl)
Methyl 3,4-Dibenzyloxycinnamate (9c) (Scheme 2)
Data for Benzyl 3,4-dibenzyloxycinnamate (5c)
1
Mp = 80–81 °C. H NMR (300.0 MHz, CDCl3): d: 5.19
(s, 2H, OCH2Ph); 5.22 (s, 2H, OCH2Ph); 5.26 (s, 2H,
OCH2Ph); 6.31 (d, 3JHH = 15.9 Hz, 1H, CH=CH–CO); 6.93
(d, 3JHH = 8.4 Hz, 1H, CHAr); 7.09 (d, 3JHH = 8.4 Hz, 1H,
CHAr);7.14(s, 1H, CHAr);7.29–7.50 (m, 15H, CHAr);7.63(d,
3JHH = 15.9 Hz, 1H, CH=CH–CO). HRMS (m/z): calculated
for C30H26O4 = 451.1909; found 451.22 [M ? H]?.
DCC (632 mg, 3.06 mmol, 2.0 equiv) and DMAP (19.5 mg,
0.23 mmol, 0.15 equiv) were added under an argon blanket to
an alcoholic solution of component 8 (500 mg, 1.53 mmol)
and the desired cinnamic acid derivative (3.06 mmol,
2.0 equiv) in dry DCM (40 mL). The mixture was stirred for
15 h at room temperature, and then distilled water (50 mL)
was added. The organic layer was removed and washed once
with distilled water (20 mL), then dried over anhydrous
magnesium sulfate, and concentrated. Finally, the crude
product was purified by a flash chromatography using silica
gel and DCM as eluant.
Synthesis of 3-Methoxy-4-benzyloxycinnamic acid (6a),
3,5-Dimethoxy-4-benzyloxycinnamic acid (6b), and
3,4-Dibenzyloxycinnamic acid (6c) (Scheme 1)
Potassium hydroxide (4.0 g, 72.0 mmol, 5.0 equiv) was
added to a solution of the corresponding benzoic ester
(14.4 mmol) in a mixture of distilled water (53 mL) and
ethanol (210 mL). The mixture was refluxed for 2 h and
the solvent evaporated under a vacuum using a rotary
evaporator. The residue was dissolved in distilled water
(200 mL) and the aqueous solution was washed twice with
diethyl ether (50 mL) and acidified with concentrated
Data for Compound 9a–9c
(60-Benzyloxy-20,50,70,80-tetramethylchroman-20-yl) methyl
3-methoxy-4-benzyloxycinnamate (9a): Aspect: White sol-
1
ids. Yield = 71%. H NMR (300.0 MHz, CDCl3): d: 1.36
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J Am Oil Chem Soc (2012) 89:55–66
Scheme 2 Reagents and
conditions used for the synthesis
of antioxidants 2a–2c. (i) BnBr,
K2CO3, DMF; (ii) LiAlH4,
THF, 0 °C; (iii) 6a-6c, DCC,
DMAP, DCM; (iv) H2, Pd/
Charcoal 10%, THF. For details
see the text
BnO
HO
BnO
(i)
(ii)
OH
OH
OBn
O
O
O
8
7
O
O
(iii)
R1
R1
HO
R2
R3
HO
R2
R3
(iv)
O
O
O
O
O
O
2a: R1 = OMe; R2 = OH; R3 = H
2b: R1 = R3 = OMe; R2 = OH
2c: R1 = R2 = OH; R3 = H
9a: R1 = OMe; R2 = OBn; R3 = H
9b: R1 = R3 = OMe; R2 = OBn
9c: R1 = R2 = OBn; R3 = H
(s, 3H, CH3);1.78–2.11 (m, 2H, C=C–CH2–CH2);2.11(s, 3H,
CH3); 2.18 (s, 3H, CH3); 2.22 (s, 3H, CH3); 2.66 (t,
3JHH = 6.9 Hz, 2H, C=C–CH2); 3.93 (s, 3H, OCH3); 4.22 (d,
(CAr); 166.8 (C=O). HRMS (m/z): calculated for
C39H42O7 = 623.3003; found 623.2977 [M ? H]?.
(60-Benzyloxy-20,50,70,80-tetramethylchroman-20-yl) methyl
3,4-dibenzyloxycinnamate (9c): Aspect: Highly viscous oil.
Yield = 88%. 1H NMR (300.0 MHz, CDCl3): d: 1.35 (s, 3H,
CH3); 1.79–2.10 (m, 2H, C=C–CH2–CH2); 2.11 (s, 3H, CH3);
2.17 (s, 3H, CH3); 2.22 (s, 3H, CH3); 2.65 (t, 3JHH = 6.9 Hz,
2H, C=C–CH2); 3.88 (s, 6H, OCH3);4.20(d, 2JHH = 11.1 Hz,
2
2JHH = 11.4 Hz, 1H, CH2OCO); 4.29 (d, JHH = 11.4 Hz,
1H, CH2OCO); 4.69 (s, 2H, OCH2Ph); 5.19 (s, 2H, OCH2Ph);
3
6.33 (d, JHH = 15.9 Hz, 1H, CH=CH–CO); 6.89 (d,
3JHH = 8.4 Hz, 1H, CHAr); 7.01–7.09 (m, 2H, CHAr);
3
7.26–7.52 (m, 10H, CHAr); 7.62 (d, JHH = 15.9 Hz, 1H,
CH=CH–CO). 13C NMR (75.0 MHz, CDCl3): d: 11.9 (CH3);
12.0 (CH3); 12.9 (CH3); 20.2 (C=C–CH2); 22.2 (CH3); 28.6
(C=C–CH2–CH2); 56.0 (OCH3); 68.7 (CH2OCO); 70.9
(OCH2Ph); 73.8 (OC–CH2OCO); 74.8 (OCH2Ph); 110.2
(CAr); 113.4 (CAr); 115.7 (CH=CH–CO); 117.3 (CAr); 122.5
(CAr); 123.2 (CAr); 126.1 (CAr); 127.2 (CAr); 127.7 (CAr);
127.8 (CAr); 127.9 (CAr); 128.0 (CAr); 128.3 (CAr); 128.5
(CAr); 128.7 (CAr); 136.6 (CAr); 137.9 (CAr); 145.0 (CH=CH–
CO); 147.4 (CAr); 148.6 (CAr); 149.8 (CAr);150.3 (CAr);167.1
(C=O). HRMS (m/z): calculated for C38H40O6 = 593.2898;
found 593.2886 [M ? H]?.
1H, CH2OCO); 4.28 (d, JHH = 11.1 Hz, 1H, CH2OCO);
2
4.68 (s, 2H, OCH2Ph); 5.18 (s, 2H, OCH2Ph); 5.19 (s, 2H,
OCH2Ph); 6.27 (d, 3JHH = 15.9 Hz, 1H, CH=CH–CO); 6.91
(d, 3JHH = 8.4 Hz, 1H, CHAr); 7.07 (d, 3JHH = 8.4 Hz, 1H,
CHAr);7.12(s, 1H, CHAr);7.28–7.51 (m, 10H, CHAr);7.57(d,
3JHH = 15.9 Hz, 1H, CH=CH–CO). 13C NMR (75.0 MHz,
CDCl3): d: 11.9 (CH3); 12.0 (CH3); 12.9 (CH3); 20.2 (C=C–
CH2); 22.2 (CH3); 28.6 (C=C–CH2–CH2); 68.7 (CH2OCO);
71.0 (OCH2Ph); 71.4 (OCH2Ph); 73.8 (OC–CH2OCO); 74.8
(OCH2Ph); 113.8 (CAr); 114.3 (CAr); 115.8 (CH=CH–CO);
117.3 (CAr); 123.0 (CAr); 123.2 (CAr); 127.2 (CAr); 127.3
(CAr); 127.7 (CAr); 127.8 (CAr); 127.9 (CAr); 128.0 (CAr);
128.3 (CAr); 128.5 (CAr); 128.6 (CAr); 136.2 (CAr); 136.3
(CAr); 136.7 (CAr); 136.9 (CAr); 137.9 (CAr); 144.9 (CH=CH–
CO); 147.4 (CAr); 148.6 (CAr); 149.0 (CAr);151.1 (CAr);153.2
(CAr); 167.0 (C=O). HRMS (m/z): calculated for C44H44O6,
669.3211 = found 669.3204 [M ? H]?.
(60-Benzyloxy-20,50,70,80-tetramethylchroman-20-yl) methyl
3,5-dimethoxy-4-benzyloxycinnamate (9b): Aspect: Highly
viscous oil. Yield = 80%. 1H NMR (300.0 MHz, CDCl3): d:
1.39 (s, 3H, CH3); 1.81–2.14 (m, 2H, C=C–CH2–CH2); 2.14
(s, 3H, CH3); 2.21 (s, 3H, CH3); 2.25 (s, 3H, CH3); 2.70
(t, 3JHH = 6.9 Hz, 2H, C=C–CH2); 3.88 (s, 6H, OCH3); 4.27
(d, 2JHH = 11.4 Hz, 1H, CH2OCO); 4.33 (d, 2JHH = 11.4 Hz,
1H, CH2OCO); 4.72 (s, 2H, OCH2Ph); 5.08 (s, 2H, OCH2Ph);
Synthesis of Antioxidants (60-Hydroxy-20,50,70,
80-tetramethylchroman-20-yl) Methyl 3-Methoxy-4-
hydroxycinnamate (2a), (60-Hydroxy-20,50,70,
80-tetramethylchroman-20-yl) Methyl 3,5-Dimethoxy-4-
hydroxycinnamate (2b), and (60-Hydroxy-20,50,70,
80-tetramethylchroman-20-yl) Methyl 3,
3
6.40 (d, JHH = 15.9 Hz, 1H, CH=CH–CO); 6.77 (s, 1H,
CHAr); 7.28–7.55 (m, 10H, CHAr); 7.65 (d, 3JHH = 15.9 Hz,
1H, CH=CH–CO). 13C NMR (75.0 MHz, CDCl3): d: 11.9
(CH3); 12.0 (CH3); 12.9 (CH3); 20.2 (C=C–CH2); 22.2
(CH3); 28.6 (C=C–CH2–CH2); 56.2 (OCH3); 68.9
(CH2OCO); 73.8 (OC–CH2OCO); 74.8 (OCH2Ph); 75.1
(OCH2Ph); 105.3 (CAr); 117.0 (CH=CH–CO); 117.3
(CAr); 123.2 (CAr); 126.1 (CAr); 127.7 (CAr); 127.9 (CAr);
128.0 (CAr); 128.2 (CAr); 128.3 (CAr); 128.4 (CAr); 128.5
(CAr); 128.6 (CAr); 130.0 (CAr); 137.5 (CAr); 137.9 (CAr);
139.1 (CH=CH–CO); 147.4 (CAr); 148.6 (CAr); 153.8
4-Dihydroxycinnamate (2c) (Scheme 2)
Palladium on charcoal (10% wt) was added to a solution of
the desired benzylated compound (1.0 mmol in 10 mL of
dry THF) then the mixture was stirred at room temperature
when purged with hydrogen for 24 h. Reactants were
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J Am Oil Chem Soc (2012) 89:55–66
59
filtered on Celite, and concentrated on a rotary evaporator
under reduced pressure. The residue was purified by flash
chromatography with silica gel, and the solvents used for
elution are described for each individual component below.
(s, 1H, CHAr); 6.73 (d, 3JHH = 8.1 Hz, 1H, CHAr). 13C NMR
(75.0 MHz, CDCl3): d: 11.3 (CH3); 11.8 (CH3); 12.2 (CH3);
20.2 (CH2CH2CCH3); 21.9 (CH3); 28.6 (CH2CH2CCH3);
30.3 (s, CH2); 36.1 (s, CH2); 68.8 (CH2OCO); 73.3
(OCCH2OCO); 115.3 (CAr); 115.4 (CAr); 117.1 (CAr); 118.7
(CAr); 120.6 (CAr); 121.4 (CAr); 122.8 (CAr); 133.2 (CAr);
142.0 (CAr); 143.4 (CAr); 144.9 (CAr); 145.0 (CAr); 173.1
(C=O). HRMS (m/z): calculated for C23H28O6 = 401.1959;
found 401.1939 [M ? H]?.
Data for Antioxidants 2a–2c
(60-Hydroxy-20,50,70,80-tetramethylchroman-20-yl) methyl
3-methoxy-4-hydroxycinnamate (2a): Eluant: ethyl acetate/
hexanes (3.5:6.5 v/v). Aspect: Highly viscous oil.
Yield = 78%. 1H NMR (300.0 MHz, CDCl3): d: 1.24 (s, 3H,
CH3); 1.65–1.95 (m, 2H, CH2CH2CCH3); 2.08 (s, 3H, CH3);
2.10 (s, 3H, CH3); 2.15 (s, 3H, CH3); 2.53–2.67 (m, 4H,
Synthesis of 6-Benzyloxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (10) (Scheme 3)
3
CH2CH2COO and CH2CH2COO); 2.89 (t, JHH = 7.5 Hz,
2H, CH2CH2CCH3); 3.86 (s, 3H, OCH3); 4.07 (d,
Carboxylic acid 10 was prepared similarly to compounds
6a, 6b, and 6c. Benzoic ester 7 (3.0 g, 6.97 mmol) and
potassium hydroxide (1.96 g, 34.85 mmol, 5.0 equiv)
produced carboxylic acid 10 (2.13 g, 6.26 mmol).
2
2JHH = 11.1 Hz, 1H, CH2OCO); 4.14 (d, JHH = 11.1 Hz,
1H, CH2OCO); 4.25 (s, 1H, OH); 5.49 (s, 1H, OH);
3
6.62–6.71 (m, 2H, CHAr); 6.82 (d, JHH = 7.8 Hz, 2H,
CHAr). 13C NMR (75.0 MHz, CDCl3): d: 11.3 (CH3); 11.8
(CH3); 12.2 (CH3); 20.2 (CH2CH2CCH3); 21.8 (CH3); 28.6
(CH2CH2CCH3); 30.7 (s, CH2); 36.2 (s, CH2); 55.9 (OCH3);
68.7 (CH2OCO); 73.3 (OCCH2OCO); 110.9 (CAr); 114.3
(CAr); 117.0 (CAr); 118.5 (CAr); 120.8 (CAr); 121.3 (CAr);
122.7 (CAr); 132.3 (CAr); 144.1 (CAr); 144.9 (CAr); 145.0
(CAr); 146.4 (CAr); 172.8 (C=O). HRMS (m/z): calculated for
C24H30O6 = 415.2115; found 415.2117 [M ? H]?.
(60-Hydroxy-20,50,70,80-tetramethylchroman-20-yl) methyl
3, 5-dimethoxy-4-hydroxycinnamate (2b): Eluant: ethyl
acetate/hexanes (2:3 v/v). Aspect: Highly viscous oil.
Yield = 83%. 1H NMR (300.0 MHz, CDCl3): d: 1.29 (s, 3H,
CH3); 1.69–1.92 (m, 2H, CH2CH2CCH3); 2.08 (s, 3H, CH3);
2.11 (s, 3H, CH3); 2.15 (s, 3H, CH3); 2.58–2.68 (m, 4H,
CH2CH2COO and CH2CH2COO); 2.89 (t, 3JHH = 7.5 Hz, 2H,
CH2CH2CCH3); 3.86 (s, 6H, OCH3); 4.07 (d, 2JHH = 11.4 Hz,
1H, CH2OCO); 4.15 (d, 2JHH = 11.4 Hz, 1H, CH2OCO); 4.24
(s, 1H, OH); 5.39 (s, 1H, OH); 6.42 (s, 2H, CHAr). 13C NMR
(75.0 MHz, CDCl3): d: 11.3 (CH3); 11.8 (CH3); 12.2 (CH3);
20.2 (CH2CH2CCH3); 21.8 (CH3); 28.6 (CH2CH2CCH3); 31.2
(s, CH2); 36.3 (s, CH2); 56.2 (OCH3); 68.7 (CH2OCO); 73.3
(OCCH2OCO); 104.8 (CAr); 117.0 (CAr); 118.5 (CAr); 121.3
(CAr); 122.7 (CAr); 131.5 (CAr); 133.1 (CAr); 144.9 (CAr); 145.0
(CAr); 147.0 (CAr); 172.8 (C=O). HRMS (m/z): calculated for
C25H32O7 = 445.2221; found 445.2208 [M ? H]?.
Data for 6-Benzyloxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (10)
Aspect: White solids. Mp = 151–153 °C. Yield = 90%. 1H
NMR (300.0 MHz, CDCl3): d: 1.64 (s, 3H, CH3); 1.88–2.02
(m, 1H, C=C–CH2–CH2); 2.15 (s, 3H, CH3); 2.17 (s, 3H,
CH3); 2.23 (s, 3H, CH3); 2.35–2.44 (m, 1H, C=C–CH2–CH2);
2.52–2.74 (m, 1H, C=C–CH2–CH2); 4.69 (s, 2H, CH2OPh);
7.28–7.53 (m, 5H, CHAr). 13C NMR (75.0 MHz, CDCl3): d:
11.9 (CH3); 12.1 (CH3); 12.9 (CH3); 20.7 (C=C–CH2); 25.2
(CH3); 30.1 (C=C–CH2–CH2); 74.7 (OCH2Ph); 117.2 (CAr);
123.0 (CAr); 126.2 (CAr); 127.8 (CAr); 127.9 (CAr); 128.5 (2*
CAr); 137.9 (CAr); 147.4 (CAr); 149.1 (CAr); 179.5 (C=O).
HRMS (m/z): calculated for C21H24O4 = 341.1747; found
341.1737 [M ? H]?.
Synthesis of (6-Benzyloxy-2,5,7,8-
tetramethylchroman-2-yl) Methyl (60-Benzyloxy-
20,50,70,80-tetramethylchroman-20-carboxylate) (11)
(Scheme 3)
Ester 11 was prepared similarly to compounds 9a, 9b, and 9c.
Alcohol 8 (0.50 g, 1.53 mmol) was mixed with carboxylic
acid 10 (1.04 g, 3.06 mmol, 2.0 equiv), DCC (0.63 g,
3.06 mmol, 2.0 equiv), and DMAP (29.0 mg, 0.23 mmol,
0.15 equiv). Compound 11 was isolated as highly viscous oil,
further purified by flash chromatography with silica gel. Two
stereoisomers were observed on the NMR spectrum.
(60-Hydroxy-20,50,70,80-tetramethylchroman-20-yl) methyl
3,4-dihydroxycinnamate (2c): Eluant: ethyl acetate/hexanes
(2:3 v/v). Aspect: highly viscous oil. Yield = 80%. 1H NMR
(300.0 MHz, CDCl3): d: 1.23 (s, 3H, CH3); 1.64–1.91 (m, 2H,
CH2CH2CCH3); 2.08 (s, 3H, CH3); 2.09 (s, 3H, CH3); 2.15
(s, 3H, CH3); 2.55–2.63 (m, 4H, CH2CH2COO and
CH2CH2COO); 2.82 (t, 3JHH = 7.5 Hz, 2H, CH2CH2CCH3);
Data for (6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl)
methyl-60-benzyloxy-20,50,70,80-
tetramethylchroman-20-carboxylate (11)
2
2
4.04 (d, JHH = 11.4 Hz, 1H, CH2OCO); 4.16 (d, JHH
11.4 Hz, 1H, CH2OCO); 4.25 (s, 1H, OH); 5.11 (s, 1H, OH);
=
Eluant: ethyl acetate/hexanes (9:1 v/v). Aspect: Highly
1
viscous oil. Yield = 73%. H NMR (300.0 MHz, CDCl3):
3
5.17 (s, 1H, OH); 6.60 (d, JHH = 8.1 Hz, 1H, CHAr); 6.62
123

60
J Am Oil Chem Soc (2012) 89:55–66
Scheme 3 Reagents and
conditions for the synthesis of
antioxidant 3: (i) KOH,
H2O/EtOH, reflux; (ii) 8, DCC,
DMAP, DCM; (iii) H2,
BnO
BnO
(i)
OBn
OH
O
O
7
10
O
O
Pd/Charcoal 10%, THF.
For details see the text
(ii)
HO
OH
BnO
OBn
(iii)
O
O
O
O
O
O
3
11
O
O
d: 1.05 and 1.15 (2, 3H, CH3); 1.40–1.60 (m, 2H, CH2);
1.65 and 1.64 (2s, 3H, CH3); 1.79–1.98 (m, 1H, CH2); 1.98,
2.03, 2.05, 2.11, 2.12, 2.13, 2.15, 2.19, 2.20 and 2.21 (10s,
18H, CH3); 2.27–2.68 (m, 5H, CH2); 3.84, 4.04, 4.16 and
4.28 (4d, 2JHH = 11.1 Hz, 2H, CH2OCO); 4.63, 4.64, 4.65,
4.67 (4s, 4H, CH2OPh); 7.27–7.53 (m, 20H, CHAr). 13C
NMR (75.0 MHz, CDCl3): d: 11.7 (CH3); 11.8 (CH3); 11.9
(CH3); 12.0 (CH3); 12.1 (CH3); 12.7 (CH3); 12.9 (CH3);
20.0 (C=C–CH2); 20.1 (C=C–CH2); 20.9 (C=C–CH2); 21.5
(CH3); 21.9 (CH3); 25.8 (CH3); 25.9 (CH3); 27.7 (C=C–
CH2–CH2); 27.8 (C=C–CH2–CH2); 30.7 (C=C–CH2–CH2);
30.8 (C=C–CH2–CH2); 68.8 (CH2OCO); 69.2 (CH2OCO);
73.4 (OC–CH3); 73.5 (OC–CH3); 74.6 (OCH2Ph); 74.7
(OCH2Ph); 117.1 (CAr); 117.3 (CAr); 122.8 (CAr); 123.0
(2s, CAr); 123.1 (CAr); 126.0 (CAr); 126.1 (CAr); 127.7 (2s,
CAr); 127.8 (CAr); 127.9 (CAr); 128.2 (CAr); 128.4 (CAr);
128.5 (CAr); 137.8 (CAr); 137.9 (CAr); 147.1 (CAr); 147.2
(CAr); 148.2 (CAr); 148.5 (CAr); 148.9 (2s, CAr); 173.9
(C=O); 174.0 (C=O). HRMS (m/z): calculated for
C42H48O6 = 649.3524; found 649.3567 [M ? H]?.
(9s, 18H, CH3); 2.22–2.65 (m, 6H, CH2); 3.80, 4.03, 4.13
2
and 4.26 (4d, JHH = 11.1 Hz, 2H, CH2OCO); 4.05, 4.13;
4.18, 4.19 (4s, 2H, OH). 13C NMR (75.0 MHz, CDCl3): d:
10.9 (CH3); 11.1 (CH3); 11.2 (CH3); 11.3 (CH3); 11.7
(CH3); 11.8 (CH3); 11.9 (CH3); 12.1 (CH3); 12.2 (CH3);
20.0, 20.1 (2s, C=C–CH2); 21.0 (CH3); 21.1, 21.6 (2s,
C=C–CH2); 25.7, 25.8 (2s, CH3); 27.7, 27.8 (2s, C=C–
CH2–CH2); 30.8, 30.9 (2s, C=C–CH2–CH2); 68.8, 69.2 (2s,
CH2OCO); 73.2 (OC–CH3); 73.3 (OC–CH3); 116.8 (CAr),
116.9 (CAr); 118.4 (CAr), 188.5 (CAr); 121.2 (CAr); 122.3
(CAr); 122.6 (CAr); 144.7 (CAr); 144.9 (CAr); 145.2 (CAr);
145.3 (CAr); 145.8 (CAr); 145.9 (CAr); 174.0, 174.1 (2s,
C=O). HRMS (m/z): calculated for C28H36O6 = 469.2585;
found 469.2587 [M ? H]?.
NMR Spectroscopy
1H and 13C NMR were recorded on a 300 MHz Bruker
Avance II spectrometer (Billerica, MA, USA) with TMS as
1
internal reference for H- and 13C-NMR chemical shifts.
Data are presented in the following order: chemical shift in
ppm, multiplicity (s, singlet; d doublet; t, triplet; m, mul-
tiplet), coupling constant in Hertz, assignment broad band
1H decoupling.
Synthesis of (6-Hydroxy-2,5,7,8-tetramethylchroman-2-yl)
Methyl (60-Hydroxy-20,50,70,80-
tetramethylchroman-20-carboxylate) 3 (Scheme 3)
Antioxidant 3 was prepared similarly to compounds 2a, 2b,
and 2c. Hydrogenation of the ester 11 (658.0 mg) using a
palladiumoncharcoalcatalyst(66.0 mg, 1.01 mmol, 10%wt)
provided compound 3 (267.0 mg, 0.57 mmol). The crude
compound was purified by flash chromatography on silica gel.
Two stereoisomers were observed on the NMR spectrum.
Melting Point
Melting points (Mp) were measured with a Barnstead Elec-
trothermal MEL-TEMP 3.0 (Barnstead; Dubuque, IA, USA).
Mass Spectrometry
Data for (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)
methyl 60-hydroxy-20,50,70,80-tetramethylchroman-
20-carboxylate (3)
High resolution mass spectra (HRMS) were obtained on a
QSTAR Elite mass spectrometer (Applied Biosystems;
Foster City, CA, USA) equipped with an electrospray and
operated in positive ion mode.
Eluant: ethyl acetate/hexanes (7/3 v/v). Aspect: White
solids. Mp = 79–81 °C. Yield = 57%. 1H NMR
(300.0 MHz, CDCl3): d: 1.05 and 1.12 (2s, 3H, CH3);
1.20–1.50 (m, 2H, CH2); 1.62, 1.63 (2s, 3H, CH3); 1.86 (s,
CH3); 2.00, 2.02, 2.03, 2.04, 2.06, 2.09, 2.12, 2.14 and 2.16
High Performance Liquid Chromatography
Tocopherols and the new antioxidants were analyzed
according to AOCS Official Method Ce 8–89 [16].
123

J Am Oil Chem Soc (2012) 89:55–66
61
Analyses were performed on a Finnigan Surveyor LC
(Thermo Electron Corp., Waltham, MA, USA) with a
Finnigan Surveyor Autosampler Plus and Finnigan Sur-
veyor FL Plus fluorescence detector. The column was a
normal phase Diol column (5 lm; 250 9 4.6 mm; Mono-
chrom, Varian, CA). For tocopherol, the mobile phase
consisted of 7% methyl-tert-butyl-ether in hexanes with a
flow rate of 0.6 mL/min and the fluorescence detector was
set for excitation at 292 nm and emission at 325 nm. For
the analysis of novel antioxidants the mobile phase was
changed to 65% methyl-tert-butyl-ether in hexane. The
fluorescence detector was set for excitation at 292 nm and
emission at 394 nm. For each sample 10 lL was injected.
37 °C for 10 min and 0.5 mL of the AAPH solution
(153.0 lM) added. The fluorescence was measured at
37 °C for 30 min at 30 s intervals. The emission and
excitation were set at 525 and 485 nm, respectively. For a
blank, phosphate buffer replaced the antioxidant solution.
Each antioxidant solution was prepared in duplicate and
three measurements were performed for each sample. A
calibration curve was generated using trolox as the refer-
ence antioxidant.
The area under the fluorescence decay curve (AUC) was
calculated as follows:
30
X
ft þ fðtꢁ0:5Þ
AUC ¼
4
t¼0:5
DPPH Radical-Scavenging Assay
where ft is the fluorescence at a time t (min).
The net AUC was calculated using the following
equation:
The DPPH assay was performed according to the method
described by Nenadis and Tsimidou [17]. Briefly, to
2,960 lL of 0.1 mM ethanolic solution of DPPH, 40 lL of
synthesized antioxidant solution in ethanol was added at
the following concentrations: 0.37, 0.74, 1.11, 1.85, 3.7,
and 5.2 mM forming the ratios between the molar amounts
of antioxidant to the molar amount of DPPH radicals at
0.05, 0.10, 0.15, 0.25, 0.5 and 0.7, respectively. The
decrease of absorbance at 516 nm was measured at 25 °C
after 20 min of reaction. The blank solution contained the
same amount of DPPH and 40 lL of ethanol. Each test was
performed in triplicate. The results are expressed as the
%DPPH inhibition calculated according to the following
equation:
AUCnet ¼ AUC ꢁ AUCblank
For each antioxidant, a regression between AUCnet and
the compound concentrations was calculated, and the
results are expressed as trolox equivalents (Tequiv).
Canola Oil Triacylglycerides Isolation
Canola oil was stripped of its endogenous minor compo-
nents including antioxidants via adsorption chromatogra-
phy, following the procedure described by Lampi and
Kamal-Eldin [19].
ðAc ꢁ AtÞ100
Accelerated Storage
%DPPH ¼
Ac
The ability of the new antioxidants to protect oil against
oxidative degradation was determined using the Schaal
Oven test. To 1 g of canola triacylglycerols (CTG) 350 lg/g
of the tested antioxidant was added in a vial (National
Scientific Target DP Vials; 2 mL, 12 9 32 mm). The
samples were stored in the dark for 5 days at 60 °C, pro-
viding a surface area to volume ratio of 0.78. Samples were
examined at 24 h intervals for the peroxide value and the
residual amounts of antioxidant. The effectiveness of the
new compounds was compared to a-tocopherol. Experi-
ments were set up with two repetitions for each tested
antioxidant, and samples from each repetition were ana-
lyzed in duplicate.
where Ac and At are the absorbances of the control and the
test sample, respectively. All standard deviations for DPPH
tests were below 3.0%. Both trolox and a-tocopherol were
used as references. Calculated IC50 represents the con-
centration of antioxidant required to decrease the initial
amount of DPPH by 50%.
ORAC Assay
ORAC assays were performed according to Szydlowska-
Czerniak et al. [18]. Briefly, the fluorescein disodium salt
and AAPH solutions were prepared in 75 mM phosphate
buffer (pH = 7.4). The antioxidant solutions, 1 mM of
each compound, were dissolved in methanol, and a specific
volume of it dissolved in the buffer to provide the required
amounts of antioxidant within a range of 3.125–25.00 lM.
Four different concentrations were tested for each anti-
oxidant. A solution of fluorescein disodium salt, 3.0 mL
(0.0816 lM) was mixed with 0.5 mL of antioxidant solu-
tion directly in a quartz cuvette. The mixture was kept at
Peroxide Value
To assess the PV, the method published by Hornero-
Mendez et al., and modified by Shantha and Decker was
used [20, 21]. Briefly, 200 mg of oil was dissolved in 5 mL
of hexane. To 200 lL of the sample solution, 5 mL of
123

62
J Am Oil Chem Soc (2012) 89:55–66
methanol/chloroform/HCl solution (1:1:0.012, v/v), then
100 lL of NH4SCN (30% w/w in water), and 100 lL of
ferrous chloride (0.4% in water) were added. After 5 min
of incubation at room temperature, the absorbance was
measured at 480 nm.
from carboxylic acids 4a, 4b, and 4c, respectively
(Scheme 1). Cinnamic acid derivatives, 4a–4c,were ben-
zylated using benzyl bromide and potassium carbonate in
dimethylformamide (DMF) to produce 5a–5c with the
following yields: 86% (5a), 92% (5b), and 91% (5c).
Saponification of 5a–5c esters with potassium hydroxide in
ethanol/water solution formed the desired cinnamic acids
derivatives with the yield of 86% (6a), 98% (6b), and 90%
(6c). Antioxidants 2a, 2b, and 2c were prepared in four
steps from trolox as described in Scheme 2. First, alcohol 8
was isolated in two steps according to a synthetic pathway
described by Percec et al. [15]. A Steglich esterification
between compound 8 and cinnamic acid derivatives 6a–6c
formed esters 9a–9c. Finally, antioxidants 2a–2c was
obtained by hydrogenation of compounds 9a–9c using
palladium as catalyst. Isolated antioxidants formed highly
viscous oils and were produced with the following yield:
78% (2a), 83% (2b), and 80% (2c).
Frying Test
The effectiveness of the developed antioxidants to protect
CTG under frying conditions was assessed using a frying test
developed in our laboratory. CTG (12.0 g), fortified with
500 lg/g of the studied compound, was weighed into a glass
beaker (Pyrex, USA). An octagonal stir bar (ThermoFischer
Scientific, USA) was placed into the vessel, altering the final
surface to volume ratio to 0.42. The vessel with sample was
heated at 185 5 °C for 10 min, and 1.2 g of formulated
food (a mixture of gelatinized potato starch with glucose and
silica gel, 4:1:1 w/w) was added. The heating was continued
for another 20 min without mixing and then was stirred at
500 rpm. Heating and stirring were afterward maintained for
another 90 min. About 0.5 g of the oil sample was withdrawn
at the 60th, 80th, 100th and 120th min of heating. Selected
sampling points reflect the frying time based on the amount of
polarcomponentsformedandcorrespondto1,3,5,and7 days
of actual frying time using an institutional fryer (General
Electric Company, NY, USA). The frying performance of oils
was assessed by the measurement of total polar components
(TPC) and the retained amounts of added antioxidant. Sam-
ples from two repetitions of frying test were analyzed in
duplicate for TPC and residual antioxidant.
In order to prepare the antioxidant 3, synthesis of pre-
cursor 10 was first carried out. Carboxylic acid 10 was
easily synthesized by saponification of ester 7 (Scheme 3).
A Steglich esterification, followed by a hydrogenation,
finally made the desired antioxidant 3 in a moderate yield
of 57% (Scheme 3).
All developed antioxidants showed good solubility in
oils and fats.
DPPH Assay
The radical scavenging properties of the synthesized anti-
oxidants 2a, 2b, 2c and 3, trolox and a-tocopherol were
evaluated by the DPPH assay. Results of this study are
given in Table 1. These results confirm that the radical
scavenging effectiveness of the novel antioxidants 2a
Total Polar Components
The amounts of polar compounds were determined by
gravimetric procedure following AOAC Method 982.27
with Schulte modification [22, 23].
Table 1 Radical scavenging activity of the novel and reference
antioxidants
Statistical Analysis
Antioxidant
DPPH Test
IC50 (mM)
ORAC Test
Data were analyzed by single factor analysis of variance
(ANOVA) and regression analyses using Minitab 2000
statistical software (Minitab Inc., PA, ver. 13.2). Signifi-
cant differences between means were determined by
Duncan’s multiple range tests. Statistically significant
differences were determined at P \ 0.05.
Inhibition
(%)
Trolox
equivalents
(lM)
2a
1.52 0.01
1.05 0.04
0.76 0.01
1.15 0.01
1.75 0.02
1.61 0.02
37.1 2.0
50.1 1.8
71.0 2.4
51.4 1.9
31.7 1.9
34.6 1.7
3.30 0.10
3.42 0.22
5.41 0.19
1.48 0.05
2b
2c
3
Troloxa
a-Tocopherola
Caffeic acida
Results and Discussion
4.46 0.22
Synthesis
Compounds 2a–2c, and 3 developed the novel antioxidants. For their
structures and properties see Fig. 1 and the text
In order to produce the new antioxidants 2a, 2b, and 2c,
precursors 6a, 6b, and 6c were first prepared in two steps
a
Reference antioxidants
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J Am Oil Chem Soc (2012) 89:55–66
63
(IC50 = 1.52 mM), 2b (IC50 = 1.05 mM), 2c (IC50
=
not included). Antioxidant capacities of each compound,
expressed in trolox equivalent (Tequiv), are given in
Table 1. According to these results, all of the synthesized
0.76 mM) and 3 (IC50 = 1.15 mM) were significantly
higher (P B 0.001) than the trolox (IC50 = 1.75 mM) and
a-tocopherol (IC50 = 1.61 mM). Antioxidant 2c, which
possesses the highest number of hydroxyl groups, was the
most efficient. Furthermore, compared to trolox (one hydro-
xyl) and a-tocopherol (one hydroxyl), the bichromanol 3 (2
hydroxyls) exhibited a significantly higher radical scavenging
activity (P B 0.001). In agreement with the present result, a
novel dimeric antioxidant containing two hydroxyl groups in
fused double chromanol rings has been reported to possess
better DPPH radical scavenging activity and provide double
reducing power compared to a-tocopherol, [24, 25].
It is well established that phenolic compounds scavenge
radicals by proton donation [6]. Consequently, the present
results are consistent with this observation because higher
number of hydroxyl group resulted in higher capacity for
proton donation. Additionally, this test established that the
presence of a second methoxy substituent in the ortho
position of the hydroxyl group significantly increases the
antioxidant effectiveness in radical scavenging. Thus,
when comparing activities for compounds 2a with 2b
where the only structural difference is in the number of
methoxy groups on the ring, 1 versus 2 methoxy groups,
respectively, the radical scavenging potency of 2b (50.1%)
was higher than 2a (37.1%). These results are in agreement
with the observation that an electron donating substituent
in the ortho position to the hydroxyl group increases the
radical scavenging activity [12, 26]. Kikuzaki et al. [26]
reported a higher radical scavenging activity for sinapic
acid compared to ferulic acid. Comparing to the previously
published results by the same authors, antioxidants 2a, 2b,
and 2c are better scavengers than the trolox hydrox-
ybenzoates (Fig. 2) [11]. Thus, as expected, the presence of
an alkyl chain between the ester group and the phenolic
rings significantly increases the radical scavenging activity
of the antioxidants.
phenolic compounds exhibited
a higher antioxidant
capacity than trolox. An improved antioxidant capacity was
reported for a synthesized compound containing hydroxy-
tyrosol attached to the chromanol ring, which inhibited the
formation of malondialdehyde in rat liver microsomal
membrane when oxidation was induced by AAPH [27].
The data obtained for caffeic acid (4.46 Tequiv) was
similar to the value published by Gomez-Ruiz et al. (4.52
Tequiv) [28]. Antioxidant 2c (5.41 Tequiv) was the most
effective radical scavenger among the studied compounds
whereas compound 3 (1.48 Tequiv) was the least active.
Similarly to the results reported in previous investigations
[11, 29], no correlations between ORAC and DPPH assays
were observed. Lack of similarities in trends between these
two antioxidative tests is related to the different radicals
used and the possible interaction between radicals and
antioxidants. Additionally, for both tests different mecha-
nism of antioxidant radical interaction is involved and with
it different response to the radicals scavenging activity
[30–32]. For instance, compound 3 (1.48 Tequiv) is less
effective than 2a (3.30 Tequiv) in the ORAC assay whereas
the opposite was observed in the DPPH test (Table 1).
Furthermore, no trend was observed regarding the nature
and position of the substituent on the ring, e.g. para posi-
tion to the hydroxyl group, when the radical scavenging
activity was measured by ORAC.
Accelerated Storage Stability (Schaal Oven Test)
Radical scavenging activity is not directly indicative of
antioxidant effectiveness in a food system, we tested the
novel compounds effectiveness in the food applications
[32]. The ability of the new compounds 2a, 2b, 2c and 3 to
protect CTG from oxidation was determined under Schaal
Oven test (SOT) conditions. The results of the SOT indi-
cated that the new antioxidants protected CTG better than
a-tocopherol (Fig. 3). Indeed, the amounts of hydroperox-
ides formed after 5 days of storage were significantly lower
for CTG fortified with 2a (39.9 mequiv/kg), 2b (39.3
mequiv/kg), 2c (15.9 mequiv/kg) or 3 (14.0 mequiv/kg)
compared to unprotected CTG (110.8 mequiv/kg).
Although no significant difference was observed even at
P B 0.12 in the protective capacities of novel antioxidant
2a, 2b and a-tocopherol, whereas antioxidants 2c and
3 were significantly more efficient (P B 0.001) than
a-tocopherol. Compound 3 was the most effective antiox-
idant among all tested compounds. These results indicate
lack of direct correlation between the radical scavenging
activity measured by DPPH and ORAC assays and the
antioxidant activity measured in the food system. A similar
ORAC Assay
The antioxidant capacities of 2a, 2b, 2c and 3 were also
evaluated by the ORAC assay. The linearity between the
AUCnet and the antioxidant (AH) concentration was
checked for each synthesized phenolic compound, and a
regression coefficient above 99% was observed (Results
R1
HO
R2
R3
1a: R1 = OCH3; R2 = OH; R3 = H
1b: R1 = R3 = OCH3; R2 = OH
1c: R1 = R2 = OH; R3 = H
O
O
O
Fig. 2 Structures of the intermediate phenolic compounds used for
the synthesis of antioxidants. For details see the text
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J Am Oil Chem Soc (2012) 89:55–66
120
100
80
60
40
20
0
30
25
20
15
10
5
CTG
2a
2b
2c
CTG
2a
2b
2c
3
3
α - Tocopherol
α -Tocopherol
0
0
1
2
3
4
5
0
20
40
60
80
100
120
Storage Time [days]
Heating Time [mins]
Fig. 3 Changes in hydroperoxides during the storage of canola
triacylglycerols with different antioxidants added at 350 lg/g. CTG
canola oil triacylglycerides. For details see the text
Fig. 4 Formation of polar components during test frying of canola
triacylglycerols with added a-tocopherol and the novel antioxidants
2a–2c and 3 at 500 lg/g. CTG canola triacylglycerides. For details
see text
lack of correlation has been reported in the literature,
ascribing it to the nature of the radicals involved and the
difference in the scavenging mechanism between DPPH
and ORAC [30–32].
Antioxidant Stability
In order to investigate the stability of 2a, 2b, 2c and 3,
the amounts of antioxidant remaining at different stages
during storage and frying were measured (Figs. 5, 6). All
the novel antioxidants exhibited higher stability than
a-tocopherol. Indeed, under accelerated storage at the end
of storage time the following amounts of antioxidant were
retained: 75.5, 69.5, 64.5, and 68.7% for 2a, 2b, 2c and 3,
respectively while only 35.2% of a-tocopherol was
observed (Fig. 5). The same superior stability was
observed during frying test (Fig. 6). After 60 min of frying
test, significantly higher amounts (P B 0.001) of antioxi-
dants 2a (56.3%), 2b (48.1%), 2c (42.1%) and 3 (20.5%)
Protection During Frying
Many compounds are known for their antioxidant activities
at ambient temperature. The extreme conditions used dur-
ing frying such as elevated temperature, prolonged expo-
sure to oxygen, adding special limitations for antioxidants.
Consequently, the antioxidant for institutional and indus-
trial frying should not only be effective at frying conditions
but also need to be: thermally stable and of low volatility to
prevent evaporation [33]. As an example, the effectiveness
of BHT under frying conditions is compromised by its
evaporative losses at elevated temperatures [34]. The
capacities of the new antioxidants to protect CTG from
degradation during frying were determined using a frying
test developed in our laboratory. Their efficiency was
assessed by measuring the TPC after different frying times.
Results are presented in Fig. 4. This study has demon-
strated that antioxidants 2a, 2b, 2c and 3 much better
protected CTG during frying, compared to a-tocopherol.
Indeed, at the end of the frying period, the TPC amounts
were: 18.3, 17.8, 15.8, and 15.4% for 2a, 2b, 2c and 3,
respectively (Fig. 4). These values are significantly lower
(P B 0.001) from unprotected CTG (25.7%). Besides,
the new antioxidants were significantly more efficient
(P B 0.004) than a-tocopherol (20.4%). Among the
synthesized antioxidant, compound 3 was the most effi-
cient. Compared to previously published results, trends
related to the difference in chemical structure were not
observed [11].
100
90
80
70
60
50
α-Tocopherol
40
30
20
2a
2b
2c
3
0
1
2
3
4
5
Storage Time (days)
Fig. 5 Percentage of remaining antioxidants during the accelerated
storage of canola triacylglycerides fortified with a-tocopherol and the
novel antioxidants 2a–2c, and 3 added at 350 lg/g. For details see the
text
123

J Am Oil Chem Soc (2012) 89:55–66
65
very valuable for high temperature applications such as
frying.
100
90
80
70
60
50
40
30
20
10
0
α - Tocopherol
2a
2b
2c
3
Further investigations are underway to develop better
understanding of the relationship between structure and
antioxidant activity, the nature of the novel antioxidants
degradation products formed, and to address any safety
issues related to these antioxidants and their degradation
products. This study was designed to prepare novel anti-
oxidants and assess their activity in an oil system, however
their potential application in various foods and regulatory
acceptance will be a matter of further development.
Acknowledgments This work was financed by the Alberta Value
Added Corporation, the Agriculture Funding Consortium and Bio-
active Oil Program.
0
10 20 30 40 50 60 70 80 90 100 110 120
Heating time (min)
Fig. 6 Percentage of remaining antioxidants during test frying canola
triacylglycerides fortified with a-tocopherol and the novel antioxi-
dants 2a–2c, and 3 added at 500 lg/g. For details see the text
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