Comparative study of the antioxidant activities of some lipase-catalyzed alkyl dihydrocaffeates synthesized in ionic liquid

Article abstract of DOI:10.1016/j.foodchem.2016.12.075

The solubility limitations of phenolic acids in many lipidic environments are now greatly improved by their enzymatic esterification in ionic liquids (ILs). Herein, four different ILs were tested for the esterification of dihydrocaffeic acid with hexanol

Full text of DOI:10.1016/j.foodchem.2016.12.075

Food Chemistry 224 (2017) 365–371
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Comparative study of the antioxidant activities of some lipase-catalyzed
alkyl dihydrocaffeates synthesized in ionic liquid
a
b
c
Somayeh Gholivand a, Ola Lasekan a, , Chin Ping Tan , Faridah Abas , Leong Sze Wei
⇑
a Department of Food Technology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
b Department of Food Science, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
c Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 August 2016
Received in revised form 17 December 2016
Accepted 20 December 2016
Available online 22 December 2016
The solubility limitations of phenolic acids in many lipidic environments are now greatly improved by
their enzymatic esterification in ionic liquids (ILs). Herein, four different ILs were tested for the esterifi-
cation of dihydrocaffeic acid with hexanol and the best IL was selected for the synthesis of four other n-
alkyl esters with different chain-lengths. The effect of alkyl chain length on the anti-oxidative properties
of the resulted purified esters was investigated using b-carotene bleaching (BCB) and free radical scav-
enging method DPPH and compared with butylated hydroxytoluene (BHT) as reference compound. All
four esters (methyl, hexyl, dodecyl and octadecyl dihydrocaffeates) exhibited relatively strong radical
scavenging abilities. The scavenging activity of the test compounds was in the following order: methyl
ester > hexyl ester P dodecyl ester > octadecyl ester > BHT while the order for the BCB anti-oxidative
activity was; BHT > octadecyl ester > dodecyl ester > hexyl ester > methyl ester.
Chemical compounds studied in this article:
1-Butyl-3-methylimidazolium bis
(trifluoromethylsulfonyl) imide (PubChem
CID: 24887144)
1-Butyl-3-methylimidazoliumhexafluoro
phosphate (PubChem CID: 24886139)
1-Hexyl-3-methylimidazoliumhexafluoro
phosphate (PubChem CID: 24889211)
3,4-Dihydroxycaffeic acid (PubChem CID:
24278290)
Ó 2016 Elsevier Ltd. All rights reserved.
Methanol (PubChem CID: 887)
Hexanol (PubChem CID: 8103)
Dodecanol (PubChem CID: 8193)
1,1-Diphenyl-2-picryl-hydrazyl (PubChem
CID: 24856989)
Linoleic acid (PubChem CID: 5280450)
Keywords:
Enzymatic esterification
Candida antarctica lipase (Novozyme 435)
Ionic liquid
Dihydrocaffeic acid
Antioxidant activity
1. Introduction
(Gulcin, Buyukokuroglu, & Kufrevioglu, 2003). Therefore, there
have been great efforts to find safe and potent natural antioxidants
Oxidative damages in the human body plays important causa-
tive roles in disease initiation and progression (Youdim & Joseph,
2001). To protect the human body from free radicals and retard
the progress of many chronic diseases as well as lipid oxidative
rancidity in foods, antioxidants such as phenolic compounds can
be used because phenolic antioxidants quench either the oxygen-
derived free radicals or the substrate-derived free radicals
from various plant sources. Phenolic acids which are broadly dis-
tributed in vegetables, fruits, spices, and aromatic herbs, are bio-
logically active antioxidants and possess various applications in
cosmetic, pharmaceutical and food industries (Figueroa-Espinoza
& Villeneuve, 2005).
Dihydrocaffeic acid (DHCA), a metabolite of caffeic acid, can be
found in many fruits, vegetables, and herbs such as coffee, arti-
choke, pear, basil, thyme, oregano, and apple. This phenolic acid
contain a catechol moiety in its structure which is responsible
⇑
Corresponding author.
E-mail address: olaniny56@gmail.com (O. Lasekan).
´
for the anti-oxidative property (Andjelkovic et al., 2006) However,
http://dx.doi.org/10.1016/j.foodchem.2016.12.075
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

366
S. Gholivand et al. / Food Chemistry 224 (2017) 365–371
due to low solubility of phenolic acids in hydrophobic media
(Figueroa-Espinoza & Villeneuve, 2005), their applications in oil-
based food and cosmetic industries are limited. To improve the
lipophilicity of phenolic acids, trans-esterification through the
enzymatic reactions in the presence of aliphatic alcohols to an
amphiphilic molecule can significantly increase their solubility in
non-aqueous media (Vafiadi, Topakas, Alissandratos, Faulds, &
Christakopoulos, 2008).
2.2. Procedure of enzymatic esterification of dihydrocaffeic acid with
hexanol in different ionic liquids
The enzymatic esterification of DHCA was carried out in differ-
ent ionic liquids using screw-capped test tubes. Substrate mole
ratio (1:4) corresponding to DHCA/hexanol was weighed into each
test tube, followed by the subsequent addition of 20 mg of pow-
dered Candida antarctica lipase in the presence of ionic liquid as
solvent medium. The tested ionic liquids were: 1-butyl-3-
methylimidazoliumbis (trifluoromethylsulfonyl) imide, 1-hexyl-3
-methylimidazoliumhexafluorophosphate, 1-butyl-3-methylimida
zoliumhexafluorophosphate and 1-octyl-3-methylimidazoliumhex
afluorophosphate. Enzymatic reactions were conducted in the
presence of 100 mg of 3 Å molecular sieves to remove the water
formed as by-product. The initial water contents in the reaction
system as determined by the volumetric Karl Fischer (KF) titration
method (Scaccia, 2005) are presented in Table 1. The reactions
were performed inside an incubator shaker at 55 °C, 250 rpm for
Phenolic acids are heat-sensitive and susceptible to oxidation
under certain pH conditions thus the chemical synthesis of their
esters is difficult. Lipase-catalyzed esterification of phenolic acids
in organic solvent media has been widely used to synthesize valu-
able esters for application in the food industry under mild condi-
tions (Croitoru et al., 2012; Jakovetic et al., 2013; Pang et al., 2013).
The low solubility of phenolic acids in most organic solvents in
addition to the toxicity effect of these media and the low activities
of enzymes are some of the technical problems preventing the gen-
eration of an efficient non aqueous enzymatic reaction system. In
recent time, organic solvents have been replaced with ionic liquids
(ILs) (Gandhi et al., 2000). ILs belong to a class of tunable designer
media with thermal stability, zero volatility, good solubility and
polarity for many polar or less polar organic components as well
as hydrophobicity (Zhao, 2010). The ionic nature of these liquids
make them good solvents for diverse reactions because they pro-
vide different reactivity and selectivity compared to conventional
organic solvents (Qiu & Texter, 2008). Therefore, ionic liquids have
continued to draw attention as replacement for organic solvents
for enzymatic trans-esterification of various components. (Chen
et al., 2011; Katsoura et al., 2009).
time period of five days. On a daily basis, 100 lL was taken from
the reaction mixture and quantified by HPLC. Following the results
from this section, the best ionic liquid was selected for other
experiments.
2.3. Enzymatic synthesis of dihydrocaffeates with different chain-
length alcohols in the selected ionic liquid
The enzymatic esterification of DHCA with methanol, hexanol,
dodecanol and octadecanol was carried out in screw-capped bot-
tles under optimal conditions that were obtained by response sur-
face methodology for hexyl dihydrocaffeate in a preliminary
experiment. Substrate mole ratio (1:2.1) corresponding to DHCA/
different chain-length alcohols was weighed into the 50 mL bot-
tles, followed by the subsequent addition of 4.16% powdered C.
antarctica lipase relative to substrates in the selected ionic liquid
The current study examined: (1) the efficiency of four different
ionic liquids (1-butyl-3-methylimidazolium bis (trifluoromethyl-
sulfonyl)
sphate, 1-hexyl-3-methylimidazoliumhexafluorophosphate, and
1-octyl-3-methyllidazoliumhexafluorophosphate) as reaction
imide,
1-butyl-3-methylimidazoliumhexafluoropho
a
medium for lipase-catalyzed esterification; (2) the ionic liquid
with the highest esterification ability was subsequently employed
in the esterification of DHCA with four different aliphatic alcohols
of different chain length; (3) In addition, the anti-oxidative proper-
ties of the alkyl esters produced were investigated.
(1-butyl-3-methylimidazoliumbis
(trifluoromethylsulfonyl)
imide). The reactions were initiated in the presence of 100 mg
3 Å molecular sieves for the removal of the water formed as by-
product. The reactions were conducted in an incubator shaker
(250 rpm) at 39.4 °C, for 77.5 h. Removal of the biocatalyst ended
the reaction. This was followed by the separation of the substrates
and products of the bio-catalytic reactions.
2. Materials and methods
2.4. Ester purification
2.1. Materials
The alkyl dihydrocaffeate esters were obtained by extracting
each reacted solution in diethyl ether. The ether layer was dried
over sodium sulfate and filtered (Borneman, Hartley, Morrison,
Akin, & Ljungdahl, 1990). In each case, the filtrate was evaporated
to dryness under reduced pressure. The extract was applied onto a
silica gel column (2.5 ꢀ 30 cm, fractions of 50 mL) washed with
200 mL of chloroform (100%) and 400 mL of (99:1–98:2%)
chloroform-methanol mixture respectively to elute the unreacted
substrates. The formed ester product was detected by thin-layer
Ionic liquids consisting: 1-butyl-3-methylimidazoliumbis (tri-
fluoromethylsulfonyl) imide with purity of 98%, 1-butyl-3-methyli
midazoliumhexafluorophosphate (97%) and 1-hexyl-3-methylimi
dazoliumhexafluorophosphate were acquired from Sigma-Aldrich
(Milan, Italy). 1-Octyl-3-methylimidazoliumhexafluorophosphate
(98%) was purchased from Fisher Scientific, Inc. (Slangerup, Den-
mark). Immobilized lipase (triacylglycerol hydrolase, EC 3.1.1.3;
Novozyme 435 from C. antarctica supported on acrylic resin beads
was obtained from Novo Nordisk Bio-industrials, Inc. (Bagsvaerd,
Denmark). 3,4-Dihydroxycaffeic acid (DHCA), methanol, hexanol,
dodecanol with the highest available purity and octadecanol with
85% purity were purchased from Sigma-Aldrich (Milan, Italy). 3 Å
molecular sieves (10–20 mesh beads) were obtained from Fluka
(Fluka, Neu-Ulm, Germany). Chloroform, diethyl ether and all sol-
vents were of analytical and HPLC grades purchased from Fisher
Scientific, Inc. (Slangerup, Denmark). Thin-layer chromatography
(TLC) with a silica gel 60F254 plate no. 5715 was obtained from
Merck (Darmstadt Germany). 1,1-Diphenyl-2-picryl-hydrazyl
(DPPH) was obtained from Tokyo Chemical Industry. BHT, linoleic
acid and b-carotene were purchased from Sigma-Aldrich. Tween-
20 was acquired from Acros Organics (New Jersey, USA).
chromatography (TLC) (Nagaoka, Banskota, Tezuka, Saiki,
Kadota, 2002) or HPLC methods.
&
2.5. Identification of alkyl dihydrocaffeates
2.5.1. HPLC analysis
The reaction components were diluted with methanol and fil-
tered using syringe filter (0.22 m). A 10 L aliquot was taken from
l
l
the reaction mixture and injected into HPLC (Agilent1200 series,
Waldbrunn, Germany) equipped with a UV detector at wavelength
range of 200–325 (which was applied for DHCA evaluation). The
injected samples were eluted between 3 and 4 min by mobile
phases consisting of 90% of solvent A (methanol) and 10% of sol-

S. Gholivand et al. / Food Chemistry 224 (2017) 365–371
367
Table 1
The initial water content (%) of the different components of reaction medium determined at 25 °C.
L1
Hexanol
Dihydrocaffeic acid
1.28 0.01
Lipase
Medium
0.18 0.01
0.28 0.01
1.60 0.01
1.67 0.01
L11-Butyl-3-methlimidazolium bis (trifluoromethylsulfonyl) imide.
vent B (distilled water containing 0.75% of acetic acid) using a C18
and also the interaction between them on the antioxidant activity
and radical scavenger of the synthesized dihydrocaffeate esters.
Each independent variable is considered as more significant when
either its F-ratio or p-value becomes larger or smaller respectively
(Montgomery, 2001). All experiments on radical scavenging assay
were carried out in duplicate and those on anti-oxidant efficiency
were conducted in triplicate respectively. The statistical analysis
was applied using the MINITAB v16 statistical package (Minitab
Inc., State College, PA, USA) software.
reverse-phase column (250 ꢀ 4.6 mm, 5
lL) (Yang, Guo, & Xu,
2012).
2.6. Determination of antioxidant activity with the b-carotene
bleaching (BCB) test
Total antioxidant activity of synthesized esters was determined
according to a slightly modified version of the b-carotene bleaching
method of (Yu et al., 2006). The b-carotene (5 mg) was added to a
boiling flask containing 10 mL chloroform, and then mixed with
linoleic acid (250 lL) and Tween 20 (2 g). The chloroform was
3. Results and discussion
removed completely by rotating the flask for 10 min on a vacuum
evaporator at 50 °C. Distilled water (500 mL) was slowly added to
the semi-solid residue and the mixture was emulsified for 5 min in
an ultrasonic bath to form emulsion ‘A’. In each tube, 1 mL of
ethanolic solution of each antioxidant (0.05–0.25 mg/mL) was
mixed with 5 mL aliquot of emulsion A. As a control solution,
1 mL of ethanol containing 5 mL of emulsion A without anti-
oxidant was used. A second emulsion ‘B’ consisting of the same
amounts of linoleic acid, Tween 20 and distilled water was also
prepared. To 1 mL ethanol, 5 mL of emulsion B was added and
the resultant solution was used for adjusting the spectrophotome-
ter as blank solution. The absorbance of all sample solutions was
measured with a spectrophotometer (Varian 100) at 470 nm dur-
ing a time period of 0–150 min (at 25 min intervals). The tubes
were placed in a water bath at 50 °C between measurements. All
determinations were performed in triplicate. The percentage inhi-
bition was calculated from the data using the following equation:
3.1. Effect of ionic liquid nature on conversion yield
Ionic liquids (ILs) are currently being used as suitable replace-
ments for the organic solvents in many reactions (Jadhav, Lee,
Koo, & Seo, 2015; Li et al., 2014). They are relatively non-toxic with
good substrate solubility and perform better in the maintenance of
enzyme activities (Forsyth & MacFarlane, 2003). The results of the
enzymatic esterification of dihydrocaffeic acid with hexanol in the
different ionic liquids after five days revealed the following: 1) In
the absence of enzyme, there was no observable conversion in all
the tested ionic liquids; 2) however, the addition of Novozyme
435 produced observable conversions with the highest yield
obtained in 1-butyl-3-methylimidazoliumbis (trifluoromethylsul-
fonyl) imide (IL1). The mechanism of this reaction was reported
previously (Uppenberg, Hansen, Patkar, & Jones, 1994). Novozyme
435 which is a catalytic triad consists of Ser-105, Hys-224 and Asp-
187. Its catalytic activity occurs in two stages. In the first step of
enzyme acylation, the active-site (Ser-105) is activated by
histidine-224 and aspartate-187 which attacks the carbonyl group
of the first substrate (dihydrocaffeic acid), forming a tetrahedral
intermediate. The acyl enzyme complex is formed when water,
as the first product, leaves the active site. The subsequent step
(deacetylation of enzyme) starts when alcohol enters the enzyme
active site and a second tetrahedral intermediate is formed. Ester
as second product exits the enzyme active site, leaving free
enzyme which is ready for another catalytic cycle (Fig. 1).
Subsequently, this IL was selected as the reaction medium. The
increasing order of conversion yield, described as a degree of ester-
ification, was: IL1 (100%) > IL2 (39%) > IL3 (26%) > IL4 (23%).
Furthermore, the results of the enzymatic esterification during
the five days period showed that the conversion yield was reaction
medium-dependent (Fig. 2A). IL1 with a [Tf2 N]-base produced the
highest conversion yield throughout the five days of reaction. This
was followed by the IL2 which has a [PF6]-base. Generally, ILs with
the [PF6]-base recorded much lower conversion yield compared
with IL1. Among the ILs with the [PF6]-base (i.e. IL2, IL3 & IL4),
the chain-length of the alkyl substitutes on their cation moiety
(i.e. attached to the imidazolium ring) seems to affect their reac-
tion conversion ability. This observation is similar to those of
Santos, Coutinho, and Ventura (2015) who reported that the vis-
cosity and hydrophobicity of ILs with the same anion are functions
of the length of alkyl group which is attached to their cation. In the
present study hexyl dihydrocaffeate production revealed an
inverse relationship with number of carbons in the alkyl group of
the ILs with [PF6]-base.
AA ¼ ½1 ꢁ ðAo ꢁ At=Aoo ꢁ Ao þ tÞꢂ ꢀ 100;
Where: AA: the antioxidant activity, Ao: the initial absorbance
of sample, At: the absorbance of sample after 150 min, Aoo: the ini-
tial absorbance of control, and Aot is the absorbance of control after
time 150 min.
2.7. Free radical scavenging method DPPH
The antioxidant activity of the samples, based on the scaveng-
ing activity of the stable DPPH free radical, was measured by the
reported method of Braca et al. (2001). In its radical form, DPPH
has an absorbance at 517 nm which disappears in the presence
of an antioxidant compound. For the test, one milliliter of
methanolic solution of each antioxidant (5, 20, 30, 50, 75 l )
g mLꢁ1
was added to 3 mL of 0.1 mM methanolic solution of DPPH. The
decreasing absorbance was measured at 517 nm after 30 min
under ambient temperature and darkness (Ae). The reference solu-
tion consisted of 1 mL of MeOH with 3 mL of DPPH and its absor-
bance at forgoing wavelength was Ao. The percent inhibition was
obtained from the following equation:
%inhibition ¼ ð1 ꢁ Ae=AoÞ ꢀ 100
2.8. Statistical analysis
All the experimental results were subjected to two-way ANOVA
(analysis of variance) to evaluate the significant (p < 0.05) main
effects of two factors namely; the chain-length and concentration

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S. Gholivand et al. / Food Chemistry 224 (2017) 365–371
Fig. 1. Proposed mechanism for lipase-catalyzed synthesis of dihydrocaffeic acid esters based on the known structure of the C. antarctica active centre and, by analogy to
other lipolytic reactions (Uppenberg et al., 1994).
3.2. Esterification of dihydrocaffeic acid with different alcohols in the
selected ionic liquid
3.3. Anti-oxidative property of alkyl dihydrocaffeate esters using free
radical scavenging DPPH assay
The esterification of dihydrocaffeic acid in 1-butyl-3-
methylimidazoliumbis (trifluoromethylsulfonyl) imide as a reac-
tion medium was further studied using various primary aliphatic
alcohols with chain lengths varying between 1 and 18 carbon
atoms. The ionic liquid used was able to solubilize relatively
large amounts of dihydrocaffeic acid and its esters. The solubility
of octadecanol in this reaction medium was low; thus, acetone
was used as co-solvent. The methyl dihydrocaffeate produced
was oily but other esters (hexyl-, dodecyl- and octadecyl dihy-
drocaffeate) were obtained in solid state. It must be noted that
the conversion yield of the reaction was about 68.7–84% after
3 days of reaction (Fig. 2B). The use of molecular sieves might
be responsible for this high reaction performance. The removal
of water with molecular sieves from the environment greatly
enhanced esterification process. The initial water content is
important in the enzymatic reaction in several ways. If the water
content is too high, hydrolysis of ester occurs instead of the syn-
thesis. Although, the level of the initial water in the reaction
medium was <0.20% (Table 1), specific measures were taken to
remove water from the environment by the use of molecular
sieves. In addition, the reaction conversion yield was higher
when the esterification was carried out with long-chain alcohols
(i.e. hexanol and dodecanol) compared with short-chain (metha-
nol). On the contrary, octadecanol produced lower conversion
yield. The low conversion yield of octadecyl- dihydrocaffeate
might be due to the low solubility of this long-chain alcohol in
the reaction medium.
Dihydrocaffeic acid (DHCA) is formed by intestinal bacteria as a
degradation product of caffeic acid (Peppercorn & Goldman, 1971)
and eriocitrin which was first isolated from lemon fruit as a flavo-
noid glycoside (Miyake, Yamamoto, & Osawa, 1997). It should also
be noted that DHCA has been detected as a catechol metabolite of
caffeic acid (CA) in human plasma (Goldstein, Stull, Markey, Marks,
& Keiser, 1984). The difference between the structure of CA and
DHCA is due to the 2,3-double bond in CA. The anti-oxidative prop-
erties of DHCA and CA have been studied by Moon and Terao
(1998). They concluded that both compounds have no significant
difference in their anti-oxidative properties and the role of 2,3-
double bond on the antioxidant activity, is environment depen-
dent. The anti-oxidative properties of CA and its derivatives have
been studied by several researchers (Goçer and Gülçin, 2011;
LeBlanc et al., 2012) but few reports have been presented on the
anti-oxidative activity of DHCA (Moon & Terao, 1998). The low sol-
ubility of DHCA prohibits its use in most media. The results of the
DPPH radical scavenging activities of the alkyl dihydrocaffeates
(i.e. methyl-, hexyl-, dodecyl- and octadecyl dihydrocaffeate) are
presented in Table 2. There was a direct proportionality between
concentration and scavenging efficiency. Increase in concentration
of the alkyl esters increased significantly their scavenging activi-
ties. For example, methyl-, hexyl- and dodecyl dihydrocaffeates
showed remarkable scavenging efficiency at concentration of
50 l
g mLꢁ1 (>88% inhibition). However, further increase in their
concentration had little effect on their scavenging efficiency. In
the case of octadecyl dihydrocaffeate, its scavenging efficiency

S. Gholivand et al. / Food Chemistry 224 (2017) 365–371
369
was higher than those of the alkyl groups (Table 2). Structural
modification of the carbonyl group of the DHCA by esterification
was probably responsible for the decrease in the radical scavenging
ability of the alkyl DHCA as compared to DHCA (Moon & Terao,
1998; Sorensen, Nielsen, Yang, Xu, & Jacobsen, 2012). In contrast,
BHT showed remarkably lower scavenging efficiency (only 18.8%
inhibition) at 75 l . In a similar study, Silva, Borges,
g mLꢁ1
Guimaraes, Lima, Matos and Reis (2000), showed that the alkyl
derivatives of dihydrocaffeic acid (methyl-, ethyl-, and propyl)
exhibited better radical scavenging activities than the reference
a-tocopherol. However, in contrast to their findings, increasing
the alkyl chain length in this study showed significant effect on
their radical scavenging activity (Table 2). The scavenging efficien-
cies of alkyl dihydrocaffeates were in the following order: Methyl-
> hexyl- > dodecyl- > octadecyl dihydrocaffeate > BHT. In addition,
the scavenging efficiency of the alkyl esters was significantly
(p < 0.05) affected by the chain length and concentration as well
as their interaction (Table 3). The high scavenging efficiencies of
alkyl dihydrocaffeates relative to BHT may be due to the presence
of catechol moiety in all the studied esters. The catechol moiety is
probably responsible for pairing off the odd electron of DPPH free
radical and thus, decreasing its absorbance.
3.4. Anti-oxidative property of alkyl dihydrocaffeate esters using b-
carotene bleaching method
In addition to the DPPH assay, the anti-oxidative activities of
the alkyl dihydrocaffeates were evaluated using the b-carotene
bleaching (BCB) approach. This method is normally used to evalu-
ate the anti-oxidant activities of compounds in emulsions. As
shown in Table 4, the anti-oxidant activities of n-alkyl esters with
long chain-lengths (dodecyl and octadecyl) were greater than
those obtained by those with shorter chain-lengths (methyl and
hexyl esters). Furthermore, all of the tested esters in the
b-carotene–linoleic acid emulsion system behaved appositively to
the DPPH assay as shown in Table 2. Octadecyl and dodecyl
dihydrocaffeates were found to have higher antioxidant activity
relative to other alkyl esters (methyl- and hexyl esters) probably
due to their high hydrophobicity in the emulsion system. The
antioxidant activity values calculated from the formula given in
Fig. 2. (A) Conversion (%) of dihydrocaffeic acid with hexanol in different ionic
liquids (IL1 (1-butyl-3-methylimidazoliumbis (trifluoromethylsulfonyl) imide), IL2
(1-butyl-3 methylimidazoliumhexafluorosphosphate), IL3 (1-hexyl-3-methylimi-
dazoliumhexafluorophosphate), IL4 (1-octyl-3-methyllidazoliumhexafluolorophos-
phate)): (B) Degree of esterification (%) of alkyl dihydrocaffeate after 3 days in (1-
butyl-3-methylimidazoliumbis (trifluoromethylsulfonyl) imide).
was quite low at 50 l
g mLꢁ1 (>68% inhibition). This might be due
to its longer chain as well as its steric hindrance which interferes
with access to DPPH. The highest scavenging efficiency of 87.9%
inhibition was obtained for octadecyl dihydrocaffeate when its
concentration increased to 75 l
g mLꢁ1. Our study has shown that
experimental part (Kaar, Jesionowski, Berberich, Moulton,
&
the radical scavenging ability of DHCA against free radical DPPH
Russell, 2003) facilitated the comparisons of the relative activity
Table 2
DPPH radical scavenging activity of alkyl dihydrocaffeate esters.
Concentration l
g mLꢁ1 Dihydrocaffeic acid Methyl dihydrocaffeate Hexyl dihydrocaffeate Dodecyldihydrocaffeate Octadecyl dihydrocaffeate BHT
aC
aB
aA
aA
aA
bB
bA
bA
bA
bA
cC
cB
bA
bA
bA
dD
eE
eE
fD
fC
5
33.60 1.55
88.38 8.56
90.12 9.87
91.27 8.95
92.32 9.77
21.760 3.73
86.307 7.24
88.106 9.69
88.355 9.92
88.361 9.23
13.609 1.08
61.008 3.23
85.372 5.52
88.243 8.37
88.633 8.41
8.915 1.27
3.871 0.24
2.048 0.18
5.227 0.08
8.814 0.53
dC
cB
bA
bA
eD
eC
cB
bA
20
30
50
75
45.220 1.51
69.473 1.52
88.112 8.41
88.130 7.63
25.661 0.84
40.537 0.86
68.721 1.23
87.828 9.85
eB
13.236 1.02
cA
18.736 0.6
(Mean SD) along the same row with different superscript are significantly different (p < 0.05).
(Mean SD) along the same column with different superscript are significantly different (p < 0.05).
Table 3
The significance of each independent variable effect shown by F-ratio and P-value.
Response variables
Independent variables
Chain length
Concentration
Interaction
Radical scavenger
P value
F ratio
0.000
216.760
0.000
217.030
0.000
14.430
Antioxidant activity
P value
F ratio
0.000
116.280
0.000
62.750
0.988
0.350

370
S. Gholivand et al. / Food Chemistry 224 (2017) 365–371
Table 4
Total antioxidant activity of alkyl dihydrocaffeate esters using b-carotene bleaching method.
Concentration
mg mLꢁ1
Dihydrocaffeic acid Methyl
dihydrocaffeate
Hexyl
dihydrocaffeate
Dodecyl
dihydrocaffeate
Octadecyl
dihydrocaffeate
BHT
dE
cD
bC
bC
aC
aC
aB
aB
5
10
15
20
25
Slope
9.78 0.56
11.266 1.71
16.552 1.97
19.818 0.31
21.368 1.42
24.246 2.95
3.078
21.897 2.49
22.934 2.73
27.660 1.03
35.468 3.47
38.243 2.64
40.070 3.95
44.723 4.49
3.873
30.003 0.58
36.237 4.04
40.460 2.71
dD
dC
eB
eA
cC
bB,C
a,bB
aB
14.78 0.43
17.89 1.45
20.19 2.14
22.86 1.75
26.543 3.45
28.921 1.90
30.556 3.99
37.938 3.82
3.610
30.697 3.12
32.571 1.82
34.079 1.76
40.233 3.22
3.798
cB,C
dA,B
dA
bB
cB
cA
bB
aB
b,cB
b,cA
a,bA,B
41.865 4.70 a,AB
a,bA
aA
47.583 2.55
4.079
(Mean SD) along the same row with different superscript are significantly different (p < 0.05).
(Mean SD) along the same column with different superscript are significantly different (p < 0.05).
Croitoru, R., Fiţiga˘u, F., Van den Broek, L., Frissen, A., Davidescu, C., Boeriu, C., &
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of different antioxidants. For instance, the slope of the linear
dependency of antioxidant activity against concentration of
methyl-, hexyl-, dodecyl- and octadecyl dihydrocaffeates varied
from 3.078 to 4.079 (Table 3) which indicated that the reaction rate
of donating radical hydrogen from antioxidant with long chain
length to peroxyl radicals was faster than those with shorter ones.
Moreover, these results also proved the dependency of the anti-
oxidant activity on the alkyl chain length. As shown in Table 2,
the chain-length had the most significant (p < 0.05) effect on the
anti-oxidant activity (F-ratio: 116.280). Conversely, the concentra-
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Figueroa-Espinoza, M.-C.,
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_
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This present study has shown that ionic liquids’ (ILs) structure
can considerably affect the conversion yield of esterification reac-
tion. Among ionic liquids tested, 1-butyl-3-methylimidazoliumbis
(trifluoromethylsulfonyl) imide was selected as the best solvent
because it has the highest degree of esterification. The antioxidant
properties of the synthesized alkyl dihydrocaffeate esters namely:
methyldihydrocaffeate, hexyldihydrocaffeate, dodecyldihydrocaf-
feate and octadecyldihydrocaffeate in the best ionic liquid were
assessed using two different anti-oxidant assays; b-carotene
bleaching and DPPH radical scavenging. The results showed that
both concentration and chain-length had significant (p < 0.05)
effects on both anti-oxidant and radical scavenging activities
respectively. In b-carotene approach, the most significant effect
on the anti-oxidant activity was the chain-length. In addition, it
was found that the long chain alkyl dihydrocaffeate esters (i.e.
dodecyl and octadecyl dihydrocaffeates) inhibited oxidation more
than the short-chain ones.
Jakovetic, S. M., Jugovic, B. Z., Gvozdenovic, M. M., Bezbradica, D. I., Antov, M. G.,
ˇ
ˇ
´
´
Mijin, D. Z., & Knezevic-Jugovic, Z. D. (2013). Synthesis of aliphatic esters of
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ˇ ´
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Acknowledgment
Montgomery, D. (2001). Design and analysis of experiments (5th ed.). New York: John
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This study was supported by University Putra Malaysia
Research Grant No: 5524558.
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