Synthesis and evaluation of antioxidant and antifungal activities of novel ricinoleate-based lipoconjugates of phenolic acids

Article abstract of DOI:10.1016/j.foodchem.2012.04.046

Syntheses of four castor oil fatty acid-based novel lipoconjugates of phenolic acids were carried out following Mitsunobu methodology. The lipid part consists of methyl ricinoleate and its saturated analogue, methyl-12- hydroxystearate and the phenolic mo

Full text of DOI:10.1016/j.foodchem.2012.04.046

Food Chemistry 134 (2012) 2201–2207
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Synthesis and evaluation of antioxidant and antifungal activities of novel
ricinoleate-based lipoconjugates of phenolic acids
⇑
Kunduru Konda Reddy, Thumu Ravinder, Sanjit Kanjilal
Centre for Lipid Research, Indian Institute of Chemical Technology, Hyderabad 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Syntheses of four castor oil fatty acid-based novel lipoconjugates of phenolic acids were carried out fol-
lowing Mitsunobu methodology. The lipid part consists of methyl ricinoleate and its saturated analogue,
methyl-12-hydroxystearate and the phenolic moieties are ferulic and vanillic acid. Synthesised com-
pounds are evaluated for antioxidant activity using three in vitro assays (DPPH radical scavenging assay,
DSC studies for oxidative induction temperature of linoleic acid and autoxidation of linoleic acid in Tween
Received 8 December 2011
Received in revised form 2 April 2012
Accepted 7 April 2012
Available online 17 April 2012
20 micellar medium) and compared with three widely used antioxidants in the food industry, BHT,
a-
Keywords:
Antioxidant
Autoxidation of linoleic acid
DSC
DPPH assay
tocopherol, and dodecyl gallate. Synthesised compounds are found to exhibit good antiradical activity.
These compounds also exhibited very good antifungal activity against studied fungal strains. All these
results suggested the applicability of the synthesised compounds as potent lipophilic antioxidants for
combating oxidative stress.
Ó 2012 Elsevier Ltd. All rights reserved.
Lipoconjugates of phenolic acids
Methyl ricinoleate
Methyl 12-hydroxystearate
1. Introduction
Development of natural antioxidants with better antioxidant
capacity and less toxicity is desirable for the prevention of diseases
Antioxidants are essential to maintain quality of human health
as well as quality of food and personal care products. Living organ-
isms are exposed to several environmental oxidative stresses
resulting in the generation of a series of oxidants such as peroxides,
hydroxyl radical, singlet oxygen species and peroxynitrites. These
reactive oxygen species (ROS) are responsible for the oxidative
modification of cell membranes, triggering altered cellular mecha-
nisms (Ames, Shigenaga, & Hagen, 1993; Cerutti, 1994; Hussain,
Hofseth, & Harris, 2003). Although living organisms are equipped
with endogenous antioxidant defence mechanisms, any imbalance
in this defence mechanism, especially during ageing, may lead to
overproduction of oxidants (Halliwell, 2001). It is necessary to
replenish the levels of antioxidants as dietary supplements. Such
exogenous antioxidant replenishment is considered a promising
approach in the prevention and therapy of diseases caused by oxi-
dants. On the other hand, antioxidants are also important in pro-
tecting the nutritive value and increasing the shelf life of
cosmetic and food products (Figueroa-Espinoza & Villeneuve,
2005). Lipids in such products, especially those enriched with poly-
unsaturated fatty acids (PUFAs) are most prone to such oxidative
damage, either by autoxidation or by thermal oxidation. For dec-
ades, natural antioxidants such as tocopherols, vitamin C and
carotenoids have been used as preservatives in the food industry.
and also for improved nutritive value and better shelf life of food
products. Natural phenolic compounds, such as flavonoids,
polyphenols and phenolic acids, are gaining interest as food preser-
vatives. Phenolics are abundant in the plant kingdom and are one of
the most well studied natural antioxidants because of their many
potent biological properties (Figueroa-Espinoza
& Villeneuve,
2005; Kondratyuk & Pezzuto, 2004). Lipophilisation of natural
phenolics can result in novel phenolipids with enhanced antioxi-
dant applications in food/cosmetics and improved bioavailability
and bioefficacy under physiological conditions (Shahidi & Zhong,
2010). Ferulic acid, cinnamic acid, sinapic acid, protocatechuic acid,
caffeic acid, etc., are some of the phenolics which have been lipophi-
lised with a view to enhance their solubility in apolar media
(Buisman et al., 1998; Gaspar et al., 2009, 2010; Guyot et al.,
2000; Jose et al., 2011; Kanjilal et al., 2008; Roleira et al., 2010;
Sabally, Karboune, Yeboah, & Kermasha, 2005; Sabally, Karboune,
St-Louis, & Kermasha, 2006). The result is drastic change in the
solubility characteristics of phenolipid without compromising the
basic core of the molecule responsible for antioxidant activity.
There are many instances where such lipophilisation resulted in
an increase of antioxidant activity, compared to the corresponding
free phenolic compound (Jose et al., 2011; Reis et al., 2010; Roleira
et al., 2010). However, such increase in activity due to lipophilisa-
tion depends on the type of medium of the assays (Reddy, Ravinder,
Prasad, & Kanjilal, 2011). Moreover, the alkyl chain length of phen-
olipid was also found to affect profoundly its antioxidant activity
⇑
Corresponding author. Tel.: +91 40 27193179; fax: +91 40 27193370.
E-mail address: sanjit@iict.res.in (S. Kanjilal).
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.04.046

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K.K. Reddy et al. / Food Chemistry 134 (2012) 2201–2207
and a nonlinear dependency of lipophilicity of phenolipid on its
antioxidant activity in an emulsion system was reported in the lit-
erature (Laguerre et al., 2009, 2010; Reddy et al., 2011). There are
also some reports on synthesis of long chain alkyl cinnamic acid es-
ters, where authors studied not only their antioxidant activity but
also evaluated their drug-likeness profile (Jose et al., 2011).
In the development of newer antioxidants with potential health
benefit for applications in cosmetic and food products, it is neces-
sary to know the physicochemical properties of the molecule, such
as hydrophobicity, affinity for the lipid substrate and ability to an-
chor in the phospholipids bilayer, which governs its antioxidant
activity (Figueroa-Espinoza & Villeneuve, 2005). The present work
mainly focused on the synthesis, characterisation, and evaluation
of four bio-based derivatives designed as lipophilic antioxidants
and antifungal agents (Fig. 1). Castor oil contains a unique fatty
acid, 12-hydroxy-9-octadecenoic acid (or ricinoleic acid), which
is known for its laxative, analgesic and anti-inflammatory effects
(Vieira et al., 2000). Two types of phenolics namely vanillic and
ferulic acids are esterified with methyl ricinoleate as well as with
its saturated analogue, methyl 12-hydroxystearate by the well-
known Mitsunobu protocol (Appendino, Minassi, Daddario, Bian-
chi, & Tron, 2002). Thus the secondary hydroxyl moiety of ricinole-
ate/12-hydroxystearate is being grafted to the phenolic moiety
resulting in a unique class of phenolipid, having a terminal ester
moiety and a pendant hydrophobic chain. This type of phenolipid
has not been synthesised and studied previously.
Mumbai, India) and identified by thin-layer chromatography
(TLC), FT-IR, MS, and NMR analysis. TLC was performed on pre-
coated silica gel 60 F254 from Merck (Darmstadt, Germany). All
1H and 13C NMR spectra were recorded at 300 and 75 MHz (Varian,
Palo Alto, CA), respectively. Mass spectra were recorded either on a
VG Auto Spec-M (Manchester, UK) or on an Agilent 5973 mass
spectrometer (Agilent, Santa Clara, CA) in EI mode. A Lambda-35
UV–Vis spectrophotometer from Perkin Elmer (Waltham, MA)
was used in the scavenging assays and also for the estimation of
conjugated diene during autoxidation of linoleic acid.
A Perkin Elmer DSC 6000 calorimeter was employed for the
thermoxidation stability measurements. The temperature scale
was calibrated using In and Zn, and the enthalpy calibration was
carried out to the heat of fusion of the same standards. The oxida-
tive stability of pure linoleic acid as well as linoleic acid spiked
with antioxidants, measured as oxidative induction temperature
(OIT) were analysed by heating samples at 5 K/min at a constant
oxygen flow of 50 mL/min. OIT is determined from the plot of heat
flow (mW/g) vs. temperature, wherein the exothermic peak indi-
cates the onset of the oxidation process.
2.2. Chemicals
Vanillic acid, ferulic acid and 1,1-diphenyl-2-picryl hydrazine
(DPPH) radical were purchased from Fluka (Buchs, Switzerland).
Methyl 12-hydroxyoctadec-9-enoate or methyl ricinoleate was
prepared from castor oil and purified by column chromatography
in the laboratory to get 99% purity. Methyl 12-hydroxystearate
was obtained from M/s Jayant Agro-Organics Ltd. (Mumbai, India).
The objective of this study is to find the influence of the pres-
ence of unsaturation in the alkyl chain and also in the side chain
of phenolic acid on the antioxidant activity. The antioxidant activ-
ities are studied using three different in vitro assays, namely,
DPPHꢀ scavenging assay in polar medium, thermal assay using
differential scanning calorimetry (DSC) to measure the oxidative
stability of linoleic acid (measured as oxidative induction temper-
ature, OIT), and inhibition of autoxidation of linoleic acid in Tween
20 micellar medium. All the prepared compounds are also evalu-
ated for their antimicrobial activities.
Linoleic acid (P99%),
a-tocopherol, dodecyl gallate and diisopro-
pylazodicarboxylate (DIAD) were purchased from Sigma–Aldrich
(St. Louis, MO). The other chemicals and solvents used were pur-
chased from SD-Fine Chem (Mumbai, India).
2.3. Synthesis
Synthesis of novel lipoconjugates of phenolic acids was carried
out by the Mitsunobu protocol (Appendino et al., 2002). Phenolic
acid (3 mM) and fatty acid methyl ester (3 mM) were taken in
dry tetrahydrofuran (THF, 20 mL) and cooled to 0–4 °C followed
by the addition of triphenylphosphine (3 mM) and diisopropylazo-
dicarboxylate (DIAD) (3 mM). The total reaction mixture was stir-
red at room temperature for 48 h. After 48 h, the organic solvent
2. Materials and methods
2.1. General
The synthesised phenolic lipids were purified by silica gel (60–
120 mesh) column chromatography (Acme Synthetic Chemicals,
O
O
OMe
OMe
HO
HO
O
O
MeO
MeO
O
O
Compound 1
Compound 2
O
O
OMe
OMe
HO
MeO
HO
O
O
MeO
O
O
Compound 3
Compound 4
Fig. 1. Structures of the synthesised lipoconjugates of phenolic acids.

K.K. Reddy et al. / Food Chemistry 134 (2012) 2201–2207
2203
was evaporated at 55 °C using a rotary evaporator and the residue
was solubilised in ethyl acetate. The organic phase was washed
successively with saturated sodium bicarbonate (2 ꢀ 50 mL), brine
(2 ꢀ 50 mL), water (2 ꢀ 50 mL), and finally dried over anhydrous
sodium sulphate. The organic solvent was evaporated at 55 °C
using a rotary evaporator to obtain the crude product, which was
purified by column chromatography using hexane/ethyl acetate
(90/10; v/v) to get the desired compounds. Spectral characteristic
of the four synthesised compounds are given below:
as reference and their concentrations were kept the same as that of
the synthesised phenolic lipids. The free radical-scavenging activ-
ity (FRSA in %) of the tested samples was evaluated by comparison
with a control (2 mL of DPPH radical solution and 1 mL of metha-
nol). Each sample was measured in triplicate and averaged. The
FRSA was calculated using the formula:
FRSA ¼ ½ðAc ꢁ AsÞ=Acꢂ ꢀ 100
where Ac is the absorbance of the control and As is the absorbance of
the tested sample after 40 min.
Methyl-12-(4-hydroxy-3-methoxyphenyl propenoate)octadec-9-
en-1-oate (compound 1): Light yellow oil (yield 58%); 1H NMR
(CDCl3, 200 MHz): d (ppm): 0.87 (t, 3H, J = 7.39 Hz), 1.28 (m,
16H), 1.54–1.69 (m, 4H), 1.97–2.10 (m, 2H), 2.23–2.42 (m, 4H),
3.66 (s, 3H), 3.92 (s, 3H), 4.93–5.08 (m, 1H), 5.30–5.56 (m, 2H),
5.92 (s, 1H), 6.28 (d, 1H, J = 16.42 Hz), 6.91 (d, 1H, J = 8.21 Hz),
7.02–7.13 (m, 2H), 7.60 (d, 1H, J = 16.42 Hz); 13C NMR (CDCl3,
75 MHz): d (ppm): 14.0, 22.6, 24.9, 25.4, 27.3, 29.1, 29.2, 29.5,
31.7, 32.0, 33.7, 34.0, 51.4, 55.9, 73.9, 109.2, 114.7, 115.9, 122.9,
124.2, 127.0, 132.6, 144.5146.7, 148.9, 166.9, 174.3; HRMS calcu-
lated for C29H44O6Na [M+Na]+ 511.3035, found 511.3015; EI MS
(m/z): 488 (M+ꢀ).
Methyl-12-(4-hydroxy-3-methoxybenzoate)octadec-9-en-1-oate
(compound 2): Light yellow oil (yield, 52%); 1H NMR (CDCl3,
300 MHz): d (ppm): 0.79 (t, 3H, J = 6.79 Hz), 1.18 (m, 16H),
1.47–1.64 (m, 4H), 1.91–1.99 (m, 2H), 2.22 (t, 2H, J = 7.74 Hz),
2.29–2.43 (m, 2H), 3.59 (s, 3H), 3.87 (s, 3H), 4.98–5.09 (m, 1H),
5.28–5.44 (m, 2H), 6.04 (br s, 1H), 6.85 (d, 1H, J = 8.31 Hz), 7.48
(s, 1H), 7.56 (d, 1H, J = 8.31 Hz); 13C NMR (CDCl3, 75 MHz): d
(ppm): 14.0, 22.5, 24.9, 25.4, 27.3, 29.0, 29.1, 29.5, 31.7, 32.0,
33.7, 34.0, 51.4, 56.0, 74.4, 111.8, 113.9, 122.9, 123.9, 124.2,
132.6, 146.1, 149.8, 166.0, 174.3; HRMS calculated for C27H42O6Na
[M+Na]+ 485.2879, found 485.2860; EI MS (m/z): 462 (M+ꢀ).
Methyl-12-(4-hydroxy-3-methoxyphenyl propenoate)octadecan-
1-oate (compound 3): Light yellow oil (49.2%); 1H NMR (CDCl3,
300 MHz): d (ppm): 0.88 (t, 3H, J = 7.55 Hz), 1.26 (m, 22H), 1.51–
1.62 (m, 6H), 2.26 (t, 2H, J = 7.55 Hz), 3.63 (s, 3H), 3.92 (s, 3H),
4.91–4.99 (m, 1H), 5.92 (br s, 1H), 6.23 (d, 1H, J = 15.86 Hz), 6.87
(d, 1H, J = 8.31 Hz), 6.99–7.08 (m, 2H), 7.54 (d, 1H, J = 15.86 Hz);
13C NMR (CDCl3, 75 MHz): d (ppm): 13.8, 22.4, 24.7, 25.1, 28.9,
29.0, 29.1, 29.3, 29.5, 31.5, 33.8, 34.1, 51.2, 55.6, 74.1, 109.2,
114.7, 115.6, 122.8, 126.7, 144.4, 146.8, 147.9, 167.0, 174.1. HRMS
calculated for C29H46O6Na [M+Na]+ 513.3192, found 513.3180. EI
MS (m/z): 490 (M+ꢀ).
2.4.2. DSC measurements
The antioxidant activity was also evaluated by differential
scanning calorimeter (DSC), using pure linoleic acid as a lipid model
system (Gaspar et al., 2010; Reis et al., 2010). All the studied antiox-
idants were taken in methanol to prepare a 1 mM solution. Samples
of linoleic acid (2.5–3.0 mg) were placed in standard aluminium
pans and spiked with 10
lL of the antioxidant solution. A blank
run of linoleic acid, spiked with 10
lL of methanol was also carried
out simultaneously to find the oxidative induction temperature
(OIT) of linoleic acid. OIT is determined from the first exothermal
peak of the plot of heat flow (mW/g) vs. temperature. All the mea-
surements for each antioxidant were run in triplicate.
2.4.3. Autoxidation of linoleic acid in Tween 20 micellar system
The rate of inhibition of autoxidation of linoleic acid in micelle
by antioxidant was measured according to the method of Chimi,
Cillard, Cillard, and Rahmani (1991) with some modifications. Lin-
oleic acid (2.5 ꢀ 10ꢁ3 M) was dispersed with 0.5% Tween 20 in
phosphate buffer at pH 6.9 containing 1 mM concentration of ref-
erence antioxidants (a-tocopherol, dodecyl gallate and BHT) or
synthesised phenolic lipids. Samples were left in the dark and in
air for 5 days at 50 °C. Samples without reference and the synthes-
ised antioxidants and the controls without linoleic acid were also
incubated under the same conditions. The autoxidation of linoleic
acid is accompanied by the generation of conjugated diene, which
was measured by UV at 234 nm. Samples were diluted twenty
times with phosphate buffer before measuring the absorbance. A
decrease in the rate of formation of conjugated diene indicates
the increased antioxidant activity of the compound added to the
micelle of linoleic acid.
2.5. Antibacterial activity
Methyl-12-(4-hydroxy-3-methoxybenzoate)octadecan-1-oate
(compound 4): Light yellow oil (50.8%); 1H NMR (CDCl3,
300 MHz): d (ppm): 0.85 (t, 3H, J = 6.79 Hz), 1.24 (m, 22H), 1.51–
1.65 (m, 6H), 2.24 (t, 2H, J = 7.55 Hz), 3.62 (s, 3H), 3.93 (s, 3H),
4.99–5.08 (m, 1H), 6.13 (br s, 1H), 6.86 (d, 1H, J = 8.31 Hz), 7.49
(s, 1H), 7.57 (d, 1H, J = 8.31 Hz); 13C NMR (CDCl3, 75 MHz): d
(ppm): 14.1, 22.6, 24.9, 25.3, 25.6, 29.1, 29.2, 29.4, 29.5, 29.7,
31.7, 31.9, 33.9, 34.3, 37.5, 51.2, 55.8, 74.3, 111.8, 114.1, 122.6,
123.9, 146.1150.0, 165.7, 173.5; HRMS calculated for C27H44O6Na
[M+Na]+ 487.3035, found 487.3025; EI MS (m/z): 464 (M+ꢀ).
The minimum inhibitory concentrations (MIC) of synthesised
phenolic lipocojugates were tested against three representative
Gram-positive organisms, Bacillus subtilis (MTCC 441), Staphylococ-
cus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 2639) and
Gram-negative organisms Escherichia coli (MTCC 443), Pseudomo-
nas aeruginosa (MTCC 741), and Klebsiella pneumoniae (MTCC
618) by broth dilution method recommended by National Commit-
tee for Clinical Laboratory (NCCL, 2000) standards. Standard anti-
bacterial agents like Penicillin and Streptomycin were also
screened under identical conditions for comparison.
2.4. Antioxidant activity assays
2.6. Antifungal activity
2.4.1. DPPH radical scavenging assay
The antioxidant activity was determined by the radical-scav-
enging ability using the stable radical DPPH (Akowuah, Zhari, Nor-
hayati, & Mariam, 2006). Briefly, 200 lL of methanolic solution of
the synthesised phenolic lipoconjugates (0.5, 1 and 2 mM) were
added to 2 mL of methanolic solution of DPPH radical (0.1 mM)
and total volume was made up to 3 mL with methanol. After
40 min incubation at 30 °C in the dark, the absorbance of the mix-
ture was measured at 517 nm against methanol as blank. Ferulic
In vitro antifungal activity of the synthesised phenolic lipocoju-
gates was studied against selected fungal strains namely, Candida
albicans (MTCC 227), Candida rugosa (NCIM 3462), Saccharomyces
cerevisiae (MTCC 36), Rhizopus oryzae (MTCC 262), Aspergillus niger
(MTCC 282) by agar well diffusion method. The ready-made potato
dextrose agar (PDA) medium (Hi-media, 39 g) was suspended in
distilled water (1000 mL) and heated to boiling until it dissolved
completely. The medium and petri dishes were autoclaved at a
pressure of 15 psi (Linday, 1962) for 20 min. The medium was
acid, vanillic acid, a-tocopherol, dodecyl gallate and BHT were used

2204
K.K. Reddy et al. / Food Chemistry 134 (2012) 2201–2207
O
O
O
OMe
OMe
MeO
HO
OH
OH
HO
O
O
MeO
OMe
Ferulic acid
Dry THF, DIAD, TPP
0 oC to RT, 48 h
O
+
Or
O
Compound 1
or
MeO
HO
OH
Methyl ricinoleate
HO
O
Vanillic acid
MeO
O
Compound 2
O
O
OMe
OMe
MeO
HO
OH
OH
HO
O
O
MeO
OMe
Ferulic acid
Dry THF, DIAD, TPP
0 oC to RT, 48 h
O
+
Or
O
Compound 3
O
or
MeO
HO
OH
Methyl-12-hydroxy stearate
HO
O
Vanillic acid
MeO
O
Compound 4
Scheme 1. Synthetic methodology used for the preparation of castor fatty acid-based lipoconjugates of ferulic and vanillic acids.
Table 1
DPPH radical scavenging activity of the synthesised phenolic lipids.
Table 2
Oxidative induction temperature (OIT) obtained for the
pure and inhibited linoleic acid (LA).
Compounda
Free radical scavenging activity (FRSA)b (%)
0.5 mM
1.0 mM
2.0 mM
Compounda
OITb (°C)
1
2
3
4
FA
VA
ATP
BHT
DDG
60.49 0.67
35.55 1.29
63.35 0.63
33.17 1.40
76.02 1.53
45.31 0.54
59.38 0.56
70.68 0.50
75.59 0.91
80.30 0.68
35.00 0.43
81.34 0.52
34.68 0.48
91.22 1.27
50.19 0.67
78.06 0.64
85.93 0.65
92.4 1.32
88.45 0.54
36.75 0.81
89.44 0.70
37.41 1.46
96.13 0.83
53.45 1.07
84.95 1.44
88.51 1.33
95.78 0.61
LA
LA + 1
LA + 2
LA + 3
112.1 1.27
162.8 2.05⁄,#
157.6 1.47#
164.5 2.54⁄
157.2 0.97#,⁄
LA + 4
⁄
LA + FA
LA + VA
LA + ATP
LA + BHT
LA + DDG
147.8 2.86
154.1 1.54#
156.2 3.72
162.1 2.79
160.1 1.07
a
1: Methyl-12-(4-hydroxy-3-methoxyphenyl propenoate)octadec-9-en-1-oate;
2: Methyl -12-(4-hydroxy-3-methoxybenzoate)octadec-9-en-1-oate; 3: Methyl-12-
(4-hydroxy-3-methoxyphenyl propenoate)octadecan-1-oate; 4: Methyl-12-(4-
hydroxy-3-methoxy benzoate)octadecan-1-oate; FA: ferulic acid; VA: vanillic acid;
a
See Table 1 for abbreviation.
Values are mean SD (n = 3), OIT of 1–4 is signifi-
b
cantly higher than their corresponding free phenolic as
shown by ⁄, p < 0.001 and #, p < 0.02. OIT of 1 is sig-
nificantly higher than 2, as shown by #, p < 0.02 and
that of 2 vs. 4, as shown by ⁄, p < 0.001.
ATP: a-tocopherol; BHT: butylated hydroxyl toluene; DDG, dodecyl gallate.
b
Values are mean SD (n = 3).
poured into sterile petri dishes under aseptic conditions in a lam-
inar air flow chamber. When the medium in the plates solidified,
0.5 mL of (week old) culture of test organism were inoculated
and uniformly spread over the agar surface with a sterile L-shaped
rod. Solutions were prepared by dissolving the compound in DMSO
and different concentrations were made. After inoculation, wells
were scooped out with a 6-mm sterile cork borer and the lids of
the dishes were replaced. To each well different concentrations
of test solutions were added. Controls were maintained. The trea-
ted samples and the controls were kept at 27 °C for 48 h. Inhibition
zones were measured and the diameter was calculated in millime-
tres. Three replicates were maintained for each treatment.

K.K. Reddy et al. / Food Chemistry 134 (2012) 2201–2207
2205
2.7. Statistical analysis
3.3. DSC measurements
All the assay results reported in the present work are the mean
of three measurements (presented as mean SD) and were ana-
lysed by a paired Student’s t-test to evaluate the level of statistical
significance. A p-value less than 0.05 was considered significant.
Oxidation of lipid, especially those rich in polyunsaturated fatty
acids affects biological systems and is an age-old problem for the
food industry. Lipid oxidation reduces not only the nutritive value
of the food products but also affects the sensory properties and
shelf life (Figueroa-Espinoza & Villeneuve, 2005). Such oxidative
modification of lipids requires energy and can be measured by
thermal analysis. DSC is one of the most versatile and suitable ther-
mal analytical methods. Using DSC, it is possible to continuously
monitor the total thermal effect of lipid oxidation in the microcal-
orimeter vessel. In the present work, pure linoleic acid was taken
as a model lipid system to study the antioxidant activity of the syn-
thesised phenolic lipoconjugates. Autoxidation of linoleic acid is an
exothermic process and is indicated by the exothermal peak
appearing in the DSC curve, the plot of heat flow vs. temperature
(Gaspar et al., 2010; Reis et al., 2010). This peak corresponds to
the onset of non-isothermal oxidation of linoleic acid and is mea-
sured as oxidative induction temperature (OIT). Thus, antioxidant
activity of the synthesised phenolic lipoconjugates was determined
3. Results and discussion
3.1. Synthesis of novel castor oil based lipoconjugates of phenolic acids
The present work describes the synthesis of four novel lipocon-
jugates of ferulic and vanillic acids. The novelty is due to the type
of fatty acids chosen for lipophilisation, methyl-12-hydroxyocta-
dec-9-enoate (methyl ricinoleate) and methyl 12-hydroxyoctade-
canoate (methyl 12-hydroxystearate). Methyl ricinoleate is the
predominant fatty acid present in castor oil and the latter is its sat-
urated analogue. The secondary hydroxyl moiety of these fatty
acids is esterified with the phenolic acid by the well-known Mits-
unobu esterification methodology (Appendino et al., 2002). The
method is simple and chemoselective. Phenolic acids, namely feru-
lic and vanillic acids were directly esterified with methyl ricinole-
ate and methyl-12-hydroxystearate in THF in the presence of
coupling agent DIAD and triphenyl phosphine to get the corre-
sponding novel phenolic lipoconjugate (Scheme 1); isolated yields
are in the range of 49–58%. All these compounds were character-
ised by NMR and MS. The difference in structure of the synthesised
compounds from the other reported phenolic lipids is their connec-
tivity through the secondary hydroxyl moiety positioned at C-12,
resulting in a pendant alkyl chain and a terminal ester moiety.
A
20
Linoleic acid
15
10
5
3.2. DPPH radical-scavenging activity
Free radical scavenging is one of the well-known mechanisms
through which antioxidants inhibits lipid oxidation. The antioxi-
dant activity of all the synthesised compounds was determined
by the radical scavenging ability, using the stable DPPH radical
method. This is a standard assay of measuring antioxidant activity
and is quite simple and rapid for screening specific compounds.
The advantage of this assay is that the DPPH radical is commer-
cially available and does not need to be generated before assay like
in other assays. The assay was conducted at three different concen-
trations of antioxidants (0.5, 1 and 2 mM) in a polar homogeneous
medium. The DPPH radical-scavenging capacity of the synthesised
compounds is shown in Table 1 along with that of dietary phenolic
acids, namely ferulic (FA) and vanillic acid (VA), and three refer-
0
0
20
40
60
80
100
120
Time (h)
B
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3
1
4
2
BHT
ATP
DDG
FA
VA
ence antioxidants, namely a-tocopherol (ATP), butylated hydroxyl
toluene (BHT) and dodecyl gallate (DDG). Results indicate de-
creased radical-scavenging activity of lipophilic phenolic com-
pounds compared to the corresponding free phenolic acid.
Decrease is significant in the case of compounds 2 and 4 compared
to compounds 1 and 3 where marginalisation of scavenging activ-
ity in comparison to corresponding free phenolic acid was ob-
served with the increased concentration of antioxidants. This
decrease in radical-scavenging activity due to lipophilisation com-
pared to free phenolic acid may be attributed to the increased
hydrophobicity of the synthesised phenolipid. Similar views were
expressed in recent articles by Laguerre et al. (2010) and Lecomte,
Giraldo, Laguerre, Barea, and Villenueve (2010). Another interest-
ing observation is the better radical-scavenging activity of lipocon-
jugated compounds prepared from ferulic acid compared to those
prepared from vanillic acid. This is true even for the free phenolic
form. This clearly demonstrates the importance of the allylic dou-
ble bond of ferulic acid in stabilising the phenoxy radical (Kanjilal
et al., 2008).
0
20
40
60
80
100
120
Time(h)
Fig. 2. Spectrophotometric determination of conjugated diene during the 120 h
autoxidation of linoleic acid (2.5 ꢀ 10ꢁ3 M) micelles with the reference antioxidant
and the synthesised phenolic lipids (1 mM): (A) linoleic acid and (B) linoleic
acid + 1, d-d; linoleic acid + 2, .-.; linoleic acid + 3, j-j; linoleic acid + 4, N-N;
linoleic acid + BHT, J-J; linoleic acid + ATP, 4-4; linoleic acid + DDG, I-I; linoleic
acid + FA, h-h; linoleic acid + VA, s-. Compounds 1–4, significantly different from
the corresponding free phenolic acids, p < 0.001 (see Table 1 for abbreviations).

2206
K.K. Reddy et al. / Food Chemistry 134 (2012) 2201–2207
Table 3
Antifungal activity of the synthesised lipoconjugates of ferulic and vanillic acids.
Compounda
Zone of inhibition (mm)
C. albicans
C. rugosa
S. cerevisiae
R. oryzae
A. nizer
100
l
g
150
lg
100
lg
150
l
g
100
lg
150
lg
100
lg
150
lg
100
l
g
150 lg
1
2
3
4
14
0
9
6
23.5
19
0
12
9
13
6
13
10
22
18
9
19
14
12
10
9
6
22
16
14
14
9
7
0
7
0
10
0
10
0
11
10
12
7
16
14
18
10
Amphotericin B (50
lg)
24
25
a See Table 1 for abbreviation.
by measuring the OIT of linoleic acid spiked with free phenolic
acids, their corresponding synthesised four lipoconjugates and also
three reference antioxidants. Identical DSC curves are obtained for
the oxidation of linoleic acid inhibited by these natural and syn-
thetic antioxidants except for the kinetics of linoleic acid oxidation.
The results for the oxidative stability of linoleic acid with and with-
out antioxidant, measured as OIT, are shown in Table 2. The OIT of
linoleic was found to be 112.1 °C. All the synthesised phenolic
lipoconjugates exhibited higher OIT than the control linoleic acid,
indicating their ability to penetrate inside the lipid matrix and
providing protection to linoleic acid. In fact, activities of all the
phenolic lipoconjugates are found to be equal to or marginally
better than reference antioxidants. However, ferulic-acid-based
lipoconjugates showed significantly higher OIT than vanillic-acid-
based lipoconjugates (p < 0.02 in the case of compound 1 vs. 2
and p < 0.001 in the case of compound 3 vs. 4). This extra stability
is due to the presence of the allylic bond in ferulic acid. The order
of reactivity of lipoconjugates is 3 > 1 > 2 = 4. The presence of
unsaturation in the hydrophobic part of the ferulic-based lipocon-
jugate was found to decrease its activity (compound 1) compared
to its saturated analogue (compound 3). However, no such effect
was observed in case of the vanillic-based lipoconjugates (com-
pounds 2 vs. 4). The activity of lipoconjugates is also found to be
enhanced significantly due to lipophilisation when compared to
the corresponding native phenolic acids (p < 0.001 in the case of
compound 1 or 3 vs. FA and p < 0.02 in the case of compound 2
or 4 vs. VA).
of conjugated diene formation by the lipoconjugates of the pheno-
lics is found to be significantly more (p < 0.001) than ferulic and
vanillic acids. This indicates appropriate orientation of the refer-
ence as well as synthesised antioxidant at the micellar interface
compared to free phenolic acids. The order of inhibitions of autox-
idation of linoleic acid by the lipoconjugates of phenolic acids
(compounds 1–4) is found to be the same as that observed in the
DSC study and are similar in value to DDG, the well-known food
grade antioxidant. In general, presence of unsaturation at the
hydrophobic part of a surfactant makes it a worse candidate for
mixed micelle formation with Tween 20, compared to its saturated
counterpart. This is somewhat evident in the case of ferulic-based
lipoconjugates (1 vs. 3), but not significantly so. No such differ-
ences were observed in the case of vanillic-based lipoconjugates
(2 vs. 4). This indicates the ease of mixed micelle formation of
these synthesised phenolipids with Tween 20, irrespective of the
presence of pendant hydrophobic chain and unsaturation at the
hydrophobic part. Thus all the studied antioxidants are appropri-
ately orientated at the micellar interface compared to free phenolic
acids. After 120 h of incubation, the order of inhibitions of autoxi-
dation of linoleic acid by all the studied antioxidants is
BHT > ATP > DDG = 3 > 1 > 2 = 4 > FA > VA.
3.5. Antifungal activity
Fatty acids and their derivatives may increase the antimicrobial
activity of certain organic molecules (Rauf, Gangal, & Sharma,
2008). But none of the synthesised phenolic lipoconjugates showed
any activity against the studied bacteria (data not shown). How-
ever, compounds 1–4 exhibited moderate to good antifungal activ-
ities against the tested fungal strains. There were some exceptions,
e.g., compound 2 did not show any activity against C. albicans and
R. oryzae (Table 3). Compounds 1 and 3 showed better antifungal
activity against all fungal strains compared to compounds 2 and
4. The reason may be the side-chain double bond present in com-
pounds 1 and 3.
3.4. Autoxidation of linoleic acid in Tween 20 micellar system
Antioxidants are widely employed in cosmetic and food prod-
ucts to inhibit lipid peroxidation, which otherwise renders the
product rancid and decreases the shelf life and nutritional quality
of the product. Most of these products exist in a formulated com-
plex emulsion system. The efficacy of an antioxidant in such a ma-
trix depends not only on its chemical reactivity as radical
scavenger but also on the orientation of its radical-scavenging nu-
cleus, interaction with other food components, and environmental
conditions (Jose et al., 2011). The assay model taken up in the pres-
ent work is the rate of autoxidation of linoleic acid in Tween 20 mi-
celle, conducted in the presence of underivatised phenolic
compounds and their synthesised lipoconjugates. The effective
association of phenolipid with Tween 20 in a mixed micellar form
is an important aspect of this assay to inhibit lipid oxidation more
efficiently (Laguerre et al., 2009). Initially the auto-oxidation of
micelles of linoleic acid is accompanied by a rapid increase of
conjugated diene, which is measured at 234 nm. In the control
sample, the formation of conjugated diene was found to reach a
maximum in 36 h (Fig. 2). The autoxidation of linoleic acid was
found to be markedly inhibited (p < 0.001), due to the addition of
reference antioxidants as well as synthesised lipoconjugates of
phenolic acids. This is evident from the negligible formation of
conjugated dienes at 36 h. After 120 h of incubation, the inhibition
4. Conclusions
In summary, four novel lipoconjugates of dietary phenolic acids
were prepared, where the lipid part is derived from castor oil. The
lipid part, ricinoleic acid methyl ester and 12-hydroxystearic acid
methyl ester is connected to the phenolic moiety, namely ferulic
acid and vanillic acids by chemoselective Mitsunobu esterification.
The antioxidant activity of the four novel phenolic esters was stud-
ied using DPPH, DSC and autoxidation of linoleic acid in micellar
medium. The prepared compounds have shown good antioxidant
activity as well as interesting antifungal properties. These novel
dietary phenolic lipoconjugates have the advantage of being syn-
thesised from castor oil derivatives, while providing a value-added
use of this oil. In conclusion, all these four novel lipoconjugates
could be used as potential antioxidants for the storage of lipids
and also to combat oxidative stresses.

K.K. Reddy et al. / Food Chemistry 134 (2012) 2201–2207
2207
Jose, C. J. M. D. S. M., Shrivallabh, P. K., Jose, A. S. C., Alexandra, G., Jorge,
G., Fernanda, B. (2011). Synthesis and antioxidant activity of long
Acknowledgements
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K.K.R. thanks the Council of Scientific and Industrial Research
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
04.046.
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