Evaluation of the antioxidant activity of capsiate analogues in polar, nonpolar, and micellar media

Article abstract of DOI:10.1021/jf104244m

Synthesis of 10 capsiate analogues was conducted by lipase-mediated (Novozyme 435) esterification of vanillyl alcohol with different fatty acids. The antioxidant activity of the synthesized capsiates was evaluated using three in vitro assays: DPPH radical

Full text of DOI:10.1021/jf104244m

564 J. Agric. Food Chem. 2011, 59, 564–569
DOI:10.1021/jf104244m
Evaluation of the Antioxidant Activity of Capsiate Analogues in
Polar, Nonpolar, and Micellar Media
K
UNDURU K. REDDY, THUMU
R
AVINDER, RACHAPUDI B. N. PRASAD
ANJILAL
,
AND
S
ANJIT
K
*
Centre for Lipid Research, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Synthesis of 10 capsiate analogues was conducted by lipase-mediated (Novozyme 435) esterification of
vanillyl alcohol with different fatty acids. The antioxidant activity of the synthesized capsiates was
evaluated using three in vitro assays: DPPH radical scavenging assay (polar medium), Rancimat assay
(nonpolar medium), and autoxidation of linoleic acid (micellar medium). The objective of this study is to
find the influence of structural characteristics of the alkyl chain of capsiate analogues on their antioxidant
activity. In these assays, BHT and R-tocopherol were used as reference compounds. Both DPPH and
Rancimat assays did not show any specific trend of antioxidant activity with the increase in lipophilicity
and also with the type of fatty acids grafted to the phenolic moiety. In the Tween 20 micellar system for
the inhibition of autoxidation of linoleic acid, vanillyl ester attached to a C18 alkyl chain (vanillyl stearate,
oleate, and ricinoleate) exhibited maximum inhibition of autoxidation of linoleic acid.
KEYWORDS: Antioxidant; autoxidation of linoleic acid; capsaiciate; capsiate; Rancimat assay; DPPH
assay
INTRODUCTION
flavor of peppers (7, 8). These are reported to possess many
potent bioactivities, such as anti-inflammatory, antimicrobial,
antimutagenic, and antitumor properties (9, 10). They are also
used as topical analgesics for treating pain and for the enhance-
ment of thermogenesis and fat consumption in mammals (8, 11).
Even though capsaicinoids show interesting antioxidant proper-
ties, pungency and obnoxious properties limit their application as
food-grade antioxidants. On the other hand, capsinoids are
nonpungent ester analogues of capsaicinoids and are known to
share many potent biological activities of capsaicinoids. Notable
among them are suppression of fat accumulation, induction of
apoptosis, and anticancer and antioxidant properties (6, 8). Be-
cause of their nonpungency, capsinoids are more palatable than
capsaicinoids and may find application in the food and beverage
industry as useful ingredients. Capsinoids are structurally similar
to capsaicinoids, the only difference being an ester bond connect-
ing the hydrophobic chain to the phenol moiety instead of an
amide bond as in capsaicinoids. Capsinoids have been isolated
from the fruit of sweet pepper cultivar, CH-19 Sweet (12). The
most naturally abundant capsinoid is capsiate (4-hydroxy-3-
methoxybenzyl (E)-8-methyl-6-nonenoate). Other capsinoids
such as dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methyl-
6-nonanoate) and nordihydrocapsiate (4-hydroxy-3-methoxy-
benzyl 7-methyloctanoate) have also been isolated (13).
Antioxidants are classified as free radical scavengers and are
important in human health. Due to exposure to radiation and
several other oxidative stresses, reactive oxygen species (ROS) are
generated in living organisms. ROS are responsible for the oxida-
tive modification of cell membranes, triggering altered cellular
mechanisms (1-3). Fatty acids, especially polyunsaturated fatty
acids (PUFAs), are most susceptible to attack by ROS. Moreover,
due to the prevalence of PUFAs in the neuronal system, ROS are
particularly active in the brain and neuronal tissue, causing many
neurodegenerative diseases (4). Although the endogenous antiox-
idant defense mechanism in living organisms prevents such oxida-
tive damages, any imbalance in this defense mechanism may lead to
overproduction of oxidants. Under such circumstances, exogenous
antioxidants such as dietary supplements are considered to be a
promising approach in the prevention and treatment of diseases
caused by oxidants. On the other hand, antioxidants are also
important for cosmetic and food products. Lipid oxidation not
only reduces the nutritive value of the food products but also affects
the sensory properties and shelf life (5). For decades antioxidants
have been used as food preservatives to control deterioration in the
form of oxidation. Some of the well-known natural antioxidants
used by the food industry are R-tocopherols, vitamin C, and
carotenoids. Natural phenolic compounds such as flavonoids,
polyphenols, and phenolic acids have also recently gained interest
as food preservatives (6).
The limited availability of natural capsinoids and their com-
plicated and costly chemical synthesis have restricted their
applications in the food and cosmetic industries. This has
necessitated the synthesis of their linear analogues. There are
few reports on the synthesis of capsiates and their analogues
following chemical as well as enzymatic approaches (10, 14, 15).
In the present work, the synthesis of a series of capsiate analogues
was conducted by lipase-mediated (Novozyme 435) esterification
Capsaicinoids and capsinoids are two families of naturally
occurring lipophilic alkaloids derived from peppers (Capsicum
annuum L.). Capsaicinoids are responsible for the pungent spicy
*Corresponding author (phone þ91 40 27193179; fax þ91 40
27193370; e-mail sanjit@iict.res.in).
pubs.acs.org/JAFC
Published on Web 12/17/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 59, No. 2, 2011 565
of vanillyl alcohol with different fatty acids. It is well-known that
the polarity of the environment strongly governs the antioxidant
activity of phenolic antioxidants (16). Accordingly, the antiox-
idant activity of all the synthesized capsiate analogues was
assayed in three different media, namely, polar, nonpolar, and
micellar media. These assays are the diphenylpicrylhydrazine
(DPPH) radical scavenging assay (polar medium), the Rancimat
assay (nonpolar medium), and autoxidation of linoleic acid
(micellar medium). The nature and type of alkyl chain grafted
to the phenolic moiety are often used as tools to change the
physical properties of the antioxidants. The objective of this study
is to find the influence of change in structural feature of alkyl
chain of capsiate analogues, especially variation in chain length,
and also inclusion of a functional group in the hydrophobic chain
on their antioxidant activity in these three in vitro assays.
66.1 (-OCH2Ph), 55.6 (-OCH3), 34.1, 33.5, 29.0, 28.9, 28.8, 28.6, 24.7
(alkyl CH2); HRMS calcd for C19H28O4Na [M þ Na]þ, 343.1885; found,
343.1895.
4-Hydroxy-3-methoxybenzyl behenate (h): 1H NMR (CDCl3, 300 MHz),
δ 0.88 (t, 3H, -CH2-CH3, J = 6.798 Hz), 1.25 (m, 36H, alkyl -CH2),
1.61(m, 2H, -CO;CH2;CH2) 2.29 (t, 2H, -CO;CH2-, J =
7.554 Hz), 3.90 (s, 3H, -OCH3), 4.99 (s, 2H, -OCH2Ph), 5.52 (s, 1H,
Ph;OH), 6.80-6.85 (m, 3H, aromatic); 13C NMR (CDCl3, 75 MHz), δ
173.7 (-CO-), 146.4, 145.7, 127.9, 121.9, 114.3, 111.2 (aromatic), 66.2
(-OCH2Ph), 55.8 (-OCH3), 34.3, 31.8, 29.7, 29.5, 29.4, 29.2, 29.0, 27.1,
24.9, 22.6 (alkyl -CH2-), 14.0 (-CH2;CH3); HRMS calcd for C30H52
-
O4Na [M þ Na]þ, 459.3763; found, 459.3767.
4-Hydroxy-3-methoxybenzyl erucate (i): 1H NMR (CDCl3, 300 MHz),
δ 0.89 (t, 3H, -CH2-CH3, J = 6.798 Hz), 1.26 (m, 32H, alkyl -CH2), 1.61
(m, 2H, -CO;CH2;CH2), 2.0 (m, 4H, -CH2;CHdCH;CH2-), 2.30
(t, 2H, -CO;CH2-, J = 7.554 Hz), 3.91 (s, 3H, -OCH3), 4.98 (s,
2H, -OCH2Ph), 5.29 (m, 2H, -CHdCH-), 5.50 (s, 1H, Ph;OH), 6.79-
6.88 (m, 3H, aromatic); 13C NMR (CDCl3, 75 MHz), δ 173.7 (-CO-),
146.4, 145.7, 127.9, 121.9, 114.3, 111.2 (aromatic), 129.8 (-CHdCH-), 66.2
(-OCH2Ph), 55.8 (-OCH3), 34.3, 31.8, 29.7, 29.5, 29.4, 29.2, 29.0, 27.1, 24.9,
22.6 (alkyl -CH2-), 14.0 (-CH2;CH3); HRMS calcd for C30H50O4Na
[M þ Na]þ, 497.3606; found, 497.3614.
MATERIALS AND METHODS
General Experimental Procedures. The synthesized capsiate ana-
logues were purified by silica gel (60-120 mesh) column chromatography
(Acme Synthetic Chemicals, Mumbai, India) and identified by thin-layer
chromatography (TLC), FT-IR, MS, and NMR analyses. TLC was
performed on precoated silica gel 60 F254 from Merck (Darmstadt,
Germany). All 1H and 13C NMR spectra were recorded on 300 and
75 MHz (Varian, Palo Alto, CA) spectrometers, respectively. Mass spectra
were recorded either on a VG Auto Spec-M (Manchester, U.K.) or on an
Agilent 5973 mass spectrometer (Palo Alto, CA) in the EI mode and are
given in mass units (m/z). A λ-35 UV-vis spectrophotometer from Perkin-
Elmer (Shelton, CT) was used in the scavenging assays and also for the
estimation of conjugated diene during autoxidation of linoleic acid. The
oxidative stability of soybean oil was measured in a Metrohm A.G. (model
743) Rancimat apparatus (Herisau, Switzerland).
4-Hydroxy-3-methoxybenzyl ricinoleate (j):1H NMR (CDCl3, 300 MHz),
δ 0.89 (t, 3H, -CH2;CH3, J = 6.987 Hz), 1.28-1.45 (m, 18H,
alkyl -CH2), 1.60 (m, 2H, -CO;CH2;CH2), 1.97-2.07 (m, 2H, CH2;
CH2;CHdCH), 2.17 (t, 2H, CHdCH;CH2, J = 6.610 Hz), 2.29 (t,
2H, -CO;CH2, J = 7.365 Hz), 3.56 (m, 1H, (-CH2(HO)CH;CH2-),
3.89 (s, 3H, -OCH3), 4.98 (s, 2H, -OCH2Ph), 5.22-5.41 (m, 1H, CHd
CH-), 5.43-5.58 (m, 1H, -CHdCH-), 6.79-6.86 (m, 3H, aromatic); 13
C
NMR (CDCl3, 75 MHz), δ 173.7 (-CO-), 146.4, 145.7, 127.9, 125.1, 121.9,
114.3, 111.2 (aromatic), 133.3 (-CHdCH-), 71.5 (-CH2(HO)CH;
CH2-), 66.2 (-OCH2Ph), 55.8 (-OCH3), 36.8, 35.3, 34.3, 31.8, 29.5,
29.3, 29.0, 27.3, 25.6, 24.9, 22.5 (alkyl;CH2-), 14.0 (-CH2;CH3);HRMS
calcd for C26H42O5Na [M þ Na]þ, 457.2929; found, 457.2949.
Chemicals. Vanillyl alcohol, DPPH radical, and fatty acids, such as
octanoic, undecanoic, 10-undecenoic, dodecanoic, hexadecanoic, octade-
canoic, and octadec-9-enoic acids, were purchased from Fluka (Buchs,
Switzerland). Erucic and behenic acids were purchased from M/s VVF
Limited (Mumbai, India). Ricinoleic acid was isolated from castor oil and
purified by column chromatography in the laboratory. Linoleic acid (g99%)
and R-tocopherol were purchased from Sigma-Aldrich (St. Louis, MO).
Immobilized lipase from Candida antarctica (Novozym 435) was purchased
from Novozymes A/S (Bagsvaerd, Denmark). Refined soybean oil, pur-
chased from the local market, has the following fatty acid composition
(in wt %), as determined by GC: palmitic acid, 11.3%; stearic acid, 5.2%;
oleic acid, 22.4%; linoleic acid, 52.6%; linolenic acid, 6.1%; arachic acid,
0.9%; behenic acid, 1.2%; and lignoceric acid, 0.3%. The rest of the chemicals
and solvents were purchased from SD-Fine Chem (Mumbai, India).
General Procedure for the Synthesis of Capsiate Analogues.
Syntheses of capsiate analogues were carried out following the method
reported by Kobata et al. (15) with little modification. Briefly, equimolar
concentrations of vanillyl alcohol and fatty acid were solubilized in tert-
butanol followed by the addition of Novozyme 435 (5% by wt of total
substrates). The reaction mixture was stirred at 55 °C, and the progress of the
reaction was monitored by TLC (eluant/30% ethyl acetate in hexane). After
maximum conversion (4 h), the reaction mixture was filtered to separate the
lipase, and the lipase was washed with ethyl acetate. The filtrate was washed
with saturated sodium bicarbonate and water. The organic phase was dried
over anhydrous sodium sulfate and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography using
hexane/ethyl acetate (96:4 v/v) to elute capsiate analogues. Isolated yields of
these synthesized compounds are in the range of 55-72%. Structural con-
firmation of all the synthesized compounds were conducted by NMR, IR, and
mass spectroscopy and matched well with those reported in the litera-
ture (10, 15, 17). The spectral data of four new analogues are given below.
4-Hydroxy-3-methoxybenzyl undecenoate (c): 1H NMR (CDCl3, 300
MHz), δ 1.28 (m, 10H, alkyl -CH2), 1.61 (m, 2H, -CO;CH2;CH2),
2.02 (m, 2H, CH2;CHdCH2), 2.29 (t, 2H, J = 7.554 Hz), 3.91 (s,
3H, -OCH3), 4.88-5.01 (dd, 2H, CHdCH2 and s, 2H, -OCH2Ph), 5.50
(s, 1H, Ph;OH), 5.67-5.83 (m, 1H), 6.80-6.85 (m, 3H, aromatic);
13C NMR (CDCl3, 75 MHz), δ 173.7 (-CO-), 146.4, 145.6, 127.6,
121.7, 114.3, 111.1 (aromatic), 138.8 (CHdCH2), 113.9 (CHdCH2),
Antioxidant Activity Assays. DPPH Radical Scavenging Assay.
The antioxidant activity was determined by the radical scavenging ability
using the stable DPPH radical (18). Briefly, 200 μL of methanolic solution of
the synthesized capsiate analogues (1 mM) was added to 2 mL of methanolic
solution of DPPH radical (0.1 mM), and the total volume was made up to
3 mL with methanol. After 40 min of incubation at 30 °C in the dark, the
absorbance of the mixture was measured at 517 nm against methanol as
blank. R-Tocopherol and BHT were used as positive controls, and their
concentrations were kept the same as that of the synthesized analogues. The
free radical scavenging activity (FRSA in %) of the tested samples was
evaluated by comparison with a control (2 mL of DPPH radical solution and
1 mL of methanol). Each sample was measured in triplicate and averaged.
The FRSA was calculated using the following formula: FRSA = [(Ac - As)/
Ac] ꢀ 100, where Ac is the absorbance of the control and As is the absorbance
of the tested sample after 40 min.
Rancimat Assay. The antioxidant activity of the synthesized capsiate
derivatives in a food matrix was also performed with soybean oil by the well-
established Rancimat method (19). The Rancimat apparatus was operated at
110 °C with a dry air flow of 20 L/h, passed through the oil sample (5 (
0.001 g) containing a 1 mM concentration of the reference antioxidants (BHT
and R-tocopherol) or synthesized capsiate analogues. All tests were per-
formed in triplicate using soybean oil of one batch to avoid batch-to-batch
variation. The volatile oxidation products generated during the oxidation of
the oil caused an increase in the electrical conductivity of the water. The time
until there was a sharp increase of conductivity value, corresponding to the
inflection point of the curve, was considered to be the induction time,
expressed in hours. An increase in the induction time indicates increased
antioxidant potency of the compound added to the soybean oil.
Autoxidation of Linoleic Acid. The rate of inhibition of autoxidation of
linoleic acid in micelle by antioxidant was measured according to the method
of Chimi et al. (20), with some modifications. Initially, phosphate buffer (pH
6.9) containing 0.5% Tween 20 was prepared. Linoleic acid (2.5 ꢀ 10-3 M)
was dispersed in the above buffer along with 1 mM concentrations of
reference antioxidants (BHT and R-tocopherol) or synthesized capsiate
analogues. Samples were left in the dark and in air for 5 days at 50 °C.
Samples containing linoleic acid but without antioxidants (reference and

566 J. Agric. Food Chem., Vol. 59, No. 2, 2011
Reddy et al.
Table 1. Synthesized Capsiate Analogues and Their Corresponding Isolated Yields
capsiate analogue
R
isolated yield (%)
a
b
c
d
e
f
-(CH2)6CH3
-(CH2)9CH3
-(CH2)8;CHdCH2
-(CH2)10CH3
-(CH2)14CH3
-(CH2)16CH3
72
70
71.4
71
65.7
70
g
h
i
-(CH2)7CHdCH;(CH2)7CH3
-(CH2)20CH3
-(CH2)11CHdCH;(CH2)7CH3
-(CH2)7CHdCH;CH2;CH(OH);(CH2)5CH3
67.4
55
61.3
58.4
j
synthesized capsiates) and the controls (containing 1 mM concentrations of
corresponding antioxidants) 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 20 times with phosphate buffer containing 0.5% Tween
20 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.
Statistical Analysis. All of the assay results reported in the present
work are the mean of three measurements (presented as mean ( SD) and
were analyzed by a paired Student’s t test to evaluate the level of statistical
significance. Differences were assessed by one-way analysis of variance. A
p value of <0.05 were considered to be significant.
Table 2. DPPH Radical Scavenging Activity of the Synthesized Capsiate
Analogues and Reference Antioxidants
capsiate analoguea
free radical scavenging activityb (FRSA) (%)
a
77.98 ( 1.14 /
78.88 ( 1.03 /
78.76 ( 1.56 †
76.91 ( 0.81 /
70.17 ( 0.99 /,§
66.65 ( 1.23 /,§
67.57 ( 0.16 /,§
59.74 ( 0.75 /,§
61.76 ( 3.11 /,§
86.8 ( 2.78 §
b
c
d
e
f
g
h
i
j
BHT
87.51 ( 0.90 /,†
79.01 ( 0.86 §
RESULTS AND DISCUSSION
R-tocopherol
a Refer to Table 1 for a -j. b Values are mean ( SD (n = 3). FRSA was
significantly different versus BHT as shown by /, p < 0.001, and †, p < 0.01. FRSA
was significantly different versus R-tocopherol as shown by §, p < 0.001.
Phenolic lipids are one of the most well-studied classes of natural
antioxidants. This is due to their many potent additional biological
activities. Both capsaicinoids and capsinoids belong to this class of
antioxidant. However, capsinoids assume significance as exogenous
antioxidant compared to capsaicinoids for their application as
dietary supplements and also as preservatives in cosmetic and food
products. This is due to the fact that capsinoids are nonpungent
ester analogues of capsaicinoids having very similar biological
activities (6). However, synthesis of natural capsinoids is compli-
cated and costly due to the very specific nature of the alkyl side
chain. To have commercial application in the food and cosmetic
industries, it is necessary to synthesize capsiate analogues with
linear alkyl chain and evaluate their antioxidant potency. In the
present work, easily available natural fatty acids are selected for the
synthesis of capsiate analogues.
Reports on the synthesis of capsiate analogues are very few in the
literature (10, 12, 14, 15). Kobata et al. (12) first reported the
chemical synthesis of capsiate, but due to poor yield as well as
lengthy workup, the same authors reported lipase-catalyzed synthe-
sis of vanillyl nonanoate, the straight-chain analogue of natural
capsiate (15). Appendino et al. (14) reported a chemoselective
Mitsunobu esterification of vanillyl alcohol with nonanoic acid.
In the present work, lipase-catalyzed esterification of vanillyl
alcohol with different fatty acids was carried out for the synthesis
of capsiates. Altogether 10 compounds were synthesized by varying
the alkyl chain length as well as the functionality in the alkyl chain
(Table 1). Equimolar concentrations of vanillyl alcohol and fatty
acid were esterified using the Candida antartica lipase (Novozyme
435) at 50 °C for 4 h. After completion of reaction, the lipase was
filtered, and the synthesized compounds were purified by column
chromatography. The protocol is simple and regioselective and
affords excellent isolated yield (55-72%).
The antioxidant activity of all the synthesized compounds was
determined by the radical scavenging ability using the stable DPPH
radical method. The advantage of this assay is that the DPPH
radical is commercially available and does not need to be generated
before the assay as in other assays. The assay was conducted at
1 mM concentration of antioxidants in a polar homogeneous
medium. The DPPH radical scavenging capacity of the synthesized
capsiate analogues, carried out in the present work, is shown in
Table 2. Results indicate good radical scavenging activity of the
synthesized compounds approaching those of the two reference
compounds. DPPH is a well-known radical scavenging assay
involving a nitrogen-centered radical, which can react with phenolic
OH either through abstracting a hydrogen atom from phenolics or
by electron transfer from phenolics and phenoxide anions depend-
ing on the polarity of the studied medium. In a polar medium, as
has been the case in the present study, the electron transfer
mechanism predominates over the H-atom abstraction mecha-
nism (21). All of the synthesized capsiates exhibited excellent radical
scavenging activity; however, reference antioxidants showed super-
iority. Very similar activity was observed among the shorter chain
homologues (a-d), and a decreasing trend was observed beyond
that (e-i). Such nonlinear dependency of lipophilicity on antiox-
idant activity is coherent with other literature reports (22, 23). The
radical scavenging activity of the shorter chain homologues (a-d) is
similar to that of R-tocopherol. As the chain length increases, the
synthesized compounds showed significant reduction in activity
(p < 0.001) compared to R-tocopherol. This decrease in radical
scavenging activity with the increase in hydrophobicity may be

Article
J. Agric. Food Chem., Vol. 59, No. 2, 2011 567
Table 3. Induction Time of the Soybean Oil Spiked with and without the
Reference Antioxidants and the Synthesized Capsiate Analogues
compounda
induction timeb (h)
SBO
a
8.76 ( 0.34
9.83 ( 0.02 /
9.59 ( 0.76
b
c
10.44 ( 0.15 /
10.60 ( 0.26 /
10.15 ( 0.58 §
9.94 ( 0.25 /
9.70 ( 0.32 §
9.84 ( 0.69
d
e
f
g
h
i
10.23 ( 0.56 §
9.78 ( 0.37 §
9.56 ( 0.49
j
BHT
R-tocopherol
9.50 ( 1.33
a Refer to Table 1 for a -j. b Values are mean ( SD (n = 3). Synthesized
capsiates exhibited significantly increased induction time versus control (soybean
oil, SBO) as shown by /, p < 0.01, and §, p < 0.02.
correlated with the decrease in solubility of the analogues in polar
medium. Among the synthesized analogues, vanillyl ricinoleate
(compound j) was found to exhibit the maximum radical scaveng-
ing activity, on par with that of BHT and significantly higher than
that of R-tocopherol (p < 0.001). This may be due to the presence
of a hydroxyl group at the hydrophobic chain, influencing their
solubility characteristics.
The Rancimat assay is an appropriate in vitro model to
evaluate the antioxidant activity of an antioxidant in vegetable
oil, and the assay mimics the traditional Indian way of cooking,
wherein oil is preheated for 2-3 min at 160-180 °C before the food
products to be fried are added. During this time, degradation
(oxidation) of oil may happen depending on the degree of un-
saturation of fatty acids present in the oil. In this assay the soybean
oil (5.0 g), spiked with antioxidant (1.0 mM) was subjected to
accelerated oxidation at 110 °C under a purified air flow rate of
20 L/h. The volatile degradation products are trapped in distilled
water, influencing its conductivity. The induction time is defined as
the time necessary to reach the inflection point of the conductivity
curve. An increase in the induction time indicates increased
oxidative stability of soybean oil. The results for the oxidative
stability of soybean oil with and without antioxidant, measured
as induction time, are shown in Table 3. All of the synthesized
capsiates inhibit the oxidation of soybean oil as is evident from their
significantly higher induction period compared to soybean oil. In
fact, the activities of the capsiate analogues are better than those of
the references. However, the Rancimat assay also does not show
any correlation between the structural characteristics of the alkyl
chain and their antioxidant activity as all synthesized capsiates
showed a nearly constant antioxidant activity. This is because of the
accelerating oxidation condition employed in this assay, which is
too drastic to find decreasing antioxidant activity with the increase
in lipophilicity, validating the rational model of “polar paradox”.
Antioxidants are widely employed in cosmetic and food
products to inhibit lipid peroxidation, which otherwise renders
the product rancid and decreases the shelf life and nutritional
quality of the product. Most of such products exist in a formulated
complex emulsion system. The efficacy of an antioxidant in such a
matrix depends not only on its chemical reactivity as radical
scavenger but also on the orientation of its radical scavenging
nucleus, interaction with other food components, and environ-
mental conditions (24). The third in vitro assay conducted in the
present work is the rate of inhibition of autoxidation of linoleic acid
by antioxidant in Tween 20-based micellar medium. Initially the
autoxidation of linoleic acid is accompanied by a rapid increase of

568 J. Agric. Food Chem., Vol. 59, No. 2, 2011
Reddy et al.
acid alkyl ester on antioxidant activity. The study is based on
conjugated autoxodizable triene (CAT) assay and observed in-
creased antioxidant activity with increased lipophilicity, reaching a
maximum for the dodecyl chain, beyond which the activity
decreases with the increase in lipophilicity. Following the same
assay, the same authors studied the rosmarinic acid alkyl esters and
reported maximum antioxidant efficiency for the corresponding
octyl ester homologue (26). To support their hypothesis of efficient
comicellization as a possible reason for antioxidant efficiency,
Lucas et al. (27) studied the surface properties of tyrosol and
hydroxytyrosol alkyl ester and found maximum surfactant effec-
tiveness in compounds having 8-10 carbon atoms. On the other
hand, hydroxytyrosol butyrate showed maximum antioxidant
activity in cell-culture DCF assay (22), and hydroxytyrosol linole-
ate showed maximum activity in brain homogenate among their
studied homologues (19). Thus, the influence of alkyl chain length
on antioxidant activity in a micellar or biological system is
contrasting in nature. All of these contrasting literature reports
indicate that the dependency of antioxidant activity on lipophilicity
is related not only to assay type and the structure of the phenolic
moiety but also to the physical location and orientation of the
radical scavenging nucleus in such a three-phase system. In the case
of synthetic capsiates, homologues with lipophilia values similar to
that of the natural one (8-10 carbon atoms) are reported to be
most active biologically (10). However, in the studied micellar
system, consisting of nonpolar micellar interior, bulk polar, and the
micellar interface, the specific partitioning of vanillyl stearate,
oleate, and ricinoleate at the interface is appropriate to counteract
the free radical initiated oxidation of linoleic acid compared to
other homologues.
In conclusion, lipase-catalyzed synthesis of vanillyl ester of
fatty acids was carried out in the present work to synthesize 10
capsiate analogues. All of the analogues exhibited excellent
antioxidant activity in the three studied in vitro antioxidant
assays. Both DPPH and Rancimat assays did not show any
specific trend of antioxidant activity with the increase in lipophi-
licity or with the type of fatty acid esterified to vanillyl alcohol. In
the Tween 20 micellar system for the inhibition of autoxidation of
linoleic acid, longer chain homologues (vanillyl stearate, oleate,
and ricinoleate) exhibited maximum inhibition of autoxidation of
linoleic acid.
Figure 1. Formation of conjugated diene over 120 h during autoxidation of
linoleic acid, measured as absorbance at 234nm versus the chain length of
fatty acids esterified to the vanillyl alcohol: (A) saturated analogues;
(B) unsaturated analogues (vanillyl ricinoleate is represented as C-19).
LITERATURE CITED
conjugated diene, which is measured by UV at 234 nm. In the
control sample, the formation of conjugated diene was found to
reach a maximum in 36 h (Table 4). The autoxidation of linoleic
acid was found to be markedly inhibited (p < 0.001) due to the
addition of reference antioxidants as well as synthesized capsiates.
This is evident from the negligible formation of conjugated diene in
36 h. When the formation of conjugated diene over 120 h, measured
as absorbance at 234 nm, is plotted against the chain length of fatty
acids esterified to the vanillyl alcohol, a minimum is obtained near
C18 chain length (Figure 1). This is observed both in the saturated
(Figure 1A) and in the monounsaturated series (Figure 1B). This
indicates that in the studied Tween 20 micellar system, vanillyl ester
attached with a C18 alkyl chain (f, g, and j) irrespective of
functional moiety exhibited maximum inhibition of autoxidation
of linoleic acid, significantly higher (p < 0.001) than that of a when
the study was conducted over a period of 5 days. Further increase in
alkyl chain length to C22 reduces the antioxidant capacity sig-
nificantly (g vs i) in the unsaturated series (Figure 1B). In the
saturated series, such a decrease in activity was observed only after
60 h (f vs h, p < 0.001), and even then the activity of h is signifi-
cantly higher than that of a(p < 0.001). Laguerre et al. (25) recently
proposed a nonlinear dependency of lipophilicity of chlorogenic
(1) Ames, N. B.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants
and the degenerative diseases of ageing. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 7915-7922.
(2) Cerutti, P. A. Oxy-radicals and cancer. Lancet 1994, 344, 862-863.
(3) Dreher, D.; Junod, A. F. Role of oxygen radicals in cancer devel-
opment. Eur. J. Cancer 1996, 32A, 30-38.
(4) Roleira, F. M. F.; Siquest, C.; Orru, E.; Garrido, E. M.; Garrido, J.;
Milhazes, N.; Podda, G.; Paiva-Martins, F.; Reis, S.; Carvalho,
R. A.; da Silva, E. J. T.; Borges, F. Lipophilic phenolic antioxidants:
correlation between antioxidant profile, partition coefficients and
redox properties. Bioorg. Med. Chem. 2010, 18, 5816-5825.
(5) Figueroa-Espinoza, M.-C.; Villeneuve, P. Phenolic acids: enzymatic
lipophilization. J. Agric. Food Chem. 2005, 53, 2779-2787.
(6) Rosa, A.; Atzeri, A.; Deina, M.; Melis, M. P.; Incani, A.; Corona, G.;
Loru, D.; Appendino, G.; Dessi, M. A. Protective effect of vanilloids
against tert-butyl hydroperoxide-induced oxidative stress in Vero
cells culture. J. Agric. Food Chem. 2008, 56, 3546-3553.
(7) Nelson, E. K.; Dawson, L. E. The constitution of capsaicin, the
pungent principle of capsicum. J. Am. Chem. Soc. 1923, 45,
2179-2181.
(8) Tanaka, Y.; Hosokawa, M.; Otsu, K.; Watanabe, T.; Yazawa, S.
Assessment of capsiconinoid composition, nonpungent capsicinoid
analogues, in Capsicum cultivars. J. Agric. Food Chem. 2009, 57,
5407-5412.

Article
J. Agric. Food Chem., Vol. 59, No. 2, 2011 569
(9) Hayman, M.; Kam, P. C. A. Capsaicin: a review of its pharmacology
and applications. Curr. Anaesth. Crit. Care 2008, 19, 338-343.
(10) Barbero, G. F.; Molinillo, J. M. G.; Varela, R. M.; Palma, M.;
Macias, F. A.; Barroso, C. G. Application of Hansch’s model to
capsaicinoids and capsinoids: a study using the quantitative structure-
activity relationship. A novel method for the synthesis of capsinoids.
J. Agric. Food Chem. 2010, 58, 3342-3349.
(11) Kawada, T.; Hagihara, K.; Iwai, K. Effects of capsaicin on lipid
metabolism in rats fed a high fat diet. J. Nutr. 1986, 116, 1272-1278.
(12) Kobata, K.; Todo, T.; Yazawa, S.; Iwai, K.; Watanabe, T. Novel
capsaicinoid-like substances, capsiate and dihydrocapsiate, from the
fruits of nonpungent cultivar, CH-19 sweet, pepper Capsicum
annuum L. J. Agric. Food Chem. 1998, 46, 1695-1697.
(13) Kobata, K.; Sutoh, K.; Todo, T.; Yazawa, S.; Iwai, K.; Watanabe, T.
Nordihydrocapsiate, a new capsinoid from the fruits of a nonpun-
gent pepper Capsicum annuum. J. Nat. Prod. 1999, 62, 335-336.
(14) Appendino, G.; Minassi, A.; Daddario, N.; Bianchi, F.; Tron, G. C.
Chemoselective esterification of phenolic acids and alcohols. Org.
Lett. 2002, 4, 3839-3841.
(15) Kobata, K.; Kawaguchi, M.; Watanabe, T. Enzymatic synthesis of a
capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives
catalyzed by lipases. Biosci., Biotechnol., Biochem. 2002, 66, 319-327.
(16) Reddy, K. K.; Shanker, K. S.; Ravinder, T.; Prasad, R. B. N.;
Kanjilal, S. Chemo-enzymatic synthesis and evaluation of novel
structured phenolic lipids as potential lipophilic antioxidants. Eur.
J. Lipid Sci. Technol. 2010, 112, 600-608.
(20) Chimi, H.; Cillard, J.; Cillard, P.; Rahmani, M. Peroxyl and
hydroxyl radical scavenging activity of some natural phenolic anti-
oxidants. J. Am. Oil Chem. Soc. 1991, 68, 307-312.
(21) Foti, M. C.; Daquino, C.; Geraci, C. Electron-transfer reaction of
cinnamic acids and their methyl esters with the DPPH• radical in
alcoholic solutions. J. Org. Chem. 2004, 69, 2309-2314.
(22) Tofani, D.; Balducci, V.; Gasperi, T.; Incerpi, S.; Gambacorta, A.
Fatty acid hydroxyltyrosol esters: structure/antioxidant activity
relationship by ABTS and in cell-culture DCF assays. J. Agric. Food
Chem. 2010, 58, 5292-5299.
(23) Medina, I.; Lois, S.; Alcantara, D.; Lucas, R.; Morales, J. C. Effect
of lipophilization of hydroxytyrosol on its antioxidant activity in fish
oils and fish oil-in-emulsions. J. Agric. Food Chem. 2009, 57,
9773-9779.
(24) Anselmi, C.; Centini, M.; Granata, P.; Sega, A.; Buonocore, A.;
Bernini, A.; Facino, R. M. Antioxidant activity of ferulic acid alkyl
ester in a heterophasic system: a mechanistic insight. J. Agric. Food
Chem. 2004, 52, 6425-6432.
(25) Laguerre, M.; Giraldo, L. J. L.; Lecomte, J.; Figueroa-Espinoza,
M.-C.; Barea, B.; Weiss, J.; Decker, E. A.; Villeneuve, P. Chain
length affects antioxidant properties of chlorogenate esters in emul-
sion: the cutoff theory behind the polar paradox. J. Agric. Food
Chem. 2009, 57, 11335-11342.
(26) Laguerre, M.; Giraldo, L. J. L.; Lecomte, J.; Figueroa-Espinoza,
M.-C.; Barea, B.; Weiss, J.; Decker, E. A.; Villeneuve, P. Relation-
ship between hydrophobicity and antioxidant ability of phenolipids
in emulsion: A parabolic effect of the chain length of rosmarinate
esters. J. Agric. Food Chem. 2010, 58, 2869-2876.
(27) Lucas, R.; Comelles, F.; Alcantara, D.; Maldonado, O. S.; Curcuroze,
M.; Parra, J. L.; Morales, J. C. Surface active properties of lipophilic
antioxidants tyrosol and hydroxytyrosol fatty acid esters: a poten-
tial explanation for the nonlinear hypothesis of the antioxidant
activity in oil-in-water emulsion. J. Agric. Food Chem. 2010, 58,
8021-8026.
(17) Alencar, J.; Gosset, G.; Robin, M.; Pique, V.; Culcasi, M.; Clement,
J.-L.; Mercier, A.; Pietri, S. Improving the stability and antioxidant
properties of sesame oil: water-soluble spray-dried emulsions from
new transesterified phenolic derivatives. J. Agric. Food Chem. 2009,
57, 7311-7323.
(18) Akowuah, G. A.; Zhari, G. A.; Norhayati, I.; Mariam, A. HPLC and
HPTLC densitometric determination of andrographolides and anti-
oxidant potential of Andrographis paniculata. J. Food Compos. Anal.
2006, 19, 118-126.
(19) Trujillo, M.; Mateos, R.; Collantes de Teran, L.; Espartero, J. L.;
Cert, R.; Jover, M.; Alcudia, F.; Bautista, J.; Cert, A.; Parrado, J.
Lipophilic hydroxytyrosyl esters. Antioxidant activity in lipid
matrices and biological systems. J. Agric. Food Chem. 2006, 54,
3779-3785.
Received for review November 2, 2010. Revised manuscript received
November 23, 2010. Accepted November 29, 2010. K.K.R. thanks the
Council of Scientific and Industrial Research (CSIR), government of
India, for the award of a Senior Research Fellowship.