Antioxidant properties of two novel lipophilic derivatives of hydroxytyrosol

Article abstract of DOI:10.1016/j.foodchem.2020.126197

Two novel lipophilic derivatives of the natural olive oil phenol, hydroxytyrosol (HT), were synthesized using 3,4-dihydroxyphenylacetic acid as starting material. Their antioxidant activities and kinetics compared to HT and TBHQ were assessed by Rancimat,

Full text of DOI:10.1016/j.foodchem.2020.126197

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Antioxidant properties of two novel lipophilic derivatives of hydroxytyrosol
Tosin M. Olajide, Tao Liu, Haian Liu, Xinchu Weng
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S0308-8146(20)30044-3
DOI:
https://doi.org/10.1016/j.foodchem.2020.126197
FOCH 126197
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29 August 2019
13 December 2019
9 January 2020
Please cite this article as: Olajide, T.M., Liu, T., Liu, H., Weng, X., Antioxidant properties of two novel lipophilic
derivatives of hydroxytyrosol, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126197
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Antioxidant properties of two novel lipophilic derivatives of hydroxytyrosol
Tosin M. OLAJIDEa,b, Tao LIUa,b, Haian LIUb and Xinchu WENGa,*
aSchool of Environmental and Chemical Engineering, Shanghai University, 333, Nanchen Road
Shanghai, 200444, China.
bSchool of Life Sciences, Shanghai University, 333, Nanchen Road, Shanghai, 200444, China
*Corresponding author: wxch@staff.shu.edu.cn
ABSTRACT
Two novel lipophilic derivatives of the natural olive oil phenol, hydroxytyrosol (HT),
were synthesized using 3,4-dihydroxyphenylacetic acid as starting material. Their antioxidant
activities and kinetics compared to HT and TBHQ were assessed by Rancimat, Schaal Oven, 2,2-
diphenyl-1-picrylhydrazyl (DPPH) and deep-frying methods. All experiments, including kinetic
data analysis based on the Arrhenius equation, utilized in assessing antioxidant activity except
the DPPH assay revealed that the new lipophilic HT derivatives exhibited much stronger
antioxidant activity than hydroxytyrosol. Tert-butylhydroquinone exhibited stronger antioxidant
activity in bulk oil at 65 °C than the new HT derivatives, but showed much lower activity at
higher temperatures (>110 °C). This demonstrates that the introduction of bulky alkyl moiety to
the ortho-diphenolic structure of HT increased its antioxidant activity. It can be concluded that
the new lipophilic HT derivatives satisfy industrial demands for bioactive compounds with
strong antioxidant potential at high temperatures.
Keywords: Antioxidant activity; Hydroxytyrosol derivatives; Kinetic parameters; Lipophilicity;
Rancimat test
1

Chemical compounds studied in this article:
Hydroxytyrosol (PubChem CID: 82755); 3,4-Dihydroxyphenylacetic acid (PubChem CID: 547);
2, 2-Diphenyl-1-picrylhydrazyl radical (PubChem CID: 15911)
1. Introduction
Food quality deterioration and off-flavor generation generally decrease nutritional value and
shelf-life of food. This condition in fat and oil is caused by lipid oxidation—a sophisticated
process involving three major steps: initiation, propagation and termination (Alamed, Chaiyasit,
McClements, & Decker, 2009). Different methods have been developed to protect against lipid
oxidation effects in foods; however, the addition of antioxidants remains the most effective
method.
Antioxidants have become a vital group of food additives mainly due to their unique
properties of elongating shelf-lives of food products without any negative effect on their sensory
and/or nutritional qualities. The phenol types such as butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT) or tert-butylhydroquinone (TBHQ) have been utilized extensively for
food preservation due to their good antioxidant capacity, but have been questioned due to their
probable side effects for human health and also for their weak potency at high temperature food
processing like deep frying (Olajide, Pasdar, & Weng, 2018).
The general aversion of consumers in the use of synthetic antioxidants has given rise to a
recent trend in which phenolic antioxidants are synthesized structurally based on efficient natural
antioxidants. Hence, the synthesis of novel antioxidants with better antioxidant potential and less
toxicity which could better preserve food or prevent certain diseases, is very desirable (de
Pinedo, Penalver, & Morales, 2007).
2

Hydroxytyrosol (HT) is the main simple phenol that gives olive oil its wide range of health
benefits such as inhibition of inflammations, microbial activities, neurological damage, tumor
growth, metabolic syndrome, cardiovascular diseases, as well as maintaining the oxidative
stability of oil (Bañares, Martin, Reglero, & Torres, 2019; de Pinedo et al., 2007; Pereira-Caro et
al., 2009; Trujillo et al., 2006). HT was recently approved as a novel food additive (Dominique
et al., 2017). It has been of high interests to food, cosmetic and/or pharmaceutical industries
(Sun, Zhou, & Shahidi, 2018), however its use is still very limited possibly due to its hydrophilic
nature, which hinders its application under lipophilic conditions, as well as its isolation and
purification from aqueous solutions (Mateos et al., 2015; Tofani, Balducci, Gasperi, Incerpi, &
Gambacorta, 2010). Another drawback, which is of particular focus in this article, is the
ineffectiveness of HT or its previously reported derivatives under high temperature food
processing (Dominique et al., 2017).
Several authors have synthesized and reported the antioxidant activities of various
derivatives of HT such as hydroxytyrosol acetate (Gordon, Paiva-Martins, & Almeida, 2001),
alkyl hydroxytyrosyl ethers (de Pinedo et al., 2007; Pereira-Caro et al., 2009), alkyl
nitrohydroxytyrosyl ether (Gallardo et al., 2016), isochromans (Mateos et al., 2015) and those
with variable lengths of saturated and unsaturation fatty acid side chains (Mateos et al., 2008;
Sun et al., 2018; Tofani et al., 2010). However, while some of these derivatives are yet to be
tested in bulk oils, the few that have been tested only showed little or no antioxidant capacity
over HT. More importantly, as of yet, there has been no report of any HT derivative that has
good antioxidant activity at high temperature despite its numerous bioactivities. In terms of
modifying phenolic compounds for better lipid solubility and antioxidant capacity at high
temperature, the influence of tert-butyl group linked to the phenyl ring is greater than long-chain
3

alkanes (Huang, Jiang, & Liao, 2014; Shi, Liao, Olajide, Liu, Jiang, & Weng, 2017; Silva et al.,
2000). Nevertheless, the analysis of kinetic parameters is useful for predicting the oxidative
stability and temperature dependence of fats and oils in the presence and absence of antioxidants
during heat processing, storage, and distribution (Hashemi et al., 2016). Also, kinetic data of
edible oils oxidation from animal sources under Rancimat conditions with ease of use and
appropriate reproducibility are scarce.
Therefore, taking into account the bioactivity attributed to hydroxytyrosol and the practical
application of a lipophilic derivative that can withstand high temperature food processing; in this
work, two novel butylated hydroxytyrosol derivatives, 3-(tert-butyl)-4,5-dihydroxyphenylacetic
acid methyl ester (BuMT) & 3-(tert-butyl)-5-(2-hydroxyethyl)benzene-1,2-diol (BuHT), were
synthesized and their antioxidant activities were assessed by Rancimat, Schaal Oven, DPPH and
deep frying tests. The kinetics of BuMT and BuHT as efficient antioxidants provided by
Arrhenius parameters, activation enthalpy (ΔH++), entropy (ΔS++), temperature coefficient (tcoeff
)
and Q10 in lard was also carried out.
2. Materials and methods
2.1. Materials
HT, 3,4-dihydroxyphenylacetic acid (DOPAC), thionyl chloride (SOCl2), lithium aluminum
hydride (LiAlH4), and p-anisidine were purchased from Shanghai Macklin Biochemical Co., Ltd
(Shanghai, China). DPPH, TBHQ, tertiary butanol, anhydrous methanol (MeOH), anhydrous
tetrahydrofuran (THF), ethanol, diethyl ether, glacial acetic acid, isooctane, dichloromethane
(DCM), phosphoric acid (H3PO4), hydrochloric acid (HCl), sodium hydroxide (NaOH),
petroleum ether (PE), ethyl acetate (EA), phenolphthalein indicator, potassium hydroxide
4

(KOH), sodium thiosulphate and starch were purchased from Sinopharm Chemical Reagent Co.
Ltd (Shanghai, China).
Lard was carefully rendered in the laboratory and stored below −18 °C for subsequent use.
Potatoes were purchased from the local market. All solvents and reagents used in this experiment
were of analytical grade and were used without further purification.
NMR spectra were recorded with a Bruker Avance 600 MHz spectrometer (USA) and data
are reported as chemical shift δ (ppm) values referenced to the solvent used. Thin-layer
chromatography (TLC) was conducted on 0.25 mm pre-coated silica gel plates with detection
under UV light at 254 nm Induction periods (IP) of the antioxidants were measured by Rancimat
743 (Metrohm, Herisau, Switzerland) and UV-2450 spectrophotometer (Shimadzu Corp, Kyoto,
Japan) for UV spectroscopy.
2.2. General synthesis of compounds
The synthesis of compounds (3 & 4) was done according to a previous method (Zhang, Xiao,
Chen, & Lian, 2010) with some improvement (Fig. 1).
DOPAC methyl ester (2): 30 mmol of DOPAC (5 g) was dissolved in anhydrous methanol
(60 mL) under stirring at 0°C in the presence of 40 mmol of SOCl2 (3 ml) as acid catalyst. The
mixture was stirred at room temperature for 9 h. After solvent was evaporated under low
pressure, desired compound 2 (94% yield) was isolated as colorless oil by silica gel column
chromatography using petroleum ether/ ethyl acetate (3:1, v/v) as eluent. Analytical data were as
reported in Zhang et al. (2010).
3-(tert-butyl)-4,5-dihydroxyphenylacetic acid methyl ester (3): 20 mmol of DOPAC methyl
ester (3 g) was added to a mixture of 200 mmol tertiary butanol (20 ml) and 98% phosphoric acid
(7 ml) under nitrogen condition. The mixture was heated under reflux for 8 h, and then
5

evaporated to dryness under low pressure to remove residual solvent. After solvent removal,
residue was washed with warm distilled water (50 ml) followed by ethyl acetate (30 ml) and the
organic layers were evaporated to dryness under vacuum. The product mixture was applied to
silica gel column chromatography using dichloromethane/methanol (20:0.1, v/v) as eluent to
afford 3 (BuMT) (71% yield) as white crystals after recrystallizing from methanol: mp 99-100
°C; 1H NMR (600 MHz, Chloroform-d) δ 6.70 (d, J = 1.9 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 3.71
13
(s, 3H), 3.51 (s, 2H), 1.39 (s, 9H). C NMR (150 MHz, Chloroform-d) δ 173.5, 143.0, 142.7,
+
136.4, 124.0, 120.1, 113.6, 52.3, 40.8, 34.6, 29.5; HRMS (ESI): Calcd for C13H19O4 (M+H)+
m/z 239.12779, found 239.12756. The purity of BuMT was determined by HPLC as 99.4%.
3-(tert-butyl)-5-(2-hydroxyethyl)benzene-1,2-diol (4): 10 mmol of BuMT (1.3 g) was added
to a stirred solution of 20 mmol lithium aluminum hydride (0.6 g) in anhydrous tetrahydrofuran
(80 ml) at 0 °C under nitrogen condition. The mixture was stirred at room temperature for 9 h,
and then a 0.5 M aqueous hydrochloric acid solution (35 ml) was added to the mixture at 0 °C.
After 30 minutes, the acidic mixture was washed with ethyl acetate (30 ml × 3), and the organic
layers were combined and evaporated to dryness under low pressure. The residue was subjected
to column chromatography using petroleum ether/ethyl acetate (2:1, v/v) as eluent to furnish 4
1
(BuHT) (91% yield) as white crystals after recrystallizing from methanol: mp 109-110 °C; H
NMR (600 MHz, Chloroform-d) δ 6.68 (d, J = 2.0 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H), 3.84 (t, J =
6.3 Hz, 1H), 2.74 (t, J = 6.3 Hz, 1H), 1.40 (s, 9H). 13C NMR (150 MHz, Chloroform-d) δ 143.1,
+
142.1, 136.7, 128.7, 119.5, 113.6, 63.9, 38.6, 34.6, 29.5; HRMS (ESI): Calcd for C12H19O3
(M+H)+ m/z 211.13287, found 211.13269. The purity of BuHT was determined by HPLC as
98.9%.
6

2.3. Rancimat test
The antioxidant activities of TBHQ, HT, BuMT and BuHT in lard were determined
according to Olajide et al. (2018) on a Rancimat 743 apparatus (Metrohm, Herisau, Switzerland).
Lard samples of 3 ± 0.05 g containing different concentrations of antioxidants (0.01, 0.02, and
0.04%) were subjected to accelerated oxidation at temperatures between 100 and 140 °C under
an air flow rate fixed at 20 L/h. Each test was carried out in duplicate and results are expressed as
induction period (IP) of lard samples with antioxidants in hours corresponding to the stability of
lard without antioxidant evaluated. The protection factors (Pf) of each antioxidant was determine
according to Bañares et al. (2019):
Pf = IPw/IPwo
where IPw and IPwo represent the IP values with and without antioxidant, respectively. Pf values
˂ 1 indicate a pro-oxidant effect of antioxidant, whereas when equal to 1 means no antioxidant
effect and values of Pf ˃ 1 are related to an antioxidant effect.
2.3.1. Kinetic parameters analysis
Temperature coefficients (tcoeff, °C–1) were determined from the slopes of the lines
obtained by regressing log (OSI) vs. temperature (T, °C):
log IP = a(T) + b
(1)
where a = tcoeff and b represent slope and intercept, respectively.
Q10 number, which is defined as the increase in rate of oxidation reaction for every 10 °C
rise in temperature, was calculated from slopes of the lines obtained above using this equation:
Q10 = 10-10a
(2)
7

Activation energies (Ea, kJ/mol) and pre-exponential or frequency factors (A, h–1) were
evaluated from the slopes and intercepts respectively, of the lines obtained by regressing ln k vs.
1/T (K−1) according to the Arrhenius equation:
ln k = ln A – (Ea/RT)
(3)
where k represents the reaction rate constant or reciprocal induction period (h–1), T is the
temperature in Kelvin, and R is the universal gas constant (8.3143 J/mol K)
Activation enthalpies (ΔH++) and entropies (ΔS++) were determined from the slope and
intercept of the lines obtained by linear regression of ln (k/T) vs. 1/T according to the equation
derived from the activated complex theory:
ln (k/T) = ln (kB/h) + (ΔS++/R) – (ΔH++/RT)
(4)
where kB represents the Boltzmann constant (1.3806586 x 10-23 J/K) and h is the Planck’s
constant (6.62607556 x 10-34 Js).
2.4. DPPH test
The DPPH radical scavenging activity of TBHQ, HT, BuMT and BuHT was determined
according to previous method reported by Jiang, Weng, Huang, Hou, and Liao (2014) with slight
modification. 0.5 ml of each antioxidant in methanol was mixed with 3 ml methanolic solution of
DPPH (0.1 mM). The final concentrations of antioxidants in the reaction mixtures were 1.5, 3, 6,
12, 24, and 48 µM. The resulting mixtures were shaken vigorously and left to react in a dark
chamber for 30 min, and then the decreasing absorbance of DPPH was read at 517 nm against
blank on a UV-2450 spectrophotometer (Shimadzu Corp, Kyoto, Japan). Methanol was used as
blank solution and 2.5 ml DPPH solution in 0.5 ml methanol served as the control. The radical
scavenging activity of the antioxidants was expressed as EC50, which is the effective
8

concentration required to obtain a 50% antioxidant capacity of a compound (Chen, Bertin, &
Froldi, 2013). DPPH radical scavenging activity was calculated using the equation below:
Scavenging activity (%) = [(Acontrol- Asample)/Acontrol] × 100
2.5. Deep frying test
The lipid matrix used in this experiment was obtained from soybean oil by purification
through a silica gel column according to the method described by Lampi, Kataja, Kamal-Eldin
and Piironen (1999) with some modification. The purification process ensured the complete
removal of endogenous pro-oxidants and antioxidants from the oil. Two replications of 30 h deep
frying trials were conducted. Each oil sample (500 g) containing 0.02% (w/w) of antioxidants
were heated to 180 ± 0.5 °C and kept at this temperature for 15 h on two consecutive days. Fresh
potato slices (50 g) were fried in the oils every hour for 8 min. Oil samples were collected and
stored below −18 °C after every frying until the conjugated dienes (CD) and acid values (AV) of
all the samples were evaluated according to the IUPAC method (Paquot, 1979). Oils were not
replenished during the frying trial.
2.6. Schaal Oven test
Soybean oil samples (50 g each) with and without antioxidant were placed in 100 ml capacity
conical flasks and heated in an oven set at 65 ± 0.5 °C. The peroxide value (PV) and p-anisidine
value (p-AV) in the samples were measured in duplicate at intervals of 0, 3, 6, 9, 12, and 15 d.
The induction period of the samples was dependent on the oil reaching a peroxide value of 80
mEq O2/kg oil (Nissiotis, & Tasioula-Margari, 2002) and an anisidine value of 10 (Maszewska et
al., 2018). After PV and p-AV were determined, total oxidation (TOTOX) values were
calculated according to the equation: TOTOX = 2PV + p-AV.
9

2.6.1. Peroxide value (PV)
The PV of oil samples was measured based on the method described in AOCS Method
Cd8b-90 with some modification.
2.6.2 p-Anisidine value (p-AV)
p-Anisidine value (p-AV) was determined according to AOCS Official Method (1995). This
method involves the spectrophotometric determination of products formed in the reaction
between p-anisidine and aldehydes in the oil. Each oil sample (1 g) was dissolved in 25 ml
isooctane and the absorbance of this test solution was read at 350 nm. Then 5ml of the above
mixture was mixed with 1 ml p-anisidine (0.25% in glacial acetic acid, w/v) and absorbance was
measured at 350 nm after 10 min. Reference solution was prepared by adding 1 ml of p-anisidine
reagent to 5 ml isooctane. The p-AV was calculated according to the equation:
p-AV = 25(1.2As-Ab)/m
Where As is the absorbance of test solution after reaction with p-anisidine reagent; Ab is the
absorbance of test solution only, and m is the mass in grams of oil samples.
2.7. Statistical analysis
All tests were performed in duplicate. Data are presented as mean ± standard deviation (SD).
Significance differences between means were examined by analysis of variance (ANOVA) using
OriginPro version 9.1 followed by Duncan’s multiple range test (P < 0.05).
3. Results and discussion
3.1. Preparation, purification and characterization of BuMT and BuHT
The new lipophilic HT derivatives were obtained as illustrated in Fig. 1. BuMT was
prepared by a two-step reaction involving the esterification of DOPAC followed by alkylation of
10

the ortho-diphenolic ring. BuHT was subsequently obtained by a one-step reduction process
from BuMT using LiAlH4 as reducing agent. Both compounds were synthesized in good yields
at a purity of 99.4% (BuHT) and 98.9% (BuMT), as determined by HPLC. Their identity was
confirmed by NMR spectroscopy and HRMS (ESI). The Rf values of HT, BuMT and BuHT
were 0.28, 0.87 and 0.50, respectively (petroleum ether/ethyl acetate, 1:1). In addition, the
theoretical partition coefficient (Log Ptheor) values of HT, BuMT and BuHT were calculated as
0.96, 2.72 and 2.66, respectively, using ChemBioDraw Ultra Software. Rf values were
coincidental with Log Ptheor values, showing the following order: HT < BuHT < BuMT. This
means that in terms of lipophilicity, HT had the highest polarity amongst all the evaluated
compounds, showing a Log P value which is in agreement with previously published data
(Gallardo et al., 2016; Mateos et al., 2015). BuMT and BuHT both showed less hydrophilic
attributes due to the presence of higher carbon skeleton density in the form of bulky hydrophobic
alkyl group bonded to their phenyl moiety. The methyl ester group attached to the aliphatic side
chain of BuMT resulted in decreased polarity compared to BuHT. The UV spectra of the
compounds were quite similar exhibiting two broad absorption bands in the range of 200–380
nm. BuMT and BuHT both had strong absorptions at 222/ 224 nm, and weak absorptions at 283/
282 nm respectively, in the UV spectra. However, when KOH was added to the solutions,
absorptions at 222 and 224 nm were strengthened and there were red shifts to 223 and 225 nm,
respectively. Meanwhile, the weak absorption of BuMT had a red shift to 295 nm and BuHT to
1
293 nm. This phenomenon confirmed that the compounds were both phenolics. In the H NMR
spectrum of BuMT, phenolic protons were not observed. Two duplets around 6.70 and 6.68 ppm
were assigned to the aromatic protons, and methyl of the ester moiety was observed at 3.71 ppm
as a sharp singlet. The methylene protons were observed at 3.51 ppm while a proton singlet at
11

13
1.39 ppm was observed for tertiary butyl group attached to the aromatic ring. In the C NMR,
the most downfield signal 173.5 ppm was assigned to the carbonyl carbon of the ester group. Six
aromatic carbon peaks were observed from 143.1 to 113.6 ppm. Methylene and methyl group
carbon signals were assigned to 52.3 and 40.8 ppm, respectively, and tertiary butyl group carbon
1
signals to 34.6 and 29.5 ppm. Similarly, phenolic protons were not observed in the H NMR
spectrum of BuHT. Two duplets around 6.68 and 6.60 ppm were assigned to the aromatic
protons. Two methylene protons were observed at 3.84 and 2.74 ppm, while a proton singlet at
13
1.40 ppm was observed for tertiary butyl group attached to the aromatic ring. In the C NMR,
six aromatic carbon peaks were observed from 143.1 to 113.6 ppm. The methylene group carbon
bearing OH and the one attached to the aromatic ring exhibited signals at 63.9 and 38.6 ppm,
respectively, and the tertiary butyl group carbon signals were assigned to 34.6 and 29.5 ppm. In
the HRMS (ESI) spectrum of BuMT, a protonated molecular ion peak was found at m/z
239.12756 for [M+H]+, whereas for BuHT, a protonated specie at m/z 211.13269 was observed,
+
which was assigned to C12H19O3 (M+H)+. All spectra data fully characterizes BuMT and BuHT,
and as far as is known, neither has been previously described in any literature.
3.2. DPPH test
The DPPH radical scavenging assay is a simple, rapid and easily reproducible method
used for evaluating the efficiency of antioxidants as radical scavengers against stable DPPH free
radicals. Scavenging activities of the evaluated antioxidants are shown in Fig. 2. Results are
expressed as EC50, which is the effective concentration required to decrease the initial DPPH
radical concentration by 50% (Chen et al., 2013; Pereira-Caro et al., 2009). Among all the tested
compounds, HT (EC50= 10.10 µM) showed the highest scavenging capacity, which was more
12

than double that of TBHQ (EC50= 20.43 µM), BuHT (EC50= 23.59 µM) and BuMT (EC50= 25.31
µM). This finding coincides with earlier reports by Sun et al. (2018), where HT had highest
scavenging activity as the most polar antioxidant. This phenomenon is explained by the polar
paradox and interface theories in which the polarity of antioxidants is proportional to their DPPH
radical scavenging activity. It is known that the presence of two or more hydroxyl groups on the
phenyl ring can increase radical-scavenging activity by allowing more hydrogen atoms of the
phenolic hydroxyl groups be donated to stabilize the free radicals (Shahidi, & Wanasundara,
1992). Herein, however, the presence of bulky tert-butyl group in the BuMT and BuHT chemical
structures decreased their radical scavenging abilities compared with HT because of steric
hindrance effects, which inhibited the ease of DPPH binding. Some researchers have also
observed this phenomenon in other phenolic antioxidants and their derivatives. For instance, Shi
et al. (2017) reported a lower free radical scavenging activity for butylated caffeic acid (BCA)
than caffeic acid itself. In a separate study by Huang et al. (2014), a methylbenzenediol
derivative, 3-(tert-butyl)-5-methylbenzene-1,2-diol (TBHPC), having a bulky alkyl group linked
to the o-position of one hydroxyl also showed lower scavenging activity compared to its mother
compound, 4-methylcatechol (HPC) which is similar to HT in structure. In summary, the
hydrogen-donating ability, molecular structure, and subsequent stabilization of phenoxyl radical
influence the antioxidant activity of the phenolic compounds (Silva et al., 2000). Thus, the steric
hindrance induced by the bulkiness of the alkyl groups varied the antioxidant capacity of the HT
derivatives in contrast with HT (Roleira et al., 2010).
3.3. Antioxidant activity in oil – Rancimat and deep-frying tests
The efficacy of BuMT and BuHT as antioxidants in comparison with HT and TBHQ was
evaluated using the Rancimat test at temperatures in the range 100–140 °C, and concentrations at
13

0.01, 0.02 and 0.04% (w/w) in air saturation conditions. The results were expressed as induction
period (IP) corresponding to the oxidative stability of lard, protection factor (Pf) of antioxidants
and Arrhenius kinetic parameters from which the effect of temperature on the rates of oxidation
was evaluated as shown in Table 1. According to Wang et al. (2000), a higher Pf value means
stronger antioxidant activity: Pf < 1 shows pro-oxidant activity; Pf = 1 shows no antioxidant
activity; 2 > Pf > 1 shows weak antioxidant activity; 3 > Pf > 2 shows potent antioxidant activity
and Pf > 3 shows the compound has a strong antioxidant activity. In this study, concentrations up
to 400 ppm were investigated to accurately evaluate the variability of antioxidant protection at a
broader range of doses. Nevertheless, there is a 215 mg/kg safety limit established for HT
(Dominique et al., 2017) and 200 mg/kg for synthetic antioxidants like TBHQ in vegetable oils
(Saad et al., 2007).
Pf values greater than 1 were observed for all evaluated antioxidants at 0.02% (w/w) in
the temperature range studied (Table 1a) which indicate a protective effect against accelerated
oxidation. The highest Pf was observed for BuMT, followed by BuHT, then the slightly less
stable, HT and finally, TBHQ. Averagely, both BuMT and BuHT showed an induction period
(IP) 51 h higher than HT and about 126 h more than TBHQ. This demonstrates that they are
much stable than both HT and the common commercial phenolic antioxidant, TBHQ. Similarly,
the newly synthesized lipophilic HT derivatives, BuMT (0.01%, Pf = 10.58; 0.04%, Pf = 32.05)
and BuHT (0.01%, Pf = 12.66; 0.04%, Pf = 27.94), showed substantially higher antioxidant
activity than HT (0.01%, Pf = 10.13; 0.04%, Pf = 17.75) or TBHQ (0.01%, Pf = 3.98; 0.04%, Pf
= 7.63) in the lipid matrix at 120 °C (Table 1b). As expected, IP and Pf values increased as
concentration of the antioxidants increased. Summarily, the antioxidant activity of the phenolic
antioxidants as a function of temperature and concentration decreased as follows: BuMT ≈
14

BuHT > HT >> TBHQ > Control. HT has been reported to display the strongest antioxidant
activity in olive oil, having higher induction period in Rancimat test than caffeic acid or gallic
acid (Ranalli, Lucera, & Contento, 2003). Nevertheless, in this experiment, the stronger
antioxidant activity of BuMT and BuHT compared to HT and TBHQ was partly due to higher
molecular weight, which reduced their partial volatilization. A similar finding has been reported
(Huang et al., 2014; Jiang et al., 2014; Olajide et al., 2018 & Shi et al., 2017). It has been
established that the catechol group is one of the main contributors to the antioxidant capacity of
phenolic compounds, enhancing their redox potential, radical scavenging ability and metal-
chelating capacities (Pereira-Caro et al., 2009). Additionally, alkylating ortho-diphenolic
structures with electron donating substituents like bulky tert-butyl group at the 2, 4 and 6-
positions can further increase their antioxidant activity (Kajiyama, & Ohkatsu, 2001; Weng, &
Huang, 2014; Zhang, Wu, & Weng, 2004). Hence, the Rancimat results demonstrate that
alkylation of HT at the o-position preserved the ortho-diphenolic group, and positively
influenced antioxidant potentials of the new HT derivatives.
The kinetic parameters obtained from Rancimat test are listed in Table 1c. Activation
energy (Ea), which is the minimal energy of molecules required to initiate oxidation reaction was
employed to evaluate the dependence of the rate of lipid oxidation on temperature. Ea of the
various oil sample oxidation ranged from 66.16 to 86.32 kJ/mol. Hashemi et al. (2016) reported
the activation energies of blended oils spiked with antioxidants obtained in a
higher temperature range 120–140 °C to be between 68.34 and 82.47 kJ/mol. The activation
energy of HT, BuMT and BuHT oil samples were lower compared to that of TBHQ. A greater
Ea value implies that a smaller temperature change is needed to increase the rate of oxidation
Bañares et al. (2019). In other words, oils containing BuMT and BuHT were more
15

resistant to oxidative degradation (Ea = 84.72 and 86.30 kJ/mol, respectively) at higher
temperature than TBHQ (Ea = 86.32 kJ/mol). Interestingly, the Ea value of control showed an
opposite trend to the result expected, but is however comparable to the study by Bañares et al.
(2019), where the kinetic parameters of Echium oil including Ea (74.1 kJ/mol) in the temperature
range 50-110 °C were lower compared to antioxidant (rosemary extract) spiked oil samples, Ea
(78.7 kJ/mol). The oxidation pathway at different temperatures may vary due to saturated and
unsaturated (MUFA + PUFA) fatty acid ratio, reactivity of metal ions, presence of antioxidants
and prooxidants and also because the solubility of oxygen in oil reduces by
25% for every 10 °C decrease in temperature (Bañares et al., 2019; Farhoosh & Hoseini‐Yazdi,
2014). The pre-exponential or frequency factor (A) for various oil sample oxidation ranged from
5.2x108 to 4.2x1010. It is the number of collisions that produce chemical change; hence, a higher
A value means higher probability of successful collisions leading to oxidation. In general,
frequency factor (A) reduces in the presence of antioxidants. The TBHQ sample had higher
frequency factor than the new antioxidants. This increase of frequency factor in the presence of
TBHQ may be ascribed to its instability at higher temperature leading to reduced hydrogen
donating ability and consequently the loss of rotational freedom in the transition state.
The ΔH++ and ΔS++ values were computed based on activated complex theory from linear
regression parameters summarized in Table 1c. ΔH++ and ΔS++ values ranged from 62.89 kJ/mol
and -88.71 J mol-1 K-1, to 84.06 kJ/mol and -52.02 J mol-1 K-1, respectively, for the oxidation of
oil samples. The positive sign of ΔH++ depicts an endothermic nature of activated complex
formation. In this work, the enthalpy and entropy of TBHQ sample were higher and lower,
respectively, than that of BuMT and BuHT, therefore the concentration of the activated complex
in oil containing the new antioxidants was lower than oil with TBHQ. The greater negative ΔS++
16

value of oil depicts a more ordered activated complex than the reactants, and hence a lower
probability of faster lipid oxidation reaction (Hashemi et al., 2016).
The temperature coefficients (tcoeff) and Q10 numbers computed from log (IP) vs. T (°C)
linear representation are shown in Table 1c. The tcoeff values ranged from -2.2 × 10-2 to -2.9 × 10-
2
°C-1 and Q10 values were between 1.66 and 1.96 for all the samples. The tcoeff and Q10 values
presented for control in this study are comparable to that reported in Farhoosh and Hoseini‐Yazdi
(2014) for lard at -2.55 × 10-2 °C-1 and 1.8, respectively. A higher Q10 number implies that a
smaller change in temperature is needed to increase the rate of lipid oxidation. Antioxidants can
decrease the dependence of the rate of lipid oxidation on temperature (Bañares et al., 2019).
Herein, the temperature dependence of lipid oxidation rate in the presence of BuHT and TBHQ
were similar, but lower than that of BuMT. This means that BuMT was fairly active than BuHT
but more active than TBHQ at high temperatures as evidenced by the IP and Pf values in Table
1a. Finally, the reaction rate constant (k), which is the reciprocal of induction period was also
employed to evaluate the protective effect of the antioxidants at temperatures between 100 and
140 °C. The higher the oxidation reaction rate constant, the lower the antioxidant protection.
Hence, the k values for the oil samples relative to antioxidant activity of the antioxidants as a
function of temperature decreased as follows: BuMT ≈ BuHT > HT >> TBHQ > Control.
Deep frying is a common process where food is wholly immersed in hot oil to produce
crispy food with better texture and taste. Conjugated diene (CD) and acid values (AV) are useful
quality control parameters used for determining the activity of antioxidants in the deep-frying
test. The results of CD measured at 233 nm are expressed in Fig. 3a. A gradual increase was
observed in CD contents for BuHT and BuMT compared to a faster rate for HT, TBHQ and
control relative to the frying time. Initial CD value was 0.53%, which at the end of 30 h deep
17

frying reached 12.10, 8.88, 7.03, 3.83 and 3.34% for control, TBHQ, HT, BuHT and BuMT
groups, respectively (p < 0.05). The continuous formation of CD may be related to the presence
of high contents of polyunsaturated fatty acids in soybean oil (Marinova et al., 2012). The
increase in CD is proportional to the uptake of oxygen and a greater level of CD means lower
oxidative stability of the oils (Bushra, Farooq, & Roman, 2007). According to Fig. 3b, the acid
value (AV) of BuHT and BuMT spiked oil samples at the end of frying trials increased slowly to
about 2.32 and 2.24 mg KOH/g, respectively, in comparison with HT, TBHQ, and control
groups, that increased to 3.44, 3.71 and 4.17 mg KOH/g of oil, respectively. This shows that the
new HT derivatives were able to suppress lipid oxidation better than HT or TBHQ leading to
lower acid values, which is an attribute of good quality oil. Thus, the CD and AV changes in oil
samples indicate that the antioxidant activities of compounds during frying decreased as follows:
BuMT ≥ BuHT > HT > TBHQ > control. This finding is in agreement with published report by
Shi et al. (2017), where the superior antioxidant activity of butylated caffeic acid (BCA) as with
BuHT and BuMT under Rancimat and deep-frying tests was due to the presence of tert-butyl
group at the ortho-position to their diphenolic structure. In addition, the presence of a catechol
moiety and an alkyl side-chain with primary alcohol linked to phenyl ring are structural factors
that can increase antioxidant activity (de Pinedo et al., 2007). These phenomena provided strong
steric hindrance influence that helped stabilized the radical resonance of the antioxidant structure
in capturing more peroxyl radicals, and contributed to an increased molecular mass that
enhanced less volatilization than HT and TBHQ.
Contrarily, the tendency found in the Rancimat and deep-frying tests for the new phenolic
antioxidants derived from HT under study was different to the one found in the DPPH test
results. This finding, which was largely due to the bulkiness of the DPPH radical as it can bind
18

easily with phenoxyl radicals with less steric hindrance like HT (Huang et al., 2014), is in
agreement with similar studies by others (Jiang et al., 2014; Olajide et al., 2018; Shi et al., 2017;
Weng & Huang, 2014).
3.4. Antioxidant activity in oil –Schaal Oven test
PV is one of the most commonly used tests for the determination of oxidative rancidity in
oils and fats. The antioxidant potential of antioxidants at 200 ppm in soybean oil evaluated in
this work is also evident from the measurement of primary oxidation products by the peroxide
value (PV) as a function of time (in days) in an oven at 65 ± 0.5 °C. Oil samples were oxidized
until a peroxide value of 80 mEq /kg was reached. As shown in Fig. 4a, the PV of fresh oil was
0.53 mEq O2/Kg oil, indicating that the oil used was in good condition for oxidative stability
determination and meets the requirements for edible oils specified by Codex Alimentarius (1999)
(PV <15 mEq O2/Kg). Except for control, the oxidation of oils containing TBHQ, HT, BuHT and
BuMT proceeded at a slower rate initially, but gradually increased dynamically with storage
time. Oil sample without antioxidant (control) reached a maximum PV of 79.2 ±0.85 mEq/kg
after 6 days of storage, whereas for HT and BuMT spiked samples it required 11 and 15 days to
reach a PV value of 80 mEq/kg, respectively. Interestingly, both TBHQ and BuHT suitably
sustained the oxidative stability of oil samples with PV values of 7.7 ± 0.13 and 27.33 ± 0.18
mEq/kg, respectively, after 15 days of storage.
The p-anisidine value (p-AV) is a good method for evaluating secondary oxidation
products formed during the oxidative degradation of oil. For a good quality oil or fat, p-AV
lower than 10 is expected (Tadesse, Ret, & Beyero, 2017). According to Fig. 4b, all samples had
a p-AV less than 10 after 15 days of storage at 65 ± 0.5 °C except for the control and HT groups.
As with PV above, TBHQ-spiked sample had the lowest p-AV level compared to other
19

antioxidants, gradually reaching a maximum of 2.24±0.04 from an initial value of 1.61±0.01,
whereas BuHT and BuMT smoothly increased to 4.71±0.23 and 7.62±0.11, respectively, after 15
days of storage.
TOTOX represents overall oxidative deterioration since it considers both peroxides and
aldehydes produced during oxidation. The lesser the TOTOX value, the better the oil quality. As
shown in Fig. 4c, similar pattern in peroxide and p-anisidine values were observed for TOTOX
values of the samples with storage time. Therefore, the antioxidant activities of compounds
studied under the Schaal Oven test decreased as follows: TBHQ > BuHT > BuMT > HT >
control. The difference in efficacy of antioxidants may be accounted for on the basis of their
chemical structures. That is, the rate of propagation of oxidation reactions in the lipid matrix was
reduced by the antioxidants ability to stabilize phenoxy radicals (Ying et al., 2010). BuMT and
BuHT both provided a much stronger protective effect than TBHQ at higher temperature
experiments (> 110 °C) such as Rancimat and deep frying, however at lower temperatures (< 65
°C) their protective effect seems to diminish. In addition, the superior antioxidant activity
demonstrated by TBHQ in comparison with HT and its new lipophilic derivatives, especially
BuMT, at lower temperature in this work is also evident in the studies done by Zhang et al.
(2004). This observation was as a result of the two para-hydroxyl groups on TBHQ which can
release hydrogen atoms to active peroxyl radicals in edible oils to interrupt the oxidative chain
reaction (Nissiotis & Tasioula-Margari, 2002).
4. Conclusion
In this work, a thorough study of the antioxidant activity of two novel lipophilic derivatives
of HT, a natural antioxidant predominantly found in olive oil, has been conducted using the
Arrhenius kinetic parameters, Rancimat, Schaal Oven, DPPH and deep-frying tests. The results
20

demonstrated that BuMT and BuHT are good antioxidants in bulk oils at high temperatures, but
their potency tends to slightly decrease at lower temperatures (< 65 °C). Although, they
exhibited a weak DPPH radical scavenging activity compared to HT and TBHQ, clearly the
butylated ortho-diphenolic structure, as well as the alkyl side-chain bearing a primary alcohol
(for BuHT) and a methyl ester (for BuMT) highly influenced their antioxidant capacity. The
polar paradox is not applicable to the antioxidant capacity found for these new phenolic
antioxidants, except from a very general perspective. Nevertheless, these new lipophilic HT
derivatives satisfy the requirements for new antioxidants with strong antioxidant potential at high
temperatures, and therefore maybe used as functional antioxidant ingredients in food, cosmetics
and pharmaceuticals after proper evaluation of their safe consumption, which will
be further studied.
Acknowledgement
The authors are thankful to Ms. Yanhong Song and Dr. Faiz-Ur Rahman of the Center for
Supramolecular Materials and Catalysis, Shanghai University, for helping with the NMR and
HRMS (ESI) analyses.
Conflict of Interest
The authors declare no conflict of interest in this experiment.
References
Alamed, J., Chaiyasit, W., McClements, D. J., & Decker, E. A. (2009). Relationship between
free radical scavenging and antioxidant activity in foods. Journal of Agricultural and
Food Chemistry, 57, 2969–2976.
AOCS (1995). Official Methods and Recommended practices of the American Oil Chemists’
Society, 4th ed. AOCS, Champaign, IL. (USA).
AOCS (2011). Official methods and recommended practices of the American Oil Chemists’
Society. AOCS Method Cd8b-90., AOCS Press, Champaign, IL. (USA).
Bañares, C., Martin, D., Reglero, G., & Torres, C. F. (2019). Protective effect of hydroxytyrosol
21

and rosemary extract in a comparative study of the oxidative stability of Echium oil.
Food Chemistry, 290, 316–323.
Bushra, S., Farooq, A., & Roman, P. (2007). Antioxidant potential of corncob extracts for
stabilization of corn oil subjected to microwave heating. Food Chemistry, 104, 997–100.
Chen, Z., Bertin, R., & Froldi, G. (2013). EC50 estimation of antioxidant activity in DPPH assay
using several statistical pro-grams. Food Chemistry, 138, 414–420.
Codex Alimentarius, C. (1999). Codex standard for named vegetable oils. Codex Stan, 210, 1–
13.
de Pinedo, A. T., Peñalver, P., & Morales, J. C. (2007). Synthesis and evaluation of new
phenolic-based antioxidants: structure–activity relationship. Food Chemistry, 103, 55–61.
Dominique, T., Jean‐Louis, B., Barbara, B., Tara, D., Susan, F. T., Marina, H., et al. (2017).
Safety of hydroxytyrosol as a novel food pursuant to regulation (EC) No 258/97. EFSA
Journal, 15(3), e04728.
Farhoosh, R., & Hoseini-Yazdi, S. Z. (2014). Evolution of oxidative values during kinetic studies
on olive oil oxidation in the Rancimat test. Journal of the American Oil
Chemists’ Society, 91(2), 281–293.
Gallardo, E., Palma-Valdés, R., Sarriá, B., Gallardo, I., de la Cruz, J. P., Bravo, L., et al. (2016).
Synthesis and antioxidant activity of alkyl nitroderivatives of hydroxytyrosol. Molecules,
21, 656.
Gordon, M. H., Paiva-Martins, F., & Almeida, M. (2001). Antioxidant activity of hydroxytyrosol
acetate compared with that of other olive oil polyphenols. Journal of Agricultural and
Food Chemistry, 49(5), 2480–2485.
Hashemi, S. M-B., Brewer, S. M., Safari, J., Nowroozi, M., Sherahi, M. H-A., Sadeghi, B., et al.
(2016). Antioxidant activity, reaction mechanisms, and kinetics of Matricaria recutita
extract in commercial blended oil oxidation. International Journal of Food Properties,
19(2), 257-271.
Huang, Y., Jiang, Z., & Liao, X. (2014). Antioxidant activities of two novel synthetic methyl-
benzenediol derivatives. Czech Journal of Food Sciences, 32, 348-353.
Jiang, Z. W., Weng, X.C., Huang, Y., Hou, J. P., & Liao, X. Y. (2014). Synthesis and antioxidant
activity of two novel tetraphenolic compounds derived from toluhydroquinone and
tertiary butylhydroquinone. Grasas Aceites, 65, e016.
Kajiyama, T., & Ohkatsu, Y. (2001). Effect of para-substituents of phenolic antioxidants.
Polymer Degradation and Stability, 71, 445–452.
Lampi, A. M., Kataja, L., Kamal-Eldin, A., & Piironen, V. (1999). Antioxidant activities of a-
and γ-tocopherols in the oxidation of rapeseed oil triacylglycerols. Journal of the
American Oil Chemists' Society, 76, 749–755.
Marinova, E. M., Seizova, K. A., Totseva, I. R., Panayotova, S. S., Marekov, I. N., &
Momchilova, S. M. (2012). Oxidative changes in some vegetable oils during heating at
frying temperature. Bulgarian Chemical Communications, 44, 57–63.
Maszewska, M., Florowska, A., Dłuzewska, E., Wroniak, M., Marciniak-Lukasiak, K., &
22

Zbikowska, A. (2018). Oxidative stability of selected edible oils. Molecules, 23, 1746.
Mateos, R., Madrona, A., Pereira-Caro, G., Domínguez, V., Cert, R. M., Parrado, J., et al.
(2015). Synthesis and antioxidant evaluation of isochroman-derivatives of
hydroxytyrosol: Structure–activity relationship. Food Chemistry, 173, 313–320.
Mateos, R., Trujillo, M., Pereira-Caro, G., Madrona, A., Cert, A., & Espartero, J. L. (2008). New
lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydro-xytyrosyl esters.
Journal of Agricultural and Food Chemistry, 56(22), 10960–10966.
Nissiotis, M., & Tasioula-Margari, M. (2002). Changes in antioxidant concentration of virgin
olive oil during thermal oxidation. Food Chemistry, 77, 371–376.
Olajide, T. M., Pasdar, H., & Weng, X. C. (2018). A novel antioxidant:6,6'-(butane-1,1-
diyl)bis(4-methylbenzene-1,2-diol). Grasas Aceites, 69 (3), e269.
Paquot, C. (1979). Standard methods for the analysis of oils, fats and derivatives. 6th Ed.
Pergamon Press, Oxford UK.
Pereira-Caro, G., Madrona, A., Bravo, L., Espartero, J. L., Alcudia, F., Cert, A., et al. (2009).
Antioxidant activity evaluation of alkyl hydroxytyrosyl ethers, a new class of
hydroxytyrosol derivatives. Food Chemistry, 115, 86–91.
Ranalli, A., Lucera, A., & Contento, S. (2003). Antioxidizing potency of phenol compounds in
olive oil mill wastewater. Journal of Agricultural and Food Chemistry, 51, 7636–7641.
Rodriguez-Morato, J., Boronat, A., Kotronoulas, A., Pujadas, M., Pastor, A., Olesti, E., Pérez-
Mañá, C., et al. (2016). Metabolic disposition and biological significance of simple
phenols of dietary origin: Hydroxytyrosol and tyrosol. Drug Metabolism Reviews, 48(2),
218–236.
Roleira, F. M., Siquet, C., Orrù, E., Garrido, E. M., Garrido, J., Milhazes, N., et al. (2010).
Lipophilic phenolic antioxidants: correlation between antioxidant profile, partition
coefficients and redox properties. Bioorganic & Medicinal Chemistry, 18(16), 5816–
5825.
Saad, B., Sing, Y. Y., Nawi, M. A., Hashim, N. H., Ali, A. S. M., Saleh, M.I., et al. Sulaiman, S.
F., (2007). Determination of synthetic phenolic antioxidants in food items using reversed-
phase HPLC. Food Chemistry, 105, 389–394.
Shahidi, F., & Wanasundara, P. K. J. P. D. (1992). Phenolic antioxidants. Critical Reviews in
Food Science and Nutrition, 32, 67–103.
Shi, G., Liao, X., Olajide, T. M., Liu, J., Jiang, X., & Weng, X. C. (2017). Butylated caffeic acid:
an efficient novel antioxidant. Grasas Aceites, 68, e201.
Silva, F. A., Borges, F., Guimarães, C., Lima, J. L., Matos, C., & Reis, S. (2000). Phenolic acids
and derivatives: Studies on the relationship among structure, radical scavenging activity,
and physicochemical parameters. Journal of Agricultural and Food Chemistry, 48(6),
2122–2126.
Sun, Y., Zhou, D., & Shahidi, F. (2018). Antioxidant properties of tyrosol and hydroxytyrosol
saturated fatty acid esters. Food Chemistry, 245, 1262–1268.
Tadesse, N., Ret, N., & Beyero, N. (2017). Level of saturation and antioxidant value of heat
23

and spice treated animal butter. Food and Public Health, 7(4), 81-90.
Tofani, D., Balducci, V., Gasperi, T., Incerpi, S., & Gambacorta, A. (2010). Fatty acid
hydroxytyrosyl esters: Structure/antioxidant activity relationship by ABTS and in cell-
culture DCF assays. Journal of Agricultural and Food Chemistry, 58, 5292–5299.
Trujillo, M., Mateos, R., Collantes de Teran, L., Espartero, J. L., Cert, R., Jover, M., et al.
(2006). Lipophilic hydroxytyrosyl esters. Antioxidant activity in lipid matrices and
biological systems. Journal of Agricultural and Food Chemistry, 54(11), 3779–3785.
Weng, X. C., & Huang, Y. (2014). Relationship structure-antioxidant activity of hindered
phenolic compounds. Grasas Aceites, 65, e051.
Ying, Z., Lei, Y., Yuangang, Z., Xiaoqiang, C., Fuji, W., & Fang, L. (2010). Oxidative stability
of sunflower oil supplemented with carnosic acid compared with synthetic antioxidants
during accelerated storage. Food Chemistry, 118, 656–662.
Zhang, C. X., Wu, H., & Weng, X. C. (2004). Two novel synthetic antioxidants for deep frying
oils. Food Chemistry, 84, 219-222.
Zhang, Z., Xiao, B., Chen, Q., & Lian, X. Y. (2010). Synthesis and biological evaluation of
caffeic acid 3,4-dihydroxyphenethyl ester. Journal of Natural Products, 73, 252–254.
24

FIGURE CAPTIONS
Fig. 1. Synthetic route leading to 3-(tert-butyl)-4,5-dihydroxyphenylacetic acid methyl ester
(BuMT, 3) and 3-(tert-butyl)-5-(2-hydroxyethyl)benzene-1,2-diol (BuHT, 4).
Fig. 2. DPPH radical scavenging activity of lipophilic hydroxytyrosol derivatives (BuMT &
BuHT), HT, and TBHQ. Values are expressed as means ± SD (n=2).
Fig. 3. Changes in conjugated diene (a) and acid values (b) of soybean oil spiked with and
without antioxidant during deep frying. Values are expressed as mean ± SD (n=2).
Fig. 4. Changes in peroxide value (a), p-anisidine value (b) and Totox (c) of soybean oil spiked
with and without antioxidant during oven test. Values are expressed as mean ± SD (n=2).
25

Figure 1
26

Figure 2
27

Figure 3
28

Figure 4
29