Full text of DOI:10.3390/molecules23020275

molecules
Article
Lipase-Produced Hydroxytyrosyl Eicosapentaenoate
is an Excellent Antioxidant for the Stabilization of
Omega-3 Bulk Oils, Emulsions and Microcapsules
ID
Taiwo Olusesan Akanbi and Colin James Barrow *
Centre for Chemistry and Biotechnology, Deakin University, Locked Bag 20000, Geelong, VIC 3220, Australia;
taiwo.akanbi@deakin.edu.au
* Correspondence: colin.barrow@deakin.edu.au; Tel.: +61-3-5227-1318
Received: 7 January 2018; Accepted: 27 January 2018; Published: 29 January 2018
Abstract: In this study, several lipophilic hydroxytyrosyl esters were prepared enzymatically using
immobilized lipase from Candida antarctica B. Oxidation tests showed that these conjugates are
excellent antioxidants in lipid-based matrices, with hydroxytyrosyl eicosapentaenoate showing the
highest antioxidant activity. Hydroxytyrosyl eicosapentaenoate effectively stabilized bulk fish oil,
fish-oil-in-water emulsions and microencapsulated fish oil. The stabilizing effect of this antioxidant
may either be because it orients itself with the omega-3 fatty acids in the oil, thereby protecting
them against oxidation, or because this unstable fatty acid can preferentially oxidise, thus providing
an additional mechanism of antioxidant protection. Hydroxytyrosyl eicosapentaenoate itself was
stable for one year when stored at −20 ◦C.
Keywords: hydroxytyrosol; eicosapentaenoic acid; antioxidant; microencapsulation; lipase;
Candida antarctica B; enzyme; conjugation
1. Introduction
Olive oil phenols, particularly hydroxytyrosol and tyrosol, are natural polyphenols with
antioxidant activity and known health benefits [1]. Hydroxytyrosol has been demonstrated in
numerous studies to have cardioprotective properties, by preventing oxidative damage to low-density
lipoproteins (LDLs), and as a result inhibits the activation of inflammatory events and prevents
endothelial damage, the major causes of atherosclerosis [
secretion of inflammatory cytokines (IL-1 , IL-1 , IL-6, IL-2, TNF-
CCL2/MCP-1) [ ]. The European Food Safety Authority (EFSA) has concluded that a cause-and-effect
relationship exists between the consumption of hydroxytyrosol and protection against oxidative
2
,3
]. It was shown to diminish the
α
β
α
) and chemokines (CXCL10/IP-10,
3
damage of blood lipoproteins [
in several countries.
4]. Hydroxytyrosol is currently marketed as a nutritional supplement
The use of synthetic antioxidants in food and other nutritional products is not accepted by
consumers and regulators in some countries, shifting the attention of manufacturers to natural
antioxidants such as polyphenols. However, there are a limited number of natural antioxidants that can
be incorporated into lipid-based food matrices [5]. The hydrophilic property of hydroxytyrosol limits
its dispersion in fats and oils, thereby reducing its effectiveness in protecting lipids against oxidation.
Lipophilic derivatives of hydroxytyrosol will have improved oil solubility and antioxidant properties
in fats and oils. Esterification of hydroxytyrosol with fatty acids has been carried out chemically and
enzymatically [5–7]. Lipase-catalysed esterification reactions can be carried out under mild conditions
and with less side reactions compared with non-enzymatic methods [8,9].
Previous studies have found that an increase in the chain length of fatty esters of hydroxytyrosol,
up to a certain chain length, correlated with an increase in antioxidant activity [5–7,10,11].
Molecules 2018, 23, 275; doi:10.3390/molecules23020275
www.mdpi.com/journal/molecules

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However, the majority of these studies focused on the antioxidant activity of short to medium chain
alkyl esters of hydroxytyrosol. Recent studies have shown significantly higher antioxidant activity of
the unsaturated fatty esters of polyphenols over their saturated counterparts [12,13], indicating that
chain length and the degree of unsaturation of fatty acids has an impact on the antioxidant activity of
polyphenol esters. For instance, it has been shown that the antioxidant activity of polyunsaturated
fatty acid derivatives of quercetin (Q3G) increased with an increasing number of double bonds [13].
Although the omega-3 esters of hydroxytyrosol have been previously synthesized [14
to stabilize omega-3 oils were not explored.
,15], their abilities
A combination of different testing systems is needed to ensure a comprehensive assessment of an
antioxidant. Studies have shown that the physical nature of lipid medium has a significant impact
on the efficacy of antioxidant [16,17]. It has also been shown that the effectiveness of some fatty acid
derivatives of polyphenols differ in bulk oil and in oil-in-water emulsions [17]. While the antioxidant
activities of phenolic esters have been widely reported, studies on their stability over extended period
of storage are lacking. The ability of hydroxytyrosyl esters to stabilize microencapsulated fish oil has
not been previously investigated. Therefore, in this study, we enzymatically synthesized and purified
lipophilic derivatives of hydroxytyrosol and tested their antioxidant activities in a range of synthetic
and lipid-based matrices, including bulk fish oil, fish oil-in-water-emulsion and microencapsulated
fish oil. The stability of these antioxidants over an extended period of storage was also investigated.
2. Results and Discussion
2.1. Lipase-Catalysed Synthesis of Hydroxytyrosol-Fatty Acid Conjugates
Esterification conditions were optimized for the synthesis of hydroxytyrosyl decanoate using
immobilized lipase from Candida antarctica B (Novozym 435) and were applied ◦to the synthesis of
all conjugates. These conditions were: Solvent (diethyl ether), temperature (40 C), molar ratio of
substrates (hydroxytyrosol-fatty acid ratios; 1:2) and reaction times (6 h). Other general conditions are
as described in the methods section. Immobilized lipase was used in preference to free lipase since the
immobilized form tends to be more robust and is more widely used at the manufacturing scale due to
its ease of recovery and reuse.
2.2. Characterisation of Hydroxytyrosyl Esters
The synthesised hydroxytyrosyl esters are shown in Table 1. As shown, the shorter the fatty
acid chain, the higher the conversion rate of hydroxytyrosol. All saturated fatty acids except stearic
acid gave higher percent conversion than unsaturated fatty acids. This may be due to the straight
chain-like structure of saturated fatty acids, which allows them to stack tightly and have more
intermolecular interactions [12]. However, with the long chain unsaturated fatty acids, there appears
not to be a relationship between chain length and conversion, as higher conversion was obtained with
eicosapentaenoic acid (EPA) (68%) than with oleic acid (46%). Although some studies have shown
that conjugation of longer chain fatty acids with polyphenols results in low yield [12,18], others have
shown the opposite [
5,19]. The variation in results may be due to lipase fatty acid selectivity primarily
differentiating degree of unsaturation rather than fatty acid chain length.

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Table 1. Identification and characterization of conjugates by liquid chromatography-mass spectrometry
(LC-MS) and proton-nuclear magnetic resonance (1H-NMR) spectroscopy.
LCMS
Fragment
1H NMR Chemical Shift (δ, ppm) **
Conversion
(%) *
Compound
A
B
C
D
(CH3)
m/z
Allylic Olefinic Bis-Allylic
(CH2) (CH2) (CH2)
Hydroxytyrosyl
decanoate
85.61 ± 2.9 e
81.61 ± 1.6 d
76.72 ± 2.7 c
75.13 ± 3.9 c
61.82 ± 3.7 b
45.91 ± 0.5 a
68.13 ± 3.5 c
[M + H]+
[M + H]+
[M + H]+
[M + H]+
[M + H]+
[M + H]+
[M + H]+
309.42 2.29
337.48 2.29
365.53 2.32
393.58 2.31
421.64 2.33
419.62 2.35
439.28 2.34
1.62
1.63
1.63
1.64
1.66
1.65
1.75
1.28
1.29
1.29
1.28
1.28
1.30
1.29
-
-
-
0.90
0.90
0.90
0.93
0.96
0.97
0.99
Hydroxytyrosyl
laurate
-
-
-
Hydroxytyrosyl
myristate
-
-
-
-
-
Hydroxytyrosyl
palmitate
-
Hydroxytyrosyl
stearate
-
-
-
-
Hydroxytyrosyl
oleate
2.05
2.09
5.36
5.37
Hydroxytyrosyl
eicosapentaenoate
2.87
* Values are mean
±
standard deviation (SD) for triplicate replications. Mean values with different superscript
letters in the same column are significantly different (p < 0.05) as determined by Tukey–Kramer honestly significant
difference (HSD). ** A, B, C and D represent
protons, respectively.
α-Carboxyl, β-Carboxyl, aliphatic and terminal methyl group
The results of the liquid chromatography-mass spectrometry (LC-MS) and proton-nuclear
magnetic resonance (1H-NMR) analyses of the conjugates are shown in Table 1. The high-performance
liquid chromatography HPLC chromatogram and LC-MS spectrum of a representative sample
(hydroxytyrosyl eicosapentaenoate) are shown in Figure 1. Results (Table 1 and Figure 1) show that
the conjugates were synthesized as anticipated, since they had the expected mass/charge ratios (m/z).
0
For 1H-NMR, the chemical shift assignments (
δ, ppm) for free hydroxytyrosol were: δ 3.69 (2H, t, H-1 ),
0
0
0
0
2.66 (2H, t, H-2 ), 6.66 (1H, d, H-4 ), 6.70 (1H, d, H-7 ) and 6.54 (1H, dd, H-8 ). Selected 1H-NMR results
are presented in Table 1. As shown, no allylic, olefinic or bis-allylic protons were detected in all the
saturate-based conjugates, whereas bis-allylic protons were found in hydroxytyrosyl eicosapentaenoate
but not in hydroxytyrosyl oleate, consistent with the predicted structures.
(a)
(b)
Figure 1. Cont.

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(c)
Figure 1. HPLC chromatograms of hydroxytyrosyl eicosapentaenoate (
purification and its (c) LCMS spectrum.
a) before and (b) after
2.3. Antioxidant Activity by Spectrophotometric Methods
2.3.1. DPPH Radical Scavenging Activity
The 2,2-diphenyl-1-picrylhydrazyl DPPH radical scavenging assay is a rapid, substrate-free
technique for determining antioxidant activity. The results of the DPPH radical scavenging assay of
conjugates are expressed as EC50, that is, the concentration necessary to lower the initial DPPH by 50%,
and are presented in Table 2. As shown, there are no significant differences (p > 0.05) between the values
obtained for hydroxytyrosol and the conjugates, indicating that hydroxytyrosol and its lipid derivatives
show effective DPPH radical scavenging activity. A similar result was also obtained for butylated
hydroxytoluene (BHT), but
α
-tocopherol showed a significantly lower (p < 0.05) activity, requiring
-tocopherol is
9.87 1.03 M to lower the initial DPPH by 50%. Previous studies have shown that α
±
µ
less active against DPPH free radicals than polyphenols and their acyl esters, with some showing two
to three times more scavenging activities than α-tocopherol [6,20].
Table 2. Antioxidant activity of conjugates by DPPH EC50, ABTS ABTS (2,2-Azino-bis-3-
ethylbenzothiazoline-6-sulphonic) EC50 and β-carotene bleaching tests.
Test Compound
DPPH EC50 (µM)
ABTS EC50 (µM)
β-Carotene Inhibition Rate (%)
6.37 ± 0.82 a
6.55 ± 0.54 a
6.42 ± 0.26 a
6.71 ± 0.42 a
7.04 ± 0.33 a
6.85 ± 0.61 a
6.65 ± 0.25 a
6.06 ± 0.31 a
6.13 ± 0.62 a
9.87 ± 1.03 b
2.02 ± 0.41 a
2.12 ± 0.22 a
2.22 ± 0.16 a
2.19 ± 0.25 a
2.64 ± 0.55 a
2.05 ± 0.12 a
2.85 ± 0.28 a
2.45 ± 0.15 a
3.88 ± 0.38 b
5.82 ± 0.65 c
57.09 ± 3.10 b
59.99 ± 3.08 c
61.73 ± 1.60 c
61.45 ± 2.79 c
61.15 ± 2.38 c
62.39 ± 2.10 c
72.21 ± 3.07 d
45.80 ± 2.81 a
56.09 ± 3.65 b
62.43 ± 1.10 c
Hydroxytyrosyl decanoate
Hydroxytyrosyl laurate
Hydroxytyrosyl myristate
Hydroxytyrosyl palmitate
Hydroxytyrosyl stearate
Hydroxytyrosyl oleate
Hydroxytyrosyl eicosapentaenoate
Hydroxytyrosol
BHT
α-tocopherol
Values are mean
±
SD for triplicate replications. Mean values with different superscript letters in the same column
are significantly different (p < 0.05) as determined by Tukey–Kramer HSD.

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2.3.2. ABTS Radical Scavenging Activity
The 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulphonic (ABTS) radical scavenging test is based
on the inhibition of ABTS radicals, which are measured spectrophotometrically in the near-infrared
region at 734 nm [21]. Results presented in Table 2 show that all conjugates and hydroxytyrosol exhibit
lower EC50 values compared to BHT and α-tocopherol. Thus, based on concentration, hydroxytyrosol
and its acyl esters are more potent antioxidants than BHT and α-tocopherol.
The results of both the DPPH and ABTS tests show that the nature of the fatty acyl side chain does
not influence the antioxidant activity of the hydroxytyrosol conjugates. However, these results may not
reflect the antioxidant activity of these conjugates in real food systems, where the lifetime of radicals
are shorter than those of the artificial DPPH and ABTS radicals [18,22]. Since it has been shown that
the lipophilicity of polyphenols often enhances their antioxidant activity in certain systems [12
,
23],
further tests are needed to obtain a better understanding of the antioxidant activities of these conjugates
in different lipid systems.
2.3.3. Antioxidant Activity in a β-Carotene-Linoleate Model System
This method measures the rate of oxidative destruction of
oxidised linoleic acid in an emulsion system. In the presence of an antioxidant, bleaching of
occurs. In this study, the antioxidant activity of conjugates, BHT and -tocopherol are expressed
as percent inhibition relative to the control and results are presented in Table 2. As shown, all the
conjugates except hydroxytyrosyl decanoate inhibited the bleaching of -carotene by up to 60%.
β
-carotene by the free radicals of
β
-carotene
α
β
Hydroxytyrosyl eicosapentaenoate in this system had the highest antioxidant activity of all the
conjugates. Although there is no report on the antioxidant activity of hydroxytyrosyl eicosapentaenoate
in an emulsion system, studies have shown that some phenolic conjugates of unsaturated fatty acids
exhibit antioxidant activities in an emulsion system [12,13]. For instance, unsaturated derivatives
of quercetin-3-O-glucoside (oleate, linoleate, eicosapentaenoate and docosahexaenoate) have shown
significantly higher antioxidant activities than quercetin-3-O-glucoside (Q3G) in fish-oil-in-water
emulsion [13]. Tyrosyl oleate has also been reported to better stabilize omega-3 rich oil-in-water
emulsion than tyrosyl stearate and tyrosol [12].
Hydroxytyrosol itself performed worse than all the test compounds, including BHT and
α
-tocopherol, in this antioxidant assay (Table 2). The behavior of hydroxytyrosol is consistent with the
“polar paradox” theory, which postulates that lipophilic antioxidants are more effective in oil-in-water
emulsions, while hydrophilic ones are more effective in bulk oil [16]. This explains why the synthesized
conjugates with improved lipophilicity showed a higher inhibitory effect on β-carotene bleaching than
did hydroxytyrosol. Several studies have shown that lyophilized polyphenols are more effective than
their parent polyphenols in oil-in-water emulsion systems [12,13,18].
2.4. Antioxidant Activity Under Rancimat Conditions
The antioxidant activities of conjugates were evaluated in anchovy oil at a concentration of
1 mmol/kg oil using the Rancimat accelerated oxidation t◦est method. BHT and
α-tocopherol were
used as standard antioxidants and a temperature of 80 C was applied to minimize the loss of
BHT by evaporation. As shown (Figure 2a), hydroxytyrosol gave the highest oxidative stability
index (OSI) value of all the test compounds. This is consistent with the “polar paradox” theory
that hydrophilic antioxidants are more effective in bulk oil than lipophilic ones [16]. The partially
oil-soluble hydroxytyrosol may be preferentially located at the oil interface and is hence more
effective in inhibiting oxidation than the conjugates that are dissolved in the lipid phase. Similar
behavior has been reported for trolox, ascorbic, gallic, caffeic and ferulic acids. They showed higher
antioxidant activities than their non-polar alkyl esters in bulk oil [17]. Among the conjugates tested,
hydroxytyrosyl eicosapentaenoate gave the highest stability, followed by hydroxytyrosyl palmitate,
which showed a slightly higher stability than other conjugates (Figure 2a). While it has been reported

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that short-to-medium-chain lipophilic esters of polyphenols are more effective antioxidants than
long-chain esters in bulk oil [17], some studies have shown equal or higher activity with long-chain
esters [
7,13]. Therefore, the antioxidant activity of lipophilic derivatives of polyphenols is not directly
related to the fatty acid chain length.
(a)
(b)
Figure 2. Effects of conjugates, butylated hydroxytoluene (BHT and
stability of anchovy oil ( ) at a concentration of 1 mmol/kg oil and (
Values are expressed as mean standard deviation (n = 3). Bars not sharing a common letter are
α
-tocopherol on the oxidation
a
b) at different concentrations.
±
significantly different (p < 0.05) within each treatment as determined by Tukey–Kramer HSD.
Since antioxidant activity is concentration-dependent and some antioxidants can exhibit
prooxidant behavior at high concentrations, we investigated the effect of concentration on the
antioxidant activity of hydroxytyrosyl eicosapentaenoate, palmitate, BHT and
α-tocopherol in
anchovy oil. The results presented in Figure 2b show that increasing the concentration of all test
compounds resulted in increased oil stability. However, beyond 2 mmol/kg oil, no further increase
was observed for BHT and α-tocopherol. Meanwhile, for the hydroxytyrosyl esters, oil stability
increased up to a concentration of 3 mmol/kg oil but only increased slightly at concentrations
above that. The significantly high stability observed with hydroxytyrosyl eicosapentaenoate over the
palmitate (p < 0.05) may either be because it orients itself with the omega-3 fatty acids in anchovy
oil, thereby protecting them against oxidation, or because this unstable fatty acid can preferentially

Molecules 2018, 23, 275
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oxidise providing an additional mechanism of antioxidant protection. A recent study has shown
that the antioxidant activity of polyunsaturated fatty acid derivatives of quercetin (Q3G) increased
with increasing double bonds [13]. The authors found that at a concentration of 0.5 mmol/L of
oil, the inhibition of secondary lipid oxidation (TBARS) in bulk fish oil were 34% (Q3G linoleate),
38% (Q3G linolenate), 46% (Q3G eicosapentaenoate) and 50% (Q3G docosahexaenoate), respectively.
The use of synthetic antioxidants such as BHT has been linked to health issues such as liver damage
and carcinogenesis [24]. Also, high levels of α-tocopherol in oil can result in prooxidation and
therefore limits the usefulness of this natural antioxidant when used on its own [25]. It is important to
identify new natural antioxidants that can be used alone or in combination with compounds such as
α
-tocopherol to improve the stability of unstable ingredients such as fish oil. Although, hydroxytyrosyl
eicosapentaenoate and palmitate showed good antioxidant activity in anchovy oil under Rancimat
conditions, further tests are needed to obtain a better understanding of their antioxidant activity in
different lipid matrices.
2.5. Antioxidant Activity under Schaal Oven Conditions
The effect of hydroxytyrosyl eicosapentaenoate and hydroxytyrosyl palmitate (3 mmol/kg oil)
on the oxidative stability of bulk anchovy oil and its oil-in-water emulsion was determined under
◦
Schaal oven conditions at 60 C for five days. Oxidized oil from the oil-in-water emulsion was
extracted using methanol:heptane mix (1:1) as previously described [26,27]. Level of oxidation was
monitored by applying conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS)
tests. CD is an indicator of the formation of primary oxidation products such as hydroperoxides,
whereas TBARS are used to determine the presence of secondary oxidation products such as aldehydes
and dialdehydes in oils [8,28].
The impact of hydroxytyrosyl eicosapentaenoate and hydroxytyrosyl palmitate on the formation
of CD and TBARS in bulk anchovy oil during extended storage is shown in Figure 3a,b, respectively.
The conjugates significantly inhibited the release of CD and TBARS throughout the 120 h of storage
compared to the control. Their abilities to suppress the formation of CD and TBARS may be attributed
to their higher radical scavenging activities [29]. The CD and TBARS values in the anchovy oil emulsion
are presented in Figure 3c,d. As shown, both conjugates inhibited CD and TBARS formation up to
72 h, but the values of these oxidation products rose afterwards. This later increase in oxidation may
partly be due to the high level of water in the emulsion, compared to that in the bulk oil, which was
relatively stable.
(a)
(b)
Figure 3. Cont.

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(c)
(d)
Figure 3. Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) values of
bulk anchovy oil ( ) and those of the oil extracted from the oil-in-water emulsion ( ). Values are
expressed as mean ± standard deviation (n = 3).
a,
b
c,d
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derivatives of quercetin-3-O-
glucoside (Q3G eicosapentaenoate and Q3G docosahexaenoate) significantly inhibited lipid
peroxidation in fish oil-in-water emulsion compared with quercetin-3-O-glucoside (Q3G) [13].
Increased concentration of Q3G eicosapentaenoate and Q3G docosahexaenoate led to an increase in the
inhibition of peroxides and TBARS formation. A 10 mmol/L concentration of Q3G docosahexaenoate
in fish-oil-in-water emulsion led to 100% inhibition of peroxidation [13]. These results are consistent
with our observations that an increase in the concentration of hydroxytyrosyl esters (hydroxytyrosyl
palmitate and hydroxytyrosyl eicosapentaenoate) resulted in increased oil stability. Although the
inhibitory effect of hydroxytyrosyl eicosapentaenoate on CD and TBARS formation in bulk oil and
oil-in-water emulsion was slightly higher than that of hydroxytyrosyl palmitate, both conjugates are
potent antioxidants.
2.6. Stability Against Oxidation of Encapsulated Anchovy Oil
Microencapsulation helps protect oil against oxidation, particularly in the presence of antioxidants.
We have previously microencapsulated fish oil with gelatin-sodium hexametaphosphate (SHMP) using
a multicore complex coacervation technique [30,31]. In this study, we used complex coacervation to
microencapsulate anchovy oil with hydroxytyrosyl palmitate and hydroxytyrosyl eicosapentaenoate
(3 mmol/kg oil) and tested their stability against oxidation. The morphology of the coacervates
in the aqueous phase after agglomeration are presented in Figure 5a, while the microcapsules of
blank anchovy oil and oil with hydroxytyrosyl palmitate and hydroxytyrosyl eicosapentaenoate are
presented in Figure 5b–d, respectively. For each sample, we obtained a high encapsulation efficiency
(between 96% and 98%) and yield (between 94% and 97%) [30,31].
(a)
(b)
Figure 4. Cont.

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(c)
(d)
(e)
Figure 5.
agglomeration and microcapsules of (
and ( ) hydroxytyrosyl eicosapentaenoate; (
anchovy oil by accelerated oxidation test using Rancimat. Values are expressed as mean
(
a
) The morphology of formed coacervates (blank anchovy oil) in the aqueous phase after
) blank anchovy oil and oil with ( ) hydroxytyrosyl palmitate
) Oxidative stability index of blank and microencapsulated
standard
b
c
d
e
±
deviation (n = 3). Mean values not sharing a common letter are significantly different (p < 0.05)
as determined by Tukey–Kramer HSD.
The accelerated oxidative stability tests (Rancimat) of microencapsulated samples are presented
in Figure 5e. As shown, the OSI value of blank non-encapsulated anchovy oil (1.9
increased after microencapsulation (18.2 2.1 h) even without an added antioxidant. Much higher
increases were observed in the presence of conjugates, with hydroxytyrosyl eicosapentaenoate giving
a higher OSI value (38.5 3.8 h) than hydroxytyrosyl palmitate (31.7 1.8 h). These results indicate
±
0.1 h) significantly
±
±
±
that lipophilic esters of polyphenols, and in particular hydroxytyrosyl eicosapentaenoate are useful
antioxidants for stabilising microencapsulated omega-3 oils.
Microencapsulated fish oil is a functional food ingredient with health benefits associated with the
EPA and DHA fatty acids. In addition to acting as an antioxidant, hydroxytyrosyl eicosapentaenoate
may also provide some additional health benefits. These may be beyond the known benefits of
hydroxytyrosol as an anti-inflammatory compound due to the synergistic benefits of the conjugated
EPA, particularly in neurodegenerative diseases [32]. However, exploring the additional health benefits
of this conjugate is outside the scope of the current study.
2.7. Stability of Conjugates After Extended Storage
The antioxidant activity of hydroxytyrosyl palmitate and hydroxytyrosyl eicosapentaenoate were
◦
tested after one year storage in a
−
20 C freezer. The results of DPPH and ABTS assays were consistent
with those obtained earlier (Table 2), although with slight differences. These were, for hydroxytyrosyl

Molecules 2018, 23, 275
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palmitate (DPPH EC50, 7.08
eicosapentaenoate (DPPH EC50, 6.18
show that fatty acid esters of hydroxytyrosol can be stored for use as antioxidants.
±
0.55
µ
M and ABTS EC50, 2.75
±
0.68
µ
M) and hydroxytyrosyl
0.45 µM). These results
±
0.82 M and ABTS EC50, 3.01
µ
±
3. Materials and Methods
3.1. Materials
Hydroxytyrosol was purchased from Biotain Pharma Co., Ltd. (Xiamen, Fujian, China)
and its purity was confirmed by HPLC, NMR and LC-MS. Anchovy oil was provided
by Lipa Pharmaceuticals Pty. Ltd. (Minto, NSW, Australia). Transglutaminase (Activa®
KS–LS) was purchased from Ajinomoto (Tokyo, Japan). Gelatin, sodium hexametaphosphate
(SHMP), 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulphonic (ABTS), alpha-tocopherol, butylated
hydroxytoluene (BHT), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), beta-carotene, immobilized lipase
from Candida antarctica B, fatty acids, hydroxytyrosol standard and gas chromatography standards
were purchased from Sigma Aldrich (Castle Hill, Australia). EPA-ethyl ester (EPA-EE) was provided
by Photonz Corporation (Auckland, New Zealand) and was converted to free fatty acid (FFA) as
previously reported [8]. Thin layer chromatography standards were purchased from Nu-Chek Prep
(Elysian, MN, USA). All other chemicals used were of analytical grade.
3.2. Enzymatic Synthesis of Hydroxytyrosyl Esters
Synthesis was first carried out using decanoic acid and hydroxytyrosol to standardize esterification
conditions. Reaction was performed in a stoppered bottle consisting 20 mL 2-methyl-2-butanol (2M2B),
2 mmol hydroxytyrosol and fatty acid (1:1), 150 mg immobilized lipase from Candida antarctica B
◦
and 150 mg molecular sieves (3 Å, 4–8 mesh). The reaction mixture was incubated at 50 C for
24 h with magnetic stirring at 250 rpm. The progress of the reaction was monitored by capillary
chromatography with flame ionization detector (Iatroscan MK5, Iatron Laboratories Inc., Tokyo, Japan).
The Iatroscan-FID settings and solvent systems were as previously described [33]. Percentage
conversion was calculated as the area of ester peak divided by the combined areas of ester peak
and hydroxytyrosol peak.
3.2.1. Optimizing Synthesis Conditions
The effects of different solvents (2-methyl-2-butanol, tert-butanol, isooctane, diethyl ether,
◦
ethyl acetate), temperatures (40 and 50 C), molar ratio of substrates (hydroxytyrosol-fatty acid
ratios; 1:1, 1:2, 1:3, 1:5) and reaction times (1–24 h) on the ester formation were investigated by varying
one factor at a time while keeping the others constant. The conditions established for the optimum
synthesis of hydroxytyrosyl decanoate were then applied for the synthesis of other esters.
3.2.2. Scaled-up Synthesis of Hydroxytyrosyl Esters
Having established the optimum conditions for the synthesis of hydroxytyrosyl esters (including
solvent type, temperature, molar ratio of hydroxytyrosol and fatty acid, and reaction time),
esterification reactions were scaled up to provide sufficient samples for subsequent experiments.
The scaled-up conditions were as follows: 15 mmol hydroxytyrosol and fatty acid (optimized mole
ratio), 150 mL solvent, 600 mg immobilized lipase from Candida antarctica B and 450 mg molecular
sieves (3 Å, 4–8 mesh). The reaction mixture was incubated with magnetic stirring (250 rpm) at
predetermined temperature and time.
3.3. Identification, Purification and Characterisation of Hydroxytyrosyl Esters
Immobilized enzyme and molecular sieves were removed by centrifugation at 5432
and the solvent from the reaction mixture was dried in vacuo. The resulting precipitate was resolved in
deionized water and heptane (1:5 v/v) after sparging with nitrogen. Mixture was shaken for 2 min and
×
g for 5 min

Molecules 2018, 23, 275
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allowed to stand for another 5 min. Hydroxytyrosyl ester and unreacted free fatty acid went into the
organic phase while the unreacted hydroxytyrosol dissolved in the water phase. Silica gel column was
then used to separate hydroxytyrosyl ester from the free fatty acid by eluting with heptane/diethyl
ether/acetic acid mixtures (50:40:2). The fractions collected were analysed by capillary chromatography
with flame ionization detector (Iatroscan-FID) to confirm separation [34]. Samples were also analysed
by Agilent 1200 Series HPLC system with the Agilent 1200 Series photodiode array detector using a C18
reversed-phase column, 4.6 mm
mobile phase 90/10 (v/v), methanol/water, flow rate 1 mL/min, temperature 27 C, injection volume
×
250 mm (Phenomenex, Torrance, CA, USA). HPLC conditions were:
◦
10 µL, detection UV detector at 210 nm. Prior to analysis, the column was equilibrated with mobile the
phase under the above conditions for 30 min. Pure reactants (fatty acids and hydroxytyrosol) were used
1
as reference standards. H NMR spectra were acquired on a Bruker Avance 500 MHz spectrometer
1
(Bruker Karlsruhe, Germany) as previously reported [
8
]. To record H NMR spectrum, 10 mg of
sample was dissolved in deuterated methanol (CD3OD; 0.4 mL). Chemical shifts were referenced to
the residual solvent peak. Mass spectrometry (MS) spectra of samples were recorded on a Thermo
NanoLC/OrbiTrap Fusion Lumos mass spectrometer (ThermoFisher Scientific) using a combination of
acetonitrile (90%) and water (10%) as mobile phase.
3.4. Measurement of Antioxidant Activity by Spectrophotometric Methods
3.4.1. DPPH Radical Scavenging Method
The DPPH radical scavenging assay was carried out following a previously reported method [35
with some modifications. Briefly, 2 mL of DPPH (40 M in methanol) solution was added to
50 L of various concentrations (0, 0.5, 1, 5, 10 and 20 M) of hydroxytyrosyl esters dissolved in
]
µ
µ
µ
methanol. Contents were thoroughly mixed, incubated at room temperature for 30 min in the dark and
absorbance was read at 517 nm in a Cary 300 Bio UV-visible Spectrophotometer (Agilent Technologies).
DPPH radical scavenging activity was calculated as:
Radical scavenging activity (%) = [(Acontrol − Asample)/Acontrol] × 100
(1)
where Acontrol and Asample are absorbance of control and sample, respectively. Control contained no
test compound.
EC50 value, which corresponds to the ester concentration necessary to lower the initial
DPPH by 50% was determined by linear regression analysis of the graph of scavenging activity
(%) vs. concentration. Low EC50 value indicates the effectiveness of the antioxidant to act as
DPPH scavenger.
3.4.2. ABTS Radical Scavenging Method
The ABTS radical scavenging activity was measured as previously described [36] with minor
modifications. ABTS radical cation (ABTS+) was generated by reacting 7 mM aqueous solution of
ABTS with 2.45 mM potassium persulphate (K2S2O8) and the mixture stood for 16 h in the dark before
use. Afterwards, solution was diluted with ethanol to an absorbance of approximately 0.7 at 734 nm
(Cary 300 Bio UV-visible Spectrophotometer, Agilent Technologies). Different concentrations (0, 0.5, 1,
5, 10 and 20 µ
M) of hydroxytyrosyl esters (2 mL) was mixed with the ethanolic ABTS+ solution (2 mL)
and absorbance was read at 734 nm after 15 min. Scavenging activity and EC50 was calculated as
described in Section 3.4.1.
3.4.3. β-Carotene Bleaching Method
The antioxidant activities of hydroxytyrosyl esters were determined using the
bleaching method [37]. Briefly, -carotene (10 mg) was dissolved in chloroform (10 mL) and an aliquot
(1 mL) of this was transferred into a round bottom flask containing linoleic acid (20 mg) and Tween
β-carotene
β

Molecules 2018, 23, 275
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40 (200 mg). Chloroform was removed in vacuo and aerated distilled water (50 mL) was added.
Contents were mixed using a magnetic stirrer and 4.8 mL of the mixture was added to test tubes
containing 0.2 mL of hydroxytyrosyl esters (1 mM in ethanol) before incubation at 50 ◦C. Absorbance
was measured at 450 nm every 20 min for 120 min. Blank sample was prepared without
β
-carotene and
control was prepared without test compounds. Percent inhibition of
was calculated as:
β-carotene-linoleic acid oxidation
Inhibition (%) = 100 × [1 − (As0 − As120)/(Ac0 − Ac120)]
(2)
where As0
−
As120 are absorbance values for test samples at 0 min and 120 min while Ac0
−
Ac120 are
absorbance values for control at 0 min and 120 min, respectively.
3.5. Accelerated Antioxidant Tests
3.5.1. Preparation of Bulk Oil and Oil-in-Water Emulsion Containing Hydroxytyrosyl Esters
For bulk oil preparation, required amount of hydroxytyrosyl esters were first dissolved in ethanol
before being mixed with oil (5 g). Ethanol was then removed in vacuo. Oil-in-water emulsion (30 mL)
was prepared by emulsifying 3 g anchovy oil, 300 mg Tween 40, required amount of hydroxytyrosyl
esters and distilled water. Mixtures were emulsified at 15,000 rpm for 15 min using a homogenizer
(Unidrive 1000, CAT Scientific, Paso Robles, CA, USA).
3.5.2. Measurement of Antioxidant Activity Using the Schaal Oven Test
The oxidative stability of bulk ◦anchovy oil and its oil-in-water emulsion was determined
under Schaal oven conditions at 60 C for five days [28]. Briefly, bulk oil (5 g) and oil-in-water
emulsion (30 mL) samples (in dark screw-capped bottles) were placed in the oven and samples
were withdrawn every 24 h for 120 h (five days). Oxidized oil from the oil-in-water emulsion was
extracted using methanol:heptane mix (1:1) as previously described [26,27]. The conjugated dienes
(CD) and thiobarbituric acid reactive substances (TBARS) values of the oil samples were measured as
previously reported [28].
3.5.3. Measurement of Antioxidant Activity by Rancimat Method
Anchovy oil (4 g) or microencapsulated products (1.5 g) were subjected to accelerated oxidation
by Rancimat (model 743, Metrohm, Switzerland) as previously reported [30]. Samples were heated
at 80 ◦C under an air flow rate of 20 L/h and induction time (IT) was recorded for each sample as
a measure of their oxidative stability index (OSI).
3.6. Microencapsulation by Coacervation
Oil samples (20 g) containing known amounts of hydroxytyrosyl esters were microencapsulated
and characterised as previously reported [30]. The formation of complex coacervates was monitored by
an Axiotron microscope equipped with a camera (Carl Zeiss GMBH, Oberkochen, Baden-Württemberg,
Germany). The encapsulation efficiency (EE), payload (PL) and encapsulation yield (EY) were
determined as previously described [30] and oxidative stability tests of microencapsulated products
were carried out immediately after freeze drying and milling.
3.7. Statistical Analysis
Experiments were carried out in triplicate and data are presented as the mean
±
standard
deviation (SD). Levene’s test of one-way analysis of variance (ANOVA) was used to verify the
homogeneity of variance. Multiple comparisons were achieved by Tukey–Kramer HSD (honestly
significant difference). Mean differences were significant when probability is less than 0.05 (p < 0.05).
Statistical analysis was performed using SPSS® statistics 22.0 software.

Molecules 2018, 23, 275
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4. Conclusions
Lipase-catalysed conjugation of hydroxytyrosol with a range of fatty acids was carried
out, followed by identification and purification of the resultant conjugates using Iatroscan-FID,
mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. All conjugates
exhibited high scavenging activity towards 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and
2,2-Azino-bis-3-ethylbenzothiazoline-6-sulphonic (ABTS) radicals,
eicosapentaenoate exhibiting the strongest inhibitory effect -carotene bleaching. In bulk oil,
hydroxytyrosyl eicosapentaenoate resulted in the highest stability, followed by hydroxytyrosyl
palmitate, with both showing higher stabilising activity than BHT and -tocopherol. Hydroxytyrosyl
with hydroxytyrosyl
β
α
eicosapentaenoate also showed superiority in stabilizing fish-oil-in-water emulsion, as determined
through CD and TBARS tests during accelerated oxidation at 60 ◦C for five days. The effectiveness of
these antioxidants, particularly hydroxytyrosyl eicosapentaenoate, for stabilizing microencapsulated
fish oil was also demonstrated. These results indicate that lipase synthesized esters of hydroxytyrosol,
particularly hydroxytyrosyl palmitate and hydroxytyrosyl eicosapentaenoate, are useful antioxidants
for stabilizing omega-3 oils such as fish oil and for addition to oil/fat-based foods. Further studies
are needed to investigate if these conjugates have similar or different bioactive properties
to hydroxytyrosol.
Acknowledgments: Authors thank Australian Research Council for funding, through the Linkage Grant
LP140100722 “Novel enzymatic processing methods and stabilization technologies for Omega-3 oils”. The authors
also acknowledge the Australian Research Council for funding Deakin University’s Nuclear Magnetic Resonance
Facility through LIEF grant LE110100141. Taiwo O. Akanbi acknowledges Pro-vice-chancellor’s equipment award
for early career researchers.
Author Contributions: T.O.A and C.J.B conceived and designed the experiments; T.O.A performed the
experiments and analysed the data; both authors wrote, revised and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Pazos, M.; Alonso, A.; Sánchez, I.; Medina, I. Hydroxytyrosol prevents oxidative deterioration in foodstuffs
rich in fish lipids. J. Agric. Food Chem. 2008, 56, 3334–3340. [CrossRef] [PubMed]
2.
Morton, L.W.; Caccetta, R.A.A.; Puddey, I.B.; Croft, K.D. Chemistry and biological effects of dietary phenolic
compounds: Relevance to cardiovascular disease. Clin. Exp. Pharmacol. Physiol. 2000, 27, 152–159. [CrossRef]
[PubMed]
3.
4.
Richard, N.; Arnold, S.; Hoeller, U.; Kilpert, C.; Wertz, K.; Schwager, J. Hydroxytyrosol is the major
anti-inflammatory compound in aqueous olive extracts and impairs cytokine and chemokine production in
macrophages. Planta Med. 2011, 77, 1890–1897. [CrossRef] [PubMed]
Turck, D.; Bresson, J.L.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; Hirsch-Ernst, K.I.;
Mangelsdorf, I.; McArdle, H.J.; Naska, A. Safety of hydroxytyrosol as a novel food pursuant to Regulation
(EC) No. 258/97. EFSA J. 2017, 15, 4728. [CrossRef]
5.
6.
Bouallagui, Z.; Bouaziz, M.; Lassoued, S.; Engasser, J.M.; Ghoul, M.; Sayadi, S. Hydroxytyrosol acyl esters:
Biosynthesis and activities. Appl. Biochem. Biotechnol. 2011, 163, 592–599. [CrossRef] [PubMed]
Pereira-Caro, G.; Madrona, A.; Bravo, L.; Espartero, J.L.; Alcudia, F.; Cert, A.; Mateos, R. Antioxidant activity
evaluation of alkyl hydroxytyrosyl ethers, a new class of hydroxytyrosol derivatives. Food Chem. 2009, 115,
86–91. [CrossRef]
7.
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. [CrossRef] [PubMed]
8.
9.
Akanbi, T.O.; Barrow, C.J. Lipase-catalysed incorporation of EPA into emu oil: Formation and characterisation
of new structured lipids. J. Funct. Foods 2015, 19, 801–809. [CrossRef]
Akanbi, T.O.; Barrow, C.J. Candida antarctica lipase a effectively concentrates DHA from fish and
thraustochytrid oils. Food Chem. 2017, 229, 509–516. [CrossRef] [PubMed]

Molecules 2018, 23, 275
14 of 15
10. Lucas, R.; Comelles, F.; Alcántara, 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 potential explanation for
the nonlinear hypothesis of the antioxidant activity in oil-in-water emulsions. J. Agric. Food Chem. 2010, 58,
8021–8026. [CrossRef] [PubMed]
11. Medina, I.; Lois, S.; Alcántara, D.; Lucas, R.; Morales, J.C. Effect of lipophilization of hydroxytyrosol on its
antioxidant activity in fish oils and fish oil-in-water emulsions. J. Agric. Food Chem. 2009, 57, 9773–9779.
[CrossRef] [PubMed]
12. Pande, G.; Akoh, C.C. Enzymatic synthesis of tyrosol-based phenolipids: Characterization and effect of alkyl
chain unsaturation on the antioxidant activities in bulk oil and oil-in-water emulsion. J. Am. Oil Chem. Soc.
2016, 93, 329–337. [CrossRef]
13. Warnakulasuriya, S.N.; Rupasinghe, H. Long chain fatty acid acylated derivatives of quercetin-3-O-glucoside
as antioxidants to prevent lipid oxidation. Biomolecules 2014, 4, 980–993. [CrossRef] [PubMed]
14. de Pinedo, A.T.; Penalver, P.; Rondon, D.; Morales, J. Efficient lipase-catalyzed synthesis of new lipid
antioxidants based on a catechol structure. Tetrahedron 2005, 61, 7654–7660. [CrossRef]
15. Zhou, D.-Y.; Sun, Y.-X.; Shahidi, F. Preparation and antioxidant activity of tyrosol and hydroxytyrosol esters.
J. Funct. Foods 2017, 37, 66–73. [CrossRef]
16. Porter, W.L.; Black, E.D.; Drolet, A.M. Use of polyamide oxidative fluorescence test on lipid emulsions:
Contrast in relative effectiveness of antioxidants in bulk versus dispersed systems. J. Agric. Food Chem. 1989
37, 615–624. [CrossRef]
,
17. Shahidi, F.; Zhong, Y. Revisiting the polar paradox theory: A critical overview. J. Agric. Food Chem. 2011, 59,
3499–3504. [CrossRef] [PubMed]
18. Wang, J.; Shahidi, F. Acidolysis of p-coumaric acid with omega-3 oils and antioxidant activity of phenolipid
products in in vitro and biological model systems. J. Agric. Food Chem. 2013, 62, 454–461. [CrossRef]
[PubMed]
19. Mateos, R.; Trujillo, M.; Pereira-Caro, G.; Madrona, A.; Cert, A.; Espartero, J.L. New lipophilic tyrosyl esters.
Comparative antioxidant evaluation with hydroxytyrosyl esters. J. Agric. Food Chem. 2008, 56, 10960–10966.
[CrossRef] [PubMed]
20. Lu, Y.; Foo, L.Y. Antioxidant and radical scavenging activities of polyphenols from apple pomace. Food Chem.
2000, 68, 81–85. [CrossRef]
21. Antolovich, M.; Prenzler, P.D.; Patsalides, E.; McDonald, S.; Robards, K. Methods for testing antioxidant
activity. Analyst 2002, 127, 183–198. [CrossRef] [PubMed]
22. Tan, Z.; Shahidi, F. Chemoenzymatic synthesis of phytosteryl ferulates and evaluation of their antioxidant
activity. J. Agric. Food Chem. 2011, 59, 12375–12383. [CrossRef] [PubMed]
23. Zhong, Y.; Shahidi, F. Lipophilized epigallocatechin gallate (EGCG) derivatives as novel antioxidants.
J. Agric. Food Chem. 2011, 59, 6526–6533. [CrossRef] [PubMed]
24. Williams, G.; Iatropoulos, M.; Whysner, J. Safety assessment of butylated hydroxyanisole and butylated
hydroxytoluene as antioxidant food additives. Food Chem. Toxicol. 1999, 37, 1027–1038. [CrossRef]
25. Martin-Rubio, A.; Sopelana, P.; Ibargoitia, M.; Guillén, M.D. Prooxidant effect of α-tocopherol on soybean
oil. Global monitoring of its oxidation process under accelerated storage conditions by 1h nuclear magnetic
resonance. Food Chem. 2018, 245, 312–323. [CrossRef] [PubMed]
26. Khan, M.A.; Shahidi, F. Oxidative stability of stripped and nonstripped borage and evening primrose oils
and their emulsions in water. J. Am. Oil Chem. Soc. 2000, 77, 963–969. [CrossRef]
27. Frankel, E.N.; Huang, S.W.; Prior, E.; Aeschbach, R. Evaluation of antioxidant activity of rosemary extracts,
carnosol and carnosic acid in bulk vegetable oils and fish oil and their emulsions. J. Sci. Food Agric. 1996, 72,
201–208. [CrossRef]
28. Hamam, F.; Shahidi, F. Synthesis of structured lipids via acidolysis of docosahexaenoic acid single cell oil
(DHASCO) with capric acid. J. Agric. Food Chem. 2004, 52, 2900–2906. [CrossRef] [PubMed]
29. Maqsood, S.; Benjakul, S. Comparative studies of four different phenolic compounds on in vitro antioxidative
activity and the preventive effect on lipid oxidation of fish oil emulsion and fish mince. Food Chem. 2010, 119,
123–132. [CrossRef]
30. Wang, B.; Adhikari, B.; Barrow, C.J. Optimisation of the microencapsulation of tuna oil in gelatin–sodium
hexametaphosphate using complex coacervation. Food Chem. 2014, 158, 358–365. [CrossRef] [PubMed]

Molecules 2018, 23, 275
15 of 15
31. Wang, B.; Vongsvivut, J.; Adhikari, B.; Barrow, C.J. Microencapsulation of tuna oil fortified with the multiple
lipophilic ingredients vitamins A, D3, E, K2, curcumin and coenzyme Q10. J. Funct. Foods 2015, 19, 893–901.
[CrossRef]
32. Mazza, M.; Pomponi, M.; Janiri, L.; Bria, P.; Mazza, S. Omega-3 fatty acids and antioxidants in neurological
and psychiatric diseases: An overview. Prog. Neuropsychopharmacol. Biol. Psychiatry 2007, 31, 12–26.
[CrossRef] [PubMed]
33. Akanbi, T.O.; Adcock, J.L.; Barrow, C.J. Selective concentration of EPA and DHA using thermomyces
lanuginosus lipase is due to fatty acid selectivity and not regioselectivity. Food Chem. 2013, 138, 615–620.
[CrossRef] [PubMed]
34. Akanbi, T.O.; Barrow, C.J. Compositional information useful for authentication of krill oil and the detection
of adulterants. Food Anal. Methods 2018, 11, 178–187. [CrossRef]
35. Nenadis, N.; Tsimidou, M. Observations on the estimation of scavenging activity of phenolic compounds
using rapid 1, 1-diphenyl-2-picrylhydrazyl (DPPH
[CrossRef]
•
) tests. J. Am. Oil Chem. Soc. 2002, 79, 1191–1195.
36. Rice-Evans, C.; Miller, N.; Paganga, G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 1997
,
2, 152–159. [CrossRef]
37. Madhujith, T.; Shahidi, F. Optimization of the extraction of antioxidative constituents of six barley cultivars
and their antioxidant properties. J. Agric. Food Chem. 2006, 54, 8048–8057. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).