Full text of DOI:10.1002/jsde.12180

J Surfact Deterg (2018)
DOI 10.1002/jsde.12180
ORIGINAL ARTICLE
Study of Antioxidative Properties of Some Mono Amino-Acid-
Type and Dipeptide-Type Surfactants
Faisal Hossain1 · Akio Ohta1 · Yumi Yamane1 · An-na Shizuka1 · Hitoshi Asakawa1 ·
Tsuyoshi Asakawa1
Received: 28 December 2017 / Revised: 5 March 2018 / Accepted: 17 March 2018
© 2018 AOCS
Abstract Surfactants of tryptophan, tyrosine, and histi-
dine were successfully synthesized. They were then modi-
fied to obtain dipeptide-type surfactants with better
solubility in both polar and nonpolar solvents, giving more
emphasis to the development of water-soluble amino-acid-
type surfactants. All developed materials were designed to
have functionality as antioxidants (AOX). The investiga-
tion of free radical scavenging capacity at pre-micellar con-
centrations revealed greater Trolox equivalent (TE, relative
scavenging capacity to Trolox) results for Trp-type surfac-
tants followed by Tyr-type and His-type. As expected, the
synthesized surfactants exhibited comparable AOX proper-
ties to their parent amino acids. The AOX behavior of these
surfactants was also checked in micellar concentrations,
and the trend was found to be analogous: Trp-type > Tyr-
type > His-type surfactants. However, all of them were act-
ing as better AOX in the micellar state than in the mono-
meric state. Investigating the synergistic effect found no
interaction between the “amino acid–surfactant” pairs or
the “surfactant–surfactant” pairs with varying ratios. They
showed total AOX activity with respect to that of each
material. This work opens up the door to the application of
some new oil and water-soluble amino-acid-type surfac-
tants having auspicious AOX activity.
Introduction
Antioxidants (AOX) have been extensively used in food
and pharmaceutical industries for decades (Packer et al.,
1999; Pattison and Winyard, 2008; Piringer and Baner,
2008; Van Meeteren et al., 2005). In the human body, inef-
ficiently coupled oxidative phosphorylation can generate a
stream of free radicals that attack mitochondria in cells with
high oxidative stress (Finley et al., 2011). This strain then
competes with body AOX defenses by enzymatic pathways
(catalase, superoxide dismutase, and glutathione peroxi-
dase) and nonenzymatic approaches (glutathione,
vitamins A, C, and E). A failure of the above processes
leads to aging and development of age-related diseases
(Finkel and Holbrook, 2000). An inequity in the redox sys-
tem of cells is also plausible and can disturb the DNA
mutation leading to various tumors, even cancers (Valko
et al., 2006). On the other hand, UV light, heat, enzymes,
microorganisms, and free radicals quicken food degrada-
tion through lipid oxidation. In both cases, AOX can delay
or even retard the oxidation process in food or oxidative
stress in the cell mitochondria by capturing the free radicals
allied with the chain reaction process hence resulting in the
termination of propagation steps (Albu et al., 2004).
TBHQ (tert-butylhydroquinone), propyl gallate, sodium
bisulfite, sodium metabisulfite, sodium sulfite (alkaline),
sodium nitrite, butylated hydroxyanisole, butylated hydro-
xytoluene, and tenox (all kinds) were historically used in
food products as AOX materials (Rehwoldt, 1986), but
there were issues with these materials because of their toxi-
cological shortcomings. Moreover, the solubility of AOX
in an “oil in water emulsion” medium is also vital for
retarding lipid oxidation in emulsion-based food, cos-
metics, and pharmaceuticals. Therefore, natural, low-toxic,
Keywords Amino-acid-type surfactant ꢀ Dipeptide-type
surfactant ꢀ Antioxidant activity ꢀ Synergistic effect
J Surfact Deterg (2018).
* Akio Ohta
akio-o@se.kanazawa-u.ac.jp
1
Faculty of Chemistry, Institute of Science and Engineering,
Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
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and highly soluble AOX are preferable for food and phar-
maceutical industries. Some AOX are now being made to
dissolve in the oil–water interface using an additional sur-
factant as a solubilizer to achieve a higher solubility
(Kiralan et al., 2014). This problem can be resolved by
means of surfactant as AOX and can be a novel way to
exchange any supplementary solubilizing materials.
Although few attempts have been made so far to prepare
surfactants from tocopherol, ascorbic acid, erythorbic acid,
and other well-known AOX, they all face problems in
water solubility (Astete et al., 2011; Silvestre et al., 2000;
Yan et al., 2007). We aimed to synthesize new AOX sur-
factants from amino acids, which will have a better solubil-
ity in both water and organic solvents.
Amino acids have been known for their AOX activity
and can be extensively used to prepare surfactants having
low toxicity and biodegradability (Christen et al., 1990;
Elias et al., 2005; Leodidis and Hatton, 1991; Moosmann
and Behl, 2000). The uses of amino-acid-type surfactants
are diverse, being used in applications such as the food,
cosmetic, and pharmaceutical industries (Bordes and Holm-
berg, 2015; Infante et al., 1997; Itoh et al., 1995; Ohta
et al., 2003). However, there is little research with emphasis
on the AOX properties of amino-acid-type surfactants with
excellent water solubility. In this study, we focused on the
water-soluble surfactants derived from three amino acids:
tryptophan, tyrosine, and histidine, which are known for
their good free radical scavenging capacities. The aim was
to prepare N-acyl amino-acid-type surfactants from the
above amino acid residues and to improve the structure of
the surfactants by modifying it into a form of a water-soluble
dipeptide. It was anticipated that modification of the peptides
to dipeptides by means of a polar amino acid (taurine) or by
leveraging stereochemical advantage (inserting alanine resi-
due) would increase the water solubility of dipeptides. The
modification was done so that the whole reaction remains
eco-friendly. Hence, the outcome of this study will be a set
of green surfactants with high antioxidative properties and
improved water solubility. After developing the surfactants,
they were analyzed for their AOX activity in both mono and
micellar states. In both the states, we also checked any syner-
gistic effect of the AOX properties among the surfactant–
amino acid pairs at different ratios.
NACALAI TESQUE INC, (Kyoto, Japan). Other amino
acids, 2,2-azobis (2-methylpropion amidine) dihydrochlor-
ide (AAPH), and ascorbic acid were collected from WAKO
pure chemical industries Ltd. (Tokyo, Japan). All other
chemicals used for purification and analysis of the final sur-
factants were of analytical grade and used without further
purification.
Synthesis of Amino-Acid-Type Surfactants
A synthetic strategy of inserting alanine residue was
employed for improving the water solubility of both Tyr-
type and Trp-type surfactants. Because of the low water
solubility of lauroyl carnosine (beta-alanyl-histidine), we
employed another synthetic strategy, i.e., ligation of a polar
amino acid (taurine). Lauroyl chloride was added dropwise
in excess histidine solution (in NaOH) and kept in an ice
bath, keeping the reaction condition at pH >11. After 2 h,
the reaction mixture was held at room temperature for
another 12 h with continuous stirring to ensure that all laur-
oyl chloride combined with His molecules (Mhaskar et al.,
1990). After completion of the reaction, the resulting white
solid was recrystallized in ethanol, and pure lauroyl histi-
dine (C12-His) was dried and then reacted with taurine to
develop the corresponding dipeptide. In this reaction, 4-
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMT-MM) was used for formation of an amide
bond between taurine and carboxylic groups of the surfac-
tant in ethanol medium (Kunishima et al., 2001) (Fig. 1).
The resulting surfactant lauroyl histidyl taurine (C12-His-
Tau) was then purified by recrystallization and separated
from impurity through a silica column. In similar pathways,
other surfactants: lauroyl tryptophan (C12-Trp), lauroyl
tyrosine (C12-Tyr), and their corresponding dipeptides:
lauroyl alanyl tryptophan (C12-Ala-Trp) and lauroyl alanyl
tyrosine (C12-Ala-Tyr) were synthesized (Fig. 2). The
identification and purity were estimated using elemental
analysis, high-performance liquid chromatography (L6320,
Hitachi Ltd, Tokyo, Japan) with a Hitachi F-1050 fluores-
cence detector, UPLC-MS with ethylene bridged hybrid
1
(BEH) C18 1.7-μm column, and H NMR and FTIR tech-
niques. The 1H NMR and IR information is given as
follows:
1
C12-His-Tau: H NMR (500 MHz, DMSO D6) 0.85 (t,
3H), 1.22 (s, 16H), 1.42 (m, 2H), 2.05 (m, 2H), 2.89 (m,
5H), 3.81 (m, 1H), 4.38 (m, 1H), 6.78 (m, 1H), 7.55 (m,
2H), 7.98 (m, 1H), and 11.14 (s, 1H); IR: 3213, 2923,
3066, 2854, 2785, 1705, 1693, 1635, 1461, 1419, 1184,
1060, 810, 767, and 586 cm−1.
Experimental Procedure
Materials
1
C12-Ala-Trp: H NMR: (500 MHz, DMSO D6) 0.75 (t,
Lauroyl chloride (≥98%) was purchased from Tokyo
Chemical Industry Co. Ltd. (Tokyo, Japan). Histidine, tryp-
tophan, tyrosine, and arginine were collected from
3H), 1.01 (m, 3H), 1.12 (m, 16H), 1.33 (m, 2H), 1.93 (t,
2H), 2.9 (o, 2H), 3.76 (m, 1H), 3.99 (m, 1H), 6.76 (t, 1H),
6.88 (m, 2H), 7.13 (d, 1H), 7.28 (d, 1H), 7.38 (d, 1H), 7.88
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O
Lauroyl chloride
NH
Lauroyl his0dine
Cl
N
(C12-His)
O
+
NH
N
COOH
H
N
+
H2N
COOH
SO3H
Taurine
H2N
Histidine
NH
N
O
H
N
N
SO3H
H
O
DMT-MM
O
Lauroyl histidyl taurine
(C12-His-Tau)
N
N+
O
N
O
N
Fig. 1 Schematic of the synthesis of C12-His-Tau
(d, 1H), and 10.53 (s, 1H); IR: 3409, 3301, 2920, 2850,
2360, 2341, 1627, 1593, 1539, 1415, 740, and 667 cm−1.
C12-Ala-Tyr: H NMR: (500 MHz, DMSO D6) 0.75 (t,
2H), 2.8 (m, 2H), 3.66 (m, 1H), 4.98 (q, 1H), 6.42 (d, 2H),
6.73 (d, 2H), 7.1 (d, 1H), 7.97 (d, 1H), and 9.17 (s, 1H);
IR: 3305, 2920, 2850, 1631, 1515, 1458, 1404, 1245,
671, and 543 cm−1.
1
3H), 1.05 (m, 3H), 1.13 (m, 16H), 1.36 (m, 2H), 1.95 (m,
Fig. 2 Structures of synthesized surfactants
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Surface Tension Measurement
400
300
200
100
0
y = 151.41x
R2 = 0.9985
The surface tension of the dipeptide-type surfactants was
measured by the Wilhelmy plate method (Elworthy and
Mysels, 1966) by using an electro surface balance (model:
ESB-V) from Kyowa Scientific Co. Ltd, Saitama, Japan.
The samples were dissolved in phosphate buffer (pH = 7.4)
at different concentrations and the critical micelle concen-
tration (CMC) was determined from the break in slope of a
plot of surface tension measured at 25ꢁC for varying con-
centrations of surfactants. The surface density, Γ (μmol
m−2), at constant temperature for uni-univalent ionic surfac-
tant is then calculated using the Gibbs adsorption isotherm
(Mukherjee et al., 2013) with the following equation:
0
0.5
1
1.5
Trolox (μM)
2
2.5
3
ꢀ
ꢁ
Fig. 4 Trolox standard curve measured at 30ꢁC for 30 min with 2 s
interval
1
∂γ
Γ = −
:
ð1Þ
2RT ∂lnC
T
Here, R is the gas constant, T is the absolute temperature,
γ is the surface tension, and C is the molarity of the
surfactants.
was mixed in a glass cell. The fluorescence intensity of the
solution was then monitored every 2 s using a fluorescence
spectrophotometer (Hitachi F 2700, Tokyo, Japan) for
30 min at a constant temperature (30ꢁC). The excitation
and emission wavelengths were 485 and 535 nm, respec-
tively. The corresponding slit widths were 20 and 2.5 nm.
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyl
ic acid) was used as a standard material (van den Berg
et al., 1999), and the standard curve was obtained by plot-
ting the net AUC (Fig. 3) against the several concentrations
measured (Fig. 4). The calculation of AUC was done as
(Dávalos et al., 2004):
Study of the AOX Capacity
The AOX properties of amino acids and other synthesized
surfactants were analyzed using a slightly modified oxygen
radical absorbance capacity (ORAC) assay (Cao et al.,
1993). This assay involves the ability of an AOX material
to neutralize free radicals generated from an initiator,
AAPH (Zulueta et al., 2009). Fluorescein is a dye used in
this method as a fluorescent species (Huang et al., 2002).
Its structure can be damaged by the radicals (ROO•) gener-
ated by AAPH and that will lead to lowering of fluores-
cence power. The AOX material can capture the radicals
and try to maintain the fluorescence intensity of the dye.
Briefly, 60 nM of fluorescein is mixed with the AOX mate-
rial with projected concentration, and then AAPH (37 mM)
AUC = Ið0Þ=Ið0Þ + Ið2Þ=Ið0Þ + Ið4Þ=Ið0Þ::… + IðnÞ=Ið0Þ
+ ::… + Ið1798Þ=Ið0Þ + Ið1800Þ=Ið0Þ
ð2Þ
where I(0) is the initial fluorescence reading at 0 s and I(n)
is the fluorescence reading at any “even time” interval in
between 0 and 1800 s. Finally, net AUC was derived as:
1
Net AUC = AUC ðAOXÞ – AUC ðBlankÞ:
ð3Þ
The AOX property was articulated by the Trolox equiva-
lent (TE) as used by Han et al. (2014):
0.75
Blank
Trolox equivalent ðTEÞ
0.5
AUC
ð4Þ
Slope of AOXmaterial ðnetAUC vsconc:Þcurve
=
:
Slope of Trolox standard curve
Sample
0.25
For the study of AOX capacity of surfactants in a micellar
medium, the medium itself was prepared from a surfactant:
lauroyl alanine (C12-Ala) (5 mM solution in phosphate
buffer, pH = 7.4) having a very low AOX property. The
measurements were then carried out as discussed above.
Synergistic effects (Leong and Shui, 2002) between
amino acids and surfactants and also in between surfactants
0
0
500
1000
1500
2000
Time (s)
Fig. 3 Area under curve between the blank and sample’s fluorescence
curve measured for 30 min
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Table 1 CMC (mM) and surface density,
Γ ) of
(μmol m−2
were studied using the preceding practice. Briefly, amino
acids and surfactants were added in equal concentration in
a 1:1 ratio and the resulting solutions were analyzed for
AOX activity. The solution’s experimental TE was com-
pared with the expected value. For surfactant pairs, they
were patterned at 1:1, 1:3, and 3:1 ratio to check any devia-
tions in the AOX property.
dipeptide-type surfactants measured at 25ꢁC
Surfactant
CMC/mM
Γ/μmol m−2
C12-Ala-Trp
C12-Ala-Tyr
C12-His-Tau
0.17
0.66
0.81
1.85
2.28
2.48
To explain the variation of AOX activities, the materials
were analyzed using molecular orbital (MO) calculations
and their individual energies and optimized structures were
determined using density functional theory (DFT) calcula-
tion with B3LYP function having basis set 6-311G(d) in
Gaussian 09W (Thilagavathi and Arivazhagan, 2011). All
other calculations were done using Microsoft Excel 2016.
is shown in Table 1. The surface tension measurement of
the amino-acid-type surfactants (C12-Trp, C12-Tyr, and
C12-His) cannot be measured with the same technique
because of their lower solubility at relatively higher
concentrations.
From the experimental findings, we found that C12-His-
Tau has the highest CMC (0.81 mM) among the surfactants
with a surface density of 2.48 μmol m−2. On the other
hand, C12-Ala-Trp has the lowest (1.85 μmol m−2) surface
density and CMC (0.17 mM). This dissimilarity in surface
density is because C12-His-Tau has more packing capacity,
which yields a higher surface density. On the other hand,
C12-Ala-Trp has a larger size and more steric hindrance
yielding, consequently, a lower surface density. C12-Ala-
Tyr with its moderate size among the surfactants has the
surface density in between C12-Ala-Trp and C12-His-Tau.
Results and Discussion
Surface Activity
One of the most significant physiochemical aspects of a
surfactant is its CMC. Usually, the surface tension of a sur-
factant solution decreases with increasing concentration,
but the CMC is the state after which the surface tension
becomes independent of the sample amount in the solution
(Patist et al., 2000). The change in surface tension with
varying concentrations for C12-Ala-Trp, C12-Ala-Tyr, and
C12-His-Tau is shown in Fig. 5. In the case of C12-Ala-
Tyr, the surface tension progressively increased after the
CMC. This phenomenon may be due to the further time
required to attain the equilibrium surface tension (Prosser
and Franses, 2001), or the presence of impurities. The
CMC and surface density for each dipeptide-type surfactant
AOX Properties of Amino Acids and Surfactants
The AOX activity of the surfactants and amino acids was
measured using the ORAC assay. In this assay, AUC indi-
cates the strength of materials to act as an AOX (Fig. 3).
The higher the AUC, the greater the AOX effect and vice
versa. The AOX capacity increases with increasing concen-
tration of materials and shows linearity for a certain con-
centration range. At first, the AOX activity of a set of seven
amino acids (Trp, Tyr, His, Car, Thn, Gln, and Arg) was
measured. Their relative strength as TE is shown in Fig. 6.
It is clear that Trp showed highest AOX activity followed
by Tyr, Car, and His. Similar observations were noted by
Garrett et al. in his study of AOX capacity of amino acids
(Garrett et al., 2014). The other amino acids had almost no
capability to be potential AOX, and hence in this study,
Trp, Tyr, and His were chosen for further investigation in
their surfactant forms. Carnosine (beta-alanyl-histidine) is
known for its AOX activity, and we found almost similar
AOX capacity (TE = 0.019) of carnosine (Car) to histidine
(His) (TE = 0.012). However, we chose “histidine” to
develop a new dipeptide-type surfactant because “carno-
sine” itself is a dipeptide amino acid (Babizhayev
et al., 1994).
65
C12-Ala-Trp
C12-Ala-Tyr
C12-His-Tau
60
55
50
45
40
35
30
–2.5
–2
–1.5
–1
–0.5
0
0.5
The synthesized C12-amino acids were then investigated
for AOX activity. Their relative AOX strengths were found
to be C12-Trp > C12-Tyr > C12-His (Fig. 7b). As expected,
the dipeptide-type surfactants also showed analogous AOX
log C (mM)
Fig. 5 Change in surface tension of C12-Ala-Trp, C12-Ala-Tyr, and
C12-His-Tau with respect to the concentration at 25ꢁC
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1.5
1.25
1
dipeptide-based surfactants showed a similar trend of AOX
activities concerning the parent amino-acid-type. A com-
plete comparison of the surfactant AOX capacity (as TE) in
mono and the micellar medium is illustrated in Fig. 9. The
final trend of AOX activity under micellar conditions was
found to be C12-Trp ≈ C12-Ala-Trp > C12-Tyr ≈
C12-Ala-Tyr > C12-His ≈ C12-His-tau.
Moreover, in the micellar medium, surfactants showed
improved AOX capability over monomeric surfactants.
Electrostatic interaction can explain this phenomenon. The
surfactant micelle is negatively charged on the head groups.
AAPH itself is positively charged and attracted to the
micelle; hence it is more difficult for AAPH to demolish
the fluorescein structure in the micellar state.
0.75
0.5
0.25
0
Arg
Car
Gln
His
Thn
Trp
Tyr
Amino acid
Fig. 6 A comparison of amino acids for their AOX capacity measured
A comparison of the neat aromatic amino acids and their
corresponding surfactants for their mean TE value is shown
in Table 2. The strength of “Trp” and “Tyr” was found to
be higher than the corresponding surfactants while “His”
had almost similar AOX capacity to its surfactants in the
mono state. Interestingly, the surfactants (both amino-acid
and dipeptide-types) retained similar strength to their parent
amino acids while in the micellar medium, it was decreased
for Trp and Tyr in the monomer state. Park et al. observed
a similar phenomenon while adding a long carbon chain to
the erythorbic acid structure to form a new surfactant with
potential AOX activity (Park et al., 2017). Therefore, these
observations justify the synthesis of dipeptides having a
similar AOX activity to the parent amino acids, but with
improved water solubility.
by the ORAC assay
properties to their monotype counterparts (Fig. 7c). Alanine
and taurine groups have lower AOX properties; hence the
inclusion of these groups in surfactant structures should
have almost no effect on the total AOX capacity of dipep-
tides. It also suggests that only the amino acid portions: Trp,
Tyr, and His should have played a vital role in showing
AOX activity in the synthesized surfactants. The final com-
parison of AOX power of surfactants was found to be:
C12-Trp ≈ C12-Ala-Trp > C12-Tyr ≈ C12-Ala-Tyr >
C12-His ≈ C12-His-Tau.
AOX Properties in the Micellar Medium
All surfactants were also subjected to analyze their AOX
properties in the micellar medium to check their behavior
under micellar conditions. Among surfactants, C12-Trp
showed the highest AOX activity followed by C12-Tyr and
C12-His. On the other hand, among dipeptide-type surfac-
tants, C12-Ala-Trp showed most potent AOX capacity
tailed by C12-Ala-Tyr and C12-His-Tau (Fig. 8). These
results also suggest that mono-amino-acid-based and
Mechanism of AOX Properties
Clearly, there was a variation in free radical scavenging
activity of surfactants having Trp, Tyr, and His residues.
The cause of such a distinction was inspected by evaluating
the reaction mechanism of surfactants with the radical initi-
ator. To do that, surfactants were individually reacted with
(a)
(b)
(c)
250
200
150
100
50
150
125
100
75
50
25
0
Trp
Tyr
His
C12-Ala-Trp
C12-Trp
120
C12-Ala-Tyr
C12-Tyr
C12-His-Tau
90
C12-His
60
30
0
0
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
Concentration (μM)
Concentration (μM)
Concentration (μM)
Fig. 7 A comparison of AOX activity of materials in their mono state; (a): For amino acids, (b): For synthesized C12-amino acids, and (c): For
modified dipeptide-type surfactants at 30ꢁC
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(a)
(b)
C12-Trp
C12-Tyr
C12-His
450
300
150
0
450
300
150
0
C12-Ala-Trp
C12-Ala-Tyr
C12-His-Tau
0
10
20
30
0
5
10
15
Concentration (μM)
Concentration (μM)
Fig. 8 Relative AOX properties of (a) C12-amino acids, and (b) C12-dipeptides in micellar state at 30ꢁC
1.2
1
0.8
0.6
0.4
0.2
0
Micellar state
Mono state
C12-Ala-Trp C12-Trp C12-Ala-Tyr C12-Tyr C12-His-Tau C12-His
Fig. 9 A complete comparison of AOX properties (as TE) for surfactants in both mono and micellar states
(i) equimolar and (ii) five times greater amount of AAPH.
The final product was analyzed using a UPLC-MS (Waters,
Ireland) instrument. Surprisingly, it was discovered that all
surfactants were oxidized with the addition of one O atom
and hence giving an idea of a simple oxidation reaction
between AAPH and surfactants. For example, C12-Tyr was
first reacted with AAPH in an equimolar amount, and then
the reaction product was instantly checked using a UPLC-
MS instrument. It was revealed that C12-Tyr was able to
capture an oxygen atom. Then, the surfactant was again
reacted with five times of AAPH to check the phenomenon,
and the result was identical (Fig. 10). A similar observation
of other surfactant and AAPH mixer proves that the reac-
tion mechanism for the surfactants with AAPH should be
the same and the variation of AOX properties might be due
to the kinetics of the reaction. Therefore, the activation
energy for converting in an oxidized form should play a
vital role in becoming more powerful AOX. Within the sur-
factants, the amino acid groups are critical functional
groups for interacting with AAPH. These residues were
analyzed for the DFT calculations for unoxidized, possible
intermediates, and oxidized structures by use of the follow-
ing two reaction models with amino acid (H-AA) and
hydrogen peroxide.
H−AA + H2O2 ! H−AA−OH + OHꢀ
! AA−OH + H2O;or,
Table 2 Antioxidative property as Trolox equivalent (TE) of amino
acids and the corresponding surfactants in mono and micellar states
H−AA + H2O2 ! AAꢀ + OH + H2O ! AA−OH + H2O:
Amino acid
Trp
TE
Surfactants
TE
TEa
1.34
C12-Trp
0.68
0.90
0.20
0.24
0.01
0.02
1.16
1.14
0.55
0.55
0.02
0.03
The result of the DFT calculations is illustrated in
Fig. 11. In the His case, it is oxidized in two ways, one
with the only single intermediate form by losing one H
atom and formation of free radicals in the carbon atom to
which O or OH will be added. The other with two interme-
diates by direct addition of OH groups to the structure. The
minimum activation energy (Ea) found from both the
C12-Ala-Trp
C12-Tyr
Tyr
His
0.48
0.01
C12-Ala-Tyr
C12-His
C12-His-Tau
a
Measured in micellar state.
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(a)
0.76
100
%
C12-Tyr + O
C12-Tyr:AAPH = 1:5
MW = 402.00; 0.5000 Da
Intensity = 1.05e5
0
0.6
0.6
0.6
0.8
0.76
1.0
1.2
1.4
1.4
1.4
1.6
1.6
1.6
1.8
2.0
2.2
(b)
100
C12-Tyr + O
C12-Tyr:AAPH = 1:2
MW = 402.00; 0.5000 Da
Intensity = 8.34e4
%
0
0.8
0.73
1.0
1.2
1.8
2.0
2.2
(c)
100
C12-Tyr
C12-Tyr
MW = 386.0; 0.5000 Da
Intensity = 4.92e4
%
0
0.8
1.0
1.2
1.8
2.0
2.2
Retention time (min)
Fig. 10 UPLC-MS chromatogram of (a) C12-Tyr, (b) equimolar mixer of AAPH and C12-Tyr, and (c) 1:5 mixer of C12-Tyr and AAPH
possible ways is 158.3 kJ mol−1. On the other hand, Trp
also has similar ways to get into oxidized form, and for
doing this, it needs at least 87.4 kJ mol−1 of energy.
Finally, Tyr needs 90.3 kJ mol−1 of Ea to get oxidized.
Hence, the order of Ea for the residues is His > Tyr > Trp.
Therefore, it can be said that Trp-type surfactants should
have more capability to capture free radicals than Tyr-type
and His-type. Moreover, the values of energy of activation
of Trp (90.2 kJ mol−1) and Tyr (87.4 kJ mol−1) are much
closer compared with His (158.3 kJ mol−1). These values
suggest that the AOX capacity of C12-Trp, C12-Ala-Trp
should be closer to that of C12-Tyr and C12-Ala-Tyr,
which resembles with the previous experimental findings.
Frontier molecular orbitals are critical for electrical prop-
erties. The energies of highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbitals
(LUMO) play a significant role in the electron transition
behavior of molecules. The three-dimensional diagrams of
the HOMO of Trp, Tyr, and His are shown in Fig. 12. The
LUMO of Trp, Tyr, and His and LUMO+1 of His are illus-
trated in Fig. 13. The energy of LUMO+1 for His is impor-
tant because it is incorporated into the π ! π* transition.
The calculated energies of HOMO and LUMO as electron
volt (eV) are tabulated in Table 3. From the calculation, it
was found that Tyr has the highest HOMO-LUMO gap and
Trp has the lowest. The HOMO-LUMO gap for His was
similar to the findings of Yoosefian and Etminan (2015). de
Leon et al. (2008) found almost similar energies to our cal-
culated values.
As all the aromatic amino acids show the UV–Visible
spectrum, we compared the experimental spectrum with the
time-dependent (TD)-DFT calculation (Compagnon et al.,
2010) for Trp, Tyr, and His. The trend of the absorption
wavelengths resembles that of the TD-DFT calculations
(Table 4). Oscillator strength and molar extinction coeffi-
cient are two important optical phenomena for molecular
absorption. Oscillator strength is proportional to the inte-
gral value of the molar extinction coefficient (Hammond
J Surfact Deterg (2018)

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Fig. 11 Energy diagram (kJ mol−1) for AOX of Trp, Tyr and His by DFT calculation with the B3LYP, 6-311G(d) basis set
and Price, 1955). It can also be seen from the table that the
trends of oscillator strength and the experimental value
of the logarithmic molar extinction coefficient
(dm3 mol−1 cm−1) are similar. Therefore, Trp has the high-
est oscillator strength with a higher absorption wavelength
followed by Tyr, then His. The excitation energy found for
the first π ! π* transition of His was 5.7910 eV followed
by Tyrosine with 5.0266 eV. Trp had the lowest excitation
energy of 4.6697 eV that was found similar (4.69 eV) to
the calculated energy by Compagnon et al. using TD-DFT
calculation with the B3LYP, 6–311++G basis set
(Compagnon et al., 2010).
similar to their mathematical sum of AOX capacity
(expected value) (Wang et al., 2011). This finding sug-
gested that the amino acids and surfactants can individually
neutralize the free radicals hence do not interfere with each
other. They do not increase or decrease the total AOX
capacity of the mixtures, thus no positive or negative syn-
ergy was found (Fig. 14). For example, 1 mL Trp has a TE
of 1.34, and 1 mL C12-Tyr has 0.2.Therefore, if they are
mixed in a 50%:50% ratio with the same concentration,
and the final volume is 2 mL, the system will have a TE
value of (0.5×1.34 + 0.5×0.20) = 0.77. As expected, the
experimental TE value was also found as 0.77
TE. Similarly, all other values were very close to their
expected values.
Synergistic Effect
The surfactants were combined in 1:1, 1:3, and 3:1 ratios
in the micellar medium (5 mM of C12-Ala) to check the
synergistic effect of surfactants. Interestingly, under micel-
lar medium the surfactant mixtures in various proportions
Initially, the three amino acids and each amino-acid-type
surfactant were mixed in pairs at the equivalent ratio for
checking their synergistic effect. The results were almost
HOMO (Tyr)
HOMO (His)
HOMO (Trp)
Fig. 12 Molecular orbital surfaces of the HOMO of Trp, His, and Tyr obtained by DFT calculation using the B3LYP, 6-311G(d) basis set
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LUMO (Trp)
LUMO +1 (His)
LUMO (Tyr)
LUMO (His)
Fig. 13 Molecular orbital surfaces of the LUMO of Trp, His, Tyr, and the second lowest-energy molecular orbital (LUMO+1) of His obtained by
DFT calculation using the B3LYP, 6-311G(d) basis set
Table 3 The highest occupied molecular orbital (HOMO) energy, the lowest unoccupied molecular (LUMO) energy, the energy gap between
HOMO-LUMO of Trp, Tyr, and His as electron volt (eV) by DFT calculation using the B3LYP/6-311G(d) basis set compared with the literature
value
Amino acid
Calculated value
ELUMO
Literature value
Basis set
EHOMO
Energy gap
Energy gap
Reference
Trp
Tyr
His
−5.62621
−6.10320
−6.06892
−0.46094
−0.32679
−0.53413
5.16527
5.77641
5.53479
5.29966
5.93720
5.510
B3LYP/3-21G
B3LYP/3-21G
B3LYP/6-31G
de Leon et al. (2008)
de Leon et al. (2008)
Yoosefian and Etminan (2015)
showed similar AOX activity as would be expected from
the calculated average; thus, again, no synergy, either posi-
tive or negative, was found. For example, when three times
of C12-Tyr is added with one time of C12-His, the
calculated
total
TE
will
be
(0.75×0.55 + 0.25×0.02) = 0.42, while the experimental
TE was also found to be 0.42 for that mixer. All the results
are summarized and shown in Table 5.
Table 4 Experimental and time dependent (TD)-DFT using the
B3LYP, 6-311G(d) basis set, calculated absorption wavelengths λmax
(nm), oscillator strength f, molar extinction coefficient (log ε) for Trp,
Tyr, and His
Conclusion
Amino-acid-type and dipeptide-type surfactants were suc-
cessfully synthesized with much easier reaction pathways
using cheap raw materials with low toxicity. The water sol-
ubility of C12-amino acids was successfully improved by
means of modification into dipeptides. Each of the dipep-
tides was found to be easily dissolved in water. ORAC
assay determination showed promising AOX activity for all
the synthesized surfactants. In comparison to the free amino
acids, the corresponding surfactants had comparable AOX
Amino acid
TD-DFT calculation
Experimental
λmax / nm
(first π!π*)
f
λmax / nm
log ε
Trp
Tyr
His
265.51
246.66
214.10
0.0617
0.0283
0.0394
278.0
274.0
207.5
3.713
3.118
3.739
Unit of ε: dm3 mol−1 cm−1
.
J Surfact Deterg (2018)

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(a)
(b)
(c)
600
600
400
Trp
Tyr
His
Trp
Tyr
His
Trp
Tyr
450
300
150
450
300
150
300
200
100
0
His
0
0
0
5
10
15
0
2.5
5
7.5
0
20
40
60
Concentration (μM)
Concentration (μM)
Concentration (μM)
Fig. 14 Synergistic effect of surfactant and amino acid pairs. (a) Synergistic effect of C12-Trp with amino acids, (b) synergistic effect of
C12-Tyr with amino acids, and (c) synergistic effect of C12-His with amino acids. Dotted lines indicate expected AOX effect and sharp lines
indicate observed effect
activity as the parent amino acids when studied at micellar
concentrations. Also, the incorporation of low AOX amino
acids (Tau and Ala) into the dipeptide surfactants did not
hamper the AOX properties. The mono and dipeptides of
similar amino acids showed similar AOX properties in both
mono and micellar states concluding that the dipeptides
could be the better choice for practical purpose. No syner-
gistic effect between the surfactant–surfactant and amino
acid–surfactant pairs was found in terms of AOX activity.
Hence, these surfactants have the potential to be used
against lipid oxidation in both water-based food products
or in emulsion-based food and pharmaceuticals simulta-
neously. In a nutshell, a set of surfactants was developed
that can either stay in the aqueous medium or has the abil-
ity to be concentrated at the oil–water interface exerting
AOX activity.
Conflict of interest The authors declare that they have no conflict
of interest.
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