Enhancing the bioconversion of azelaic acid to its derivatives by response surface methodology

Article abstract of DOI:10.3390/molecules23020397

Azelaic acid (AzA) and its derivatives have been known to be effective in the treatment of acne and various cutaneous hyperpigmentary disorders. The esterification of azelaic acid with lauryl alcohol (LA) to produce dilaurylazelate using immobilized lipas

Full text of DOI:10.3390/molecules23020397

molecules
Article
Enhancing the Bioconversion of Azelaic Acid to Its
Derivatives by Response Surface Methodology
Nurshafira Khairudin 1, Mahiran Basri 1,*, Hamid Reza Fard Masoumi 2,*, Shazwani Samson 1
and Siti Efliza Ashari 1,
*
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia;
nurshafirakhairudin@yahoo.com (N.K.); dorawanie_89@yahoo.com (S.S.)
Department of Biomaterials, Iran Polymer and Petrochemical Institute, Tehran 14977-13115, Iran
Correspondence: mahiran@upm.edu.my (M.B.); fardmasoumi@yahoo.com (H.R.F.M.);
ctefliza@upm.edu.my (S.E.A.); Tel.: +60-3-894-677-266 (M.B.); Fax: +60-3-8943-2508 (M.B.)
2
*
Received: 25 September 2017; Accepted: 29 November 2017; Published: 13 February 2018
Abstract: Azelaic acid (AzA) and its derivatives have been known to be effective in the treatment of
acne and various cutaneous hyperpigmentary disorders. The esterification of azelaic acid with lauryl
alcohol (LA) to produce dilaurylazelate using immobilized lipase B from Candida antarctica (Novozym
435) is reported. Response surface methodology was selected to optimize the reaction conditions.
A well-fitting quadratic polynomial regression model for the acid conversion was established
with regards to several parameters, including reaction time and temperature, enzyme amount,
and substrate molar ratios. The regression equation obtained by the central composite design of RSM
predicted that the optimal reaction conditions included a reaction time of 360 min, 0.14 g of enzyme,
a reaction temperature of 46 ◦C, and a molar ratio of substrates of 1:4.1. The results from the model
were in good agreement with the experimental data and were within the experimental range (R2 of
0.9732).The inhibition zone can be seen at dilaurylazelate ester with diameter 9.0 0.1 mm activities
±
against Staphylococcus epidermidis S273. The normal fibroblasts cell line (3T3) was used to assess the
cytotoxicity activity of AzA and AzA derivative, which is dilaurylazelate ester. The comparison
of the IC50 (50% inhibition of cell viability) value for AzA and AzA derivative was demonstrated.
The IC50 value for AzA was 85.28
µg/mL, whereas the IC50 value for AzA derivative was more than
100 g/mL. The 3T3 cell was still able to survive without any sign of toxicity from the AzA derivative;
µ
thus, it was proven to be non-toxic in this MTT assay when compared with AzA.
Keywords: azelaic acid; anti-acne; enzymatic reaction; Novozym 435; response surface
methodology (RSM)
1. Introduction
Azelaic acid (AzA; HOOC-(CH2)7-COOH), also known as 1,7-heptanedicarboxylic acid,
is a naturally occurring saturated C9-dicarboxylic acid. Azelaic acid possesses antimicrobial,
anti-inflammatory, and comedolytic actions with a well-established role in the treatment of acne
vulgaris [
treatment of acne vulgaris (AV) when applied twice daily, and it has been approved for use by the
Food and Drug Administration (FDA) [ ]. These formulations have been shown to be effective in the
1]. Currently, Azelaic acid is available in the market as a 20% azelaic acid cream for the
2
treatment of inflammatory (nodulocystic, papulopustular, and nodular) acne and comedonal acne, as
well as in various cutaneous hyperpigmentary disorders in pharmacological applications. However,
azelaic acid is limited in terms of cosmaceutical and pharmaceutical application because it has a
high melting point, poor solubility properties, and requires a high dosage. The latter increases the
incidence of side effects such as local irritation, stinging, burning, and redness of the skin associated
with exposure to high levels of undissolved, dispersed AzA that has an inherent low pH [3].
Molecules 2018, 23, 397; doi:10.3390/molecules23020397
www.mdpi.com/journal/molecules

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A derivative of azelaic acid (dilaurylazelate ester) with even better characteristics than the
original starting material may be produced through the modification to azelaic acid, especially the
efficacy of azelaic acid derivatives in the treatment of various dermatological and cosmetic disorders,
which involve inflammation and bacterial and fungal infection [4]. Modification of azelaic acid by
attachment through covalent ester linkages and converting at least one of the carboxylic acid groups
into the ester group has enhanced not only the technical property of azelaic acid but also the functional
performance of the resulting modified azelaic acid [4].
The modified AzA as a prodrug showed the efficacy of a relatively low dosage of AzA derivatives
in the treatment of various cutaneous hyperpigmentary disorders and acne vulgaris [4]. The prodrug
has been extensively improved by modifying the drug’s hydrophilic–lipophilic balance to improve the
bioavailability of many drugs. Many prodrugs were synthesized, resulting in successfully improved
bioavailability [
physicochemical properties to increase the rate of diffusion into the skin barrier. A promising approach
in this respect is the development of prodrugs [ ]. Prodrugs can facilitate the transfer of a drug into
5]. To improve the efficiency of drugs by the dermal route would be to manipulate the
6,7
or across the skin through the addition of a cleavable chemical group that typically increases a drug’s
lipophilicity. Because the prodrug approach is based on altering a drug’s structure, prodrugs usually
produce no skin irritation [8].
Hsieh et al. (2012) also introduced a co-drug of azelaic acid and conjugated hydroquinone to
enhance topical skin targeting and decrease penetration through the skin. Their studies demonstrated
that esterification of azelaic acid incorporated with hydroquinone contains two hydroxyl functionalities,
which allow the formation of an ester co-drug using Dimethyl cyclohexylcarbodiimide (DCC)
and 4-dimethyl aminopyridine (DMAP),which is chemical catalyst that obtains only 52% yield [9].
Ethanolic ester of azelaic acid was synthesized by Manosroi et al. (2016) to study its impact on
cytotoxicity activity and its effect on azelaic acid diffusion through the silicone membrane [5].
Production of such esters from AzA by esterification reactions requires either a chemical catalyst
or an enzyme. A chemical catalyst normally requires high pressure and high temperatures, as well
as corrosion-resistant equipment, which often results in undesired by-products. The preparation
of the fatty acid substrates, for example, produces many impurities, producing a dark-coloured
product. Subsequent downstream processes such as distillation and bleaching are needed to obtain
lighter-coloured and purer products [10]. In addition, due to their high capability of solvating the raw
materials, most of chemical syntheses employ toxic organic solvents as reaction media. The product
esters containing toxic solvent residue are not permitted in the pharmaceutical industries [11].
Lipases are the most versatile biocatalysts and have been extensively employed for the direct
synthesis of many esters [12]. Lipases contain a helical oligopeptide unit called “lid” or “flap” that
shields the active site. The active site is composed of serine, histidine, aspartate, and a carboxylic acid
residue (aspartic or glutamic acid). The lid, upon interaction with a hydrophobic interface such as
a lipid droplet, undergoes a conformational change in such a way that exposes the catalytic site to
free access of the reaction substrate. However, Candida antarctica lipase B (CalB) possesses an open
active site and does not have a real lid. Therefore, CalB does not display any interfacial activations or
conformational changes in which a lid region removes to uncover the active site. Among the lipases,
Candida antarctica lipase B has been reported to show a high catalytic activity for esterification of
dicarboxylic acids [13].
The synthesis of AzA derivatives was carried out using immobilized Candida antarctica lipase B as
a biocatalyst. Zisis et al. (2015) reported that CalB was recently reclassified as Pseudozymaantartica
lipase B (PalB) [14]. This lipase has been shown in numerous publications to be a particularly efficient
enzyme, catalyzing a great number of different reactions including both regio- and enantio-selective
synthesis. This lipase demonstrates that enzymes can be not only very effective but also highly
robust tools within organic synthesis [15]. In this study, the effect of different variables on the degree
of conversion of AzA was investigated using RSM and the optimization of the reaction variables
including the reaction times (90, 180, 270, 360, and 450 min), enzyme amounts (0.05, 0.15, 0.25, 0.35,

Molecules 2018, 23, 397
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and 0.45 g),reaction temperatures (40, 46, 52, 58, and 64 ◦C), and substrates molar ratios (1:3,1:4.5, 1:6,
1:7.5, and 1:9 mole, AzA:LA) is reported. The impact of cytotoxicity activity AzA derivatives on the
fibroblasts cell line (3T3), and its antibacterial activity against on the bacteria Staphylococcus epidermidis
S273, was studied.
2. Results and Discussion
2.1. Identification of Dilauryl Azelate Ester
The identification of the dilaurylazelate ester was determined by TLC, FT-IR, GCMS, and NMR.
Thin layer chromatography (TLC) was used to confirm the production of the dilaurylazelate ester
in the initial biocatalyst screening. The least polar component, dilaurylazelate ester (Rf = 0.57), was
eluted first, followed by lauryl alcohol (Rf = 0.25). Azelaic acid with a higher polarity was the last
component eluted (Rf = 0), as it was absorbed onto the stationary phase more than the dilaurylazelate
ester and lauryl alcohol; thus, it moved through the stationary phase more slowly. Figure 1 illustrates
the FT-IR spectra of azelate acid, lauryl alcohol, and dilaurylazelate ester. The FTIR spectra of azelaic
acid showed an absorption at Vmax 1692.26 cm−1 (C=O carboxylic acid), whereas lauryl alcohol had
an absorption at Vmax 3317.01 cm−1 (O-H alcohol stretch), and the product had an absorption at
Vmax 1730.10 cm−1 (C=O ester), indicating the successful formation of the ester.
Figure 1. Infrared spectrum of dilaurylazelate ester.
The gas chromatography mass spectroscopy (GCMS) spectrum of the dilaurylazelate ester showed
a molecular ion peak at m/z 525 [M + H]+ with base peak = 171, which matched the molecular weight
1
of the dilaurylazelate ester (C33H64O4). H NMR and 13C NMR indicated the presence of dilauryl
1
alcohol linked to azelaic acid and the respective carbon and hydrogen positions. H NMR ((CD3)2CO,
TMS): 4.05 (t, 4H, J = 10Hz), 2.30 (t, 4H, J = 10 Hz), 1.62 (m, 8H), 1.31 (m, 42H), 0.90 (t, 6H, J = 10 Hz).
13C NMR: 13.49, 22.46, 24.78, 25.80, 28.58, 28.84, 28.96, 29.07, 29.21, 29.41, 29.44, 29.49, 29.51, 31.77,
33.75, 63.69, 172.76.
2.2. Mathematical Model and Analysis of Variance (ANOVA)
The efficacy of Novozym 435 was tested in the esterification of azelaic acid with lauryl alcohol by
selecting four variables, as shown in Table 1. The experiments were programmed as per the CCRD
(central composite rotatable design) layout, and the degree of conversion of AzA was selected as the
response variable ((design expert software version 7.0) Stat-Ease, Minneapolis, MN, USA) involving

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30 different combinations of the coded variables (Table 2). In order to demonstrate reproducibility,
each experimental run was conducted in triplicate. The optimal process conditions corresponding to
maximum dilaurylazelate ester yields and minimum enzyme amounts were then computed using
mathematical models in the Design Expert software. Optimization of the process parameters by RSM
techniques has been reported by several research reports [16–18].
Table 1. Variables and their levels employed in the central composite rotatable design.
Coded Level of Variables
Variables
Units
−2
−1
0
1
2
X1
X2
X3
X4
Enzyme amount
Reaction time
Reaction temperature
Molar ratio of substrates
gram
min
C
0.05
90
40
0.15
180
46
0.25
270
52
0.35
360
58
0.45
450
64
◦
AA:LA (mole:mole)
1:3
1:4.5
1:6
1:7.5
1:9
The experimental conversion values at each point according to the experimental design are
shown in Table 2. Based on the central composite rotatable design and results of the experiments,
the coefficients of the regression equation (Equation (1)) were calculated using the Design Expert
7.0 software (Stat-Ease, Minneapolis, MN, USA). The following second-order polynomial equation
was established to obtain the degree of conversion of AzA. Where A is enzyme amount, B is the
reaction time, C is the reaction temperature, and D is the molar ratio of substrates. The positive sign in
front of the terms indicates a synergistic effect, whereas the negative sign indicates an antagonistic
effect. Negative values of a coefficient estimate denote a negative influence of parameters on the
reaction yield.
Y(Degree of Conversion%)
(1)
= +86.24 + 0.11 A + 3.83 B + 1.29 C − 1.63 D + 0.64 AB + 1.16 AC
− 1.79 BC − 0.89 BD + 1.75 + 0.65 B2 + 0.74 C2 − 0.92 D2
The mathematical model found after fitting the function to the data can sometimes not
satisfactorily describe the experimental domain studied. The more reliable way to evaluate the quality
of the model fitted is by applying analysis of variance (ANOVA). ANOVA analysis was necessary
in order to determine whether or not the second-order polynomial model was significant. Table 3
indicated that the quadratic polynomial model was statistically significant (p-value < 0.0001) and
adequate to represent the actual relationship between the response (degree of conversion, %) and
the significant variables. The coefficient of determination (R2) and the R2 of the model obtained
adj
were 0.9732 and 0.9542, respectively, which indicated that the accuracy of the polynomial model was
adequate. The “Lack of Fit F-value” of 0.7794 implied the lack of fit was not significant relative to the
pure error.
Table 2. Central composite rotatable design matrix and result for the model of dilaurylazelate
ester synthesis.
Degree of Conversion %
Molar Ratio of
Substrates, AzA:LA
(mole)
Enzyme
Amount (g)
Reaction
Temperature ( C)
Reaction
Time (min)
Run No.
◦
Actual
Value
Predicted
Value
1
2
3
4
5
0.15
0.15
0.25
0.35
0.25
180
360
270
180
90
58
58
52
58
52
1:4.5
1:4.5
1:6
1:7.5
1:6
87.83
93.51
86.61
87.89
80.80
87.82
92.40
86.24
87.60
81.19

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Table 2. Cont.
Degree of Conversion %
Molar Ratio of
Substrates, AzA:LA
(mole)
Enzyme
Amount (g)
Reaction
Temperature ( C)
Reaction
Time (min)
Run No.
◦
Actual
Value
Predicted
Value
6
7
8
9
0.35
0.35
0.25
0.25
0.35
0.35
0.25
0.15
0.25
0.35
0.25
0.15
0.25
0.35
0.25
0.15
0.05
0.15
0.25
0.15
0.25
0.35
0.15
0.25
0.45
180
360
270
270
360
360
450
360
270
360
270
180
270
180
270
360
270
180
270
360
270
180
180
270
270
46
58
52
52
46
46
52
58
40
58
52
46
52
46
52
46
52
58
52
46
64
58
46
52
52
1:4.5
1:4.5
1:9
80.62
95.22
79.69
87.95
95.40
90.83
96.74
86.73
85.71
90.69
85.27
83.91
85.24
78.76
84.38
95.38
93.20
84.98
86.61
90.13
92.52
89.78
84.14
86.61
93.17
80.59
96.21
79.28
86.24
94.88
89.84
96.49
87.36
86.60
91.16
86.24
82.50
85.79
79.12
86.24
95.71
93.04
86.35
86.24
90.67
91.77
89.07
83.96
86.24
93.47
1:6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1:4.5
1:7.5
1:6
1:7.5
1:6
1:7.5
1:6
1:7.5
1:3
1:7.5
1:6
1:4.5
1:6
1:7.5
1:6
1:7.5
1:6
1:4.5
1:4.5
1:6
1:6
The p-value was also used to check the significance of each coefficient. The smaller the p-value,
the more significant was the corresponding coefficient. It can be seen from the table that the coefficients
of B, C, D, AB, AD, BD, CD, A2, B2, C2, and D2 were statistically significant, with p-values below
0.05. The most significant variable was the reaction time (p-value of 0.0001
ꢀ
0.05), followed by the
molar ratio of substrates and reaction temperature. The immobilized catalyst dosage, as one of main
variables, did not show any significant effect, but when multiplied by itself (A2) it played a very
important role in the model (F-value 75.60, p-value 0.0001).
Table 3. Analysis of variance (ANOVA) for the quadratic model developed for synthesis of
dilaurylazelate ester.
Source
Sum of Squares
DF *
Mean Square
F-Value
p-Value Prob > F
Model
A-Novozym 435 amount
B-Reaction time
C-Reaction temperature
688.58
0.27
351.47
40.23
63.52
6.52
21.40
51.28
12.77
84.45
11.62
14.91
23.47
18.99
11.25
7.74
12
1
1
1
1
1
1
1
1
1
1
1
1
17
12
5
57.38
0.27
351.47
40.23
63.52
6.52
21.40
51.28
12.77
84.45
11.62
14.91
23.47
1.12
51.37
0.24
314.67
36.02
56.87
5.84
19.16
45.92
11.43
75.60
10.40
13.35
21.01
-
<0.0001
0.6308
<0.0001
<0.0001
<0.0001
0.0272
0.0004
<0.0001
0.0035
<0.0001
0.0050
0.0020
0.0003
-
D-Molar ratio of substrates
AB
AC
BC
BD
A2
B2
C2
D2
Residual
Lack of fit
Pure error
0.94
1.55
0.61
-
0.7794
-

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Table 3. Cont.
Source
Sum of Squares
DF *
Mean Square
F-Value
p-Value Prob > F
Standard deviation
1.06
54.47
0.9732
0.9542
0.9230
1.20
PRESS
R2
Adjusted R2
Predicted R2
Coefficient of variation
Adequate Precision
24.973
* DF: Degree of Freedom.
The residual plot is an important diagnostic tool to detect the systematic departures from the
assumptions used in developing the regression equations [19]. As shown in Figure 2a, the data
points on this plot were close to a straight line, confirming the assumption of normal distribution
and the independence of the residuals. Figure 2b shows the relationship between the actual and
predicted values for the conversion of AzA. The actual values represented the measured response data
for a particular run, and the predicted values were generated by the model. As observed in Figure 2b,
the predicted values correlated with the actual values with an R2 of 0.9732. This indicated the accuracy of
the model in determining the correlation between the conversion and the independent reaction variables.
Figure 2.
(a) The residual plot of runs from central composite rotatable design; (b) scatter plot of
predicted conversion% value versus actual conversion% value.

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2.3. Mutual Effects of Process Parameters
The terms in Equation (1) showed that the interactions between variables had significant effects
on the degree of conversion% of the enzyme-catalyzed reaction. Therefore, the interactions between
variables could be further investigated to clarify their significance and importance for a more
comprehensive optimization, instead of studying single variables individually. Figure 3a shows
the interaction between enzyme amounts and reaction temperatures on the degree of AzA conversion.
As reaction temperature increased, the degree of AzA conversion correspondingly increased till a
further extension in the temperature variable was not possible due to the boiling point of the solvent
◦
(n-Hexane, bp
≈
68 C). However, denaturation of the enzyme could also occur at higher reaction
temperatures [20,21].
Figure 3b shows the effect of reaction times and enzyme amounts on the degree of AzA conversion.
A response surface plot for the interaction between enzyme amounts and reaction times was generated
with the other variables kept constant. The data obtained proved that the amount of enzyme did
not significantly affect the degree of conversion%. However, at any given enzyme amount between
0.15–0.35 g, the degree of conversion% increased with increased reaction time.
◦
Figure 3. Response surface plots: (
time (min) versus enzyme amount (g); (
) reaction time (min) versus molar ratio of substrates (AzA:LA) (mole) on the percentage conversion
a
) reaction temperature ( C) versus enzyme amount (g); (
b
) reaction
c
) reaction time (min) versus reaction temperature (◦C);
(
d
as a response.
The predicted response surface plots for the interaction of reaction time and reaction temperature
are presented in Figure 3c. At any given reaction time from 90 to 450 min and any given reaction
temperature from 40 to 60 ◦C, the percentage of conversion increased to maximum value. The figure
showed that there was an increase in the percentage of conversion with an increase in both variables,
but a reaction with longer reaction time and lower reaction temperature could increase the conversion
of ester to a higher percentage. This indicated that the conversion of ester was greatly affected by

Molecules 2018, 23, 397
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both reaction time and reaction temperature. Figure 3d depicts the effect of reaction time and molar
ratio of substrates on the percentage of conversion when the other variables were kept constant. It can
be observed that the maximum conversion of AzA was achieved when the reaction time increased
while the molar ratio of substrates decreased. From the data obtained, it was expected that very low
levels of esterification were observed at shorter reaction times. Reactions with longer reaction time
could allow for an increased conversion of ester. It was also observed that the degree of conversion of
AzA increased until the substrate molar ratio reached a 1:4.1 molar ratio. This observation was also
similar to data obtained by Basyaruddin et al. (2008) who reported the optimized enzymatic synthesis
of dilauryladipate ester using response surface methodology [22].
2.4. Reaction Optimization and Model Validation for Dilauryl Azelate Ester
Optimum reaction conditions are needed to achieve the desired degree of conversion of AzA.
Optimum conditions allow for the highest degree of conversion and have the lowest enzyme
amounts, faster reaction times, and lower reaction temperatures. It would be favorable to obtain
the highest conversion percentage for better productivity and improved efficiency, while the lowest
possible enzyme amounts were used in order to save costs due to the economic concerns involved.
With reference to Table 2, experiment number 12 yielded a higher degree of conversion compared
to the optimum conditions, but the amount of enzyme used was also higher (0.25 g). To achieve the
maximum conversion of substrate, it was preferable to use the minimum enzyme amount [23,24].
It is impractical to increase the conversion percentage by utilizing a higher amount of enzyme and a
heavier cost of production, as immobilized lipase (Novozym 435) is more expensive than the other
substrates; thus, it was desirable that a higher reaction conversion was attained by a lower amount of
enzyme. Moreover, lower reaction temperatures and shorter reaction times were considered in the
optimization process to avoid possible enzyme denaturation.
Optimization was carried out by identifying the optimum conditions for a higher reaction
conversion percentage as shown in Table 4. From multivariate analysis, the optimum reaction variables
◦
were: 360 min of reaction time, 0.14 g of enzyme, a reaction temperature of 46 C, and a mole of
substrates molar ratio (AzA:LA) of 1:4.1. The experimental reaction gave a reasonable percentage
conversion of 95.38%. The experimental data confirmed the model validity, and the data achieved was
quite close to the predicted value (96.32%), implying that RSM based on CCRD experimental design
was an accurate and reliable method to optimize the reaction conditions.
In addition, five random reaction conditions were prepared to validate the model. The adequacy
of the final model was checked by comparing the predicted values with the experimental values.
The recommended optimum conditions were also conducted to verify the optimum conversion
percentages that were predicted by the model. The percentage of residual standard error (RSE%) was
calculated for the response.
Table 4. Optimum conditions derived by response surface methodology (RSM) and validation set for
synthesis of dilaurylazelate ester.
Independent Variables
Conversion%
Enzyme
Amount
(gram)
Reaction
Time (min)
Reaction Molar Ratio of Substrates, Actual Predicted RSE
◦
Temperature ( C)
AA:LA (mole)
Value
Value
(%)
0.20
0.30
0.20
0.16
0.10
180
300
360
250
360
50
52
52
55
52
1:6
1:5
1:6.5
1:5.5
1:6
84.23
91.19
90.99
87.17
95.10
83.01
89.05
89.84
87.75
93.54
1.47
2.40
1.28
0.66
1.66
Validation
Set
Optimal
conditions
0.14
360
46
1:4.1
95.38
96.23
0.88

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2.5. Evaluation of the Importance of the Variables on the Reaction Conversion
The important influence of effective variables on the conversion percentages was illustrated by
Pareto analysis in Figure 4. The Pareto analysis describes the effect of each variable, in percentages, on
the response according to the coefficient of the coded variable in Equation (2) [18,25].
!
bi2
b2
P =
i
× 100 (i = 0)
(2)
∑
i
Figure 4. Pareto graphic analysis describes the effect of each of the variables (enzyme amount, reaction
time, reaction temperature and molar ratio of substrates) in percentages (%).
The most important factor that affected the conversion percentage was the reaction time (B) at
49.50%, followed by the interaction between reaction times and reaction temperatures (BC) at 10.80%
and the square form of the enzyme amount factor (A2) at 10.34%.
2.6. Antibacterial Assay of Dilauryl Azelate Ester
The mechanism of the reported bacterial activity of AzA remains to be confirmed. However,
effects on enzyme activities, macromolecular synthesis, and intracellular pH have been suggested.
No bacterial resistances to azelaic acid have as yet been reported. The pH is crucial for the
bacteriological activity of AzA, which is only active at specific values of pH 4–6 Thiboutot et al., 2008.
Normal skin pH has been estimated to be 5.0–6.0. A pH of 5.6 was chosen as representative, following
the lead of others who have conducted tests similar to those in the present study.
The antibacterial activity against the pathogen bacteria Staphylococcus epidermidis S273 of AzA
and dilaurylazelate ester was estimated by the disc and well diffusion agar methods at pH 5.6, and the
results are shown in Table 5. The size of the inhibition zone indicated the antibacterial effect of AzA and
dilaurylazelate ester [
Staphylococcus epidermidis S273. Based on the result obtained, it showed the inhibition zone can be seen
at AzA with diameter 11.5 0.1 mm, while the dilaurylazelate ester also gave the diameter inhibition
zone 9.0 0.1 mm, as well as AzA. This effect is dependent on the concentration of dilaurylazelate
ester and AzA, as well as any putative condition causing a local increase in skin pH.
1]. Figure 5 represented the inhibition zone of dilaurylazelate ester on bacteria
±
±

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Figure 5. The inhibition zone of AzA and dilaurylazelate ester on bacteria Staphylococcus epidermidis
S273, (−ve is Acetonitrile and +ve is Standard (Streptomycin)).
Table 5. The size of the inhibition zone indicated the antibacterial effect of AzA and dilaurylazelate
ester on bacteria Staphylococcus epidermidis S273.
Diameter Zone Inhibition (mm)on Bacteria
Samples
Staphylococcus epidermidis S273
Azelaic acid
Dilauryl azelate ester
Standard (Streptomycin)
11.5 ± 0.1 mm
9.0 ± 0.1 mm
28.0 ± 0.1 mm
2.7. Cytotoxicity Assay of Dilauryl Azelate Ester
Normal fibroblasts cell line (3T3) was used to assess the toxicity of AzA and AzA derivative,
which is dilaurylazelate ester. Concentration cytotoxicity of both compounds was evaluated by MTT
colorimetric assay to test cellular response of the 3T3 cell (Figure 6). It was showed the comparison
of IC50 (50% inhibition of cell viability) value for AzA and AzA derivative. IC50 value for AzA was
85.28
AzA derivative did not reach up to the highest concentration tested. (Gad, 2009) reported that IC50
value lower than 10 g/mL is very toxic, while 10–100 g/mL is toxic. In this study, the cells were still
µg/mL, whereas AzA derivative was more than 100 µg/mL. Based on the result, IC50 value of
µ
µ
able to survive without any sign of toxicity for dilaurylazelate ester; thus, it was proven to be non-toxic
in this MTT assay compared with AzA and is safe to be used in pharmaceutical applications [26].

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Figure 6. Comparison the toxicity effect of azelaic acid (AzA) and AzA derivatives (dilaurylazelate
ester) on normal fibroblasts cell line (3T3). Data represented in percentage (%) of cell viability (IC50).
3. Materials and Methods
3.1. Materials
Lauryl alcohol and azelaic acid were obtained from Merck (Darmstad, Germany). Immobilized
Candida antarctica lipase B (Novozym 435) was purchased from Novo Nordisk Industries (Bagsvaerd,
Denmark). All solvents and reagents were of analytical high-performance liquid chromatography
(HPLC) grade. n-Hexane, chloroform, dichloromethane, acetone, ethanol, acetonitrile, and diethyl
ether obtained from J.T.Baker (Center Valley, PA, USA) was used as the organic solvent. All other
reagents used were of analytical grade.
3.2. Method
3.2.1. Enzymatic Esterification of Azelaic Acid and Analysis of Samples
In a typical experiment, azelaic acid (1.59 mmol), a proportional amount of lauryl alcohol,
and different amounts of Novozym 435 in n-hexane (5 mL) were placed in 10 mL screw-capped
glass vials. The 3Å molecular sieves (Merck) (0.03g) that were added in the reaction mixture helped to
remove water from organic media. The mixture was stirred (150 rpm) in a horizontal water bath shaker.
When the AzA substrate was entirely converted according to the adjusted reaction times, the mixture
was filtered to remove the enzyme.
A titration method using 0.1 M NaOH solution was applied to determine the percentage of AzA
conversion. The AzA conversions were calculated from the titration volumes of the NaOH solution
for the reaction samples, with enzyme (test) and without enzyme (control). The product formed was
expressed as equivalent to the conversion of acid [27,28]. The percentage of the degree of conversion
of AzA was calculated by the consumption of NaOH solution (0.1 M) for both the experiment and the
control. The degree of conversion was determined using Equation (3).
For purification of the azelate ester, after termination of the reaction, the enzyme was filtered and
the solvent removed by evaporator under reduced pressure. Product in the remaining mixture were
separated via silica gel (Kieselgel 60, Merck, particle size 0.063–0.200 mm) column chromatography
(15 cm
×
20 mm) using a chloroform/dichloromethane (95/5, v/v) mixture as eluent. A sample
made up of 1:1 (w/w) ratio of silica gel and the free solvent reaction mixture was deposited at the

Molecules 2018, 23, 397
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top of the column previously equilibrated with chloroform/dichloromethane (95/5, v/v) mixture.
Five millilitre fractions were collected and tested using thin layer chromatography to identify the
product-rich portions. Such fractions were pooled and the solvent evaporated by a rotary evaporator.
The purity of product was then checked with TLC before FTIR, GCMS, and NMR.
A solution of dichloromethane:chloroform (5:95, v/v) was selected as the mobile phase for the
thin-layer chromatography (TLC) analysis. Further identification of the ester formed was determined
by FTIR (Perkin Elmer, Bridgeville, PA, USA); gas chromatography mass spectroscopy (GCMS), model
MS QP5050A; Shimadzu, Tokyo, Japan; and NMR spectra (1H and 13C) were obtained by using a JEOL
FTNMR 500 MHz transformed spectrometer in CDCl3 or CD3OD and using tetramethylsilane (TMS)
as the internal standard 500 MHz for both 1H NMR and 13C NMR, respectively.
V
− V
test
control
Degree o f Conversion% =
and V are NaOH (0.1 M) volumes used for neutralizing the excess acid in the
test
control
× 100
(3)
V
control
In which
V
test (with enzyme) and control (without enzyme) experiments, respectively.
3.2.2. Antibacterial Assay
The antibacterial effect of azelaic acid derivatives (dilaurylazelate ester) against Staphylococcus
epidermidis S273 were studied by means of agar diffusion method. The test is carried out by placing
6 mm diameter of paper disc containing antibiotic onto a plate on which microbes are growing.
The microbe culture is standardized to 0.5 McFarland standard, which is approximately 108 cells
(Mueller Hinton agar pH 7.3). Phosphate buffer (NaH2PO4) was added until pH 5.6. Streptomycin
standard was used for each bacteria. The plates are inverted and incubated at 30–37◦C for 18–24 h
using a target organism. After incubation, zone of inhibition appeared and each plate was examined.
The diameters of zones of complete inhibition are measured including the diameter of disc. Zones are
measured to the nearest whole millimeter using sliding calipers, which are held on the back of the
inverted petri plate.
3.2.3. Cytotoxicity Assay
Azelaic acid and azelaic acid derivative (dilaurylazelate ester) were tested further for their
cytotoxicity activity. Both of the samples were tested on 3T3 normal fibroblast cells. All cells were
maintained in RPMI medium supplemented with 10% fetus calf serum (FCS), 100 unit/mL penicillin,
and 0.1 mg/mL streptomycin. Initially, cell culture with a concentration of 1
prepared and plated (100
×
105 cells/mL was
◦
µ
L/well) onto 96-wellplates. The cell was incubated for 24 h at 37 , 5% CO2.
After incubation, the diluted ranges of samples (1.56, 3.125, 6.25, 12.5, 25, 50, 100 and 120 µg/mL)
were added to each well and incubated for another 24 h. Then, MTT solution at 2 mg/mL was added
to each well and left to stand for 1 h before being measured at 570 nm. The cytotoxicity result was
expressed as IC50, defined as the concentration of sample that caused inhibition of 50% cell growth.
4. Conclusions
In general, the optimization of a lipase (Novozym 435) catalyzed esterification reaction to
produce dilaurylazelate ester by applying response surface methodology was successfully carried
out. The effect of four main reaction parameters (reaction times, amounts of enzyme, reaction
temperatures, and substrate molar ratio) were evaluated using an empirical model. The coefficient of
the determination value (R2) of 0.9732 was desirable and demonstrated a good fit with the experimental
data. This fitted model could be applied to find the yields of production of dilaurylazelate ester under
any given conditions within the experimental range. Experimentally, a high percentage of conversion
(95.38%) was achieved, which also matched well with the predicted value of 96.23%, by applying only
a minimal enzyme amount. Therefore, the scaled-up enzymatic production of dilaurylazelate ester,
taking into consideration the economical and environmental aspects, can be carried out in the future

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by applying the RSM techniques. In addition, the cytotoxicity was tested on 3T3 normal fibroblast
cells; the results showed that dilaurylazelate ester is non-toxic compared with AzA and safe to be used
in pharmaceutical applications, and it has interesting antibacterial properties which are worthy of
further investigation in clinical trials of a relevant group of test subjects.
Acknowledgments: The financial assistance provided by Universiti Putra Malaysia under the Research University
Grant Scheme (RUGS) is gratefully acknowledged.
Author Contributions: N.K., M.B., and H.R.F.M. conceived and designed the experiments; N.K. performed the
experiments; N.K., H.R.F.M., S.E.A., and S.S. analyzed the data. N.K., and H.R.F.M. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Kosmadaki, M.; Katsambas, A. Topical treatments for acne. Clin. Dermatol. 2017, 35, 173–178. [CrossRef]
[PubMed]
2.
Khairudin, N.; Basri, M.; Masoumi, H.R.F.; Samiun, W.S.; Samson, S. Lipase-catalyzed synthesis of dilauryl
azelate ester: Process optimization by artificial neural networks and reusability study. RSC Adv. 2015, 5,
94909–94918. [CrossRef]
3.
4.
5.
Al-Marabeh, S.; Khalil, E.; Khanfar, M.; Al-Bakri, A.G.; Alzweiri, M. A prodrug approach to enhance azelaic
acid percutaneous availability. Pharm. Dev. Technol. 2016, 22, 1–9. [CrossRef] [PubMed]
Tamarkin, D. Novel Conjugate Compounds and Dermatological Compositions Thereof. U.S. Patent
20,040,191,196, 30 September 2004.
Manosroi, A.; Panyosak, A.; Rojanasakul, Y.; Manosroi, J. Characteristics and anti-proliferative activity of
azelaic acid and its derivatives entrapped in bilayer vesicles in cancer cell lines. J. Drug Target. 2007, 15,
334–341. [CrossRef] [PubMed]
6.
7.
8.
9.
Fang, J.-Y.; Leu, Y.-L. Prodrug strategy for enhancing drug delivery via skin. Curr. Drug Discov. Technol. 2006
3, 211–224. [CrossRef] [PubMed]
Svensson, C.K. Biotransformation of drugs in human skin. Drug Metab. Dispos. 2009, 37, 247–253. [CrossRef]
[PubMed]
Prausnitz, M.R.; Langer, R. Transdermal drug delivery. Nat. Biotechnol. 2008, 26, 1261–1268. [CrossRef]
[PubMed]
Hsieh, P.-W.; Al-Suwayeh, S.A.; Fang, C.-L.; Lin, C.-F.; Chen, C.-C.; Fang, J.-Y. The co-drug of conjugated
hydroquinone and azelaic acid to enhance topical skin targeting and decrease penetration through the skin.
Eur. J. Pharm. Biopharm. 2012, 81, 369–378. [CrossRef] [PubMed]
,
10. Narula, O. Treatise on Fats, Fatty Acids & Oleochemicals; Industrial Consultants: New Delhi, India, 1995.
11. Cao, L.; Fischer, A.; Bornscheuer, U.T.; Schmid, R.D. Lipase-catalyzed solid phase synthesis of sugar fatty
acid esters. Biocatal. Biotransform. 1996, 14, 269–283. [CrossRef]
12. Chaibakhsh, N.; Abdul Rahman, M.B.; Basri, M.; Salleh, A.B.; Rahman, R.N.Z.R.A. Effect of alcohol chain
length on the optimum conditions for lipase-catalyzed synthesis of adipate esters. Biocatal. Biotransform.
2009, 27, 303–308. [CrossRef]
13. Syamsul, K.; Salina, M.; Siti, S.; Hanina, M.; Basyaruddin, M.; Jusoff, K. Green synthesis of lauryl palmitate
via lipase-catalyzed reaction. World Appl. Sci. J. 2010, 11, 401–407.
14. Zisis, T.; Freddolino, P.L.; Turunen, P.; van Teeseling, M.C.; Rowan, A.E.; Blank, K.G. Interfacial activation
of Candida antarctica lipase b: Combined evidence from experiment and simulation. Biochemistry 2015, 54,
5969–5979. [CrossRef] [PubMed]
15. Anderson, E.M.; Larsson, K.M.; Kirk, O. One biocatalyst—Many applications: The use of Candida antarctica
b-lipase in organic synthesis. Biocatal. Biotransform. 1998, 16, 181–204. [CrossRef]
16. Ashari, S.E.; Mohamad, R.; Ariff, A.; Basri, M.; Salleh, A.B. Optimization of enzymatic synthesis of
palm-based kojic acid ester using response surface methodology. J. Oleo Sci. 2009, 58, 503–510. [CrossRef]
[PubMed]
17. Khandanlou, R.; Ahmad, M.B.; Masoumi, H.R.F.; Shameli, K.; Basri, M.; Kalantari, K. Rapid adsorption of
copper (ii) and lead (ii) by rice straw/fe 3 o 4 nanocomposite: Optimization, equilibrium isotherms, and
adsorption kinetics study. PLoS ONE 2015, 10. [CrossRef] [PubMed]

Molecules 2018, 23, 397
14 of 14
18. Sohrabi, M.R.; Amiri, S.; Masoumi, H.R.F.; Moghri, M. Optimization of direct yellow 12 dye removal by
nanoscale zero-valent iron using response surface methodology. J. Ind. Eng. Chem. 2014, 20, 2535–2542.
[CrossRef]
19. Yetilmezsoy, K.; Demirel, S.; Vanderbei, R.J. Response surface modeling of pb (ii) removal from aqueous
solution by Pistacia vera L.: Box—Behnken experimental design. J. Hazard. Mater. 2009, 171, 551–562.
[CrossRef] [PubMed]
20. Krishna, S.H.; Divakar, S.; Prapulla, S.G.; Karanth, N.G. Enzymatic synthesis of isoamyl acetate using
immobilized lipase from rhizomucor miehei. J. Biotechnol. 2001, 87, 193–201. [CrossRef]
21. Radzi, S.M.; Basri, M.; Salleh, A.B.; Ariff, A.; Mohamad, R.; Abdul Rahman, M.B.; Abdul Rahman, R.N.Z.
Large scale production of liquid wax ester by immobilized lipase. J. Oleo Sci. 2005, 54, 203–209. [CrossRef]
22. Basyaruddin, A.R.M.; Chaibakhsh, N.; Basri, M.; Rahman, R.N.Z.R.A.; Salleh, A.B.; Radzi, S.M. Modeling
and optimization of lipase-catalyzed synthesis of dilauryl adipate ester by response surface methodology.
J. Chem. Technol. Biotechnol. 2008, 83, 1534–1540.
23. Hari Krishna, S.; Sattur, A.P.; Karanth, N.G. Lipase-catalysed synthesis of isoamly isobutyrate-optimisation
using a central composite rotatable design. Process Biochem. 2001, 37, 9–16. [CrossRef]
24. Masoumi, H.R.F.; Kassim, A.; Basri, M.; Abdullah, D.K.; Haron, M.J. Multivariate optimization in the
biosynthesis of a triethanolamine (tea)-based esterquat cationic surfactant using an artificial neural network.
Molecules 2011, 16, 5538–5549. [CrossRef] [PubMed]
25. Ngan, C.L.; Basri, M.; Lye, F.F.; Fard Masoumi, H.R.; Tripathy, M.; Abedi Karjiban, R.; Abdul-Malek, E.
Comparison of box—Behnken and central composite designs in optimization of fullerene loaded palm-based
nano-emulsions for cosmeceutical application. Ind. Crop Prod. 2014, 59, 309–317. [CrossRef]
26. Gad, S.C. Alternatives to in vivo studies in toxicology. In General, Applied and Systems Toxicology, 2nd ed.;
Ballantyne, B., Marvis, Y., Turner, P., Eds.; Macmillan: New York, NY, USA, 2009.
27. Chaibakhsh, N.; Rahman, M.B.A.; Abd-Aziz, S.; Basri, M.; Salleh, A.B.; Rahman, R.N.Z.R.A. Optimized
lipase-catalyzed synthesis of adipate ester in a solvent-free system. J. Ind. Microbiol. Biotechnol. 2009, 36,
1149–1155. [CrossRef] [PubMed]
28. Fard Masoumi, H.R.; Basri, M.; Kassim, A.; Kuang Abdullah, D.; Abdollahi, Y.; Abd Gani, S.S.; Rezaee, M.
Statistical optimization of process parameters for lipase-catalyzed synthesis of triethanolamine-based
esterquats using response surface methodology in 2-liter bioreactor. Sci. World J. 2013, 2013. [CrossRef]
[PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
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