Ultrasound assisted enzyme catalyzed degradation of Cetirizine dihydrochloride

Article abstract of DOI:10.1016/j.ultsonch.2014.10.016

Cetirizine dihydrochloride, a pharmaceutical drug of the class antihistamines is frequently detected in wastewater samples. In the present work, the degradation of Cetirizine dihydrochloride is carried out using a novel technique of laccase enzyme as a catalyst under the influence of ultrasound irradiation. Effect of various process parameters such as enzyme loading, temperature, power, duty cycle, frequency and speed of agitation has been studied along with identification of the degradation intermediates. The maximum degradation of 91% is achieved at optimized experimental parameters such as 0.02% enzyme loading (w/v), 50°C temperature, power input of 100 W, 25 kHz frequency and 50% duty cycle with agitation speed of 200 rpm. It is observed that enzymatic degradation of Cetirizine dihydrochloride under the influence of ultrasound irradiation not only enhances the degradation but also reduces the time of degradation as compared to conventional enzymatic degradation technique.

Full text of DOI:10.1016/j.ultsonch.2014.10.016

Accepted Manuscript
Ultrasound assisted enzyme catalyzed degradation of Cetirizine dihydrochloride
Rahul S. Sutar, Virendra K. Rathod
PII:
S1350-4177(14)00322-8
DOI:
http://dx.doi.org/10.1016/j.ultsonch.2014.10.016
ULTSON 2721
Reference:
Ultrasonics Sonochemistry
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Accepted Date:
2 July 2014
17 September 2014
13 October 2014
Please cite this article as: R.S. Sutar, V.K. Rathod, Ultrasound assisted enzyme catalyzed degradation of Cetirizine
dihydrochloride, Ultrasonics Sonochemistry (2014), doi: http://dx.doi.org/10.1016/j.ultsonch.2014.10.016
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Ultrasound assisted enzyme catalyzed degradation of Cetirizine dihydrochloride
Rahul S. Sutar, Virendra K. Rathod*
Chemical Engineering Department
Institute of Chemical Technology,
Matunga, Mumbai-400019,
India.
* Corresponding Author: E-mail: vk.rathod@ictmumbai.edu.in, Phone: 91-22-33612020,
Fax: 91-22-24145614

Abstract
Cetirizine dihydrochloride, a pharmaceutical drug of the class antihistamines is frequently
detected in wastewater samples. In the present work, the degradation of Cetirizine
dihydrochloride is carried out using a novel technique of laccase enzyme as a catalyst under
the influence of ultrasound irradiation. Effect of various process parameters such as enzyme
loading, temperature, power, duty cycle, frequency and speed of agitation has been studied
along with identification of the degradation intermediates. The maximum degradation of 91%
is achieved at optimized experimental parameters such as 0.02% enzyme loading (w/v), 50°C
temperature, power input of 100 W, 25 kHz frequency and 50% duty cycle with agitation
speed of 200 rpm. It is observed that enzymatic degradation of Cetirizine dihydrochloride
under the influence of ultrasound irradiation not only enhances the degradation but also
reduces the time of degradation as compared to conventional enzymatic degradation
technique.
Keywords: Degradation, Cetirizine dihydrochloride, Laccase, Ultrasound irradiation.

1. Introduction
In recent years, pharmaceutical pollutants are given much attention due to their
multiple adverse effects. The pharmaceuticals arriving into an aquatic environment from
sewage effluents is a rising problem [1]. Pharmaceuticals include numerous therapeutic drugs
having diverse physicochemical properties. The continuous discharge of pharmaceuticals into
the environment attributes to their adverse effect on human health and environment. The
concentration of pharmaceuticals present in wastewater varies from nanograms per liter to
micrograms per liter [2]. Owing to elicitation of biological response, pharmaceuticals show
detrimental effects to the aquatic ecosystems including endocrine disrupting and other
hazardous side effects [3]. Pharmaceuticals are existed in aquatic environment through
several routes comprising their incomplete removal in conventional wastewater treatment
methods, human as well as animal excretions, direct disposal of unneeded medicines and also
the release of them during the process of manufacturing of the drug [4].
Presence of Cetirizine in the wastewater was investigated in variable concentration
range [5, 6]. In India, pharmaceuticals have been detected at elevated levels of concentration
(up to 1,400 micrograms per liter) in the effluent acquired from a wastewater treatment plant
near Hydrabad MIDC [1]. Cetirizine dihydrochloride append to the piperazine class of
second generation antihistamines used in the treatment of allergies, hay fever, angioedema
and urticaria. Chemical name of Cetirizine dihydrochloride is 2-(2-(4-(4-chlorophenyl)
phenylmethyl) piperazin-1-yl) ethoxy) acetic acid Dihydrochloride [7]. In order to
circumvent the adverse effects of such pharmaceuticals on the environment, wastewater
treatment is essential for their removal.
Over the last decades, various methods have been attempted for the treatment of
wastewater. For demand of water quality standards considering the release of industrial
wastewater and also water decontamination; the concern has been improved considerably
towards enzyme application for the degradation of contaminants detected in the wastewater.

Enzyme possess many advantages over conventional biological treatment and/or chemical
oxidation which includes absence of an acclimatization period, easy process control, low
energy requirement, react at higher concentrations of compounds, no toxic effects and
absence of unwanted product generation [8, 9]. Due to these benefits, enzymatic treatment
can be deliberated as one of the alternatives over conventional wastewater treatment. Enzyme
Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) belongs to the class of oxidases
containing four copper atoms in its catalytic site with extensive range of substrate specificity.
Oxidation of a variety of compounds including phenolic compounds like polyphenols,
methoxy-substituted monophenols, aromatic amine, diamines is catalyzed by laccase with
concomitant reduction of molecular oxygen to water [10, 11]. As laccase catalyzes oxidation,
degradation of several drugs has been reported in the literature. However, conventional
enzymatic degradation necessitates higher time for degradation of pharmaceuticals due to
slower reaction rate and also exhibits less degradation for some pharmaceuticals [2]. These
disadvantages of conventional method can be overcome by concurrent application of
ultrasound with enzymes [9].
Formation, subsequent growth and violent collapse of bubbles or cavities in the
medium, is known as acoustic cavitation resulting due to ultrasonic irradiation [12]. Release
of energy due to cavitation enhances mass transfer rate owing to enhanced interaction
between enzyme and substrate molecules [13]. Enzyme stability and catalytic activity
amplified by the application of ultrasound in case of enzyme-based applications [9]. Use of
ultrasound has been reported for the enhancement of various important bio-processes such as
enzymatic transformations, environmental remediation, fermentations, anaerobic digestion,
food processing and enzyme assisted chemical synthesis [14].
To the best of our knowledge, the degradation of Cetirizine dihydrochloride has not
reported neither by enzymatic method nor by ultrasound assisted enzymatic method. As

Cetirizine dihydrochloride belongs to class of antihistamines, it causes various side effects
such as sleepiness, dry mouth, fatigue fever, sedation, stomach upset, blurred vision when
ingested accidentally to human body [15, 16]. At higher concentration, cumulative health
effects may be produced due to long-term occupational exposure of Cetirizine
dihydrochloride as suggested in limited evidences [15]. In the present work, the degradation
of Cetirizine dihydrochloride was carried out using laccase enzyme under the influence of
ultrasonic irradiation. Various process parameters such as enzyme loading, temperature,
speed of agitation, ultrasonic power, ultrasonic frequency and duty cycle were optimized
along with identification of intermediates and comparison has also been done with
conventional stirring method.
2. Materials and Methods
2.1 Materials
Laccase from Aspergillus oryzae in a free form was received as a gift sample from
Advanced Enzymes Pvt. Ltd, Thane. Cetirizine dihydrochloride was obtained from Sigma
Aldrich. All other chemicals such as Acetonitrile (Thomas Baker, HPLC grade), Guaiacol
were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai.
2.2 Laccase-catalyzed degradation of Cetirizine dihydrochloride
Ultrasound assisted enzymatic degradation of Cetirizine dihydrochloride using
Laccase as a catalyst was carried in an ultrasonic thermostatic water bath (Model No. 6-
SL200H/DTC/DF – manufactured by Dakshin India Pvt. Ltd., 4.5 L capacity) with four
transducers placed at the bottom of the bath. The reaction was carried out in a flat bottom
baffle glass reactor of 50 mL (i.d. 4.5 cm), capacity with 3 blade turbine glass impeller
connected with motor and reflux condenser. The agitation was provided with the help of
electric motor having the provision of speed control. The whole reactor assembly was placed

in the ultrasonic water bath. The ultrasonic thermostatic water bath was maintained at desired
temperature with an accuracy of 1oC by circulating water. Throughout all the experiments,
the reactor was placed at the position where maximum efficiency of ultrasonic irradiation was
obtained as reported by Kulkarni et al., (2014) [17].
In order to study the degradation, 20 mL of Cetirizine dihydrochloride (5 mg/L) in
phosphate Buffer (0.05 M) was added in the reactor along with 0.025% (w/v) enzyme
laccase. The reaction mixture was agitated at 200 rpm and the temperature of reaction
mixture was maintained at 60oC. By considering the addition of enzyme as 0 min, samples
were withdrawn periodically at an interval of 30 minute and subsequently analyzed by using
High Pressure Liquid Chromatography (HPLC).
2.3 Analytical Method
2.3.1 High Pressure Liquid Chromatography
The samples withdrawn periodically from the reaction mixture were analyzed using High
Pressure Liquid Chromatography. The HPCL equipment (Agilent Technologies) consists of
1260 Quat pump VL and 1260 VWD VL as a UV detector. The column used for the analysis
was C18 (Cosmosil Packed Column) of dimensions 4.6 ID × 250 mm. The wavelength was
set at 230 nm. Sample volume of 20 µL was injected in each run. The samples were analyzed
using mobile phase of Acetonitrile: Water (40:60, pH 3.0) at a flow rate of 1 mL/min.
2.3.2 Assay of laccase activity
Enzyme activity was determined using Guaiacol as the substrate. Amount of enzyme
catalyzing the production of colored product per min per mL is known as enzyme activity
which was measured in U/mL [18].

∆Aꢀ/ꢀminꢀ × ꢀ4ꢀ × ꢀVtꢀ × ꢀdilutionꢀfactor
€ꢀ × ꢀVs
ꢁ
ꢂ
Volumeꢀactivityꢀ U/ml =
Where, Vt = final volume of reaction mixture (mL)
Vs = sample volume (mL)
€ = extinction co-efficient of guaiacol = 6,740 /M/cm
3. Results and discussion
3.1 Optimum pH and optimum temperature for enzyme Laccase
In order to determine the optimum pH for the laccase enzyme, various buffers were prepared
with pH ranging from 4 to 8 while to determine the optimum temperature, the temperature
was also varied from 30oC to 70oC. By using Guaiacol as the substrate, enzyme activity was
determined which is expressed as percent relative activity. Relative activity is defined as the
ratio of the activity at each pH to the maximum attained at pH 6.0 [2].
Results obtained are depicted in the Fig. 1 and Fig. 2 which shows that the optimum pH and
temperature for the laccase enzyme was 6.0 was 60oC respectively.
3.2 Effect of enzyme loading
The quantity of enzyme required for maximum degradation is the main economic factor in
enzymatic degradation study for its industrial application. To select the optimum amount of
enzyme required for the maximum degradation, experiments were carried out in the range of
0.015% – 0.03% (w/v) enzyme loading, keeping power 50 W, duty cycle 50% and frequency
25 kHz, temperature 60oC and agitation speed of 200 rpm. With various enzyme loadings, the
degradation results obtained are depicted in Fig. 3. It can be seen from the figure that an
increase in enzyme concentration from 0.015% to 0.02% (w/v) results in an increase in the
degradation from 48% to 73%. However, the degradation is decreased with further increase
in enzyme loading. Rapid formation of enzyme–substrate complexes initially with increasing

enzyme loading attributes to enhancement of reaction rate and the degradation. The reduction
in degradation at higher enzyme loading is possibly due to increase in viscosity of solution
and formation of enzyme aggregates. The increase in viscosity of solution generates problem
of mixing while enzyme aggregates reduces the mass transfer by diminishing surface area to
reactant [13, 19]. Therefore, the results show that an optimum enzyme loading of 0.02% w/v
exists beyond which any further improvement was not observed for increasing enzyme
concentration.
3.3 Effect of temperature
A higher temperature leads to higher reaction rate, enhancing interactions between catalytic
particles and substrates by reducing the viscosity of the reaction mixture [19, 20]. However,
when the temperature rises above the optimum temperature of enzyme, the disruption of the
active conformation of enzyme takes place at increasing temperature which causes the loss of
activity and selectivity due to denaturation [20]. Considering the enzyme feasibility and
maximum cavitation effect, it is likely that the degradation reaction must be carried out at an
optimum temperature. Effect of reaction temperature on the degradation was carried out
under ultrasonic irradiation at different operating temperatures ranging from 40oC to 70oC.
The other parameters i.e. power 50 W, duty cycle 50% and frequency 25 kHz, agitation of
200 rpm and catalyst loading of 0.02 % were kept constant. The results obtained have been
depicted in Fig. 4 which shows that at lower temperature the extent of degradation is lower as
compared to higher temperature range. With an increase in reaction temperature from 40°C to
50°C, the degradation obtained after 7 h was increased from 58 to 84%. The cavitation effect
starts decreasing as the temperature was further increased to 60°C. The cavitation intensity
gets lower down with an increase in temperature owing to reduction in cavitation intensity at
higher temperatures [21]. Bubbles collapse with high intensity at lower temperature,

increasing the mass transfer while at higher temperature more bubbles are formed which
collapse with less intensity [13, 21]. Also in optimization study of temperature for enzyme,
activity was decreased comparatively at higher temperature as shown in Fig. 2. Due to
thermal deactivation of enzyme at higher temperature and also decrease in the cavitation
intensity, the degradation was reduced at reaction temperature of 70°C as compared to other
temperatures. Therefore, the optimum temperature of 50°C was selected where maximum
extent of degradation was attained.
3.4 Effect of ultrasound power and frequency
Effect of irradiation power on degradation was investigated by varying the power in the range
of 30W-150W, keeping other parameters constant. The degradation was less at lower
irradiation power of 30 W; as compared to higher irradiation power (Fig. 5). When the
irradiation power was increased to 100 W, the degradation was also enhanced. As the
irradiation power was increased, bubbles formed imploded energetically which resulted into
the increase in the degradation. More bubbles were formed with further increase in irradiation
power, hampering the propagation of shock waves so that degradation gets decreased at 150
W [21]. Also at higher irradiation power, the bubbles coalesce to form bigger bubbles causing
weak implosion so that degradation was reduced. Therefore, the irradiation power of 100 W
was considered as optimum. Calorimetric method was used for the calculation of power
dissipation in reactor by varying input power at 25 kHz frequency. It has been observed that
power dissipation was 13.95 W, 33.13 W, 48.83 W and 64.53 W for 30 W, 50 W, 100 W and
150W respectively [13].
In order to investigate effect of frequency, the degradation of Cetirizine dihydrochloride was
carried out at same energy dissipation in the reactor (approximately 32 W); at two different
frequencies i.e., 25 kHz and 40 kHz. Results obtained are depicted in Fig. 6 which showed

higher degradation at 25 kHz than 40 kHz. At higher frequency, the cavitation decreases
because smaller and less energetic bubbles are formed which leads to decreased cavitation
zone violence. It has been observed that at same energy dissipation in the reactor; a
significant improvement in the degradation was observed at 25 kHz as compared to 40 kHz
frequency. Due to less scattering and attenuation of sound waves at lower frequencies as well
as easy cavitation at lower frequency, comparatively higher degradation was obtained [13,
17]. Kasaai et al., 2013 also reported that insufficient bubble growth takes place at higher
frequency due to very short rarefaction (and compression) cycles [22]. Hence, 25 kHz was
considered as optimum frequency for further experiments.
3.5 Effect of Duty Cycle
Duty cycle is an important factor in ultrasound assisted enzymatic degradation as continuous
exposure of sound waves may lead to denaturation of the enzyme [23]. This forces the use of
ultrasound with an appropriate duty cycle. Total time of exposure of ultrasonic waves on the
reaction in one cycle is referred as duty cycle which is expressed as percent duty cycle. By
varying on time and off time of ultrasound, the percent duty cycle can be changed. The effect
of duty cycle on the degradation of Cetirizine dihydrochloride was studied at 40%, 50% and
60% duty cycles. In order to obtain 50% duty cycle for a 20 min cycle, the ultrasound was
kept on for 10 min and then 10 min off. Similarly for 40% duty cycle, on time was 8 min and
off time was 12 min. However, for 60% duty cycle, on time was 12 min and off time was 8
min. As the percent duty cycle increases from 40% to 50%, the degradation increases from
50% to 91% as depicted in Fig. 7. When the percent duty cycle was further increased to 60%
the degradation was decreased to 44% possibly due to the denaturation of enzyme at higher
duty cycle. Hence the appropriate on and off time should be determined for the degradation
reaction to avoid denaturation of the enzyme due to excessive heating at higher duty cycle as

well as unnecessary electrical energy consumption [13, 24]. Therefore, the duty cycle of 50%
was considered as optimum duty cycle for further experiments.
3.6 Effect of speed of agitation
Speed of agitation is also an important factor in degradation reaction as it affects the mass
transfer resistance. In order to study the effect of speed of agitation on the degradation,
reactions were carried out under ultrasonic irradiation ranging from 0 rpm to 300 rpm.
Results obtained are shown in the Fig. 8. It has been observed that the degradation increases
from 42 % to 90% with an increase in the speed of agitation from 0 rpm to 200 rpm due to
increase in mass transfer. While further increase in the speed i.e. at 300 rpm, the rate of
reaction was higher but did not give any further improvement in degradation. To minimize
unnecessary electrical energy consumption, the 200 rpm was taken as the optimum speed of
agitation for the degradation reaction.
3.7 Comparative analysis of Ultrasonic irradiation Vs. Conventional stirring
method
Experiments were performed under optimized conditions for enzyme catalyzed degradation
i.e. ultrasonication with and without stirring, conventional stirring method and also with
ultrasonication without enzyme. The results obtained are shown in Table 1. It has been
observed that when ultrasound coupled with stirring, the degradation of Cetirizine
dihydrochloride was 91 % in 7 h while with conventional stirring method the degradation was
very less about 13 % only in 24 h. For the experiment of ultrasound without stirring,
degradation was less i.e. 42 % after 7 h. It has been observed that although speed of agitation
is increased beyond 200 rpm in the conventional stirring method, no improvement in the
degradation was observed. While the degradation was reduced to 6 % at speed 300 rpm.
Therefore, the lower degradation has been attributed to denaturation of enzyme at higher

speed of agitation. Excessive agitation produces shearing effect along with large amount of
heat generation which leads to denaturation of enzyme [25-27]. As laccase belongs to the
oxidoreductase class of enzyme, during oxidation reaction with substrates, it requires oxygen
as final electron acceptor for its reduction into water. Therefore, the oxygen concentration
was also determined and results obtained are represented in Table 2. This study was
performed under three different conditions, ultrasonication combined with stirring,
ultrasonication without stirring and conventional stirring method. It has been observed that
even though change in oxygen concentration is less in case of conventional stirring method, it
does not show higher degradation due less interaction between enzyme and substrate
molecules. In the case of ultrasonication with stirring, the change in oxygen concentration is
comparatively more so increased interaction between enzyme and substrate particles
enhances the degradation percentage. However, ultrasonication without stirring does not
produce significant change in oxygen concentration but due to lower mass transfer, less
degradation has been obtained. Rokhina et al., 2009 also reported that ultrasound perturbs the
loop and domain regions of enzyme which produces effect on enzyme activity. Hence, this
might be reason for the increased degradation in case of ultrasound coupled with stirring.
From results it can be concluded that in the case of ultrasonication coupled with stirring gives
higher degradation compared to conventional stirring method. Therefore, the drawback of
enzyme catalyzed reaction i.e. slow reaction rate can be overcome by the use of ultrasound.
3.8 Identification of intermediates
In order to determine the Cetirizine dihydrochloride degradation products, the withdrawn
samples at optimized conditions were analyzed using Thermo Electron Corporation MS
instrument (Finnigen LCQ Advantage Max). The samples were analyzed using MS analysis
where maximum degradation of 91% is achieved at optimized experimental parameters such

as 0.02% enzyme loading (w/v), 500C temperature, agitation speed of 200 rpm, power input
of 100 W, 25 kHz frequency and 50% duty cycle.
Laccase carries out oxidation of substrate with concomitant reduction of molecular oxygen to
water. Hence, according to the reaction mechanism, Laccase first transfers electron form
Cetirizine dihydrochloride producing (2-(4-(4-chlorophenyl) phenylmethyl) piperazin-1-yl)
ethanol which may further get degraded into 1-benzyl-4-chlorobenzene and 2-(piperazin-1-
yl) acetaldehyde. The pathway of degradation of Cetirizine dihydrochloride is shown in Fig.
9 and these products are confirmed by m/z values obtained by MS analysis.
Three prominent peaks were identified from LC–MS analysis at m/z 330.5, m/z 202.5 and
m/z 128 corresponding to 2-(2-(4-(4-chlorophenyl) phenylmethyl) piperazin-1-yl) ethanol, 1-
benzyl-4-chlorobenzene and 2-(piperazin-1-yl) acetaldehyde respectively as shown in Table
3.

4. Conclusions
For the first time degradation of Cetirizine dihydrochloride using laccase as a bio-
catalyst under the influence of ultrasound irradiation was studied. Optimization of various
process parameters such as enzyme loading, temperature, speed of agitation, ultrasound
power, frequency and duty cycle were carried out successfully. With various optimized
parameters, the degradation of Cetirizine dihydrochloride achieved was 91%. Comparison of
ultrasound assisted enzymatic degradation with conventional method interprets importance of
ultrasound for increased degradation percentage at a shorter time. Thus, enhanced
degradation with comparatively less time possesses an attractive candidature for ultrasound
assisted enzymatic degradation which can be further used for various pharmaceuticals.
Acknowledgment
The authors would like to acknowledge financial assistance of University Grants Commission
(UGC) for the research work.

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Figure Captions
Fig.1. Effect of pH on laccase activity (Reaction conditions: 100 mM buffers of pH ranging
from pH 4 to pH 8; 10 mM Guaiacol as a substrate, Room temperature reaction).
Fig.2. Effect of temperature on laccase activity (Reaction conditions: 100 mM phosphate
buffer of pH 6.0; Temperature 40oC- 70oC: 10 mM Guaiacol as a substrate).
Fig.3. Effect of enzyme loading (Reaction conditions: Cetirizine dihydrochloride (5 mg/L) in
phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.015% - 0.03% (w/v); temperature 60oC; Under ultrasound waves with
frequency 25 kHz; power 50W; 50% duty cycle).
Fig.4. Effect of temperature (Reaction conditions: Cetirizine dihydrochloride (5 mg/L) in
phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.02% (w/v); temperature 40oC – 70oC; Under ultrasound waves with
frequency 25 kHz; power 50W; 50% duty cycle).
Fig.5. Effect of ultrasonic power (Reaction conditions: Cetirizine dihydrochloride (5 mg/L)
in phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with frequency 25
kHz; power 30W - 150W; 50% duty cycle).
Fig.6. Effect of ultrasonic frequency (Reaction conditions: Cetirizine dihydrochloride (5
mg/L) in phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200
rpm; enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with
frequency 25 kHz – 40 kHz; 50% duty cycle).
Fig.7. Effect of duty cycle (Reaction conditions: Cetirizine dihydrochloride (5 mg/L) in
phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;

enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with frequency 25
kHz; power 100W; 40% - 60% duty cycle).
Fig.8. Effect of speed of agitation (Reaction conditions: Cetirizine dihydrochloride (5 mg/L)
in phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 0- 300 rpm;
enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with frequency 25
kHz; power 100W; 50% duty cycle).
Fig.9. Degradation pathway for Cetirizine dihydrochloride

100
80
60
40
20
0
4
5
6
7
8
pH
Fig.1. Effect of pH on laccase activity (Reaction conditions: 100 mM buffers of pH ranging
from pH 4 to pH 8; 10 mM Guaiacol as a substrate, Room temperature reaction).

100
80
60
40
20
0
30
40
50
60
70
Temperature (°C)
Fig.2. Effect of temperature on laccase activity (Reaction conditions: 100 mM phosphate
buffer of pH 6.0; Temperature 40oC- 70oC: 10 mM Guaiacol as a substrate).

80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
0.025% (w/v)
0.015% (w/v)
0.02% (w/v)
0.03% (w/v)
Fig.3. Effect of enzyme loading (Reaction conditions: Cetirizine dihydrochloride (5 mg/L) in
phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.015% - 0.03% (w/v); temperature 60oC; Under ultrasound waves with
frequency 25 kHz; power 50W; 50% duty cycle).

90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
40°C 50°C 60°C 70°C
Fig.4. Effect of temperature (Reaction conditions: Cetirizine dihydrochloride (5 mg/L) in
phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.02% (w/v); temperature 40oC – 70oC; Under ultrasound waves with
frequency 25 kHz; power 50W; 50% duty cycle).

100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
100 W
30 W
50 W
150 W
Fig.5. Effect of ultrasonic power (Reaction conditions: Cetirizine dihydrochloride (5 mg/L)
in phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with frequency 25
kHz; power 30W - 150W; 50% duty cycle).

100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
40 kHz
25 kHz
Fig.6. Effect of ultrasonic frequency (Reaction conditions: Cetirizine dihydrochloride (5
mg/L) in phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200
rpm; enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with
frequency 25 kHz – 40 kHz; 50% duty cycle).

100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
40% 50% 60%
Fig.7. Effect of duty cycle (Reaction conditions: Cetirizine dihydrochloride (5 mg/L) in
phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 200 rpm;
enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with frequency 25
kHz; power 100W; 40% - 60% duty cycle).

100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
200 rpm
0 rpm
100 rpm
300 rpm
Fig.8. Effect of speed of agitation (Reaction conditions: Cetirizine dihydrochloride (5 mg/L)
in phosphate buffer of pH 6.0 ; total reaction volume 20 mL; speed of agitation 0- 300 rpm;
enzyme loading 0.02% (w/v); temperature 50oC; Under ultrasound waves with frequency 25
kHz; power 100W; 50% duty cycle).

OH
2 HCl
N
O
N
O
Cl
2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)acetic acid dihydrochloride
Laccase
N
OH
N
Cl
2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethanol
HN
O
N
CH
2-(piperazin-1-yl)acetaldehyde
Cl
1-benzyl-4-chlorobenzene
Fig.9. Degradation pathway for Cetirizine dihydrochloride