Vol. 17 | No.

2 |381-390| April - June | 2024

ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP
http://www.rasayanjournal.com
http://www.rasayanjournal.co.in

BLUE CRAB SHELL WASTE VALORIZATION AS A SOURCE

OF HYDROXYAPATITE FOR BIOCOMPATIBLE DENTAL
IMPLANT COATING FABRICATION
Eva Oktavia Ningrum1,, Imam Safari Azhar2 and Ardila Hayu Tiwikrama3
1
Department of Industrial Chemical Engineering, Faculty of Vocational Studies, Institut
Teknologi Sepuluh Nopember, Kampus ITS Sukolilo, Surabaya, 60111, Indonesia
2
Department of Prosthodontics, Faculty of Dental Medicine,
Universitas Airlangga Surabaya, Indonesia
3
National Taipei University of Technology, 1, Section 3, Zhong-Xiao East Road, Da’an District,
Taipei City, Taiwan 10608

Corresponding Author: eva-oktavia@chem-eng.its.ac.id
ABSTRACT
In the presented work, the effect of hydroxyapatite/polycaprolactone (HAp/PCL) ratio and the immersion/withdrawal
speed on the corrosion rate and antibacterial properties of Ti-6Al-4V ELI dental implant coating was investigated. Ti-
6Al-4V ELI implants are coated with different HAp-PCL ratios and immersion speeds. The functional groups of
product hydroxyapatite were confirmed using the HAp standard JCPDS no. 09-0432, with crystallinity of 88.32% and
ratio Ca/P at 2.12. The result shows that the higher concentrations of hydroxyapatite result in the decrease of corrosion
rate and increased antibacterial activity reflected in larger inhibition zones. Additionally, the higher immersion and
withdrawal speeds during dip-coating lead to lower corrosion rates. Furthermore, decreased corrosion rates may
enhance the antibacterial properties of HAp-PCL coatings. These results demonstrate that the HAp/PCL composite
can be considered as a prospective candidate for dental implant Ti-6Al-4V ELI coating.
Keywords: Hydroxyapatite/Polycaprolactone, Corrosion Rate, Antibacterial, Ti-6Al-4V ELI, Coating.
RASĀYAN J. Chem., Vol. 17, No. 2, 2024

INTRODUCTION
One of the most significant and often neglected global public health problems are diseases of the oral cavity.
An example of a disease of the oral cavity is tooth loss or loss of teeth. Tooth loss or loss of teeth is an
indicator of damage that is mostly caused by dental caries and periodontal disease.1 In addition, according
to the 2009 UK Adult Dental Health Survey, 5% of adults aged 55-64 and 15% of adults aged 65-74 were
edentulous.2 Dental implants, removable dentures, and bridges are among the options for replacing lost
teeth. Due to their improved esthetics and comfort, dental implants are the most popular procedure. This
has increased the demand for dental implants and led to a growth in materials and biomedical health
research. Metals and their alloys are often used for bone implants.3 Furthermore, the metal implants must
be biocompatible and corrosion-resistant.4,5 Titanium alloy, Ti-6Al-4V ELI is commonly used for in the
knee and hip joints, orthopedic implants, and dental implants, as it offers excellent biocompatibility,
mechanical properties and corrosion resistance.6 However, in dental implant applications, the oral cavity
remains a perfect habitat for corrosion.7,8 This is because the temperature, quality and pH of saliva affect
the stability of metal ions. Moreover, the sulfur of organic acid in food also promotes corrosion. 9 Corrosion
causes the roughness of the metal surface, the ions released in the implant alloy, and toxic responses such
as oral edema, perioral stomatitis, and gingivitis.10 In addition to the corrosion risk posed by Ti-6Al-4V ELI
dental implants, the impact of microbial infections is a major concern in the medical community. 11
Microbial infections caused by Escherichia coli and Staphylococcus aureus attack the corroded implant
surface and continuously release proteins, enzymes, and polysaccharides to form and grow biofilms. 12 The
biofilm layer of pathogenic bacteria leads to severe clinical infections and delayed healing of the patient. 11
If the bond between metal and soft tissue is not strong enough, germs penetrate and cause irritation and
implantation failure.13 Hydroxyapatite (HAp) is a polymeric biocomposite that is often used as a coating
for metal implants due to its chemical stability and similarity to natural bone structure. In addition, HAp
Rasayan J. Chem., 17(2), 381-390(2024)
http://doi.org/10.31788/RJC.2024.1728696 This work is licensed under a CC BY 4.0 license.
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has good biodegradability, biocompatibility, and osteoconductive properties and forms strong bonds with
human hard tissue.11 The HAp has been shown to improve the density of osteoblast cells in implants and
make the implants more stable. HAp nanoparticles are particularly effective in inhibiting microbial growth
due to the interaction between the HAp filler and the microbial membrane, which slows the bacterial growth
cycle.14,15 HAp coatings require a suitable morphology to increase the bone contact area for implant-to-
bone bonding, have high stability and dissolution resistance, and have sufficient surface adhesion. 16-17 In
addition, HAp has several limitations, including brittleness, structural instability in contact with human
fluids such as blood, and poor mechanical properties.18,19 Therefore, the combination of HAp with the
polymer binder polycaprolactone (PCL), which has excellent biodegradability, mechanical properties, low
molecular weight, and low price, is crucial. Due to its excellent properties, PCL is widely used in anticancer
drugs and implant coatings. Furthermore, PCL can improve the stability and efficacy of antibacterial
activities.20 Based on this background; it is necessary to carry out further research regarding the coating of
Ti-6Al-4V ELI dental implants using hydroxyapatite from crab shell (Portunus pelagicus) in combination
with polycaprolactone to meet the prerequisites as an antibacterial bone implant. Therefore, the current
research aims to investigate the effect of the HAp/PCL ratio and immersion/withdrawal speed by the dip-
coating method on the corrosion rate and antibacterial properties of Ti-6Al-4V ELI dental implants.
EXPERIMENTAL
Materials
Crab shells of Portunus pelagicus and ortho-phosphoric acid (H3PO4) precursor of 85% were utilized to
synthesize hydroxyapatite (HAp). The material employed for coating is an alloy-dental implant screw of
Ti-6Al-4V ELI. A binding agent of polycaprolactone (PCL) (CAPA-6800, Solvay Chemicals, Inc.) and a
CH3COOH solvent (Merck) were also used to produce a HAp/PCL solution. Various concentrations of
HAp/PCL solutions and immersion/withdrawal speeds used in the coating process are shown in Table-1.
Table-1: The Concentrations of HAp/PC Solutions and Immersion/Withdrawal Speed of the Coating Process
Immersion and Withdrawal Speed
HAp: PCl (%wt)
(mm/min)
100%: 0 20
90%: 10% 40
80%: 20% 60
70%: 30% 80
60%: 40% 100

Synthesis of Hydroxyapatite from Crab Shells

Pre-Treatment
During pre-treatment, the crab shells were washed to remove any remaining impurities. This step is repeated
until the solution is clear. The crab shells are then dried in an oven at 80 oC for 30 minutes to remove the
water. The shells were then ground in a mortar before calcination.
Calcination
Calcination was carried out by heating crushed crab shells to 1000℃ for five hours to remove organic
material and convert calcium carbonate into calcium oxide by reaction:
CaCO3 (s) → CaO (s) + CO2 (g)
Nano Hydroxyapatite Synthesis
A 0.5 M H3PO4 solution was prepared by dissolving 3.4 mL of 85% H3PO4 in 100 mL of distilled water.
Then dissolve 5.164 g of CaO in 500 mL of boiling distilled water to prepare a Ca(OH) 2 solution. Then mix
the Ca(OH)2 solution with 100 mL of H3PO4 solution by slow dripping in a heated sonicator bath to form
hydroxyapatite as follows:
CaO (s) + H2O (l) → Ca(OH)2 (aq)
6 H3PO4 (aq) + 10Ca(OH)2 (aq) → Ca10(PO4)6(OH)2 (s) + 18H2O (l)
To ensure homogeneous conditions, this solution was stirred for 2 hours. It was then filtered to obtain a
HAp precipitate, which was subsequently washed three times with distilled water to remove any remaining
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impurities. Finally, the filtered precipitate was heated at 110 oC for one hour and sintered at 800 oC for 4
hours.

Ti-6Al-4V ELI Dental Implant Coating by Dip-Coating Method

Material Preparation
Sandpaper with a grade of 1200 grit was used to sand the Ti-6Al-4V ELI dental implants. The dental
implants were then disinfected by immersing them in methanol for 15 minutes and then in acetone for 15
minutes, washed and dried in a 110 °C oven for 10 minutes.

Preparation of Hydroxyapatite and Polycaprolactone Suspension

The suspension used in this coating process consists of hydroxyapatite and polycaprolactone.
Hydroxyapatite and polycaprolactone are weighed according to a predetermined amount and placed in a
beaker. The HAp-PCL suspension was then dissolved with the specified amount of glacial acetic acid. The
suspension was then shaken with a magnetic stirrer at 300 rpm for 20 hours until it was homogeneous.
Dip-Coating
First, a sterile Ti-6Al-4V ELI dental implant is mounted on an immersion coating device and a beaker is
placed under the immersion device. The immersion device is positioned under the beaker. After starting the
immersion coating device, the substrate is immersed in the suspension. After immersing the substrate,
please switch off the device and leave it in the suspension solution for the desired immersion time before
removing the suspended substrate. The substrate is then dried in the oven for 30 minutes and sintered at
700°C for an hour.
Characterization
Analysis of Hydroxyapatite
After the dip-coating process was carried out on the Ti-6Al-4V ELI dental implant, several analyses were
used to determine the characteristics of the HAp and coating. The Ca content of the calcined Blue crab
shells was analyzed by X-ray fluorescence (XRF, Panalitical, Prodigi). The phosphate, carbonate, and
hydroxyl groups of HAp were examined via Fourier Transform Infra-Red (FT-IR) analysis (Agilent Cary
630 instrument), especially in the wavenumber 4000-600/cm. The element compositions of the sample were
analyzed by Energy Dispersive X-Ray (EDX 7000, Shimadzu, Japan). The phase composition and
crystallinity of the HAp were analyzed by X-ray diffraction (XRD, X’Pert PRO PANalytical).
Potentiodynamic Polarization Analysis
The corrosion properties of Ti-6Al-4V ELI samples after dip coating were determined by Potentiodynamic
Polarization Analysis, which consists of three electrodes: a reference electrode (SCE, Saturated Calomel
Electrode), a working electrode (titanium sample) and an auxiliary electrode (graphite). The corrosion rate
of Ti-6Al-4V ELI dental implants coated with HAp/PCL was measured in a Fuayama Mayer Artificial
Saliva solution, the composition of which is shown in Table-2.
Table-2: Fuayama Mayer Artificial Saliva Solution Composition
Component Composition (g/L)
NaCl 0.4
KCl 0.4
CaCl2 0.6004
NaH2PO4.2H2O 0.78
KSCN 0.3
Na2S.9H2O 0.005
Urea 1
Disk Diffusion Analysis
To determine the antibacterial properties of HAp/PCL coated Ti-6Al-4V ELI, a disk diffusion analysis was
performed. In this analysis, the formation of an inhibition surface for the bacterial growth of Staphylococcus
aureus was observed. Staphylococcus aureus bacteria were cultured in Nutrient Agar (NA) and incubated
at 37 oC for 24 hours. After culture, the bacteria were collected with a sterile loop and then placed in an
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analyzer tube containing 5 ml PBS. The bacteria were incubated at 37 oC for 2 hours until the turbidity met
the 1.0 McFarland standard (equivalent to 3 x 108 cells/ml) and the bacterial count was 105-108/ml. 21,22
The bacteria were inoculated into 0.1 ml of Muller Hinton Agar (MHA) medium, which was flattened and
dried. The sample was then placed on the paper disk in the center of the MHA medium and incubated at
37°C for 24 hours. The complete zone of inhibition and the diameter of the paper were measured using a
caliper.
RESULTS AND DISCUSSION
To meet the requirements as an antibacterial material in the form of HAp/PCL, the properties of synthesized
HAp from blue crab shells were analyzed before mixing.

Ca/P Ratio of Hydroxyapatite

According to previous research, hydroxyapatite with a Ca/P ratio close to 1.67 (the hydroxyapatite
stoichiometry) allows narrower and more uniform pores, may increase the contact between surface area
and HAp tissue, and improve the implant osteoconduction.23 The Ca/P ratio of HAp synthesized from crab
shells is displayed in Table-3.
Table-3: EDX Analysis of Hydroxyapatite (Portunus pelagicus) Shell
Component Molecular weight %wt
Ca 40 71.65%
P 30.97 26.13%
Ca/P ratio 2.12
Table-3 shows that the Ca/P ratio of hydroxyapatite is higher than 1.67 with an amplitude of 100%. This
may be due to the fact that synthetic hydroxyapatite contains such calcium compounds, namely CaCO 3 and
CaO.24 Carbonate groups are formed due to an interaction between the CaO phase and free CO2 in the air
during the synthesis process or heat treatment/sintering, resulting in an increased Ca content in the final
Hap.25 The increase in the H3PO4 concentration also leads to a decrease in the pH value, which increases
the concentration of H+ and PO43- ions in the reaction solution. This is also due to a significant decrease in
Ca. The value of the Ca/P ratio for the variable 1.1 M H3PO4 concentration is 2.12, which is close to the
standard value, i.e. 1.67.26-28
Functional Groups of Hydroxyapatites
The hydroxyapatite FTIR spectrum is shown in Table-4 and Fig.-1. As can be seen in Fig.-1, the hydroxyl
group is observed at 3500 cm-1. Zhang et al.29 have made a similar observation. In addition, the synthesized
HAps show bands at 960 and cm-1, indicating the presence of phosphate functional groups. 30
Previous research has shown that the PO43- functional group of commercial HAp, i.e., P-O stretching, both
symmetric and asymmetric, found around wavenumbers 560, 600, and 960-1090 cm -1.31 Along with the
XRD investigation, these two functional groups in IR spectra strongly support the formation of the HAp
phase. Between 1200 and 1250 cm-1, the carbonate group can be identified as Ca10(PO4)6CO3 and
Ca10(PO4)6CO3(OH)2.32 During the synthesis process or heat treatment/sintering, a contact between the
CaO phase and free CO2 in the air forms carbonate groups.25 Even though carbonate compounds may appear
as contaminants, they still have no detrimental consequences on HAp performance. 30
Table-4: Spectrum of FTIR Analysis
Wavenumber (cm-1)
Functional groups
commercial Crab shell
OH- 3570.78 3652.58
CO32- 1250 1241
950 – 965 960
PO43-
1100 – 1030 1021.29

XRD Analysis of Hydroxyapatite

The produced HAp's phase shift, crystal structure, and degree of crystallinity were examined using XRD
(X-ray diffraction) analysis. The diffraction patterns of HAp are demonstrated in Fig.-2.
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Synthesized HAp

Commercial HAp

Fig.-1: FTIR Spectra of HAp with Calcination Temperature of 1000°C

JCPDS No.00-009-0432
Ca5(PO4)3(OH)
Intensity (%)

10 20 30 40 50 60 70 80 90
2 theta (degree)

Fig.-2: XRD Diffraction Pattern of HAp Synthesis from Crab Shells

The representations of HAp are shown in three primary peaks at 31.77, 32.90, and 32.19° based on the
reference of JCPDS 09-0432. These peaks can be seen in all samples, which identify HAp with a hexagonal
crystal structure (a = b = 9.423 Å and c = 6.8801 Å) similar to commercial Hap. 33-34 The compliance of the
HAp spectrum of the crab shells with JCPDS standard No.00-009-0432 suggests that the synthesis products
are indicated as hydroxyapatite. Using Origin Graphing Analysis, a curve fitting algorithm divided the
diffraction profile into three crystalline reflections and an amorphous halo. In this case, the degree of
crystallinity of 88.43% was obtained. The high crystallinity value of HAp obtained in this study is expected
to have increased stability of the coating onto the Ti-6Al-4V ELI implant, consistent with previous reports. 35
Effect of Immersion/Withdrawal Speed on Corrosion Behavior
The Tafel curve is displayed in Fig.-3 based on the potentiodynamic analysis performed. The Tafel curve
displays the corrosion current strength (Icorr), corrosion potential (Ecorr), and corrosion rate (mmpy). The
Tafel curve clearly shows how Ecorr and Icorr affect corrosion rate. The correlation between current Icorr
(A/cm2) and potential Ecorr (volts) is depicted in Fig.-3 by the Tafel curve from the potentiodynamic study.
Its Icorr and corrosion rate are closely proportional.
The corrosion rate increases as I corr increases. For each titanium implant specimen, the corrosion rate
values are shown in Table-5 for various immersion rates during the dip-coating process. Equation 1 is used
to calculate the corrosion rate.
CR = (K1 . I corr . EW) / ρ (1)
Where
EW = equivalent weight
ρ = density (g/cm3)
CR = corrosion rate (mm/yr) for icorr (μA/cm2)
K1 = 3.27 x 10-3 mm g/μA cm yr
Icorr = exchange current density
The potentiodynamic polarization analysis demonstrates that the variable immersion speed in the
dip-coating process significantly impacts the corrosion behavior of titanium implants. Table-5
shows that when the withdrawal and immersion speed increased, the corrosion rate of the Ti-6Al-
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4V ELI implant with the variable reduced. The lowest speed of 20 mm/min had the highest
corrosion rate by 0.2572 mmpy.

Fig.-3: Tafel Curve of Various Withdrawal/Immersion Speed (a) 20, (b) 40, (c) 60, (d) 80, and (e) 100 mm/min

Table-5: Corrosion Rate of Coated Ti-6Al-4V ELI Implant in Various Withdrawal/Immersion Speed
Withdrawal/ Immersion
Speed Variable Ecorr (Volts) Icorr (A/cm2) Corrosion Rate (mmpy)
(mm/min)
20 -0.25628 3.3618E-06 0.2572
40 -0.31804 2.9847E-06 0.2284
60 -0.38364 2.5911E-06 0.1982
80 -0.27328 2.3473E-06 0.1796
100 -0.32086 2.7754E-08 0.0021
In addition, the lowest corrosion rate was reached at 100 mm/min by 0.0021 mmpy. According to previous
studies, the faster the withdrawal/immersion, the more uniformly the layer developed on the implant surface
and the more firmly it adhered to the implant. This causes the corrosion rate to be less. In addition, the
hydroxyapatite layer thickness develops on the surface of the Ti-6Al-4V ELI implant increases when the
speed of withdrawal and immersion utilized in the dip-coating process is increased.
Effect of HAp/PCL Ratios of Coating Solution on Corrosion Rate
The various concentrations of the HAp/PCL solutions were utilized to assess the corrosion properties of the
coated Ti-6Al-4V ELI implant. The correlation between current Icorr (A/cm 2) and potential Ecorr (volts)
at various concentrations of HAp: PCL solutions is shown by the Tafel curve in Fig.-4.
The Tafel curve clearly shows how the concentration of HAp/PCL affects the rate of corrosion. It was found
that as the HAp content in the coating solution increased, the corrosion rate decreased. The corrosion rate
for each titanium implant specimen with various amounts of polycaprolactone and crab shell hydroxyapatite
is shown in Table-6.
Table-6 demonstrates that the corrosion rate decreases as hydroxyapatite concentration rises. The lowest
corrosion rate was attained at HAp/PCL ratios of 90:10, by 0.0439 mmpy. Additionally, at HAp/PCL ratios
of 60:40, the titanium specimen with the highest corrosion rate of 0.3912 mmpy was obtained. According
to European medical application guidelines, implants have corrosion rate values thresholds of 0.475 mpy
to prevent toxicity and corrosion in the body. In this case, the corrosion rate value obtained complies with
these requirements. This result also agrees with recent studies that demonstrate implants coated by
hydroxyapatite have improved corrosion resistance and can induce a beneficial biological response when
implanted into the body.36,37 Moreover, it can be observed that the rate of corrosion of HAp/PCL ratios of
100:0 is slightly higher compared with that of 90:10. This is due to the filament with the absence of
polycaprolactone, the coating layer develops on the implant surface being weaker. Polycaprolactone is used
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due to its biocompatible characteristics and ability to alleviate the brittle hydroxyapatite layers formed. In
addition, it acts as a binding agent and adhesive between the coated layer and the implant. 38

Fig.-4: Tafel Curve of HAp: PCL with Various Ratios of (a) 60:40, (b) 70:30, (c) 80:20, (d) 90:10, (e) 100:0
Table-6: Corrosion Rate of Coated Ti-6Al-4V ELI Implant in Various HAp/PCL Ratios
HAp Comparison Variable:
Ecorr (Volts) Icorr (A/cm2) Corrosion Rate (mmpy)
PCL
60:40 -0.42787 5.1172E-06 0.3912
70:30 -0.34361 5.1054E-06 0.3907
80:20 -0.57099 4.3722E-06 0.3346
90:10 -0.5351 5.742E-07 0.0439
100:0 -0.31415 1.5146E-06 0.1159
Inhibition of Bacteria
The inhibition zone developed in each Ti-6Al-4V ELI implant specimen was measured in various HAp/PCL
ratios in the coating solution during the dip-coating process. The inhibition zone showed bactericidal
activity on the bacterial strain of Staphylococcus aureus as shown in Fig.-5 and Table-7.

(a) (b) (c)

(d) (e)
Fig.-5: Clear Inhibition Zones of HAp: PCL ratios of (a) 60:40, (b) 70:30, (c) 80: 20, (d) 90:10, and (e) 100:0
Against Staphylococcus aureus

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Table-7: Antibacterial Activity in Various HAp/PCl Ratios

Clear Zone
Disc Diameter Inhibition Zone
HAp: PCL Diameter
(mm) Diameter (mm)
(mm)
60:40 45 15 30
70:30 47 15 31
80:20 48 15 33
90:10 50 15 35
100:0 50 15 35
Table-7 shows that higher antibacterial activity and a wider inhibition zone are produced by hydroxyapatite
at higher concentrations.39 Nano-hydroxyapatite is attractive due to its biocompatibility40,41, solubility in
vivo, and capacity to penetrate cell membranes. This is because the OH- concentration in hydroxyapatite,
which has free radical properties, is highly reactive and kills microorganisms. When hydroxyapatite OH-
ions interact with bacteria's mitochondria and cell membranes, it causes an irreversible change in
their structure.42 The HAp/PCL ratios of 90:10 resulted in the optimum inhibitory zone of 35 cm, as
demonstrated by the broadest inhibition against Staphylococcus aureus, and 60:40 is the lowest with the
inhibitory zone of 30 cm. This study has an important contribution to the development of HAp/PCL-shaped
antibacterial materials with low corrosion properties and strong antibacterial effects, which have wide
application potential in the fields of dentistry and orthopedics. The findings also highlight the importance
of controlling synthesis and parameters to produce materials with optimal performance. According to the
regulations established for European medical applications, implant requirements must not cause corrosion
and be hazardous to the body with a corrosion rate value of less than 0.475 mpy, therefore HAp/PCL ratio
of 90:10 variable complies with these standards. As discussed above, HAp/PCL has proven to have a good
antibacterial effect. Moreover, the reduced corrosion rate will also decrease the inhibition zone or increase
antibacterial properties. According to the regulations established for European medical applications,
implant requirements must not cause corrosion and be hazardous to the body with a corrosion rate value of
less than 0.475 mpy, therefore HAp/PCL ratio of 90:10 variable complies with these standards.
CONCLUSION
Hydroxyapatites with a Ca/P ratio of 2.12, crystallinity of 88.43%, and the presence of e PO 43- functional
group and the OH- functional group obtained in this study. The XRD analysis findings also indicated
agreement with the pure HAp peaks from the JCPDS standard No.00-009-0432. was also attained in
addition. The potentiodynamic polarization analysis demonstrates that the variable immersion speed in the
dip-coating process significantly impacts the corrosion behavior of titanium implants. With increasing
withdrawal and immersion speed, the corrosion rate of the Ti-6Al-4V ELI implant was reduced. Moreover,
when the concentration of hydroxyapatite increases, the corrosion rate decreases. The disk diffusion
analysis shows the inhibition zone grew with increasing hydroxyapatite concentration. HAp/PCL produced
in this study has proven to have a good antibacterial effect and, when applied as a coating, meets the
regulations established for European medical applications.
ACKNOWLEDGMENTS
The work was supported by Research, Innovation, and Entrepreneurship Proyek Higher Education for
Technology and Innovation (HETI) ADB loan number 4110-INO 2023, Institut Teknologi Sepuluh
Nopember, International Collaboration Research (ICR) Scheme under contact number 0007/01.PKS/PPK-
HETI/ITS/2023.
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest.
AUTHOR CONTRIBUTIONS
All the authors contributed significantly to this manuscript, participated in reviewing/editing, and approved
the final draft for publication. The research profile of the authors can be verified from their ORCID IDs,
given below:
Eva Oktavia Ningrum https://orcid.org/0000-0003-0581-3070
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Imam Safari Azhar https://orcid.org/0000-0002-0615-4321

Ardila Hayu Tiwikrama https://orcid.org/0000-0002-0393-6774
Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s)
and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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