INTRODUCTION:

Cancer is the second leading disease that cause of deaths overall¹. Approximately about 9.6 million deaths case, in year 2018 has become one of the six leading causes of fatalities in the world². Furthermore, HCC (hepatocellular carcinoma) has become the most common liver disease and became leading cause of death in the worldwide^3,4.

Several studies that have been conducted on HCC liver cancer show that many genetic factors alterations occur, in the development of HCC liver cancer, there are several genes involved in the role of signaling pathway, namely p53, Ras/ERK, PI3K/Akt, also MAPK (Mitogen Activated Protein Kinase) pathway⁵.The pathogenesis of HCC liver cancer has been described in recent studies, most of which are limited to the function of the underlying signaling pathway during the initiation of HCC. The increasing and decreasing activity of p53 can be caused by the possible interaction between the p53 protein and other proteins.

Activation of p53 can cause apoptosis and arrest of cell cycle. The p53 protein (53 kDa) is an important transcription factor that plays several antiproliferative roles to response to many stressors like DNA damage, the activation of oncogenes, and nutrient deprivation^6,7. It induces p21, CDK inhibitor that can cause cell cycle arrest at G1 phase of growth⁸. Furthermore, it inhibits cyclin-CDK1, cyclin-CDK2 and also cyclin-CDK 4/6 during checkpoint in the G1/S phase⁹. When DNA is damaged, p53 expression promotes the synthesis of p21 and activates repair processes before entering the S phase^6,10. The damage is uncontrolled p53 protein activates the apoptosis (programmed cell death) mechanism⁶, which is primarily the main mechanism of cell death in mammalian cells. In addition, it induces Bcl-2 Homology (BH) protein which causes increased mitochondrial outer membrane permeability. Increased permeability allows releasing of cytochrome c from intermembrane space. Then it binds with Adenosine Triphosphate (ATP) and Apaf-1 to form apoptosome complexes that activate caspase 9. After series of reactions, the formed caspase 9 activates caspase 3 which is an executor of apoptosis¹¹.

It has been established that functional interactions can occur between p53 and MAPK pathways, such as in the stress-activated protein kinase SAPK/c-Jun N-terminal protein kinase (JNK), protein kinase (MAPK) mitogen-activated by p38, and the extracellular signal related kinase (ERK). p53 is phosphorylated and activated by MAP kinases to response to the stressful stimuli, which in turn leads to cellular responses of p53. Recent studies showed that p53 has been proposed has a role as an upstream activator that regulates MAPK signaling by activating transcriptional members of the phosphatase family member with dual specificity. A further study identified JNK as a functional inducer that has role to activate p53 transcriptional activity. Also, to mediate p53 phosphorylation to response DNA damage, JNK could do the regulation in p53 stability in non-stressed cells using mechanism of MDM2-independent. Furthermore, an upstream regulator of JNK, namely MAPK/ERK kinase kinase 1 (MEKK-1), has also regulated the stability of p53. Generally, growth factors activate ERK¹². The Ras/Raf pathway is also activated upon growth factor stimuli and further activates MEK1/2 kinase, which then activates ERK 1/2. Subsequently, the activation of ERK 1/2 also leads to the process of activation for several transcriptions factors¹³. ERK acts as an upstream activator to phosphorylate p53 at residues of threonine and serine, leading activation of p53 and the subsequent of cellular responses¹⁴.

Trisindoline was isolated from cultured bacterium Vibrio sp. separated from marine sponge called Hyrtios altum and this can be a new discovery of newer drugs¹⁵. Trisindoline can show the major bioactive compound like antibacterial activity against some of these bacteria Escherichia coli, Bacillus subtilis, and Staphylococcus aureus as well as anticancer cytotoxic activity ¹⁶. The trisindoline group has been widely developed in recent anti-cancer research due to the success of synthesis methods and its high cytotoxic potential. It also provides better availability of new products with therapeutic possibilities and benefits for patients¹⁷. Santoso and Mursyidah successfully synthesized trisindoline into 4 compounds, namely compound 1: 5'-nitro-[3,3':3',3"-terindoline]-2'-one which is the result of the synthesis of trisindoline with the addition of a nitro group, compound 2: 1,1”-dimethyl-5'-nitro-[3,3':3',3”-terindoline]-2'-one which is the result of the synthesis of trisindoline with the addition of a dimethyl group, compound 3: 5,5”,7,7”-tetrabromo-[3,3':3',3”-terindoline]-2'-one which is the result of the synthesis of trisindoline with the addition of a bromo group, and compound 4: 5'-chloro-1,1”-diethyl-1H,1”H-[3,3':3',3”-terindole]-2'(1'H)-one which is the result of the synthesis of trisindoline with the addition of a chloro group¹⁸. Cytotoxic tests were carried out to assess the cytotoxic activity of the trisindoline 1, 2, 3, and 4 compounds against the MCF-7 cell line. Trisindoline 2 showed no activity, while the trisindoline 1, 3, and 4 compounds showed high activity with the values of IC50 is 2.059 µM, 3.9759 µM, and 15.46 µM. Trisindoline 1 showed the highest cytotoxic activity, which is showed by the lowest IC₅₀ value. This may be due to the addition of a nitro group (NO2) which decreases the expression of cyclin E1, an activator of (Cdk) 2 or cyclin-dependent kinase. Therefore, the cyclin E-Cdk2 complex, an essential regulator of progression through the G1 phase of the cell cycle, is not formed and cell growth stops¹⁹. Furthermore, the other studies also tested the cytotoxic activity of Trisindoline 1 against several cancer cell lines studies²⁰. such as HepG2 (a human liver cancer cell line), HeLa (a cervical cancer cell line), T47D (a breast cancer cell line), WiDr (a colon cancer cell line), Raji (nasopharyngeal carcinoma cancer cell line), 4T1 (a breast cancer cell line), and Vero (a kidney epithetical cell line), and obtained IC50 values of 0.183 µg/ml, 1.532 µg/ml, 1.293 µg/ml, 1.431 µg/ml, 1.374 µg/ml, 0.392 µg/ml, and 0.392 µg/ml, respectively⁸. This show that HepG2 cell line is the most sensitive to Trisindoline 1. The HepG2 cell line are used as a human in vitro model in anticancer drug research. In addition, HepG2 cells can be used to study the regulation of drug-metabolizing enzymes²¹.

The purpose of this study is to determine the potential of trisindoline 1 as an inhibitor of the MAPK pathway in liver cancer through silico and in vitro analysis. The induction of apoptosis in HepG2 cells via p53 expression by the trisindoline 1 compound (5'-nitro-[3,3':3',3”-terindoline]-2'-one) is investigated. Gene expression was carried out on unphosphorylated p53 protein as a target of anticancer activity through a proliferation inhibition mechanism. Considerable progress of this field can be attributed to the using of computational methods which is able to provide the valuable information of the structural characteristic for both kinase or ligand that are very important for beneficial interactions and the presence of the desired inhibitory activity. Therefore, there is a need for an exploration of cancer drugs that are capable of inhibiting cell proliferation without prompting MDR and apoptosis, and substances from natural resources as anticancer candidates²².

MATERIALS AND METHODS:

Materials:

Cell lines HepG2, RPMI (Roswell Park Memorial Institute), ELISA reader, protein p53, cyclin D1, cytochrome c, and caspase 3, Flow cytometry, Calibur FACS flow cytometer and light microscope equipped with OptiLab. Autodock Vina, ChemDraw, PyRx, Biovia Discovery Studio 2019 and SwissADME were used. MAPKs (ERK2, JNK1, and p38) were selected and the comparison compound selected was Doxorubicin. The structures were obtained with the codes as follows: (ERK2:5NHF, JNK1:1UKI, p38:2QD9, Doxorubicin:3NS9)²³.

Physicochemical and Pharmacophore:

a) Physicochemical Pharmacophore Analysis of Trisindoline 1 Using Swiss ADME:

In this study, we used a scoring function named the ADMET-score to evaluate the drug-likeness of a compound. The scoring function was defined because of 18 ADMET. The weight of each property in the ADMET-score was determined by three parameters, namely the accuracy rate of the model, the importance of the endpoint in the process of pharmacokinetics, and the usefulness index. The FDA-approved drugs from Drug Bank, the small molecules from ChEMBL and the old drugs withdrawn from the market due to safety concerns were used to evaluate the performance of the ADMET-score⁷.

In Vitro Analysis:

a) Cytotoxicity Test was carried out using the MTT Assay Method:

The concentration series of Trisindoline 1 used were 3.125, 6.25, 12.5, 25, 50, 100; 200 g/ml with 3 repetitions at each concentration. Furthermore, the obtained IC50 values were used for the cell proliferation tests.

b) Proliferation test ware carried out using the MTT Assay Method:

The IC50 values from cytotoxicity test were used for the cell proliferation tests. The concentrations were in the proliferation test were 0.5 IC50, IC50, 2IC50. The incubation times used were 24, 48, and 72 hours.

c) Apoptosis Test using Flow Cytometry:

Three concentration series of Trisindoline 1 were used (0.5 IC50; IC50; 2IC50) with 3 repetitions. As control cells, 100 μl of cell suspension was added to well that contained 100 μl of complete media. Incubation was carried out for 24 hours at 37°C in an incubator with 5% CO2 flow. Readings with the FACSCalibur flow cytometer are used to determine the apoptotic profile per 10,000 cells.

In Silico to Protein-Protein Pro-Apotosis:

Molecular Docking:

a) Data Mining:

The structure of the MAPKs (ERK2, JNK1, and p38) and Doxorubicin with the following codes: (ERK2:5NHF, JNK1:1UKI, p38:2QD9, Doxorubicin:3NS9), were taken from the Protein Data Bank (PDB) and PubChem.

b) Preparation of Receptor and Ligands Molecules:

Preparation of the receptor was done using Discovery Studio 2019 software, while preparation of the ligand was done using the PyRx software.

c) Evaluation and Interpretation of Results:

The molecular docking was copied to directory C:/docking_plants for the YASARA tool. Docking results are calculated with reference to the experimental results using Root Mean Square Distances (RMSD) by Analyze > RMSD of > Molecules. Calculations of RMSD are carried out for evaluating validation. An RMSD value of < 2.0 Å is used as a criterion for a successful docking method. If the RMSD value is < 2,0 Å, then it can be used as a docking protocol for the following virtual screening of samples²⁴.

d) Visualization of Docking Results with Discovery Studio:

To visualize the docking results, the docked ligand and receptor files are accessed in Discovery studio. Visualization of the docking results are shown via the amino acids that play a role in the bond between the ligand and target protein. For example, LYS 54 is the code for the amino acid, lysine 54, that plays a role in the bond the ligand and target protein²⁵.

RESULT:

a) Physicochemical and Pharmacokinetic Analysis of Trisindoline 1 using Swiss ADME:

Radar bioavailability is a quick assessment of the drug-likeness of a molecule that considers six physicochemical properties. The pink area indicates the optimal range for each physicochemical properties, namely lipophilicity: XLOGP3 is between − 0.7 and + 5.0, size: the molecular weight is between 150 and 500 g/mol, the polarity: TPSA is between 20 and 130 Å2, the solubility: log S is no more than 6, the saturation: the fraction of carbon at sp3 hybridization is not less than 0.25, and the flexibility: no more than 9 rotatable bonds²⁶. Based on the six physicochemical properties, Trisindoline 1 met all the properties of drug-likeness. Predictions of physicochemical and pharmacokinetic properties of trisindoline 1 and doxorubicin are shown in Table 1.

Table 1. Predicted Physicochemical Pharmacokinetic of Trisindoline 1 and Doxorubicin^27,29.

Physicochemical Characteristics	Trisindoline 1	Doxorubicin	Optimum Score
Formula (chemical formula)	C₂₄H₁₆N₄O₃	C₂₇H₂₉O₁₁
Molecular weight (MW)	408.41 g/mol	543.52 g/mol	≤ 600
Number of heavy atoms	31	39	20-70
Number of aromatic heavy atom	24	12	≤4
Fraction Csp3 (Fraction of carbons in the sp3 hybridization)	0.04	0.44	0.25
Number of rotatable bonds	3	5	≤10
Number of H-bond acceptor (HBA)	3	12	≤10
Number of H-bond donor (HBD)	3	6	≤5
Molar refractivity (MR)	123.13	132.66	≤155
Topological polar surface area (TPSA)	106.50 ⁰A (Low toxic)	206.07 ⁰A (High toxic)	≤ 150 (low toxic)

In vitro Test:

a) Trisindoline 1 Cytotoxic Activity Against HepG2 Cells:

Trisindoline 1 was tested for cytotoxic activity against HepG2 and Vero cells and showed IC50 values of 2.837 µg/ml and 7.301µg/ml, respectively. Doxorubicin was also tested for cytotoxic activity against HepG2 and Vero cells and showed IC50 values of 0.194µg/ml and 182.736µg/ml. The results are shown in Table 2. Trisindoline 1 has anticancer activity based on the criteria that established by the American National Cancer Institute (the IC50 < 30g/mL)³⁰. The results of the cytotoxic activity against HepG2 and Vero Cells are shown in Table 2.

Table 2. Results of Trisindoline 1 and Doxorubicin Cytotoxic Activity Test Against HepG2 and Vero Cells

Cell Types	IC₅₀ (µg/ml)
		_{Trisindoline 1}	_Doxorubicin
Liver Cancer	HepG2	2.837	0.194
_{Normal Cell}	_Vero	_7.301	_182.736

Effect of Trisindoline 1 on the proliferation test of HepG2:

Trisindoline 1 at a concentration of 2 IC50 for an incubation of 24hours inhibited the proliferation of HepG2 cells. For trisindoline 1 concentrations of 0.5 IC₅₀ and IC₅₀, an increase in the number of HepG2 cells was exhibited after 48hours of incubation. Increasing the number of cells indicates that HepG2 cells are resistant to trisindoline 1 at a concentration of 0.5 IC₅₀ and IC₅₀. The effect of trisindoline and doxorubicin at different concentrations on the doubling time of HepG2 cells is shown in Figure 1.

Figure 1. The effect of trisindoline concentrations on the doubling time of HepG2 cells.

b) Trisindoline 1 Apoptosis Test Against HepG2 Cells:

The cells were treated for 24 h to determine whether Trisindoline 1 reduced cell viability via activation of apoptotic processes, with increasing Trisindoline concentrations of 0.5 IC50, IC50, 2 IC50 (IC50 = 2.837 g/ml) and stained with Annexin-V/7-AAD³¹. The results show that treatment with Trisindoline 1 at a concentration of 0.5 IC50 and 2 IC50 can increase the number of living and necrotic cells but decrease the number of apoptotic cells. IC50 induced early apoptosis from 64.92 to 89.50% and decreased late apoptosis from 2.20 to 0.83%, and further reduced necrosis from 3.44 to 0.38%.

Figure 2. Flow Cytometry test of the Trisindoline 1 compound against HepG2 cells using the double staining method with Annexin V-FITC (yellow) and PI (red), live cell population (R1; Annexin V-FITC and PI negative), early apoptosis (R2; Annexin V-FITC positive and PI negative), late apoptosis (R3; Annexin V-FITC and PI positive), and necrosis (R4; Annexin V-FITC negative and PI positive).

Molecular Docking:

a) Data Mining:

The structures of the MAPKs (ERK2, JNK1, and p38) are shown in Supplementary Fig S1.

b) Preparation of Ligands and Receptors:

Ligand preparation was carried out using the PyRx software shown in Supplementary Fig S2.

c) Molecular Docking with Autodock Vina:

The scores of the docking results between the MAPK pathway proteins and drugs are shown in Table 3 (more conformations are shown in Supplementary Table)

Table 3. ERK2, JNK1, p38 and Ligand Docking Results Confirmation

Conformation	Protein	Trisindoline 1 (Kcal/mol)	Doxorubicin (Kcal/mol)
entry_00001_conf_01	ERK2	-9.8	-9.5
entry_00001_conf_01	JNK1	-8.7	-8.7
entry_00001_conf_01	p38	-8.7	-7.3

d) Validation of Docking Studies:

9 conformations were obtained from the docking results. From the scoring results, the lowest docking score was compared to the energy affinity that was obtained based on RMSD as a validation for the docking process. An RMSD value < 2Å indicates the structure that has the lowest energy and most like the structure resulted from the docking^32,33. The lowest docking scores of Trisindoline 1 ligand against ERK2, JNK1, and p38 were -9.8, -8.7, and -8.7 Kcal/mol, respectively in Table 4.

Table 4. Molecular Docking Score

Ligand

ERK Score

(Kcal/mol)

JNK1 Score

(Kcal/mol)

P38 Score

(Kcal/mol)

Trisindoline 1

-9.8

-8.7

Doxorubicin

-9.5

-8.7

-7.3

Based on the obtained docking scores, trisindoline 1 was able to bind with the MAPKs (ERK2, JNK1, and p38) with an energy affinity value equal to or less than that of Doxorubicin.

e) Visualization of Docking Results with Discovery Studio:

Visualization of the amino acids attached to the ligands and receptors was done through 2D and 3D visualization. More explanation about the result obtained from the results of visualization of the amino acids attached to ligands and target receptors are shown in Table 5 and Figure 3.

Table 5. Visualization of the amino acids attached to ligands and target receptors (MAPKs)

Ligand dan targeted receptor	Amino Acid Visualization	*Binding* *site*
ERK2 and Trisindoline 1	Glu33, Gln105, Cys166, Val39, Ala52, Leu156, Gly34, Lys54.	Lys54, Met108, Lys114, Cys154, Cys166, Cys167
ERK2 and Doxorubicin	Lys54, Lys151, Ser153, Glu33, Asp111, Leu156, Val39, Ile84, Ile31, Ala52, Met108, Asp106 and Gln105.	Lys54, Met108, Lys114, Cys154, Cys166, Cys167
JNK1 and Trisindoline 1	Ser155, Ser34, Gly38, Leu168, Val40, Ile32.	Ile32, Gly33, Gly35, Gln37, Gly38, Val40, Asn114, Val151, Ala55
JNK1 and Doxorubicin	Gln37, Leu168, Ala53, Val40, Ile32, Gly35 and Gly33.
p38 and Trisindoline 1	Lys295, Glu245, Val239, Asp292, Leu246, Leu289, Leu291, Phe270, Glu286, Val290, Gly243, Lys267, Pro242, Thr241, Thr241, Gly240, Pro266 dan Val 239.	Val30, Ala34, Val38, Tyr35, Lys53, Arg71, Met109, Ser154, Leu168, Pro242
p38 and Doxorubicin	Arg67, Arg70, Tyr35, Leu171, Ala172, Arg149 and Glu328.

Figure 3. 2D visualization; (A) ERK2 and Trisindoline 1; (B) ERK2 and Doxorubicin; (C) JNK1 and Trisindoline 1; (D) JNK1 and Doxorubicin; (E) p38 and Trisindoline 1; (F) p38 and Doxorubicin.

DISCUSSION:

In the physicochemical and pharmacokinetic analysis, trisindoline 1 met the parameters of drug-likeness properties because it satisfied the recommended optimum value. Trisindoline 1 also has a lower toxicity level than doxorubicin based on the TPSA value. In addition, if a compound meets all the criteria stated in Lipinski’s rule of five, it can increase pharmacokinetic properties and increase bioavailability in the metabolic processes of organisms. Doxorubicin does not meet all the criteria stated in Lipinski's rule of five, in which the recommended optimal values of 3 rules are exceeded, namely the number of hydrogen bond acceptors, the number of hydrogen bond donors and the TPSA value. The TPSA value of Doxorubicin tends to be high and exceeds the optimum value and so its toxicity level is considered high. The trisindoline 1 compound was shown to be better than Doxorubicin through physicochemical analysis. Trisindoline 1 is included in the class of indole alkaloids with the addition of nitro groups that possess similarities with the isatin group and its derivatives due to the presence of heterocyclic rings³⁴. Isatin and its metabolites are natural constituents of many natural compounds as well as components of synthetic compounds that exhibit antitumor and antiangiogenic activity^35,36. Isatin activity can affect MAPK pathways.

Trisindoline 1 is a naturally contain alkaloid compound that can prevent the growth of the cells even after cessation of the growth provoking signals³⁷. In this study, we can evaluate the possible pro-apoptotic activities of trisindoline in the HepG2 cell line through in silico analysis. The results indicate that trisindoline possesses promising anticancer properties and can is as effective as doxorubicin for inhibiting growth of HepG2 cell.

In this study, apoptosis induction in HepG2 cells was observed from cell proliferation, apoptosis and has the potential to become an inhibitor of ERK2, JNK1, and p38 MAPK pathways. The ERK activation needs the active transcriptionally of p53, due to p53 mutants could lack the fail transcriptional activity to ERK activation. The result implies p53 could promote the transcription of MAPK or ERK signaling. Previous studies also consider activation the survival pathways by p53 maintains the balance both death and life of cells. Trisindoline 1 exhibit potential binding with strong affinity at the significant sites which can be suggest as alternative therapeutic compound³⁸. The p53 involves cell survival by targeting DDR1, which is MAPK pathways stimulatory component. Stressful stimuli by the outcomes of p53 can be caused by type, context of the cell or stress duration.

The addition of a nitro group to trisindoline 1 is known to increase its cytotoxic effect, because ROS are formed from the reduction of the nitro group, resulting in nitro radical anion compounds which, when bound to biomolecules, can cause cytotoxicity^39,40. The cytotoxic activity of Doxorubicin is thought to be because Doxorubicin can inhibit DNA transcription through inhibition of DNA-dependent polymerase and DNA Topoisomerase II enzymes. This inhibition causes DNA chains to be damaged and ROS or free radicals are formed which can kill cells. From the tests on HepG2 cell doubling time, it was shown that trisindoline 1 was able to inhibit the proliferation of HepG2 cells. The inhibition was showed after 24 hours of incubation. The ability to inhibit cell proliferation can be attributed to the cell cycle arrest mechanism²⁹.

Based on the obtained docking scores, trisindoline 1 was able to successfully bind to the target proteins (ERK2, JNK1 and p38) in the MAPK pathway with an energy affinity value equal to or less than the that of doxorubicin. The lower the docking score, the easier it is for a compound to bind. Therefore, based on the in-silico approach, the trisindoline 1 compound can be considered as a potential inhibitor of MAPK pathway in liver cancer. Furthermore, binding sites on the ligand and receptors were shown in the visualization of amino acids. Trisindoline 1 inhibits ERK2 through the bonding between the amino acids, Lys 54 (lysine number 54) and Cys 166 (cysteine number 166). Doxorubicin inhibits ERK2 through the bonding of the amino acid Met 108 (methionine number 108) with the alkyl group. Trisindoline 1 inhibits JNK1 through the bonds in the amino acid Gly38 (glycine number 38) through the hydrogen bonds, Val40 (valine number 40), and Ile32 (isoleucine number 32) in the alkyl group. Doxorubicin inhibits JNK1 through the bonds in the amino acid Gln37 (glutamine number 37) through the electron donors, Val40 (valine number 40) and Ile32 (isoleucine number 32) in the alkyl group, and Gly35 (glycine number 35) and Gly33 (glycine number 33) through hydrogen bonds. Trisindoline 1 inhibits p38 through the bonds in the amino acid Pro242 (proline number 242) through hydrogen bond. Doxorubicin can inhibit p38 through the bonds in the amino acid Tyr35 (tyrosine number 35) through hydrogen bonds.

Figure 4. Mechanism of Trisindoline 1 on the induction of apoptosis in HepG2 cells through the p53 pathway

Trisindoline 1 can activate p53 protein and increase p21 expression as a cdk inhibitor, and as a result, the cdk4-cyclin d1 complex is not formed and the cells stop at the G1/S phase. Trisindoline 1 inhibits the expression of Bcl-2, causing the release of cytochrome c from the mitochondria to the cytoplasm. In the cytoplasm, cytochrome c binds with Apaf-1, forming a complex known as apoptosome. The apoptosome activates caspase 9, which induces cell death by apoptosis^41-43. Trisindoline 1 (5'-nitro-[3,3':3',3"-terindoline]-2'-one) is an indole derivative compound with the addition of a nitrogen group that causes the formation of ROS from the reduction reaction of nitro groups, resulting in nitro radical anion compounds. Cell stress due to Trisindoline 1 treatment will in turn activate p53 protein (phosphorylated p53) (Figure 4).

CONCLUSION:

Trisindoline 1 was able to induce growth arrest and apoptosis. Cyclin D1 expression was inhibited but the expression of cytochrome c and caspase 3 was induced. The docking results show that trisindoline 1 has the potential to be an inhibitor of the ERK2, JNK1 and p38 MAPK pathways. Trisindoline 1 met the properties of drug-likeness and has lower toxicity compared to Doxorubicin. These results suggest that trisindoline 1 has the potential as an anticancer drug.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We thank the Ministry of Research, Technology and Higher Education of the Republic of Indonesia and LPPM ITS for providing the higher education research grant.

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Received on 11.09.2022 Revised on 18.12.2023

Accepted on 07.10.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):765-772.

DOI: 10.52711/0974-360X.2025.00113

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