Molecular Docking Studies and In-silico ADMET Profile Analysis of Triphala Plant constituents Morin and 9, 10-anthraquinone as Potential Inhibitors of human Estrogen Receptor Alpha

Satyanarayana Murthy Malladi¹*,Surya Prabha Sadhu², Devendra Kumar Pandey³,

Nagendra Sastry Yarla⁴

¹Department of Botany, Research Scholar, Lovely Professional University, Phagwara, Jalandhar, Punjab, India.

²Research Scholar, AU College of Pharmaceutical Sciences, Department of Pharmacology,

Andhra University, Visakhapatnam, India.

³Department of Biotechnology, Lovely Professional University, Phagwara, Jalandhar, Punjab, India.

⁴Department of Life Science, University of Hyderabad, Hyderabad, India.

*Corresponding Author E-mail: drmalladi53@gmail.com

ABSTRACT:

The expanding global burden of cancer demands novel treatment options. Herbal medicine offers a viable alternative to conventional cancer treatment. Breast cancer is frequently diagnosed cancer among women all over the world. Normal breast cells and specific breast cancer cells containestrogen and progesterone receptors. Estrogen and progesterone bind to receptors and stimulate cancer cell proliferation and expansion with growth factors (e.g., oncogenes and mutated gene suppressors). Tamoxifen, raloxifene, andtoremifene are among the most frequently used drugs for breast cancer, whereas estrogen continues to be produced in breast cancer cells. These medications primarily function against theestrogen binding to theestrogenreceptors on these cells. Protein-ligand interaction plays a major role in structural drug design. In this study, the molecular interactions between theHuman estrogen receptor alpha (PDB ID: 2IOK), and the two ligands isolated from Triphala Ayurvedic formulation morin, and 9,10-anthraquinone, an endogenous estrogen receptor ligand, estradiol, and the three economically available breast cancer drug, tamoxifen, raloxifene, and toremifene using the AutodockVina software tool. The ADMET properties of these substances were determinedusing popular web-based software tools that include preADMET, admetSAR, Molinspiration, and SwissADME.The present available anti-cancer drugs have so many side effects. The present work is aimed at determining the efficacy ofestrogen receptor inhibitors and the safety profile of morin, anthraquinone molecules isolated from Triphalapolyherbal formulation by in-silico methods. The binding energies (Kcal/mol) of ligands with human estrogen receptor were calculated as follows: morin (-9.0), 9,10-anthraquinone (-8.7), estradiol (-10.1), tamoxifen (-9.6), raloxifene (-9.8) and toremifene (-8.9). The resultssupported the drug-like properties of the molecules tested and they are likely to have a therapeutic effect.Further,in vivo and pre-clinical trials of the most active compound are also worthwhile for producing effective inhibitors.

KEYWORDS: Breast cancer, Triphala, Morin, 9, 10-anthraquinone, Molecular docking, ADMET.

INTRODUCTION:

Cancer is a leading cause of death and a substantial impediment to improving life expectancy in every country.

Breast cancer is the most commonly diagnosed cancer in women in 159 nations around the world, according to GLOBOCON estimates of Global cancer statistics 2020, and cervical cancer is the most commonly diagnosed disease in around 23 countries. Female breast cancer surpassed lung cancer as the leading cause of cancer in 2020, accounting for 11.7 percent of all cancer cases, with a projected 2.3 million new cases. It is the seventh leading cause of cancer death, with 685,000 fatalities globally. Early menarche, late menopause, fewer children, less breastfeeding, menopausal hormone therapy, contraceptive pills, and lifestyle risk factors such as alcohol consumption, excess body weight, and physical inactivity, as well as improved cancer prediction accuracy, account for the higher incidence rates in most countries¹.

The prevalence of breast cancers in men is 100 times less frequent than in women because of the small amount of breast tissue and different hormonal environment in males. The tissue which is affected, and the factors that influence malignant changes in breast cancer are identical in both men and women^2,3. Breast tumors are classified into three categories based on their receptor status: hormone receptor-positive (HR+), human epidermalfactor receptor-2 overexpressed (HER2+), and triple-negative (TN)⁴. The estrogen and progesterone receptors present in hormone-positive tumors are identical to those present in the normal cells, the hormone receptor-positive breast cancer accounts for about two out of every three cases. These hormones stimulate cancer cell proliferation through growth factors such as oncogenes. Hormone therapy for breast cancer is available in a variety of forms. The majority of hormone therapies either reduce estrogen levels in the body or prevent estrogen from assisting breast cancer cells in their growth^5,6.

Estrogen and progesterone respond more likely to hormone therapy drugs like tamoxifen, raloxifene, and toremifene for a healthier prognosis⁷. The activity of estrogen interferes with the drug tamoxifen (Nolvadex) when taken orally. It causes side effects like uterine cancer, cataract, seizures, and blood clots which are quite common⁸. Raloxifene, on the other hand, rarely causes blood clots in the eyes, lungs, or legs, but it can cause pain in the legs or swelling, vision disorders, chest pain, or difficulty in breathing^9,10. Toremifeneis also an antiestrogen drug that reduces tumor growth, but it causes serious side effects not limited to cardiac arrhythmias, blood clots in the brain, heart, lungs, and legs, allergic reactions, increased risk of uterine cancer, and liver diseases¹¹. Thus, there is an immense need todevelop a potent and safe alternative lead molecule that can function as antiestrogen drugs.

Triphala is a triherbal ayurvedic formulation consisting of powdered Terminalia Chebula, Terminalia belerica, and Emblicaofficinalis in equal proportions. In Ayurveda, it is commonly prescribed to balance tridoshas^12,13. Even thoughTriphala was being used for generations to treat a variety of illnesses, recent in vitro, in vivo, and human studies have proved its safety and efficacy in the treatment of a variety of disorders, including infectious diseases and cancer^14,15. Triphala has been demonstrated to have antitumor, radioprotective, and chemoprotective activities in experimental trials throughout the last decade by modulating various signaling molecules¹⁶. In the present study, two isolated molecules from triphala formulation, morin, and 9,10-anthraquinone were selected to determine their anti-estrogen activity by in-silico approach.

Bioinformatics uses computation methods to speed up the drug development process after the process of drug synthesis and make it cost-effective tool¹⁷. Drug docking is a type of molecular modelling that predicts ligands' preferred orientation at their target proteins, where it form a stable complex¹⁸. The present study was done to determine the binding affinities of morin and 9,10-anthraquinone towards the human estrogen receptor alpha protein (ERα) and compare the results with the endogenous ligand “estradiol” and the commercially available antiestrogen drugs tamoxifen, raloxifene, and toremifene. The evaluation of molecular properties, drug-like properties determination, and the ADMET profile analysis of morin and 9,10-anthraquinonewas done by using the online tools PreADMET, admetSAR, SwissADME and Molinspiration software.

MATERIALS AND METHODS:

Tools used:

We have used bioinformatics methods for the current studies. Biological databases such as Pub Med, PDB (Protein Data Bank), Drug Bank, and applications such as Chem Sketch drawing and graphics kit that was built to rapidly and easily draw compounds, schematic diagrams, and calculate chemical properties were used.

DrugBank is a cheminformatics/bioinformatics database that integrates extensive drug target (protein) data with detailed drug data. Each Drug Card entry comprises approximately 80 data fields, with half of them dedicated to drug/chemical information and the other half to drug target or protein information. Drug Bank is one of the extensively used resources in-silico drug design, target discovery molecular docking, and the prediction of drug interactions, drug metabolism, and similar applications¹⁹.

In 1971, it was founded as a repository for biological macromolecular crystal structures. PDB depositors use a variety of techniques, including X-ray crystal structure elucidation, NMR, cryoelectron microscopy, and theoretical modelling²⁰.

PubMed provides free access to approximately 19 million citations for biomedical studies from MEDLINE and life science journals. The National Library of Medicine in the United States provides this free online service (NLM)²¹.

Calculation ofPhysicochemical properties, bioavailability, and ADMETox profile analysis:

During the early phases of the drug development process, absorption, distribution, metabolism, and excretion (ADME) testing is becoming more common, but the number of drug samples accessible is limited. Computer models can be utilised instead of experiments in this scenario²². Computer models have been credited as a feasible alternative to experimental methodologies for ADME prediction, particularly at the early phases of drug development. Early ADME estimation in the discovery phase has been demonstrated to reduce pharmacokinetics-related dropout in the clinical phases considerably²³.

The physicochemical properties of morin, 9,10-anthraquinone, estradiol, tamoxifen, raloxifene, and toremifenewere computed by SwissADME using OpenBabel version 2.3.0.²⁴. Molinspiration software was used to determine the bioactivity score.PreADMET and admetSAR, SwissADME, and Molinspiration software were used for calculating the ADMET profile of all the drugs included in the study.

Preparation of protein structure:

The human estrogen receptor alpha (PDB ID: 2IOK) structure was extracted from the Protein Databank (PDB) website as .pdb file. The receptor protein was a homodimer consisting of identical ERαA and ERαB chains. Thus, to concentrate on the locations where docked receptors will be found, one monomer, chain A, was chosen.The protein wassimplified by deleting the co-crystallized ligands, chain B, and water molecules using BIOVIA Discovery Studio Visualizer v.4.5 (Accelrys).

Preparation of ligands:

The 3D structures of ligands were downloaded as .sdf files from Zinc Library and saved as .pdb files after optimization. The Protein PDBQT file is prepared by using AutoDock Tools v. 1.5.6rc3. This involves reading the protein macromolecule in PDB format and adding Kollman Charges, Compute Gasteiger charges, and assigning the AD4 type atoms to the target protein. The energy minimization was done and the root meansthe square value is ensured to be less than 0.001Kcal/mol. The prepared ligands and target proteins were subjected to protein-ligand docking using the AutodockVina software. The ligand-receptor affinity in AutodockVina is estimated by an empirical scoring function, which is based on the X-score function^25,26. The protein-ligand interactions were visualized using a discovery studio visualizer.

Fig. 1. The molecular structures of ligands.

RESULTS AND DISCUSSION:

Physicochemical properties:

In the traditional drug discovery process, the ADME-Tox properties were determined in the late phase. However, various in-silico tools were developed in the recent past, which enable predicting these properties during the early phase, thus, aiding the drug discovery process. About 60% of drug molecules fail during the drug development process due to their poor ADMET Profile^27,28. So, predicting these properties during the early phase of drug discovery would save time and reduce the expenditure significantly.

The physicochemical properties of molecules used in the study were presented in Table 1. The PSA was determined using a fragmental technique known as topological polar surface area (TPSA) with polar atoms sulfur and phosphorus. This has shown to be a valuable attribute in many models and rules for fast estimating some ADME qualities, particularly biological barrier crossing properties like absorption and brain access.

Table 1: Physicochemical properties of the moleculesanalyzed by Swiss ADME

ID	Morin	9,10-anthraquinone	Estradiol	Tamoxifen	Raloxifene	Toremifene
Molecular Formula	C₁₅H₁₀O₇	C₁₄H₈O₂	C₁₈H₂₄O₂	C₂₆H₂₉NO	C₂₈H₂₇NO₄S	C₂₆H₂₈ClNO
Molecular weight (g/mol)	302.24	208.21	272.38	371.51	473.58	405.96
Num. heavy atoms	22	16	20	28	34	29
Num. arom. Heavy atoms	16	12	6	18	21	18
Num. rotatable bonds	1	0	0	8	7	9
Num. H-bond acceptors	7	2	2	2	5	2
Num. H-bond donors	5	0	2	0	2	0
Molar refractivity	78.03	59.75	81.03	119.72	141.21	124.52
TPSA (Å²)	131.36	31.14	40.46	12.47	98.24	12.47
LogP	1.20	2.64	3.40	5.77	5.05	5.98

Druglikeness assessment:

Compared to the commercially available drugs tamoxifen, raloxifene, and toremifene, the test molecules morin and 9,10-anthraquinone are in compliance with CMC rule, leadlike rule, MDDR like rule, Rule of Five, and WDI like rule with no violations. Both the morin and 9,10-anthraquinone showed similar results, whereas all the commercial drugs tested showed similar results, while estradiol did not have quality CMC rule, leadlike rule, and MDDR like rule. However, the test molecules did not violate any of these rules, thus,appealing their drug-like properties (Supplementary file 1).

Assessment of ADMEproperties:

PreADMET and SwissADME software were used to calculate the ADME properties of the test compounds. For evaluating the intestinal absorption of the test molecules, the PreADMET server considers in vitro results and other criteria. Caco2 (human colon cancer cells) and MDCK (Madin-Darby canine kidney) cell models have been indicated as credible in vitro models for oral medication absorption prediction. Additionally, data on HIA (human intestine absorption) and skin permeability can be used to forecast and choose between oral and transdermal medication delivery. Skin permeability serves as a risk evaluation measure for any substance that comes in close contact with the skin. The data from Plasma Protein Binding (PPB) and Blood-Brain Barrier Penetration (BBB) can be used to predict whether a drug molecule will be available to bind with its pharmacological target after being absorbed into the systemic circulation, as well as the likelihood of accessing the central nervous system. The PreADMET service selects important 2D descriptors using a genetic functional approximation, then predicts ADME data using Resilient back-propagation (Rprop) neural network analysis²⁹.

Supplementary file 2 shows a comparison of the pharmacokinetic properties of morin, 9,10-anthraquinone, estradiol, tamoxifen, raloxifene, and toremifene.To access their pharmacological target, biological substances or drugs must pass through the intestinal barrier. 9,10-anthraquinone showed better intestinal absorption (99.05) and oral bioavailability compared to all other compounds. However, morin showed less oral bioavailability and intestinal absorption than the other compounds. The hepatic enzyme inhibition showed mixed results for all the compounds. The skin permeability of morin (-4.42) and 9,10-anthraquinone (-3.05) was found to be poor than all other compounds. It is worth mentioning the plasma protein binding of morin (91.62%) and 9,10-anthraquinone (93.60%) that is less than estradiol (100%), tamoxifen (94.74%), and raloxifene (100%), which indicates their better bioavailability. A higher bioavailability reduces the drug dose and thus, the adverse effects of the drugs.

Toxicity prediction:

In-silico toxicity, prediction is a method of toxicity assessment that analyses, simulates, or predicts chemical toxicity utilizing computational resources (algorithms, software, and data). All of the compounds in the study had their toxicity predicted in-silico using the preADMET and admetSAR software.

The toxicity prediction results (Supplementary file 3) indicated that morin, 9,10-anthraquinone, raloxifene, and toremifeneare mutagenic as per the Ames-test prediction, while estradiol and tamoxifen were found to be non-mutagenic. Tamoxifen showed a positive result for the mouse carcinogenicity test, while morin and raloxifene were found to be negative to this test. Carcinogenicity on rats test was negative for tamoxifen, raloxifene, and toremifene but positive for morin and 9,10-anthraquinone. Except for 9,10 anthraquinone, all the test molecules showed positive reproductive and respiratory toxicity. All the test molecules were found to bind to the androgen and estrogen receptors. Overall, the findings suggest mixed toxicity results for morin and 9,10-anthraquinone and also for the standard drugs tamoxifen, raloxifene, and toremifene.

Prediction of bioactivity:

The bioactivity scores of the molecules were predicted for a variety of criteria, including binding to GPCR and nuclear receptors, altering the ion channel permeability, tyrosine kinase receptor inhibition, protease inhibition, and inhibiting the enzyme activity. The Molinspiration software program was used to determine all of the parameters.

Molecules with higher activity scores are more likely to be active. The predicted results suggest that estradiol (0.95), tamoxifen (0.57), raloxifene (0.63), and toremifene (0.50) all showed the highest activity scores as nuclear receptor ligands than any other drug targets tested. Similarly, morin also showed the highest activity score (0.34) as a nuclear receptor ligand. However, 9,10-anthraquinone showed negative scores for all the drug targets tested including the nuclear receptor (-0.45). The highest bioactivity score obtained with 9,10-anthraquinone was -0.15 as an ion channel modulator. These results indicate the possible nuclear receptor inhibitory activity of morin.

Table 2: Bioactivity scores predicted by the Molinspiration software.

Parameter	Morin	9,10-anthraquinone	Estradiol	Tamoxifen	Raloxifene	Toremifene
GPCR ligand	-0.09	-0.40	0.18	0.30	0.26	0.18
Ion channel modulator	-0.22	-0.15	0.20	0.00	0.01	-0.10
Kinase inhibitor	0.22	-0.30	-0.36	-0.01	0.29	-0.06
Nuclear receptor ligand	0.34	-0.45	0.95	0.57	0.63	0.50
Protease inhibitor	-0.27	-0.51	-0.02	0.04	0.15	-0.02
Enzyme inhibitor	0.28	-0.03	0.61	0.32	0.20	0.22

Table 3: The molecular docking results with human estrogen receptor alpha.

Ligand	Binding energy (-Kcal/mol)	Interacting amino acids – bond length (Å)
Morin	-9.0	Leu387 (3.9), Phe404 (4.9), Leu346 (4.5), Ala350 (5.4), Leu525 (4.8), Leu391 (5.02)
9,10 - anthraquinone	-8.7	Leu387 (3.9), Phe404(5.1), Leu346(5.4), Ala350(4.8), Leu391(5.4), Ile424 (5.4)
Estradiol	-10.1	Leu387 (4.2), Phe404 (5.4), Ala350 (5.1), Leu391 (5.0), Ile424 (4.9), Arg394 (2.3), Met388 (5.4)
Tamoxifen	-9.6	Leu387(3.9), Phe404(5.0), Leu346(5.2), Ala350(3.9), Leu525(4.6), Leu391(5.2), Thr347(2.7), Met421(5.7), Leu428(5.17)
Raloxifene	-9.8	Leu387(5.4), Phe404(5.0), Ala350(3.5), Leu525(5.1), Arg394(2.4), Gly521(2.4), Asp351(3.5), Leu354(5.1), Leu536(4.7), Leu384(5.3), Ile424(4.9)
Toremifene	-8.9	Leu387(4.9). Phe404(4.9), Leu346(4.8), Ala350(4.2), Leu525 (4.7), Leu428(5.3), Met421(5.2)

Molecular docking studies:

The active sites of proteins often include the structural pockets having a high affinity for candidate drugs. The catalytic domain of the ERα was determined using data from the literature. In the three-dimensional structure of the ERα, the amino acids Leu346, Ala350, Leu384, Leu387, Phe404, Val418, Met421, Ile424, His524, and Leu525 serve as catalytic residues, according to computational study and experimental evidence³⁰. The results obtained are in line with earlier findings (Table 3).

Estradiol is a natural ligand to the ERαreceptors. Its structural features i.e. the rigid hydrophobic backbone, aromatic rings, hydroxyl groups that enable hydrogen bond interactions, facilitate binding at ERα agonist pharmacophore with a greater affinity³¹. The amino acids of estradiol found interacting with the estrogen receptor include, Leu387 (4.2), Phe404, Ala350, Leu391, Ile424, Arg394 (hydrogen bond with the 3-OH group), Met388. Except for Arg394, all other amino acids formed hydrophobic interactions, which stabilize the position of estradiol within the active site pocket that resulting in greater binding energy (-10.1 Kcal/mol) (Fig. 2C; Table 3).

Tamoxifen works by competitively binding to the oestrogen receptor, displacing estrogen and so reducing estrogen's breast cancer-promoting effect³². It occupied the same binding space in the ERα pocket as estradiol. Tamoxifen formed hydrophobic interactions with amino acids Leu387, Phe404, Leu346, Ala359, Leu525, Leu391, Met421, Leu428, and hydrogen bond with Thr347 (Fig. 2D). Thr347 act as an HB donor and tamoxifen acts as an acceptor.

Raloxifene is a selective modulator of the estrogen receptors that produce anti-estrogen effects on the uterine endometrium and breast tissue by binding to the estrogen receptors³³. The hydrophobic interactions of raloxifene are observed with the amino acids Phe404, Ala350, Leu525, Leu354, Leu536, Leu384, and Ile424 of ERα receptor (Fig. 2E). The amino acids Leu387, Arg394, Gly521, Asp351 served as hydrogen donors and formed HB with raloxifene in the active site pocket. The common interacting amino acids for raloxifene and estradiolwere found to be Leu387, Phe404, Ala350, Arg394, and Ile424. It showed more amino acid interactions than all other test molecules that might be responsible for its greater binding affinity (-9.8 Kcal/mol), but it is less than estradiol (-10.1Kcal/mol) (Table 3).

Toremifene, like tamoxifen and raloxifene, is an estrogen receptor modulator, and its anti-estrogen properties are made it a safe replacement for tamoxifen ¹¹. The docking results showed that toremifene binds to the ERα receptor with the binding energy -8.9Kcal/mol (Table 3). All of its interactions with the ERαreceptor were found to be hydrophobic. The interacting amino acids are Leu387, Phe404, Leu346, Ala350, Leu525, Leu428, Met421 (Fig. 2F). All these amino acids are also found to be interacting with tamoxifen, however, tamoxifen had additional hydrophobic interaction with Leu391 and a hydrogen bond with Thr347 that might contribute to its greater binding energy (-9.6 Kcal/mol) (Table 3).

Molecular docking studies revealed that the morin occupied the same binding space in the ERα pocket as estradiol, tamoxifen, raloxifene, and toremifene. Morin showed significant homology with estradiol, tamoxifen, and toremifene while interacting with amino acids Leu387, Phe404, Leu346, Ala350, Leu525, and Leu391 of ERα (Fig. 2A). All the six amino acid interactions of morin coincide with the tamoxifen, except for the HB-forming residue. Except for interaction at Leu346, all other five amino acid interactions of morin were observed with estradiol also, indicating the strong binding orientation of morin with the ERα receptor. There are four common amino acids involved in interactions with morin and raloxifene, i.e.Leu387, Leu346, Ala350, Leu525 (Fig. 2A; Fig. 2E). Morin formed a hydrogen bond with Leu387 similar to toremifene. Morin and toremifene shared five common amino acid interactions, except for the Leu391, which might have contributed to the greater binding affinity of morin with ERα receptor than toremifenewhich might contribute to its good anti-estrogenic potential. The binding energy of morin was found to be -9.0 Kcal/mol that is greater than 9,10-anthraquinone (-8.7 Kcal/mol) and toremifene (-8.9 Kcal/mol), but less than estradiol (-10.1 Kcal/mol), tamoxifen (-9.6 Kcal/mol), and raloxifene (-9.8 Kcal/mol) (Table 3). The strong interaction of morin with the hydrophobic amino acids might contribute to its stabilization in the active site pocket of the receptor, which can ensure a longer duration of action.

9,10-anthraquinone interacted deeply within the hydrophobic pocket of ERα and formed strong hydrophobic bonds with Leu387, Phe404, Leu346, Ala350, Leu391, Ile424 similar to the endogenous ligand estradiol (except Leu346) and the synthetic ligands tamoxifen (except Ile424), raloxifene (except Leu346 and Leu391), and toremifene (except Leu391, and Ile424) (Fig. 2F; Table 3). Similar to toremifene, 9,10-anthraquinone interacted with ERα only through hydrophobic bonds. These findings suggest the potential anti-estrogen activity of 9,10-anthraquinone.

Fig. 2. Molecular docking studies of ligands on ERα crystal protein (PDB ID: IOK). (A).The 2D structure shows the interactions of morin with the active site amino acids (B). The 2D structure shows the interactions of 9,10-anthraquinone with the active site amino acids (C). The 2D structure shows the interactions of estradiol with the active site amino acids (D). The 2D structure shows the interactions of tamoxifen with the active site amino acids. (E). The 2D structure shows the interactions of raloxifene with the active site amino acids. (F). The 2D structure shows the interactions of toremifene with the active site amino acids.

CONCLUSION:

In conclusion, the current investigation showed that test compounds morin and 9,10-anthraquinone an endogenous ligand of estrogen receptor estradiol, and three synthetic anticancer drugs tamoxifen, raloxifene, and toremifeneoccupy the same space in the active pocket ERα receptor. Morin, estradiol, tamoxifen, and raloxifene interact with ERα through hydrogen bonds and hydrophobic interactions, whereas 9,10-anthraquinone and toremifene interact through hydrophobic bonds only. Both morin and 9,10-anthraquinone qualified all the druglikeness tests without any violations and showed a greater association with nuclear receptors than any other drug targets. These predictions support the therapeutic potential of morin and 9,10-anthraquinone against hormone-sensitive breast cancer. This study strongly recommends further research on morin and 9,10-anthraquinone by in vitro and in vivo methods to evaluate their anti-breast cancer potential and safety profile.

ACKNOWLEDGMENTS:

We thank the LPU team members for their continued support and cooperation. Our heartfelt thanks to Dr M K Arunasree madam, Department of life science, Hyderabad Central University, Hyderabad India for providing necessary guidelines to complete the work.

CONFLICT OF INTEREST STATEMENT:

The authors declare no conflict of interest.

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Received on 03.05.2022 Modified on 06.09.2022

Research J. Pharm. and Tech 2023; 16(8):3759-3766.

DOI: 10.52711/0974-360X.2023.00621