Antiemetic activity of abietic acid possibly through the 5HT3 and muscarinic receptors interaction pathways

The present study was designed to evaluate the antiemetic activity of abietic acid (AA) using in vivo and in silico studies. To assess the effect, doses of 50 mg/kg b.w. copper sulfate (CuSO4⋅5H2O) were given orally to 2-day-old chicks. The test compound (AA) was given orally at two doses of 20 and 40 mg/kg b.w. On the other hand, aprepitant (16 mg/kg), domperidone (6 mg/kg), diphenhydramine (10 mg/kg), hyoscine (21 mg/kg), and ondansetron (5 mg/kg) were administered orally as positive controls (PCs). The vehicle was used as a control group. Combination therapies with the referral drugs were also given to three separate groups of animals to see the synergistic and antagonizing activity of the test compound. Molecular docking and visualization of ligand-receptor interaction were performed using different computational tools against various emesis-inducing receptors (D2, D3, 5HT3, H1, and M1–M5). Furthermore, the pharmacokinetics and toxicity properties of the selected ligands were predicted by using the SwissADME and Protox-II online servers. Findings indicated that AA dose-dependently enhances the latency of emetic retching and reduces the number of retching compared to the vehicle group. Among the different treatments, animals treated with AA (40 mg/kg) exhibited the highest latency (98 ± 2.44 s) and reduced the number of retching (11.66 ± 2.52 times) compared to the control groups. Additionally, the molecular docking study indicated that AA exhibits the highest binding affinity (− 10.2 kcal/mol) toward the M4 receptors and an elevated binding affinity toward the receptors 5HT3 (− 8.1 kcal/mol), M1 (− 7.7 kcal/mol), M2 (− 8.7 kcal/mol), and H1 (− 8.5 kcal/mol) than the referral ligands. Taken together, our study suggests that AA has potent antiemetic effects by interacting with the 5TH3 and muscarinic receptor interaction pathways. However, additional extensive pre-clinical and clinical studies are required to evaluate the efficacy and toxicity of AA.


Selection and preparation of test and control groups
Based on a review of the literature, we selected two concentrations of the test sample (lower and higher).We prepared the sample's mother solution at a concentration of 50 mg/kg by dissolving it in distilled water (DW) and a small amount of Tween 80 (0.5%) used as a co-solvent.The mother solution was then diluted at concentrations of 20 and 40 mg/kg.In contrast, doses of the referral drugs were chosen by converting human doses to animal doses supported by animal dose calculation protocol and literature procedures 8,36 .The reference drug's solutions were also prepared by thoroughly mixing them into DW (where a small amount of Tween 80 was used as a cosolvent) at concentrations of 16, 6, 10, 21, and 5 mg/kg for the drugs APT, DOM, DHM, HYS and OND, respectively.Three combined doses of AA (40 mg/kg), DOM, HYS, and OND were also prepared for the co-treatments.

In vivo protocol
The procedures outlined by Akita et al. 32 were used to conduct the study with a few minor modifications.The chicks were divided into eleven groups of six each.Before receiving the treatments, each bird was retained in a sizable, clear plastic container for 10 min.Using DW, the test sample (AA) was prepared in two different doses (20 and 40 mg/kg), which were then administered orally.The reference drugs APT, DOM, DHM, HYS, and OND were orally given at doses of 16, 6, 10, 21, and 5 mg/kg b.w.The lower dose (20 mg/kg) of AA did not exhibit any significant synergistic or antagonistic effects in the combination therapy; therefore, the three reference medicines, DOM, HYS, and OND, were combined with AA at a dose of 40 mg/kg.Then, animals were orally given these combined doses to assess their synergistic or antagonistic effects.The other two referral drugs were omitted for combination therapy due to their inadequate antiemetic properties when given alone to the animals.DW with a small amount of tween 80 (0.5%) was used as a control group (vehicle).It was given orally at a dose of 150 mL/ kg b.w.Each chick specimen had a 30-min treatment period before having emesis caused by the oral gavage of CuSO 4 ⋅5H 2 O at a dose of 50 mg/kg of b.w.The latency period is the duration of time between the administration of the CuSO 4 ⋅5H 2 O treatment and the occurrence of the first retch, then, the total number of retches within 10 min of receiving CuSO 4 ⋅5H 2 O treatment and the latency were carefully noted.Compared to the vehicle group, we calculated the percentage reduction in retches and prolongation in latency according to the following formula: where M: is the mean of latency in seconds in standard and test groups, N: is the mean of latency in seconds in the vehicle group, C: is the mean of retches in the vehicle group, and D: the mean of retches in the standard and test groups.

Statistical analysis
The values of the antiemetic efficacy are expressed as the mean and standard error of the mean (SEM).The Graph Pad Prism (version 6.0) is a statistical computer application that was used to estimate the variations' statistical significance which was determined at a 95% confidence limit.p values of < 0.05 are considered significant, whereas p values of p < 0.0001 are very significant.

Homology model and preparation of receptors
Based on published research, we selected nine receptors to perform molecular docking and ligand-receptor visualization.We developed a homology model because the human 5HT 3 receptor's 3D structure wasn't available in the RCSB Protein Data Bank 37 .Human 5HT 3 receptor homology modeling was achieved using the SWISS-MODEL 38 .The UniProt database (http:// www.unipr ot.org) was used to retrieve the protein's sequence, and the NCBI BLAST program was employed to conduct a BLAST analysis to determine the best template 39 .The GMQE 40 and a Ramachandran plot using ProCheck 41 methods were used to evaluate the 5HT 3 homology modeling structures.D 2 (PDB ID: 6LUQ) 42 , D 3 (PDB ID: 3PBL) 43 , H 1 (PDB ID: 3RZE) 44 , M 1 (PDB ID: 6WJC), M 2 (PDB ID: 5ZK8), M 3 (PDB ID: 4U15), M 4 (PDB ID: 7V6A), M 5 (PDB ID: 6OL9) 45 , and NK 1 (PDB ID: 6HLO) 46 were obtained from the RCSB Protein Data Bank (https:// www.rcsb.org/).After collecting receptors, the PyMol software program (v2.4.1) was used to remove any extraneous molecules, such as lipids, heteroatoms, and water molecules, from the protein sequence to optimize the receptors and prevent docking interference.Finally, using the SwissPDB Viewer software program and the GROMOS96 force field, the receptors' shape and energy were optimized.The PDB file was then saved for use in molecular docking.

Collection and preparation of ligands
Based on the literature, we chose several well-known and commercially available antiemetic medications as reference ligands to compare the binding energy and molecular interaction with our test ligand (AA), with the focus on different emesis-causing receptors, to understand the root cause of the antiemetic mechanism.Afterward, the following were collected using the PubChem chemical database in SDF format (https:// pubch em.ncbi.nlm.nih.gov/): several receptors, molecular docking, and prediction of pharmacokinetic features of the 3D conformers of abietic acid (Compound CID: 10569), aprepitant (Compound CID: 135413536), diphenhydramine www.nature.com/scientificreports/(Compound CID: 3100), domperidone (Compound CID: 3151), hyoscine (Compound CID: 3000322), and ondansetron (Compound CID: 4595).Then, using the Chem3D 16.0 computer application, which is used for performing molecular docking and anticipating pharmacokinetics, the 3D conformers of the chemical agents were minimized, stored as SDF files, and transformed into MOL files, respectively.Finally, using the Gaussian View program (v5.0), all the ligands were optimized.Displayed in Fig. 1 are the chemical structures of AA and standard drugs.

Molecular docking study
Molecular docking was conducted using the PyRx software tool to predict the active binding energy of the drugs toward the active sites of receptors.For successful docking, the grid box dimensions were set at 85 × 80 × 75 Å along the x-, y-, and z-axes, respectively, and the calculation required 2000 steps 47,48 .The docking potential result is saved in 'CSV' format, and the ligand-protein complex is collected in PDB format to collect the ligand in PDBQT format.The interactions between ligand-receptors and the receptor's active site were seen using the computer programs PyMol (v2.4.1) and Discovery Studio Visualizer (v21.1.020298).Then, the types of bonds, the number and length of hydrogen bonds, and each ligand-receptor interaction's amino acid residues are documented.

Prediction of drug-likeness and pharmacokinetics
Drug-likeness is a qualitative assessment used to evaluate a molecule's potential to be discovered and developed into an orally administered drug.A structural or physicochemical investigation was conducted to show similarities between the compounds and existing medications that were advanced enough in the research phase to be considered potential treatment options 49 .A chemical agent's pharmacokinetics and drug-likeness may be calculated using a variety of web servers and applications.In this investigation, with the help of SwissADME, we discussed numerous criteria for evaluating the physicochemical characteristics of the test compound (http:// www.swiss adme.ch/ index.php).

Toxicity prediction
To predict various toxicity parameters of any compound, ProTox-II online servers can be used.The ProTox-II web server is used to assess the safety profile of a chemical or compound by analyzing multiple toxicity endpoints, for instance, hepatotoxicity, carcinogenicity, mutagenicity, acute toxicity, immunogenicity, and cytotoxicity 50 .
To evaluate the toxicity parameters, the Canonical SMILES were entered into the ProTox-II server (http:// tox.chari te.de/ protox_ II), which was collected from PubChem.The toxicity parameters of the selected compounds are listed in Table 1.

In vivo investigation
In our experiment, animals in the control (vehicle) group exhibited their first retching at 7.50 ± 0.92 s, whereas animals in the reference groups showed an elevated latency compared to the control group.A remarkable reduction in the number of retches in animals treated with reference drugs such as APT, DOM, DHM, HYS, and OND was observed as compared to the vehicle, where DOM exhibited the lowest (10.00 ± 1.46) retches among the selected reference drugs, even across all the treatment groups.The values of the number of retching for APT, DHM, HYS, and OND are 58.66 ± 6.03, 51.00 ± 4.87, 46.33 ± 3.70, and 34.00 ± 2.26, respectively.In the case of the test sample, there was a significant dose-dependent decrease in the number of retches, and the animals in the AA-20 and AA-40 groups displayed 20.33 ± 2.04 and 11.66 ± 2.52 retches, respectively.In the combination groups, the lowest number of retches exhibited was in the OND + AA-40 group (15.50 ± 1.76).The total number of retches for all treatment groups is shown in Fig. 3.
Table 1.Different treatments and their doses were investigated in animals.Animals belonging to the AA-20 and AA-40 groups showed an increase in percentage of latency compared to the vehicle group, which was 74.27 and 92.34%, respectively.Findings indicated that the latency period is elevated with the increase in doses in the test groups.However, animals treated with combined therapies also showed a significant elevation in the latency percentage; among the several combination treatments, DOM + AA-40 exhibited the highest latency percentage of 89.70%.In the case of a percentage decrease in retches, treatment with the test compound demonstrated a dose-dependent percentage decrease in retching.The highest percentage decrease in retching was observed in the DOM group (85.03%), though the drug's combination therapy with AA showed a reduction in retching (75.56%).Our findings showed that the percentage decrease in retching for other combination groups are 58.10 and 76.80% for the HYS + AA-40 and OND + AA-40 groups, respectively.The percentage decrease in retching and the rise in the latency period for each treatment group are displayed in Table 2.

In silico study
Homology modeling of human 5HT 3 protein Findings from the homology modeling show that the desired sequence and the template sequence of 4PIR (PDB ID), an X-ray crystallographic structure of the mouse 5HT 3 receptor, have similar sequences.The target protein sequence shares 95% coverage and 86.95% identity with the template sequence, which also has a 58% sequence similarity.With a QMEAN of − 3.91 and a GMQE score of 0.72, the homology model of human 5HT 3 was developed, suggesting high quality and consistency.To verify the accuracy and reliability of the residues' Psi and Phi angles, the Ramachandran plot was designed.The plot revealed 1.81% Ramachandran outliers and 91.65% Ramachandran preferences in Fig. 4.

Molecular docking
A molecular docking approach was used to predict the possible binding energy between ligand and protein.Our in silico study revealed that the test ligand (AA) shows the highest docking score (− 10.2 kcal/mol) toward the M 4 receptor among the selected emesis-inducing receptors, whereas the referral ligand HYS exhibited a reduced docking score for the test ligand against the same receptor.The test ligand also showed a higher docking score than HYS toward the other subtypes (M 1 , M 2 , and M 5 ) of mAChRs except M 3 (Table 3).AA also demonstrated higher binding affinity toward 5HT 3 and H 1 receptors, and the docking scores are − 8.1 and − 8.5 kcal/mol, respectively.While the selected referral ligands OND and DHM expressed binding affinity of − 6.9 and − 6.3 kcal/ mol with the 5HT 3 and H 1 receptors, respectively, In the case of the dopamine receptor, the selected antagonist DOM elicited higher docking scores than AA toward its emesis-inducing subunits D 2 and D 3 , and the values are − 9.6 and − 9.9 kcal/mol, respectively.This study also revealed that APT binds with the NK 1 receptor by showing a remarkable binding interaction of − 12.7 kcal/mol, while AA exhibited a lower binding interaction   3.

Prediction of non-bond interactions between protein-ligand complexes
Findings from the in silico study demonstrated that ligands interact with receptors by establishing a variety of bonds, including hydrogen bonds (HB) (both conventional HB and carbon HB) and other types of bonds, including alkyl, pi-alkyl, sigma, pi-pi T-shaped, pi-sulfur, pi-cation, and pi-pi stacked bonds.For the 5HT 3 receptor, AA showed a higher docking value of − 8.1 kcal/mol, while the standard drug OND revealed a docking value of − 6.9 kcal/mol.AA binds with the 5HT 3 receptor by forming one hydrogen bond residue (HB), namely ILE98, in addition to showing several hydrophobic bonds (HP) with amino acid residues of PRO113, LYS25, PRO89, VAL95, and TYR114.In contrast, OND did not bind with the 5HT 3 receptor through HBs but formed numerous numbers of HP bonds with specific amino acid residues of LEU260, LEU259, VAL237, LEU234, and VAL264.DOM exhibits strong antagonistic action against the D2 receptor with a docking score of − 9.6 kcal/mol by generating 4 HBs, namely THR433, SER430, HIS414, and ASP114.AA exhibited a docking value of − 9.2 kcal/mol and formed one HB, DOM also interacted with the D 3 receptor by showing a higher docking value of − 9.9 kcal/ mol with three HBs of VAL111, ASP110, and CYS181, whereas AA displayed one HBs with a certain amino acid residue of SER366 and a binding affinity of − 9.2 kcal/mol.Due to the interaction between the DHM and H 1 receptor, which displayed a docking score of − 6.3 kcal/ mol with no HBs, it formed several HP bonds, including particular amino acid residues of PHE116, PHE119, PRO202, ILE120, and ALA151.On the contrary, two HBs are formed, including ILE148 and SER68 amino acid residues, and they also obtained a greater binding energy of − 8.5 kcal/mol after docking AA with the H 1 receptor.On the other hand, the binding scores of AA for the M 1 , M 2 , M 3 , M 4 , and M 5 receptors were − 7.7, − 8.7, − 7.6, − 10.2, and − 8.9 kcal/mol, respectively.It is obvious that AA exhibited the highest binding affinity against the M 4 receptor among all emesis-inducing receptors.Interaction is established between the AA and M 4 receptors by the formation of one HB with a particular amino acid residue of PHE186 and four HP bonds, namely ASP432, TYR439, PHE186, and TRP435.In contrast, the standard drug HYS demonstrated docking values against M 1 , M 2 , M 3 , M 4 , and M 5 receptors of − 6.7, − 7.7, − 9.1, − 8.9, and − 8.8 kcal/mol, respectively.However, two HBs are formed due to the interaction between the HYS and M 4 receptors with the amino acid residues of TYR92 and ASP432.Additionally, HYS formed three HP bonds with specific amino acid residues of TYR439, PHE186, and TRP435.The highest level of docking value (− 12.7 kcal/mol) occurs from the interaction between the APT and NK 1 receptor.It also revealed four HBs with the amino acid residues of ASN89, TRP184, GLN165, and HIS265.Furthermore, APT exhibited numerous numbers of HP bonds.Moreover, AA showed two HB namely HIS265 and THR201 and several HP bonds after binding with the NK 1 receptor.The number of HBs, ligands, receptors' bond types, HB lengths, amino acid residues, and the interacted ligand-receptor pockets are represented in Table 4 and Fig. 5.

Estimation of in silico pharmacokinetics and drug-likeness (ADME)
Drug-likeness is an important characteristic of a drug candidate and involves developing a chemical substance into a medication and assessing its pharmacokinetics.The in silico ADMET method play key roles in drug discovery and development.A high-quality drug candidate should not only have sufficient efficacy against the therapeutic target, but also show appropriate ADMET properties at a therapeutic dose.Hydrogen bond donors (HBD), molecular weight (MW), hydrogen bond acceptor (HBA), molar refractivity (MR), and Log P are the primary parameters used to assess drug-likeness.According to the in silico ADMET results, all the drugs used Results also showed that AA has all the pharmacokinetics and physiochemical properties to be a drug-like compound.The compound also followed the Egan, Ghose, and Veber rules to assure drug-likeness but violates the Lipinski rules because its MLOGP is less than 4.15.Other parameters, for instance, P-gp substrate, TPSA, www.nature.com/scientificreports/CYP2C19 inhibitor, BBB permeability, and bioavailability score of AA and reference drugs are given in Table 5 and a graphical representation in Fig. 6.
In silico toxicity of the selected compounds Toxicological assessment of small molecules is crucial in predicting their acceptability for use in animal and human models.The toxicity parameters of a drug candidate can be predicted using the online server Protox-II.According to our in silico toxicity assessment, DOM, HYS, and AA are categorized into toxicity class 4 (harmful if swallowed, 300 < LD50 ≤ 2000).On the other hand, OND and DHM fall into toxicity class 3 (toxic if swallowed, 50 < LD50 ≤ 300).In the case of organ (liver) toxicity, our findings predict that all the referral drugs are inactive, whereas AA expressed a positive result (active).Results of the risk assessments of the selected compound were also carried out by using toxicity end point estimation, where HYS, DHM, and AA exhibited no toxicity in the cases of carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity, but the prediction showed a positive result (active) for the drugs DOM and OND in the cases of immunotoxicity and mutagenicity, respectively, and the other mentioned toxicity parameters are inactive for these two drugs.The different toxicity parameters and their status or values for our selected chemical compounds are given in Table 6.

Discussion
Orally consumed poisonous CuSO 4 can trigger a particular vagal-induced vomiting reaction and can injure the mucous membranes in the GIT since CuSO 4 is effective as an oxidizing and corrosive agent 51,52 .The GIT's visceral afferent nerve fibers are stimulated by peripheral processes, which subsequently transmit the stimulation toward the VC, causing the act of vomiting 53,54 .The principal mediator of emesis is a CTZ in the medulla that is located outside the blood-brain barrier (BBB).It works by triggering a second region of VC 55 .Once initiated, vomiting www.nature.com/scientificreports/proceeds in two stages, including retching and ejection.A VC or a central pattern generator may be in the area postrema, and the nearby NTS controls the muscles that are responsible for that series of events 56 .In addition, emesis is caused by local neuronal release of 5HT in the area postrema, triggering different subtypes of 5HT such as 5HT 3 and 5HT 4 receptors 57 .Several other receptors, such as H 1 58 , mAChRs (M 1 -M 5 ) 59 , NK 1 60 , and different subtypes (D 2 and D 3 ) of dopamine receptors are also involved in the emetic process 18 , which have a significant impact on stimulating CTZ for inducing emesis.
APT is a highly selective antagonist of the NK 1 receptor used to manage and treat chemotherapy-induced and postoperative nausea and vomiting 61 .Our findings showed that APT exhibits a lower efficacy to reduce the emetic symptoms of the animals as the number of retches and onset of the retching period were comparatively close to those of the vehicle group.The referral drug DOM is widely used for the treatment of nausea and vomiting because it is a selective systemic antagonist of dopamine D 2 and D 3 receptors, which reduces the activity of these receptors at the CTZ in the brain to alleviate the emetic symptoms 62 .Our findings from the in vivo investigation  www.nature.com/scientificreports/demonstrated that the animals given DOM revealed 10.00 ± 1.46 retches, while the animals belonging to the vehicle group showed 66.83 ± 3.58 retches, indicating the drug's notable emesis diminishing capability.Additionally, DOM remarkably elevated the latency period (63.16 ± 3.99 s) compared to the control group (7.5 ± 0.92 s), which is also evidence of the drug's remarkable antiemetic properties.In this context, histamine plays an essential role in sending signals from the GI system related to food allergies and histamine seafood poisoning to the brain, leading to vomiting 63,64 .Antihistamine drugs such as DHM play an important role in minimizing the emetic process by antagonizing the H 1 receptor 23,65 .In our study, DHM-treated animals exhibited comparatively lower efficacy than other treatment groups and failed to manage CuSO 4 ⋅5H 2 O-mediated emesis.In the case of the 5HT 3 receptors, they are implicated in the process of causing vomiting through interpreting information from the digestive system.These receptors significantly influence the enteric nervous system's ability to control bowel movements and peristalsis 66 .5HT 3 antagonists like OND hinder the activity of the receptor and alleviate vomiting.In this in vivo test, the OND and HYS-treated animal groups reduced the number of retches compared to the vehicle group to 34.00 ± 2.26 and 46.33 ± 3.70, respectively.Furthermore, the OND and HYS-ingested groups also showed an elevated latency period of 14.83 ± 2.27 and 11.83 ± 1.37 s, respectively.All these findings indicate the potent antiemetic features of drugs.In this respect, the test compound (AA) also has significant capability to alleviate the emetic condition, as the animals treated with AA demonstrated an incredibly reduced number of retches and an elevation in the onset of retching.These results show that AA exhibits better antiemetic activity than the referral drugs APT, DHM, HYS, and OND to mitigate CuSO 4 ⋅5H 2 O induced-emesis in the in vivo experiment, as animals given AA expressed a lower number of retching and an elevated onset period.In addition, findings revealed that AA shows a dose-dependent antiemetic response.The higher dose of the test compound showed longer latency (98.00 ± 2.44) than the referral drug DOM, and the number of retching was also comparatively similar (10 ± 1.46 and 11.66 ± 2.52 for DOM and AA-40, respectively).These findings indicate remarkable potency compared to DOM to mitigate the emetic process.A synergistic effect was observed in this study by the combination drug therapy, which resulted in fewer retches and a longer latency period in chicks 67 .In our in vivo experiment, the combined group of (OND + AA-40) exhibited a significant percentage decrease in retches and an increase in latency period of 76.80% and 82.28%, respectively in comparison to the vehicle group.However, AA increases the antiemetic effect of DOM and HYS in the combination groups by showing a lower number of retching and elevated latency compared to the compound administered alone into the experimental animals.Depicted in Fig. 7 is the Suggested anti-emetic mechanism of the standard medications and test compound, AA.The molecular docking approach attempts to predict the most effective orientation of a compound to its macromolecular target (receptor) when these molecules are bonded together to form an enduring complex 8,68 .Recently, computational investigations have made it possible to create, screen, and develop medication candidates in a novel way.This cuts down on expenses related to animals and laboratories as well as overall evaluation time 69 .Molecular affinity is employed to estimate the level of binding (interaction) between a ligand and a targeted protein 70 .Findings from our in silico study revealed that the test ligand AA exhibits comparatively higher affinity than the selected referral ligands against different types of receptors that are liable for stimulating emesis, such as H 1 , 5HT 3 , and various subtypes of muscarinic receptors (M 1 -M 5 ).The M 4 receptor, which is an essential part of the cholinergic system, can control the release of several neurotransmitters, including dopamine, in the brainstem's CTZ, which evokes emesis 71 .Among several emesis-inducing muscarinic subtype receptors, the tested ligand (AA) exhibited the highest binding value (− 10.2 kcal/mol) against the M 4 receptor and blocked the receptor activity.On the other hand, HYS yielded a binding score of − 8.9 kcal/mol toward the M 4 receptor.Among several muscarinic subtypes' receptors, M 3 receptors are activated by acetylcholine, which is under systematic control.These receptors are abundant in smooth muscle and the GIT and are responsible for the contraction of the GI and gallbladder smooth muscles 72,73 .The M 4 receptors are in the cortex and hippocampus among other parts of the brain, but they are most noticeable in the striatum, where it is hypothesized that they regulate dopamine production and locomotor activity 74,75 .The ligand (e.g., the neurotransmitter acetylcholine), which binds to the active site of the M 4 receptor, starts the receptor activity.In this case, the number of amino acid residues that compose the binding site of the M 4 receptor has not yet been fully identified, but this study has found some significant residues.
Based on our in silico study, and due to the binding of the tested ligand and reference medications with various receptors, numerous identical amino acid residues are formed, including THR433, VAL91, PHE410 for D 2 , VAL86, LEU89, VAL107, ILE183, PHE106 for D 3 , TYR404 for M 1 , TYR426 for M 2 , PHE186, TYR92, TRP435, TYR439 for M 4 , and HIS265, ILE113, PHE264, PHE268 for NK 1 .This signifies that they interact with the identically highlighted amino acid residues to form a coupling at the same area on the receptors.The highest docking score for the experimental ligand (AA) toward the M 4 receptor is caused by the formation of one HB bond and multiple additional hydrophobic bonds.On the other hand, the standard drug HYS formed a lower number of hydrophobic bonds than the experimental ligand.Our findings also showed that the HB distance of AA is 2.24 Å, whereas it is 2.87 Å and 2.77 Å for HYS, which indicates that AA binds more closely to the receptor than HYS.Therefore, we anticipate that PHE186, TYR92, TYR439, and TRP435 are the key residues which implicated in the antagonizing action of AA against the M 4 receptor.However, the solitary tract nucleus (STN) and the CTZ of the central nervous system have elevated concentrations of 5HT 3 receptors 76 .It triggers nausea and vomiting by activating the appropriate emetic receptors on the vagal afferents 77 .The 5HT 3 antagonists (e.g., ondansetron) prevent 5HT from activating both centrally in the CTZ and peripherally on GI vagal nerve terminals.This hindrance exerts potent antiemetic activity 78 .Our in silico investigation also revealed that AA exhibited a higher binding affinity against the 5HT 3 receptor compared to the standard medication OND, the binding affinity is − 8.1 kcal/ mol and − 6.9 kcal/mol, respectively.The tested ligand interacts with the 5HT 3 receptor by forming one HB of ILE98 amino acid residue and several hydrophobic bonds with specific amino acid residues of PRO113, LYS25, PRO89, VAL95, TYR114 whereas, OND did not form any HB.Therefore, our findings show that AA exhibits potential antiemetic activity by blocking both the muscarinic and 5HT 3 receptor pathways.
Drug-likeness is a fundamental guideline in the context of drug development and discovery, and it provides qualitative predictions about the probability that a chemical compound would be used in an oral medication in terms of sufficient bioavailability.It identifies the drug's nature-related pharmacokinetics by assessing the drug's physicochemical characteristics 8,22,79 .Lipinski's rule of five is broadly used in predicting pharmacokinetics and drug-likeness.According to Lipinski's rule of five, a drug candidate ought to have a MW of 500 g/mol or less, five or fewer HBD, ten or fewer HBA, and a lipophilicity (LogPo/w) of no more than five 80 .All ligands are predicted to have superior pharmacokinetic characteristics and are within the range of becoming medicines under Lipinski's criterion.Our chosen test ligand meets each requirement of Lipinski's rule of five and establishes improved pharmacokinetic characteristics.
For the development of secure and reasonably priced drugs, in silico toxicology studies are essential and critical 81 .Evaluating the effectiveness of possible medication candidates is the main goal of toxicology studies regarding the process of developing new drugs.The ultimate objective is to interpret animal responses to determine the risk to human subjects [82][83][84] .Toxicology testing is also crucial for determining any possible adverse effects that compounds may have.For instance, persistent chemical exposure in humans typically results in genotoxicity, immunotoxicity, carcinogenicity, and developmental and reproductive toxicity 85,86 .Results from this investigation showed that AA does not exhibit immunotoxicity, mutagenicity, carcinogenicity, or cytotoxicity-related toxic effects.However, it did show toxic effects in terms of hepatotoxicity.Due to its ability to antagonize muscarinic acetylcholine and 5HT 3 receptors, our findings demonstrated that AA exhibits significant antiemetic activity against the CuSO 4 ⋅5H 2 O-induced emesis.The synthetic antiemetics that are now on the market have been shown in several trials to exhibit a multitude of adverse effects, including diarrhea or constipation, lethargy, malaise, headache, visual changes, lightheadedness, and dry mouth 87,88 .In contrast, alternative antiemetic medications, particularly those made of natural ingredients, showed comparatively fewer adverse effects and effective therapeutic advantages 89,90 .
Studies utilizing specific laboratory animals give crucial information on the positive and negative effects of novel drug candidates as well as potential biopharmaceutical issues 91 .Consequently, each pre-clinical investigation supports medical researchers in assessing the potential of biologically active compounds for clinical trials.This study showed limitations such as a lack of clinical trials and results based on the behavioral representation of the animals.The probable antiemetic mechanism of AA in this study is based on the in silico and in vivo studies, and it does not present any actual antiemetic mechanism.Taken together, our findings revealed that AA exhibits a potent antiemetic effect in experimental animals by reducing the number of retching and elevating the latency of emesis.The in-silico investigation manifested the reasons behind the antiemetic effects of AA, possibly through the interaction of AA with 5HT 3 and different subunits of muscarinic receptors.

Conclusions
In conclusion, findings from this investigation indicated that AA exhibits remarkable dose-dependent anti-emetic activity with a diminishing retching of 11.66 ± 2.52 and an elevating latency period of 98.00 ± 2.44 s for 40 mg/ kg in CuSO 4 ⋅5H 2 O-induced emetic animals compared to the vehicle group of 66.83 ± 3.58 and 7.50 ± 0.92 s, respectively.On the other hand, the emetic symptoms were also notably attenuated in the experimental animals treated with the selected standards (DOM, HYS, and OND), but the efficacy of APT and DHM is comparatively low.In addition, findings from the in silico investigation show that AA successfully meets all the parameters of drug-likeness, and the molecular docking study revealed that the ligand AA has a greater binding affinity against muscarinic receptors, particularly the subtype M 4 with a docking score of (− 10.2 kcal/mol) and 5HT 3 with a docking score of (− 8.1 kcal/mol) compared to selected standards for these receptors, with docking scores of HYS (− 8.9 kcal/mol) and OND (− 6.9 kcal/mol) for M 4 and 5HT 3 , respectively.Our results also showed that AA exhibits a synergistic effect when given with the selected referral drugs targeting various receptors liable for initiating emesis.The toxicological study also revealed that AA shows no toxic characteristics except hepatotoxicity.However, more investigations are suggested to identify the actual toxic mechanisms of AA.Furthermore, investigations are also required to establish a proper dose for humans through clinical trials and to investigate the exact mechanisms of action of AA in relieving vomiting and nausea brought on by several different reasons.

Figure 4 .
Figure 4. (i) The Swiss Model-built 3D structure of the human 5HT 3 receptor, (ii) Ramachandran plot of the homology model 5HT 3 protein for all non-glycine/proline residues.

Figure 5 .
Figure 5. 3D and 2D view of protein-ligand interaction and their binding sites with related amino acid residues.

Figure 7 .
Figure 7.The suggested anti-emetic mechanism of the test compound (abietic acid) compared to the selected standard drugs.[This Fig illustrates the anti-emetic mechanisms of APT, DOM, HYS, and OND, as well as a probable anti-emetic mechanism of AA, based on their affinity for binding to the muscarinic, D 2 , D 3 , 5HT 3 , and NK 1 receptors.In this case, AA acts as an inhibitor of D 2 , D 3 , 5HT 3 , M 4 , and NK 1 receptors, while DOM, APT, OND, and HYS inhibit D 2 , NK 1 , 5HT 3 , and muscarinic receptors, respectively.The vomiting center (medulla oblongata) is kept from being triggered when these stomach receptors are blocked, preventing muscular contraction, GIT contraction, and the outcome of no emesis].

Table 4 .
Amino acid residues, number of hydrogen bonds, and hydrogen bond length of non-bond interactions between the selected ligands and receptors.HB hydrogen bond, AA abietic acid, DOM domperidone, OND ondansetron, HYS hyoscine, DHM diphenhydramine, APT aprepitant.

Table 6 .
Prediction of different toxicity parameters of Abietic acid and selected referral drugs using Protox-II online tools.DOM domperidone, OND ondansetron, HYS hyoscine hydrobromide, DHM diphenhydramine, AA abietic acid.