Introduction

Ticks belonging to the Ixodidae family are ectoparasites that feed on blood, holding significant importance in the medical and veterinary fields1,2. The relevance of these ticks lies in the potential adverse effects associated with their infestations in both humans and animals, including issues such as blood loss and harmful effects resulting from the introduction of toxic substances and microorganisms during their feeding process. The most substantial economic impact arises from the transmission of tick-borne pathogens, particularly in tropical and subtropical regions worldwide3.

Hyalomma anatolicum is a tick species belonging to the genus Hyalomma within Parasitiformes, Ixodoidea, Ixodidae4,5. The range of these ticks is worldwide, including portions of Eastern Europe, Central Asia, North Africa, the former Soviet Union, India, Nepal, and Pakistan6. They parasitize various animals, including cattle, sheep, camels, horses, donkeys, and some wild species, acting as vectors for diseases like Crimean–Congo hemorrhagic fever7. Additionally, they are reported to carry the bacterium Coxiella burnetii, causing Q fever, and contribute to the spread of brucellosis and piriformis disease8.

The cow tick, or Rhipicephalus (Boophilus) microplus, is widely recognized, is a blood-feeding arthropod with a global distribution primarily found in tropical and subtropical regions like Pakistan9. This tick species exhibits monoxenous behavior, relying primarily on cattle as its host10. It holds significant importance in livestock management due to its substantial economic impact. Rhipicephalus (Boophilus) microplus has the potential to act as a vector for various pathogens, including Babesia bovis, B. bigemina, and Anaplasma marginale, leading to tick-borne diseases as elucidated11. Ailanthus altissima (Mill.) Swingle, a deciduous tree native to Pakistan12. India, and Southeast Asia, is often grown for its decorative qualities. It has been widely planted in Africa, Oceania, and Europe13. Additionally, it is frequently used in paper production to help reduce environmental contamination14,15 To date, 221 substances, including triterpenoids, volatile oils, phenylpropanoids, quassinoids, and alkaloids, have been extracted and characterized from A. altissima16. Modern pharmacological research has shown that its crude extracts and isolated components possess anticancer, anti-inflammatory, insecticidal, and herbicidal properties16,17,18. Tick infestations, particularly by Hyalomma anatolicum and Rhipicephalus microplus, pose a significant threat to cattle health and productivity. Given the rising resistance to synthetic acaricides, exploring medicinal plants as safer and eco-friendly alternatives is crucial for sustainable tick control19. Acaricidal activity against Rhipicephalus microplus was assessed using citrus limetta seed oil (CLO), which showed 100% mortality in vitro and a notable tick decrease in vivo without causing any negative side effects. The presence of bioactive fatty acids shown by GC-HRMS analysis supports its potential as a secure and efficient tick control substitute19.

Glutathione S-transferases (GSTs) are essential enzymes for ticks, aiding in detoxifying and eliminating harmful substances like xenobiotics and reactive oxygen species encountered during blood-feeding or exposure to the host’s environment. These enzymes are crucial for tick survival, helping them feed on hosts while reducing the adverse effects of host defenses and oxidative stress20. Targeting specific GSTs that hinder the tick’s ability to resist acaricide treatment is key to developing an effective tick control strategy21. Additionally, acetylcholinesterase (AChE) is a vital enzyme in ticks’ nervous systems and is the primary target for organophosphate and carbamate pesticides22. In this context, the current study aims to evaluate the in vitro tick-inhibitory capacity of A. altissima plant extract and investigate, through in-silico methods, the inhibitory interactions of the plant’s phytochemicals with acetylcholine esterase from R. (B.) microplus and glutathione S-transferases from H. anatolicum. The acaricidal potential of A. altissima plant extract against tick species has not been previously studied, and no prior docking experiments have been conducted to understand the interactions of these specific phytochemicals with target proteins in ticks. This research is significant as it contributes to the growing understanding of A. altissima’s potential application in pest control and elucidates the underlying molecular pathways.

Results

GCMS analysis

The use of plants for the control of ticks depends on their phytochemical constituents. The results of our studies have shown that crude methanol extract was the most suitable for the identification of the active constituents. Thus, the methanol extract was the only one, which was used in GC–MS. Gas chromatography–mass spectrometry (GC–MS) of the methanol leaf extract of A. altissima has been seen to have 29 bioactive compounds, each with a different retention time, and 12 of them have been selected for in silico analysis.

The following bioactive compounds were found in the methanolic extracts: Cytisine (RT: 13.066 min), Methanone (RT: 14.258 min), 9-Octadecyne (RT: 16.450 min), n-Hexadecanoic acid (RT: 17.300 min), Diisooctyl adipate (RT: 21.061 min), 4-Aminobutyramide (RT: 21.612 min), Diisooctyl phthalate (RT: 22.134 min), 4-Quinolinecarboxylic acid (RT: 23.154 min), Gamma Tocopherol (27.133 min), Aminocarb (RT: 27.133 min), Benzaldehyde (RT: 35.705 min), and Indolizine (RT: 35.705 min) (Fig. 1).

Fig. 1
figure 1

GCMS analysis.

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Adult immersion test

The adult immersion test was employed to assess the impact of varied concentrations (25, 50, 75, and 100 mg/mL) of A. altissima plant extract on the mortality rates of two tick species, R. (B.) microplus and H. anatolicum, at different time intervals (0.5, 1, and 2 h). The findings, presented in Table 1 (Fig. 2C,F), reveal a concentration-dependent increase in mortality for both species over time. The control group, subjected to positive control permethrin and distilled water, exhibited minimal mortality, underscoring the superior performance of the plant extract against the test tick species.

Table 1 The mean percent mortality and its standard deviation of the plant extract against tested tick species.
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Fig. 2
figure 2

(A,D) represents the lethal concentration (LC50 and 90) of A. altissima against R. microplus and H. anatolicum respectively, similarly (B,E) represents the lethal time (LT50 and 90) of A. altissima against R. microplus and H. anatolicum respectively. (C,F) represents the overall mortality of R. microplus and H. anatolicum respectively at various concentration and time interval. Same alphabets on the error bars represents significance according to One Way ANOVA test.

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Table 2 provides lethal concentration values (LC50 and LC90) with 95% confidence limits, accompanied by statistical parameters like Chi Square, Slope, Intercept, and p value, offering insights into concentration-specific effects at different time points (Fig. 2A,B). Meanwhile, Table 3 (Fig. 2D,E) presents lethal time values (LT50 and LT90) with 95% confidence limits, providing further understanding of the temporal dynamics of mortality at various concentrations. Together, these tables offer comprehensive insights into the concentration and time-dependent effects of the tested substance on the mortality of R. (B.) microplus and H. anatolicum ticks. Notably, no larval packet test (LPT) was conducted, as the plant extract induced almost immediate mortalities in the test ticks, with no ticks surviving beyond 5 h post the assay.

Table 2 Respresents the lethal concentration “LC50 and LC90” values along with its other statistical parameters (chi square, slope, intercept and p value) of the plants extract at various time intervals against ticks.
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Table 3 Respresents the lethal time “LT50 and LT90” values along with its other statistical parameters (chi sqaure, slope, intercept and p value) of the plants extract at varying concentrations against ticks.
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Homology modelling and validation

Given the absence of crystallized 3D structures for Acetylcholine esterase of R. (B.) microplus and H. anatolicum glutathione S-transferase proteins in the Protein Data Bank (http://www.Rcsg.org, accessed on September 11, 2023), we employed homology modeling, a predictive method based on structurally similar templates, to construct protein models. The accuracy and reliability of predicted models are well established to depend largely on the degree of similarity with the template structures. Thus, before proceeding with protein modeling, an extensive template search was conducted, following the methodology outlined by Dalton and Jackson23.

SWISS-MODEL was the tool of choice, and model quality estimation guided the selection of the most suitable templates, offering an approximate assessment of model accuracy. The sequences of RmAChE3 and HaGST, obtained from NCBI, were uploaded to SWISS-MODEL. A single model was generated, yielding a GMQE (Global Model Quality Estimation) score of 0.84 for RmAChE3 and 0.98 for HaGST (Fig. 3). For HaGST, the template Rattus rattus 6GSV.1.A, with a resolution of 1.75 Å, was selected as the best option for building a 3-D model due to its 52.07% sequence identity. Meanwhile, for RmAChE3, the D7RXY2.1.A model from AlphaFold DB, demonstrating an impressive 94.17% sequence identity, was employed.

Fig. 3
figure 3

The three-dimensional homology modeled structures of (A) R. microplus Acetylcholine esterase and (B) H. anatolicum’s glutathione S transferase.

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The quality assessment of the 3D models for RmAChE3 and HaGST involved the use of the Ramachandran plot in PROCHECK software (Fig. 3). The plots indicated that 87.6% of residues for RmAChE3 (Fig. 4A,B) and 93.7% for HaGST (Fig. 5A) fell within the most favorable region, with 11.5% and 5.8% residing in the allowed region, affirming the good quality of the predicted models. Additionally, ERRAT, an overall quality factor assessing non bonded atomic interactions, provided scores above 50, signifying high quality models. Specifically, the ERRAT scores were 95.104 for RmAChE3 (Fig. 3B) and 93.720 for HaGST (Fig. 5B).

Fig. 4
figure 4

Validation plots for the Rhipicephalus microplus acetylcholine esterase protein 3D structure; (A) the Ramachandran plot, in which the most favorable, favorable and disallowed regions are represented by the red, yellow, and black colors respectively. Additionally, the torsion angles are represented by the phi and psi linkages which indicate potential peptide shapes. (B) Shows the overall quality factor values of the ERRAT server whereas, (C,D) represents the Z score values of the PROSA server.

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Fig. 5
figure 5

Validation plots for Hyalomma anatolicum’s glutathione S transferase protein 3D structure; (A) the Ramachandran plot, in which the most favorable, favorable and disallowed regions are represented by the red, yellow, and black colors respectively. Additionally, the torsion angles are represented by the phi and psi linkages which indicate potential peptide shapes. (B) Shows the overall quality factor values of the ERRAT server whereas, (C,D) represents the Z score values of the PROSA server.

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Furthermore, the PROSA server was employed to scrutinize the three-dimensional models of the proteins under study for potential errors. The Z-score, which is a measure of the structure’s total energy divergence from an energy distribution obtained from random conformations, is provided by PROSA as a general model quality indicator. Analysis of RmAChE3 and HaGST with PROSA revealed Z-scores of − 8.68 and − 8.08 for RmAChE3 (Fig. 4C,D) and HaGST (Fig. 5C,D), respectively. Collectively, these validation tools strongly support the acceptance of both proposed 3-D models as reliable, with a high degree of confidence.

Inhibition of acetylcholine esterase

Our docking experiments unveiled a distinct binding affinity order for the compounds, with 4-Quinolinecarboxylic acid exhibiting the highest energy of − 6.6 kcal/mol, followed by Gamma Tocopherol, Cytisine, 9-Octadecyne, n-Hexadecanoic acid, Diisooctyl adipate, 4-Aminobutyramide, Diisooctyl phthalate, 4-Quinolinecarboxylic acid, Aminocarb, Benzaldehyde, Indolizine and Methanone.

4-Quinolinecarboxylic acid demonstrated remarkable interactions during the molecular docking process, forming three hydrophobic bonds, including three pi–pi stacked, pi–pi T-shaped bonds, and one carbon–hydrogen bond. Furthermore, 4-Quinolinecarboxylic acid established one hydrogen bond with specific amino acids in the target protein, namely HIS 532, residues (Fig. 6A–C).

Fig. 6
figure 6

(A–C) Shows the 3D and 2D interactions of 4-Quinolinecarboxylic acid with Rhipicephalus microplus Acetylcholine esterase, whereas (D–F) shows the 3D and 2D interactions of Gamma Tocopherol with the glutathione S transferase protein of Hyalomma anatolicum.

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Inhibition of glutathione S transferase

All isolated compounds exhibited robust binding affinity, ranging from − 5.0 to − 6.7 kcal/mol, towards the glutathione S-transferase enzyme of H. anatolicum. The order of binding affinity for these compounds was as follows: Gamma Tocopherol > Diisooctyl phthalate > Cytisine > 9-Octadecyne > n-Hexadecanoic acid > Diisooctyl adipate > 4-Aminobutyramide > 4-Quinolinecarboxylic acid > Aminocarb > Benzaldehyde > Indolizine and Methanone.

During the molecular docking of Gamma Tocopherol (− 6.7 kcal/mol) with the target protein, a total of six hydrophobic interactions were observed, including one alkyl interaction, pi–alkyl interaction, pi–pi stacked interaction, pi–sigma interaction, one pi–Donor hydrogen bond, and one carbon–hydrogen bond. Additionally, Gamma Tocopherol formed three hydrogen bonds with specific amino acids in the target protein, namely TYR-7, TYR-116, and GLY-210 residues (as shown in Fig. 6D–F).

ADMET calculation

The most critical and challenging stage in the drug discovery and development process is DMPK (drug metabolism and pharmacokinetics) research, also known as ADMET. This stage accounts for approximately 60% of drug failures during clinical phases24. ADMET stands for “absorption, distribution, metabolism, excretion, and toxicity”, and it provides insights into how a drug compound is distributed and processed within the body. ADMET Predictor is a specialized computer program designed to estimate the pharmacokinetic characteristics and features of drug-like substances based on their molecular structures25.

A freely accessible online program called Swiss ADME is used to forecast the basic physicochemical characteristics of compounds as well as their absorption, distribution, metabolism, elimination, and pharmacokinetic features. These properties are vital for evaluating a compound’s potential for clinical trials. The tool assesses six key physicochemical properties: lipophilicity, flexibility, saturation, polarity, solubility, and size26.

The ADMET results provide insights into the physicochemical properties of the synthesized compounds and indicate whether they meet the “Rule of Five” criteria (molecular weight, iLOGP, hydrogen bond acceptors, and hydrogen bond donors). In addition, the study takes into account additional characteristics like the molecular polar surface area (TPSA), the number of rotatable bonds (ROTBs), the count of aromatic heavy atoms, and the existence of alerts for undesired substructures (such as Brenk and PAINS alerts). Table 4 provides a summary of these results.

Table 4 Calculated ADME parameters of the selected compounds.
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Many parameters are measured, such as the number of rotatable bonds (RB), the number of hydrogen donors (HBD), the number of hydrogen acceptors (HBA), the number of aromatic heavy atoms (nAH), the number of molar refractivities (MR), the topological polar surface area (TPSA), the octanol/water partition coefficient (iLOGP), and the number of alerts for undesirable substructures (Brenk alerts and PAINS alerts), are among the important properties of the designed compounds that are displayed in Table 4. Based on Table 1’s explanation of the QED principle and Lipinski’s rule of five, every developed compound satisfies the predetermined standards. However, it’s worth noting that some compounds exhibited more than one violation of these criteria. More specifically, all of the following properties are within acceptable bounds: MW, RB, HBD, HBA, TPSA, iLOGP, nAH, and MR Additionally, there were five alerts for Brenk in some compounds, indicating that the compounds fall within an average range. In conclusion, based on these findings, it can be stated that the designed compounds, which are highly acaricidal, exhibit a favorable pharmacokinetic profile, with most properties conforming to Lipinski’s rule of five and the QED concept. However, a few compounds may have minor deviations from these rules, but overall, their pharmacokinetic attributes are promising.

Discussion

Ticks, as external parasites, pose substantial threats to both animal and human health by transmitting various diseases caused by protozoa, viruses, and bacteria27,28. The use of chemical acaricides has several drawbacks, including negative environmental impacts, the development of resistance, and high costs29. As a result, researchers are actively exploring safe and environmentally friendly alternatives30. Herbal control methods are considered ideal substitutes for chemical measures due to their minimal harmful effects on ecosystems and mammals, as well as their cost-effectiveness31. This approach shows promise for managing ectoparasites such as ticks32.

Natural products from plants have attracted considerable interest as potential sources for new therapeutic agents. Researchers have investigated the medicinal properties of plants because of their strong pharmacological effects, low toxicity, and cost-effectiveness. Additionally, many clinically effective drugs have natural origins, highlighting the importance of studying medicinal plants to discover active ingredients for disease treatment. Once these active components are identified, they can be synthesized in the laboratory33,34. In this context, the plant A. altissima has been studied for its acaricidal properties against R. (B.) microplus and H. anatolicum ticks, including in silico molecular docking studies and ADME analysis.

In this study, the methanolic extract of A. altissima exhibited greater lethality to the test ticks compared to the positive control, permethrin. This finding is notable as it is the first research to examine the acaricidal activity of A. altissima extract against R. (B.) microplus and H. anatolicum ticks. The study confirms the acaricidal effect of the A. altissima extract on various stages of R. (B.) microplus and H. anatolicum ticks. The tick mortality rate was dose-dependent across all tested concentrations, with the highest concentration (100 mg/mL) achieving 100% mortality in all replicates. These results are consistent with previous studies on the acaricidal activity of different plants, such as Monotheca buxifolia35. Camellia sinensis36. Pinus roxburghii37, and Acacia nilotica38.

Previous research has investigated the effectiveness of various plant extracts in combating ticks, including Artemisia monosperma and Haplophyllum tuberculatum39. Melia azedarach40, and Azadirachta indica41. The current study introduces Artemisia atlanta as a promising and safe plant extract with potential acaricidal activity against R. (B.) microplus and H. anatolicum ticks. This finding is significant for developing environmentally friendly tick control methods that can serve as alternatives to chemical acaricides. Although the results are encouraging, it is important to recognize the limitations of this study. Verification of the extract’s safety and effectiveness in real-world settings is hampered by the absence of in vivo tests. Moreover, there are several obstacles in converting test results into workable, extensive tick control plans. Because geographical and seasonal variations might affect the phytochemical content and, in turn, the extract’s efficacy, standardization and scalability of extract production continue to be major concerns42. Additional research in formulation science is also necessary to develop stable and effective formulations that are appropriate for field application. Additionally, regulatory approval procedures for plant-based acaricides are frequently intricate and time-consuming, especially when assessments of environmental effects and safety for non-target organisms are required43. The understanding and approval of end users, such as farmers and veterinarians, is also essential for the successful implementation of such botanical remedies. Therefore, in order to enable the development of safe, efficient, and environmentally friendly acaricidal products derived from Artemisia atlanta, future research should concentrate on the chemical profiling of more active ingredients, in vivo validation, and extensive field trials.

The study also explores the molecular interactions between specific compounds and proteins, providing insights into the molecular mechanisms driving their acaricidal activity. Molecular docking analysis showed that 4-Quinolinecarboxylic acid (− 6.6 kcal/mol) and Gamma Tocopherol (− 6.7 kcal/mol) achieved impressive docking scores, indicating 4-Quinolinecarboxylic acid and Gamma Tocopherol have strong binding affinity and low binding energy with the target proteins. These results suggest that are promising candidates as potential anti-tick agents.

To gain a thorough understanding, the study first evaluated the physicochemical properties, drug suitability, and ADME (absorption, distribution, metabolism, and excretion) characteristics of all phytochemicals. The results showed that all phytochemicals met the criteria for potential drug candidates, exhibiting favorable solubility, drug-likeness, and non-toxic ADME properties. Following this, docking studies used blind docking to investigate how these phytochemicals interacted with two different parasite enzymes. The observed lower binding energies for all phytochemicals suggested higher affinities for the active binding sites, indicating their potential effectiveness. Additionally, the 2D bond interactions within the protein–ligand complexes showed strong binding, including conventional hydrogen bonds. These strong affinities of plant-derived compounds for the target receptors support their potential as effective anti-tick agents in the studied drugs.

Material and methods

Plant collection, identification, and extract preparation

The plant parts selected for the study were collected from District Buner in Khyber Pakhtunkhwa, Pakistan (coordinates: 34.1917° N, 72.0347° E). The collection of plant material is in accordance with institutional guidelines and legislation. The plant is fast growing and can be easily cultivated in the area and there is no risk of extinction. Following collection, the plant materials underwent thorough cleaning and inspection for any damage. The leaves of the plant were subsequently identified as A. altissima by Dr. Imtiaz ahmad, Lecturer and herbarium in charge at the department of Botany, Bacha Khan university Charsadda BKUC. The identified plant was placed for reference in the Herbarium, department of Botany, BKUC under the voucher number Hbkuc-2935. After identification, the leaves were air dried for 3 weeks. Following a 22-day drying period, the leaves were finely ground into a powder. Stock solutions were prepared by dissolving 50 g of the powdered leaves in 500 mL of 80% methanol. The resulting mixture was incubated in a shaking incubator at 37 °C and 250 rpm for 72 h. Subsequently, it underwent a three-time filtration process and was evaporated at 48 °C using a Water Bath, untill a completely dried extract (solid/powder form) was obtained. To assess the extract’s effectiveness, the resulting powdered extract was diluted to concentrations of 25, 50, 75, and 100 mg/mL.

Gas chromatography–mass spectrometry (GC–MS) analysis

A Thermo Scientific Trace GC1310-ISQ mass spectrometer with a TG–5MS capillary column was used to analyze the chemical composition of the samples. The column oven temperature started at 50 °C, gradually increased to 230 °C at 5 °C/min, held for 2 min, then further increased to 290 °C at 30 °C/min and held for another 2 min. The injector and MS interface temperatures were set at 260 °C and 250 °C, respectively. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. A 1 μL sample was injected in split mode using an autosampler, with a 3-min solvent delay. Mass spectra were generated using electron ionization (EI) at 70 eV, covering a range of m/z 40–1000, with an ion source temperature of 200 °C. Compounds were identified by comparing their retention times and mass spectra with the WILEY and NIST spectral databases.

Ticks’ collection and identification

Following the guidelines set forth by the “World Association for the Advancement of Veterinary Parasitology”44, adult fully engorged R. (B.) microplus and H. anatolicum ticks were meticulously collected from wild sheep, goats, and cattle across multiple regions in District Buner, Khyber Pakhtunkhwa (coordinates: 34.1986° N, 72.0404° E). These animals were grazing freely in open lands and had not been treated with acaricides. Therefore, the ticks collected were assumed to be non-acaricide-resistant strains. To ensure consistency, all collected ticks were in the same feeding status and were detached from the animals around the same time. After collection, the ticks underwent a thorough cleaning process by rinsing them in distilled water. Finally, they were identified using standard tick identification keys under a microscope45. A total of approximately 3000 adult ticks were judiciously selected to be included in the study. The selected ticks were ensured to be approximately of the same weight and size. Subsequently, the adult immersion test was conducted using these ticks, with the primary objective of assessing the acaricidal properties of the selected plant extract.

Adult immersion test

The adult immersion test (AIT) was conducted to evaluate the efficacy of the extract against the targeted tick species. In brief, each tick species (R. (B.) microplus and H. anatolicum) was divided into two distinct groups, each consisting of the same tick species. Subsequently, each of the R. (B.) microplus and H. anatolicum groups was further divided into four subgroups, with each subgroup comprising 100 ticks submerged in varying extract concentrations for 5 min. After immersion, the ticks were placed on petri plates lined with filter paper chips. The petri plates were then incubated at 27 °C with 75% R.H (relative humidity) in an incubator with 12 h daylight cycle. Inspection of the ticks occurred at intervals of 30 min, 1 h, and 2 h. Ticks were deemed deceased if they exhibited no movement when stimulated with light and a needle. Furthermore, a control group was established using permethrin as the positive control and distilled water as the negative control to facilitate a comparison of the extract’s results. All experiments were replicated three times on separate days, each with newly collected tick species.

Target sequence retrieval

The protein sequences for R. (B.) microplus acetylcholine esterase (RmAChE3) with the accession number ALD51332.1 and H. anatolicum glutathione S transferases (HaGST) protein with the accession number WIF29840.1 were obtained in FASTA format from the National Centre for Biotechnology Information (NCBI) database. These sequences served as the foundation for subsequent analyses, including the construction of a homology model.

Protein template search and homology modelling

A sequence similarity search for R. (B.) microplus acetylcholine esterase (RmAChE3) and H. anatolicum glutathione S transferases (HaGST) was conducted using the PSI-BLAST method, utilizing the procedures described by Chamizo-González46. In particular, the search was conducted using the PSI-BLAST tool, which can be found at https://blast.ncbi.nlm.nih.gov/Blast.cgi, and the HHpred online server, which can be found at https://toolkit.tuebingen.mpg.de/tools/hhpred, also on September 11, 2023. The species of the template, the percentage of sequence identity, the E-value, and the percentage of sequence coverage/probability were among the quality characteristics that were used to choose the best templates. The best template structures were then obtained by downloading them from the Protein Data Bank (PDB). The SWISS-MODEL server, available at https://swissmodel.expasy.org/, was used to create the 3D structures of both target receptors. This was done on September 11, 2023.

Validation of the modeled structure

To validate the modeled structures, their quality was assessed using PROCHECK, available at https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/, accessed on September 12, 2023, and ERRAT, accessible at https://servicesn.mbi.ucla.edu/ERRAT/, also accessed on September 12, 2023. Additionally, Z-scores for both proteins were calculated using the ProSA web server.

Blind docking analysis

For the blind docking study aimed at unraveling the interactions between ligands and macromolecules, the molecular docking investigations were carried out using AutoDock Vina (version 1.1.2). On September 15, 2023, a graphical user interface (GUI) for AutoDock Vina called AutoDockTools (ADT) was accessed at http://vina.scripps.edu/download.html. Furthermore, an extensive literature survey was conducted to evaluate the phytochemical profile of A. altissima plants. The selection of phytochemicals was based on studies assessing the plant’s phytochemicals through GC–MS, LC–MS, and/or HPLC. Only studies from regions with climatic conditions similar to Pakistan were considered in the selection process47,48,49,50,51. Fifteen phytochemicals derived from A. altissima were chosen based on prior studies supporting their known possible biological, pharmacological, and biochemical actions against many insects and parasites. These selected compounds underwent docking using AutoDock Vina with default parameters against the RmAChE3 and HaGST proteins. The docking specifications included a 40 × 60 × 40 grid box with a 0.375 Å grid point spacing for the x, y, and z grid points. The coordinates 18.79 Å, 17.62 Å, and 30.608 Å were utilized to define the grid’s central square. Each ligand produced nine different conformations during the docking process, and AutoDock Vina scoring tools were employed to compute and rank their binding energies. Post-docking analysis was conducted using the Discovery Studio Visualizer to investigate target receptor–ligand interactions. Ligand torsions were enabled, and PDBQT files were created for the processed protein and ligand structures, assuming proteins to be stiff. Completeness of 2000 was employed in the docking process to cover the entire protein with the receptor grid.

Theoretical prediction of ADMET parameters

ADMET predictor, a specialized computer program, is utilized to predict the characteristics and pharmacokinetic parameters of molecules resembling drugs based on their molecular structures. As elucidated by Singh, Gupta52, ADMET encompasses absorption, distribution, metabolism, excretion/elimination, and toxicity. While possessing high bioactivity and low toxicity are crucial criteria in assessing the suitability of a drug or drug-like compound, these factors alone are insufficient to qualify a compound as a strong candidate. An evaluation of the compound’s pharmacokinetic profile is essential in the drug discovery process. Therefore, predicting the ADMET properties of new compounds in advance holds great significance to avoid wasting valuable time and resources. In this study, we employed the SWISS ADME online software developed by Daina, Michielin53 to predict the ADMET properties of fifteen designed compounds. The “Rule of Five”, introduced by Lipinski, Lombardo54, comprises four ADMET properties, serving as a widely recognized rule-based filter for assessing drug-likeness and oral absorption. A few of these requirements are: (1) Molecular weight (MW) ≤ 500, (2) The partition coefficient of octanol/water (iLOGP = A log P) ≤ 5. (3) There must be no more than five hydrogen bond donors (HBDs) and no more than ten hydrogen bond acceptors (HBAs). (4) If a molecule does not break two or more of these rules, it is deemed orally active/absorbable by the Rule of Five, However, some complex natural products may not conform to these rules, leading to the proposal of alternative drug-likeness rules and filters, as suggested by Bhal, Kassam55.

Bickerton, Paolini56 introduced the concept of Quantitative Estimate of Drug-likeness (QED), which considers eight physicochemical properties. The number of rotatable bonds (ROTBs), the number of aromatic rings (nAROMs), the presence of alerts for undesirable substructures (ALERTs, such as PAINS #alert and Brenk #alert), and thermal polar surface area (TPSA) are the four additional parameters added to the original Rule of Five criteria (MW, iLOGP, HBAs, and HBDs). Compared to traditional drug-likeness regulations, the QED idea is more adaptable and extensively used. To evaluate the pharmacokinetic characteristics of the developed drugs, Chemdraw Ultra (version 12.0) was utilized to create two-dimensional structures, and the SMILES notation of each structure was input into the SwissADME interface at http://swissadme.ch/, accessed on 20 September 2023. The SwissADME analysis was conducted, and resulting ADMET properties and parameters were generated, following the methodology outlined by Mishra and Dahima24.

Statistical analysis

R and RStudio were utilized for all statistical analyses. Prior to importing into the R programming environment for additional statistical analysis, data were organized in Microsoft Excel (v 2302). Descriptive statistics, including mean ± standard deviation, were generated in R. A one-way analysis of variance (ANOVA) was employed to compute the significance difference between various concentrations, and the Tukey honestly significant difference (HSD) test was used to confirm the results. Additionally, the R “ecotox” package was employed to calculate the 50% and 90% lethal concentration and lethal time (LC50, LC90, LT50, LT90). The “ggplot2 and ggpubr” R packages were utilized for visually representing the data.

Conclusions

In conclusion, this study highlights the acaricidal potential of the methanolic extract of A. altissima against R. (B.) microplus and H. anatolicum ticks. The observed dose-dependent mortality rates and the superior effectiveness compared to permethrin suggest that A. altissima could be a promising natural alternative for tick control. Molecular docking analyses identified specific compounds, such as, 4-Quinolinecarboxylic acid and Gamma Tocopherol which demonstrated strong binding affinities to target proteins, providing insights into the biochemical mechanisms behind their anti-tick properties. The evaluation of physicochemical properties and ADME characteristics further supports these phytochemicals as potential non-toxic anti-tick agents. With their favorable drug-likeness and solubility profiles, these compounds offer a foundation for developing environmentally friendly tick control strategies. This research provides valuable insights into tick management and emphasizes the potential of A. altissima as a source of effective and sustainable tick control agents. Future studies, however, ought to concentrate on carrying out field tests to evaluate the extract’s efficacy in actual environmental settings. Furthermore, to ensure the sustainability of utilizing plant-based acaricides in integrated pest management systems, long-term safety studies, which include assessments of possible effects on non-target organisms and the environment, should be given top priority. It may strengthen the acaricidal capability of A. altissima and aid long-lasting tick control methods by filling in these research gaps.