In silico investigation and experimental validation of pistagremic acid isolated from Pistacia integerrima against Rhipicephalus microplus and Sarcoptesscabiei

Abstract

Rhipicephalus microplus and Sarcoptes scabiei are major ectoparasites affecting both humans and animals, causing significant economic losses to the dairy and agricultural sectors. This study aimed to evaluate the in vitro and in silico efficacy of pistagremic acid (PA), a natural compound isolated from Pistacia integerrima, against these parasites. Different concentrations of the compound were tested using the adult immersion test (AIT) and larval packet test (LPT) to assess their in vitro effects on mites and various tick life stages. Molecular docking was conducted to examine the interactions of PA with glutathione S-transferases (GSTs) of S. scabiei and R. microplus proteins. The results demonstrated high in vitro acaricidal activity, with significant efficacy in both AIT and LPT assays. In silico studies identified PA as a key bioactive compound, showing strong binding interactions with S. scabiei GST (binding energy: 10.0 kcal/mol) compared to permethrin (−8.1 kcal/mol) and with R. microplus GST (docking score: 7.8 kcal/mol) compared to ivermectin (−8.3 kcal/mol). Overall, PA shows strong potential as a plant-derived alternative for managing tick and mite infestations, supporting its further exploration as a novel acaricidal agent.

Graphical abstract

Highlights

• •First report on the acaricidal potential of pistagremic acid (PA) from P. integerrima against R. microplus and S. scabiei.
• •In vitro bioassays (adult immersion test and larval packet test) confirmed strong dose-dependent acaricidal activity of PA.
• •PA showed superior binding affinity to S. scabiei glutathione S-transferase compared to permethrin.
• •PA also showed comparable docking affinity to R. microplus glutathione S-transferase relative to ivermectin.

1. Introduction

About 80% of the world’s population uses medicinal herbs for their fundamental health requirements. The history of mankind may be summed up by the interaction between humans, plants, and medications made from plants (Gurib-Fakim, 2006). The primary source of natural medicines is plants. It is believed that plants contain substances with the potential to be utilized in contemporary medicine for the treatment of incurable illnesses (Sofowora et al., 2013). The historical value of natural products as a source of pharmaceuticals with therapeutic potential has been established (David et al., 2015). The primary source of the drugs’ early discovery was plants, particularly those used in traditional medicine. Available data indicate that ethnicity makes up 80% of the 122 herbal products (Pan et al., 2014). The most popular medications, including vincristine, morphine, and paclitaxel, are manufactured from natural ingredients (Alamgir, 2017). Natural ingredients make up about 40% of all contemporary medications in use today. Biological screening of plant-based compounds has revealed several potent drugs (Najmi et al., 2022).

Pistacia integerrima (Family of Anacardiaceae), commonly known as Kakar Singhi or Karkatshringi, is distributed in various countries, including India, Afghanistan, and Pakistan (Grover, 2021). The primary uses of P. integerrima include blood purification, gastrointestinal diseases, and anti-inflammatory, and antidiabetic effects (Jeet and Baldi, 2019). Pistacia integerrima is a medicinal plant composed of a variety of bioactive secondary metabolites (Singla et al., 2022) and is used to alleviate inflammation as a traditional herbal medicine (Rauf et al., 2017). The ethanolic and aqueous extracts have sedative, muscle relaxant, analgesic, and anti-inflammatory effects (Rauf et al., 2022). Varieties of bioactive compounds are found in the extract of this plant, which are responsible for its pharmacological activities (Irfan et al., 2021). There have been reports of strong antibacterial, enzyme-inhibitory, and antipyretic properties of pistagremic acid (PA) extracted from several P. integerrima plant sections (Grover, 2021).

Despite extensive research on medicinal plants, ectoparasite control remains a critical challenge, especially due to growing resistance to synthetic acaricides. The emergence of acaricide-resistant pests, such as Rhipicephalus microplus (cattle tick) and Sarcoptes scabiei (scabies mite), demands alternative and eco-friendly control measures. Given the increasing demand for natural alternatives in pest control, PA presents a promising candidate for managing ectoparasites such as R. microplus and S. scabiei. Rhipicephalus microplus, commonly known as the cattle tick, is a major ectoparasite affecting livestock, leading to significant economic losses in the agricultural sector due to reduced productivity and vector-borne diseases. Sarcoptes scabiei, the causative agent of scabies, is responsible for highly contagious skin infestations in humans and animals, often resulting in severe dermatological complications (Jain et al., 2020). In recent years, in silico approaches have played a crucial role in elucidating the molecular mechanisms of bioactive compounds against specific targets. Computational methods such as molecular docking and pharmacokinetic assessments provide valuable insights into the potential interactions between PA and key biomolecular targets associated with ectoparasitic infestations (Jiao et al., 2021). However, computational predictions require experimental validation to confirm the bioefficacy of the compound.

Therefore, this study investigates the acaricidal activity of PA from P. integerrima against R. microplus and S. scabiei, using a combined computational and in vitro approach. Our findings aim to contribute to the development of safe, sustainable, and plant-based alternatives to conventional acaricides.

2. Materials and methods

2.1. Plant collection, extraction, and isolation

The plant Pistacia integerrima was collected from Razagram, District Dir, Pakistan. Its identification was confirmed by Prof. Dr. A. Rashid from the Department of Botany, University of Peshawar (UOP), and a voucher specimen (No. Bot.20037 (PUP)) has been deposited in the herbarium of the same department. The collected plant materials were shade-dried, crushed, and ground. The shade-dried and finely crushed bark of P. integerrima (14 kg) was subjected to repeated maceration with methanol at room temperature. The resulting methanolic extract (600 g) was concentrated under reduced pressure to remove the solvent and subsequently dissolved in distilled water. Sequential liquid-liquid partitioning was performed using organic solvents of increasing polarity, including n-hexane, CHCl₃, EtOAc, and n-BuOH. The chloroform fraction (15.2 g), which exhibited a prominent green residue, was further purified using silica gel column chromatography. Elution was performed using a gradient solvent system of methanol and chloroform (100:0 to 0:95). The collected fractions were analysed using thin-layer chromatography (TLC) and assembled into ten distinct sub-fractions (PS-1 to PS-10).

Among these, fraction PS-10, obtained using a methanol-chloroform system (100:0 to 5:99), was subjected to successive rounds of column chromatography and TLC purification using a more refined solvent gradient (100:0 to 2:98). This process ultimately led to the isolation and characterisation of the pure compound, identified as PA (Fig. 1). The structure of the isolated compound was previously identified by our research group (Uddin et al., 2012).

Fig. 1.

Chemical structure of pistagremic acid (PA) isolated from Pistacia integerrima.

2.2. Adult immersion test

Fully engorged adult female R. microplus ticks were collected from naturally infested cattle and from the ground in cattle sheds located on various farms in and around the Dir District, Khyber Pakhtunkhwa, Pakistan. After collection, the ticks were washed with clean water to remove dirt and debris and then dried using absorbent paper. The adult immersion test was conducted to evaluate the acaricidal activity as per the Food and Agriculture Organization (FAO) protocol (Sharma et al., 2012). Dose-response assays were performed using concentrations of 200, 150, 100, 50, and 25 ppm. For each concentration, five fully engorged adult female R. microplus were immersed in separate solutions for 5 min, with three replicates per concentration, totalling 75 ticks (15 × 5). Deionised water and ethanol were used as negative and solvent controls, respectively, while trichlorofon served as the positive control. After immersion, the ticks were removed from the solutions, dried and placed individually into plastic specimen vials. These vials were maintained in a bio-oxygen demand (BOD) incubator at 28.0 °C and a relative humidity of 80–85%. The average weight of eggs laid to the average weight of females was determined to calculate the IE. After that, the eggs were weighed, placed in glass jars, and given 21 days to hatch. The inhibition of oviposition (IO as a percentage) was calculated using the formula: IO (%) = (IE_{control group} – IE_{treated group})/IE_{control group} × 100, where IE represents the index of egg-laying.

2.3. Larval packet test

The larval packet test was conducted with slight modifications based on FAO standards (FAO, 1984; Adehan et al., 2016). PA with concentrations of 25–200 ppm was applied to rectangular Whatman filter papers at a volume of 1 ml per rectangle. The treated filter papers were dried at 37 °C, folded in half, and secured on the sides with adhesive tape to form open-ended packets. These packets were designed to hold tick larvae. Approximately 100 larvae were introduced into each packet, and the open ends were sealed. The packets were then placed in a desiccator (Fisher Scientific, Pittsburgh, Pennsylvania, USA) maintained at 28 ± 1 °C and a relative humidity of 85 ± 5% for 24 h. After the exposure period, the packets were removed, and larval mortality was assessed by counting the live and dead larvae. Each acaricide concentration was tested in triplicate.

2.4. Contact bioassay

This assay was conducted to evaluate the effectiveness of plant extracts in controlling mite populations by assessing their mortality following direct exposure to different concentrations of the extract. Infected rabbits (likely with mite infestations such as S. scabiei) were raised at the rabbit farm in accordance with the Guide for the Care and Use of Laboratory Animals, and skin scraps were collected (Albus, 2012). A sterile surgical blade was used to scrape the infected skin into a micro-Petri plate after it had been thoroughly cleaned, till the skin turned red. Skin scraps were placed in a Petri dish and incubated at 37 °C until the mites emerged. In the experimental setting, 10 mites were inoculated using a fine needle into separate Petri plates. After that, the mites in the Petri plates were directly exposed to 0.5 ml samples of the plant extract. Each concentration of the extract underwent this process independently, and each concentration was repeated three times.

2.5. In silico screening

In silico screening was conducted to evaluate the potential interactions of plant-derived compounds with glutathione S-transferase (GST) proteins from R. microplus and S. scabiei, with the aim of identifying possible inhibitors for these proteins.

2.5.1. Target protein selection and homology modelling

Target protein selection was conducted to select the appropriate target proteins and generate their 3D models to facilitate further analysis of potential binding interactions. GSTs were chosen as molecular targets due to their well-documented role in the detoxification processes of arthropods, including ticks and mites (Pavlidi et al., 2018). Among the proteins, the GSTs of R. microplus and S. scabiei were not available from the RCSB Protein Data Bank, but with the help of the Swiss Model Server, they were established (Waterhouse et al., 2018). The target protein sequences for R. microplus and S. scabiei (Uniprot IDs: A0A9J6E730_RHIMP and D2IFM1_SARSC, respectively) were downloaded in FASTA format from the UniProt website. The amino-acid sequences of the target proteins were submitted to the SWISS-MODEL server for the construction of 3D models. Finding the template structure for the SWISS-MODEL template library was possible with the aid of BLAST and HHblits techniques. The protein model accuracy was checked through the SAVES online server (https://saves.mbi.ucla.edu/; last accessed on 28 June 2024).

2.5.2. Ligand modelling

Ligand modelling analysis aims to model the ligands (identified plant-derived compounds) for docking studies, ensuring they were in an appropriate 3D format for interaction with the target proteins. Using ChemDraw, the structure of the compounds (to be used as ligands) was sketched. The mol files was saved in pdf using Open Babel (O’Boyle et al., 2011). The Gasteiger charges and merging of non-polar hydrogens were done with AutoDock4 v.2.17 (last accessed on 28 June 2024).

2.5.3. Active site prediction

The CASTp (last accessed on 28 June 2024) was utilized to forecast the active sites found in the protein structure that was modelled. CASTp is an online server used for the identification and measurement of voids present in proteins in 3D (Dundas et al., 2006). The 3D protein that was modelled was the one submitted to the server, and the necessary binding amino acids were visualized to predict the interactions. For this study, the binding sites with the best volume, surface area, and draggability score were the key considerations.

2.5.4. Molecular docking

Docking was done using Autodock Vina. The active site coordinates were determined with the dimensions of x = 38.451, y = −10.356, z = 58.119 for GST of R. microplus, and x = −5.126, y = −9.324, z = 17.215 for GST of S. scabiei. A grid box with a radius of 17, 17, 17 Å for R. microplus and 21, 21, 21 Å for S. scabiei and a grid point spacing of 0.3 Å were generated for both the proteins. The protein-ligand docking was performed, and the lowest binding energy (kcal/mol) for the best docked pose was visualized using BIOVIA Discovery Studio software (last accessed on 29 June 2024).

2.6. Statistical analysis

Results were compared using one-way ANOVA, while mortality (LC₅₀) was calculated using probit analysis; all analyses were performed using IBM SPSS Statistics (v.25).

3. Results

3.1. In vitro adult immersion test for ovicidal and acaricidal activity

The toxicity assay results revealed varying levels of inhibition of oviposition (IO) across different concentrations of the tested substance. At a concentration of 25 ppm, the mean IO was 21.67 ± 3.99%, with significantly higher inhibition rates observed at elevated concentrations. Notably, the highest concentration tested, 200 ppm, produced IO value (57.37 ± 0.79%) comparable to the positive control acaricide (trichlorfon). Additionally, the calculated LC₅₀ was 29.513 ppm, with 95% confidence limits ranging from 29.513 to 36.068 ppm. These findings underscore the dose-dependent efficacy of the tested substance in inhibiting oviposition, highlighting its potential as an effective acaricide (Table 1, Fig. 2B).

Table 1.

Effects of P. integerrima extract on oviposition and mortality of R. microplus in adult immersion and larval packet assays.

Concentration (ppm)	IO (%)	Larval mortality (%)
	Mean ± SD	Mean ± SD
25	21.67 ± 3.99∗	51.33 ± 0.58∗
50	23.95 ± 0.70∗	56.67 ± 0.58∗
100	45.17 ± 1.54∗	64.67 ± 2.08∗
150	48.25 ± 0.80∗	78.67 ± 2.08∗
200	57.37 ± 0.79∗	86.33 ± 2.08∗
Distilled water	−1.14 ± 0.80	2.00 ± 1.00
Trichlorfon	61.39 ± 1.21∗	97.67 ± 0.58∗

Note: Asterisks indicate significant differences from the negative control at P < 0.05.

Abbreviations: IO, inhibition of oviposition (engorged female ticks); SD, standard deviation.

Fig. 2.

Effect of pistagremic acid (PA) on larval mortality (A) and oviposition inhibition (B) of Rhipicephalus microplus. Error bars represent the standard error; the columns with the same letters show no significant difference at P < 0.05.

3.2. Larval packet test

A concentration-dependent rise in mortality was noted in the larval packet test against R. microplus larvae. The mortality rate at 25 ppm was 51.33 ± 0.58%, and it progressively rose to 56.67 ± 0.58% at 50 ppm and 64.67 ± 2.08% at 100 ppm. Mortality increased dramatically with further dose increases, reaching 78.67 ± 2.08% at 150 ppm and 86.33 ± 2.08% at 200 ppm. The observed larvicidal effects were confirmed to be caused by the tested treatments, as the negative control group, which was treated with distilled water, showed just 2.00 ± 1.00% mortality (Fig. 2A). The positive control (trichlorfon) demonstrated potent acaricidal action with the highest mortality rate of 97.67 ± 0.58% (Table 1). The LC₅₀ and LC₉₀ values for PA were calculated as 29.513 ppm and 40.374 ppm, respectively, indicating a strong larvicidal potential of PA. These findings highlight the promising acaricidal activity of the plant extracts against tick larvae. A dose-dependent relationship between concentration and mortality rates was observed, with higher mortalities recorded at increased concentrations, aligning with the results from the adult immersion test.

3.3. Mite contact bioassay

The mite contact bioassay results demonstrated a dose-dependent increase in mite mortality upon treatment with PA. At 25 ppm, the mean mortality rate was 81.00%, increased to 90.67% at 50 ppm, and reached 100% at 100 ppm and above. The LT₅₀ values further confirmed this trend, showing a significant reduction with increasing concentrations, from 2.112 h at 25 ppm to 0.701 h at 200 ppm. Notably, the efficacy of PA at 200 ppm (LT₅₀ = 0.701 h) was even higher than that of permethrin (LT₅₀ = 0.913 h), suggesting strong acaricidal potential. The statistical parameters further validated these findings, as the slope values increased with concentration, indicating a stronger dose-response relationship. The chi-square values were also significant, particularly at higher concentrations (e.g. 44.802 at 200 ppm), reinforcing the reliability of the observed mortality rates. In contrast, the distilled water control showed only 19.33% mortality, with an LT₅₀ of 2.983 h, confirming that the observed effects were due to the bioactive compound rather than external factors (Table 2).

Table 2.

Lethal time (LT₅₀) and mortality rates of Sarcoptes scabiei at various concentrations of P. integerrima extract and control treatments.

Concentration (ppm)	Mean mortality ± SD (%)	LT50	95% CI	Slope	χ2-value
25	81.00 ± 4.34	2.112	1.949–2.291	2.026	6.298
50	90.67 ± 2.52	1.338	1.230–1.449	2.113	14.985
100	100 ± 0	1.008	0.855–1.162	2.244	34.023
200	100 ± 0	0.701	0.576–0.821	2.721	44.802
Permethrin	100 ± 0	0.913	0.485–0.632	2.978	46.572
Distilled water	19.33 ± 2.31	2.983	2.312–2.968	1.986	5.127

Abbreviations: SD, standard deviation; CI, confidence interval.

In a similar manner, the data shown in Table 3 demonstrate the calculated lethal concentration (LC₅₀) values. A clear trend was observed wherein the LC₅₀ values progressively declined with increasing exposure time. This inverse relationship suggests that the extract exhibits greater toxicological effectiveness over time. Notably, after a 6-h exposure period, the LC₅₀ was determined to be approximately 0.119 g/ml, signifying enhanced bioactivity of the extract.

Table 3.

LC₅₀ values of P. integerrima extract against S. scabiei over time.

Time (h)	LC50	95% CI	χ2-value	Slope ± SE	Intercept ± SE	P-value
0.5	2.752	2.134–3.965	1.885	1.155 ± 0.123	−0.508 ± 0.042	0.99
1	1.659	1.310–2.335	2.567	0.887 ± 0.113	−0.195 ± 0.040	0.99
2	0.238	0.183–0.289	8.606	1.298 ± 0.123	0.810 ± 0.048	0.57
4	0.109	0.043–0.174	23.752	1.493 ± 0.157	1.436 ± 0.066	0.008
6	0.119	0.083–0.150	14.602	2.483 ± 0.289	2.298 ± 0.143	0.15

Abbreviations: SE, standard error; CI, confidence interval.

3.4. Homology modelling and structure validation of modelled protein

The 3D structures of GSTs from both species were modelled using SWISS-MODEL. For R. microplus, the structure was based on the template 1gsu.1.A (Avian class-Mu GST), with a GMQE of 0.39, QMEAN of 0.73, and 56.00% sequence identity. In the case of S. scabiei, the template 4q5q.1.A was selected, yielding a GMQE of 0.90, QMEAN of 0.85, and 67.90% sequence identity. These values suggest that both models are of good quality and structurally reliable, as further supported by the Ramachandran plot and its statistical evaluation (Fig. 3, Fig. 4) obtained from the SAVES 6.0 online server, showing the distribution of amino-acid residues according to their phi (Φ) and psi (Ψ) backbone dihedral angles.

Fig. 3.

Structure validation of glutathione S-transferase (GST) from Rhipicephalus microplus using the SAVES online server. Abbreviations: [A, B, L], residues in most favoured regions; [a, b, l, p], residues in additional allowed regions; [∼a, ∼b, ∼l, ∼p], residues in generously allowed regions.

Fig. 4.

Structure validation of glutathione S-transferase (GST) from Sarcoptes scabiei using the SAVES online server. Abbreviations: [A, B, L], residues in most favoured regions; [a, b, l, p], residues in additional allowed regions; [∼a, ∼b, ∼l, ∼p], residues in generously allowed regions.

The Ramachandran plot analysis for R. microplus revealed that 91.0% of residues fall within the most favoured regions (red zones in Figs. 3), 8.2% were located in allowed regions (yellow zones in Figs. 3), and 0.3% in the generously allowed regions (light yellow zones in Fig. 3), indicating acceptable though less common conformations. Only 0.6% of residues appeared in disallowed regions, indicating minimal structural strain.

The Ramachandran plot analysis of the modelled 3D structure of S. scabiei revealed that 92.0% of its amino-acid residues fall within the most favoured regions (red zones in Fig. 4), indicating optimal backbone conformations. An additional 7.0% were located in the allowed regions (yellow zones in Figs. 4), 1.0% in the generously allowed regions (light yellow zones in Fig. 4), and none in disallowed regions, reflecting a high-quality and reliable model.

These data also validate that the modelled 3D structures are good quality models, as generally, a reliable model is expected to have more than 90% of residues in the most favoured regions and minimal residues in disallowed regions. The program creates a graph between the residue and the error value once each of the predicted sequences for R. microplus and S. scabiei is submitted separately as an input file. The ERRAT plots highlighted areas with potential errors (in red and yellow in Fig. 3, Fig. 4) and normal regions (in white in Fig. 3, Fig. 4), allowing for rapid visual assessment of model quality. Finding residues with error values higher than 95% and 99% is made simple by plot analysis. The result is displayed in the form of an overall quality factor with a value of 92.895 for R. microplus (Fig. 3) and 91.349 for S. scabiei (Fig. 4); both values are considered a good quality score.

3.5. Active site identification

The CASTp (last accessed on 28 June 2024) was utilised to forecast the active sites found in the protein structure modelled. From the active site prediction, pockets were identified with an area (SA) of 466.308 Å² and a volume (V) of 437.169 Å³ for R. microplus. A pocket area of 1136.556 Å² and a volume of 1637.878 Å³ were predicted for S. scabiei. However, because of the commonalities found when the modelled structures aligned with the template structures, the following locations were selected as more advantageous for the docking assessments: TRP8, ASP9, ILE10, GLY12, TYR33, THR34, CYS35, GLY36, PRO37, PRO38, PRO39, ASP40, PHE41, ASP42, ARG43, TRP111, VAL112, CYS115, and TYR116 for R. microplus; and LEU1, VAL6, ASP7, PHE8, VAL9, ASP10, LYS11, ARG12, TYR13, GLU29, LEU33, LEU40, PRO41, TYR42, TYR43, ILE44, ASP45, SER53, ILE56, LEU57, and LEU60 for S. scabiei as shown in Fig. 5.

Fig. 5.

CASTp predicted active sites of glutathione S-transferase (GST) from Sarcoptes scabiei and Rhipicephalus microplus. Colours indicate different pockets or cavities; same colour regions indicate the same cavities, both surface and interior.

3.6. Molecular docking

The entire docking process was carried out in a sequential fashion. The GSTs of S. scabiei and R. microplus were docked with the herbal phytochemical components of P. integerrima independently. The docking energy value and the interaction at the binding sites are the only factors that go into the outcomes. The compound is more stable and has a higher binding affinity when the value is more negative. The energy funnel hypothesis states that a more stable conformation is represented by less energy. Therefore, a considerable dissociation energy would be required to break the complex.

3.6.1. Inhibition of GST of Rhipicephalus microplus

All the selected ligands were docked with GST of R. microplus. PA had the highest binding energy (−7.8 kcal/mol) as compared to the standard drug ivermectin (−8.3 kcal/mol), second to that of naringenin, which also scored high (−7.0 kcal/mol). PA showed the strongest inhibitory effect, as the hydrogen bonds and hydrophobic interactions it formed suggest a stable binding complex with high catalytic inhibition. The amino-acid residues forming hydrogen bonds with PA were TYR7, LEU13 VAL112; TYR 116 and TRP8 formed alkyl bonds; ILE10, CYS35, and PHE41 formed Pi-alkyl bonds; and TRP8 formed Pi-sigma bond as shown in Fig. 6D, E, F.

Fig. 6.

3D view and 2D view of the interactions of pistagremic acid (PA) with glutathione S-transferase (GST) of Sarcoptes scabiei (A, B, C) and Rhipicephalus microplus (D, E, F).

3.6.2. Inhibition of GST of Sarcoptes scabiei

All the selected ligands were docked with the GST of S. scabiei. Results showed that PA had the highest binding energy (−10.0 kcal/mol) as compared to the standard drug permethrin (−8.1 kcal/mol), making PA a more competent inhibitor against S. scabiei; the second drug, quercetin, also scored well (−8.1 kcal/mol). These results showed that PA is a potent inhibitor when compared with the standard drugs. The amino-acid residues with which PA formed hydrogen bonds were SER53; LEU1, PHE8, ARG12, TYR13, TYR15, LEU40, PRO41, ILE56, LEU57; LEU60 formed alkyl and Pi-alkyl bonds with the target protein as shown in Fig. 6A, B, C.

4. Discussion

Chemical acaricides for controlling R. microplus in cattle farming have become increasingly limited due to the high selective pressure, leading to the emergence of resistant strains in several dairy farms (de Oliveira Souza Higa et al., 2015; Klafke et al., 2017). Consequently, there is a growing need for a sustainable, less harmful, and more effective alternative approach to managing R. microplus. One promising strategy is the identification and characterisation of plant-based extracts with acaricidal properties, particularly when addressing resistant tick strains (Borges et al., 2011; Ghosh et al., 2015). Compared to synthetic products, herbal medicines exhibit a variety of actions on parasites, are less toxic to mammals and the environment, and are less likely to induce the selection of resistant strains (Wanzala, 2017). Resistance in mites often develops through mutations in the target site of the acaricide or the upregulation of detoxification enzyme genes (Al Khoury et al., 2020), highlighting the urgency for new bioactive agents with high efficiency, species selectivity, and safety. Natural products have gained significant attention for their role in treating various ailments in humans and animals and serve as promising sources for small-molecule drug discovery in acaricide development (Zhu et al., 2020). This underscores the need to explore new botanical specimens for their potential in controlling R. microplus.

The present study demonstrated that the mean mortality of adult R. microplus ticks increased significantly with higher dosages (concentrations) and longer exposure times following in vitro treatment with PA. A similar pattern was observed in S. scabiei mites, where mortality was also dependent on both dose and exposure duration. These findings align with the results of previous studies (Malak et al., 2022; Hakami et al., 2023), which reported that the mortality effects of plant extracts are influenced by dosage, concentration, and exposure time. Furthermore, our study showed that all methanolic extracts of the bark exhibited a significant acaricidal effect against S. scabiei and R. microplus compared to the negative control.

The in vitro findings of this study revealed that at a concentration of 25 ppm, the mean IO for R. microplus was 21.67 ± 3.99%, with significantly higher inhibition rates observed at increased concentrations. Notably, the highest tested concentration, 200 ppm, resulted in an IO of 57.37 ± 0.79%, comparable to the positive control, the acaricide trichlorfon. Additionally, higher mortality rates were recorded in the larval packet test with increasing concentrations, consistent with the outcomes of the adult immersion test.

In parallel, significant mortality was also observed in S. scabiei mites, where 25 ppm resulted in 81.00 ± 4.34% mortality and 100–200 ppm achieved 100% mortality within shorter exposure times. The LT₅₀ values progressively decreased with increasing concentrations, ranging from 2.112 h at 25 ppm to 0.701 h at 200 ppm, demonstrating rapid acaricidal activity. Notably, permethrin, a standard chemical acaricide, also achieved 100% mortality but with an LT₅₀ of 0.913 h, slightly longer than PA at 200 ppm.

Molecular docking serves as a valuable tool for predicting interactions between bioactive compounds and their biological targets, helping to elucidate potential mechanisms of action and binding behaviour within enzyme active sites. In the present study, PA isolated from P. integerrima was subjected to docking analysis against two GST proteins, one from S. scabiei (UniProt ID: D2IFM1_SARSC), and one from R. microplus (UniProt ID: A0A9J6E730_RHIMP).

Docking analysis with S. scabiei GST revealed that PA exhibited the highest binding energy (−10.0 kcal/mol), outperforming the standard drug permethrin, which had a binding energy of −8.1 kcal/mol. This suggests that PA is a more potent inhibitor of S. scabiei, potentially contributing to the anti-mite activity of P. integerrima through its interactions with the target protein. Similarly, in the anti-tick docking study, the compound was analysed against R. microplus GST, yielding docking scores between −5.6 kcal/mol and −7.8 kcal/mol. PA again demonstrated the highest binding energy (−7.8 kcal/mol), although it was slightly lower than that of the standard drug ivermectin (−8.3 kcal/mol). The docking interactions and orientations of the ligands were carefully examined, highlighting their potential as acaricidal agents. Thus, our study offers computational evidence supporting the potential inhibition of glutathione S-transferase proteins.

While the present study demonstrates promising in vitro and in silico acaricidal activity of PA, translating these findings into practical applications requires careful consideration. In vivo validation is essential to assess the PA bioavailability, metabolic stability, and potential toxicity in host organisms. Challenges such as formulation for optimal delivery, dosing, and sustained efficacy under field conditions must also be addressed. Encapsulation in nano-carriers or the development of topical formulations could enhance stability and targeted delivery. Furthermore, PA could potentially be integrated into current parasite management programmes as a complementary phytochemical agent, offering a sustainable and resistance-mitigating alternative to conventional acaricides. Future research should prioritise assessing the clinical efficacy of PA, which may aid in the development of innovative solutions for managing species of Acari.

5. Conclusions

The acaricidal activity of P. integerrima against the tick R. microplus and the mite S. scabiei examined in vitro showed high larvicidal and adulticidal efficacy. Our study thus highlights the ability of PA to target various life stages of R. microplus and S. scabiei. PA emerged as a particularly effective and specific inhibitor of R. microplus and S. scabiei GST. This compound exhibited remarkable inhibitory activity, with docking scores of −7.8 kcal/mol and −10.0 kcal/mol, respectively, outperforming the standard drugs. These findings suggest that PA holds promise as a valuable acaricide and a potential lead compound for developing new acaricides with a novel mode of action. Future research should focus on evaluating its efficacy under in vivo and field conditions, testing against acaricide-resistant strains, and exploring its stability and performance under diverse environmental conditions to support its integration into sustainable parasite control strategies.

CRediT authorship contribution statement

Imtiaz Ahmad: Conceptualization, Methodology, Writing – review & editing. Rahaf Ajaj: Investigation, Data curation, Writing – original draft, Writing – review & editing. Abdur Rauf: Investigation, Data curation, Writing – original draft, Writing – review & editing. Adil Khan: Visualization, Investigation, Writing – review & editing. Nosheen Malak: Validation, Writing – review & editing. Saima Naz: Validation, Writing – review & editing. Zubair Ahmad: Writing – review & editing.

Ethical approval

Not applicable.

Funding

This study received funding from the Pakistan Science Foundation (PSF), Project no. PSF/NSLP/KP-UoS (892).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix. A. Supplementary data

The following are the Supplementary data to this article:

Data availability

The data supporting the conclusions of this article are included within the article and its supplementary files.