Evaluation of the larvicidal activity against Culex pipiens L. larvae and phytochemical characterization of Pulicaria odora L. extractABSTRACT
Mosquitoes, particularly Culex pipiens, are critical vectors of diseases, posing global health risks. The overuse of synthetic insecticides has led to environmental and ecological concerns, prompting the search for eco-friendly alternatives. This study evaluates the larvicidal efficacy of Pulicaria odora extract against Cx. pipiens larvae and analyzes its phytochemical composition using LC-ESI-MS/MS-MRM techniques. P. odora was collected, processed, and subjected to 70% methanol Extraction. Larvicidal activity was assessed according to WHO protocol. Phytochemical profiling identified bioactive compounds, and molecular docking studies explored protein-ligand interactions. The extract demonstrated significant larvicidal activity, with an LD50 of 0.69 mg/mL after 72 hours. Mortality increased with concentration and exposure, peaking at 66.66% at 1mg/mL. LC-ESI-MS/MS analysis identified 12 polyphenolic compounds, including kaempferol, chrysin, and hesperetin. Molecular docking revealed that kaempferol has the strongest binding affinity (-8.8kcal/mol) to the target protein (AChE1), with kaempferol forming multiple hydrogen bonds and π-stacking interactions. This study highlights P. odora's value in sustainable mosquito management, offering a natural alternative to synthetic insecticides. Future work should focus on optimizing its application in integrated pest management systems.
Keywords:
Pulicaria odor; Culex pipiens; molecular docking; acetylcholinesterase; LC-ESI-MS
RESUMO
Os mosquitos, especialmente o Culex pipiens, são vetores essenciais de doenças, o que representa um risco global para a saúde. O uso excessivo de inseticidas sintéticos gerou preocupações ambientais e ecológicas, o que levou à busca de alternativas ecologicamente corretas. Este estudo avalia a eficácia larvicida do extrato de Pulicaria odora contra larvas de Cx. pipiens e analisa sua composição fitoquímica usando as técnicas LC-ESI-MS/MS-MRM. A P. odora foi coletada, processada e submetida à extração com metanol a 70%. A atividade larvicida foi avaliada de acordo com o protocolo da OMS. O perfil fitoquímico identificou compostos bioativos e os estudos de acoplamento molecular exploraram as interações proteína-ligante. O extrato demonstrou uma atividade larvicida significativa, com um LD50 de 0,69 mg/mL após 72 horas. A mortalidade aumentou com a concentração e a exposição, atingindo um pico de 66,66% a 1mg/mL. A análise LC-ESI-MS/MS identificou 12 compostos polifenólicos, incluindo o kaempferol, a crisina e a hesperetina. O acoplamento molecular revelou que o kaempferol tem a maior afinidade de ligação (-8,8 kcal/mol) à proteína-alvo (AChE1), com o kaempferol formando várias ligações de hidrogênio e interações de empilhamento π. Este estudo destaca o valor da P. odora no controle sustentável de mosquitos, oferecendo uma alternativa natural aos inseticidas sintéticos. Trabalhos futuros devem se concentrar na otimização de sua aplicação em sistemas de manejo integrado de pragas.
Palavras-chave:
Pulicaria odora; Culex pipiens; acoplamento molecular; acetilcolinesterase; LC-ESI-MS
INTRODUCTION
Mosquitoes are acknowledged as major vectors of economically and medically relevant diseases (Roth et al., 2010; Weaver and Reisen, 2010). Their extensive distribution and significant ability to transmit various diseases result in millions of fatalities each year (Roth et al., 2010; Guidelines…, 2015). In 2019, malaria impacted 229 million individuals, leading to around 409,000 deaths globally (World…, 2023).
Culex pipiens (Diptera: Culicidae) is a recognized vector that transmits various severe human diseases (Salem et al., 2024). In the Eastern Hemisphere, several mosquitoes of the Cx. pipiens complex serve as essential enzootic vectors of the West Nile virus, disseminating the virus throughout avian populations, while some strains are also recognized for feeding on humans and other mammals (Fonseca et al., 2004).
The management of Cx. pipiens predominantly depends on synthetic insecticides. Nonetheless, their widespread, localized application might negatively affect wildlife, aquatic ecosystems, and the environment (Chakraborty, 2023). Bioactive plant extracts have been suggested as effective alternatives for mosquito control to mitigate these problems. These plant-derived solutions provide many bioactivities, including growth regulation, fecundity suppression, male sterility, diminished flying capability, immunological suppression, and enzyme inhibition (Abdulmajeed and Badia'a, 2017; Shehata et al., 2020). Bioactive plant extracts demonstrate significant potential in the prevention and management of disease-vector insects, especially in low-income and developing areas. They offer an environmentally sustainable alternative to synthetic substances, which persist in presenting health, environmental, and economic issues. Regrettably, numerous plants continue to be devalued or unrecognized, while others confront risks of neglect and extinction (Bararunyeretse et al., 2024). Historically, in 1947, Roark documented that 1200 plant species had insecticidal activity, but Sukumar et al. (1991) identified and examined 344 plant species with specialized mosquitocidal activity.
The genus Pulicaria (Asteraceae) includes more than 80 species found in Europe, North Africa, and Asia (Williams et al., 2003). P. odora is recognized for its medicinal characteristics and is utilized in traditional medicine to address different diseases, including respiratory, digestive, and inflammatory disorders (Bilgin and Gürdal, 2021). P. odora, native to North Africa, the Middle East, and certain areas of Asia, typically thrives in dry and semi-arid environments. Numerous research has underscored its bioactive potential, exhibiting antibacterial, antioxidant, and anti-inflammatory effects (Touati et al., 2018; Zefzoufi et al., 2020; Saidani et al., 2023). Nonetheless, its promise as a natural mosquito larvicide remains inadequately investigated.
This research aims to examine the larvicidal effectiveness of P. odora extract on Cx. pipiens larvae, and to identify its phytochemical composition by LC-ESI-MS/MS-MRM techniques. This study may provide a significant insight into bioactive substances with larvicidal properties, promoting the use of safer and environmentally sustainable methods for mosquito control. The phytochemical profiling of P. odora could highlight its polyphenolic content, enhancing its potential application in integrated pest management systems.
MATERIAL AND METHODS
Pulicaria odora was collected at its optimal flowering stage from Ain Kechra, Skikda, northern Algeria, at an elevation of 282 meters (36°46'03.4" N, 6°26'52.8" E). The plant specimen was authenticated by Dr. Sakhraoui, a botanist at the Faculty of Sciences at a local university. Following collection, the stems were separated from the leaves, which were thoroughly washed with tap water to remove surface impurities. The cleaned leaves were air-dried in a shaded area at ambient temperature for 15 days, then finely ground using a laboratory mill to obtain a uniform powder.
For the extraction, 50 grams of P. odora leaf powder was macerated in a 500 mL solution of 70% methanol and water, stirred continuously on a magnetic stirrer for 24 hours. The mixture was filtered through Whatman No. 1 paper, and the solvent was evaporated under vacuum at 40°C to ensure complete removal. The resulting dried extract was stored in glass bottles at 4°C for subsequent research applications, including larvicidal assays against Cx. pipiens larvae.
The Cx. pipiens larvae were collected from stagnant water sources with dense vegetation at the University Messoud Boukhadoume campus in Skikda and reared for multiple generations in the Technology Laboratory at the University of Skikda. They were kept in plastic trays (30 x 20 x 10 cm) containing a 1:3 mixture of dechlorinated water and site water to minimize environmental shock. The larvae were fed daily with fish food powder, and laboratory conditions were controlled at 27±2°C, 70-80% relative humidity, with a 12:12 light-dark cycle. Upon reaching the pupal stage, pupae were carefully transferred to small containers (about 5 x 5 x 5cm) and placed in larger adult rearing cages (50 x 50 x 50cm) for emergence. Adults were provided with a 10% sugar solution, while females were given access to mouse blood for egg production. Third-instar larvae from each generation were used to test the larvicidal effects of P. odora leaf extract.
The larvicidal activity of crude P. odora leaf extracts was evaluated following World Health Organization protocols (WHO, 1996). For each assay, ten active third-instar Cx. pipiens larvae were introduced into testing cups containing 100mL of deionized water. Target concentrations of the extract (0.125, 0.25, 0.5, and 1mg/mL) were prepared by adding precise volumes of stock solution to the water. Each concentration was tested in five replicates, totaling 50 larvae per concentration. A single control group was included, containing only 100mL of deionized water without the extract. The assays were conducted under controlled conditions of 26 ± 2°C, relative humidity of 60 ± 10%, and a 12-hour light/dark photoperiod. Larval mortality was recorded after 24, 48, and 72 hours.
The LC-ESI-MS/MS-MRM analysis was performed using a Shimadzu 8040 Ultra-High Sensitivity Instrument featuring UFMS Technology and a binary pump (Nexera XR LC-20AD). To optimize polyphenol standards, a direct injection method without a column was applied, utilizing two mobile phases: mobile phase A (water with 0.1% formic acid) and mobile phase B (methanol, HPLC grade ≥99.9%; Honeywell Seelze, Germany). The gradient elution program was set as follows: 80% A from 0-1 minute, reduced to 20% A from 1-30 minutes, further reduced to 0% A from 30-45 minutes, and returned to 80% A from 45-60 minutes. The analysis used an injection volume of 5 μL, a column temperature of 25°C, and a flow rate of 0.3 mL/min, employing an Ultra-force C18 column (2.5 mm ID × 100 mm, 1.8 µm particle size; Restek) for separation. ESI parameters were set with a CID gas pressure of 230 kPa, conversion dynode voltage of -6.00 kV, interface temperature of 350°C, desolvation line (DL) temperature of 250°C, nebulization gas flow rate of 3.0 L/min, heat block temperature of 400°C, and drying gas flow rate of 15.0L/min. Polyphenol standards were prepared in methanol at a concentration of 500µg/L, and the mass spectrometry analysis employed multiple reaction monitoring (MRM) in both positive and negative ion modes using an ion trap analyzer.
For our study, the three-dimensional structure of the target protein was predicted using Phyre2 (www.sbg.bio.ic.ac.uk/phyre2) based on its amino acid sequence obtained from the UniProt database (UniProt ID: Q86GC8). The "Intensive" mode of Phyre2 was used to optimize model accuracy, and the output structure was validated using several tools. The quality of the model was assessed using swissmodel.expasy (https://swissmodel.expasy.org/assess), the SAVES website (https://saves.mbi.ucla.edu/) for validating protein structures in PDB (Protein Data Bank) format. It includes several programs like ERRAT, VERIFY 3D, PROVE, PROCHECK, and WHATCHECK, which is used to assess the quality of protein models by analyzing atomic interactions, deviations from standard volumes, and overall structural integrity. Energy minimization was carried out in Chimera with the AMBER ff14SB force field to refine the model further, using the steepest descent and conjugate gradient methods until convergence.
The receptor was prepared using AutoDock Tools version 1.5.6 (Morris et al., 2009), with hydrogens added, charges assigned, and the grid box centers defined. Files were saved in pdbqt format. Docking simulations were conducted with AutoDock Vina (Eberhardt et al., 2021), using a 40 × 40 × 40 grid box and specific coordinates (center x = -65.385, center y = 34.001, center z = 89.282). The results were analyzed using PyMOL and Discovery Studio Visualizer to study ligand interactions and conformations in the active site.
All data were analyzed in triplicate, with results reported as mean ± standard deviation (SD). IC50 values for antioxidant assays were determined using linear regression analysis. Statistical analyses were performed with GraphPad Prism version 9.5.1, using ANOVA followed by Tukey's post hoc test to compare group differences, with significance levels set at P<0.05, P<0.01, and P<0.001.
RESULTS
This study examined the effectiveness of P. odora extract at various concentrations (0.125, 0.25, 0.5, and 1mg/mL) against Cx. pipiens third-stage larvae. Toxicity tests were conducted at specific time intervals: 24, 48, and 72 hours. After 72 hours of exposure, the LD50 value for the L3 larval stage was determined to be 0.69mg/mL. The results were intriguing; starting from a concentration of 0.25 mg/mL, the mortality rate after 24 hours of exposure was relatively low at 16%. This trend continued with a mortality rate of 17.67% after 48 hours and 29.56% after 72 hours.
Among the concentrations tested, statistical analysis revealed that the 0.5mg/mL and 0.25mg/mL concentrations showed similar mortality rates, with no significant difference. However, at the highest concentration of 1mg/mL, the mortality rate reached 30% after 24 hours, increased to 33.33% after 48 hours, and rose significantly to 66.66% after 72 hours. It is evident that toxicity increased with higher extract concentrations and longer exposure durations. ANOVA analysis showed a statistically significant difference between the 1 mg/mL concentration and the lower concentrations of 0.125, 0.25, and 0.5mg/mL. However, there was no significant difference between the 0.125, 0.25, and 0.5mg/mL concentrations. After 48 hours, similar findings were observed, but after 72 hours, there was a notable difference between the 0.125mg/mL and 1mg/mL concentrations, as well as between 0.125mg/mL and 0.25mg/mL, and 0.5mg/mL compared to 1mg/mL. The concentrations of 0.25mg/mL and 0.5mg/mL did not differ significantly in terms of mortality rates (Fig. 1).
The variance of corrected mortalities of larvae after 24 hours and 48 hours of toxicity tests and treatment with various doses of P. odora extract.
The LC-ESI-MS/MS-MRM analysis identified 12 polyphenolic compounds in P. odora using multiple reactions monitoring (MRM) mode. These compounds displayed distinct retention times (Rt), molecular weights, precursor and production ion mass-to-charge ratios (m/z), charges, voltages, and maximum intensities. Among the identified compounds, Chrysin exhibited the highest maximum intensity (170,000), with a retention time of 47.7 minutes, a molecular weight of 254.24, a precursor ion m/z of 255.1, and a production ion m/z of 223.3. Quercetine also showed high intensity (127,000), with a retention time of 51.5 minutes and a molecular weight of 303.23. Keampferol, Hesperetin, and Cinnamic acid demonstrated significant intensities, indicating their presence in substantial quantities within the extract. Each compound had a unique set of parameters. For instance, Esculin had a retention time of 30.9 minutes, a molecular weight of 340.28, a precursor ion m/z of 341.3, and a production ion m/z of 309.4, with a positive charge and voltage of -7.0 V. Vanillic acid, on the other hand, had a retention time of 48 minutes and displayed lower intensity (30,000), with a precursor ion m/z of 153.1 and a production ion m/z of 71.15.
Overall, the analysis successfully quantified various polyphenolic compounds, with notable variations in retention times, intensities, and ionization properties, reflecting the diverse chemical composition of P. odora (Table1 and Fig. 2).
LC-ESI-MS/MS-MRM profile of polyphenols identified from hydro-methanolic extract of P. odora
LC-ESI-MS/MS-MRM chromatograms of 12 polyphenol compounds in P. odora in multiple reactions monitoring (MRM) mode.
The quality assessment of the protein model, based on QMEANDisCo, MolProbity, and Ramachandran analyses, indicates a high structural integrity and reliability level. The QMEANDisCo global score of 0.93 ± 0.05 reflects strong similarity to experimentally validated protein structures, with high local quality scores across most residues and only minor deviations. Furthermore, the MolProbity score of 1.33 places the model within the quality range expected for high-resolution structures, reinforcing its accuracy. The Ramachandran favored percentage of 95.51% shows that most residues adopt energetically favorable conformations, with very few outliers (0.19%). These metrics confirm that the model is structurally robust, accurate, and suitable for reporting and further analysis (Fig 3)
Model Quality Assessment and Ligand Binding Site. (a) Comparison of the AChE1 model to a non-redundant set of PDB structures. (b) Local quality assessment of the model. (c) Binding pocket of the AChE1 model with identified compounds. (d) Ramachandran plot illustrating the backbone dihedral angles.
The docking analysis revealed a spectrum of binding affinities among the analyzed molecules, with kaempferol demonstrating the strongest binding affinity (-8.8kcal/mol) and vanillic acid the weakest (-5.9kcal/mol). Flavonoids such as kaempferol, chrysin (-8.5kcal/mol), and hesperetin (-8.5kcal/mol) exhibited notably high binding affinities, indicating their potential for interaction with the target protein. Compounds with moderate docking scores, such as caffeic acid (-6.5kcal/mol) and paracoumaric acid (-6.3 kcal/mol), reflected a comparatively lower binding potential. This variation in binding affinities underscores the diverse interaction profiles of the molecules, with those exhibiting more negative binding scores emerging as promising candidates for further investigation. Detailed interaction analyses highlighted specific binding patterns of kaempferol, chrysin, hesperetin, and esculin with the target protein. Kaempferol, which exhibited the strongest binding affinity (-8.8 kcal/mol), formed hydrogen bonds with ASP200A, ASN213A, SER250A, and GLU326A, as well as hydrophobic interactions with ASP200A, TRP212A, and TYR456A, along with π-stacking interactions involving TRP212A and TYR249A. Similarly, chrysin demonstrated a binding affinity of -8.5 kcal/mol, forming hydrogen bonds with ILE198A and GLY251A, alongside comparable hydrophobic and π-stacking interactions. Hesperetin displayed a binding affinity of -8.5 kcal/mol, engaging in hydrogen bonding with residues SER554A, LYS555A, GLY556A, ASN557A, TRP559A, GLY584A, and GLU589A, while forming π-stacking interactions with TYR585A. Esculin, with a binding affinity of -8.3 kcal/mol, formed extensive hydrogen bonds with residues such as PRO207A, GLY208A, and TRP212A, among others, and engaged in π-stacking with TYR585A (Table 2 and figures 4 and 5).
The molecular docking results for various compounds, including their docking scores, hydrogen bonds, hydrophobic interactions, and π-stacking interactions with the AChE1 model of Cx. pipiens
The heatmap illustrates the docking scores of various molecules, highlighting their predicted binding affinities with the target. The gradient of cool tones visually represents these differences, with deeper blue shades corresponding to stronger binding affinities.
illustrates the molecular docking interactions of Kaempferol, Chrysin, and Hesperetin with the modeled Cx. pipiens AChE1 protein. The protein structure is depicted in a gray cartoon representation, while the ligands are shown in stick representation. Non-covalent interactions, including hydrophobic interactions (dashed black lines), hydrogen bonds (dashed blue lines), and π-stacking interactions (dashed and dotted green lines), are highlighted to visualize the binding modes of the ligands to the protein.
DISCUSSION
Given the prevalence of insecticide resistance, this study aims to identify natural compounds, or phytochemicals, that could serve as effective alternatives. Numerous plants and their phytochemicals are known to potentially cause larval mortality. Shaalan et al. (2005) and Noorazlan et al. (2024) have comprehensively discussed various phytochemicals with mosquitocidal potential in their reviews. Many of these compounds demonstrate efficacy comparable to that of commercially available synthetic larvicides, which has led to their promotion as herbal alternatives.
The larvicidal assay results in this study indicate that P. odora extract exhibits a dose-dependent and time-dependent effect on mosquito larvae mortality, with increasing concentrations and longer exposure times yielding higher mortality rates. This finding aligns with previous research on Artemisia herba alba, a related Asteraceae family plant, which also demonstrated a strong dose-dependent larvicidal effect. A study by Berchi & Aouati (Berchi and Aouati, 2017) reported a 50% mortality rate at a 0.5mg/mL concentration of A. herba alba after 72 hours, higher than the 29.16% mortality observed in our study at the same concentration and time. This discrepancy in efficacy might be attributed to variations in the active compound composition or mosquito species used in each study. Additionally, the LD50 of P. odora (0.6945mg/mL) was notably higher than the 0.347mg/mL for A. herba alba, further suggesting that P. odora requires a higher concentration to achieve similar mortality levels.
Comparatively, Laurus nobilis extract at a concentration of 0.25mg/mL caused only 9% mortality in Cx.pipiens larvae after 72 hours (Zouaoui, 2017), much lower than our result of 29.16% under similar conditions. The LD50 for L. nobilis was close to that of P. odora, indicating that both plants might have comparable efficacy at a similar dosage range. However, the comparatively lower mortality suggests that P. odora possesses more effective larvicidal compounds. This efficacy is further highlighted when comparing with Nerium oleander extract, which required much higher LD50 and LD90 values (57.57mg/mL and 166.35mg/mL, respectively) to impact Cx. pipiens (El-Akhal et al., 2015). The significantly lower LD50 and LD90 values for P. odora (0.6945mg/mL and 35.86mg/mL, respectively) demonstrate its potent activity at relatively lower doses.
The polyphenolic profile identified in P. odora may be responsible for its larvicidal efficacy, as polyphenols are known for their insecticidal properties. This finding aligns with Rouari et al (Rouari et al., 2022), who identified compounds such as retin, chlorogenic acid, and caffeoylquinic acid as key contributors to the larvicidal activity of Oudney Africana against Cx. pipiens. Additionally, Hadidy et al. (2022) linked the larvicidal activity of Ageratum houtonianum to compounds like phytol, coumarin, rosmarinic acid, rutin, and chlorogenic acid. These studies support the potential role of polyphenolic compounds in larvicidal mechanisms, particularly those involving AchE inhibition.
The bioactive compounds in P. odora likely contribute to larvicidal action through multiple mechanisms. According to Al-Solami (Al-Solami, 2021), AchE inhibition is a major factor, as it leads to acetylcholine buildup in synaptic clefts, causing prolonged neuroexcitation, paralysis, and death. Furthermore, a reduction in GST activity might impair stress physiology and metabolic pathways, contributing to larval death. Hadidy et al. (2022) also suggested that polyphenolic compounds inhibit CYP-450 and CarE, enzymes involved in detoxification and metabolic degradation, further supporting the efficacy of polyphenols in mosquito control.
The maceration extraction method using hydro-methanol enabled a yield of 17.1% polyphenols from P. odora leaves, close to the reported yields for P. crispa and P. undulata (Mohamed et al., 2020). The polyphenolic compounds identified in our study such as escalin, vanillic acid, naringenin, kaempferol, quercitrin, chrysin, rutin, hesperetin, and 4-hydroxy-coumarin-represent a diverse chemical profile with likely insecticidal properties. This is the first report on these specific phenolic compounds in P. odora. Related species, such as P. inuloides (Al-Hajj et al., 2021) and P. undulata (Alshehri and Ghobashy, 2020), have shown similar profiles, with common compounds like kaempferol, quercetin, and rutin, underscoring the potential of P. odora as a source of bioactive polyphenols for larvicidal applications.
Common insecticides, such as carbamates and organophosphates, primarily function as acetylcholinesterase (AChE) inhibitors. By inhibiting AChE, these chemicals disrupt nerve signal transmission, leading to an accumulation of acetylcholine and hyperactivity in the nervous system. This results in symptoms such as restlessness, tremors, convulsions, and paralysis. In the present study, we employed molecular docking to investigate the mechanisms of action of identified polyphenols and found that these compounds are promising candidates for use as insecticidal agents (Baz et al., 2024; Yousuf et al., 2024). Our findings suggest that they may also act as potential AChE inhibitors.
For instance, kaempferol, a flavonoid, has been recognized as a potent AChE inhibitor. Li et al. (2023) reported that kaempferol demonstrated significant AChE inhibitory activity, consistent with the strong binding affinity observed in our study (dock score of -8.8kcal/moL). The compound’s ability to bind tightly to AChE likely contributes to its insecticidal properties. Similarly, chrysin has been studied for its AChE inhibitory effects. Li et al. (2017) found that chrysin effectively inhibited AChE activity, further supporting its potential as an insecticidal agent, aligning with our results (dock score of -8.5kcal/mol).
These studies, in conjunction with our findings, underscore the AChE inhibitory potential of compounds such as kaempferol, chrysin, hesperetin, and esculin. The molecular docking scores obtained in our study are in line with previous research, suggesting that these phytochemicals hold promise as natural larvicidal agents. By targeting AChE, they could serve as effective candidates for pest control, offering an alternative to traditional insecticides.
CONCLUSION
The results of this study demonstrate the potential of P. odora extract as an effective larvicide against Cx. pipiens larvae. The extract exhibited dose- and time-dependent toxicity, with the highest concentration (1 mg/mL) yielding the most significant larval mortality after 72 hours. LC-ESI-MS/MS-MRM analysis identified key polyphenolic compounds which contribute to the observed bioactivity. Molecular docking studies further revealed that kaempferol had the strongest binding affinity to the target protein, suggesting it as a promising candidate for further investigation in the development of natural insecticides. These findings underscore the potential of P. odora as a sustainable alternative to chemical insecticides, with its polyphenolic compounds playing a key role in its bioactivity.
ACKNOWLEDGEMENT
The authors express their sincere appreciation to Ongoing Research Funding program - Research Chairs (ORF-RC-2025-0902), King Saud University, Riyadh, Saudi Arabia
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