Chitosan nanoparticles loaded with Cosmos bipinnatus essential oil for sustainable management of plant parasitic nematodes

Abstract

The sustainable management of plant-parasitic nematodes is a growing concern in agriculture due to the limitations of synthetic nematicides, such as environmental toxicity and resistance development. This study reports, for the first time, the encapsulation of Cosmos bipinnatus essential oil (CAO) into chitosan nanoparticles (CSNPs) for nematicidal application against Meloidogyne incognita. GC–MS analysis identified cis-β-ocimene, cosmene, germacrene D, sabinene, and β-pinene as the dominant constituents. The resulting CSNPs exhibited favourable physicochemical characteristics (mean diameter: 91.2 nm; encapsulation efficiency: 83%) and a pH-responsive controlled release profile. In vitro assays revealed that at the highest concentration (1000 µg/mL), CSNPs demonstrated significantly higher nematode mortality (100%) and egg hatching inhibition (100%) than CAO (77% mortality and 89.04% egg hatching inhibition) and comparable to the commercial nematicide Nimitz at 96 h. Enhanced acetylcholinesterase (AChE) inhibition by CSNPs (IC₅₀ = 16.14 ± 0.05 µg/mL) was observed, with IC₅₀ value approaching that of the standard physostigmine (IC₅₀ = 8.57 ± 0.08 µg/mL). Docking studies confirmed favorable interactions of germacrene D (− 6.8 kcal/mol), sabinene (-6.3 kcal/mol), caryophyllene (− 6.2 kcal/mol), β-pinene − 6.1 kcal/mol), and endo-arbozol (− 5.6 kcal/mol) with AChE’s active site, suggesting a neurotoxic mode of action.The study also evaluated ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of major terpenoids, confirming favorable pharmacokinetic profiles for potential agrochemical development. These results highlight the efficacy and mechanistic relevance of chitosan-based nanoformulations of C. bipinnatus EO as a promising biopesticide alternative for integrated nematode control.

Introduction

The global agricultural industry continues to grapple with the challenge of plant-parasitic nematodes, particularly Meloidogyne incognita, commonly known as root-knot nematodes. These microscopic pests are infamous for their capacity to inflict significant harm on various crops, resulting in considerable economic losses [1]. Conventional chemical nematicides have been commonly employed to control these pests, but their widespread application has sparked serious environmental and health issues. Issues such as soil and water contamination, non-target species toxicity, and the development of nematode resistance have driven the demand for safer and more sustainable alternatives [2].

In the quest for eco-friendly nematicides, plant-derived essential oils (EOs) have gained attention due to their rich content of bioactive compounds with diverse biological activities [3–5]. Essential oils are complex mixtures of volatile compounds, including terpenes, phenolics, and aldehydes, which have been shown to possess significant nematicidal properties [6, 7]. Bioactive constituents of essential oils, mainly terpenes, are responsible for nematicidal activity. Their lipophilic nature, particularly phenols, aldehydes, and alcohols, disrupts nematode plasma membrane integrity, leading to cytoplasmic leakage, while terpenes may also exert toxicity through biochemical interactions [8]. Specific monoterpenes disrupt biomolecules like polysaccharides and phospholipids or depolarize mitochondrial membranes, linking their mode of action to their chemical structure [9]. Acetylcholinesterase (AChE), an essential enzyme responsible for hydrolyzing acetylcholine, plays a vital role in nematode neural signaling and muscle function. Inhibiting AChE disrupts this signaling pathway, resulting in paralysis and eventual death of nematodes [9–11]. Many EO constituents are believed to target AChE enzyme [12, 13]. However, the practical application of essential oils in agriculture is often hampered by their inherent limitations, such as volatility, low water solubility, and potential phytotoxicity when applied at higher concentrations [14].

To overcome these challenges, the encapsulation of essential oils into nanoparticles has emerged as a viable strategy. Nanoparticles can protect the essential oil from degradation, enhance its stability, and allow for controlled release, thereby improving its efficacy as a nematicide [15]. Various polymers, including alginate, gelatin, pectin, cellulose derivatives, and synthetic polymers like PLGA, are commonly used for encapsulating essential oils [16], but chitosan is frequently regarded as the most effective. Chitosan, a natural biopolymer derived from chitin, has been widely explored as a nanoparticle carrier due to its biocompatibility, biodegradability, and strong interaction with cell membranes that enhances bioactive delivery. Additionally, its inherent antimicrobial properties may further contribute to the overall efficacy of the formulation. [17, 18]. Importantly, chitosan itself has been reported to exhibit direct nematicidal activity, with low molecular weight chitosan showing significant toxicity against root-knot nematodes such as Meloidogyne incognita, reducing nematode mortality, egg mass formation, and root galling in both in vitro and greenhouse studies [19]. Thus, chitosan serves as an efficient coating biopolymer for anionic emulsions, improving stability in acidic conditions and boosting the nematicidal activity of natural pesticides.

Cosmos bipinnatus, commonly known as Mexican aster, is a flowering plant from the Asteraceae family with significant traditional medicinal uses for ailments such as headaches, jaundice, stomach aches, and malarial fever [20, 21]. Phytochemical analysis has revealed a diverse array of bioactive compounds, including flavonoids, phenolic acids, triterpenoids, essential oils, and steroids. These compounds are responsible for various biological effects, including antioxidant, antimicrobial, anti-inflammatory, protective against genetic damage, larvicidal, and liver-protective activities [21–26]. The essential oil of C. bipinnatus contains key compounds like β-caryophyllene, germacrene D, sabinene, β-ocimene, p-cymene, cosmene, and α-cadinol [27–29]. Additionally, flavonoids such as chrysoeriol, helianol, lupeol, and luteolin highlight its medicinal value [27, 30]. Despite being a common ornamental plant, C. bipinnatus is largely unexplored for pesticidal uses. Its ease of cultivation, tolerance to diverse conditions, and rapid growth make it a sustainable resource, filling a significant research gap.

This study aims to evaluate, for the first time, the nematicidal activity of C. bipinnatus essential oil (CAO) against M. incognita and to enhance its efficacy through chitosan based nanoencapsulation. In addition to evaluating nematicidal activity, this study investigates the mechanism of action of both free and encapsulated essential oil by assessing their inhibition on acetylcholinesterase (AChE) activity and through. molecular docking analysis to elucidate the interactions between bioactive compounds from the essential oil and the AChE enzyme at the molecular level.

Material and methods

Chemicals

Chitosan (CAS#9012–76–4, degree of deacetylation >  = 75%) was purchased from HiMedia Labs Pvt Ltd. (India). Acetic acid (CAS # 64–19–7) and dichloromethane (CAS # 75–09–2) were purchased from MOLYCHEM India and Tween80 (CAS# 9005–65–6) and sodium tripolyphosphate (CAS#7758–29–4) and other chemicals used in this study were purchased from CDH, Ltd. India, all of which were stored at 4 °C.

Plant material collection

The fresh aerial parts (leaves, twigs, and flowers) of Cosmos bipinnatus were collected during its blooming stage in September 2022 from Almora, Uttarakhand, India (Latitude: 29°38′35″ N, Longitude: 79°39′42″ E, Elevation: 1956 m). The species was taxonomically identified by Dr. Dharmendra Singh Rawat (one of the authors), Department of Biological Sciences, GBPUAT, Pantnagar and the identification was confirmed by comparison with descriptions in The World Flora Online (WFO, 2025, http://www.worldfloraonline.org/taxon/wfo-0000066682, Accessed on: 31 Dec 2025), A voucher specimen (GBPUH-1601) was prepared and deposited in the herbarium of the Department of Biological Sciences, GBPUAT, Pantnagar. The collection and use of the plant material complied with all relevant local and national guidelines for plant research.

Essential oil (EO) extraction

The essential oils from the aerial parts of C. bipinnatus (CAO) were extracted through hydro-distillation (700 gm aerial parts in 3000 ml distill water) for four h using a Clevenger-type apparatus [31]. After extraction, the oil was dehydrated with anhydrous sodium sulfate (Na₂SO_4, 0.4 g) to eliminate any moisture and then stored in amber glass vials at a low temperature (4 °C in a refrigerator) for future use.

GC–MS analysis

The essential oil was analyzed using a Shimadzu GCMS-QP2010 Plus system, equipped with a thermal desorption unit, Ultra DB-5, and GCMS-QP2010 Ultra Rtx-5MS column. The analysis was conducted under the following conditions: the column oven temperature was initially set at 50.0 °C, with an injection temperature of 260.0 °C using split injection mode. The flow control was set to linear velocity, with a pressure of 69.0 kPa, a total flow rate of 28.4 mL/min, column flow of 1.21 mL/min, linear velocity of 39.9 cm/sec, and a purge flow rate of 3.0 mL/min. The split ratio was set to 20.0, with high-pressure injection, carrier gas saver, and splitter hold all disabled. The oven temperature program started at 50.0 °C for 2 min, then increased at 3.00 °C/min to 210.0 °C, held for 2 min, and finally ramped at 6.00 °C/min to 280.0 °C, holding for 11 min. The mass spectrometer had an ion source temperature of 220.0 °C and an interface temperature of 270.0 °C. The components of the essential oil were identified based on their retention time and comparison of the retention index (RI) with the C₁₀-C₄₀ n-alkane homologous series under identical conditions. The relative percentage of each component was calculated by peak area normalization (%Area) of the GC–MS total ion chromatogram. Identification was further confirmed by matching mass spectra with the NIST (version 2.1) and WILEY (7th edition) libraries, and by comparing fragmentation patterns with those found in the literature [32]. Compound identification was accepted when the mass spectral similarity index was ≥ 90% in the NIST (version 2.1) and WILEY (7th edition) libraries and the calculated retention index differed by less than ± 10 units from literature values.

Nanoencapsulation of essential oil

The oil-loaded chitosan nanoparticles (CSNPs) were prepared with slight modifications to a previously established method [29]. Initially, 0.5 g of chitosan (medium molecular weight, ≥ 75% deacetylation) was dissolved in a 1.0% (v/v) acetic acid solution, stirred continuously for 12 h at room temperature until a clear and uniform solution was achieved. The solution was then vacuum filtered through several Whatman grade 1 filter papers. Following this, 80 mL of the clear polymer solution was transferred to a conical flask, and 1.0 g of Tween-80 was added dropwise while the solution was stirred at 60 °C for 2 h, resulting in a transparent polymer solution. The organic phase was prepared by dissolving 1 mL of essential oil in 5 mL of dichloromethane [33, 34]. This organic phase was then added dropwise to the aqueous polymer solution while stirring rapidly at room temperature, forming a stable oil-in-water emulsion. The emulsion was stirred for 1.5 h to completely evaporate the solvent, resulting in a milky mixture. Once the stable emulsion was formed, 15.0 mL of a sodium tripolyphosphate (TPP, 1% w/v) solution was added dropwise under continuous stirring. The TPP acted as a crosslinking agent, inducing ionic gelation of the chitosan and leading to the formation of oil-loaded chitosan nanoparticles. The suspension was stirred for an additional 60 min to ensure uniform particle size. The nanoparticles were then collected by centrifugation at 10,000 rpm for 15 min at 4 °C. The resulting pellet was washed several times with deionized water and 1% Tween-80 to remove unreacted substances and residual acetic acid. Finally, the nanoparticles were redispersed in water and freeze-dried to obtain a stable powder. The dried nanoparticles were stored in airtight containers at − 4 °C for further characterization and use in subsequent experiments.

Characterisation of CSNPs

Determination of encapsulation efficiency (EE) and loading capacity (LC)

The encapsulation efficiency (EE%) and loading capacity (LC%) of CSNP were evaluated using a centrifugation method. The freshly prepared nanoparticles were centrifuged at 12,000 rpm for 30 min at 4 °C to separate the unencapsulated essential oil from the encapsulated portion. The supernatant, which contained the free essential oil, was collected and analyzed using a UV–vis spectrophotometer (Thermo Fisher Scientific, GENESYS 10S) at 260 nm. A pre-established calibration curve for CAO was used to quantify the amount of free essential oil. EE and LC were calculated using the following equation:

All experiments were conducted in triplicates, and the average values were reported.

Physicochemical characterisation of CSNPs

The synthesized nanoparticles were characterized using multiple analytical techniques to evaluate their physicochemical properties. These comprehensive analyses confirmed the nanoparticles’ structural, compositional, and thermal properties.

Dynamic light scattering analysis (DLS)

The hydrodynamic particle size and polydispersity index (PDI) of CSNPs were measured by dynamic light scattering (DLS) using the Nano-ZS90 Malvern Zetasizer (UK). For the analysis, CSNPs were prepared at a concentration of 200 ppm in distilled water and placed in a quartz cuvette. The measurements were conducted at room temperature with a scattering angle of 90˚ and a refractive index of 1.70.

Scanning electron microscopy and energy dispersive X-rays analysis (SEM–EDX)

The size, morphology and elemental composition of CSNPs were determined by scanning electron microscopy-EDX (JSM 6510LV model (JEOL, Japan)) at an accelerating voltage of 15 kV. For analysis, sample was prepared by stirring and sonicating the lyophilized particles in 1% Acetic acid in water for 15 min under ice bath then drying the solution onto a glass slide. Then the slide was gold coated using a sputter coater.

Fourier transforms infrared analysis (FTIR)

Fourier transform infrared (FTIR) spectroscopy was performed to identify the functional groups present in both the essential oil and CSNP, as well as to investigate potential interactions between the components. The spectra were recorded within the range of 4000 to 600 cm⁻¹ with a resolution of 5 cm⁻¹ using a Perkin Elmer FTIR spectrophotometer (model L1280127).

X-ray diffraction analysis

X-ray diffraction (XRD) analysis was performed to evaluate the crystallinity of the CSNPs. The XRD pattern was recorded using a Bruker D8 ADVANCE ECO X-ray diffractometer at 10 kV and 30 mA with a Cu anode, over a 2θ range of 0 to 40°.

Thermogravimetric analysis

The thermal stability of CSNPs was assessed using a Thermo-Gravimetric Analyzer (Pyris TGA 9, N5210032) under a nitrogen flow rate of 30 ml/min. The temperature was gradually increased from room temperature to 500 °C at a heating rate of 20 °C/min.

Controlled release of CSNPs

The in-vitro release profile of essential oil encapsulated in chitosan nanoparticles was evaluated in phosphate buffer media at pH 7.4 and pH 6, following the method described by [35]. Lyophilized CSNP (50 mg) were dispersed in 100 mL of release media, consisting of 80 mL phosphate buffer and 20 mL acetone, and incubated in a shaker incubator at 30 °C for 60 h. At regular intervals (every 2–4 h), 5 mL samples were taken from the release media and replaced with an equal volume of fresh media to maintain a constant volume throughout the experiment. The collected samples were centrifuged at 10,000 rpm for 10 min to remove any particulate matter, and the supernatant was analyzed using a UV–vis spectrophotometer (Thermo Fisher Scientific, GENESYS 10S) at 260 nm. The concentration of essential oil in the samples was determined using a pre-established calibration curve, and the cumulative release of essential oil over time was calculated. The release percentage was plotted against time to generate the release profile.

Nematicidal activity

In-vitro nematicidal activity was carried out following the method outlined by [36]. The nematode culture (Meloidogyne incognita) was maintained on infected tomato plants in a greenhouse at the Department of Plant Pathology, G.B.P.U.A.&T., Pantnagar, Uttarakhand. Roots were washed thoroughly, and the galls were macerated and passed through a series of sieves (e.g., 150 μm and 500 μm) to separate egg mashes from plant debris. The eggs were then bleached with 0.5% NaOCl for 2–3 min to remove adherent organic matter and rinsed several times with distilled water. A portion of the egg suspension was transferred onto a moisture chamber prepared with dampened cloth in Petri plates to maintain humidity and facilitate hatching. Plates were incubated at 25 ± 1 °C under ambient light and humidity. A stock suspension of hatched nematodes was prepared, containing approximately 100–110 s-stage juvenile nematodes (J2) per mL. Tween 20 was used as a surfactant to facilitate uniform dispersion of the essential oil and CSNPs in the aqueous nematode suspension (1 mL distilled water). Since essential oils are hydrophobic and CSNPs may aggregate in water, the use of Tween 20 ensured a stable and homogeneous emulsion, allowing consistent exposure of second-stage juvenile nematodes to the test formulations. Essential oil and CSNP emulsions were prepared using a 1.00% (v/v) Tween 20 surfactant solution. All concentrations were expressed as the amount of formulated product per unit volume of the test solution (μg/mL), not as active ingredient equivalents. A primary stock solution of 2000 μg/mL was made and then serially diluted to secondary concentrations of 250, 500, and 1000 μg/mL.

Mortality assay

Approximately 100 J2 nematodes per mL were placed in gridded Petri plates (50 × 12 mm). Prior to treatment, J2s were observed under a 40 × stereomicroscope and only motile, healthy nematodes were used. Essential oil and nanoparticle solutions were prepared separately at concentrations of 250, 500, and 1000 µg/mL. Each solution was adjusted to a total volume of 2.00 mL per plate for the bioassay, ensuring uniformity across all the three replicates. A 1.0% Tween 20 solution was included as a solvent control to account for any effects of the surfactant on nematode survival. Plates were incubated at 25 ± 1 °C under ambient light and humidity. Nematodes that were motionless with a straight body were further tested by gentle probing with a fine needle under 40 × magnification. Only nematodes that failed to respond to mechanical stimulation were considered dead, distinguishing true mortality from temporary paralysis. Observations were recorded at 24, 48, 72, and 96 h under 40 × magnification. Nimitz (Fluensulfone 2%) was taken as the standard nematicide at the same concentrations. Percent mortality was calculated using Abbott’s formula [37]:

LC₅₀ values for the free essential oil, encapsulated oil, and those for the standard, Nimitz was obtained from concentration-mortality data subjected to Probit analysis.

Egg hatching inhibition assay

For the egg hatching assay, 1 mL of egg suspension containing approximately 100 eggs was used per treatment. A stock egg suspension was prepared by counting the number of eggs in 1 mL aliquots across three replicates using a stereomicroscope. Based on the average egg count, the suspension was adjusted by dilution with distilled water to obtain a standardized concentration of approximately 100 eggs per mL, which was used consistently across all experimental replicates. Eggs were exposed to the same concentrations of essential oil, and CSNPs as described for the juvenile mortality assay. Plates were incubated under the same conditions (25 ± 1 °C, ambient light and humidity). Egg hatching was monitored at 24, 48, 72, and 96 h using a stereomicroscope at 40 × magnification.

The percentage of egg hatching inhibition was calculated using the following formula [37]:

Possible mechanism of action for nematicidal activity

In-vitro acetylcholinesterase inhibitory activity

The acetylcholinesterase (AChE) inhibitory activity of CAO and CSNPs was evaluated using a modified spectrophotometric assay, following methods from [38] and [39]. Meloidogyne incognita larvae were homogenized in ice-cold 50 mM phosphate buffer (pH 8.0) and centrifuged at 1000 rpm for 5 min at 4 °C. The supernatant, containing AChE, served as the enzyme source. Prior to inhibition studies, AChE activity in the crude enzyme extract was determined spectrophotometrically using acetylthiocholine iodide as substrate, and the enzyme solution was standardized to a final activity of 0.1 units/mL. Five different concentrations of CAO and CSNPs (10, 20, 30, 40, and 50 µg/mL) were prepared through serial dilutions. The reaction mixture for each assay included 20 μL of the sample solution (dissolved in phosphate buffer with up to 10% methanol), 150 μL of 0.1 M sodium phosphate buffer (pH 8.0), and 20 μL of the AChE enzyme solution (0.1 units/mL). The mixtures were incubated at 25 °C for 15 min. Following incubation, 10 μL of 10 mM DTNB (Ellman reagent) was added to each mixture. The enzymatic reaction was initiated by adding 10 μL of 1 mM acetylthiocholine iodide (ATChI), and the reaction continued for 30 min, during which ATChI was hydrolyzed into thiocholine and acetate. Thiocholine reacted with DTNB to produce a yellow-colored compound. Absorbance was measured at 405 nm using a UV–Vis Spectrophotometer. The assay controls included a blank (10% methanol in buffer), a negative control (phosphate buffer without acetylcholine), and a positive control (physostigmine salicylate). Five concentrations of CAO and CSNPs (10, 20, 30, 40, and 50 µg/mL) were prepared by serial dilution and tested in triplicate and the inhibitory activity was expressed as IC₅₀ values (µg/mL), which represent the concentration required to inhibit 50% of AChE activity. The percentage inhibition was calculated using the formula:

where Ao and At refer to the absorbance values of the control and the tested samples, respectively. The percentage inhibition was plotted against varying concentrations of both the samples and the standard. IC₅₀ values were calculated using linear regression analysis of the dose–response curves (y = mx + c), and the concentration corresponding to 50% inhibition was interpolated. The coefficient of determination (R²) was used to assess the goodness-of-fit of the regression model.

In silico molecular docking study

A molecular docking study was conducted to explore the binding interactions between the components of CAO and the acetylcholinesterase (AChE) crystal structure from Meloidogyne incognita. AChE plays a key role in regulating synaptic transmission and movement in nematodes. For the docking studies, homology modeling of AChE was carried out following the method outlined by Rajasekharan et al., (2020) [40], using the protein sequence obtained from the NCBI GenBank database [41]. The SWISS-MODEL web server was utilized to generate the protein model, which was then used for docking through PyRx software. The 3D structures of the major components of CAO (> 2%) were retrieved from the PubChem database in SDF format, with Physostigmine (CID 5983) being used as the standard inhibitor for comparison.

The ligands choosen were cis-β-ocimene (CID 5320250), germacrene D (CID 5317570), cosmene (CID 5368451), sabinene (CID 10887971), endo-arbozol (CID 55250309), β-pinene (CID 14896), and caryophyllene (CID 5281515). The ligands were then imported into PyRx using the built-in Open Babel tool, followed by energy minimization, which included charge addition and structural optimization using the universal force field. The ligands were subsequently converted into AutoDock ligand format (PDBQT). Molecular docking was carried out using the Vina Wizard tool in PyRx, which calculated the binding affinities and identified potential interactions between the ligands and the receptor. The docking process was initiated through the "Run Vina" function, and the results were exported as CSV files for further analysis. To visualize the 2D and 3D interaction patterns of the docked poses, Biovia Discovery Studio-2024 Client was utilized.

Absorption, distribution, metabolism, excretion, toxicity (ADMET) profiling

ADME analysis is vital for predicting the pharmacokinetic behavior, bioavailability, and safety of bioactive compounds, ensuring their potential as effective and environmentally sustainable alternatives. It aids in evaluating key parameters such as absorption, distribution, metabolism, excretion, and toxicity, which are essential for determining their suitability for practical applications. The chemical structures of the selected major compounds form CAO were sourced from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and converted into SMILES format for computational assessment. SwissADME (http://www.swissadme.ch/) was utilized to predict ADME properties, including gastrointestinal absorption, blood–brain barrier permeability, lipophilicity, and solubility. Toxicity predictions, including oral toxicity (LD₅₀), hepatotoxicity, mutagenicity, carcinogenicity, and immunotoxicity, were conducted using the ProTox-II webserver (http://tox.charite.de/protox_II).

Statistical analysis

All in vitro bioassays were performed in triplicate. Data were analyzed using two-factor ANOVA in OriginPro 2024 (64-bit) SR19.9.5.171 (student trial version) with significance set at p < 0.05. Assumptions of normality and homogeneity of variances were checked, and data were transformed when necessary. Post-hoc comparisons of treatment means were made using Tukey’s test. LC₅₀ values were calculated from concentration–mortality data using Probit analysis via the OPSTAT webtool (http://opstat.somee.com/Probit/probit.html), reporting 95% confidence intervals (CIs), slopes, and goodness-of-fit statistics.

Results and discussion

Chemical composition of C. bipinnatus essential oil

The aerial parts of C. bipinnatus (700 g) were hydrodistilled (in 3000 ml distilled water) using a Clevenger apparatus, yielding essential oil at 0.105 ± 0.01% (v/w). GC–MS analysis of CAO identified 46 compounds, accounting for 99.12% of the oil. The major compounds were cis–β–ocimene (35.73%), germacrene D (12.05%), cosmene (11.20%), sabinene (9.48%), endo-arbozol (5.57%), β-pinene (2.75%), and caryophyllene (2.36%). The other compounds were detected with a percentage between 0 and 1% as shown in Table 1 and Fig. 1.

Table 1.

Chemical composition of essential oil of Cosmos bipinnatus

Peak	RT (min)	%Area	RIDB	RICALC	Compound
Monoterpene hydrocarbons including: 65.25%
	6.928	0.66	939	933	α-Pinene
	7.490	0.21	954	950	Camphene
	8.390	9.48	975	969	Sabinene
	8.535	2.75	979	974	β-Pinene
	9.052	1.73	990	988	Myrcene
	10.453	0.55	1024	1024	para-Cymene
	10.641	0.40	1029	1030	Limonene
	11.004	1.76	1050	1050	trans-β-Ocimene
	11.567	35.73	1037	1037	cis-β-Ocimene
	11.917	0.42	1059	1060	γ-Terpinene
	11.864	0.36	1110	1107	1,3,8-p-menthatriene
	14.770	11.20	1130	1130	Cosmene
Oxygenated Monoterpenes including: 11.41%
	10.742	0.80	1031	1030	Eucalyptol
	13.435	0.46	1093	1093	α-Naginatene
	13.879	0.82	1096	1096	Linalool
	15.592	0.22	1135	1135	Z-Myroxide
	17.421	1.75	1177	1181	(-)-Terpinen-4-ol
	18.005	0.09	1195	1194	Myrtenal
	18.144	0.31	1349	1350	(R)-α-Terpinyl acetate
	19.563	0.22	1430	1430	α-Ionone
	45.735	5.57	1434	1433	endo-Arbozol
	46.995	1.17	1454	1454	exo-Arbozol
Sesquiterpene hydrocarbons including: 18.52%
	25.907	0.26	1376	1373	α-Copaene
	26.552	0.20	1390	1387	β-elemene
	27.728	2.36	1419	1418	Caryophyllene
	28.376	0.16	1434	1434	Trans-α-Bergamotene
	28.797	0.30	1489	1488	Cis-Eudesma-6,11-diene
	29.195	0.41	1454	1452	α-Humulene
	30.259	0.24	1455	1455	Alloaromadendrene
	30.346	12.05	1481	1481	Germacrene D
	30.588	1.08	1490	1488	β-Selinene
	31.390	0.75	1505	1503	α-Farnesene
	31.837	0.71	1523	1520	δ-cadinene
Oxygenated Sesquiterpenes including: 3.26%
	32.154	0.12	1530	1528	Kessane
	34.121	0.21	1578	1570	Spathulenol
	34.240	1.66	1583	1582	Caryophyllene oxide
	35.012	0.06	1590	1595	(-)-Globulol
	35.105	0.22	1607	1606	β-Oplopenone
	35.297	0.09	1608	1608	Humulene epoxide II
	37.129	0.47	1654	1654	α-Cadinol
	39.031	0.03	1756	1752	α-Sinensal
	39.599	0.06	1595	1590	Cubeban-11-ol
	41.436	0.06	1641	1643	Caryophylla-3,8(13)-dien-5.β-ol
	41.677	0.09	1666	1670	Ylangenol
	42.154	0.19	1803	1802	14-Hydroxy-δ-cadinene
Oxygenated diterpene including: 0.68%
	52.312	0.68	1943	1943	Phytol
Total		99.12

RT, Retention time (min); RI^CALC, calculated retention indices; RI^DB, Literature retention indices value on a DB−5 MS column

Fig. 1.

Percentage area of the identified compounds in C. bipinnatus essential oil

There are limited studies on the chemical composition of Cosmos bipinnatus essential oil. The major compound of CAO, cosmene (a polyene compound) has previously been identified in the essential oils from the flowers and leaves of C. bipinnatus [27] and in Erigeron trichophylla and Erigeron orientalis essential oils [39]. Additionally, key constituents of CAO, including E-β-ocimene, germacrene D, and sabinene, have been reported from different geographical regions, such as Cairo, Egypt, Harrismith, South Africa [22], Portugal [29] and Chittagong, Bangladesh [42]. These previous reports align well with the findings of this study. Endo-arbozol, another compound identified in significant amounts in CAO in this study, has also been previously reported in Artemisia absinthium [43], A. judaica [44], and A. herba-alba [45], all of which belong to the same family Asteraceae. Thus, the presence of endo-arbozol in CAO, along with its recurrence in various Artemisia species, underscores its potential taxonomic significance. However, a significant gap in the literature exists as no studies have yet detailed the chemical composition of C. bipinnatus essential oil from India. This research, therefore, provides the first comprehensive GC–MS analysis of C. bipinnatus essential oil sourced from India, shedding light on its chemical profile and expanding the understanding of this species’ essential oil across different regions.

Characterization of CSNPs

Encapsulation efficiency and loading capacity of CAO in CSNPs

Encapsulation efficiency (EE) and loading capacity (LC) are essential parameters for assessing the effective incorporation of core material into nanoparticles. In this study, the EE of the chitosan nanoparticles was determined to be 83%, while the LC was 28.6%, indicating successful encapsulation of the essential oil. The high encapsulation efficiency (83%) suggests that the chitosan matrix effectively retained the essential oil, minimizing losses during the preparation process. The loading capacity of 28.6% further highlights the ability of the nanoparticles to incorporate a significant amount of the oil.

Hydrodynamic particle size of nanoparticles

The hydrodynamic size of the nanoparticles was analysed using the dynamic light scattering (DLS) technique. As shown in Fig. 2A, CSNP exhibited an average diameter of 91.2 nm with a polydispersity index (PDI) of 0.379, indicating a relatively narrow size distribution. The average size of 91.2 nm falls within the optimal range for nanoparticles intended for biological applications, such as enhanced permeability and retention (EPR) effects. The PDI value of 0.379 also suggests moderate uniformity in size distribution, which is critical for ensuring consistent behaviour in applications like drug delivery and controlled release.

Fig. 2.

(A). DLS analysis of colloidal CSNPs; (B). ATR-FTIR analysis of (a) Chitosan, (b) CAO, (c) CSNPs

Chemical characterization using FTIR analysis

Fourier Transform Infrared (FTIR) spectroscopy was used to analyze the chemical properties of pure chitosan, pure CAO, and CSNPs (Fig. 2B). The FTIR spectrum of pure chitosan showed a broad peak between 3500–3200 cm⁻¹, attributed to the combined stretching vibrations of hydrogen-bonded –O–H and > N–H groups. Distinct peaks were observed at 1640 cm⁻¹ and 1560 cm⁻¹, corresponding to > C=O stretching and > N–H bending of residual N-acetyl groups, respectively. Additionally, a band at 1400 cm⁻¹ indicated C–N stretching in the –CONH₂ group, while another at 1051 cm⁻¹ was attributed to C–O stretching. These observations are consistent with previously published findings. The FTIR spectrum of pure CAO revealed sharp characteristic peaks, including one at 2928 cm⁻¹ due to sp³ C-H stretching and another strong peak at 890 cm⁻¹ linked to vinyl = C-H bending vibrations. Notably, the FTIR spectra of CSNPs included all characteristic peaks of chitosan and pure CAO, with noticeable peak shifts likely resulting from interactions between CAO and chitosan. These FTIR results confirm the successful encapsulation of CAO within the chitosan-TPP matrix.

Size, morphological and elemental analysis of lyophilized nanoparticles

The size and morphology of the lyophilized nanoparticles were analysed using a Scanning Electron Microscope (SEM), with elemental composition further assessed through EDX analysis. SEM analysis of CSNP revealed that the morphology of the nanoparticles was the spherical as well semi-spherical shape. The average particle size ranged from 200–700 nm (Fig. 3A). The size obtained from SEM was higher than that of obtained from DLS. This size change may be attributed to the fact that DLS was performed on the nanoparticle whereas the SEM was performed on the lyophilized nanoparticles. This increase in size may be due to agglomeration of nanoparticles and due to swelling of nanoparticles in presence of water during freeze drying.

Fig. 3.

(A). SEM image of colloidal CSNPs at 10,000 magnification; (B). EDX spectra of CSNPs

The elemental analysis data from EDX revealed that the major part of lyophilized nanoparticles was composed of carbon (45.77%), nitrogen (16.15%), oxygen (34.30%) with some sodium (2.62%) and Phosphorus (1.16%). The C,N,O peaks were due to chitosan and organic phytoconstituents, P peak due to crosslinker sodium tripolyphosphate and Na peak due to NaOH used for pH correction (Fig. 3B).

TGA/DTG analysis

Thermogravimetric analysis (TGA), as shown in the Fig. 4A, was conducted to evaluate the thermal stability of chitosan and CSNP, with the derivative thermogravimetry (DTG) curve as shown in Fig. 4B, providing the degradation temperatures (Td) corresponding to the highest rates of weight loss in Fig. 6. The TGA thermogram of chitosan revealed two distinct degradation steps: the first between 80 and 100 °C attributed to moisture evaporation, and the second between 250 and 350 °C, corresponding to dehydration and thermal degradation. In contrast, the TGA thermogram of chitosan nanoparticles displayed three degradation steps: the first between 50 and 70 °C, representing the evaporation of volatiles and moisture; the second between 180 and 220 °C, corresponding to the evaporation-degradation of phytochemicals from the essential oil; and the third, a major mass loss between 350 and 400 °C, indicating the degradation and dehydration of chitosan. CSNP exhibited two additional degradation peaks compared to chitosan, and the degradation step of chitosan shifted from 250–350 °C to 350–400 °C, suggesting interactions between the chitosan matrix and phytochemicals from the essential oil. The Td values for chitosan and CSNP were determined to be 260 °C and 370 °C, respectively, demonstrating the enhanced thermal stability of CSNPs. While data for the free essential oil was unavailable, its GC–MS profile indicates volatile phytochemicals likely result in significant mass loss at lower temperatures, implying lower thermal stability compared to both chitosan and CSNP. These findings underscore that the nanoencapsulation enhances the thermal stability of the essential oil, with chitosan exhibiting the highest overall stability.

Fig. 4.

(A). TGA thermogram of chitosan and CSNPs; (B). DTG thermogram of chitosan and CSNPs

Fig. 6.

Concentration- and time-dependent mortality of second-stage juveniles (J2) of Meloidogyne incognita exposed to CAO, CSNP, and Nimitz. Values represent mean ± SD (n = 3). Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05). Different superscript letters indicate significant differences among treatments within the same exposure time

Structural analysis using XRD analysis

The crystallographic structures of native chitosan and chitosan nanoparticles (CSNPs) were analyzed using X-ray diffraction (XRD) (Fig. 5A). Native chitosan exhibited two characteristic diffraction peaks at 2θ ≈ 10.2° and 20.9°, corresponding to hydrated crystalline regions and intermolecular hydrogen-bonded crystalline domains, respectively, in agreement with previous reports [44, 45]. The principal peak at ~ 21° showed a full width at half maximum (FWHM) of 2.18°, indicating the semi-crystalline nature of chitosan. The crystallinity index (CrI) of native chitosan was 52.6%.

Fig. 5.

(A). XRD spectrogram of chitosan and CSNPs; (B). In-vitro release of CAO from CSNPs at different pH

In contrast, the XRD pattern of CSNPs showed a marked decrease in peak intensity and significant peak broadening. The diffraction peak at ~ 10° almost completely disappeared, while the peak at ~ 20° became broader and less intense, with an increased FWHM of 4.71°. Correspondingly, the CrI of CSNPs decreased to 27.4%, confirming a substantial reduction in crystallinity following nanoparticle formation.

The reduction in crystallinity can be attributed to ionic crosslinking between chitosan and tripolyphosphate (TPP) during nanoparticle formation. The penetration of TPP counterions between chitosan polymer chains disrupts intermolecular hydrogen bonding and regular chain packing, leading to loss of long-range molecular order and a more amorphous structure [46]. Similar reductions in crystallinity following ionic gelation have been widely reported for chitosan-based nanoparticles.

In-vitro release studies

The in-vitro release profile of the essential oil from the CSNPs showed an initial burst release, likely caused by the rapid dissolution of free oil present on the nanoparticle surface. This was followed by a sustained release phase, attributed to the slow diffusion of encapsulated oil through the polymer matrix. The release rate was pH-dependent, with faster and higher release observed at lower pH levels. Notably, the initial burst release was more pronounced at pH 6.0, likely due to increased dissolution of the polymer matrix in acidic conditions. After 60 h, 65% of the encapsulated oil was released at pH 7.2, while 74% was released at pH 6.0. (Fig. 5B).

Nematicidal activity

Mortality of second-stage juveniles (J2) of Meloidogyne incognita

The percentage mortality of second-stage juveniles (J2) of Meloidogyne incognita is presented in Fig 6. Figure 6 demonstrates distinct variations among pure oil (CAO), encapsulated oil (CSNP), and the standard nematicide Nimitz across different concentrations (250–1000 µg/mL) and time intervals (24–96 h). At the lowest concentration (250 µg/mL), CSNP exhibited superior activity with 40.33–88.0% mortality over 24–96 h, greater than CAO (24.67–41.33%) and comparable with Nimitz (48.67–98%). At higher concentrations (500 and 1000 µg/mL), both CSNP and Nimitz achieved 100% inhibition by 48 h, while CAO with 61% and 77% inhibition, respectively, at 96 h. This trend was further supported by Probit-derived LC₅₀ values (Table 2), which decreased progressively from 24 to 96 h, indicating a time-dependent mortality effect. The LC₅₀ value for CAO at 24 h was estimated as 1227.51 µg/mL using probit regression analysis. However, since the highest concentration tested experimentally was 1000 µg/mL and 50% mortality was not achieved at this dose, the calculated LC₅₀ lies outside the tested concentration range and therefore represents an extrapolation of the dose–response relationship beyond the observed data. The LC₅₀ values of CAO, CSNP, and Nimitz over 24, 48, 72, and 96 h demonstrate significant differences, highlighting the enhanced efficacy of the encapsulated formulation. CSNP consistently exhibited lower LC₅₀ values (290.64–12.88 µg/mL) compared to the pure oil (CAO) (1227.51–455.84 µg/mL) and was comparable to the commercial standard Nimitz (259.43–0.046 µg/mL) across the tested intervals.

Table 2.

Probit analysis of nematicidal activity against second-stage juveniles (J2) of Meloidogyne incognita

Sample	Exposure time (h)	LC50 (µg/mL)	95% CI (LL–UL)	Probit parameters	Model fit
CAO	24	(> 1000)	155.33–9700.25	β = 1.069, Intercept =  − 3.30	χ2 = 0.83, p = 0.361
	48	722.01	127.72–4081.44	β = 1.25, Intercept =  − 1.25	χ2 = 0.38, p = 0.536
	72	498.69	98.98–2512.41	β = 1.33, Intercept =  − 3.60	χ2 = 0.04, p = 0.838
	96	455.84	455.84–1504.986	β = 1.84, Intercept =  − 0.489	χ2 = 0.08, p = 0.769
CSNP	24	290.64	142.49–592.83	β = 3.68, Intercept =  − 9.07	χ2 = 0.01, p = 0.922
	48	239.53	108.55–528.54	β = 1.54, Intercept =  − 0.214	χ2 = 1.54, p = 0.21
	72	133.72	40.30–443.711	β = 2.59, Intercept =  − 5.51	χ2 = 4.26, p = 0.039
	96	12.88	0.42–388.98	β = 1.06, Intercept =  − 1.18	χ2 = 0.93, p = 0.334
Nimitz	24	259.43	85.67–785.65	β = 2.12, Intercept =  − 5.12	χ2 = 0.00, p = 0.987
	48	112.54	25.78–491.167	β = 1.91, Intercept =  − 3.93	χ2 = 0.64, p = 0.421
	72	33.23	2.67–412.28	β = 1.29, Intercept =  − 1.97	χ2 = 0.02, p = 0.872
	96	0.046	0.00–337.23	β = 0.44, Intercept = 0.59	χ2 = 0.17, p = 0.675

LC₅₀ values were calculated using Probit analysis based on concentration–mortality data obtained from three tested concentrations with triplicate measurements. LL and UL represent the lower and upper limits of the 95% confidence interval. β represents the Probit slope. χ² and p-values indicate goodness-of-fit of the Probit model. LC₅₀ values exceeding the maximum tested concentration is expressed as > 1000 µg/mL. p < 0.05 indicates marginal deviation from model fit and value T.

Egg-hatching inhibition of Meloidogyne incognita

He egg hatching inhibition activity of CAO and CSNP was evaluated against Meloidogyne incognita over a 96-h period. The results indicated both concentration- and time-dependent inhibitory effects (Table 3). At the highest concentration of 1000 µg/mL, CSNP achieved complete inhibition (100%) as early as 24 h, maintaining this effect throughout the 96-h duration. In contrast, CAO at the same concentration exhibited 94.44% inhibition at 24 h, which gradually decreased to 89.04% at 96 h. At lower concentrations (250 and 500 µg/mL), CSNP demonstrated superior efficacy compared to CAO. For instance, at 250 µg/mL, CSNP exhibited a steady increase in egg hatching inhibition from 61.11% at 24 h to 85.53% at 96 h, while CAO showed a declining trend, starting at 83.33% and reducing to 76.71% over the same period. Similarly, at 500 µg/mL, CSNP achieved higher inhibition rates than CAO across all time points, emphasizing the enhanced bioactivity conferred by encapsulation.

Table 3.

Percent egg hatching inhibition (%) for CAO and CSNP against M. incognita

Samples	Concentration (µg/mL)	Percent egg hatching inhibition (%)
		24 h	48 h	72 h	96 h
CAO	250	83.33 ± 0.00a	82.86 ± 0.00bc	81.13 ± 0.47c	76.71 ± 0.47b
	500	88.89 ± 0.47a	88.57 ± 0.47abc	86.79 ± 0.47bc	84.93 ± 0.47b
	1000	94.44 ± 0.47a	91.43 ± 0.00ab	90.57 ± 0.47ab	89.04 ± 0.47ab
CSNP	250	61.11 ± 0.47b	77.42 ± 0.47c	81.63 ± 0.00c	85.53 ± 0.47c
	500	83.33 ± 0.00a	87.10 ± 0.47abc	89.80 ± 0.47abc	90.79 ± 0.47ab
	1000	100.00 ± 0.00a	96.67 ± 0.47a	95.83 ± 0.47a	94.74 ± 0.00a

Values represent mean ± SD (n = 3). Egg hatching inhibition (%) was calculated relative to the untreated control. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05). Different superscript letters within the same column indicate significant differences among treatments

Previous studies have suggested that the nematicidal activity of essential oils is closely linked to their chemical composition, with terpenoids being the main bioactive constituents [9]. For instance, major compounds of CAO under investigation herein; sabinene, caryophyllene, and myrcene, when present predominantly in the essential oil of Ocimum basilicum, have demonstrated significant nematicidal and gall-reducing effects against Meloidogyne incognita [46]. Similarly, another major compound of CAO, z-β-ocimene, abundant in the EOs of Ridolfia segetum and Trifolium incarnatum, exhibits nematicidal properties, including juvenile mortality, mobility inhibition, and egg-hatching suppression against M. incognita and M. javanica [46, 47]. Isolated z-β-ocimene from Tagetes minuta oil has also been reported to show strong nematicidal activity [48]. Moreover, another predominant compound in CAO; germacrene D, a prominent component in the EO of Seseli mairei, has demonstrated notable activity against Bursaphelenchus xylophilus [49]. However, no studies to date have specifically evaluated the nematicidal potential of cosmene, the predominant compound in CAO. Interestingly, cosmene-rich EO from Eupatorium adenophorum has shown potent antimicrobial activity against plant-pathogenic fungi and bacteria [50, 51], suggesting potential for pesticidal applications. Additionally, the minor components of C. bipinnatus EO, such as α-pinene, cymene, linalool, and eucalyptol, which have been previously reported to possess nematicidal activities [9, 52, 53], may contribute synergistically to its overall efficacy. These results emphasize the significance of both the major and minor components in influencing the nematicidal activity of the essential oil. The present study highlights the superior nematicidal activity of chitosan-encapsulated essential oil (CSNP) compared to the pure EO against Meloidogyne incognita. The enhanced efficacy of the nanoparticles can be attributed to improved stability, controlled release, and better bioavailability. While the pure EO showed promising results, its rapid volatilization and degradation under environmental conditions likely reduced its efficacy over time. In contrast, the chitosan matrix protected the active components, enabling their gradual release and prolonging their nematicidal effects. This finding aligns with previous studies that demonstrated the advantages of nanoencapsulation in pest management. For instance, Castillo et al., (2023) [54] reported that chitosan nanoparticles loaded with Ruta oil exhibited enhanced nematicidal activity due to sustained release and increased stability of the oil’s bioactive compounds. Similarly, [55] observed that encapsulating cinnamon bark extracts in polymeric nanoparticles significantly improved its efficacy against Meloidogyne incognita and Pratylenchus coffeae. These studies support our observation that encapsulation enhances the performance of essential oils by overcoming their inherent limitations such as volatility and instability. Furthermore, chitosan itself has been reported to exhibit antimicrobial, nematicidal and pesticidal properties [56, 57], which may synergize with the EO’s activity. This dual action likely contributed to the significantly lower LC₅₀ values observed for CSNP compared to the pure EO in this study. The nanoscale size of the chitosan nanoparticles likely facilitated better penetration into the nematode cuticle, enhancing the delivery of active compounds to target sites. Hence, this study pioneers the use of C. bipinnatus essential oil, formulated with chitosan nanoparticles, for nematicidal activity. Despite its widespread cultivation as an ornamental plant, its pesticidal properties remain untapped. The plant’s adaptability and rapid growth make it a practical, sustainable resource, with the nanoencapsulation presenting significant effects.

Possible mechanism of action

In-vitro acetylcholinesterase (AChE) inhibitory activity

The AChE inhibitory activity of CAO, its chitosan nanoparticle formulation (CSNP), and the standard physostigmine was evaluated at 10–50 µg/mL (Fig. 7). A concentration-dependent increase in inhibition was observed across all samples with CAO showing moderate activity (36.90%–66.73%) and CSNP exhibiting significantly enhanced inhibition (41.21%–76.77%). CSNP consistently outperformed CAO at all concentrations and approached the activity of physostigmine, which ranged from 49.63% to 81.75%. At higher concentrations (40–50 µg/mL), CSNP showed activity comparable to the standard drug. Further, the IC₅₀ values of the samples were calculated to assess their inhibitory potency. Physostigmine, as expected, exhibited the lowest IC₅₀ value (8.57 ± 0.08 µg/mL), indicating its superior inhibitory activity. CSNP demonstrated a significantly improved IC₅₀ value (16.14 ± 0.05 µg/mL) compared to CAO, highlighting the enhancement in activity achieved through nanoencapsulation. CAO showed the highest IC₅₀value (27.69 ± 0.2 µg/mL), reflecting its comparatively moderate activity in its pure form.

Fig. 7.

Concentration-dependent acetylcholinesterase (AChE) inhibitory activity of CAO, CSNP, and physostigmine. Values represent mean ± SD (n = 3). Acetylcholinesterase (AChE) inhibition (%) was determined at concentrations of 10–50 µg/mL. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05). Different superscript letters indicate significant differences among treatments and concentrations. Physostigmine was used as the positive control

Molecular docking study

To assess the interactions between ligands and the protein, and to elucidate the potential inhibition mechanism, molecular docking studies were conducted for all selected (major) compounds of CAO against the AChE enzyme of Meloidogyne incognita (Fig. 8A). Generally, a more negative binding energy (ΔGᵇⁱⁿᵈ) indicates a more stable ligand-receptor complex, reflecting higher activity and potency of the ligand. Root mean square deviation (RMSD) was used to quantify variations in the position and conformation of a ligand across different docking poses, providing an indication of the stability and reliability of ligand–receptor interactions. In this study, RMSD values were calculated for each compound based on nine docking poses, with lower RMSD values reflecting more stable and consistent binding conformations. The binding energies of the ligands were observed in the following order: Germacrene D (− 6.8 kcal/mol), Sabinene − 6.3 kcal/mol), Caryophyllene (− 6.2 kcal/mol), β-Pinene − 6.1 kcal/mol), Endo-arbozol − 5.6 kcal/mol), cis-β-Ocimene − 4.8 kcal/mol), and Cosmene (− 4.7 kcal/mol). The standard inhibitor, physostigmine, had a docking score of − 6.2 kcal/mol, which was lower than several of the tested ligands, indicating that some of these compounds exhibit superior inhibitory potential. Germacrene D (Fig. 8B) demonstrated key van der Waals interactions with residues ASN284, TRP285, PRO289, SER292, LYS288, THR287, MET346, and VAL338 of the AChE active site. Notably, a Pi-Alkyl interaction was observed with PHE434, indicating its ability to stabilize within the hydrophobic pocket. These interactions highlight the compound’s potential to modulate AChE activity by occupying its active site. Sabinene (Fig. 8C) exhibited a higher affinity for the hydrophobic pocket of AChE, forming van der Waals interactions with residues like GLY169, GLY170, TYR182, GLU251, TRP138, GLY515, HIS514, and TYR516. Significant Pi-Alkyl interactions with TRP138 and TYR390 further emphasized the compound’s anchoring within the active site. Caryophyllene (Fig. 8D) formed van der Waals interactions with residues ASN284, PRO289, LYS288, VAL338, SER341, SER292, and GLN340. Additionally, a Pi-Sigma interaction with PHE434 was observed, suggesting a strong aromatic interaction contributing to its binding affinity. The 2D interaction diagrams of the selected ligands within the binding pocket of acetylcholinesterase as illustrated in Fig. 8B–H, provide a detailed visualization of the molecular interactions. Interestingly, the major compounds of the essential oil demonstrated lower docking scores compared to other ligands, suggesting that the inhibition may not necessarily be driven by the most abundant components of the oil. The other compounds also contribute to the inhibitory effect.

Fig. 8.

Molecular docking (2D) interaction between selected ligands and enzyme (A). The docked conformations are: (B): AChE-Germacrene D, (C): AChE-Sabinene, (D): AChE-Caryophyllene, (E): AChE-β-pinene, (F): AChE-Endo-arbozol, (G): AChE-cis-β-ocimene, (H): AChE-Cosmene

AChE is a crucial detoxification enzyme in nematodes like Meloidogyne incognita, essential for maintaining proper neural function. Its inhibition disrupts acetylcholine breakdown, causing synaptic accumulation, overstimulation of post-synaptic membranes, and paralysis, ultimately leading to nematode death. This makes AChE a valuable target for assessing the toxicity of natural and synthetic nematicides. Major compounds of CAO, including germacrene D [58], sabinene [59], caryophyllene [60], and β-pinene [59], have been previously reported for their potent AChE inhibitory activity. These bioactive terpenoids are likely responsible for the enhanced activity observed in the oil, as their synergistic interactions with other minor components may also amplify the inhibitory potential. Terpenoids are known to block the AChE enzyme by binding to its active site, primarily through hydrophobic interactions and hydrogen bonding. This binding interferes with the hydrolysis of acetylcholine by preventing substrate access to the catalytic triad, leading to the accumulation of acetylcholine at synaptic junctions [10]. Encapsulation of CAO into nanoparticles resulted in a marked enhancement of AChE inhibitory activity in vitro, achieving inhibition levels closely aligned with the standard inhibitor, physostigmine and significantly outperforming the pure oil. This notable improvement can be attributed to several factors inherent to the encapsulation process. The nanoencapsulation likely enhanced the solubility and stability of the essential oil’s bioactive constituents, preventing their rapid degradation or volatilization. Moreover, the nanoparticles provided a controlled and sustained release of these active compounds, ensuring prolonged interaction with the enzyme. The nanoscale size of the particles may have facilitated more efficient penetration and binding at the enzyme’s active site, leading to improved inhibitory efficacy. The current findings are consistent with previous research on peppermint essential oil, which demonstrated significant AChE inhibitory potential and notable insecticidal activity against stored grain pest [61]. The findings of this study are in agreement with those of [54], where encapsulated Ruta essential oil exhibited comparable or enhanced nematicidal activity against Meloidogyne spp. The docking study further supported the results of the in vitro assay, revealing that the compounds exhibited strong interactions with the AChE enzyme and demonstrated favorable binding affinities. For instance, compounds such as germacrene D, which exhibited strong interactions in the docking study, have also been reported in previous studies to show significant AChE inhibition [62]. These findings highlight the compatibility of the compounds with the enzyme’s active site, reinforcing their potential as effective AChE inhibitors.

ADMET study

ADMET analysis is a crucial aspect not only in pharmacology but also in the development of effective and safe pesticides. Understanding the ADMET properties of bioactive compounds helps predict their environmental fate, persistence, bioavailability, and potential toxicity to non-target organisms. For botanical pesticides, such as essential oils, these properties determine their efficacy in pest management while minimizing ecological risks. The ADMET analysis of the major constituents of Cosmos bipinnatus essential oil revealed key pharmacokinetic and toxicological properties relevant to their nematicidal potential (Table 4). Most compounds exhibited low gastrointestinal absorption, except for endo-Arbozol, which had high absorption, suggesting their primary activity might be as contact nematicides. Several compounds, including cis-β-Ocimene, Cosmene, Sabinene, endo-Arbozol, and β-Pinene, demonstrated blood–brain barrier permeability, indicating possible neurotoxic effects on nematodes. None of the constituents were P-glycoprotein substrates, and while Germacrene D, β-Pinene, and Caryophyllene inhibited CYP2C9, only Caryophyllene inhibited CYP2C19, suggesting a limited impact on metabolic pathways. The lipophilicity (WLOGP 2.92–4.89) suggests a strong affinity for lipid-rich nematode membranes, potentially enhancing penetration and bioavailability at the target site. Additionally, the moderate skin permeability (log Kp − 4.94 to − 4.01 cm/s) indicates efficient transcuticular diffusion, supporting contact-based nematicidal action. The combination of these properties suggests that the essential oil components can effectively integrate into nematode cuticles, disrupting physiological processes. Toxicity assessments indicated no hepatotoxicity, mutagenicity, or cytotoxicity, though Cosmene showed potential carcinogenicity, and Germacrene D and Caryophyllene exhibited immunotoxicity. Predicted LD₅₀ values ranged from 113 mg/kg (cis-β-Ocimene, Class III) to 5300 mg/kg (Germacrene D, Caryophyllene, Class V), suggesting relatively low acute toxicity. Overall, these findings indicate that the major constituents of C. bipinnatus essential oil possess favorable ADMET properties, including high lipophilicity, moderate skin permeability, and low toxicity risks (Table 4).

Table 4.

ADMET analysis of major components of C. bipinnatus essential oil

Molecule	cis-β-Ocimene	Germacrene D	Cosmene	Sabinene	endo-Arbozol	β-Pinene	Caryophyllene
Molecular weight	136.23	204.35	134.22	136.23	192.3	136.23	204.35
Rotatable bonds	3	1	3	1	2	0	0
H-bond acceptors	0	0	0	0	1	0	0
H-bond donors	0	0	0	0	1	0	0
TPSA	0	0	0	0	20.23	0	0
WLOGP	3.48	4.89	3.25	3	2.92	3	4.73
Water Solubility	Soluble	Soluble	Soluble	Soluble	Soluble	Soluble	Soluble
GI absorption	Low	Low	Low	Low	High	Low	Low
BBB permeant	Yes	No	Yes	Yes	Yes	Yes	No
Pgp substrate	No	No	No	No	No	No	No
CYP1A2 inhibitor	No	No	No	No	No	No	No
CYP2C19 inhibitor	No	No	No	No	No	No	Yes
CYP2C9 inhibitor	No	Yes	No	No	No	Yes	Yes
CYP2D6 inhibitor	No	No	No	No	No	No	No
CYP3A4 inhibitor	No	No	No	No	No	No	No
log Kp (cm/s)	− 4.11	− 4.18	− 4.01	− 4.94	− 5.71	− 4.18	− 4.44
Lipinski violations	0	1	0	1	0	1	1
Bioavailability Score	0.55	0.55	0.55	0.55	0.55	0.55	0.55
Hepatotoxicity	No	No	No	No	No	No	No
Carcinogenicity	No	No	Yes	No	No	No	No
Cytotoxicity	No	No	No	No	No	No	No
Immunotoxicity	No	Yes	No	No	No	No	Yes
Mutagenicity	No	No	No	No	No	No	No
Predicted LD50 (mg/kg)	113	5300	1900	5000	4300	4700	5300
Toxicity class	III	V	IV	V	V	V	V

Conclusion

This study demonstrates that Cosmos bipinnatus essential oil exhibits nematicidal activity against Meloidogyne incognita, and that chitosan-based nanoencapsulation (CSNP) enhances its performance. The nanoencapsulation significantly improved the stability, bioavailability, and nematicidal potency of the essential oil, surpassing both the free oil and comparable to the synthetic nematicide, Nimitz. The CSNPs exhibited controlled, pH-responsive release kinetics and enhanced enzymatic inhibition of acetylcholinesterase, a key neurotoxic target enzyme in nematodes. Molecular docking corroborated these findings, revealing strong interactions between CAO constituents and the enzyme’s active site. The in silico ADMET analysis confirmed favorable pharmacokinetic and physicochemical profiles for the major compounds of the essential oil, particularly endo-arbozol and germacrene; however, these results are preliminary and need experimental validation. Overall, the findings highlight the potential of chitosan-encapsulated C. bipinnatus essential oil as a plant-based nematicidal formulation and provide a foundation for further studies, including in vivo efficacy, environmental safety, and formulation optimization.

Author contributions

H.K., S.J., and P.B. contributed to the writing – original draft, methodology, and experimental procedures. H.K. also conducted the investigation, formal analysis, and conceptualization. R.K. and M.S.O. provided supervision, with R.K. also responsible for review, project administration, and conceptualization, and M.S.O. also contributing to formal analysis alongside F.C. S.J. and P.B. contributed to validation and experimental work. S.G. was responsible for the experimental design of the encapsulation process and provided facilities. S.R. designed and provided facilities for in vitro activities. D.S.R. was responsible for identifying the plant material and providing the voucher herbarium number. All authors reviewed and approved the final manuscript.

Funding

This manuscript did not receive funding.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. All relevant data are also included within the manuscript.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.