Synthesis and bio-efficacy evaluation of castor seed oil-based nanoemulsion against diamondback moth (Plutella xylostella) in cabbage

Introduction

Cabbage (Brassica oleracea var. capitata L.) is a widely cultivated cole crop in India, valued for its nutritional and economic importance. However, its production is severely affected by the diamondback moth (Plutella xylostella L.), a highly destructive pest causing yield losses of up to 52% and significant quality degradation through foliage injury and head contamination (Paudel et al. 2022; Shamsi et al. 2026) [20,23] . Its epidemiological success is attributed to its short life cycle (14-21 days), high fecundity (150-300 eggs per female), and rapid resistance evolution, enabling persistent outbreaks across cropping seasons (Sultana et al. 2025) [28] . Seasonal dynamics in Indian agro-ecosystems reveal peak infestation during warm and moderately humid periods, particularly in early summer and post-monsoon seasons. Moreover, the larval stage is highly destructive, with feeding localized to mesophyll tissues, resulting in "windowing" symptoms that later develop into shot holes, significantly reducing photosynthetic efficiency (Talekar and Shelton 1993; Furlong et al. 2013) [30,7] . The pre-heading stage of cabbage has been identified as a critical window where DBM infestation causes maximum yield loss, necessitating timely intervention (Farias et al. 2021) [6] . Management of DBM has predominantly relied on synthetic insecticides; however, quantitative assessments indicate that while chemical control can be effective, its performance is highly variable and often unsustainable. For instance, newer insecticides such as spinosad, chlorantraniliprole, and indoxacarb have demonstrated 75.96-83.20% reduction in larval population under field conditions (Patra et al. 2015; Kumar et al. 2025) [19,13] , with spinosad alone achieving up to 89.97% suppression over control (Dotasara et al. 2017) [5] . Similarly, chlorantraniliprole has shown approximately 81% reduction in larval population, outperforming several conventional chemicals (Chowdary and Sharma 2019) [2] . Despite these high efficacy levels, repeated and indiscriminate use has led to rapid resistance development in DBM populations, making it one of the most insecticideresistant pests globally (Shehzad et al. 2023) [24] . Even biologically derived agents such as Bacillus thuringiensis (Bt), once considered highly effective, now show comparatively lower efficacy (45% reduction in some cases) due to resistance evolution (Guo et al. 2021) [9] . This underscores a critical need for next-generation, environmentally benign pest control strategies that are both effective and sustainable. In this context, plant-derived bioactive compounds have gained attention due to their biodegradability and reduced ecological footprint. Castor (Ricinus communis L.) seed oil, rich in ricinoleic acid and other bioactive constituents, has been reported to possess insecticidal, antifeedant, and oviposition-deterrent properties (Ramos-López et al. 2010; Kodjo et al. 2011; Mirzaei and Omara 2024) [21,12,16] . However, its practical application is constrained by poor dispersion, instability, and limited persistence on leaf surfaces. Recent advances in nanotechnology, particularly nanoemulsion-based delivery systems, provide a transformative approach to enhance the efficacy of botanical pesticides. Nanoemulsions, with droplet sizes typically ranging from 20-200 nm, offer enhanced surface area, improved wettability, better adhesion, and controlled release of active compounds, thereby increasing their bioavailability and insecticidal potential (Mustafa and Hussein 2020; Garcia et al. 2022) [18,8] . The reduction of droplet size to the nanometric scale significantly enhances dispersion stability, surface area, and interaction with biological membranes, facilitating improved uptake and bioefficacy (Abesekara and Chau 2022; Garcia et al. 2022) [1,8] . Therefore, the present study focuses on the development and optimization of a castor oil-based nanoemulsion and its mechanistic evaluation against Plutella xylostella. By integrating detailed physicochemical characterization (e.g., droplet size distribution, interfacial stability, and functional group interactions) with bioefficacy assessment, the study aims to elucidate the relationship between nanoformulation properties and insecticidal performance. This work establishes a chemistry-driven framework for nano-enabled botanical pesticide design, offering a sustainable, residueminimizing, and environmentally compatible alternative for effective diamondback moth management in cabbage.

Materials and Methods

Extraction of castor seed oil

Mature seeds of castor (Ricinus communis L.) were procured from a local market in Udaipur, Rajasthan, and thoroughly cleaned to remove extraneous materials prior to pulverization into a fine powder using a mechanical grinder. The powdered material was subjected to hydro-distillation using a Clevenger-type apparatus following standard protocols described by Harborne (1998) [11] . Briefly, distilled water was heated to generate steam, which permeated the plant matrix and facilitated the volatilization and co-distillation of oil constituents. The vapor mixture of steam and oil was subsequently condensed, and the distillate was collected, allowing phase separation of the oil from the aqueous layer based on density differences. The recovered oil fraction was dried over anhydrous Na 2 SO 4 to eliminate residual moisture, followed by filtration and storage in airtight containers under controlled conditions for subsequent analyses.

Nanoemulsion synthesis and characterization

The oil-in-water nanoemulsions were formulated using castor oil, a blend of surfactants and co-surfactants (Tween 80 and Span-20), along with double-distilled water using a magnetic stirrer, and further subjected to ultrasonic emulsification using a 20kHz sonicator (Ultrasonics, USA) with a power output of 750 W. Energy input was given through a sonotrode containing a piezoelectric crystal with a probe diameter of 13 mm. The major surfactant Tween 80 (Sisco Research Laboratories Pvt. Ltd., Maharashtra, India) was used along with co-surfactant span-20 (Sisco Research Laboratories Pvt. Ltd., Maharashtra, India). Other important chemical compounds like propylene glycol (PPG) were sourced from SRL, Maharashtra, India, while ethanol (99.9% pure) was purchased from Genteq Laboratories Pvt. Ltd., Delhi, India. The laboratory extracted castor seed oil was utilized as the organic phase for the nanoemulsion formulation, and laboratory-made double-distilled water (pH 6.5) was used as the aqueous phase and for the preparation of all the reagents.

Synthesis

Oil-in-water (O/W) nanoemulsions were formulated using castor oil as the dispersed phase at concentrations of 0, 2, 4, 6, 8, and 10% (v/v) (Table 1). The nanoemulsion systems were prepared through phase-wise mixing of aqueous and lipid components under controlled thermal and stirring conditions. The aqueous phase (Mass I) consisted of Tween 80, distilled water, and propylene glycol, which were heated to 50 °C under continuous magnetic stirring until a clear and thermodynamically uniform phase was obtained. Simultaneously, the oil phase (Mass II) was prepared by dissolving Span-20 in ethanol, followed by the gradual dropwise incorporation of castor oil under constant agitation to achieve a transparent isotropic mixture. Subsequently, the oil phase was slowly titrated into the aqueous phase under continuous stirring to facilitate spontaneous interfacial emulsification and nanoscale droplet formation. The resulting formulations were further subjected to magnetic stirring at 1000 rpm for 72 h to ensure complete homogenization, reduction of interfacial tension, and stabilization of the dispersed nanodroplets. The prepared nanoemulsions (designated as CSNE) were subsequently characterized for their physicochemical attributes and monitored for colloidal stability over a storage period of one month.

Characterization

The developed nanoemulsions (CSNE) were characterized for their physicochemical properties to assess stability and functionality. The particle size distribution, polydispersity index (PDI), and zeta potential were determined by dynamic light scattering (DLS) using Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) at 25 °C after appropriate dilution with deionized water, where PDI indicated size distribution uniformity and zeta potential reflected colloidal stability (Mukherjee and Baruah 2025) [17] . The viscosity was measured using a rotational viscometer at controlled temperature to evaluate flow behavior and its influence on formulation stability (Tadros et al. 2004) [29] . The pH of the formulations was recorded using a calibrated digital pH meter to ensure stability and compatibility (de Oca-Ávalos et al. 2017) [3] .

Experimental details

An in vitro study was conducted to evaluate the larvicidal activities of the laboratory-formulated CSNEs against DBM. The experiment was carried out in a complete randomized design (CRD) with eight treatments and three replications. To estimate the relative efficacy index (REI) of CSNEs, Novaluron 10% EC (Conventional insecticide, C.I) was employed as the standard reference due to its established efficacy against DBM. All the experimental details have been summarized in Table 2. [13] .

Relative Efficacy (%) = ( Mortality in CSNE Mortality in C.I.

Statistical analysis

All experimental data were subjected to statistical analysis using one-way analysis of variance (ANOVA) performed in IBM SPSS Statistics. Treatment means were compared using Tukey's honestly significant difference (HSD) test at a significance level of p<0.05. Percentage data were arcsinetransformed prior to analysis to stabilize variance and satisfy assumptions of normality.

Results and Discussion

Synthesis and characterization of castor seed oil based nanoemulsion

The O/W nanoemulsion system demonstrated efficient nanoscale emulsification and colloidal stabilization through synergistic interactions between castor oil, Tween 80, Span-20, ethanol, and propylene glycol. Controlled thermal mixing (50 °C) and gradual phase titration promoted spontaneous interfacial emulsification by reducing interfacial free energy and facilitating uniform nanodroplet formation (Liu et al. 2024) [14] . The combined non-ionic surfactant system provided an optimized hydrophiliclipophilic balance (HLB), enhancing interfacial film stability and preventing droplet coalescence through steric stabilization (Miranda-Olvera et al. 2025) [15] . Ethanol and propylene glycol further improved phase miscibility and reduced interfacial tension, contributing to transparent isotropic nano-dispersion. Extended magnetic stirring at 1000 rpm for 72 h enhanced homogenization, minimized phase separation, and promoted stable nanoscale droplet distribution. Increasing castor seed oil concentration likely influenced dispersed phase viscosity and droplet packing behavior; however, the optimized surfactant-co-surfactant matrix maintained nanoemulsion stability and physicochemical uniformity. The synthesized nanoemulsions exhibited favorable physicochemical characteristics immediately after formulation, with pH values ranging from 6.01-6.70 and viscosity below 100 cP (Table 3), indicating low-resistance flow behavior and compatibility for application on cabbage leaf surfaces during leaf dipping. Dynamic light scattering (DLS) analysis revealed concentration-dependent variations in hydrodynamic diameter, polydispersity index (PDI), and zeta potential (Table 3), reflecting the influence of dispersed oil phase loading on interfacial organization and colloidal stability (Solans and Solé 2012) [26] . Among the formulations, 6, 8, and 10% CSNE remained optically transparent and free from phase separation, confirming successful nanoscale dispersion and kinetic stability. Notably, the 6% CSNE exhibited the most uniform nanodroplet distribution with a low PDI (0.248) (Figure 1) and high negative surface charge (-38.4 mV), suggesting enhanced electrostatic stabilization, reduced droplet aggregation, and superior colloidal homogeneity within the nano-range. Larval mortality progressively increased with both concentration and exposure duration across all CSNE formulations, demonstrating a pronounced time-dependent toxicological response (Table 4). Among the tested nanoemulsions, 6% CSNE emerged as the most potent formulation, particularly at 1500 ppm, where mortality escalated from 3.3% at 2 HAT (hours after treatment) to 76.7% at 48 HAT, substantially outperforming the commercial standard Novaluron 10% EC (46.7%). The enhanced efficacy of 6% CSNE may be attributed to the optimized surfactant-oil balance, which likely improved nano-droplet stability, dispersion uniformity, and penetration of bioactive ricinoleic acid-rich constituents through the larval cuticle (Shekade et al. 2023) [25] . In contrast, the 0% CSNE formulation exhibited comparatively poor larvicidal activity, indicating that the castor seed oil phase played a critical role in toxicity enhancement. Interestingly, increasing oil loading beyond 6% (8-10% CSNE) did not proportionally improve mortality, suggesting possible droplet aggregation, interfacial saturation, or reduced physicochemical stability at higher oil concentrations, ultimately limiting bioavailability. The delayed but steep rise in mortality between 24 and 48 HAT further indicates a sustained-release or cumulative mode of action of the nanoemulsion system rather than immediate knockdown toxicity (Donsì et al. 2012; Sugumar et al.

2014) [4,27] . Collectively, these findings highlight that precise optimization of nanoemulsion composition critically governs larvicidal efficiency, with 6% CSNE representing the most physiochemically balanced and biologically effective formulation for sustained insecticidal activity. The Relative Efficacy Index (REI) data further revealed that larvicidal performance was governed by nanoemulsion optimization rather than concentration alone, with 6% CSNE consistently exhibiting the highest efficacy at both 24 and 48 HAT, particularly at 1500 ppm (Figure 2). The superior REI of 6% CSNE suggests an optimum oilsurfactant balance that enhanced nano-droplet stability, interfacial interaction, and trans-cuticular delivery of ricinoleic acid-rich bioactive compounds (Gutiérrez et al.

2008) [10] . In contrast, lower efficacy in 0-2% CSNE indicated inadequate oil-mediated bioactive delivery, while the absence of proportional REI enhancement in 8-10% CSNE suggests possible interfacial saturation, droplet aggregation, or reduced colloidal stability at higher oil loading. The sustained superiority of 6% CSNE over time further indicates improved residual activity and controlled release behavior, highlighting it as the most physiochemically stable and biologically efficient formulation.

Conclusion

The study successfully developed a stable castor seed oil nanoemulsion with significant larvicidal activity against Diamondback moth. Among the formulations, 6% CSNE exhibited optimal physicochemical properties and superior bioefficacy, achieving higher larval mortality than the conventional insecticide standard. Improved performance was associated with enhanced colloidal stability, nanoscale droplet distribution, and efficient delivery of ricinoleic acidrich bioactive compounds. These findings demonstrate the potential of castor oil nanoemulsions as a sustainable, residue-minimizing, and environmentally compatible strategy for DBM management in cabbage cultivation.

References

1. Abesekara MS, Chau Y. Recent advances in surface modification of micro-and nano-scale biomaterials with biological membranes and biomolecules. Front Bioeng Biotechnol. 2022 Oct 12;10:972790.
2. Chowdary NM, Sharma PC. Bioefficacy of newer insecticides against Plutella xylostella (L.) infesting cabbage. Himachal J Agric Res. 2019;46:46-50.
3. de Oca-Ávalos JM, Candal RJ, Herrera ML. Nanoemulsions: Stability and physical properties. Curr Opin Food Sci. 2017 Aug 1;16:1-6.
4. Donsì F, Annunziata M, Vincensi M, Ferrari G. Design of nanoemulsion-based delivery systems of natural antimicrobials: Effect of the emulsifier. J Biotechnol. 2012 Jun 30;159(4):342-350.
5. Dotasara SK, Agrawal N, Swami D. Efficacy of some new insecticides against diamond back moth (Plutella xylostella L.) on cauliflower. Indian Soc Agric Sci. 2017:227.
6. Farias ED, Sant'ana LC, Melo JB, Santana PA Jr, Picanço MC. Impact of diamondback moth density and infestation timing on broccoli yield. Neotrop Entomol. 2021 Apr;50(2):298-302.
7. Furlong MJ, Wright DJ, Dosdall LM. Diamondback moth ecology and management: Problems, progress, and prospects. Annu Rev Entomol. 2013 Jan 7;58:517541.
8. Garcia CR, Malik MH, Biswas S, Tam VH, Rumbaugh KP, Li W, et al. Nanoemulsion delivery systems for enhanced efficacy of antimicrobials and essential oils. Biomater Sci. 2022;10(3):633-653.
9. Guo Z, Kang S, Wu Q, Wang S, Crickmore N, Zhou X, et al. The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host. PLoS Pathog. 2021 Sep 8;17(9):e1009917.
10. Gutiérrez JM, González C, Maestro A, Solè IM, Pey CM, Nolla J. Nano-emulsions: New applications and optimization of their preparation. Curr Opin Colloid Interface Sci. 2008 Aug 1;13(4):245-251.
11. Harborne AJ. Phytochemical methods: A guide to modern techniques of plant analysis. Berlin: Springer Science & Business Media; 1998. pp. 1-302.
12. Kodjo TA, Gbénonchi M, Sadate A, Komi A, Yaovi G, Dieudonné M, et al. Bio-insecticidal effects of plant extracts and oil emulsions of Ricinus communis L. (Malpighiales: Euphorbiaceae) on the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) under laboratory and semi-field conditions. J Appl Biosci. 2011 Jan;43:2899-2914.
13. Kumar A, Choudhary JK, Singh TK, Singh R, Shukla M, Singh S, et al. Efficacy of novel insecticides and bio-pesticides for diamond back moth (Plutella xylostella) management in cabbage. Veg Sci. 2025 Jun 20;52(1):191-195.
14. Liu J, Li Y, Lun Z, Zhang Y, Yang P, Tang X, et al. Factors, mechanisms, and kinetics of spontaneous emulsification for heavy oil-in-water emulsions. Molecules. 2024 Jun 24;29(13):2998.
15. Miranda-Olvera AD, Domínguez-Esquivel JM, Martinez J. Hydrophilic-lipophilic balance (HLB) correlation method for the selection of ionic liquid surfactant modifiers of the viscosity and emulsion stability of heavy oils. Langmuir. 2025 Mar 28;41(13):8753-8765.
16. Mirzaei S, Omara Z. Plant-derived insecticides for pest management. J Ethno-Pharm Prod. 2024 Dec 1;4(1):4058.
17. Mukherjee P, Baruah KN. Characterization of nanoemulsion: Particle size, polydispersity index, zeta potential, thermal stability, and kinetic stability. In: Essential Oil Extraction from Food By-Products. New York: Springer US; 2025. p. 273-288.
18. Mustafa IF, Hussein MZ. Synthesis and technology of nanoemulsion-based pesticide formulation. Nanomaterials. 2020 Aug 17;10(8):1608.
19. Patra B, Das BC, Alam SF, Dhote V, Patra S, Chatterjee ML, et al. Evaluation of new insecticides against diamond back moth, Plutella xylostella (L.) on red cabbage. Int J Bio-Resour Stress Manag. 2015 Apr 1;6(2):1-5.
20. Paudel A, Yadav PK, Karna P. Diamondback moth Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae): A real menace to crucifers and its integrated management tactics. Turk J Agric Food Sci Technol. 2022 Dec 20;10(12):2504-2515.
21. Ramos-López MA, Pérez S, Rodríguez-Hernández GC, Guevara-Fefer P, Zavala-Sanchez MA. Activity of Ricinus communis (Euphorbiaceae) against Spodoptera frugiperda (Lepidoptera: Noctuidae). Afr J Biotechnol. 2010;9(9):1359-1365.
22. Shakeel F, Ramadan W, Ahmed MA. Investigation of true nanoemulsions for transdermal potential of indomethacin: Characterization, rheological characteristics, and ex vivo skin permeation studies. J Drug Target. 2009 Jul 1;17(6):435-441.
23. Shamsi IH, Fu W, Askri SM, Waris S, Ibrahim Z, Ali A, et al. Interactive mechanisms and management challenges of Plutella xylostella (diamondback moth) in Brassica. Insect-Plant Interact. 2026 Jun 9:143-170.
24. Shehzad M, Bodlah I, Siddiqui JA, Bodlah MA, Fareen AG, Islam W. Recent insights into pesticide resistance mechanisms in Plutella xylostella and possible management strategies. Environ Sci Pollut Res. 2023 Sep;30(42):95296-95311.
25. Shekade SV, Shirolkar SV, Deshkar SS, Giram PS. Phytochemical, pharmacognostic, and pharmacological aspects of Ricinus communis seed oil: An overview. Nat Prod J. 2023 May 1;13(3):31-47.
26. Solans C, Solé I. Nano-emulsions: Formation by lowenergy methods. Curr Opin Colloid Interface Sci. 2012 Oct 1;17(5):246-254.
27. Sugumar S, Clarke SK, Nirmala MJ, Tyagi BK, Mukherjee A, Chandrasekaran N. Nanoemulsion of eucalyptus oil and its larvicidal activity against Culex quinquefasciatus. Bull Entomol Res. 2014 Jun;104(3):393-402.
28. Sultana R, Ansari MS. Effects of constant temperature regimes on the life table parameters of Plutella xylostella (Lepidoptera: Plutellidae) (Linnaeus). J Exp Agric Int. 2025 Jun 30;47(6):764-775.
29. Tadros T, Izquierdo P, Esquena J, Solans C. Formation and stability of nano-emulsions. Adv Colloid Interface Sci. 2004 May 20;108:303-318.
30. Talekar NS, Shelton AM. Biology, ecology, and management of the diamondback moth. Annu Rev Entomol. 1993 Jan 1;38(1):275-301.