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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

In-vitro Assessment and Characterization of Pongamia Seed Oil-based Nano-Emulsion and ZnO Nano-formulations as Environmentally Sustainable Alternatives for the Control of Aulocophora foveicollis Lucas (Coleoptera-Chrysomelidae)

R
R. Jeethasri1
V
V. Sherene Victoria1
D
D. Surya1
A
Ananthi Rachel Livingstone1,*
0000-0002-7603-1220
1Department of Zoology, Madras Christian College (Autonomous), Tambaram-600 059, Tamil Nadu, India.

ABSTRACT

Background: Nano-emulsion formulation and ZnO-loaded nano-emulsion system was developed using the seed oil of Pongamia pinnata (L) to control the serious pest of cucurbit crops Aulocophora foveicollis Lucas.

Methods: The formulations were prepared using high energy emulsification method (utrasonication) and were further characterized to determine the droplet size, polydispersity index, zeta potential using dynamic light scattering (DLS). Further, thermodynamic stress studies, viscosity, pH, UV- Visible spectroscopic analysis, TEM, ATR-FTIR analysis were carried out to evaluate the stability of nano-emulsion formulations. The efficacy of the prepared formulations is tested against the target pest (red pumpkin beetle) in vitro.

Result: The results of characterization confirm the stability of nano-emulsion systems. The insecticidal efficacy of ZnO-loaded nano-emulsion system showed highest mortality (73.31%) and LC50 value was found to be 11.24% v/v. These results suggests that the formulated nano-emulsion system from the seed oil of pongamia holds potential to reduce the reliance on synthetic pesticides and offer environmentally sustainable alternative for the control of the insect pest.

INTRODUCTION

Cucurbits of Family-cucurbitaceae comprise economically important crops rich in dietary fibre, vitamins and minerals that supports metabolism and growth (Lal et al., 2014; Kaur et al., 2020 and Mondal et al., 2020). However, these crops are highly susceptible to insect pests, among which Aulocophora foveicollis Lucas, the most destructive pest affecting leaves, cotyledons causing stunted growth and death of the plant with infestation ranging from 30% to 100% (Sahu and Samal 2020; Dhillon et al., 2005). Despite the fact that pesticides reduce yield loss, their comprehensive usage pose serious threat to environment (Aktar et al., 2009). In this context, plant-based formulations and extracts exhibit potent eco-friendly bioactivity for effective pest management, contributing to sustainable and integrated agricultural practices (Shinde et al., 2025; Kikraliya et al., 2024; Iqbal et al., 2024). Pongamia pinnata (L) (Family- Fabaceae), commonly known as karanj (Kumar et al., 2006), produces seed oil that contains karanjin, pongamol, glabrin and has proven to exhibit insecticidal properties Kumar et al., 2006; Gadge et al., 2022). Although there is still lack of detailed study on its nano-emulsion and ZnO-loaded nano-emulsion formulations, especially regarding their stability and potential against insect pest, red pumpkin beetle. Nano-emulsions formulated from such seed oils and integration of nanoparticles provide greater efficacy against insect pests as they provide stability, controlled release, penetration for effective pest (Ghormade et al., 2011; Parisi et al., 2015). Therefore, this study aims.
► To analyse the chemical composition of pongamia seed oil using GC-MS analysis.
►  To formulate and evaluate the insecticidal efficacy of bulk emulsion of pongamia oil against the target pest A. foveicollis Lucas in laboratory conditions.
►  To formulate, characterise and assess the insecticidal potential of oil-in-water nano-emulsion and ZnO-loaded nanoemulsion of pongamia oil on the target pest in laboratory conditions.

MATERIALS AND METHODS

Sample collection and chemicals used
 
Seed oil of Pongamia pinnata (L) was commercially purchased for the formulation of nano-emulsion. Analytical grade chemicals like absolute ethanol (99.9%) (Changshu Hongsheng Fine Chemicals Co.Ltd.), Tween 80 (Spectrochem Pvt. Ltd., India), Sodium Lauryl Sulphate (SLS/SDS- 90% extrapure) (SRL Pvt. Ltd., Mumbai, India), Zinc oxide nanoparticles (99% purity) (ADZnO Nanopowder, ADnano Technologies, Karnataka, India), Polyethylene glycol (PEG 6000, Bangalore Fine Chemicals, Bangalore, India) and double distilled water were used throughout the study. The study was carried out in the period of December 2024 to May 2025 in the Department of Zoology, Madras Christian College.
 
GCMS analysis
 
The oil sample was analysed using Gas Chromatography- Mass Spectrometry (GCMS) in electron ionization (EI) mode on a capillary GC column using helium as carrier gas. 1.0 µL of sample was injected with initial temperature at 50oC (2 mins), ramping at 10oC/minute to 300oC. The total run time was 30 minutes with ion source and interference temperature at 230oC and 280oC, respectively and mass spectra ranged between 40 and 600 m/z.
 
Preparation of bulk emulsion
 
The oil-in-water bulk emulsion was prepared using pongamia seed oil (10% v/v), distilled water (68% v/v), tween 80 (15% v/v) (non-ionic surfactant), ethanol (7% v/v) (co-surfactant). The components were mixed thoroughly using the magnetic stirrer at 500 rpm for 20 minutes.
 
Preparation of nano-emulsion
 
The oil-in-water nano-emulsion of pongamia oil was prepared by ultrasonication method. Sodium dodecyl sulphate (SDS) at 1% w/v was dissolved in water and magnetic stirred for 15 mins at 500 rpm. Tween 80 was added dropwise while stirring followed by ethanol and oil as shown in Table 2. The resulting pre-emulsion was ultrasonicated for 30 mins (33kHz, 650watts, 60% amplitude) and the formulation was subjected to thermodynamic stability tests (Fig 1a).

Fig 1 (a), (b): Schematic representation of the development and characterization of Nano-emulsion system of pongamia oil.



Thermodynamic stability test
 
To evaluate the stability of the oil-in-water nano-emulsion formulations, different stress tests described by Shafiq et al., (2007) were performed. All the tests were triplicated.
 
a. Centrifugation test: The oil-in-water nano-emulsion formulations were subjected to centrifugation at 3000 rpm for 15 minutes.
 
b. Heating-cooling cycle: The formulation was kept at 4oC and 45oC for 48 hours each. Then the cycle was repeated thrice and checked for phase separation.
 
c. Freeze- thaw stress: This was performed by subjecting the formulation at -21oC and +25oC for 48 hours each.
 
Formulation with no phase separation observed was selected and further characterized.
 
Preparation of ZnO-loaded nano-emulsion
 
ZnO-loaded nano-emulsion was prepared by using 1% w/v SDS and 1% w/v PEG dissolved in distilled water. ZnO nanoparticles (0.25% w/v) were added and the pH is adjusted to 9.0 and the mixture is ultrasonicated for 30 mins. Separately, a pre-emulsion is prepared by using tween 80 (8% v/v), ethanol (3% v/v), pongamia seed oil (4% v/v) and added to ZnO mixture and then subjected to ultrasonication (33kHz, 650watts, 60% amplitude) for 30 mins. The resultant nano-emulsion were further characterized (Fig 1b).
 
Characterization of Nano-emulsion and ZnO-loaded Nano-emulsion system of Pongamia seed oil
 
pH Measurement
 
The pH of the nano-emulsion and ZnO-loaded Nano-emulsion was measured using a digital pH meter.
 
Viscosity
 
The viscosity of both oil-in-water nano-emulsion and ZnO-loaded nano-emulsion was measured using Brookfield viscometer with spindle number 18 at rotation speed of 5.0 and a torque setting maintained around 28% to ensure accurate measurement.
 
Particle size, zeta potential and polydispersity index (PDI)
 
The droplet size of the nano-emulsion, zeta potential and polydispersity index (PDI) were measured using Dynamic Light Scattering (DLS), 200 µL of sample was introduced into a clean PTFE sample cell at room temperature using the instrument’s 180o backscatter configuration. Each measurement was performed in triplicates.
 
UV- visible spectroscopic analysis
 
For UV visible spectroscopic analysis, the samples were diluted 100-fold with distilled water at room temperature and measured using quartz cuvettes of 1 cm path length. The absorption spectra were recorded across a wavelength range of 200-800 nm. Distilled water was used a blank.
 
Transmission electron microscopic (TEM) analysis
 
The samples were diluted 1:100 with distilled water. A small aliquot (10 µL) of sample was placed on TEM grid and stained using 2% uranyl acetate followed by air drying at room temperature. Then the samples were observed under TEM.
 
Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) analysis
 
The chemical structure and molecular interactions of ZnO-loaded nano-emulsion system of pongamia oil was examined using FTIR-ATR, equipped with Sp10 software for data processing. A small volume of sample was placed onto the ATR crystal and the spectra were recorded over a range of 4000 cm-1 to 400 cm-1 at room temperature.
 
Insecticidal assessment of bulk emulsion, Nano-emulsion and ZnO-loaded Nano-emulsion system and statistical analysis
 
Adult beetles were reared in sterile plastic containers and the efficacy of bulk emulsion, nano-emulsion and ZnO-loaded nano-emulsion was evaluated by treating the target pests with each nano-emulsion system individually in following concentrations, 5%, 10%, 15%, 20% v/v for each treatment 10 insects were treated and the experiment is triplicated. Distilled water treated leaves were given as feed for control groups. Mortality was recorded for every 24 hours and the experiment was carried out for 120 hours. Results were statistically analysed using SPSS and MS OFFICE EXCEL. LC50 values were derived using Probit analysis. Significant differences among the treatment groups were analysed using one-way ANOVA and Tukey’s Post hoc HSD test.

RESULTS AND DISCUSSION

GCMS analysis
 
The GCMS profile of pongamia seed oil sample identifies the presence of a diverse array of compounds namely fatty acids and their esters, aliphatic hydrocarbons, aromatic compounds and derivatives (Fig 2). The chemical compounds in Table 1 exhibit almost a similar trend to the findings of Abdul and Singh (2024); Purkait et al., (2021) and Seenuvasan et al., (2013). The compounds hexadecenoic acid, methyl ester (area- 2.09%, 6 octadecenoic acid, methyl ester (area- 8.06%), methyl stearate (1.27%), octadecanoic acid (3.73%) are found to have insecticidal and larvicidal properties according to Farag et al. (2021); Zayed et al. (2016); Ismail et al. (2022); Kannathasan et al. (2008) and Okonkwo et al. (2017).

Table 1: Chemical constituents of pongamia oil (Pongamia pinnata L) identified by GCMS analysis.



Fig 2: Chromatographic profile of pongamia oil (Pongamia pinnata) obtained by GCMS analysis.


 
Thermodynamic stability test of prepared Nano-emulsion formulations
 
The oil-in-water nano-emulsion pongamia seed oil passed thermodynamic stability stress conditions (Table 2). No phase separation, creaming was seen in the formulation (F4) of the nano-emulsion. Therefore, the formulation (F4) was selected for characterization.

Table 2: Composition of Nano-emulsions formulation of pongamia oil.


 
Characterization of Nano-emulsion and ZnO-loaded Nano-emulsion system of pongamia seed oil
 
Particle size, zeta potential and polydispersity index (PDI)
 
The particle size, zeta potential and polydispersity index (PDI) (Table 3) are one of the key factors for the stability of nano-emulsion (Anjali et al., 2012). The droplet size of nano-emulsion is between 20-500 nm (Pagar et al., 2019; Zhang et al., 2014). The average droplet size of nano-emulsion and ZnO-loaded nano-emulsion system of pongamia seed oil was found to be 216 nm and 210 nm respectively, contributing to the stability of the nano-emulsion system. Poly dispersity index (PDI) is a measure of homogeneity of droplet size in nano-emulsion. The PDI value ranging from 0-0.2 generally indicates strong narrow range size distribution of droplets in nano-emulsion system according to Baboota et al. (2007) and Sampathi et al. (2015). The PDI values resulted from the current study indicated narrow size distribution of droplets. The measure of electrical charge (zeta potential) on the surface of the droplets of nano-emulsion system influences its stability. The zeta potential value above ±30 mV is considered to be stable (Khalid et al., 2023). In this case the average zeta potential values -32.07 mV and -72.40 mV of samples indicated that the nano-emulsions were stable.

Table 3: pH, viscosity, DLS analysis of Nano-emulsion and ZnO-loaded Nano-emulsion system of pongamia seed oil.


 
UV- visible spectroscopic analysis
 
The UV-Visible spectroscopic analysis of oil-in-water nano-emulsion of pongamia seed oil exhibited a characteristic absorption peak at 264 nm (Fig 3). This suggests a uniform and homogenous nano-emulsion system consistent with the reports of Ullah et al., (2022). The UV-visible absorption spectrum of ZnO-loaded nano-emulsion showed two distinct peaks at 289 nm and 370 nm (Fig  4). These findings confirm the successful incorporation and stable dispersion of ZnO nanoparticles in the emulsion system. This is in alignment with the previous study of Shamhari et al., (2018) and Enkhtuya et al., (2016).

Fig 3: UV-visible absorption spectrum of oil-in-water Nano-emulsion of pongamia oil.



Fig 4: UV-visible absorption spectrum of ZnO-ioaded Nano-emulsion system of pongamia oil.


 
Transmission electron microscopic (TEM) analysis
 
TEM analysis revealed spherical, well dispersed droplets in consistent with the results of DLS for both oil-in-water nano-emulsion and ZnO-loaded nano-emulsion system of pongamia seed oil (Fig 5 and Fig 6). The increased contrast and lack of free zinc oxide nanoparticles outside the droplets as observed in Fig 6 are indicative of the effective entrapment of zinc oxide nanoparticles within the droplets of nano-emulsion system.

Fig 5: TEM image of Oil-in-water Nano-emulsion droplets of pongamia oil at 100 nm magnification (Scale bar:100 nm).



Fig 6: TEM image of ZnO-ioaded Nano-emulsion droplets of pongamia oil (Scale bar: 1 µm).


 
Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) analysis
 
The FTIR spectrum (Fig 7) of ZnO-loaded nano-emulsion system of pongamia seed oil shows a characteristic peak indicating the presence of ZnO nanoparticles and other components of nano-emulsion system. Around 3400 cm-1, the broad O-H stretching band shows the presence of surface hydroxyl group and water in aqueous phase. A peak near 1630-1650 cm-1 represents C=O stretching in ester or carbonyl groups, indicating the organic constituents of nano-emulsions. The absorption band in 470-500 cm-1 corresponds to Zn-O stretching vibrations confirming the presence of ZnO nanoparticles. The strong Zn-O stretching vibration observed below 600 cm-1 confirms the presence of ZnO nanoparticles (Malaikozhundan et al., 2017). In another study by El-Saadony et al., (2024), the FTIR analysis confirmed the bond formation with absorption bands between 409- 588 cm-1. Subsequently, the presence of bands around 2850- 2950 cm-1 and 1740 cm-1 corresponds to C-H and C=O stretching respectively, associated with oil phase of nano-emulsion system (Rajiv et al., 2013; Ramesh et al., 2021).

Fig 7: ATR-FTIR spectrum of ZnO-ioaded nanoemulsion system of pongamia oil.


 
Insecticidal assessment of bulk emulsion, Nano-emulsion and ZnO-loaded Nano-emulsion system of pongamia oil
 
The bulk emulsion showed minimal mortality of 26.65% at 20% v/v and 16.65% at 15% v/v, while lower concentration showed least efficacy. One-way ANOVA exhibits significant differences among treatments (F(3,8) = 6.250; p= 0.017 < 0.05) and Tukey’s HSD revealed 20% v/v as significantly distinct (Table 4, Fig 8). The efficacy of oil-in-water nano-emulsion at 20% v/v was found to be 43.32% and demonstrated a dose dependent effect (F (3,8) = 8.889, p=0.006 < 0.05), recording LC50 at 33.45% v/v. ZnO-loaded nano-emulsion exhibited highest mortality and outperformed both bulk and oil-in-water nano-emulsion with 73.31% mortality rate at 20% v/v and lowest LC50 value (11.24% v/v). One-way ANOVA revealed that there is significant difference among treatment groups (F (3,8) = 7.608, p= 0.010< 0.05), also reflecting dose-dependent mortality effect (Table 4, Fig 8).

Table 4: Insecticidal bioassay of pongamia oil emulsions against Aulocophora foveicollis Lucas, serious pest of cucurbits.



Fig 8: Comparative mortality rates (%) of Aulocophora foveicollis Lucas treated with Bulk emulsion, Nano-emulsion and ZnO-loaded Nano-emulsion of pongamia oil.


       
A study by Stepanycheva et al. (2020) revealed that pongamia oil exhibited 100% mortality at 3% concentration on Frankliniella occidentalis. Similar study by Uçak et al. (2014) and Tran et al., (2017) demonstrated that karanj oil and leaf extract caused 100% mortality in F. occidentalis and Spodoptera litura respectively. Pongamia oil in combination with neem oil (PONNEEM) caused upto 90.78% feeding deterrence and DNA damage in Helicoverpa armigera at 2 ppm concentration Packiam et al. (2015). Malaikozhundan et al., (2017) demonstrated ZnO nanoparticles coated with Bacillus thuringiensis caused 100% mortality in Callosobruchus maculatus at 25 µg/mL concentration. Jameel et al. (2020) reported that ZnO nanoparticles induced oxidative stress in Spodoptera litura by generating reactive oxygen species and inhibiting key antioxidant enzymes. Similar study by Pittarate et al. (2021) demonstrated that ZnO nanoparticles caused oxidative stress in S. frugiperda, which led to body deformities, reduced fecundity and impaired life cycle progression. These studies consistently reveal that pongamia oil and its nano-emulsions particularly enhance by loading ZnO nanoparticles show potent insecticidal and larvicidal effect on target pests.
 
Practical implications and future prospects
 
Understanding the physiological and molecular mechanisms of insecticidal action of pongamia oil-based nano-emulsion formulations will provide essential insights. This will aid in developing more effective and targeted bio-pesticides, reducing the dependence on synthetic chemicals. These advances promote sustainable pest management by enabling eco-friendly and field applicable botanical formulations with minimal environmental impact.

CONCLUSION

The nano-emulsion system of pongamia oil formulated and characterized in this study provided excellent stability and greater efficacy against red pumpkin beetle, the destructive pest of cucurbits. This in-vitro study strongly supports the use of pongamia seed oil-based nano-emulsion formulation as an alternative approach to control the target pest reducing the reliance on synthetic pesticide.

ACKNOWLEDGEMENT

The present study was supported by the University of Madras, Madras Christian College (Autonomous), Department of Zoology, Centre for Nanomaterial Research and Innovations (CNRI) MCC MRF, Madras Christian College (Autonomous), Vels Institute of Science, Technology and Advanced studies (VISTAS), Chennai, CIC Madurai Kamaraj University.

Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Ethical approval
 
Not applicable, as the study involved only pest species and no human subjects, cell lines, or animals.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article and no funding was received for this study.

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    Full Research Article

    In-vitro Assessment and Characterization of Pongamia Seed Oil-based Nano-Emulsion and ZnO Nano-formulations as Environmentally Sustainable Alternatives for the Control of Aulocophora foveicollis Lucas (Coleoptera-Chrysomelidae)

    R
    R. Jeethasri1
    V
    V. Sherene Victoria1
    D
    D. Surya1
    A
    Ananthi Rachel Livingstone1,*
    0000-0002-7603-1220
    1Department of Zoology, Madras Christian College (Autonomous), Tambaram-600 059, Tamil Nadu, India.

    ABSTRACT

    Background: Nano-emulsion formulation and ZnO-loaded nano-emulsion system was developed using the seed oil of Pongamia pinnata (L) to control the serious pest of cucurbit crops Aulocophora foveicollis Lucas.

    Methods: The formulations were prepared using high energy emulsification method (utrasonication) and were further characterized to determine the droplet size, polydispersity index, zeta potential using dynamic light scattering (DLS). Further, thermodynamic stress studies, viscosity, pH, UV- Visible spectroscopic analysis, TEM, ATR-FTIR analysis were carried out to evaluate the stability of nano-emulsion formulations. The efficacy of the prepared formulations is tested against the target pest (red pumpkin beetle) in vitro.

    Result: The results of characterization confirm the stability of nano-emulsion systems. The insecticidal efficacy of ZnO-loaded nano-emulsion system showed highest mortality (73.31%) and LC50 value was found to be 11.24% v/v. These results suggests that the formulated nano-emulsion system from the seed oil of pongamia holds potential to reduce the reliance on synthetic pesticides and offer environmentally sustainable alternative for the control of the insect pest.

    INTRODUCTION

    Cucurbits of Family-cucurbitaceae comprise economically important crops rich in dietary fibre, vitamins and minerals that supports metabolism and growth (Lal et al., 2014; Kaur et al., 2020 and Mondal et al., 2020). However, these crops are highly susceptible to insect pests, among which Aulocophora foveicollis Lucas, the most destructive pest affecting leaves, cotyledons causing stunted growth and death of the plant with infestation ranging from 30% to 100% (Sahu and Samal 2020; Dhillon et al., 2005). Despite the fact that pesticides reduce yield loss, their comprehensive usage pose serious threat to environment (Aktar et al., 2009). In this context, plant-based formulations and extracts exhibit potent eco-friendly bioactivity for effective pest management, contributing to sustainable and integrated agricultural practices (Shinde et al., 2025; Kikraliya et al., 2024; Iqbal et al., 2024). Pongamia pinnata (L) (Family- Fabaceae), commonly known as karanj (Kumar et al., 2006), produces seed oil that contains karanjin, pongamol, glabrin and has proven to exhibit insecticidal properties Kumar et al., 2006; Gadge et al., 2022). Although there is still lack of detailed study on its nano-emulsion and ZnO-loaded nano-emulsion formulations, especially regarding their stability and potential against insect pest, red pumpkin beetle. Nano-emulsions formulated from such seed oils and integration of nanoparticles provide greater efficacy against insect pests as they provide stability, controlled release, penetration for effective pest (Ghormade et al., 2011; Parisi et al., 2015). Therefore, this study aims.
    ► To analyse the chemical composition of pongamia seed oil using GC-MS analysis.
    ►  To formulate and evaluate the insecticidal efficacy of bulk emulsion of pongamia oil against the target pest A. foveicollis Lucas in laboratory conditions.
    ►  To formulate, characterise and assess the insecticidal potential of oil-in-water nano-emulsion and ZnO-loaded nanoemulsion of pongamia oil on the target pest in laboratory conditions.

    MATERIALS AND METHODS

    Sample collection and chemicals used
     
    Seed oil of Pongamia pinnata (L) was commercially purchased for the formulation of nano-emulsion. Analytical grade chemicals like absolute ethanol (99.9%) (Changshu Hongsheng Fine Chemicals Co.Ltd.), Tween 80 (Spectrochem Pvt. Ltd., India), Sodium Lauryl Sulphate (SLS/SDS- 90% extrapure) (SRL Pvt. Ltd., Mumbai, India), Zinc oxide nanoparticles (99% purity) (ADZnO Nanopowder, ADnano Technologies, Karnataka, India), Polyethylene glycol (PEG 6000, Bangalore Fine Chemicals, Bangalore, India) and double distilled water were used throughout the study. The study was carried out in the period of December 2024 to May 2025 in the Department of Zoology, Madras Christian College.
     
    GCMS analysis
     
    The oil sample was analysed using Gas Chromatography- Mass Spectrometry (GCMS) in electron ionization (EI) mode on a capillary GC column using helium as carrier gas. 1.0 µL of sample was injected with initial temperature at 50oC (2 mins), ramping at 10oC/minute to 300oC. The total run time was 30 minutes with ion source and interference temperature at 230oC and 280oC, respectively and mass spectra ranged between 40 and 600 m/z.
     
    Preparation of bulk emulsion
     
    The oil-in-water bulk emulsion was prepared using pongamia seed oil (10% v/v), distilled water (68% v/v), tween 80 (15% v/v) (non-ionic surfactant), ethanol (7% v/v) (co-surfactant). The components were mixed thoroughly using the magnetic stirrer at 500 rpm for 20 minutes.
     
    Preparation of nano-emulsion
     
    The oil-in-water nano-emulsion of pongamia oil was prepared by ultrasonication method. Sodium dodecyl sulphate (SDS) at 1% w/v was dissolved in water and magnetic stirred for 15 mins at 500 rpm. Tween 80 was added dropwise while stirring followed by ethanol and oil as shown in Table 2. The resulting pre-emulsion was ultrasonicated for 30 mins (33kHz, 650watts, 60% amplitude) and the formulation was subjected to thermodynamic stability tests (Fig 1a).

    Fig 1 (a), (b): Schematic representation of the development and characterization of Nano-emulsion system of pongamia oil.



    Thermodynamic stability test
     
    To evaluate the stability of the oil-in-water nano-emulsion formulations, different stress tests described by Shafiq et al., (2007) were performed. All the tests were triplicated.
     
    a. Centrifugation test: The oil-in-water nano-emulsion formulations were subjected to centrifugation at 3000 rpm for 15 minutes.
     
    b. Heating-cooling cycle: The formulation was kept at 4oC and 45oC for 48 hours each. Then the cycle was repeated thrice and checked for phase separation.
     
    c. Freeze- thaw stress: This was performed by subjecting the formulation at -21oC and +25oC for 48 hours each.
     
    Formulation with no phase separation observed was selected and further characterized.
     
    Preparation of ZnO-loaded nano-emulsion
     
    ZnO-loaded nano-emulsion was prepared by using 1% w/v SDS and 1% w/v PEG dissolved in distilled water. ZnO nanoparticles (0.25% w/v) were added and the pH is adjusted to 9.0 and the mixture is ultrasonicated for 30 mins. Separately, a pre-emulsion is prepared by using tween 80 (8% v/v), ethanol (3% v/v), pongamia seed oil (4% v/v) and added to ZnO mixture and then subjected to ultrasonication (33kHz, 650watts, 60% amplitude) for 30 mins. The resultant nano-emulsion were further characterized (Fig 1b).
     
    Characterization of Nano-emulsion and ZnO-loaded Nano-emulsion system of Pongamia seed oil
     
    pH Measurement
     
    The pH of the nano-emulsion and ZnO-loaded Nano-emulsion was measured using a digital pH meter.
     
    Viscosity
     
    The viscosity of both oil-in-water nano-emulsion and ZnO-loaded nano-emulsion was measured using Brookfield viscometer with spindle number 18 at rotation speed of 5.0 and a torque setting maintained around 28% to ensure accurate measurement.
     
    Particle size, zeta potential and polydispersity index (PDI)
     
    The droplet size of the nano-emulsion, zeta potential and polydispersity index (PDI) were measured using Dynamic Light Scattering (DLS), 200 µL of sample was introduced into a clean PTFE sample cell at room temperature using the instrument’s 180o backscatter configuration. Each measurement was performed in triplicates.
     
    UV- visible spectroscopic analysis
     
    For UV visible spectroscopic analysis, the samples were diluted 100-fold with distilled water at room temperature and measured using quartz cuvettes of 1 cm path length. The absorption spectra were recorded across a wavelength range of 200-800 nm. Distilled water was used a blank.
     
    Transmission electron microscopic (TEM) analysis
     
    The samples were diluted 1:100 with distilled water. A small aliquot (10 µL) of sample was placed on TEM grid and stained using 2% uranyl acetate followed by air drying at room temperature. Then the samples were observed under TEM.
     
    Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) analysis
     
    The chemical structure and molecular interactions of ZnO-loaded nano-emulsion system of pongamia oil was examined using FTIR-ATR, equipped with Sp10 software for data processing. A small volume of sample was placed onto the ATR crystal and the spectra were recorded over a range of 4000 cm-1 to 400 cm-1 at room temperature.
     
    Insecticidal assessment of bulk emulsion, Nano-emulsion and ZnO-loaded Nano-emulsion system and statistical analysis
     
    Adult beetles were reared in sterile plastic containers and the efficacy of bulk emulsion, nano-emulsion and ZnO-loaded nano-emulsion was evaluated by treating the target pests with each nano-emulsion system individually in following concentrations, 5%, 10%, 15%, 20% v/v for each treatment 10 insects were treated and the experiment is triplicated. Distilled water treated leaves were given as feed for control groups. Mortality was recorded for every 24 hours and the experiment was carried out for 120 hours. Results were statistically analysed using SPSS and MS OFFICE EXCEL. LC50 values were derived using Probit analysis. Significant differences among the treatment groups were analysed using one-way ANOVA and Tukey’s Post hoc HSD test.

    RESULTS AND DISCUSSION

    GCMS analysis
     
    The GCMS profile of pongamia seed oil sample identifies the presence of a diverse array of compounds namely fatty acids and their esters, aliphatic hydrocarbons, aromatic compounds and derivatives (Fig 2). The chemical compounds in Table 1 exhibit almost a similar trend to the findings of Abdul and Singh (2024); Purkait et al., (2021) and Seenuvasan et al., (2013). The compounds hexadecenoic acid, methyl ester (area- 2.09%, 6 octadecenoic acid, methyl ester (area- 8.06%), methyl stearate (1.27%), octadecanoic acid (3.73%) are found to have insecticidal and larvicidal properties according to Farag et al. (2021); Zayed et al. (2016); Ismail et al. (2022); Kannathasan et al. (2008) and Okonkwo et al. (2017).

    Table 1: Chemical constituents of pongamia oil (Pongamia pinnata L) identified by GCMS analysis.



    Fig 2: Chromatographic profile of pongamia oil (Pongamia pinnata) obtained by GCMS analysis.


     
    Thermodynamic stability test of prepared Nano-emulsion formulations
     
    The oil-in-water nano-emulsion pongamia seed oil passed thermodynamic stability stress conditions (Table 2). No phase separation, creaming was seen in the formulation (F4) of the nano-emulsion. Therefore, the formulation (F4) was selected for characterization.

    Table 2: Composition of Nano-emulsions formulation of pongamia oil.


     
    Characterization of Nano-emulsion and ZnO-loaded Nano-emulsion system of pongamia seed oil
     
    Particle size, zeta potential and polydispersity index (PDI)
     
    The particle size, zeta potential and polydispersity index (PDI) (Table 3) are one of the key factors for the stability of nano-emulsion (Anjali et al., 2012). The droplet size of nano-emulsion is between 20-500 nm (Pagar et al., 2019; Zhang et al., 2014). The average droplet size of nano-emulsion and ZnO-loaded nano-emulsion system of pongamia seed oil was found to be 216 nm and 210 nm respectively, contributing to the stability of the nano-emulsion system. Poly dispersity index (PDI) is a measure of homogeneity of droplet size in nano-emulsion. The PDI value ranging from 0-0.2 generally indicates strong narrow range size distribution of droplets in nano-emulsion system according to Baboota et al. (2007) and Sampathi et al. (2015). The PDI values resulted from the current study indicated narrow size distribution of droplets. The measure of electrical charge (zeta potential) on the surface of the droplets of nano-emulsion system influences its stability. The zeta potential value above ±30 mV is considered to be stable (Khalid et al., 2023). In this case the average zeta potential values -32.07 mV and -72.40 mV of samples indicated that the nano-emulsions were stable.

    Table 3: pH, viscosity, DLS analysis of Nano-emulsion and ZnO-loaded Nano-emulsion system of pongamia seed oil.


     
    UV- visible spectroscopic analysis
     
    The UV-Visible spectroscopic analysis of oil-in-water nano-emulsion of pongamia seed oil exhibited a characteristic absorption peak at 264 nm (Fig 3). This suggests a uniform and homogenous nano-emulsion system consistent with the reports of Ullah et al., (2022). The UV-visible absorption spectrum of ZnO-loaded nano-emulsion showed two distinct peaks at 289 nm and 370 nm (Fig  4). These findings confirm the successful incorporation and stable dispersion of ZnO nanoparticles in the emulsion system. This is in alignment with the previous study of Shamhari et al., (2018) and Enkhtuya et al., (2016).

    Fig 3: UV-visible absorption spectrum of oil-in-water Nano-emulsion of pongamia oil.



    Fig 4: UV-visible absorption spectrum of ZnO-ioaded Nano-emulsion system of pongamia oil.


     
    Transmission electron microscopic (TEM) analysis
     
    TEM analysis revealed spherical, well dispersed droplets in consistent with the results of DLS for both oil-in-water nano-emulsion and ZnO-loaded nano-emulsion system of pongamia seed oil (Fig 5 and Fig 6). The increased contrast and lack of free zinc oxide nanoparticles outside the droplets as observed in Fig 6 are indicative of the effective entrapment of zinc oxide nanoparticles within the droplets of nano-emulsion system.

    Fig 5: TEM image of Oil-in-water Nano-emulsion droplets of pongamia oil at 100 nm magnification (Scale bar:100 nm).



    Fig 6: TEM image of ZnO-ioaded Nano-emulsion droplets of pongamia oil (Scale bar: 1 µm).


     
    Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) analysis
     
    The FTIR spectrum (Fig 7) of ZnO-loaded nano-emulsion system of pongamia seed oil shows a characteristic peak indicating the presence of ZnO nanoparticles and other components of nano-emulsion system. Around 3400 cm-1, the broad O-H stretching band shows the presence of surface hydroxyl group and water in aqueous phase. A peak near 1630-1650 cm-1 represents C=O stretching in ester or carbonyl groups, indicating the organic constituents of nano-emulsions. The absorption band in 470-500 cm-1 corresponds to Zn-O stretching vibrations confirming the presence of ZnO nanoparticles. The strong Zn-O stretching vibration observed below 600 cm-1 confirms the presence of ZnO nanoparticles (Malaikozhundan et al., 2017). In another study by El-Saadony et al., (2024), the FTIR analysis confirmed the bond formation with absorption bands between 409- 588 cm-1. Subsequently, the presence of bands around 2850- 2950 cm-1 and 1740 cm-1 corresponds to C-H and C=O stretching respectively, associated with oil phase of nano-emulsion system (Rajiv et al., 2013; Ramesh et al., 2021).

    Fig 7: ATR-FTIR spectrum of ZnO-ioaded nanoemulsion system of pongamia oil.


     
    Insecticidal assessment of bulk emulsion, Nano-emulsion and ZnO-loaded Nano-emulsion system of pongamia oil
     
    The bulk emulsion showed minimal mortality of 26.65% at 20% v/v and 16.65% at 15% v/v, while lower concentration showed least efficacy. One-way ANOVA exhibits significant differences among treatments (F(3,8) = 6.250; p= 0.017 < 0.05) and Tukey’s HSD revealed 20% v/v as significantly distinct (Table 4, Fig 8). The efficacy of oil-in-water nano-emulsion at 20% v/v was found to be 43.32% and demonstrated a dose dependent effect (F (3,8) = 8.889, p=0.006 < 0.05), recording LC50 at 33.45% v/v. ZnO-loaded nano-emulsion exhibited highest mortality and outperformed both bulk and oil-in-water nano-emulsion with 73.31% mortality rate at 20% v/v and lowest LC50 value (11.24% v/v). One-way ANOVA revealed that there is significant difference among treatment groups (F (3,8) = 7.608, p= 0.010< 0.05), also reflecting dose-dependent mortality effect (Table 4, Fig 8).

    Table 4: Insecticidal bioassay of pongamia oil emulsions against Aulocophora foveicollis Lucas, serious pest of cucurbits.



    Fig 8: Comparative mortality rates (%) of Aulocophora foveicollis Lucas treated with Bulk emulsion, Nano-emulsion and ZnO-loaded Nano-emulsion of pongamia oil.


           
    A study by Stepanycheva et al. (2020) revealed that pongamia oil exhibited 100% mortality at 3% concentration on Frankliniella occidentalis. Similar study by Uçak et al. (2014) and Tran et al., (2017) demonstrated that karanj oil and leaf extract caused 100% mortality in F. occidentalis and Spodoptera litura respectively. Pongamia oil in combination with neem oil (PONNEEM) caused upto 90.78% feeding deterrence and DNA damage in Helicoverpa armigera at 2 ppm concentration Packiam et al. (2015). Malaikozhundan et al., (2017) demonstrated ZnO nanoparticles coated with Bacillus thuringiensis caused 100% mortality in Callosobruchus maculatus at 25 µg/mL concentration. Jameel et al. (2020) reported that ZnO nanoparticles induced oxidative stress in Spodoptera litura by generating reactive oxygen species and inhibiting key antioxidant enzymes. Similar study by Pittarate et al. (2021) demonstrated that ZnO nanoparticles caused oxidative stress in S. frugiperda, which led to body deformities, reduced fecundity and impaired life cycle progression. These studies consistently reveal that pongamia oil and its nano-emulsions particularly enhance by loading ZnO nanoparticles show potent insecticidal and larvicidal effect on target pests.
     
    Practical implications and future prospects
     
    Understanding the physiological and molecular mechanisms of insecticidal action of pongamia oil-based nano-emulsion formulations will provide essential insights. This will aid in developing more effective and targeted bio-pesticides, reducing the dependence on synthetic chemicals. These advances promote sustainable pest management by enabling eco-friendly and field applicable botanical formulations with minimal environmental impact.

    CONCLUSION

    The nano-emulsion system of pongamia oil formulated and characterized in this study provided excellent stability and greater efficacy against red pumpkin beetle, the destructive pest of cucurbits. This in-vitro study strongly supports the use of pongamia seed oil-based nano-emulsion formulation as an alternative approach to control the target pest reducing the reliance on synthetic pesticide.

    ACKNOWLEDGEMENT

    The present study was supported by the University of Madras, Madras Christian College (Autonomous), Department of Zoology, Centre for Nanomaterial Research and Innovations (CNRI) MCC MRF, Madras Christian College (Autonomous), Vels Institute of Science, Technology and Advanced studies (VISTAS), Chennai, CIC Madurai Kamaraj University.

    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Ethical approval
     
    Not applicable, as the study involved only pest species and no human subjects, cell lines, or animals.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article and no funding was received for this study.

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